production and composition of milk from 10 60 days...

Production and composition of milk from 10 – 60

days of lactation in mothers who delivered

prematurely

By

Ching Tat Lai (B.Sc., PostGrad. Dip. Sc., M.Sc.)

This thesis is presented for the degree of

Doctor of Philosophy

of

The University of Western Australia

School of Biomedical, Biomolecular and Chemical Sciences

Faculty of Life and Physical Sciences

The University of Western Australia

35 Stirling Highway Crawley, Western Australia, 6009.

Australia

2007

i

Preface

The work in this thesis was supervised by Professor Peter Hartmann, School of

Biomedical, Biomolecular and Chemical Sciences and Professor Karen Simmer,

School of Women’s and Infants Health, The University of Western Australia. I was

supported by a scholarship provided by Medela AG (June 2002- June 2005).

Financial assistance for the experimental work was provided by Medela AG.

Parts of this these have been presented at international conferences (2- 12) or

published as abstract (1 and 13), as follows:

1) Ching Tat Lai, Leon Mitoulas, Dorota Doherty, Karen Simmer and Peter E.

Hartmann (2006) Expression regimes and milk yield of preterm mothers in

Western Australia, 13th

International Conference for the International Society for

Research in Human Milk and Lactation conference, Niagara-on-the-Lake, Ontario,

Canada, (Sep, 2006)

2) Ching Tat Lai, (2006) Milk synthesis, expression, and composition of preterm

mothers, 4th

Amarillo conference, Richmond, Virginia, USA (Sep, 2006)

3) Ching Tat Lai (2006) Variation in milk composition during initiation and

establishment of lactation in mothers who deliver prematurely, Annual ILCA

conference for International Lactation Consultant Association, Philadelphia,

Pennsylvania, USA, (Sep, 2006)

4) Ching Tat Lai, Leon Mituolas, Karen Simmer and Peter Hartmann (2006)

Expression frequency and milk yield after preterm birth, 10th

Annual Congress for

Perinatal Society of Australia and New Zealand, Perth, Western Australia,

Australia, (Apr, 2006)

5) Ching Tat Lai (2005) Optimizing donor milk for babies with special needs,

3rd

Amarillo Conference, Amarillo, Texas, USA (Oct, 2005)

6) Ching Tat Lai (2005) Synthesis and secretion of breastmilk and the measurement

of milk production, 2nd

Amarillo Conference, Amarillo, Texas, USA (Oct, 2005)

7) Ching Tat Lai (2005) Breastmilk fortification for preterm infants, Conference for

New Zealand Lactation Consultants Association, Auckland, New Zealand (May,

2005)

8) Ching Tat Lai (2005) Breastmilk production and composition in pre-term mothers,

Conference for New Zealand Lactation Consultants Association, Auckland, New

Zealand (May, 2005)

9) Ching Tat Lai (2004) Variation in the composition of breastmilk and its

fortification for pre-term babies, 2nd

Amarillo Conference, Amarillo, Texas, USA

(Oct, 2004)

ii

10) Ching Tat Lai (2004) Variation in the composition of breastmilk and its

fortification for pre-term babies, Conference for Breastmilk production,

composition and evidence based assessment of human lactation, Toronto, Ontario,

Canada (Oct, 2004)

11) Ching Tat Lai (2004) 'Giving the best of the breast’- the new way in fortifying

mother’s own milk, 7th

Biennial Conference for Australian Lactation

Consultants’ Association Ltd, Parramatta, New South Wales, Australia (Sep,

2004)

12) Ching Tat Lai (2004) Tailoring mother’s own milk for her pre-term babies,

Annual conference for College of Lactation Consultants, Scarborough, Western

Australia, Australia (Sep, 2004)

13) Ching Tat Lai, Tom Hale, Peter E. Hartmann and Jacqueline Kent (2004) The

hourly rate of milk synthesis, 12th

International Conference for the International

Society for Research in Human Milk and Lactation conference, Cambridge,

United Kingdom, (Sep, 2004)

iii

Acknowledgments

I don’t know how I would have completed this thesis without the assistance of

following people and it is here that I would like to thank and acknowledge them for

their help.

I am indebted to Professor Peter E. Hartmann and Professor Karen Simmer for

their guidance and assistance. Especially during the time when I had doubts about the

project, they always showed me different ways to approach the question and let me

see possibility out of impossibility.

I would like to gratefully acknowledge the generous financial support of

Medela AG for my research.

Almost all of the work presented in this research required the participation of

volunteer mothers in the studies and without these mothers this work would not have

been possible.

I would like to thank Ms Sharon Zuiderduyn, Ms Michelle Lewis and Ms

Bronwyn Davis for the recruitment of mother in the NICU.

I would like to acknowledge Professor Thomas W. Hale and Professor Peter E.

Hartmann for their initial idea of the hourly pumping experiment on lactating mothers.

The collaboration between Professor Hale (Texas Tech University) and us

(UWA/KEMH) on this study further enhance our knowledge about the effect of breast

milk removal on the synthesis of milk in the breasts.

I am very grateful to Dr. Dorota Doherty for the statistical advice provided for

the statistical analysis of my research. I also thank her for her enthusiasm and interest

in the study.

I would like to thank Ms Tracey Williams for the help in sample preparation

and the fat analysis of 2000+ samples. I also like to acknowledge the assistant from

her and Dr. Jacqueline Kent for the hourly pumping experiment.

I would like to thank Dr Naomi Trengove for her guidance in the completion

of this thesis.

A million thanks to my colleagues in the lab for all their humor, wisdom and

support.

My thanks to my wife, Dr. Lisa Ng for all the love, patience and support.

My thanks to mum, dad and my sister for all their support and patience.

iv

Table of Contents

Preface i

Acknowledgements iii

Table of contents iv

List of tables vii

List of figures ix

List of abbreviations xii

Abstract xiii

Chapter 1: Literature review

1.1 Introduction 1

1.2 Anatomy of the breast 3

1.3 Synthesis of milk 4

1.3.1 Lactogensis I 4

1.3.2 Lactogensis II 4

1.4 Regulation of milk synthesis 5

1.4.1 Endocrine Control 5

1.4.1.1 Progesterone 6

1.4.1.2 Glucocorticoids 6

1.4.1.3 Prolactin 6

1.4.1.4 Human Growth Hormone 7

1.4.1.5 Insulin 8

1.4.1.6 Oxytocin 8

1.4.2 Autocrine Control 9

1.4.2.1 Feedback Inhibition of Lactation 10

1.5 Milk composition 10

1.5.1 Fat 11

1.5.2 Nitrogen 12

1.5.2.1 Non-protein Nitrogen 12

1.5.2.2 Protein 12

Casein 13

Whey 14

Immunoglobulins 14

Lactoferrin 14

Lysozyme 15

Alpha- lactalbumin 16

1.5.3 Carbohydrates 16

1.5.3.1 Lactose 17

1.5.3.2 Oligosaccharides 17

1.6 Preterm mothers 18

1.6.1 Milk production 19

1.6.2 Milk composition 20

1.7 Aims of the project 22

v

Chapter 2: Materials and methods

2.1 Materials 24

2.2 Equipment 26

2.3 Human subjects 27

2.4 Sample Treatment 27

2.5 Biochemical Analyses 27

2.5.1 Lactose 27

2.5.2 Total protein assay 28

2.5.3 Enzyme Linked Immunosorbent Assay (E.L.I.S.A) 30

2.5.3.1 sIgA 30

2.5.3.2 Lactoferrin 31

2.5.4 Esterified Fat Assay 32

2.6 Statistical analysis 33

Chapter 3: Frequent expression of breastmilk and milk synthesis in mothers of term

babies

3.1 Introduction 34

3.2 Methods 35

3.2.1 Subjects 35

3.2.2 Criteria for recruitment 36

3.2.3 Determination of hourly pumping 36

3.2.4 Determination of 24 hour milk production 36

3.2.5 Statistical analysis 36

3.3 Results 37

3.3.1 Subjects 37

3.3.2 Milk production per 24 hours 37

3.3.3 Milk production per hour 37

3.4 Discussion 45

Chapter 4: Milk production of preterm mothers between days 15 and 20 postpartum

4.1 Introduction 50

4.2 Methods 51

4.2.1 Subjects 51


4.2.3 Data recording 51

4.2.4 Maternal record 52

4.2.5 Graphic presentation and statistical analysis 52

4.2.6 Calculation 52

4.3 Results 52

4.3.1 Expression regimes and milk production of preterm mothers 52

vi

4.3.2 Variability of milk volume, interval since last expression and

rate of milk removal

53

4.3.3 Proportion of 24hr milk yield and interval since previous

expression

60

4.4 Discussion 76

Chapter 5: Longitudinal changes of macronutrients and milk production of preterm mothers’ milk from day 10 to 60 postpartum

5.1 Introduction 83

5.2 Methods 84

5.2.1 Subjects 84


5.2.3 Data recording 84

5.2.4 Maternal record 85

5.2.5 Biochemical analysis 85

5.2.6 Graphical presentation and statistical analysis 85

5.2.7 Calculation 86

5.3 Results 87

5.3.1 Subjects 87

5.3.2 Frequency of expression/breasfeeding , milk production and

composition

88

5.3.2.1 Circadian changes 88

5.3.2.2 Consecutive day changes- days 15 to 20 postpartum 101

5.3.2.3 Longitudinal changes- days 10, 15-20, 30, 40, 50 and 60

postpartum

114

5.4 Discussion 133

Chapter 6: General discussion and clinical implications

6.1 General discussion 142

6.2 Clinical implications 147

References 154

vii

List of tables


2.1 Enzymes for biochemical assays 24

2.2 Anibodies for ELISA 24

2.3 Other chemical reagents 25

2.4 Equipment 26

Chapter 3: Frequent expression of breastmilk and milk synthesis in women

3.1 Age and stage of lactation all participating women 38

3.2 Breastfeeding data of all participating women 39

3.3 Hourly pumping data of all participating women (left breast) 40

3.4 Hourly pumping data of all participating women (right breast) 41


4.1Overall CVs of milk volume, interval since previous expression and

hourly rate between days 15 and 20 from each breast of all mothers 61

4.2 Within mother variability 62

4.3 Number of breasts paired and unpaired in each group 64

4.4 Mean daily milk production, interval between expressions and duration

of expression per breast at days 15 - 20 postpartum grouped according to

the R-square for the proportional milk yield

66


5.1 Abbreviations for variables used in the statistical analysis 86

5.2 Demographic and obstetric data of preterm mothers

and their babies (continuous variables)

87

5.3 Demographic and obstetric data of preterm mothers

and their babies (class variables)

87

5.4 Number of mothers who were exclusively expressing, partially

expressing/breastfeeding and exclusively breastfeeding at day 10, 15-20,

030, 40, 50 and 60 postpartum

88

viii

5.5 Median, IQR and range of milk production and component of left breast

during the day

90

5.6 Median, IQR and range of milk production and component of right breast

during the day

91

5.7 Median, IQR and range of milk production of left breast from day 15 to

20 postpartum

102

5.8 Median, IQR and range of milk production of right breast from day 15 to

20 postpartum

102

5.9 Median, IQR and range of milk component of left breast from day 15 to

20 postpartum

103

5.10 Median, IQR and range of milk component of right breast from day 15

to 20 postpartum

104

5.11 Median, IQR and range of sIgA and lactoferrin of left breast from day

15 to 20 postpartum

105

5.12 Median, IQR and range of sIga and lactoferrin of right breast from day

15 to 20 postpartum

105

5.13 Median, IQR and range of milk production of left breast from day 10 to

60 postpartum

118

5.14 Median, IQR and range of milk production of right breast from day 10

to 60 postpartum

118

5.15 Median, IQR and range of milk component of left breast from day 10 to

60 postpartum

119

5.16 Median, IQR and range of milk component of right breast from day 10

to 60 postpartum

120

5.17 Median, IQR and range of sIgA and lactoferrin of left breast from day

10 to 60 postpartum

121

5.18 Median, IQR and range of sIga and lactoferrin of right breast from day

10 to 60 postpartum

121

5.19 Statistical difference in the concentration of lactoferrin between days

10, 15-20, 30, 40, 50 and 60 postpartum (left breast). 122

5.20 Statistical difference in the concentration of lactoferrin between days

10, 15-20, 30, 40, 50 and 60 postpartum (right breast).

122

5.21 Fat content of preterm milk at day 15, 30 and 60 postpartum by current

and other studies

137

5.22 Protein concentration of preterm milk at day 15, 30 and 60 postpartum

by current and other studies

138

5.23 Lactose concentration of preterm milk at day 15, 30 and 60 postpartum

by current and other studies

139

5.24 Energy content of preterm milk at day 15, 30 and 60 postpartum by

current and other studies

140

ix

List of figures


2.1 Standard curve of human milk protein using the Bio-Rad protein assay 29

Chapter 3: Frequent expression of breastmilk and milk synthesis in women

3.1 Milk volume at each pumping in an hourly pumping session 42

3.2 Milk volume for each pumping of a mother (M4) at three different hourly

pumping sessions

43

3.3 Average hourly rate of milk production from hourly pumping and from

24-h production

44


4.1 Total volume per day per breast from day 15 to 20 postpartum 54

4.2 Frequency of expression from day 15 to 20 postpartum 55

4.3 Duration of an expression per day 56

4.4 Frequency of expression and total milk volume per breast of individual

mothers (C01, C02, C17 and C31) for days 15 to 20 postpartum

57

4.5 Mean volume and frequency of expression per day for days 15-20 58

4.6 Volume of milk per expression for days 15 to 20 postpartum 59

4.7 Milk volume and hour rate during day 15-20 postpartum 63

4.8 Group 1, mother C08 67







4.15 Group 1 (left breast) and group 3 (right breast), mother C11 69

4.16 Group 1 (right breast) and group 2 (left breast), mother C18 70


4.18 Group 2, motherC02 70








x



4.28 Group 3 (left breast) and group 4 (right breast), mothe C19 74



4.31 mother C10 75

4.32 mother C26 75


5.1 Frequency of expression/breastfeed during the day 92

5.2 Milk volume per expression /breastfeed during the day 93

5.3 Fat content per expression /breastfeed during the day 94

5.4 Total milk protein concentration per expression /breastfeed during the

day

95

5.5 Nutritive protein per expression /breastfeed during the day 96

5.6 Lactose concentration per expression /breastfeed during the day 97

5.7 Energy per expression /breastfeed during the day 98

5.8 sIgA concentration per expression /breastfeed during the day 99

5.9 Lactoferrin concentration per expression /breastfeed during the day 100

5.10 Percentage of difference in daily milk production between left and right

breasts

106

5.11 Fat content per breast per mother for days 15 to 20 postpartum 107

5.12 Total protein concentration per breast per mother for days 15 to 20

postpartum

108

5.13 Nutritive protein per breast per mother for days 15 to 20 postpartum 109

5.14 Lactose concentration per breast per mother for days 15 to 20

postpartum

110

5.15 Energy content per breast per mother for days 15 to 20 postpartum 111

5.16 sIgA concentration per breast per mother for days 15 to 20 postpartum 112

5.17 Lactoferrin concentration per breast per mother for days 15 to 20

postpartum

113

5.18 Frequency of expression/breastfeed per breast per day per mother from

day 10 to 60 postpartum

123

5.19 Milk volume per breast per day per mother from day 10 to 60

postpartum

124

5.20 Fat content per breast per day per mother from day 10 to 60 postpartum 125

5.21 Total protein concentration per breast per day per mother from day 10 to

60 postpartum

126

5.22 Nutritive protein per breast per day per mother from day 10 to 60

postpartum

127

5.23 Lactose concentration per breast per day per mother from day 10 to 60

postpartum

128

5.24 Energy per breast per day per mother from day 10 to 60 postpartum 129

xi

5.25 sIgA concentration per breast per day per mother from day 10 to 60

postpartum

130

5.26 Lactoferrin concentration per breast per day per mother from day 10 to

60 postpartum

131

5.27 The regression analysis between sIgA (g/l) and lactoferrin (g/l) 132

xii

List of abbreviations

2,2’-azino-di-[3-ethylbanzthiaoline sulfonate] ABTS

Acyl-CoA Synthetase ACS

ß-Galactosidase ß-Gal

Bio- Rad protein assay dye reagent concentrate Protein dye

Bovine serum albumin (fatty acid free) BSA

Coefficient of variation CV

Correlation coefficient r

Di-potassium hydrogen orthophosphate 3-hydrate K2HPO4

Feedback Inhibitor of Lactation FIL

Ferric chloride FeCl3

Glucose Oxidase GOD

Human lactoferrin H- lactoferrin

Human sIgA H- sIgA

Hydrochloric acid HCl

Hydrogen peroxide H2O2

Inter-quartile range IQR

Lanthanum chloride LaCl3

Necrotizing enterocolitis NEC

Nitric Acid HNO3

Orthophosphoric acid H3PO4

Peroxidase POD

Polyoxyethylenesorbitan monolaurat Tween 20

Potassium chloride KCl

Potassium dihydrogen orthophosphate KH2PO4

Potassium hydroxide KOH

Potassium phosphate K3PO4

R- square R2

Standard deviation SD

Standard error of the mean SEM

Sodium azide NaN3

Sodium chloride NaCl

Sodium hydroxide NaOH

Sodium phosphate, dibasic, heptahydrate Na2HPO4.7H2O

Sulphuric acid H2SO4

Trichloroacetic acid TCA

xiii

Abstract

Mothers who deliver prematurely often have a delay in lactogenesis II and

subsequent milk supply. Furthermore, due to the inability of their babies to breastfeed

immediately after birth, these mothers are ‘pump dependent’ during both initiation

and establishment of lactation. Apparently, there are no evidence based guidelines for

the expression regime but some data suggesting that expression regimes for both

breasts should be at least five times per day and at least 100 minutes expressing time

per day.

The project was set out to document the self selected current expression

regimes of the preterm mothers from day 10 to 60 postpartum. It defined how various

aspects of breast expression, such as frequency and interval, impact on the synthesis

and production of milk. In addition, it determined the variations in the composition of

preterm mother’s milk.

The collection of 24hr expression data and milk samples at each expression of

each breast, each day, of 25 preterm mothers (<32 gestation age) from the neonatal

intensive care unit in King Edward Memorial Hospital, Western Australia on day 10,

15-20, 30, 40, 50 and 60 postpartum showed that during the ‘pump dependent’ period

(day 10 – 20), the frequency of expression for both breasts was 6, 6-7, 3-9 times per

day (median, IQR, range) and total duration with the pump was 115, 80-160, 32-320

minute per day (median, IQR, range). Furthermore, during the ‘transition from

exclusively expressing to exclusively breastfeeding’ period (day 30-60), frequency of

expression/breastfeed and total duration of milk removal (both expressing and

breastfeeding) for both breasts were 6, 5-7, 1-9 and 135, 75-170, 25-320, respectively

(median, IQR, range). This indicated that these mothers were within the current

expression recommendation. However, the intervals between expressions were 3.33,

2.67- 4.00, 0.50- 13.80 hour and 3.33, 2.37- 4.15, 0.01- 19.50 hour (median, IQR,

range), on day 10-20 and day 30-60 respectively, suggesting that mothers were self-

selecting their expression regime within the recommended frequency and duration of

expression.

Previous studies had only related the expression regime to the daily milk

production of the mothers. However, due to the fact that the physiology of each

breast can be different within the same mother, it is important to study the effect of

the expression regime on the milk yield of individual breast in order to fully

xiv

understand the impact of the expression regime on mother’s daily milk production.

Milk synthesis is regulated by both systemic and autocrine (local) mechanisms, the

latter being related to volume of milk removed during an expression. Thus, there is a

general perception that increased frequency of expression will result in an increased

volume of milk. However, when term mothers (n=19) were expressing at hourly

intervals in a controlled environment, there was no increase in milk yield per breast

from subsequent breast expression during the day but rather a constant yield of milk

was obtained from the 3rd

hourly expressions onward up to 8hr). I have called this the

intrinsic rate of milk synthesis. Furthermore, the intrinsic rate of milk synthesis was

equivalent to the hourly rate of milk synthesis by 24hr test weight method suggesting

that there is a constant rate of milk synthesis for an individual breast under conditions

that minimize the influence of the autocrine (local) mechanism. Therefore, the timing

of frequency (interval between expressions) rather than frequency of expression itself

would have a greater impact on milk production of the mothers because the interval

between expressions could directly affect the level of autocrine regulation on milk

synthesis which masks the real synthetic capacity of the breast.

There was a large variation in milk production per breast from day 10 to 60

postpartum and it was very difficult to assess the effect of the interval between

expressions on milk production. However, when milk production was expressed as a

proportion (%) of 24hr milk production (actual milk yield), it permitted the analysis of

the impact of the interval since previous expression on milk yield of each breast.

There was a significant correlation between the interval since previous expression and

the actual milk yield for 40 of the 46 breasts (87%). However the proportion of the

actual milk yield that could be explained by variation in the interval since previous

expression ranged from 12 to 73%, suggesting that some breasts would be more

responsive to interval changes than other breasts.

Optimizing milk production of preterm mothers ensures the supply of essential

macro- and micro- nutrients for the growth and development of their babies. The

intensive milk sampling used in this thesis showed the content of fat and

concentration of lactose gradually increased and reached a plateau from day 30

postpartum, while concentrations of total protein, nutritive protein, sIgA and

lactoferrin declined as lactation progressed. There were large variations in the

concentration of macronutrients (fat, total protein and lactose) between mothers

from day 10 – 60 postpartum. The content (median, IQR and range) of fat was 44.6,

xv

37.2-53.5, 0.65-182.7g/l; total protein was 16.9, 13.3-21.5, 3.5-83.3g/l and lactose

was 65.9, 58.7-73.8, 29.5-115.9g/l. These nutrients make up the energy content of

milk, thus the energy content of milk also varied greatly between mothers. Therefore,

milk from individual preterm mothers varies greatly for individual values for fat, total

protein, lactose and energy and this should be taken into account when calculating the

level of fortification required for individual babies. The results suggest that when

fortifying mother’s milk, weekly measurement of fat and protein in milk would

provide good estimates on which to base fortification requirements.

The concentration of sIgA plus lactoferrin formed 32% of the total proteins in

breastmilk. However there was large variations in the concentration of sIgA and

lactoferrin (median, IQR, range: 0.82, 0.59-1.13, 0.05-2.93g/l and 2.41, 1.52-3.52,

0.04-8.82g/l, respectively) between mothers. Therefore the level of protection

provided by these two proteins could differ greatly between babies. Further research

on the relationship between the concentration of sIgA and lactoferrin in milk and the

onset of infection would indicate the minimum amount of these proteins needed for

the babies to benefit from the immune protection provided by their mother’s milk.

The hourly breast expression method and regression analysis of actual milk

yield and interval since previous expression provides information that identifies the

potential milk synthesis capacity of the breasts of the mothers and the impact of the

interval between expressions on the milk production of the mothers. This information

can be applied to individualize the interval between expression regimes to optimise

milk production and minimize the demand on the mother. In addition, determining

the changes in the milk composition of individual mothers would provide a more

precise base to fortify their milk for their preterm babies.

1

Chapter 1

Literature Review

1.1 Introduction

Most of the components of human milk are synthesized in mammary glands

(breasts) of mothers and the rate of synthesis is regulated by the hormonal (endocrine

and autocrine) changes during pregnancy and lactation (Knight et al., 1998; Wilde et

al., 1998). Human milk contains carbohydrate, protein and fat; and concentrations

of these macronutrients are optimal for the growth and development of the term baby.

In addition, human milk contains immune properties, such as secretory IgA (sIgA)

and lactoferrin, that protect the baby against infections and diseases (Hanson et al.,

1985b; Telemo & Hanson, 1996; Tanaka et al., 1999; Lonnerdal & Lien, 2003).

Preterm mothers often have a normal pregnancy up until the third trimester but

deliver their babies at less than 37 weeks gestation (Joffe & Wright, 2002). This

early delivery disrupts the pattern of hormonal changes that regulate breast

development and milk synthesis. Therefore preterm mothers often have difficulty

initiating and maintaining their lactation. Furthermore, their babies, especially those

less than 32 weeks gestation, are often sick and do not fully develop the co-ordination

of suck, swallow and breathe. Thus these mothers cannot directly breastfeed their

babies and have to exclusively express their milk from their breasts either by hand or

with a breast pump (manually or electric).

There is no particular expression regime for preterm mothers to express milk

from their breasts. However the positive relationship between the frequency of

expression and milk production found in both dairy (Wilde & Knight, 1990) and

human (DeCarvalho et al., 1985) studies suggests that preterm mothers should be

encouraged to express as often as possible to obtain an adequate production of milk.

In general, mothers are advised to express their breasts at least 5 times per day with

approximately 100 minutes of total expression time per day (Hopkinson et al., 1988).

With this practice, most preterm mothers can obtain an adequate production of milk,

but some mothers still have trouble producing enough milk for their babies. Mothers

with low milk supply are often advised to increase the frequency of expression.

However an increase in the frequency of expression is very demanding and puts

additional pressure on mothers at a time of considerable stress.

2

There has been no evolutionally pressure for mothers of preterm babies to

produce milk of a composition and quantity that is optimal for preterm babies because

these babies did not survive. However, in the past 30 years, medical advancements

have resulted in the survival of babies born as early as 23 weeks of gestation. The

provision of optimal nutrition is crucial for both the survival and maintenance,

desirable growth and development of preterm babies. It was first suggested that

preterm mothers’ own milk with minimal supplementation would be enough to

sustain the life of preterm babies (Atkinson et al., 1980b). However the theoretical

estimation of the protein requirement for preterm babies by Foman et al. (1977)

together with the slow weight gain of babies fed unfortified human milk (Davies,

1977) indicated that human milk alone provided inadequate nourishment for the

growth and development of preterm babies (Arslanoglu et al., 2006; Rigo & Senterre,

2006). Therefore, it is common to fortify preterm mothers’ milk with human milk

fortifiers to meet the needs of preterm babies. Currently fortification protocols are

based on an assumed composition of mother’s own milk and exclude adjustments for

possible variation in milk composition between mothers largely because the extent of

the variation between mothers has not been documented in detail. The average

values used as references for the fortification were derived from milk analysis of

either single or pooled milk samples of mothers between 26 to 37 weeks of gestation

(Atkinson et al., 1980b; Lemons et al., 1982; Butte et al., 1984), therefore they maybe

unsuitable references to cover a wider range of gestation age (23-37 weeks). Although

reports suggest that the composition of preterm mother's milk, like term mother's

milk, changes as lactation progresses (Anderson et al., 1983; Butte et al., 1984;

Lepage et al., 1984; Paul et al., 1997; Maas et al., 1998), the use of average values

does not account for any possible variation in composition of milk for mothers at

different stages of lactation once full enteral feeding has been established. Therefore

without considering such variation, the fortified human milk may not meet the target

level of nutrients that are required by these babies.

Most components in human milk provide essential nutrients for human babies,

but many components carry out specific functions that assist the protection and

development of babies. SIgA and lactoferrin are two proteins in human milk that

provide protection against a variety of infections and diseases (Hanson et al., 1985b;

Hanson et al., 1995). The concentration of sIgA plus lactoferrin makes up

approximately 32% of total protein in term mothers’ milk during established lactation

(Lonnerdal & Atkinson, 1995). Furthermore, the concentrations of these proteins in

3

preterm mothers’ milk are similar to that in term mothers’ milk (Montagne et al.,

1999). In this connection, these proteins may be important for the survival of the

preterm babies because their immunity is extremely immature and so they are

susceptible to developing life-threatening infections, such as sepsis and necrotizing

enterocolitis. Therefore, it is also important to consider variation in the

concentration of these proteins between mothers and with stage of lactation.

1.2 Anatomy of the breast

Briefly, the breast is located, vertically, between the second and sixth ribs and,

horizontally, between the parasternal and midaxillary line (Vorherr, 1974).

Externally the nipple and areola of the breast are apparent. Internally the breast

consists of glandular, connective and adipose tissues with little nervous tissue present

except that found associated with the nipple and areola (Vorherr, 1974). The smallest

elements of the glandular tissue are the lactocytes (mammary secretory epithelial

cells). The lactocytes are cuboidal to columnar in shape and are morphologically

and functionally polarised (Pitelka & Hamamoto, 1983). They are arranged in a

single layer around a lumen, forming an alveolus. Each alveolus is surrounded by a

network of starlike, branching myoepithelial cells responsible for contracting and

ejecting the milk from the alveoli lumen into very small ducts leading from each

alveolus (Hartmann, 1991). The very small ducts join to form larger ducts draining

the lobules. These larger ducts then merge into one milk duct for each lobe and

there are approximately nine milk ducts that extend to the nipple in each lactating

breast (Ramsay et al., 2004). The adipose tissue in the breast can be found between

and around the lobes whilst fibrous, connective ligaments hold the adipose and

glandular tissues together, giving the breast its structure (Cooper, 1840; Riordan,

2005a).

The breast is highly vascularized and ninety percent of its blood is supplied

through the internal mammary and lateral thoracic arteries (Vorherr, 1974). Blood

supply is not constant and changes in response to certain stimuli e.g. breastfeeding

(Janbu et al., 1985). Innervation of the breast glandular tissue is minimal when

compared to the nipple and areola where sensory innervation is extensive and consists

of both autonomic and sensory nerves (Lawrence & Lawrence, 1999). Apart from

some nerve endings found in the mammary lobules nearest the major ducts, the

glandular tissue is virtually devoid of nerve fibres (Hartmann, 1991) with the majority

of nerves following arteries and arterioles (Lawrence & Lawrence, 1999).

4

Furthermore, there is no evidence of either motor secretory innervation or of ganglia

in the breast nor of innervation of the myoepithelial cells (Lawrence & Lawrence,

1999). These observations suggest that milk synthesis and secretion are both

independent of neural stimulation (Zaks, 1962).

1.3 Synthesis of milk

The synthesis of milk in the mammary gland starts during late pregnancy in

women. Certain hormonal changes in pregnancy cause the gland to proliferate and

the lactocytes in the gland begin to synthesize milk constituents. There are two

stages in the development of milk synthesis in women: lactogensis I and II.

1.3.1 Lactogenesis I

In the weeks after conception, mammogenesis presents the first changes in the

breast which results from the hormonal stimulation of the existing structure of the

breast, i.e. lengthening the milk ducts and cell differentiation (Vorherr, 1974). The

use of the Computerized Breast Measurement (CBM) system has shown, on average,

that an increase in breast size commenced at week 10 of pregnancy (Cox et al., 1999).

Lactogensis I is defined as the time when the breast develops the capacity to

synthesize the specific milk components (lactose, casein, and alpha-lactalbumin) and

these components begin to accumulate in the breast (Kulski & Hartmann, 1981).

Since the tight junctions between lactocytes remain open during pregnancy, these

components, especially lactose, can escape into the bloodstream through the

paracellular pathway (Neville & Allen, 1983; Neville & Casey, 1986). Hence by

measuring the lactose concentration in urine, Cox et al (1999) were able to determine

that lactogenesis I occurred about mid pregnancy in women.

1.3.2 Lactogenesis II

Lactogenesis II is defined as the initiation of copious milk secretion and it

commences 30-40 hours post-partum in healthy women who had delivered at term

(Arthur et al., 1989b). About a day after this time women often experience a sudden

feeling of breast fullness and their milk supply is said to be "coming in" and it has

long been assumed that this was a marker of the initiation of lactation in women

(Rendle-Short & Rendle-Short, 1966). Due to the closure of the tight junctions

between lactocytes at lactogensis II, milk components that are synthesized in the

5

lactocytes, are then predominantly retained in the alveolar lumen before they are

transferred to the nipple during either a breastfeed or breast expression (Neville &

Allen, 1983). Therefore the measurement of milk components (lactose, fat, citrate,

sodium, potassium and protein) as well as the daily milk production are means of

determining the time of lactogensis II in lactating women (Hartmann & Arthur, 1986;

Arthur et al., 1989a).

In the early stage of lactogensis II (ie the first few days of postpartum), the

milk is defined as colostrum. It is a yellowish and thick fluid which contains high

concentrations of protein, sodium, potassium and chloride but low in fat, citrate and

lactose.

Colostrum is rich in antibodies, that provide protection against pathogenic

bacteria and viruses that are present in the birth canal and associated with other

human and environmental contact. It also facilitates the establishment of bifidus

flora in the digestive tract of the infant and antioxidants which may function as traps

for neutrophil generated reactive oxygen metabolites (Buescher & McIlheran, 1988).

At about 1-2 days after lactogensis II, the milk becomes thinner with pale

bluish colour as the result of a ‘fully’ productive breast. The concentration of

protective proteins is decreased while fat and lactose increases. Even with the

production of mature milk, there are large variations in composition and production of

milk within and between women (Mitoulas et al., 2000). The variation continues

until approximately day 30 postpartum when the composition and production of milk

become more stabilized (Anderson, 1985).

1.4 Regulation of milk synthesis

1.4.1 Endocrine Control

The breast is one of the major endocrine target organs of the body. Hormones

secreted from the pituitary, adrenals, thyroid, ovaries and placenta will all have some

effect on the breast at different stages of a woman's life. During pregnancy and

established lactation the major hormones acting on the breast are those involved with

the initiation and maintenance of milk synthesis. These hormones include

progesterone, estrogen, glucocorticoids, human placental lactogen, prolactin, growth

hormone, insulin and oxytocin.

6

1.4.1.1 Progesterone

Lactogenesis II in rats was first shown to be triggered by the withdrawal of

progesterone (Kuhn 1969) and asimilar control mechanism occurs in other animals

including women (Kuhn, 1969; Hartmann et al., 1995). Rosen et al. (1978) found that

progesterone inhibited casein mRNA and polysomal casein synthesis in the rat

mammary gland during pregnancy. In women, the retention of placental fragments

after delivery was observed to inhibit lactogenesis II, thus suggesting that it is the

progesterone synthesised by the placenta that inhibits lactogenesis II during

pregnancy (Neifert et al., 1981). Hence the delivery of the placenta at parturition leads

to the reduction of progesterone in the maternal blood, which in term triggers the

synthesis of lactose in the lactocytes (Kulski et al., 1977; Cox et al., 1999; Neville,

1999). Since it is the delivery of the placenta that initiates the fall in progesterone,

lactogenesis II is therefore delayed until progesterone clears from the circulation,

some 30 to 40 hours post-partum.

1.4.1.2 Glucocorticoids

In the rat, glucocorticoids have been shown to play a regulatory role in the

synthesis of fat, lactose, and protein (Korsrud & Baldwin, 1972b, 1972c, 1972a).

Experiments using adrenalectomized rats deprived of the foetoplacental unit showed

that without corticosteroids in the late pregnancy, the concentration of lactose in the

mammary tissue and the total wet weight of mammary tissue were significantly

decreased. When these rats were treated with corticosteroids, milk secretion was

restored (Nicholas & Hartmann, 1981). Furthermore in mice, corticosterone has

been shown to increase the ability of the lactocytes to bind prolactin (Sakai et al.,

1979) and that without corticosterone the expression of messenger RNA for the

prolactin receptor within the lactocytes is low (Mizoguchi et al., 1997).

1.4.1.3 Prolactin

Prolactin is a 23 kDa peptide, mainly synthesised in lactotrophic cells of the

anterior pituitary (Henninghausen & Robinson, 1997). Prolactin is critical for the

establishment and maintenance of lactation in women and other mammals (Ostrom,

1990). This has been clearly illustrated by the immediate fall in milk production in

rabbits after the administration of the ergot alkaloid bromocriptine, an inhibitor of

7

prolactin release from the anterior pituitary (Cowie et al., 1980) and the suppression

of the initiation of lactation by bromocryptine in women (Kulski et al., 1978).

Levels of prolactin in the blood are highest in early lactation (Jacobs, 1977)

and decline as lactation progresses (Cox et al., 1996). This observation suggests that

the function of prolactin may differ during the course of lactation. Prolactin

concentration in the blood has been found to follow a circadian rhythm, being higher

at night when compared to the day (Neville, 1999). In addition, prolactin is secreted

in response to suckling, peaking 45 minutes after the start of the feed (Noel et al.,

1974). It has been shown that nipple stimulation in the absence of breastfeeding will

result in the release of prolactin in lactating and non-lactating women (Frantz, 1977).

Prolactin also has been found in breast milk (Cox et al., 1996). Prolactin binds to an

integral membrane receptor on epithelial cells before being transported into the milk

through the secretory cell (Ollivier-Bousquet et al., 1993).

In women, mammary engorgement, milk ejection and an increase in milk fat

content have all been observed with increased prolactin levels in the blood (Tyson et

al., 1975). Prolactin stimulates lipogenic activity in the mammary gland (Martyn &

Falconer, 1985) by increasing the rate of synthesis of key lipogenic enzymes (Ros et

al., 1990). In addition, it is believed to play a key role in the coordination of fat

metabolism between adipose tissue and the mammary gland (Ros et al., 1990).

Studies have shown that the administration of drugs that increase prolactin levels can

increase milk synthesis in women in the declining phase of lactation and stimulate

milk production in women suffering lactational insufficiency. However, even in

these cases prolactin has not always proven to be very successful (Tyson et al., 1975).

In this regard, Cox et al. (1996) determined that prolactin in the blood did not regulate

the short- or long-term rates of milk synthesis, leading to the conclusion that the

requirement for blood prolactin for lactation was permissive rather than regulatory

(Cregan & Hartmann, 1999; Neville, 1999).

1.4.1.4 Human Growth Hormone

Human growth hormone (hGH) is a 23 kDa single chain polypeptide secreted

from the eosinophilic cells of the anterior pituitary. It is one of the key hormones

involved in breast development during puberty (Kleinberg, 1997). In lactating

women both high and low levels of hGH have been observed. Normal lactation in

ateliotic dwarf women who are deficient in hGH has shown that lactation is possible

in its absence (Rimoin & Holzman, 1968). Furthermore, acute interruption of hGH

8

secretion does not interfere with milk secretion (Wehrenberg & Gaillard, 1989). In

contrast, milk production was stimulated in cows treated with either recombinant

growth hormone (Oldenbroek et al., 1993) or bovine growth hormone (McCutcheon

& Bauman, 1986; Sechen et al., 1990).

1.4.1.5 Insulin

The role of insulin in the development of the breast during pregnancy and in

the functioning of the breast during lactation remains poorly understood. Insulin is

required in in vitro systems to maintain mammary epithelium functionality (Neville &

Picciano, 1997). However, although insulin is required for the development of the

breast during pregnancy, it is not required for either ductal or alveolar growth (Topper

& Freeman, 1980). Despite insulin levels being generally decreased in lactating

compared to non-lactating women (Neville et al., 1993), during established lactation it

has been shown to affect several enzymes involved in carbohydrate and lipid

metabolism (Neville & Picciano, 1997). It has been hypothesised that a major role

for insulin in lactating woman is in the regulation of nutrient partitioning, and that

these effects may either act directly on the mammary gland or provide systemic

homeorhetic adaptations to support the dominant status of lactation (Hartmann et al.,

1998).

1.4.1.6 Oxytocin

Oxytocin is an octapeptide hormone, synthesised mainly in specialised

magnocellular neurons in the supraoptic and paraventricular nucleus of the

hypothalamus (Neville & Allen, 1983) and is released from nerve terminals in the

median eminence into the pituitary portal vasculature. The release of oxytocin is a

conditional reflex and can be initiated either by nipple stimulation or through the

activity of other sensory pathways, such as visual, tactile, olfactory and auditory

signals (Newton, 1992b).

Oxytocin binds to receptors on myoepithelial cells causing them to contract

(Cowie et al., 1980). The contraction of the myoepithelial network around the

alveoli causes the ejection of milk from the lumina into the ductules. This event is

known as milk ejection and is required to allow the removal of milk from the breast as

simple negative pressure applied to the nipple alone is not effective in achieving milk

removal (Neville, 1999). In women, pressure increases induced by myoepithelial

cell contractions (milk ejection contractions) last for approximately 1 minute and can

9

occur 4 to 10 times in a 10 minute period with the pulsatile release of oxytocin

corresponding to the contractile episodes (Neville, 1999). This mechanism for milk

ejection may have evolved to prevent milk loss from the mammary gland as well as to

regulate milk availability to the ability of the infant to consume it (Neville, 1999).

An extreme case being that of the sow where piglet growth is rapid and is highly

dependent on milk intake thus milk is only available to be removed for approximately

15 second periods preventing any unnecessary milk loss at sucking prior to milk

ejection.

It has also been suggested that oxytocin may possess some galactopoietic

effects as Bencini (1993) showed increased rates of milk secretion in ewes that were

injected with oxytocin after milking. This galactopoietic effect also has been

observed in cows (Nostrand et al., 1991) and in goats (Linzell & Peaker, 1971).

Ballou et al. (1993) found equal increases in milk yield with oxytocin injections both

before and after milking in cows, suggesting that the increase in milk yield is due to

increased gland output, the mechanism of which is unknown. However, Knight

(1994) found increased milk output only in the gland injected with oxytocin prior to

milking, supporting the established view that the galactopoietic effects of oxytocin are

mediated indirectly through the removal of an inhibitor of milk production from the

gland.

1.4.2 Autocrine Control

It is well understood that the milking of a goat three times daily produces more

milk than milking only twice daily (Wilde et al., 1987a). The response to a more

frequent pattern of milking is rapid and lasts for as long as the pattern is continued

(Wilde & Peaker, 1990). Henderson and Peaker (1984) found if only one half of the

udder in the goat is milked three times a day only its milk production is increased.

No effect can be observed on the other half of the udder indicating that there was a

level of local (autocrine) control in each half of the udder.

Originally it was thought that the mechanism of control was merely the

physical pressure of milk within each full gland. However, Henderson and Peaker

(1984) found that the rates of milk synthesis in glands with the expressed milk

replaced with iso-osmotic sucrose was similar to the other thrice milked udders.

This suggested that physical distension was not the inhibiting factor. This was

further reinforced by the finding that diluting the milk in the udder with an inert

isotonic solution increased the rate of secretion (Wilde et al., 1987b). These results

10

suggested that it was the removal of a factor in the milk which caused the increase in

milk synthesis.

1.4.2.1 Feedback Inhibition of Lactation

The Feedback Inhibitor of Lactation (FIL) is a 7.5 kDa acidic whey protein

produced by the lactocytes and has been shown to acutely regulate milk synthesis and

secretion in a dose dependent manner (Wilde et al., 1995). Rennison et al. (1992)

showed FIL decreased protein synthesis and Wilde et al. (1995) showed FIL

decreased lactose and casein synthesis. These effects were mediated by the

disruption of the endoplasmic reticulum and the trans-Golgi network (Rennison et al.,

1992) and were specific to FIL (Wilde et al., 1998).

FIL has been identified in the milks of goats (Wilde et al., 1995), women and

cows (Wilde et al., 1998). However, it is uncertain whether the inhibitor is secreted

into the milk in an active form or if it is secreted as a pro-inhibitor. The

concentration dependent nature of the inhibition suggests an increase in FIL occurs as

milk accumulates in the gland, and is relieved upon milk removal. However, this

model does not allow for increased milk synthesis in residual milk where FIL levels

would still be elevated (Wilde et al., 1995). One possibility that has gained favour is

that the active inhibitor is secreted into the milk from the gland and is inactivated in a

first order process by an enzyme in the milk. With the continuous secretion of

inhibitor the degradation process is saturated and active inhibitor levels increase and

produce their effects (Wilde & Peaker, 1990), which may be mediated by receptors on

the apical membrane of the lactocytes (Wilde et al., 1987b). Although good

evidence for the local control effects of FIL exists, the purification and sequence data

and mechanism of action still remain unclear.

1.5 Milk composition

Human milk contains a large variety of components that provide energy (e.g.

fat, protein and carbohydrates), developmental factors (e.g. growth factors, long chain

polyunsaturated fatty acid, oligosaccharides, cytokines and enzymes) and protective

factors (sIgA, lactoferrin, and lysozyme etc) as well as vitamins and minerals to the

babies. Furthermore, milk composition is not uniform as it changes with the stage of

lactation, over the day and over the course of a breastfeed or breast expression.

11

1.5.1 Fat

Fat is the most variable component of human milk and the content changes

within a feed, over the course of the day, with stage of lactation, between breasts, with

parity, age and between mothers (Nims et al., 1932; Hytten, 1954; Hall, 1979;

Prentice et al., 1981a, 1981b; Jackson et al., 1988a; Jackson et al., 1988b; Daly et al.,

1993; Emmett & Rogers, 1997; Jensen, 1999). Despite this variation, the fat in milk

is very important to the maturation of the newborn infant. Fat provides

approximately 50% of the energy in milk, essential fatty acids (EFA) and also serves

as a vehicle for the delivery of fat soluble vitamins to the infant (Riordan, 2005b). In

addition, particular fatty acids, monoacylglycerols and the fat globule membrane

provide protection against microbial pathogens.

The fat fraction of human milk consists of several different classes.

Triacylglycerols are the most abundant, representing 98-99% of total fat. The

remaining 1-2% consists of di- and monoacylglycerols, nonesterified fatty acids,

phospholipids, cholesterol and cholesterol esters. The relative proportion of the fat

classes changes slightly over the course of lactation, for example, levels of cholesterol

and cholesterol esters decrease and triacylglycerols increase (Bitman et al., 1983).

Triacylglycerols in milk are readily broken down to free fatty acids and

glycerol by lipase which is commonly found in infant's intestine as well as in milk.

Although, over 200 different types of fatty acids have been described for human milk,

only 7-9 fatty acids are present with proportions greater than 1% (Jensen, 1999).

The fatty acids of human milk are a complex mixture of saturated, mono- and

polyunsaturated, branched and cyclic fatty acid (Jensen, 1999). They can either be

synthesised within the gland or imported from the blood stream where they are

derived from the diet, mobilised body fat or synthesised by the liver.

Short chain fatty acids (c4-c6) are rarely found in human milk. Medium

chain fatty acids (c12-c14) are synthesised with the gland via de novo synthesis.

They account for approximately 15% of fatty acid found in human milk. The

lactocytes in the mammary gland contain a unique thioesterase (thioesterase II) which

catalyses the termination of FA synthesis at the 14th

carbon (Thompson & Smith,

1985). Long chain polyunsaturated fatty acids account for approximately 12% of the

total fatty acid in mature milk. Since the mammary gland is unable to synthesis long

chain polyunsaturated fatty acid, they are imported from the blood stream and are

greatly influenced by the maternal diet (Jensen et al., 1995).

12

1.5.2 Nitrogen

The nitrogen concentration of mature human milk is approximately 1.71 ±

0.31 (mean ± SD) g/L (Hambraeus et al., 1978). Nitrogen in human milk derives

from two sources, protein and other non-protein components.

1.5.2.1 Non-protein nitrogen

Non-protein nitrogen is derived from free amino acids, peptides, nucleic acids

and nucleotides, creatine and creatinine, urea, uric acid, ammonia, amino sugars,

polyamines, carnitine and other biologically active compounds (Atkinson, 1989).

Many of these components are thought to be either metabolic breakdown products

directly from the maternal blood or derived from the mammary gland itself (Atkinson,

1989). In general, however, the contribution each component plays in either the

non-energy roles or developmental value of breastmilk remains to be fully explained.

1.5.2.2 Protein

Milk proteins are divided into two main groups, casein and whey proteins

based on the precipitation of the casein fraction at pH 4.5 leaving the whey proteins in

solution (McKenzie, 1967; Anderson et al., 1982). Casein and several whey proteins

(lactoferrin, alpha- lactalbumin and protein associated with the fat globule membrane)

in human milk are synthesised within the lactocytes by the rough endoplasmic

reticulum from free amino acids (Burgoyne & Duncan, 1998). The newly

synthesised proteins are then packaged into Golgi secretory vesicles and secreted into

milk by exocytosis (Neville & Allen, 1983). Additional milk proteins

(immunoglobulins, albumin and transferrin) are derived from extracellular fluid

(Burgoyne & Duncan, 1998). These proteins are taken up by endocytosis at the

basolateral membrane, transported through the lactocyte and either released directly

into the alveolar lumen or secreted by exocytosis with other milk proteins (Mather &

Keenan, 1983).

Most proteins are nutritive proteins that provide essential nutrients for human

babies. However about 3-10% of total protein, which is largely made up of sIgA and

lactoferrin, was found in the fecal extracts of term babies (Davidson & Lonnerdal,

1987). These undigested proteins carry out specific functions that assist the

protection and development of babies (Lonnerdal, 2003).

13

Casein

Casein is a phosphoprotein that is synthesized in the rough endoplasmic

reticulum and secreted through the Golgi apparatus of the lactocyte. It is packaged

into secretary vesicles together with lactose at the Golgi apparatus, and secreted into

the alveolar lumina by exocytosis (Kinsella, 1984). Casein contains high levels of

calcium and phosphate as well as other trace elements. Unlike other proteins, casein

does not have well defined secondary and tertiary structures; rather it exists as a large

colloidal micelle, composed of a number of sub-micelles.

The sub-micelle is made up from alpha, beta and kappa caseins. All three

sub-types of caseins are found in cow's milk, but mainly beta and kappa caseins are

present in human milk (Wong et al., 1996). In cow's milk, each sub-micelle has a

hydrophobic core that is made up of alpha and beta caseins. The hydrophilic regions

of this casein submicelle are located on the surface and the hydrophobic region is at

the core. The kappa casein, due to self- association, is restricted to one area on the

surface of the submicelle. Therefore, on the surface of the submicelle, there are two

separate regions: a hydrophilic macropeptide portion of associated kappa caseins and

the phosphate- rich area of the other caseins. The phosphate groups on the surface

can interact with calcium- phosphate bridges. The sub-micelle with calcium will

then bind with phosphate to form a colloidal calcium phosphate. Each colloidal

calcium phosphate binds to two sub-micelles and eventually a large casein micelle is

formed. These micelles are large enough to scatter light and therefore skim cow’s

milk with its high casein content as a dense white appearance.

As the micelle grows, the kappa casein ratio on the micelle surface increases,

with decreasing content of the phosphoserine residues for phosphate clusters.

Micelle growth stops when the micelle surface is predominantly occupied by kappa

casein (Holt & Sawyer, 1988; Holt, 1992; Imafidon et al., 1997). The unusual

structure and formation of casein can easily be altered by altering the pH, temperature

and by the action of digestive proteases (Bloomfield & Mead, 1975). Human casein

can easily be hydrolyzed by the infant’s digestive system and the amino acids

absorbed by the infant. Thus, the major role of casein is to provide amino acid

substrates and trace elements (calcium, phosphate and zinc). However, biologically

active peptides are also released during the hydrolysis of casein (McKenzie, 1967;

Raiha et al., 1986; Graham et al., 1993; Cuilliere et al., 1999). The concentration of

casein in human milk is about a tenth of the concentration in cow milk, thus skim

14

human milk has a pale blue appearance rather than the dense white appearance of

skim milk from cows.

Whey

Whey contains the proteins that remain soluble after the precipitation of

caseins with acid. The whey proteins are a diverse group, including enzymes,

immunoglobulins, lactoferrin, lysozyme, alpha-lactalbumin, hormones and binding

proteins (Kinsella & Whitehead, 1989). These proteins are either synthesized in the

mammary gland, or are derived from the blood.

Immunoglobulins

The major immunoglobulin in human milk is sIgA that has a molecular weight

of 173 kDa, and is a dimer of two-serum IgA molecules associated with a secretory

and J chain components (Roitt, 1997). Like other classes of immunoglobulins, a

single IgA molecule is a glycoprotein that has two light and two heavy chains. The J

chain component connects two IgA molecules to form a dimer IgA. The dimer IgA

binds to a secretory component that gives the dimer IgA an ability to move freely in

and out of the cells by endo/exocytosis. Secretory IgA is highly associated with

mucosal tissues (gastrointestinal lymphoid tissue, respiratory tract, salivary glands

and female reproductive tract). Its relatively high avidity for antigens promotes its

antigen- binding function. It binds to microorganisms to reduce the ability of the

microbes to attach to and enter the epithelium of the mucosae, which reduces the

chance of infection (Hanson et al., 1985a; Hanson et al., 1990; Kutteh & Mestecky,

1994; Hanson et al., 1995; Telemo & Hanson, 1996).

Lactoferrin

Lactoferrin is a glycoprotein with a molecular mass of 80 kDa, consisting of a

single polypeptidic chain of 703 amino acids composed of two homologous domains.

It is an iron transport protein that can bind two ferric ions in association with a

bicarbonate ion, but only a small part of this iron binding capacity is utilized by

lactoferrin in human milk (Lonnerdal & Atkinson, 1995). Several other biological

functions have been proposed for lactoferrin in human milk. A receptor for

lactoferrin has been found in the intestine of newborns, and it therefore has been

proposed that lactoferrin has a role in iron absorption, to provide iron and other trace

elements for the infant. Due to its high affinity with iron, it was initially proposed

15

that lactoferrin competed with siderophilic pathogens for iron thus impeding their

proliferation (Hanson et al., 1985b; Tanaka et al., 1999). However it has become

clear that many microorganisms can reduce the strong iron binding effect of

lactoferrin, either by secreting siderophores that can compete with lactoferrin for iron,

or by developing receptors to lactoferrin (Tomita et al., 1994). Another type of

antimicrobial activity has been proposed. When lactoferrin is hydrolysed, an active

peptide known as lactoferricin is released. Either lactoferricin or

lactoferrin/lactoferricin complex has been shown to bind with lipopolysaccharide,

leading to increased permeability of the cell membrane and cell death (Tomita et al.,

1994).

Another biological function of lactoferrin is its anti-inflammatory reactivity.

It was proposed that lactoferrin might protect lymphocyte function at sites of

inflammation by sequestering potentially toxic iron arising from tissue destruction

(Hanson et al., 1995; Levay & Viljoen, 1995). However the mechanisms involved

are still unclear.

Lysozyme

Lysozyme is a minor whey component of human milk compared with sIgA,

lactoferrin and alpha-lactoalbumin. It is a 15 kDa, single chain protein of 123 amino

acids. Lysozyme is present in many human cells and tissues as well as in soluble

form in various body fluids, such as serum, umbilical cord serum, urine, cerebrospinal

and amniotic fluid and most secretions, including gastric juice, bile, saliva, tears,

seminal fluid and milk. Lysozyme is found in human milk at a concentration higher

than in milk from other species such as cow and pig (McKenzie, 1996). Although

the reported concentration of lysozyme in human milk varies between authors, the

general trend was relatively consistent throughout lactation (Goldman et al., 1982;

Prentice et al., 1984; Lewis-Jones et al., 1985; Montagne et al., 1998).

Lysozyme is a glycoyl hydrolase, cleaving the beta- 1,4 bonds between N-

acetylglucosamine and N-acetylmuramic acid. By hydrolyzing the peptidoglycans

of procaryote cell walls, human milk lysozyme has a bacteriolytic function and plays

a role, together with sIgA and lactoferrin, in the innate protection of breast fed

newborns (McKenzie, 1996).

Human milk lysozyme and alpha- lactalbumin are also evolutionarily related,

with conservation of the position of four disulfide bonds and with 40% amino acid

sequence homology between the two proteins (McKenzie & White, 1991).

16

Alpha- lactalbumin

Alpha- lactalbumin is a major whey protein in milk across species (Findlay &

Brew, 1972; Hopper & McKenzie, 1973; Bell et al., 1981; Bell et al., 1981b; Prasad

et al., 1982; Halliday et al., 1990). In human milk, it makes up 10-20% of the total

protein. It is a 14.1 kDa protein that consists of a single polypeptide with 123 amino

acids (Nagasawa et al., 1973). Although, alpha- lactalbumin and lysozyme have

40% sequence homology, the function of alpha- lactalbumin is totally different.

Together with galactosyltransferase, alpha- lactalbumin forms a unique enzyme

known as lactose synthase (McKenzie, 1996). This enzyme catalyses the synthesis

of the beta- 1,4 glycopyranosyl linkage between UDP- galactose and glucose to

produce lactose (Phillips & Jenness, 1971; McKenzie, 1996).

The action of this enzyme is similar to that of lysozyme but in the reverse

direction. Much research has been conducted to determine whether alpha-

lactalbumin (part of lactose synthase) can act the same as lysozyme and vice versa

(McKenzie, 1996). Until recently there was no solid evidence to suggest that they

shared any common functionality.

Recently studies have shown that alpha- lactalbumin has the ability to induce

apoptosis in abnormal cell types (Hakansson et al., 1995). Under appropriate acidic

condition in the presence of free oleic acid (C18:1, 9cis), alpha- lactalbumin changes

its normal conformation and converts into the apoptosis- inducing form, HAMLET

(human alpha-lactalbumin made lethal to tumor cells) (Hakansson et al., 1999;

Svensson et al., 2000; Kohler et al., 2001). In animal models, HAMLET inhibited a

broad array of malignant tumors and in humans topical treatment with HAMLET

reduced the volume of >95% of skin papillomas compared with 20% of papillomas

reduced by placebo (Duringer et al., 2003; Svanborg et al., 2003; Fischer et al., 2004;

Gustafsson et al., 2004; Gustafsson et al., 2005).

1.5.3 Carbohydrates

The major carbohydrate in human milk is the disaccharide, lactose. In

addition to lactose, human milk also contains monosaccharides (mainly glucose and

galactose) and over 130 different oligosaccharides (McVeagh & Miller, 1997).

Lactose is the major osmole of human milk, and in most terrestrial species the rate of

lactose synthesis is strongly related to the volume of milk produced (Neville & Allen,

1983).

17

1.5.3.1 Lactose

Lactose is a disaccharide comprised of galactose and glucose joined by a beta-

1, 4 -glycosidic bond. Glucose and UDP-galactose are transported into the Golgi

secretory vesicle system of the alveolar cell and lactose formed by way of lactose

synthase (Neville & Allen, 1983). This enzyme complex consists of a membrane

bound enzyme, galactosyl transferase, and a regulatory protein, alpha- lactalbumin.

The binding of alpha- lactalbumin to galactosyl transferase increases the affinity of

glucose to the latter and allows the formation of lactose under physiological

conditions (Ebner & Schanbacher, 1974).

Human milk contains one of the highest concentrations of lactose of any

species, contributing to approximately 40 % of the energy delivered to the infant

(Hambraeus et al., 1984). Lactose is hydrolysed to the monosaccharides, glucose

and galactose, by the brush border enzyme lactase in the intestine. Although both

can be used by the brain for energy, galactose is also used to make galactolipids (e.g.

cerebroside) that are essential for the developing central nervous system. In

addition, lactose has also been shown to improve the absorption of calcium by the

infant (Riordan, 2005b).

1.5.3.2 Oligosaccharides

Oligosaccharides are complex carbohydrates consisting of glucose, galactose,

N-acetylglucosamine, fucose, sialic acid (Coppa et al., 1993) with lactose usually

found at the reducing end (McVeagh & Miller, 1997). They are synthesised in the

Golgi and range from three to ten monosaccharide units in length (McVeagh &

Miller, 1997). Unlike lactose, oligosaccharides are not believed to provide a major

proportion of the energy requirements of the infant as they are not readily digested in

the small intestine (McVeagh & Miller, 1997). Instead they can protect the infant

from infection by preventing bacterial adhesion to epithelial surfaces (Coppa et al.,

1993) and by promoting the growth of the symbiotic bacterium, Bifidobacterium

bifidum (McVeagh & Miller, 1997). In addition to their protective role,

oligosaccharides are an important source of sialic acids that, in turn, are an integral

part of the gangliosides found in the infant cerebral and cerebellar grey matter

(McVeagh & Miller, 1997).

18

1.6 Preterm mothers

Preterm mothers often have a normal pregnancy up until the third trimester,

then due to certain factors, such as maternal health status, multiple pregnancy or

physical insults, they deliver their babies at less than 37 weeks of gestation (Joffe &

Wright, 2002). The early delivery means an early removal of placenta and

withdrawal of progesterone which in turn triggers an early onset of lactogenesis II.

However breast development of these mothers may not be complete and lactogenesis I

may not have occurred. Furthermore, hormone changes discussed in section 1.4.1

may not be optimal. Therefore preterm mothers may have difficulty initiating their

lactation. Cregan et al. (2002) measured the concentrations of several lactogenic

markers (lactose, protein, citrate and sodium) in milk and demonstrated that many

preterm mothers had a delay in the initiation of lactogenesis II. If the concentrations

of these markers were not in the range for term mothers, a delay in the initiation of

lactogenesis II occurred. He found that 82% of preterm mothers at 5 days

postpartum had a delay in lactogenesis II, in that the concentration of 1, 2 or 3

markers in the milk had not reached term levels and that milk production in these

mothers was progressively lower depending on the number of abnormal markers.

However, the study did not control for the parity of the preterm mothers.

On the basis of the subjective assessment of milk ‘coming in’

Hildebrandt (1999) reported that multiparous term mothers with uncomplicated

vaginal delivery entered lactogenesis II approximately eleven hours earlier than

primiparous term mothers. He also concluded that caesarian section delivery also

delayed lactogenesis II by approximately 6 hours. However, Kulski et al. (1981)

found no difference in the timing of lactogenesis II between vaginal and caesarian

sectioned mothers using both subjective assessment by the mother and detailed

analysis of the changes in milk composition. Therefore, although the findings from

Cregan et al. (2002) were not controlled for parity it is unlikely that the large

differences in milk composition observed could be accounted for by an imbalance in

parity in the subject groups.

19

1.6.1 Milk production

Most preterm mothers use a breast pump (manual or electric) for milk

removal, because their babies are either too sick or too premature (< 34 weeks

gestational age) to develop sucking, breathing and swallowing co-ordination. The

volume of milk that these mothers express varies from 1.7 to 1300 ml/day (Hill et al.,

2005c). To date, the causes of this large variation in milk production are poorly

understood. It may be due to the expression regime, the efficiency of the pump,

stress or a combination of these factors.

Different modes of milk expression generate various different pressure (hands)

or vacuum (manual or electric) profiles that removed different amount of milk from

the breasts (Green et al., 1982; Boutte et al., 1985). Mitoulas et al. (2002a) found in

term mothers that some vacuum profiles were able to remove more milk from the

breasts in a shorter period than other tested profiles. In addition, they also identified

a small group of mothers, who had no problem breastfeeding their babies, but

expressed very low volume of milk from their breasts with any tested profiles. This

suggested that mechanical conditions of the breast pump could affect the milk yield of

the mothers.

It is well established in dairy animals that increased milking frequency results

in an increase in milk production and this response is, in part, controlled by local

(autocrine) factors (Wilde et al., 1987a; Wilde et al., 1995). Studies in women

support a similar response. For example, Eglis et al (1961) reported an increase in the

milk production of a lactating mother when she increased feeding frequency from 3 to

6 times a day. In a randomize trial, preterm mothers obtained high milk yield per

day when they increased expression frequency from <4 times per day (week 1) to >6

times per day (week 2) (DeCarvalho et al., 1985). In addition, preterm mothers who

expressed greater than 6 times per day and with the sum of daily expression duration

around 100 minutes had an observable increase in total milk production (Hill &

Aldag, 2005d). This positive association between milk production and frequency of

expression might be related to the increased concentrations of prolactin and oxytocin

in the serum caused by either infant suckling or breast expression (Noel et al., 1974;

Frantz, 1977; Cowie et al., 1980; Newton, 1992b). Prolactin is responsible for the

establishment and maintenance of lactation (Ostrom, 1990), while oxytocin is

responsible for the facilitation of the milk ejection reflex (Neville, 1999).

Furthermore, the frequent milk removal minimized the autocrine inhibition of milk

production by reducing the accumulation of FIL in the gland (Wilde et al., 1998).

20

This would result in higher milk production. The use of higher frequency of

expression to obtain higher milk production is now a standard guideline in the

management of low milk supply in preterm mothers.

Preterm mothers are generally recommended to express their breasts at least 5

times per day with approximately 100 minutes of total expression time per day

(Hopkinson et al., 1988). However some preterm mothers still have low milk supply

and are advised to increase the frequency of expression. An increase in the

frequency of expression is very demanding and puts additional pressure on mothers at

a time of considerable stress. Sisk et al. (2006) reported that the maternal anxiety

trait scores of preterm mothers were inversely correlated with infant breast milk

intake. Furthermore, Nissen et al. (1998) found that anxiety trait scores were also

inversely related to levels of prolactin and oxytocin in mothers of term infants who

were delivered by cesarean section compared with mothers who delivered vaginally.

In this connection, the extra stress from high frequency of expression could make

preterm mothers become more anxious. This would reduce the levels of prolactin

and oxytocin in serum of these mothers that could affect their milk supply.

In addition, there is lack of information about the performance of left and right

breasts, as recent studies of breastfeeding mothers showed that one breast is more

often productive than the other in breastfeeding mothers (Kent et al., 2006). In

addition, the recent anatomy studies of lactating breast indicate that the structure of

the breast is different between sides (Ramsay et al., 2005a). Even though breasts

have simultaneous milk ejections, the amount of milk expressed can vary from each

breast. Furthermore the autocrine control mechanism clearly functions at the level of

the individual breast. However very few studies have measured milk production

from each breast, rather data is recorded as the sum of both the left and right and

breasts. The lack of information about the physiological differences between left

and right breasts makes it very difficult to determine if it is necessary to develop

expression regimes to optimize milk synthesis for individual breasts.

1.6.2 Milk composition

Milk composition of preterm mothers has the same constituents as that of term

mother’s milk. Once lactation is established in term mothers, the fat content and

protein concentration gradually decrease while the concentration of lactose remains

constant over the first 6 months of lactation (Gross et al., 1980; Lemons et al., 1982;

Mitoulas et al., 2000). The main difference between preterm and term mothers’ milk

21

is the concentration of total nitrogen at the early stage of lactation (Atkinson et al.,

1980a). Over the first 4 weeks of lactation, the absolute concentration of protein

nitrogen was significantly higher in preterm milk, while the proportion of non-protein

nitrogen was similar in both term and preterm milk (Atkinson et al., 1980a). The

additional nitrogen provided by protein in preterm milk was thought to provide

additional substrates for synthesis of tissue protein (Snyderman et al., 1962).

The use of human milk as a sole source of nutrients for preterm babies has

been the subject of controversy and debate during recent years (Anderson & Bryan,

1982). Early preterm milk (the first 4 weeks postpartum) is denser in nutrients than

milk from mothers delivered at term. This observation supports the position that

such milk should be considered as optimal primary nutrition for preterm babies. In

addition to the nutritional properties, human milk also provdes anti-infective benefits

for the babies. Babies fed their own mother's milk have a lower risk of necrotizing

enterocolits (Lucas & Cole, 1990) and even short-term use of preterm milk may be

associated with long term advantages for intellectual development (Lucas et al.,

1992). However, it was shown that preterm milk is not completely adequate as a

sole source of nourishment for preterm babies, in particular the energy, protein,

minerals and some vitamins are in inadequate concentrations (Forbes, 1989; Rigo &

Senterre, 2006). Therefore, it is common to fortify preterm mothers’ milk with human

milk fortifiers to meet the nutritional needs of preterm babies.

Currently fortification protocols are based on an assumed composition of

mother’s own milk and exclude adjustments for possible variation in milk

composition between mothers largely because the extent of the variation between

mothers has not been documented in detail. Milk composition of preterm mothers in

previous studies has been determined using either single or pooled milk samples of

mothers (Atkinson et al., 1980b; Lemons et al., 1982; Butte et al., 1984). Although

this method of sampling allowed the determination of average values of milk

constituents at particular gestational age, it does not permit the assessment of either

circadian or longitudinal variations in milk composition of preterm mothers. Fat

content, in particular, is extremely variable in term mothers’ milk. It changes within

a feed, over the course of the day, with stage of lactation, between breasts, with parity,

age and between mothers (Nims et al., 1932; Hytten, 1954; Hall, 1979; Prentice et al.,

1981a, 1981b; Jackson et al., 1988a; Jackson et al., 1988b; Daly et al., 1993; Emmett

& Rogers, 1997; Jensen, 1999). This variation would also be expected in milk

composition of preterm mothers. Indeed, reports suggest that the composition of

22

preterm mother's milk, like term mother's milk, changes as lactation progress

(Anderson et al., 1983; Butte et al., 1984; Lepage et al., 1984; Paul et al., 1997; Maas

et al., 1998), but due to the method of milk sampling, the changes in milk

composition are inclusive. Without adjustments for these variations, the fortification

of mother’s own milk may not meet the target level of the nutrients required by the

preterm baby.

In addition, current fortification protocols overlook the protective properties

offered by human milk. SIgA and lactoferrin are two proteins in human milk that

provide protection against a variety of infections and diseases (Hanson et al., 1985b;

Hanson et al., 1995). The concentration of sIgA plus lactoferrin makes up

approximately 32% of total protein in term mothers’ milk (Lonnerdal & Atkinson,

1995). Furthermore, the concentrations of these proteins in preterm mothers’ milk

are similar to that in term mothers’ milk (Montagne et al., 1999). The concentration

of sIgA (14.8 ± 17.2g/l) and lactoferrin (6.5 ± 5.4g/l) in colostrum of preterm milk

is high, but declines to 1.7 ± 0.9 g/l (sIgA) and 2.7 ± 0.7 g/l (lactoferrin) as lactation

progresses (Montagne et al., 1999). These proteins are important for the survival of

the preterm babies because their immune systems are extremely immature and hence,

preterm babies are susceptible to develop life-threatening infections, such as sepsis

and necrotizing enterocolitis. Therefore it is also important to consider variation in

the concentration of these proteins between mothers and with stage of lactation.

1.7 Aims of the project

There is a greater need to understand the impact on the current expression

regime on the quality and quantity of preterm mothers’ milk in order to improve the

current method of optimizing the milk production and milk fortification to meet the

demands for growth and development of preterm infants.

The project set out to investigate the rate of milk synthesis of term mothers in

established lactation, and milk production and composition of preterm mothers for

24hr periods at 10, 15, 16, 17, 18, 19, 20, 30, 40, 50, and 60 days of lactation.

The aims of this investigation were to determine:

1) The impact of hourly expression on the rate of milk synthesis of term

mothers.

2) The impact of expression regime on the milk production of preterm

mothers between day 15 to 20 postpartum.

23

3) The circadian and longitudinal variations in the milk composition of

preterm mothers during the first two months of lactation.

4) The variations of sIgA and lactoferrin in milk of preterm mothers.

24

Chapter 2

Materials and methods

2.1 Materials

The enzymes, antibodies and purified chemical reagents that were used in the

experiments described in this thesis with their respective manufacturers are presented

in Tables 2.1, 2.2 and 2.3. Those supplied as enzyme suspensions were used without

further treatment. Those supplied as lyophilised enzymes were solubilized with the

appropriate buffer prior to use.

Reagents were made up with double deionized water (DDI) prepared by a

circulating water deionsier, and microfilter (NANOpure, Subron/Barnsted,

Massachusetts, USA) unless otherwise stated.

Table 2.1 Enzymes for biochemical assays

Enzyme Source EC number Abbreviation

Glucose Oxidasea Aspergillus niger 1.1.3.4 GOD

Peroxidasea Horse radish 1.11.1.7 POD

Acyl-CoA Synthetaseb Pseudomonas fragi 6.2.1.3 ACS

ß-Galactosidaseb Escherichia coli 3.2.1.23 ß-Gal

a- Sigma-Aldrich PTY. LTD, Castle Hill, NSW, Australia b- Boehringer Mannheim Australia, North Ryde, NSW, Australia

Table 2.2 Antibodies for ELISA

Antibodies Source

Anti-human sIgA Rabbit

Anti-human sIgA, horseradish peroxidase conjugated Rabbit

Anti-human Lactoferrin Rabbit

Anti-human Lactoferrin, horseradish peroxidase conjugated Rabbit

ICN Biomedicals, Inc., Aurora, Ohio.

25

Table 2.3 Other chemical reagents

Chemical Abbreviation

Bio- Rad protein assay dye reagent concentratea Protein dye

Human sIgAb H- sIgA

Human lactoferrinb H- lactoferrin

Potassium phosphateb K3PO4

Sodium phosphate, dibasic, heptahydrateb Na2HPO4.7H2O

Polyoxyethylenesorbitan monolaurateb Tween 20

Lactoseb

Bovine serum albumin (fatty acid free) b BSA

Trichloroacetic acidb TCA

Trioleinb

Hydrozylamine hydrochlorideb

di- Sodium hydrogen orthophosphate dihydratec

di- Potassium hydrogenc

Sodium hydroxidec NaOH

Hydrogen peroxide (30%v/v) c H2O2

Sulphuric acidc H2SO4

Lanthanum Chloridec LaCl3

Di-potassium hydrogen orthophosphate 3-hydratec K2HPO4

Heptane (HPLC grade) c

Potassium dihydrogen orthophosphatec KH2PO4

Sodium sulphate (anhydrous) c Na2SO4

Sodium Chlorided NaCl

Sodium azided NaN3

Potassium Chlorided KCl

Citric Acidd C6H8O7

Nitric Acidd HNO3

Ferric chlorided FeCl3

Hydrochloric acid (32% w/w) d HCl

Orthophosphoric acid (85%)d H3PO4

Potassium hydroxided KOH

2,2’-azino-di-[3-ethylbanzthiaoline sulfonate] e ABTS

Air (industrial) f

Acetylene f

Skim Milk powderg

a- Bio-Rad laboratories PTY. LTD, Regents park, NSW, Australia b- Sigma-Aldrich PTY. LTD, Castle Hill, NSW, Australia c- Merck (BDH) LTD, Kilsyth, Victoria, Australia d- Ajax Chemicals PTY LTD., Melbourne, Victoria, Australia e- Boehringer Mannheim Australia PTY LTD., Castle Hill, NSW, Australia f- BOC Gases Australia, Chatswood, NSW, Australia g- Diploma, Fonterra Corporate Centre, Mt Waverley, Victoria, Australia

26

2.2. Equipment

The equipment that was used in the experiments described in this thesis with

the respective manufacturer is presented in Table 2.4.

Table 2.4 Equipment

Equipment

Titertek® microtitration multi- well plate (96 wells)a

Linbro® plate sealer a

Immulon® 2 HB microtiter® plate (96 wells) b

EDP- Plus electronic pipette c

EDP- Plus E8 electronic pipette c

M90 pH meter d

Techne DicBlack D8-3 incubater e

Microfuge 11 f

Titertek Multiskan® MCC/340 plate reader g

Titertek® microplate washer S8/12 g

Whatman #1 filter paper h

Minisart 0.20m sterile, non-pyrogenic, hydrophilic filter i

Sartorius balance j

WellMix2 plate mixer k

Varian Techtron Model 1200 Atomic Absorption Spectrophotometer l

a- ICN Biomedicals, Inc., Aurora, Ohio. b- DYNEX Technologies, Inc, Chantilly, VA. c- Rainin Instrument Co. inc., Woburn, MA d- Ciba Corning diagnostics LTD, Halstead, Essex, England e- Techne (Cambridge) LTD, Duxford, Cambridge, England f- Beckman, Woodville, Adelaide, Australia g- Flow Laboratories, McLean, VA h- Whatman International LTD, Springfield Mill, Maidstone, Kent, England i- Sartorius, Gottingen, Germany j- Selby Biolab, Mylgrove, Victoria, Australia k- Denley Welltech WellMix 2, Cytosystems, Castle Hill, Australia l- Varian Techtron Pty. Ltd., Melbourne, Australia

27

2.3 Human subjects

Ethics approval for the project was obtained from the Human Research Ethics

Committee of The University of Western Australia (part 1), Texas Tech University

(part 1) and King Edward Memorial Hospital (part 2-4) for women.

Twenty mothers who delivered at term were recruited for part 1 of this project.

Thirty- nine mothers who delivered prematurely were recruited for part 2 of

this project. Twenty- five mothers completed the study. Nine mothers withdrew

from the study for personal reasons. Data of five mothers were incomplete.

All mothers supplied written informed consent to participate in the studies.

2.4 Sample Treatments

Milk samples were thawed at room temperature, mixed and centrifuged at

10000 RPM (MicrofugeTM

11, Beckman) for 10 min in 250l polypropylene tubes.

The fat layer was removed by slicing the tube with a dog nail clipper at the bottom of

the fat layer. The defatted milk (skim milk layer) was transferred into a new tube

and stored at -20C.

2.5 Biochemical Analyses 2.5.1 Lactose

Adapted from: Kuhn (1969)

Modified by: Arthur et al. (1989a)

Assay principle:

Lactose + H20 galactose + glucose

Glucose + O2 + H2O gluconic acid + H2O2

H2O2 + ABTS(reduced) H2O + ABTS(oxidized, green)

Reagents:

1. Lactase reagent: 0.1mM MgCl2, 0.01M Potassium phosphate buffer

(K2HPO4/KH2PO4, pH 7.2), 8 U/mL β-Galactosidase

2. Glucose reagent: 0.1 M Potassium phosphate buffer (as above), 9.6 U/mL

Glucose Oxidase, 2.5 U/mL Peroxidase, 500 µg/mL ABTS (reduced)

β-Gal

GOD

POD

28

Procedures:

Defatted milk samples and lactose standards (0-300 mM) were diluted 1 in

150 with distilled deionised water. Duplicates of diluted standards and samples (5

µL) were pipetted into wells on a flat bottom 96-well microtitre plate (Flow

Laboratories, McLean, Virginia, USA). Lactase reagent (50 µL) was added to each

well and the plate mixed (Wellmixx 2, Denley, England) and incubated at 37 ˚C for

60 min. Following this, glucose reagent (200 µL) was added to each well and the

absorbance measured at 405 nm on a plate spectrophotometer (Titertek Multiskan®

MCC/340, Flow Laboratories, McLean, Virginia, USA) at 5 min intervals until a peak

absorbance was reached at approximately 45 min.

Validation:

The recovery of a known amount of lactose added to defatted milk samples

was 95.8 ± 7.4 % (n = 13). The detection limit of the assay was 7.5 μM (n = 20) and

the interassay coefficient of variation was 4.45 % (n = 20).

2.5.2 Total protein assay

Assay principle:

The protein assay measured the binding of the dye to the primarily basic and

aromatic amino acid residues of the proteins. The assay was carried out according to

the “Bio-Rad Protein Assay” technical instructions.

Reagents:

Bio- Rad protein assay dye reagent concentrate

Human milk protein standard

Procedures:

The Bio- Rad protein assay dye reagent concentrate was diluted in double,

deionized water (1:4 v/v) and filtered with Whatman #1 filter paper prior to use.

Human milk protein standard was determined by the modified version of

Kjeldahl method (Sherriff, 1994). This method determines the amount of nitrogen

from milk protein through a series of organic reactions. In brief, protein was

digested at high temperature by a strong acid, which caused the release of nitrogen.

The solution was then made alkaline to convert the nitrogen into free ammonia. The

ammonia was then removed by distillation and trapped in a boric acid solution to

prevent the loss of ammonia. The amount of ammonia was determined by titration

and the percentage of nitrogen present in the milk sample was calculated.

29

During the process, nitrogen could also be derived from other non-protein

sources. Thus another pre-treated milk sample was required in order to determine

the true percentage of nitrogen from protein. The treatment involved the removal of

protein from milk by either acid precipitation or by dialysis. Then the same

procedures were carried out with the pre-treated sample to determine the percentage

of nitrogen. The difference between the first and the pre-treated milk samples was

multiplied by 6.38 (Kjeldahl protein nitrogen value) (1984) to obtain the true

percentage of nitrogen from protein.

For this assay the concentration of the protein of human milk standard was

found to be 8.6g/l. The concentration range of the protein concentration standard

curve was 0 to 0.131g/l (Fig 2.2).

The skim milk samples were diluted 60-fold for the assay.

Standards and diluted samples (5l) were pipetted into a microtiter plate.

Dye reagent (250l) was added and mixed with the plate mixer (WellMix2). The

absorbance of each sample/standard was measured at 620nm with the Multiskan plate

reader until the max absorbance was reached in approximately 20 minutes.

y = 4 . 7 0 4 8 x + 0 . 3 4 9 9

R2 = 0 . 9 9 5 1

0

0 . 2

0 . 4

0 . 6

0 . 8

1

1 . 2

0 0 . 0 2 0 . 0 4 0 . 0 6 0 . 0 8 0 . 1 0 . 1 2 0 . 1 4

Figure 2.1 Standard curve of human milk protein using the Bio-Rad protein assay

Validation:

The recovery of a known amount of protein added to defatted milk samples

was 96.0 ± 3.0 % (n = 7). The detection limit of the assay was 0.06g/l (n = 20) and

the interassay coefficient of variation was 5.15 % (n = 20).

Human milk protein standard concentration (g/l)

Abso

rban

ce A

62

0

30

2.5.3 Enzyme Linked Immunosorbent Assay (E.L.I.S.A)

2.5.3.1 sIgA

Method adapted from: Tijssen (1985)

Assay Principle:

Polystyrene support – Abx- Ag- ABz*E

Where Abx = Rabbit anti-human secretory IgA IgG (polyclonal)

Ag = IgA (milk sample or standard)

ABz*E = Rabbit anti-human secretory IgA IgG conjugated to horseradish

peroxidase (*E)

Reagents:

Phosphate buffered saline (PBS)

Phosphate buffered saline with Tween (PBS Tween)

Skim milk powder

Bovine serum album (BSA)

Standard sIgA

Rabbit anti-human - sIgA IgG

Rabbit anti-human - sIgA IgG conjugated to horseradish peroxidase

Colour reagent (reduced ABTS)

Sulphuric acid

Procedures:

The phosphate buffered saline (PBS) was prepared with potassium phosphate

(1.46mM), di- sodium hydrogen orthophosphate dihydrate (6.46mM), potassium

chloride (2.68mM) and sodium chloride (136mM) in double deionised water. The

pH of the solution was adjusted to 7.4 with 1.0M HCl before it was made up to the

final volume. In the case of phosphate buffered saline with Tween (PBS Tween),

Tween (0.05% w/v) was added before it was made up to the final volume. The skim

milk powder in PBS Tween (50g/l) was used as the blocking solution. Standard

human IgA was prepared in PBS Tween (range from 0 to 0.52g/ml). Milk samples

were diluted 5000 fold in PBS Tween for analysis.

Rabbit anti-human - IgA IgG was diluted 2000 fold in PBS. Rabbit anti-

human - IgA IgG conjugated to horseradish peroxidase was diluted 1000 fold in PBS

Tween containing 1g/l BSA. The use of BSA prevented any non-specific absorption

31

during the reaction. Colour reagent was prepared containing hydrogen peroxide (6%

v/v), reduced ABTS (1% v/v) in citric acid- sodium phosphate buffer (pH 5.2, 67mM

citric acid, 77mM di-sodium hydrogen phosphate. All antibody solutions and the

colour reagent were prepared just prior to use. Blocking Buffer was prepared by

dissolving 25g of skim milk powder with PBS Tween. Sulphuric acid (3M) was

made up in double deionised water.

Rabbit anti-human IgA solution (250l) was coated onto the Immulon® 2 HB

microtiter® plate (96 wells) and was left in the wells either overnight at 4C or 1 hour

at 37C. The solution was then discarded by inverting the plate over a sink (use a

throwing motion) and gently tapping onto an absorbent paper towel to remove any

adhering drops.

Blocking solution (300l) was added to each well and incubated for 30

minutes at room temperature. The plate was then washed three times with PBS

Tween, and was tapped as described above.

The standards, samples and controls (200l) were added to the wells. Each

standard, sample and control were analyzed in duplicate. The plate was sealed and

incubated for 1 hour at 37C. After the plate was washed as described above, rabbit

anti-human - IgA IgG conjugated to horseradish peroxidase solution (200l) was

added and incubated at 37C for 1 hour.

The plate was then washed six times with PBS Tween and rinsed three times

with double deionised water. Colour reagent (200l) was added and incubated at

room temperature for 30min. The reaction was stopped by the addition of sulphuric

acid (100l). The absorbance of the plate was read at 405nm by Titertek

Multiskan® MCC/340 plate reader.

Validation:

The recovery of a known amount of human sIgA added to defatted milk

samples was 100 ± 10 % (n = 12). The detection limit of the assay was 0.03 g/l (n =

20) and the interassay coefficient of variation was 19 % (n = 20).

2.5.3.2 Lactoferrin

All solutions, except standard, rabbit anti-human lactoferrin and rabbit anti-

human lactoferrin conjugated to horseradish peroxidase, were prepared in the same

way as for sIgA. Human lactoferrin standard ranged from 0 to 5μg/l. The samples

32

were diluted 200,000 fold in PBS Tween. Rabbit anti-human lactoferrin IgG was

diluted 6000 fold in PBS. Rabbit anti-human lactoferrin IgG conjugated to

horseradish peroxidase was diluted 5000 fold in PBS Tween containing 1g/l BSA.

Procedures for lactoferrin ELISA assay were the same as those for sIgA.

Validation:

The recovery of a known amount of lactoferrin added to defatted milk samples

was 99 ± 12 % (n = 8). The detection limit of the assay was 0.09 g/l (n = 20) and the

interassay coefficient of variation was 26 % (n = 20).

2.5.4 Esterified Fat Assay

Adapted from: Stern and Shapiro (1953)

Modified by: Atwood & Hartmann (1992)

Assay principle:

In alkaline solution carboxylic acid esters react with hydroxylamine hydrochloride to

produce hydroxamic acids. These acids react with ferric chloride to give a red to

violet end product that can be measured spectrophotometrically.

Reagents:

1. 2 M Hydroxylamine hydrochloride

2. 3.5 M NaOH

3. 4 M HCl

4. 0.37 M FeCl3 in 0.1 M HCl

5. 7.5 g Trichloroacetic acid (TCA) in 10 mL reagent 4

Fat standards: Triolein in heptane

Procedures:

Milk samples (warmed to 37 ˚C) and standards (0-200 mM) were aliquoted

(2.5 µL) into 1.5 mL polypropylene tubes (Disposable Products Pty Ltd., Adelaide,

SA, Australia) containing redistilled ethanol (600 µL) and mixed for 10 seconds using

a vortex mixer. Reagent 1 (100 µL) and reagent 2 (100 µL) were then added to each

tube and the tubes mixed and left to stand at room temperature for 20 minutes. Each

tube was acidified by the addition of reagent 3 (100 µL) and colour production

achieved by the addition of reagent 5 (100 µL). The tubes were mixed and 250 µL

of each tube was pipetted into duplicate wells on a 96-well microtitre plate (Flow

Laboratories, McLean, Virginia, USA). Absorbance of each well was determined at

33

540 nm using a plate spectrophotometer (Titertek Multiskan MCC/340, Flow

Laboratories, McLean, Virginia, USA).

Validation:

The detection limit of this assay was 0.45 ± 0.41 g/L (n = 13) and the interassay

coefficient of variation was 8.1 % (n = 13).

2.6 Statistical analysis

The statistical analysis was calculated by the statistic function of SAS (version

9.0, SAS inc, Cary, IL). Unless otherwise stated, all results are presented as mean

SD. P values <0.05 are considered significant.

34

Chapter 3

Frequent expression of breastmilk and milk synthesis in mothers of term babies

3.1 Introduction

Methods have been developed to assess the synthetic activity of the mammary

glands of laboratory and dairy animals, but many of these methods are invasive and

not appropriate for human studies (Popjak et al., 1950; Campbell & Work, 1952;

Barry, 1952a). However, several indirect methods have been used to measure the

milk synthesis in lactating mothers. First, either the mothers or their babies can be

weighed before and after each breastfeed over a period of 24-hours and the

differences summed to obtain 24-hour milk production (Arthur et al., 1987).

Secondly, milk intake can be indirectly measured by the administration of deuterium

oxide to the mother and measuring the turnover of body water in the baby over ~14

days to estimate the average volume of milk consumed by the baby (Coward et al.,

1982; Butte et al., 1988). Although this is a very good method for determining the

average intake of breastmilk for infant nutrition studies, it does not provide

information on the short-term control of milk synthesis by the mother. Thirdly, in

mothers who are fully expressing their milk, for example for their pre-term babies, it

is possible to measure the amount of milk produced at each expression for up to 24-

hour and longer. Whereas breast expression has the potential to measure the capacity

of the breast to synthesize milk, the measurement of milk intake by healthy babies is

usually a measure of the baby’s appetite rather than the mother’s physiological

capacity to synthesize milk (Daly & Hartmann, 1995b, 1995a). Fourthly, the rate of

increase in breast volume between breastfeeds has been used to measure the short-

term rate of milk synthesis in mothers (Daly et al., 1996). However, this method is

only practical for periods of up to 24-hours.

Changes in milk synthesis in mothers from one breastfeed to the next were

observed (Daly et al. 1996). Over a 24-hour period, when a breast was full of milk it

had a lower rate of milk synthesis than when it was drained. These findings are

consistent with the presence of an autocrine (local) inhibition component, possibly the

Feedback Inhibitor of Lactation (FIL; Peaker and Wilde, 1998) that has been shown in

35

animals to slow milk production when the mammary gland is full of milk. On the

other hand, increasing the frequency of either suckling or milk expression usually

results in an increase in milk production within one to two days in both dairy animals

(Linzell, 1967) and women (Auerbach, 1990; Lacy-Hulbert et al., 1999). However,

the relationship between the autocrine inhibition of milk production in a full

mammary gland and increased milk synthesis stimulated by increasing the frequency

of milk removal is not well understood. The aim of my study was to minimize the

influence of the autocrine inhibition of milk production in mothers by the frequent

expression of their breasts (breasts expressed each hour) and then to observe the effect

of this increased frequency of breast expression for periods of up to 7 hours.

3.2 Method

3.2.1 Subjects

This study was a joint collaboration between the University of Western

Australia (Australia) and Texas Tech University (USA). Mothers (n = 14) of healthy

term babies were recruited through the lactation clinic at Texas Tech University

(USA) and six mothers were recruited through the Australian Breastfeeding

Association (Australia). All mothers supplied written informed consent to participate

in the studies, which were approved by the Human Research Ethics Committee of The

University of Western Australia, Australia and the Institutional Review Board at

Texas Tech University, USA. The same experiment protocol was applied at both sites.

Of the 20 recruited mothers, ten mothers completed hourly pumping on their

left and right breasts as well as 24hr milk production procedures. Of the remaining

mothers, three mothers completed the hourly pumping on both breasts (M1, 2, 6) and

six (M8, M16-20) completed hourly pumping only on their left breast. One mother

(M9) completed the hourly pumping for both breasts but only completed the 24hr

milk production for her left breast. One mother (M8) had a missing record at the first

session of the 24hr milk production for the right breast.

36

3.2.2 Criteria for recruitment of mothers

Inclusion criteria: Mothers aged over 18, delivered at term, intended to

breastfeed and had initiated their lactation; and their babies accepted expressed human

milk from bottles were recruited into the study.

Exclusion criteria: Mothers who did not intend to provide breastmilk for their

infants and mothers of infants who were ill were excluded from recruitment.

3.2.3 Determination of hourly pumping

Mothers were invited to come to the research laboratory for a day visit.

During the visit, the mother would express milk from each breast for 10-15 minutes,

every hour for up to 7 hours using an electric breast pump (Symphony, Medela AG,

Switzerland). The expressed milk was given back to the mother to feed her baby.

The protocol for this study was quite demanding for the mothers because it

was found that to obtain reliable results it was necessary for the mothers to remain at

the study site for the duration of each session. Only women whose babies could be fed

from a bottle were approached for the study. This ensured that the babies were not

deprived of breastmilk over the duration of breast expression sessions.

3.2.4 Determination of 24 hour milk production

Milk yield was determined for each breast by test weighing the infant at each

breastfeed from each breast over a 24 hour period using an electronic balance

(BabyWeighScales, Medela AG, Switzerland) at each mother’s home within

approximately 1 week prior to undertaking the study. Mothers were instructed on the

use of the balance, provided with written instructions, and were required to

demonstrate the use of the balance to the visiting researcher. Mothers also recorded

the duration and frequency of breastfeeds from each breast.

3.2.5 Statistical analysis

Figures were generated by Excel (Microsoft). Univariate repeated- measures

analysis of variance (SuperANOVA, Abacus Concepts Inc) was used to analyze the

hourly milk yields. Unless otherwise stated, results are presented as mean SD. All

37

statistical analysis of the data was completed by Ching Tat Lai (The University of

Western Australia).

3.3 Results

3.3.1 Subjects

The age of the mothers for whom data were available (n = 14) was 31 ± 5

years. The stage of lactation was 6 ± 2 months (Table 3.1). These mothers had

adequate supply of milk at the time of the study as shown by the volumes obtained in

Table 3.2. All mothers produced >275g per breast over 24hr period. Eight mothers

chose not to complete the 24 hour milk production because of the high demand

involved in the 24 hour test weighing procedures. One mother completed the 24 hour

milk production for her left breast only and one mother had a missing record at the

first session of the right breast. The hourly pumping method was also very

demanding method of data collection. Thus nine of twenty mothers only participated

in one session, and six of these nine mothers completed the hourly pumping method

for their left breast. The remaining eleven mothers participated in more than one

session for both breasts. Furthermore, only three mothers did the hourly pumping

method up to eight hours in a session, while most mothers did the hourly pumping

method up to six hours in a session. Although the dataset from these mothers seems

incomplete, it had enough statistical power to demonstrate the relationship between

the milk production obtained from the 24 hour milk production and the hourly

pumping methods.

3.3.2 Milk production per 24 hours

In the 24hr milk production, the average frequency of breastfeeds was 6.9 ±

1.3 and 6.7 ± 1.1 times, and the average of milk volume produced were 462 ± 154

g/24hr and 517 ± 154g/24hr, for left (n = 11) and right (n = 10) breasts respectively

(Table 3.2).

3.3.3 Milk production per hour

In the hourly pumping session, the overall average number of breast

expressions for that day was 6 ± 1 times for both breasts, and the overall average

38

volume per expression was 22.7 ± 27.7g and 23.8 ± 19.8g, left (n=216) and right

(n=178) breasts respectively. The average milk volume of the hourly pumping

session were 142 ± 90g and 148 ± 68g, left (n=35) and right (n=29) breasts

respectively (Table 3.3 & 3.4).

Typical patterns for the hourly pumping sessions for these women are shown

in Figure 3.1. Higher volumes of milk were pumped at the first expression, followed

by a decline in volume in the next expressions then a plateau was observed for

subsequent expressions (Table 3.2 & 3.3).

In Fig 3.2, three hourly pumping sessions were carried out on one mother

(M4), each session was approximately one month apart. The first two sessions

commenced in the morning and the third session commenced in the evening.

Table 3.1- Age and stage of lactation of participating women.

Mum ID Age Stage of lactation (month)

M1

M2

M3 31 7

M4 38 5

M5 34 6

M6

M7 27 5

M8 25 7

M9 25 3

M10

M11 24 5

M12 32 7

M13 23 5

M14 34 5

M15 39 5

M16 35 9

M17 35 12

M18

M19 34 4

M20

The missing data was indicated as empty space in the table.

39

Table 3.2 Breastfeeding data of all participating women. The values presented are the

volumes (g) of milk at each breastfeed over 24hr and the mean, SD and n of each

breastfeed over 24-hour period.

Breastfeed (24hr data collection, left breast)

Mum

ID

1 2 3 4 5 6 7 8 9 Total/

24hr

n

M3 60.0 55.0 50.0 10.0 30.0 30.0 70.0 305 7

M4 56.7 30.0 40.0 40.0 60.0 55.0 45.0 30.0 357 8

M5 34.1 130.4 76.1 38.0 68.0 61.6 48.6 457 7

M7 120 90.0 50.0 80.0 80.0 125 35.0 580 7

M8 55.0 45.0 15.0 15.0 50.0 50.0 40.0 90.0 360 8

M9 97.0 41.0 55.0 66.0 56.0 75.0 14.0 86.0 164 654 9

M11 220 54.0 65.0 50.0 78.0 90.0 55.0 612 7

M12 91.0 98.3 67.0 51.4 308 4

M13 60.0 15.0 40.0 50.0 45.0 65.0 275 6

M14 95.0 90.0 25.0 45.0 45.0 25.0 140 465 7

M15 108 83.0 219 114 71.0 112 707 6

Mean 462

SD 154

n 11

Breastfeed (24hr data collection, right breast)

Mum

ID

1 2 3 4 5 6 7 8 9 Total/

24hr

n

M3 90.0 40.0 40.0 35.0 20.0 75.0 25.0 325 7

M4 68.0 90.0 85.0 55.0 35.0 75.0 65.0 64.0 537 8

M5 74.6 90.7 96.2 56.5 117 92.8 83.1 611 7

M7 65.0 60.0 70.0 85.0 165 65.0 85.0 595 7

M8 65.0 65.0 60.0 40.0 30.0 90.0 50.0 30.0 430 8

M11 75.0 70.0 65.0 62.0 65.0 135 472 6

M12 111.8 82.8 69.2 59.2 323 4

M13 125 85.0 65.0 115 85.0 475 5

M14 180 50.0 55.0 55.0 60.0 55.0 100 555 7

M15 108 188 90.0 67.0 86.0 102 92.0 116 849 8

Mean 517

SD 154

n 10

M9 only completed the 24hr data collection for left breast. M1, M2, M6, M10, M16-

20 did not participate in this part of study. M8 had a missing record at the first session

of the right breast. The number of breastfeed represented the order of breastfeed after

midnight during that 24hr period, i.e. 1 = 1st feed after midnight.

40

Table 3.3 Hourly expression data of all participating women (left breast). The values

presented are the volumes (g) of milk at each hourly expression in each session and

the mean, SD and n of each hourly expression.

Expression (hourly session, left breast)

Mum

ID

Session 1 2 3 4 5 6 7 8 Total/

24hr

n

M1 1 31.9 13.3 8.20 11.8 5.70 7.0 79 7

2 33.3 10.8 8.80 9.40 5.70 7.70 78 7

M2 1 76.8 30.8 15.3 10.3 13.3 12.7 11.6 13.6 185 9

2 38.9 22.3 18.4 12.6 14.4 8.4 117 7

M3 1 13.2 15.0 12.3 5.6 17.2 17.2 82 7

2 79.0 37.2 20.6 13.6 13.0 14.0 179 7

3 36.3 26.1 16.7 13.0 4.80 7.80 11.5 119 8

M4 1 39.7 15.4 15.2 10.3 12.1 9.90 12.6 116 8

2 31.0 22.4 16.0 15.1 12.3 12.6 111 7

3 41.0 16.7 16.4 12.9 12.1 16.1 13.8 132 8

M5 1 45.3 20.4 10.2 17.6 11.9 11 117 7

2 37.2 11.7 9.7 14.5 75 5

3 26.0 14.3 18 9.7 19.9 16.8 14.6 17.4 140 9

M6 1 9.0 6.0 2.8 3.5 22 5

2 31.2 4.90 4.70 7.20 3.50 54 6

M7 1 84.0 49.3 13.5 24.7 27.4 16.2 216 7

2 117.4 45.7 29.2 31.6 23.0 19.2 268 7

3 45.0 31.8 20.4 10.4 20.0 13.6 19.6 164 8

M8 1 23.5 11.4 3.90 6.70 4.30 16.6 67 7

M9 1 19.5 28.5 10.4 17.2 77 5

2 30.0 26 23.3 27 108 5

M10 1 38.9 22.3 18.4 12.6 14.4 8.40 116 7

2 76.8 30.8 15.3 10.3 13.3 12.7 11.6 13.6 186 9

M11 1 34.1 22.5 16.1 11.3 14.8 14.9 16.2 131 8

2 45.0 19.6 15.8 17.7 35.6 35.4 171 7

M12 1 15.8 8.80 6.40 5.90 6.30 3.60 48 7

2 61.0 18.2 8.80 7.20 10.4 11.2 6.20 125 8

M13 1 70.5 30.0 17.7 13.4 11.8 12.7 157 7

M14 1 53.8 22.6 14.8 11.4 12.8 12.7 129 7

M15 1 74.0 31.0 24.2 16.9 20.9 24.4 22.7 215 8

M16 1 318 51.6 30.0 26.7 9.90 24.5 462 7

M17 1 158.4 80.7 59.9 31.8 25.9 24.1 25.1 407 8

M18 1 14.1 8.80 10.9 15.7 6.40 14.6 72 7

M19 1 27.6 15.8 9.80 13.9 9.60 10.6 10.2 99 8

M20 1 58.4 20.7 20.2 10.7 19.0 13.9 144 7

Mean 55.3 24.0 16.1 13.6 14.2 14.3 15.6 14.9 142

SD 54.9 15.3 10.0 6.67 7.44 6.40 6.23 2.19 90

n 35 34 35 34 32 30 13 3 35

41

Table 3.4 Hourly expressions of all participating women (right breast). The values

presented are the volumes (g) of milk at each hourly expression in each session and

the mean, SD and n of each hourly expression.

Expression (hourly session, right breast)

Mum

ID

Session 1 2 3 4 5 6 7 8 Total/

24hr

n

M1 1 29.5 18.4 3.80 6.30 10.5 9.80 79 7

2 35.7 14.5 14.0 12.7 8.90 14.6 102 7

M2 1 31.7 22.9 11.8 13.7 13.8 12.0 12.5 12.0 131 9

2 43.7 10.1 10.8 9.70 11.3 8.80 96 7

M3 1 19.7 22.2 14.5 11.0 18.1 16.6 103 7

2 28.7 22.1 15.5 12.0 8.40 8.90 98 7

3 14.7 20.0 13.5 8.20 7.50 8.50 15.1 91 8

M4 1 56.3 28.2 21.0 19.7 19.8 19.5 19.4 185 8

2 58.8 39.7 26.9 23.4 17.9 20.6 189 7

3 94.3 34.3 24.0 24.0 21.7 25.1 21.1 248 8

M5 1 47.0 28.1 15.3 13.1 12.1 16.2 133 7

2 25.5 23.7 17.8 20.5 90 5

3 42.5 17.9 21.4 12.4 25.7 25.1 18.8 24.8 192 9

M6 1 5.80 1.40 0.9 0.6 10 5

2 23.2 6.0 3.1 2.7 1.9 39 6

M7 1 116 46.6 29.0 33.4 29.7 21.5 277 7

2 86.4 35.0 29.1 27.2 26.6 21.5 228 7

3 66.6 39.5 28.4 16.9 26.1 16.2 25 222 8

M9 1 21.8 38.3 27.6 31.9 121 5

2 60.1 48.8 46.3 50.1 207 5

M10 1 43.7 10.1 10.8 9.70 11.3 8.80 95 7

2 31.7 22.9 11.8 13.7 13.8 12.0 12.5 12.0 132 9

M11 1 44.1 30.0 15.0 11.7 12.4 14.6 18.2 147 8

2 24.8 11.6 10.0 12.8 30.4 30.1 122 7

M12 1 25.2 25.1 11.0 9.60 9.0 5.50 86 7

2 53.8 41.2 19.4 10.2 18.0 12.1 10.1 167 8

M13 1 148 18.0 28.1 25.7 19.1 18.7 259 7

M14 1 103 36.9 21.0 16.3 16.5 17.0 212 7

M15 1 78.3 30.9 19.2 32.7 24.3 23.0 22.9 232 8

Mean 50.3 24.8 18.1 15.8 17.7 16.2 20.5 16.2 148

SD 33.1 11.5 9.86 8.69 9.42 6.43 10.8 7.39 68

n 29 28 29 28 26 24 11 3 29

M8, M16-20 did not participate the hourly pumping session with their right breast.

42

Figure 3.1 Milk volumes at each pumping in an hourly pumping session. All sessions

began in the morning.

Time (24hr Clock)

Vo

lum

e (

g)

8 10 12 14 16 0

80

160

0

80

160 0

80

160

M15

M14

M13

43

Figure 3.2 Milk volumes for each pumping of a mother (M4) at three different hourly

pumping sessions. Sessions 1, 2, 3 were approximately 1 month apart. The first two

sessions began in the morning and the third began in the evening.

Time (24hr Clock)

Vo

lum

e (

g)

0 6 12 24 0

60

100 0

60

100

0

60

100

Session 3

Session 2

Session 1

18

44

Figure 3.3 Average hourly rate of milk production from hourly pumping and from 24-

h production. The light blue dot at time 0 is the mean volume (ml) of the first

pumping session. The mean hourly rates (ml/h) of the subsequent pumping session of

the left and right breasts are represented as the blue and pink lines in the graph. The

blue and pink bars are the average hourly rate of milk production from 24-h milk

production. The error bar is the standard error mean: left, 0-5h & 24h (n=10), 6h

(n=7); right, 0-5h (n=8), 24h (n=9), 6h (n=6). ** & * are different from intervals 2 to

7 (p < 0.05).

Vo

lum

e (

g)

0

50

100 0

50

100

Left

Interval (hour)

0 1 2 4 3

Ra

te (

g/h

)

5 6 24hr 7

Right

**

**

*

*

45

The mean (± SEM) hourly rates for the 24-hour milk production were 18.4 ±

2.0 g/h and 15.5 ± 1.7 g/h for the right and left breasts, respectively. The overall

mean (± SEM) hourly milk productions for the hourly milk expressions from 2-6

hours were 18.0 ± 0.5 g/h (n = 38) and 14.0 ± 0.7 g/h (n = 47) for the right and left

breasts, respectively.

Repeated measures ANOVA showed that the hourly rate of milk production from the

24- hour milk production (n=10) was significantly less than the hourly rate recorded

between zero and one hour milk expression. However, there were no significant

differences (p > 0.05) between the hourly rates of milk production from the 24- hour

milk production for each breast and the hourly expression rates recorded between 1

and 2, 2 and 3, 3 and 4, 4 and 5, and 5 and 6, hours (Fig 3.3).

3.4 Discussion

Previous studies of milk production following hourly milking in dairy animals

and women have used exogenous oxytocin as it was assumed that this would ensure

that residual milk did not represent an unduly large proportion of the milk yield. For

example, Linzell (1967) administered a minimum effective dose of oxytocin (2-

4mU/kg) for hourly milk removal in lactating goats in an attempt to avoid the

pharmacological side effects of oxytocin on glucose metabolism. Furthermore, there

is also evidence that exogenous oxytocin has indirect and direct effects on mammary

gland metabolism that result in an increase in milk synthesis (Lollivier et al., 2002).

Thus exogenous oxytocin could influence the results obtained from hourly breast

expression and, therefore, the current study relied on the release of endogenous

oxytocin to facilitate milk removal. Ramsay et al. (2005b) observed that in women,

consistent and effective increases in milk flow were associated with the release of

oxytocin in response to the stimulation provided by an electric breast pump. These

findings suggest that the physiological release of oxytocin should be sufficient for

effective milk removal in women and the consistency of the milk volume obtained

from the 3rd

to 7th

breast expressions support this conclusion (Fig 3.1 and 3.2).

The volume of milk obtained at the first expression of an hourly pumping

session was always the highest, the volume declined at the second expression and

reached stable levels at the third and subsequent hourly expressions (Fig 3.1). The

higher volume of milk obtained in the first expression, was expected since the time of

46

the mothers previous either breastfeed or breast expression was not prescribed but was

more than one hour before the start of the session. This observation was consistent

with previous studies that have shown that on average during either a breastfeed or

breast expression about 67% of the available milk was removed (Kent et al., 2006).

Since the volume of milk expressed at the second expression was significantly higher

than the subsequent expressions, it is possible that an additional proportion of the

‘residual’ milk was removed at this expression. However, there was no significant

difference in the volume of milk expressed between the third and subsequent

expressions (Fig 3.3) suggesting that the ‘residual’ milk remained constant after the

second expression and that, as in goats (following oxytocin injections) (Linzell, 1967),

the volumes of milk removed represented the volume of milk synthesized in the

previous interval from the 3rd

to 7th

hourly expressions.

Experiments with dairy animals have shown that higher milk yield was

achieved when milking three or more times per day (Lacy-Hulbert et al., 1999; Nudda

et al., 2002; Ayadi et al., 2003; Dahl et al., 2004). Clinically, in women, it is widely

held that inadequate milk production is often associated with a low frequency of

breastfeeding (< five times/24h) and that increasing the frequency of breastfeeding

usually increases milk production (Lawrence and Lawrence 1999). Contrary to what

might be expected from these considerations, mothers who were expressing every

hour did not lead to a progressive increase in milk yield (Fig 3.3). These findings are

supported by similar experiments where hourly milking with exogenous oxytocin for

periods of up to 10-12 hours resulted in little if any stimulation of milk synthesis in

goats (Linzell, 1967). These conflicting results may be explained by different

mechanisms being involved in the control of milk synthesis and secretion.

Daly et al. (1996) observed that significant changes in the rate of milk

synthesis occurred from one breastfeed to the next and that these changes depended

on whether the breast was either full (low rate of synthesis) or drained (high rate of

synthesis) of milk. In this connection it is of interest that Molenaar et. al. (1992; 1996)

used immuno-histochemistry and in situ hybridization to localize the sites of mRNA

synthesis for -lactalbumin and S1-casein in ewes and cows and concluded that the

synthesis of these milk proteins was the most active in lobules containing alveoli that

had been drained of milk and the least active in adjacent lobules containing alveoli

that were distended with milk. Thus the functional unit for the autocrine control

47

mechanism may well be individual lobules rather than individual mammary glands.

Furthermore, the results of in vitro and in vivo experiments have demonstrated that a

factor was synthesized by the mammary gland that inhibited the short-term rate of

milk production in dairy and laboratory animals (Section 1.4.2). This autocrine

response was associated with an acidic glycoprotein protein termed Feedback

Inhibitor of Lactation (FIL) that was first discovered and partially purified from goat’s

milk and subsequently identified in bovine and human milk (Wilde et al., 1995).

Experiments using Pulse-chase radiolabelling of milk proteins showed that the

inhibition by FIL was at the early stages of the secretory pathway, that is, at the level

of protein transport from endoplasmic reticulum to Golgi vesicle (Rennison et al.,

1993). The inhibition of FIL became apparent when milk had accumulated in the

mammary gland and its effect on secretion could be detected in as little as 1 hour

(Rennison et al, 1993). Such an autocrine inhibitory control mechanism would

explain the breast-specific, short-term regulation of milk synthesis observed from

breastfeed to breastfeed by Daly et al. (1996) because milk secretion would have been

inhibited as the breasts filled with milk over longer suckling intervals (>6 hours). On

the other hand, such a response would not be expected to occur in the hourly pumping

sessions because only relatively small volumes of newly synthesized milk could

accumulate in the gland and this would not be sufficient to invoke the autocrine

inhibitory response. This conclusion is consistent with the constant rates of milk

production which have been observed from the 3rd

to 7th

expressions (Fig 3.3).

It was found that the constant rates of milk production from the 3rd

to 7th

expressions could occur at any time of day as well as at different stages of lactation

(Fig 3.2). The hourly rates of milk production from the 3rd

to 7th

pumping were not

significantly different from the average hourly rate of milk production calculated from

the 24-hour measurement of milk production using the test weighing procedure (Fig

3.3). Furthermore, the hourly rates of milk production from the 3rd

to 7th

pumping

obtained on different days for the same mother were not significantly different (Fig

3.2) and the values obtained for one mother from two morning sessions were not

significantly different to those obtained at an evening session (Fig 3.2). Thus it is

apparent that the underlying rate of milk production from the 3rd

to 7th

pumping

represented the intrinsic synthetic capacity of the breast. Therefore, by expressing the

breast each hour, the 3rd

expression provides an estimate of the average hourly milk

48

production for the 24-hour period. This procedure could provide a very useful

method of measuring daily milk production in women, as it is much easier than test

weighing each breastfeed over a 24-hour period.

The response time for changes in milk synthesis appears to be longer than

those for milk secretion. Radioactive tracer studies in goats showed that it took at

least 1.5 hours for milk casein to be synthesized in mammary gland. Barry (1952b)

and Popjak (1950) found that the complete synthesis of milk fat in the mammary

gland took approximately 4-6 hours. It is clear from a number of studies in dairy

animals, that increasing the frequency of milking from two to four times per day

increases milk production and that the response is observed in 6 to 12 hours (Wilde et

al., 1987a). Furthermore, compensatory changes in milk production have been

documented in goats, cows, sheep rabbits and mice (Knight et al., 1998). For

example, if a goat was milked 4 times per day and then one gland was milked twice

per day, milk yield dropped in that gland, but a compensatory increase in yield

occurred in the gland that continued to be milked 4 times per day (Wilde et al 1987).

These findings strongly suggest that milk synthesis responds to a systemic effector.

Furthermore, Dewey and Lonnerdal (1986) found that milk production increased by

an average of 124ml/24-hour when women expressed their breasts after each

breastfeed for a period of two weeks. This response was obtained by ensuring the

breasts were drained of milk by expressing the remaining milk after each breastfeed

rather than by increasing the frequency of breastfeeding. These considerations suggest

that when milk accumulates in the mammary gland local factors inhibit milk secretion

and when the gland is released from this inhibitory effect, separate systemic factors

can stimulate an increase in milk synthesis. Indeed, Wilde et al. (1987) reported that

there was a significant increase in activity of several key enzymes required for milk

synthesis within two weeks of the start of thrice daily milking. These findings suggest

that it is first necessary to reduce the effects of the local inhibitory factor before an

increase in the activity of key enzymes involved in milk synthesis can occur. It is of

interest that if increased stimulatory pressure (increased frequency of removal of

breastmilk by either the baby sucking or breast expression) is maintained over a

longer period of time it can lead to increased cell division and growth of mammary

secretory tissue (Wilde et al., 1987b; Knight et al., 1998). Indeed, suckling pressure is

able to induce lactation in non-pregnant, non-lactating women (Kolodny et al., 1972).

49

It can be concluded that increasing the stimulatory pressure on the mammary gland by

increasing the frequency of breastfeeding/breast expression mediates a series of

responses that first decreases local inhibition, then stimulates metabolic activity and

proliferation of the mammary parenchyma leading to increased milk synthesis and

secretion. If these considerations are correct the longest interval between

breastfeeds/breast expressions may be a more important determinant of milk

production than the total number of breastfeeds/breast expressions per day.

50

Chapter 4

Milk production of preterm mothers between days 15 and 20 postpartum

4.1 Introduction

It is still unclear how the human breast regulates milk synthesis to meet the

irregular demand of the baby (Kent et al., 2006). As discussed, additional pumping

after a breastfeed was shown to increase milk production of term mothers (Dewey &

Lonnerdal, 1986) and increasing the frequency of expression increased the production

of milk in cows (Lacy-Hulbert et al., 1999) and goats (Nudda et al., 2002). This

increase has been associated with the removal of the FIL that inhibits milk synthesis

by the inhibition of milk secretion (Wilde et al., 1998). However, as suggested in

chapter 3, an increase in milk synthesis may be associated with some other unknown

systemic factors, and the inhibitory effect of FIL on milk secretion only becomes

effective when it is present in a high concentration in the alveolar lumen.

Preterm babies who are born at less than 34 weeks gestation are generally unable

to breastfeed due to the difficulty in the coordination of the suck, swallow and breathe

reflex. Therefore their mothers have to initiate and maintain their milk production

using either hand or mechanical breast expression. DeCarvalho et al. (1985)

reported that milk production increased when frequency of expression increased from

<4 (week 1) to >6 (week 2) times per day. Hopkinson et al. (1988) found that

frequency of expression correlated (r = 0.49) with the change in milk volumes of

preterm mothers between week 2 and 4 postpartum. Preterm mothers who expressed

>7 times per day soon after birth and during the initiation of lactation also maximized

milk production (Hill et al., 2001). Thus the general advice given to preterm

mothers with a low milk supply is to ‘increase frequency of expression to increase

milk production’ (Tupling, 1988; Phillips, 1991). However not all preterm mothers

were able to improve their milk production with this advice. This suggests that

frequency of expression may not be the only factor that can control milk production in

preterm mothers.

The aim of this study was to observe milk production from days 15 to 20

postpartum in preterm mothers who were self selecting their expression regimes, and

to examine the impact of these expression regimes on milk production.

51

4.2 Methods

4.2.1 Subjects

Mothers (n= 25) recruited prior to the 10th

day after birth and recorded their

milk production from each breast at each expression. The milk production data from

day 15 to 20 is presented in this chapter.

All mothers gave written informed consent to participate in the study, which

was approved by the Human Research Ethics Committee of King Edward Memorial

Hospital, Western Australia.

All mothers, except C10, C26 and C33, completed the data recording for all

six consecutive days from 15 to 20 days postpartum. Mother C10 completed the

data recording for day 15-16 & 19-20 postpartum, while mother C26 completed the

data recording for day 15-16 postpartum. Mother C33 completed the data recording

for day 15-19.

4.2.2 Criteria for recruitment

Inclusion criteria: Mothers aged over 18, delivered before 32 weeks of

gestation, intended to breastfeed and had initiated their lactation by day 8 post partum

and were expressing breast milk for their baby but had not begun breastfeeding, were

recruited into the study.


infants for at least two months post partum and mothers of infants who were not

expected to survive were excluded from recruitment.

4.2.3 Data recording

Mothers were recruited prior to the 10th

day after birth and asked to keep a log

of their expression on days 15-20 postpartum. The record included the starting and

finishing time of each expression from each breast, the volume of milk (by weighing

the bottle before and after an expression) of each expression from each breast per

day.

Overall there were 1724 breast expression observations from the mothers’

breasts during this data collection period. All the observations (including data from

mothers C10 and C26) were included in the analyses.

52

4.2.4 Maternal record

Demographic and obstetric details were collected for the mothers who

participated for the study and are shown in chapter 5.

4.2.5 Graphic presentation and statistical analysis

Figures were generated by Excel (Microsoft) and SAS Enterprise Guide 4

(SAS inc, Cary, IL). Results are presented as mean ± SD unless when they failed

the Shapiro-Wilk (1965) normality test. The results for total volume, volume,

frequency, duration, hourly rate, CV and actual percentage of 24hr milk yield failed

this test and therefore are presented as median, IQR and range.

The relationship between the proportion (%) of 24hr milk yield and interval

since previous expression was plotted against the expected percentage of daily milk

yield for hourly intervals of up to 14hr.

4.2.6 Calculation

Twenty-four hour milk production per breast- the sum of the volumes of

each expression falling within the 24-hour period from midnight on one day to

midnight on the next day.

Hourly rate- the volume of milk from one expression divided by the interval

since the previous expression.

Coefficient of variation (CV)- SD divided by mean and multiplied by 100.

For each mother, coefficient of variation was calculated for milk volume, interval

between expressions and hourly rate.

Actual percentage of 24hr milk yield for each breast - the volume of milk

from one expression divided by the 24hr milk production and multiplied by 100.

The expected percentage of 24hr milk yield- the percentage of total daily

milk yield divided by 24 and multiplied by time interval.

4.3 Results

4.3.1 Subjects

The core interest of this chapter was to examine the expression regime in

relation to the milk production of preterm mothers from day 15 to 20 postpartum and

did not involve the demographic data of mothers. The demographic data is presented

53

in Chapter 5 in combination with milk composition and milk production from day 10

to day 60 postpartum.

All mothers were exclusively expressing milk from day 15 to 20 postpartum.

However, one mother attempted to breastfeed in one session on day 20 postpartum

and milk volumes of the breastfeeds were recorded. Furthermore, there were

missing data records for C10 (day 17-18); C26 (day 17-20) and C33 (day 20).

Despite the missing records, all data was included in the analyses.

4.3.2 Expression regimes and milk production of preterm mothers

Within each mother, the total volumes per day from the left and right breasts

were very similar but there were large variations between mothers (Fig 4.1). The

median, IQR and range for the left breast was 339, 261-526, 130-723g/day and for the

right breast was 358, 235-440, 65-822g/day. The expression regimes of the left and

right breasts of preterm mothers were similar. The median, IQR and range of the

frequencies of expression of both breasts were 6, 6-7, 3-9 expressions/day (Fig 4.2).

Furthermore duration of expressions was 17, 15-25, 4-80 minutes/expression and 18,

15-25, 5-80 minutes/expression, for left and right breasts respectively (Fig 4.3). One

mother (C01, Fig 4.4) had a constant frequency of expression per day per breast

during this period. The mother who obtained the highest frequency of expression

per day per breast (C01) also obtained the highest volume of milk per day.

However, overall the mean volume per day per breast was not correlated with the

mean frequency of expression per day per breast (Fig 4.5). The average volume of

milk per expression for days 15 to 20 postpartum was 53, 37.3-81, 5.1- 254.3g and 54,

37- 79, 3.2- 257g, for left and right breasts, respectively (Fig 4.6).

54

Figure 4.1 Total milk volumes per day per breast for days 15 to 20 postpartum. Each colour represents one mother. Same colour used for the same mother’s data.

Postpartum (days)

To

tal V

olu

me/d

ay (

g)

15 16 17 18 19 20

Left

Right

0

300

600

900

0

300

600

900

55

Figure 4.2 Frequency of expression for days 15 to 20 postpartum. The top and bottom edges of the box are the values of 75

th and 25

th percentiles. The whiskers are

the highest and lowest values of the day. Median (black line) is the line inside the box. The median of days 15, 16, 17, 19 and 20 was the same as the values of 25

th

percentile for that day of observation.

Postpartum (Days)

5

7

3

Nu

mb

er

of

ex

pre

ss

ion

s/2

4h

r

15 16 17 18 19 20

Left

Right

9

5

7

3

9

56

Figure 4.3 Duration of each expression per day. The top and bottom edges of the box are the values of 75

th and 25

th percentiles. The whiskers are the highest and

lowest values of the day. Median (black line) is the line inside the box. The median of day 17 is same as the values of 25

th percentile for that day.

Postpartum (Days)

Du

rati

on

of

an

ex

pre

ss

ion

(m

inu

tes

)

15 16 17 18 19 20

Left

Right

20

40

0

60

80

20

40

0

60

80

57

Figure 4.4 Frequency of expression and total milk volume per breast of individual mothers (C01, C02, C17 and C31) for days 15 to 20 postpartum. Each colour represents one mother. Same colour is used for the same mother’s data.

3

6

15 16 17 18 19 20

Left & right 9

Postpartum (days)

0

300

600

0

Nu

mb

er

of

exp

ressio

ns

Left

Right

300

600

0

To

tal

mil

k v

olu

me/d

ay (

g)

58

Figure 4.5 Mean volume and frequency of expression per day for days 15-20 postpartum. Left breast Right breast

Mean frequency of expression

Me

an

vo

lum

e p

er

da

y (

g)

4 5 6 7 8

0

200

600

1000

59

Figure 4.6 Volume of milk per expression for days 15 to 20 postpartum. The top and bottom edges of the box were the values of 75

th and 25

th percentile. The whiskers

are the highest and lowest values of the day. Median (black line) is the line inside the box.

Postpartum (Days)

100

200

0

Vo

lum

e p

er

ex

pre

ssio

n (

g)

15 16 17 18 19 20

Left

Right

300

100

200

0

300

60

4.3.3 Variability of milk volume, interval since last expression and rate of milk

removal

The descriptive data for all mothers is shown in Table 4.1. Medians, IQR,

range and CV for milk volume, interval and hourly rate of left and right breasts of five

mothers are given in Table 4.2. These five mothers (C08, C17, C01, C02 and C31)

are used to illustrate the pattern of variation of milk expression observed between

mothers.

C08 had the lowest CV for the hourly rate for both breasts (Fig 4.7a). The

milk volume curves were less consistent than the hourly rate curves throughout this

period of lactation. C17 had the lowest CV for the milk volume for both breasts (Fig

4.7b). The hourly rate curves were consistent throughout this period of lactation.

The volume and hourly rate curves of one breast (left in the example, C01) were more

consistent than that of the other breast (right) throughout this period of lactation (Fig

4.7c). Mothers with high CV of rate, volume and low CV of interval of both breasts

(C02) had similar milk volume and rate of milk removal between breasts (Fig 4.7d).

Both volume and hourly rate curves varied throughout this period of lactation while

the interval between breast expressions was fairly consistent. It appeared that the

volume and hourly rate curves formed a horizontal mirror image with each other at

the longer interval between expressions (e.g. the expressions at about 72 hours on Fig

4.7d). Furthermore, the volume and hourly rate curves were more synchronized at

shorter interval between expressions (e.g. the expressions at about 108 hours on the

Fig 4.7d).

C31 represented mothers with high CV of rate of milk removal and milk

volume and high CV of interval of both breasts (Fig 4.7e). Compared to the Figure

of C02 (both breasts), the volume and hourly rate curves for both breasts of C31 were

much more synchronized, i.e. when the volume curve was high, the hourly rate curve

was also high. The large variations in the milk volume and interval since previous

expression could decrease in some mothers by representing milk yield at each

expression as hourly rate (i.e. milk yield at an expression divided by the preceding

interval in hours). Therefore it was necessary to examine other methods of

presenting the data.

61

Table 4.1 Overall CVs of milk volume, interval since previous expression and hourly

rate between days 15 and 20 from each breast of all mothers

CV (%) Breast Median IQR Range

Milk volume Left 26.0 20.3- 29.3 11.5- 70.0

Right 23.4 20.5- 29.1 12.6- 52.7

Interval since previous expression Left 23.2 20.1- 25.9 0- 43

Right 24.0 18.1- 26.3 3.04- 44.6

Hourly rate Left 21.0 15.9- 25.9 12.3- 62.3

Right 20.2 17.8- 26.3 10.3- 48.6

Table 4.2 Within mother variability

Milk volume (g) Interval (hr) Hourly rate (g/h)

Mother Breast Median IQR Range CV(%) Median IQR Range CV(%) Median IQR Range CV(%)

C08 Left 131 107-164 79-180 24 5 4-7 3-7 30 28 26-29 24-33 9 Right 152 122-190 91-198 23 5 4-7 3-7 30 32 30-34 27-37 9 C17 Left 72 66-75 46-89 15 4 3-4 2-5 22 19 18-21 16-28 16 Right 65 61-72 48-84 14 4 3-4 2-5 22 18 17-19 15-28 19 C01 Left 64 53-79 38-103 27 2 2-3 2-4 24 24 22-29 18-44 24 Right 55 43-70 8-112 37 2 2-3 2-4 24 20 17-28 3-41 43 C02 Left 35 27-49 23-53 30 5 3-8 1-14 66 7 6-14 3-16 52 Right 30 21-34 12-54 39 5 3-8 1-13 65 6 3-9 2-13 55 C31 Left 15 10-31 5-72 77 3 2-3 2-5 26 5 4-8 2-23 73 Right 40 21-53 6-100 60 3 2-3 2-5 26 15 8-18 2-22 47 CVs of all data between days 15 and 20 from each breast of each mother

62

63

Figure 4.7 milk volume and hourly rate during day 15-20 postpartum. Time is expressed in hour where 0 was midnight of day 15 and 144 was the last hour of day 20. Each cross or cone represents the value obtained in one expression. a) C08, b) C17, c) C01, d) C02, e) C31

0

80

160

240

20

40

60

0

Left Right

0

80

160

240

20

40

60

0

0

80

160

240

20

40

60

0

0 24 72 96 120 48 144 0 24 72 96 120 48 144

0

80

160

240

20

40

60

0

Hours

0

80

160

240

20

40

60

0

Vo

lum

e (

g)

Rate

(g/h

)

e)

d)

c)

b)

a)

64

4.3.4 Actual percentage of 24hr milk yield and interval since previous

expression

Milk production at each expression from each breast was expressed as actual

percentage of the 24hr milk yield. Two mothers (C10 and C26) were excluded from

the analysis because they did not complete data recording for all six days (Fig 4.31

and 4.32). The data was expressed in left and right breasts separately and in total

data for 46 breasts are presented. The relationship between the actual percentage of

the 24hr milk yield and interval since previous expression for each breast of the 23

mothers is presented in Fig 4.8 to 4.30. Forty of 46 breasts had a range of significant

correlations (r = 0.05 to 0.85, left breast; and 0.03 to 0.84, right breasts) between the

proportion of 24hr milk yield and the interval since previous expression.

I divided the individual breast into 4 groups based on an arbitrary range of R-

square obtained from the results of the data. The groups were as follows: Group 1(n=

17 breasts) with significant R-square >0.5 from the regression analysis (Figure 4.8-

4.17). Group 2 (n=13 breasts) breasts with significant R-square in-between 0.5 and

0.25 (Figure 4.16- 4.25). Group 3 (n = 10 breasts) with significant R-square less

than 0.25 (Figure 4.15- 4.27) and group 4 (n= 6 breasts) with no significant

correlation (Figure 4.21- 4.30).

This method of grouping indicated that a proportion of mothers had their left

and right breasts paired in the same group, while some mothers’ breasts fell into two

different groups (Table 4.3).

Table 4.3 Number of breasts paired and unpaired in each group

Groups Paired Unpaired n

1 14 3 17

2 6 7 13

3 4 8 10

4 2 4 6

Total 26 20 46

There was a trend for a relationship between the R-square for the left and right

breasts of individual mothers (r = 0.61, n = 46, p= 0.05) with 14 of 17 breasts in group

1, 6 of 13 breasts in group 2, and 4 of 10 breast in group 3 being paired left and right

breasts.

The mean daily milk production per breast at day 15 to 20 postpartum of

group 1 breasts was significantly higher than for groups 2 and 3 (Table 4.4).

65

However, in milk production for group 4, the breasts with non-significant correlations

between interval since previous expression and milk yield, was not significantly

different from group 1 breasts. There was no significant difference in the interval

between expressions and duration per expression between the groups.

If milk was synthesized within the breast at a constant rate over the 24 hr

period, then milk yield should relate to the interval since previous expression

according to the equation mp= xt (where mp = milk yield (%), x = slope and t =

interval since previous expression), such that the expected percentage of 24hr milk

yield is 100% if the interval between the last expression was 24hr (Daly et al., 1996).

Each breast of each mother would have its own expected milk regression line

(mp=xt), and the average x for the left breast was 4.18 and for the right breast was

4.19.

To aid in the interpretations of the results, 95% confidence intervals have been

fitted to the actual regression lines (the actual percentages of 24hr milk yield) and

compared to the expected regression lines (the expected percentages of 24hr milk

yield). For the majority of breasts the expected regression line was within the 95%

confidence limits for the actual milk yields. However, for five breasts (C29; LR C18;

LR; C38, R), some actual milk yields were outside the intercept of the expected milk

yield and the 95% confidence intervals and for a further nine breasts (C24, LR; C02,

LR: C06, LR; C11, L; C20, R, C13, L) some points were marginally outside the

intercept. In all breasts, except for those of one mother (C31), the actual regression

line had either a similar slope and course to the expected regression line (see Fig. 4.9)

or had a shallower slope and bisected the expected regression line (see Fig. 4.17). For

mother (C31) the slope of the actual line was greater than the expected line and the

longest interval between breast expressions was only 4 hr over the six-day period

(Fig. 4.19). Of the 40 breasts with significant correlations between milk yield and

interval between breast expressions, the slope of the actual regression line was similar

to that of the expected line in eight breasts, that is, the actual slope was at least 80%

of the expected slope. For the remaining breasts (except mother C31) with significant

correlations the actual slope was between 24-76% of the expected slope and the mean

intercept between the actual regression lines and the expected regression lines was 4.3

±0.9 hours.

66

Table 4.4 Mean daily milk production, interval between expression and duration of expression per breast at day 15 to 20 postpartum grouped according to the R-square

for the proportional milk yield. a,b

means with the same superscript were not significantly different NS- non significant R-square

Group N Daily milk production (g)

Interval between expression (hr)

Duration of expression (min)

1) >0.50 17 506198a 3.720.58 19.4910.20

2) 0.50 to 0.25 13 303140b 3.621.16 19.726.37

3) <0.25 10 289136b 3.380.31 19.634.91

4) NS 6 419231a 3.590.90 18.243.77

67

Figure 4.8 to 4.32 illustrate the regression graph with the same layout. Milk yield at each expression expressed as percentage of the 24hr milk yield for each breast and the interval since the previous expression. The colour dots, the trend line and 95% confidence interval were the actual percentage of 24hr milk yield at the interval (hour) from the previous expression during day 15-20 postpartum. The black dotted line was the expected percentage of 24hr milk yield. The mean daily milk yield (g/24hr) per breast and R-square for each breast is also given.

Figure 4.8 Group 1, mother C08.


0 4 8 12 0 4 8 12 0

50

Interval since previous expression (hour)

% o

f to

tal m

ilk y

ield

/day

Left Right C04

25

C04

R2= 0.58

72474g/24hr

R2= 0.55

76683g/24hr

0 4 8 12 0 4 8 12 0

50

Interval since previous expression (hour) (hour)

% o

f to

tal m

ilk y

ield

/day

Left Right C08

25

C08

R2= 0.71

65898g/24hr

R2= 0.68

58169g/24hr

68




0 4 8 12 0 4 8 12 0

50


% o

f to

tal m

ilk y

ield

/day

Left Right C35

25

C35

R2= 0.51

37625g/24hr

R2= 0.67

35721g/24hr

0 4 8 12 0 4 8 12 0

50


% o

f to

tal m

ilk y

ield

/day

yie

ld/d

ay

Left Right C34

25

C34

R2= 0.59

22427g/24hr

R2= 0.64

28734g/24hr

0 4 8 12 0 4 8 12 0

50


% o

f to

tal m

ilk y

ield

/day

yie

ld/d

ay

Left Right C16

25

C16

R2= 0.58

55052g/24hr

R2= 0.64

40117g/24hr

69



Figure 4.15 Group 1 (left breast) and group 3 (right breast), mother C11.

0 4 8 12 0 4 8 12 0

50


% o

f to

tal m

ilk y

ield

/day

yie

ld/d

ay

Left Right C11

25

C11

R2= 0.22

35129g/24hr

R2= 0.58

50436g/24hr

0 4 8 12 0 4 8 12 0

50


% o

f to

tal m

ilk y

ield

/day

Left Right C06

25

C06

R2= 0.67

89737g/24hr

R2= 0.70

78683g/24hr

0 4 8 12 0 4 8 12 0

50


% o

f to

tal m

ilk y

ield

/day

Left Right C17

25

C17

R2= 0.51

4118g/24hr

R2= 0.61

42913g/24hr

70

Figure 4.16 Group 1 (right breast) and group 2 (left breast), mother C18.



0 4 8 12 0 4 8 12 0

50


% o

f to

tal m

ilk y

ield

/day

Left Right C02

25

C02

R2= 0.42

11139g/24hr

R2= 0.31

13515g/24hr

0 4 8 12 0 4 8 12 0

50


% o

f to

tal m

ilk y

ield

/day

Left Right C20

25

C20

R2= 0.30

21622g/24hr

R2= 0.73

27421g/24hr

0 4 8 12 0 4 8 12 0

50


% o

f to

tal m

ilk y

ield

/day

yie

ld/d

ay

Left Right C18

25

C18

R2= 0.63

40036g/24hr

R2= 0.33

28614g/24hr

71




0 4 8 12 0 4 8 12 0

50


% o

f to

tal m

ilk y

ield

/day

yie

ld/d

ay

Left Right C33

25

C33

26075g/24hr

R2= 0.43

35429g/24hr

0 4 8 12 0 4 8 12 0

50


% o

f to

tal m

ilk y

ield

/day

Left Right C37

25

C37

R2= 0.39

30439g/24hr

R2= 0.47

32133g/24hr

0 4 8 12 0 4 8 12 0

50


% o

f to

tal m

ilk y

ield

/day

Left Right C31

25

C31

R2= 0.37

28366g/24hr

R2= 0.38

17532g/24hr

72




0 4 8 12 0 4 8 12 0

50


% o

f to

tal m

ilk y

ield

/day

yie

ld/d

ay

Left Right C24

25

C24

R2= 0.38

49659g/24hr

R2= 0.12

46132g/24hr

0 4 8 12 0 4 8 12 0

50


% o

f to

tal m

ilk y

ield

/day

yie

ld/d

ay

Left Right C22

25

C22

R2= 0.34

50912g/24hr

R2= 0.20

43521g/24hr

0 4 8 12 0 4 8 12 0

50


% o

f to

tal m

ilk y

ield

/day

Left Right C13

25

C13

R2= 0.19

1389g/24hr

R2= 0.45

20918g/24hr

73




0 4 8 12 0 4 8 12 0

50


% o

f to

tal m

ilk y

ield

/day

Left Right C29

25

C29

R2= 0.13

15526g/24hr

R2= 0.15

168 29g/24hr

0 4 8 12 0 4 8 12 0

50


% o

f to

tal m

ilk y

ield

/day

Left Right C30

25

C30

R2= 0.21

27231g/24hr

R2= 0.22

23933g/24hr

0 4 8 12 0 4 8 12 0

50


% o

f to

tal m

ilk y

ield

/day

Left Right C01

25

C01

44643g/24hr

R2= 0.35

54146g/24hr

74




0 4 8 12 0 4 8 12 0

50


% o

f to

tal m

ilk y

ield

/day

Left Right C07

25

C07

715135g/24hr 64567g/24hr

0 4 8 12 0 4 8 12 0

50


% o

f to

tal m

ilk y

ield

/day

yie

ld/d

ay

Left Right C38

25

C38

24213g/24hr

R2= 0.18

37430g/24hr

0 4 8 12 0 4 8 12 0

50


% o

f to

tal m

ilk y

ield

/day

Left Right C19

25

C19

18125g/24hr

R2= 0.21

25269g/24hr

75

Figure 4.31 mother C10.

Figure 4.32 mother C26.

0 4 8 12 0 4 8 12 0

50


% o

f to

tal m

ilk y

ield

/day

Left Right C26

25

C26

22966g/24hr 458.0g/24hr

0 4 8 12 0 4 8 12 0

50


% o

f to

tal m

ilk y

ield

/day

Left Right C10

25

C10

31129g/24hr 37860g/24hr

76

4.4 Discussion

The mean daily milk production from day 15 to 20 postpartum for the preterm

mothers (n=25) was 769 382g/24hr. This mean value was higher than those

published previously. The milk production at week 3 postpartum were previously

reported to be 606 369g (Hopkinson et al., 1988), 627 517g (Hopkinson et al.,

1992), 398 227 (Hill et al., 1999) and 551 428g (Hill et al., 2005c). The higher

daily milk production (Fig 4.1) compared to the earlier studies may be due to the

timing of subject recruitment. Hill et al. (1999; 2005c) and Hopkinson et al. (1988;

1992) recruited the mothers in the first week postpartum whereas I recruited mothers

within the first 10 days postpartum. Therefore it is possible that my study excluded

more of the mothers who were having difficulty in establishing their lactation and this

resulted in a higher average daily milk production. While a 1.0kg preterm baby only

requires approximately 160ml/day of breastmilk, the adequacy of the mothers’ milk

production has focused on the need of the babies for their long-term growth and

development. Meier (personal communication) found that preterm mothers who had

a daily milk production of 350g/24hr had a better success in establishing and

maintaining their lactations. Furthermore Hill et al. (2005c) found that preterm

mothers were 2.81 times more at risk of not producing adequate milk at 3 months if

they did not produce 500ml of milk per day at week 2 postpartum. In addition

Kent et al. (2006) and my study (see Chapter 3) found that the daily milk production

of term mothers from 1-6 months of lactation was 788 169g/24hr and

992g/24hr, respectively. From these considerations, if mothers in the present

study maintained their milk production of 769 383g/24hr, they would be able to

meet their babies’ long-term developmental requirements. However, the large

variation about the mean in the preterm mothers’ daily milk production indicated that

25% of mothers were producing less than 485g per day for their babies (Fig 4.3) and

therefore may be at risk of not maintaining breast milk feeding in the longer term.

Hopkinson et al. (1988) concluded that preterm mothers could optimize milk

production using an electric breast pump by expressing their breasts at least 5 times

each day with a total expression time of about 100 minutes. The mean frequency of

breast expression (Fig 4.2) was 6 1 time per day with a total expression time of 120

28 minutes (Fig 4.3). In this study, the frequency of expression and expression

time was similar to the data presented by Hopkinson et al. (1988) and Hill et al.

77

(2005c). However, the frequency of expression varied greatly ranging from 3 to 9

times per day which was similar to the variation observed by Hopkinson et al. (1998)

(3-7 times per day) and Hill et al. (2005c) (2-13 times per day). The influence of

this variation in frequency of expression was not clear as some mothers with high

frequency of expression produced lower volumes of milk while others with a

relatively low frequency of expression produced higher volumes of milk (Fig 4.4).

Since the mothers almost always double pumped their breasts, the frequency

and duration of expression for the left and right breasts were similar. However the

milk yield obtained per day per breast was different between mothers as well as

between breasts (Fig 4.4). Furthermore, I found that the mean milk volume per day

per breast was not directly correlated with frequency of expression (Fig 4.5). This

indicated that the frequency of expression was not the only factor influencing milk

yield/24hr. This finding agreed with Daly et al. (1996). Daily milk production per

breast was the sum of milk volume from each milk expression over the day. The

large variation of milk volume per expression may be related to the variation in the

interval since the previous expression (Fig 4.7) with a higher yield of milk after a

longer interval. In an attempt to correct for this variation, the hourly rate of milk

removal was calculated by dividing the milk volume at an expression by the number

of hours since the previous expression was calculated. However, for one third of the

breasts, there was a large variation in the hourly rate (CV >25%) indicating that

factors other than interval between expressions can affect milk volume at an

expression. Furthermore, a progressive plot of time course of milk volume per

expression and hourly rate of milk removal from day 15 to 20 showed that in some

breasts the hourly rate of milk removal decreased with extended intervals (Fig 4.7a &

b), but in other breasts higher milk volumes were associated with higher hourly rates

of milk removal (Fig 4.7c-d). This variation in milk volume per expression per

breast between and within mothers has not been closely examined in past studies as

the main concern was the impact of frequency of expression on total daily milk

production (sum of both breasts).

Wilde et al. (1998) demonstrated that milking one of a goat’s glands four

times per day produced more milk than milking other glands twice per day. Similar

results were also obtained with dairy cows (Knight et al., 1998). This phenomenon

was found to be associated with the local inhibitory control of milk synthesis and was

caused by a local inhibitory factor, FIL (section 1.4.2). Daly et al. (1996) reported

that term mothers who were fully expressing milk for their babies produced different

78

proportion (%) of 24hr milk yield per breast at each expression during a day and that

this difference was related to the interval between expressions. Shorter intervals

(<6hr) since the last expression produced a higher proportion of daily milk production

per breast than the longer interval (>6hr) between expressions. These findings

clearly showed that the interval between breast expressions influenced milk

production at the level of the individual breast. Therefore, it is not possible to

examine the influences of the frequency of breast expression on milk production of

preterm mothers from the studies of Hopkinson et al. (1988); Hill et al. (1999, 2001)

because the daily milk productions were presented as the sum of both left and right

breasts. Hence it was important to examine the milk production from each breast

separately (Fig 4.4) to gain a better understanding of how the variation in the

frequency of expression and interval since the previous expression impacted on the

local control of milk production of each breast.

Hopkinson et al. (1988) found that mothers who delivered premature infants

increased their average milk volumes from 493 to 606 ml/24hr between week 2 and

week 4 of lactation. However this increase was not related to either the absolute

frequency of pumping, duration of pumping, or the longest interval between pumping.

Similarly, there was no relationship between milk production and frequency from day

15-20 (Fig 4.5). This may be partially explained by the fact that women who had a

low milk production were encouraged to express their breasts more frequently to

increase their milk supply. In this context it is of interest that Hopkinson et al. found

that the change in milk production from week 2 to week 4 was correlated with

absolute frequency and duration of pumping (r = 0.49 and 0.42, respectively).

Daly et al. (1996) determined the regression for the actual milk yield

(expressed as actual percentage of the 24hr milk yield) at each breast expression over

the day. They also calculated a theoretical regression of the expected yield assuming

24 hr milk was synthesized at a constant rate over the 24 hr period. Thus 100% of

milk was expressed if the theoretical interval was 24hr and 50% was expressed if the

theoretical interval was 12hr (as shown in Fig 4.8-4.32). For term mothers, the slope

of the actual regression line was less than that of the expected regression line, and the

intercept of the two regression lines was at an interval between breast expressions of

6-7 hr. This showed that milk synthesis occurred at a higher rate at expression

intervals of <6-7hr and at lower rates if the interval was >6-7 hr, suggesting that the

accumulation of milk in the breast over longer intervals between breast expressions

inhibited milk synthesis (Daly et al., 1996). Similar observation was obtained when

79

my results for pre-term mothers were analysis using this regression method. Milk

synthesis of the breasts of pre-term mothers was higher when interval between breast

expressions was <5-6 hr.

Furthermore, the regression analysis with my results showed that there were

significant correlations (p<0.05) between the interval from the previous expression

and the actual milk yield for 40 of the 46 breasts. However, the association between

the actual percentage of 24hr milk yield at an expression and the interval since

previous expression varied greatly (R2 ranged from 0.73, Fig 4.17 to 0.12, Fig 4.23)

and the additional source of the variation has not been identified (Daly et al., 1996).

However, it may be related to the mother’s ability to consistently remove all of the

available milk at each breast expression. In this connection it is of interest that when

mothers expressed their breasts every hour (Chapter 3), consistent volumes of milk

were not expressed until the third breast expression. On average 37% more milk was

obtained at the second expression compared to subsequent hourly expressions.

Mothers can experience multiple milk ejections during both breastfeeding and breast

expression (Ramsay et al., 2004). It is possible that variation in the number of milk

ejections could lead to variations in the amount of the available milk expressed and

that this effect diminished when there was less available milk in the breast. On the

other hand one mother expressed consistently at 4 hr intervals over the six day period

and she had considerable variation (CV 20%) around her mean milk yield at each

expression (Fig 4.31). This may suggest that variation in the mother’s pumping

efficiency could be a potential cause of the unidentified variation. Milk production

was higher in breasts where the variation in the interval between breastfeeds

accounted for more than 50% of the total variation (Table 4.2). Furthermore, there

was a significant but relatively weak correlation between the R2

values for left and

right breast pairs. This demonstrated a tendency for both breasts of a mother to

respond in a similar manner. However, it is important to point out that some breasts

(C07, L,R and C01,R) with no correlation between actual milk yield and interval

between breast expressions (group 4) had high milk productions (Table 4.2).

No standard statistical methods are available to compare the actual milk yield

regression lines to the ‘expected’ regression lines for the breasts that had significant

correlations between milk yield and the interval since previous expressions.

Nevertheless, a comparison of these regression lines has very important implications

for expressing mothers. It is clear that the slopes of regression lines of the actual milk

yields were greater than that of the expected milk yield in the left and right breast of

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only one mother (Fig 4.21). In eight breasts the slopes of actual and expected milk

yields were similar (within 80% of each other). Therefore, the longest interval

chosen for the expression of these breasts would not jeopardize the daily milk

production compromising the milk yield. Of the eight breasts, four were the left and

right breasts of four mothers. In the remaining 32 breasts the actual milk yields

intersected the expected milk yields regression lines such that at shorter intervals

between breast expressions, milk yield was higher than expected and at longer

intervals it was lower than expected. This finding is consistent with both the findings

for term mothers (Daly et al., 1996) and the autocrine control hypothesis (Wilde et al,

1998) that as milk accumulates in the breast the rate of milk synthesis is inhibited.

Breasts with these regression patterns would be expected to respond to decreasing the

longer intervals between breast expressions if low milk supply was of concern.

Although the above discussion has focused on individual breasts, from a

practical point of view it would be difficult to apply different pumping regimes to

each of the mother’s breast. However, bearing this in mind, the current findings can

be used to suggest ways to optimize pumping regimes for pre-term mothers. The

standard advice for increasing milk production in pre-term mothers is to increase the

frequency of breast of expression (Tupling 1988, Phillips, 1991). In view of the

current findings this advice is rather simplistic and may impose unnecessary demands

on the mother. It is of interest that most of the mothers studied had successfully

initiated their lactation and were expressing their breasts at the recommended

frequency (Hopkinson et al., 1988; Fig 4.2). Ten mothers had milk productions above

the average production of 790ml/24hr for exclusively breastfeeding term mothers

from 1-6 months of lactation (Kent et al., 2006). In some of these mothers the slopes

of the actual and expected regression lines were similar for both breasts suggesting

that milk production was not inhibited by the longest intervals between expressions

chosen by these mothers. Therefore, the interval between breast expressions could

be extended to the maximum interval used by the mothers without compromising

daily milk production. For example, the mean interval between breast expressions

for mother C08 was 4.8 hr, but my results suggest that the interval could be extended

to 7.5hr without compromising milk production. This would significantly reduce the

amount of effort required for this mother to sustain her lactation. Similar conclusions

can be made for mothers C04, and C16. Although, for six of the other mothers

extended expression intervals were associated with lower milk yields, daily milk

production was above the average that would be required by a 1-6 month old term

81

baby, therefore these mothers could continue with their current patterns of expression.

However, if the milk production began to decline for mothers C06, C11, C17 and

C24, decreasing the longer intervals between expressions would be expected to

reverse the declining production in both breasts and in mother C22 for her right

breast. Mother C01 expressed her breasts frequently (mean interval 2.55 hr) over the

study period and it is possible that this high frequency of breast expression prevented

the identification of the effect of lengthening the expression interval on milk yield.

It was not possible to draw any conclusions from the results for mother C07 because

there was not a significant relationship between milk yield and interval between

breast expressions. Three mothers had milk productions greater than the highest

volumes (1298ml/24hr) recorded for exclusively breastfeeding term mothers from 1-6

months of lactation (Kent et al., 2006). Therefore, it might be possible to down

regulate their milk production by extension of the expression interval. However, only

the results of one of these mothers (C06) supports this conclusion and care would

need to be taken that such a recommendation did not increase the susceptibility to

mastitis.

Five mothers had milk productions less than the lowest volumes

(<478ml/24hr) recorded for exclusively breastfeeding term mothers from 1-6 months

of lactation (Kent et al., 2006). Decreasing the longer intervals between breast

expressions for mothers C02, C13 and C10 (left breast) would be expected to increase

milk production. Mother C31 expressed her breast frequently (<4hr) and extending

the interval increased rather than decreased milk yield. Further studies are required

to determine if inconsistency in pumping efficiency is influencing her milk

production. Similar conclusions can be made for mother C29 because she expressed

her milk frequently (<4hr) for all but two occasions and these extended intervals

influence the regression relationship. For the remaining mothers, milk production

ranged between 478 and 790ml/24hr. Decreasing the longer intervals between breast

expressions for mothers C18, C20, C34, C35, C37, and C38 (right breast) would be

expected to increase milk production. Mother C30 was interesting because she usually

expressed at 4 hr intervals and had very consistent milk yield, however, on occasions

she either extended the interval or pumped less efficiently. On the other hand the

regression slopes were similar for the actual and expected milk yields for the left

breast mother C33 and there was no correlation for the right breast. Therefore, it is not

possible to predict an outcome for changing the expression interval for these two

mothers.

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The large variations in the daily milk production and frequency of expression

between preterm mothers has also been reported in previous studies (Hill et al., 2005;

Hopkinson et al., 1988), but these studies did not provide a clear understanding of the

source of this variation. Presenting the data from the mothers as milk yield from

individual expressions for each breast showed that the large variation in daily milk

production was the result of variation in the milk yield at each expression over the

day. The variation in the milk yield per expression for 87% breasts could be

explained in part by the interval since the previous expression and these findings are

consistent with the hypothesis of local inhibitory autocrine control of milk synthesis

and secretion (Chapter 1, section 1.4.2). While left and right breasts responded

similarly in 13 women, in other mothers their breast responded differently to the

interval between breast expressions. Based on these findings it was possible to predict

that some mothers could lengthen the intervals between breast expressions without

compromising milk yield. It was predicted that in other mothers decreasing the longer

intervals between breast expressions could increase milk yield. However, such action

was not required at this time for most of these mothers as they already had adequate

milk production.

In 13% of the breasts, the interval since previous expression was not related to

milk yield at a breast expression and some of these mothers also had high milk

production. These findings demonstrate that at least two factors are associated with

variation in milk yield from pumping mothers, first longer intervals between breast

expressions and secondly an unknown factor that appeared to be related to variation in

the efficiency of milk expression. The relative importance of these two factors varied

between breasts with the interval accounting for from 0 to 73% of the total variation

in milk yield between expressions. Understanding the interaction between these

sources of variation could optimize milk yield while minimizing the demands on the

mother in regards to pumping frequency.

83

Chapter 5

Longitudinal changes of macronutrients and milk production of preterm mother’s milk from day 10 to 60

postpartum

5.1 Introduction

The concentrations of macronutrients (fat, protein, lactose and energy) in the

milk of preterm mothers are known to be different to that of term mothers. Atkinson

et al. (1978) reported that milk from preterm mothers had a 20% higher concentration

of total nitrogen than milk from term mothers from 2 to 29 days postpartum. Despite

the differences in the experiment protocols, Gross et al. (1980), Sann et al. (1981),

Anderson et al. (1981), Lemons et al. (1982), and Butte et al. (1984) also

demonstrated that the concentrations of macronutrients in milk of preterm mothers

were either higher or similar to those of term mothers at the same stage of lactation.

In addition, they all suggested that mother’s own milk with minimal supplementation

would be enough to sustain the life of preterm babies (Atkinson et al., 1980).

However, the theoretical estimation on the protein requirement for preterm babies

created by Fomon et al. (1977) and the slow weight gain of preterm babies fed

unfortified human milk (Davies, 1977) indicated that human milk provided

inadequate nourishment for the growth and development of preterm babies

(Arslanoglu et al., 2006; Rigo & Senterre, 2006).

Currently preterm babies receive either preterm formula or fortified human

milk, with the latter favored because it is not only provides essential nutrients but also

bioactive molecules, such as sIgA and lactoferrin, that reduce the onset of sepsis and

necrotizing enterocolitis (Lucas & Cole, 1990b) as well as lipase and growth hormone

to assist the growth and development (Schanler & Atkinson, 1999; Schanler et al.,

1999). However, fortifying human milk for preterm babies is not a straight-forward

calculation, because of the enormous variability in the composition of expressed

breastmilk (Gross et al., 1980; Michaelsen et al., 1990; Weber et al., 2001). Although

preterm mothers’ milk has been characterized at various different stages of lactation,

the short- and long-term variation in milk production and composition of preterm

mothers’ milk on a daily basis has not been described. Analysis of these variations

84

would provide a better understanding of what preterm mothers’ milk can or cannot

provide to their babies.

5.2 Method

5.2.1 Subjects

Mothers (n= 25) were recruited prior to the 10th

day after birth and recorded

their milk production from each breast at each expression on days 10, 15-20, 30, 40,

50 and 60 postpartum.

All mothers gave written informed consent to participate in the study, which

was approved by the Human Research Ethics Committee of King Edward Memorial

Hospital, Western Australia.

The analyses presented in this chapter were involved all the daily records on

days 10, 15-20, 30, 40, 50 and 60 postpartum. The results for 25 mothers are

presented for the volume and frequency analysis while results for 20 mothers are

presented for the milk composition analysis. Mother: C26, C29, C31, C37 and C38

were not included in the milk composition analysis because they did not complete the

milk sample collection protocol.

5.2.2 Criteria for recruitment

Inclusion criteria: Mothers aged over 18, delivered before 32 weeks of

gestation, who intended to breastfeed and had initiated their lactation by day 8 post

partum and were expressing breast milk for their baby but had not begun

breastfeeding, were recruited into the study.


infants for at least two months post partum and mothers of infants who were not

expected to survive were excluded from recruitment.

5.2.3 Data recording

Mothers were recruited prior to the 10th

day after birth and asked to keep a log

of their expression on days 10, 15-20, 30, 40, 50 and 60 postpartum. The record

included the starting and finishing time of each expression from each breast, the

volume of milk (by weighing the bottle before and after an expression) of each

expression from each breast. When mothers began to breastfeed their babies, the

babies were test weighed before and after each breastfeed and milk intake recorded

85

for each breastfeed from each breast. A milk sample (~1.0ml, mid) from each breast

was collected separately at each expression session on those days.

On days 15 and 20 postpartum, additional fore- and hind- milk samples from

each breast were collected by hand expression at each expression session. In a

breastfeeding session, fore- and hind- milk samples were collected instead. Milk

samples were stored at -20C prior to biochemical analysis.

Since the core interest of this study was the variation in milk production and

composition of preterm mothers per day per breast, each milk expression or

breastfeed per day per breast was counted as one episode of milk removal over a 24 hr

period. Hence the data was expressed as ‘per expression/breastfeed’ instead of ‘per

expression’. Overall there were 2861 observations from all mothers’ breasts during

the entire data collection period. All data were included in the analyses.

5.2.4 Maternal record

Maternal demographic and obstetric details were collected for the women who

participated in the study.

5.2.5 Biochemical analysis

Fat, protein and lactose assays were used to determine the concentration of the

marco-nutrients in the milk samples. The assays involved either enzymatic or

chemical reactions to determine the concentrations of macronutrients (Chapter 2,

section 2.5). ELISA assay which involved the use of specific antibodies was used to

determine the concentration of sIgA and lactoferrin in the milk samples. Details of

the methods were listed in chapter 2, section 2.5.3.

5.2.6 Graphical presentation and statistical analysis

Unless otherwise stated, each mother is represented by the same colour when

presenting individual mother’s plots.

The results of production variables (volume, frequency, duration) and

composition variables (fat, protein, nutritive protein, lactose, sIgA and lactoferrin) are

presented as median, IQR, and range because they did not pass the Shapiro-Wilk

(1965) normality test. However, mean ± SD was used to present the demographic

variables (mother’s age, height, weight and BMI; gestational age, babies’ birth weight)

86

as they passed the Shapiro-Wilk (1965) normality test. Furthermore mean was used

for graphical representation for individual mothers and mean ± SD was used when

comparing the results to previous studies.

Observations on each preterm mother were weighted according to frequency

of expression, so that the weight per observation was the same for each mother, thus

the analysis was not biased toward mothers who have a high frequency of expression.

One- way analysis of variance was performed with ANOVA and Analysis of

variance with repeated measures was performed with PROC MIXED in SAS

software (SAS inc, Cary, IL, version 9.1). The covariates for the analysis include:

mother’s age, height, weight and BMI; intention to breastfeed; parity; multiple birth;

antenatal steroids; method of delivery and baby gestational age as well as variables

listed in table 5.1

Table 5.1 Abbreviations for variables used in the statistical analysis.

Abbreviations descriptions

Day Day of expression (from day 10, 15-20, 30, 40, 50

and 60 postpartum); a day was defined as a 24 hr

period starting and ending at 12 midnight

Order of expression (1st…5

th) 1

st expression is the first expression after midnight

Duration Duration of an expression

Frequency Number of expression per day

Interval Interval since previous expression

Time of day The distribution of expression within a period of

time during a day (morning: 0401-1000; day: 1001-

1600; evening: 1601-2200; night: 2201-0400)

5.2.7 Calculation

Energy (Cal/30ml) = (concentration of fat x 9 + concentration of protein x 4

+ concentration of lactose x 4) x 30/1000

Nutritive protein (g/ 24hr) = absolute amount of total protein – (absolute

amount of sIgA x 24% + absolute amount of lactoferrin x 2%). The percentages of

excreted sIgA and lactoferrin were based on the finding from Davidson & Lonnerdal

(1987).

87

5.3 Results

5.3.1 Subjects

Demographic data for 25 mothers were recorded from the given questionnaire.

However, some data were found to be missing in the questionnaire. Thus the ‘n’ in

table 5.2 and 5.3 represents the number of mothers for whom the information was

available in each variable.

Table 5.2 Demographic and obstetric data of preterm mothers and

their babies (continuous variables).

Demographic & obstetric

data

n Mean SD Range

Age 23 34.2 4.60 23-44

Height (cm) 18 165 8.30 151-180

Weight (kg) 20 70.7 8.40 58-90

BMI 17 25.8 4.20 18.8-35.1

Intend to breastfeed (month) 18 24 12.3 2-24

Baby gestational age (week) 25 28.8 2.40 24-32

Baby birth weight (kg)

Singleton 17 1.34 0.39 0.50-2.15

Twins 14 1.10 0.41 0.63-1.84

triplet 3 1.03 0.22 0.76-1.10

Continuous variables were indicated with their mean ± SD, range

Table 5.3 Demographic and obstetric data of

preterm mothers and their babies (class variables).

Demographic & obstetric data n Frequency (%)

Primiparity 25 48

Births

Singleton 17 68

Twins 7 28

triplet 1 4

Antenatal Steroids (completed) 25 20

Method of delivery (CS) 25 68

Class variables were expressed as frequency.

All mothers were exclusively expressing milk from day 10 to 20 postpartum

(Table 5.4). However, one mother was attempting to breastfeed in one session at day

10 and 20 postpartum and milk volumes of the breastfeeds were recorded.

Furthermore, there were missing data records for C10 (day 17-18); C26 (day 17-20)

and C33 (day 20). From day 30 onwards, mothers began to progress from exclusively

expressing milk to exclusively breastfeeding (Table 5.4). Of the 25 mothers at day 15,

only 5 were still exclusively expressing milk at day 60 postpartum. Eight were

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expressing and breastfeeding, 3 were exclusively breastfeeding and 9 mothers had

withdrawn from the study by day 60 postpartum.

Table 5.4 Number of mothers who were exclusively expressing, partially

expression/breastfeed and exclusively breastfeed at day 10, 15-20, 30, 40, 50

and 60 postpartum

Day n Exclusively

expressing

Partially

expressing/breastfeeding

Exclusively

breastfeeding

10 19 18 1

15 25 25

16 25 25

17 23 23

18 23 22 1

19 24 24

20 23 23

30 23 19 3 1

40 21 13 6 2

50 18 9 7 2

60 16 5 8 3

5.3.2 Frequency of expression/breastfeeding, milk production and composition

One-way analysis of variance was performed to examine the differences in

means of the outcomes (milk volume and concentration of milk components per

expression/breastfeed) between each time periods throughout a day and each data

record day.

Analysis of variance with repeated measures was performed to examine the

degree of influence on the outcomes from the mothers’ demographic data and their

expression/breastfeeding regime.

5.3.2.1 Circadian changes

The analysis of circadian changes was involved the data on days 10, 15-20, 30,

40, 50 and 60 postpartum. A day was divided into 4 time periods (morning: 0401-

1000; day: 1001-1600; evening: 1601-2200; night: 2201-0400). The division was

based on the published work by Kent et al. (2006) for term breastfeeding studies. The

frequency of expression/breastfeeding was similar between left and right breasts as

well as between the time periods (Fig 5.1). Relative to the morning number of

expressions/breastfeeds, Proc Mixed analysis showed that significantly more

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expressions/breastfeeds (p <0.05) occurred during the day and evening. On the other

hand, there were fewer expressions/breastfeeds at night (Table 5.5 & 5.6).

The morning expressions/breastfeeds had the highest and most variable milk

volume compared to other time periods (Fig 5.2). Milk volume per

expression/breastfeed for the left breast was significantly lower (p<0.05) during the

day, evening and night, relative to the morning expression/breastfeed while milk

volume per expression/breastfeed for right breast was significantly lower (p<0.05)

during day and evening, relative to the morning expression/breastfeed (Table 5.5 &

5.6). During the day, the milk volume between individual expressions/breastfeeds

was very variable and ranged from 45 to 91g per expression/breastfeed per breast.

The longer the duration of expression/breastfeed and interval since previous

expression/breastfeed, the higher the milk volume per expression/breastfeed obtained.

A mother with triplets had the highest milk volume per expression

expression/breastfeed per breast.

Fat content, concentration of total protein, nutritive protein, lactose, energy,

sIgA and lactoferrin were similar and not statistically different between each time

period (Table 5.5 & 5.6). The changes of each milk component were steady within

each mother from midnight to midnight (Fig 5.3-9).

90

Table 5.5 Median, IQR and range of milk production and component

of left breast during the day

Left breast

N Median IQR Range

Frequency Morning 355 2 1-2 1-3 Day 390 2 2-2 1-4 Evening 399 2 2-2 1-4 Night 299 2 1-2 1-3

Vol/exp Morning 355 58 39-85 1-254 or bf Day 390 49 33-72 2-196

Evening 399 49 33-73 2-226

Night 299 54 37-82 1-234

Fat Morning 257 44.1 37.7-54.7 15.8-122.5 (g/l) Day 274 46.1 38.1-56.8 14.5-158.5

Evening 271 44.7 35.9-52.9 1.6-182.7 Night 221 44.0 36.4-51.7 6.2-140.5

Total Morning 257 16.7 13.2-21.9 6.8-69.8 Protein Day 274 17.1 13.1-21.1 6.0-68.0

(g/l) Evening 274 16.8 13.1-21.4 5.6-57.4 Night 221 16.9 13.8-21.4 5.0-83.3

Nutritive Morning 257 1.08 0.70-1.61 0.02-6.75 Protein Day 274 0.84 0.64-1.35 0.08-6.35 (g/24hr) Evening 272 0.86 0.61-1.40 0.14-4.58

Night 220 0.91 0.65-1.49 0.14-6.54

Lactose Morning 257 67.1 58.5-74.6 34.7-115.9 (g/l) Day 275 65.2 59.5-73.9 40.1-95.7

Evening 273 66.4 58.5-74.1 38.8-96.0 Night 222 66.9 59.7-73.2 41.7-96.4

Energy Morning 257 22.7 20.0-24.9 12.5-44.1 (Cal/30ml) Day 276 22.5 20.1-25.6 7.9-53.4

Evening 274 21.5 19.7-24.5 1.5-63.3 Night 222 22.1 19.8-24.6 8.7-50.6

sIgA Morning 257 0.80 0.591.10 0.05-2.90

(g/l) Day 274 0.83 0.59-1.18 0.08-2.63

Evening 272 0.82 0.59-1.12 0.07-2.28

Night 223 0.85 0.61-1.08 0.05-2.38

Lactoferrin Morning 257 2.37 1.51-3.52 0.12-7.93

(g/l) Day 274 2.56 1.58-3.53 0.07-6.13

Evening 272 2.30 1.48-3.45 0.13-7.42

Night 223 2.48 1.55-3.67 0.09-7.46

N- number of observation

Vol/exp or bf- milk volume per expression/breastfeed

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Table 5.6 Median, IQR and range of milk production and component of

right breast during the day

Right breast

N Median IQR Range

Frequency Morning 349 2 1-2 1-3 Day 384 2 2-2 1-4 Evening 390 2 2-2 1-4 Night 292 2 1-2 1-3

Vol/exp Morning 349 59 38-86 1-257 or bf Day 386 48 34-72 4-199

Evening 389 51 34-71 3-263

Night 292 55 37-79 5-257

Fat Morning 257 44.3 36.9-52.6 9.9-124.4 (g/l) Day 269 45.6 37.3-55.7 2.4-167.9

Evening 263 44.3 35.3-52.6 2.9-155.2 Night 212 44.6 38.8-51.3 0.7-123.5

Total Morning 259 16.7 13.2-21.3 5.3-61.6 Protein Day 268 16.7 12.6-21.7 3.5-55.0

(g/l) Evening 261 16.9 13.4-21.8 6.3-75.6 Night 212 17.2 12.8-21.1 6.4-59.6

Nutritive Morning 259 0.99 0.60-1.62 0.02-7.26 Protein Day 268 0.85 0.57-1.35 0.06-4.42 (g/24hr) Evening 261 0.88 0.58-1.50 0.06-4.74

Night 212 0.94 0.63-1.58 0.18-8.06

Lactose Morning 258 66.3 58.7-74.5 38.7-89.0 (g/l) Day 267 65.7 57.9-73.6 39.5-105.4

Evening 259 65.1 58.5-73.8 31.8-109.7 Night 214 66.3 58.7-73.7 29.3-98.7

Energy Morning 259 21.9 19.5-24.5 9.9-44.6 (Cal/30ml) Day 271 22.2 19.7-25.3 6.7-57.6

Evening 266 21.6 19.0-24.4 9.0-54.2 Night 215 22.2 19.8-24.5 8.7-45.2

sIgA Morning 259 0.79 0.58-1.14 0.08-2.14

(g/l) Day 269 0.83 0.61-1.14 0.08-2.50

Evening 262 0.81 0.57-1.16 0.11-2.93

Night 215 0.82 0.58-1.13 0.10-2.01

Lactoferrin Morning 259 2.45 1.50-3.42 0.14-6.37

(g/l) Day 269 2.39 1.55-3.39 0.04-8.82

Evening 262 2.33 1.49-3.48 0.04-6.80

Night 215 2.45 1.52-3.64 0.25-6.83



92

Figure 5.1 Frequency of expression/breastfeed during the day. Each point is the mean

frequency of expression/breastfeed per time period for all days.

day evening morning

Time of the day

night

0

1

2

3

0

1

2

3 F

req

uen

cy

of

ex

pre

ss

ion

/bre

as

tfe

ed

Left

Right

93

Figure 5.2 Milk volume per expression/breastfeed during the day. Each point is the

mean milk volume per expression/breastfeed per time period for all days.

Right

50

100

150

0

200

Left

50

100

150

200

day evening morning

Time of the day

night

0

Mil

k v

olu

me

(g

/ex

pre

ss

ion

/bre

as

tfe

ed

)

94

Figure 5.3 Fat content per expression/breastfeed during the day. Each point is the

mean of milk fat content per expression/breastfeed per time period for all days.

day evening morning

60

0

Left

Right

Time of the day

120

night

60

120

day evening morning

60

Mil

k f

at

(g/l)

0

0

95

Figure 5.4 Total protein concentration per expression/breastfeed during the day. Each

point is the mean of total protein concentration per expression/breastfeed per time

period for all days.

day evening morning

Left

Right

Time of the day

30

60

0

night

Mil

k t

ota

l p

rote

in (

g/l

)

30

60

0

96

Figure 5.5 Nutritive protein per expression/breastfeed during the day. Each point is

the mean amount of nutritive protein per expression/breastfeed per time period for all

days.

Left

Right

3

5

Nu

trit

ive

pro

tein

(g

/24

hr)

3

5

0

day evening morning

Time of the day

0

night

1

1

97

Figure 5.6 Lactose concentration per expression/breastfeed during the day. Each point

is the mean of lactose concentration per expression/breastfeed per time period for all

days.

day evening morning

Left

Right

Time of the day

60

100

0

night

Mil

k L

ac

tose

(g

/l) 20

60

100

0

20

98

Figure 5.7 Energy per expression/breastfeed during the day. Each point is the mean of

energy per expression/breastfeed per time period for all days.

day evening morning

Left

Right

Time of the day

40

0

night

Mil

k e

nerg

y (

Cal/3

0m

l)

20

40

0

20

99

Figure 5.8 sIgA concentration per expression/breastfeed during the day. Each point is

the mean of sIgA concentration per expression/breastfeed per time period for all days.

day evening morning

Left

Right

Time of the day

2.5

0

night

sIg

A (

g/l

)

1.5

2.5

0

1.5

100

Figure 5.9 Lactoferrin concentration per expression/breastfeed during the day. Each

point is the mean of lactoferrin concentration per expression/breastfeed per time

period for all days.

day evening morning

Left

Right

Time of the day

6

0

night

Lac

tfe

rrin

(g

/l)

4

6

0

4

2

2

101

5.3.2.2 Consecutive day changes- days 15 to 20 postpartum

The data for frequency of expression and milk volume per

expression/breastfeed was presented in chapter 4. The data presented in this chapter

focuses on the changes in the milk composition during these 6 consecutive days.

The median, IQR and range of frequency of expression and milk volume per

expression/breastfeed are described in Table 5.7-8. Frequency of

expression/breastfeed was found to be significantly different (p<0.05) between day 15

and 18, 15 and 20, 16 and 18 for both breasts. The overall median, IQR and range of

the frequencies of expression of both breasts were 6, 6-7, 3-9 expressions/day (n=863,

left; n=861, right). Milk volume per expression/breastfeed was significantly different

between these six days. The overall median, IQR and range of milk volume per

expression/breastfeed from days 15 to 20 postpartum were 53, 37.3-81, 5.1- 254.3g

(n=863) and 54, 37- 79, 3.2- 257g (n=859), left and right breasts, respectively. The

differences in the daily milk production between the left and right breasts (right – left)

were calculated in grams and as a percentage of the total daily milk production (sum

of left and right). Figure 5.10 showed that the differences in the daily milk

production were evenly distributed between the left and right breasts.

The changes in the fat content, concentrations of total protein, nutritive protein,

lactose and energy were very steady for each mother from day 15 to 20 postpartum

(Fig 5.11-17). However one mother (C01) was found to have a fat content about 2-

fold higher than other mothers on two occasions (day 15 and 20) and the

concentration of total protein about 2-fold higher than other mothers, but the

concentration of lactose in her milk was in range of other mothers. Only fat content

and energy for the left breast was found to be significantly different, other milk

components were not significantly different between days (Fig 5.11, 5.15). The

changes in concentration of sIgA and lactoferrin were very steady for each mother

from day 15 to 20 postpartum (Fig 5.16, 5.17). There was no significant difference in

the concentration of sIgA for both breasts between day 15-20 postpartum, but

significant differences were found for the concentration of lactoferrin between day 15

and 19, 15 and 20, 16 and 20 for left breast and day 15 and 19, 15 and 20, 16 and 19,

16 and 20, 17 and 20, 18 and 20 for the right breast. The median, IQR and range of

each milk components are described in Table 5.9-12.

102

Table 5.7 Median, IQR and range of milk production of left breast

from day 15 to 20 postpartum

Left breast

Day N Median IQR Range

Frequency 15 158 7 6-7 4-8 16 153 6 6-7 3-9 17 139 6 6-7 3-8 18 132 6 5-7 3-8

19 145 6 6-7 4-8 20 136 6 6-7 3-8

Vol/exp 15 158 50 35-73 5-199 or bf 16 153 52 39-72 5-220

17 139 57 38-82 5-254 18 132 58 42-84 9-221 19 145 55 37-89 12-243 20 136 51 35-85 9-226



Table 5.8 Median, IQR and range of milk production of right breast


Right breast


Frequency 15 158 7 6-7 4-8

16 153 6 6-7 3-9

17 139 6 6-7 3-8

18 131 6 5-7 3-8

19 145 6 6-7 4-8

20 135 6 6-7 3-8

Vol/exp 15 158 45 30-70 3-257

or bf 16 153 52 35-71 5-212

17 137 54 37-86 6-229

18 131 58 40-80 14-237

19 145 56 42-80 8-257

20 135 56 37-81 12-199



103

Table 5.9 Median, IQR and range of milk component of left breast


Left breast


Fat 15 122 47.7 39.1-60.4 19.8-140.5 (g/l) 16 116 45.1 37.0-52.4 1.6-98.1

17 110 45.4 37.2-56.7 21.3-93.7 18 106 45.6 37.3-53.3 18.6-82.7 19 114 44.5 36.7-53.8 11.4-139.7 20 102 43.8 38.4-51.4 22.2-182.7

Total 15 122 19.1 15.5-24.0 5.6-45.4 Protein 16 118 18.5 13.8-22.9 7.2-57.0

(g/l) 17 110 17.6 14.0-21.0 6.3-56.5 18 106 17.3 14.0-20.9 7.2-69.8 19 113 18.1 14.3-23.4 9.6-83.3 20 102 18.1 14.2-23.0 7.9-53.3

Nutritive 15 122 1.04 0.24- 4.51 0.69- 1.48

Protein 16 117 1.03 0.28- 5.20 0.71- 1.46

(g/24hr) 17 110 1.00 0.26- 5.01 0.71- 1.63

18 105 1.09 0.36- 6.75 0.77- 1.59

19 113 1.18 0.34- 6.65 0.76- 1.77

20 102 1.04 0.20- 6.54 0.69- 1.79

Lactose 15 122 67.2 61.5-73.8 43.7-92.9 (g/l) 16 117 67.7 58.8-73.2 46.8-95.7

17 110 67.1 59.6-73.2 50.1-89.0 18 106 66.3 60.1-74.0 51.7-96.4 19 113 64.9 58.1-73.4 43.0-93.5 20 103 65.7 59.0-74.6 38.8-94.2

Energy 15 122 23.4 21.1-27.1 13.5-50.6

(Cal/30ml) 16 118 22.2 19.9-24.9 1.5-36.5

17 110 22.7 20.1-25.8 14.6-35.5

18 106 22.4 20.2-25.0 14.5-35.5

19 114 22.6 19.8-25.1 11.4-48.7

20 103 21.9 20.1-24.4 8.7-63.3


104

Table 5.10 Median, IQR and range of milk component of right

breast from day 15 to 20 postpartum

Right breast


Fat 15 120 45.5 40.8-56.1 9.7-136.1

(g/l) 16 113 45.9 38.5-56.0 7.6-109.8

17 109 45.4 36.9-54.5 0.7-90.7

18 104 44.8 39.8-52.8 2.9-92.3

19 111 43.6 37.8-51.4 15-158

20 101 44.7 37.7-53.5 23.2-167.9

Total 15 120 18.9 15.2-25.0 8.0-49.5

Protein 16 111 18.8 15.7-24.0 5.5-64.9

(g/l) 17 108 17.4 14.4-20.9 5.4-54.8

18 105 17.3 13.6-22.6 6.5-61.6

19 112 17.6 14.5-23.1 6.6-75.6

20 101 18.2 15.4-22.5 7.3-47.8

Nutritive 15 120 0.97 0.29- 8.06 0.70- 1.59

Protein 16 111 1.02 0.17- 6.62 0.67- 1.71

(g/24hr) 17 108 1.08 0.25- 4.79 0.66- 1.56

18 105 1.00 0.32- 4.17 0.68- 1.67

19 112 1.08 0.13- 7.26 0.68- 1.78

20 101 1.05 0.23- 4.20 0.70- 1.96

Lactose 15 120 66.6 58.1-74.9 40.9-89.0

(g/l) 16 112 66.0 58.8-72.1 29.3-84.2

17 109 66.8 60.8-73.0 40.4-94.7

18 104 65.0 57.9-75.4 41.7-105.4

19 109 64.3 58.5-74.5 33.8-101.7

20 102 65.3 59.1-74.1 44.5-91.0

Energy 15 120 23.1 20.8-25.9 9.5-50.3

(Cal/30ml) 16 115 22.4 20.3-25.5 6.7-41.2

17 110 22.3 20.2-24.5 8.0-35.7

18 105 22.2 20.5-25.1 10.1-39.7

19 113 22.1 19.4-24.7 10.0-39.2

20 102 22.1 20.1-25.3 8.7-57.6


105

Table 5.11 Median, IQR and range of sIgA and lactoferrin of left


Left Breast Day N Median IQR Range

sIgA 15 122 0.83 0.61-1.18 0.22-2.90

(g/l) 16 117 0.87 0.63-1.13 0.22-2.13

17 110 0.80 0.57-1.05 0.24-1.95

18 105 0.83 0.59-1.05 0.13-2.25

19 113 0.87 0.60-1.07 0.13-1.80

20 103 0.78 0.57-1.08 0.10-1.96

Lactoferrin 15 122 3.15 1.93-4.06 0.58-6.12

(g/l) 16 117 2.87 1.75-3.66 0.60-7.42

17 110 2.74 1.70-3.51 0.90-5.38

18 105 2.73 1.68-3.76 0.91-6.23

19 113 2.21 1.37-3.42 0.43-5.72

20 103 2.14 1.31-3.33 0.34-5.99


Table 5.12 Median, IQR and range of sIgA and lactoferrin of right


Right Breast Day N Median IQR Range

sIgA 15 120 0.83 0.60-1.17 0.09-2.07

(g/l) 16 112 0.93 0.57-1.13 0.24-2.15

17 108 0.80 0.57-1.14 0.31-1.80

18 105 0.82 0.58-1.05 0.22-1.85

19 112 0.86 0.61-1.08 0.10-1.97

20 102 0.76 0.50-1.11 0.11-1.63

Lactoferrin 15 120 3.11 1.80-3.90 0.25-6.57

(g/l) 16 112 2.95 1.70-3.77 0.51-5.92

17 108 2.70 1.73-3.56 0.68-4.81

18 105 2.55 1.57-3.67 0.82-6.03

19 112 2.30 1.50-2.93 0.72-5.76

20 102 2.13 1.14-3.10 0.06-4.87


106

Figure 5.10 Percentage of difference in daily milk production between left and right

breasts. The yellow line is the normal distribution curve. Positive value- when the

right breast had higher milk production. Negative value- when the left breast had

higher milk production

Dis

trib

uti

on

Percentage of difference

0

30

40

50

20

10

-70 -50 -30 -10 10 30 50 70 90

107

Figure 5.11 Fat content per breast per mother for days 15 to 20 postpartum. Each

point is the mean of milk fat content per day per mother.

Postpartum (days)

Mil

k F

at

(g/l)

Left

Right

15 16 17 18 19 20

80

0

40

120

80

0

40

120

108

Figure 5.12 Total protein concentration per breast per mother for days 15 to 20

postpartum. Each point is the mean of total protein concentration per day per mother.

Left

Right

Postpartum (days)

Mil

k P

rote

in (

g/l

)

15 16 17 18 19 20

40

0

20

60

40

0

20

60

109

Figure 5.13 Nutritive protein per breast per mother for days 15 to 20 postpartum. Each

point was the mean amount of nutritive milk protein per day per mother.

Nu

trit

ive

pro

tein

(g

/24

hr)

Left

Right

5

3

0

1

5

3

0

1

Postpartum (days)

15 16 17 18 19 20

110

Figure 5.14 Lactose concentration per breast per mother for days 15 to 20 postpartum.

Each point is the mean of lactose concentration per day per mother.

Postpartum (days)

Mil

k L

ac

tose

(g

/l)

Left

Right

15 16 17 18 19 20

60

0

100

60

0

100

20

20

111

Figure 5.15 Energy per breast per mother from day 15 to 20 postpartum. Each point is

the mean energy per day per mother.

Postpartum (days)

Mil

k e

nerg

y (

Cal/3

0m

l)

Left

Right

15 16 17 18 19 20

30

0

10

50

30

0

10

50

112

Figure 5.16 sIgA concentration per breast per mother for days 15 to 20 postpartum.

Each point is the mean sIgA concentration per day per mother.

Postpartum (days)

sIg

A (

g/l

)

Left

Right

15 16 17 18 19 20

0

1.5

0.5

2.5

0

1.5

0.5

2.5

113

Figure 5.17 Lactoferrin concentration per breast per mother for days 15 to 20

postpartum. Each point is the mean lactoferrin concentration per day per mother.

Postpartum (days)

Lact

ofe

rri

n (

g/l

) Left

Right

15 16 17 18 19 20

2

4

0

6

2

4

0

6

114

5.3.2.3 Longitudinal changes - days 10, 15-20, 30, 40, 50 and 60 postpartum

Due to the complexity of the data set, the graphic presentation in this section

only includes data at days 10, 20, 30, 40, 50 and 60 postpartum. However the

statistical analysis involved the entire data set.

The frequency of expression/breastfeed varied greatly between mothers (Fig

5.18). However, the median, IQR and range of frequency of expression/breast per

day as shown in Table 5.13 and 5.14 where similar to the result for days 15-20

postpartum. The frequency of expression/breastfeed on day 10 was different (p<0.05)

to day 17, 18, 19, 20, 30, 40 and 60 for both breasts and also day 30 and 50 for right

breast only.

Mean milk volume per expression/breastfeed for about 5 mothers’ breasts

changed greatly from day to day, while others remained steady (Fig 5.19). There was

no statistical difference between milk volume per expression/breastfeed from the left

breast and that from the right breast of the mothers. The median, IQR and range of

overall milk volume per expression/breastfeed for all days were 51, 35-79, 0.9-254.3g

(n=1443) and 52.4, 35-76.2, 1.2-263g (n=1416), left and right breasts, respectively.

The mean milk volume per expression/breastfeed was different (p<0.05) between day

10 and 17, 10 and 18, 10 and 19, 10 and 20 for both breasts as well as 10 and 30 for

right breast only (Table 5.9, 5.10, 5.13 & 5.14). Milk volume per

expression/breastfeed was strongly related to day (p <0.0001), order of expression (p

<0.0001), duration of expression (p <0.0001), interval since last expression (p

<0.0001) and multiple-birth (p <0.0001).

Fat content of milk varied greatly between mothers but the change from day to

day was very steady for each mother’s breasts (Fig 5.20). The overall median, IQR

and range of fat for all days were 44.7, 37-53.9, 1.5-182g/l (n=1023) and 44.6, 37.2-

52.7, 0.65-167g/l (n=1002), left and right breasts respectively. It was different

(p<0.05) between day 15 and 10, 15 and 16, 15 and 19, 15 and 40, 20 and 40 for left

breast and day 15 and 30, 15 and 40, 20 and 30, 20 and 40 for right breast (Table 5.9

& 5.10). Fat content per expression/breastfeed was strongly related to day (p =

0.0001), milk volume (p <0.0001), interval since last expression (p <0.0001) and

duration of expression (p = 0.0098). The fat concentration of milk gradually declined

from day 10 to 60 postpartum. The lowest fat contents were on day 40 and 50

postpartum for left breast but 30 and 40 postpartum for right breast. Higher milk

115

volume obtained in an expression or breastfeed was associated with a high fat

concentration. However, longer interval since last expression and duration of

expression were related to the decrease in the concentration of fat per

expression/breastfeed.

There was a trend in the concentration of total protein and the absolute amount

of nutritive protein per day that showed a decrease from day 10 to 60 postpartum for

all mothers (Fig 5.21 and 5.22). The overall median, IQR and range of total protein

for all days were 16.8, 13.3-21.4, 4.96-83.3g/l (n=1026) and 16.8, 12.9-21.5, 3.51-

75.6g/l (n=1001); nutritive protein (g/24hr) were 0.97, 0.65-1.45, 0.02-6.75 (n=1023)

and 0.89, 0.60-1.51, 0.02-8.06 (n=999), left and right breasts respectively. The

concentrations of total protein and the absolute amount of nutritive protein of day 10,

and 15-20 was different (p<0.05) to day 30, 40, 50 and 60 postpartum for both breasts

(Fig 5.34 and 5.36). Total protein concentration per expression/breastfeed was

strongly related to day (p < 0.0001), milk volume (p= 0.003) and frequency of

expression (p= 0.0039). In addition, nutritive protein per expression/breastfeed was

strongly related to day (p <0.0001), milk volume (p< 0.0001) and frequency of

expression (p = 0.0316). The concentration of total protein and the absolute amount

of nutritive protein declined rapidly from day 10 to 60 postpartum. Increased in

volume of milk per expression/breastfeed was related to a slight decrease in the

concentration of total protein (-0.027g/l per expression/breastfeed) but a slight

increase in nutritive protein (0.013g/24hr per expression/breastfeed). Increase

frequency of expression was related to a slight increase in total protein (~1.41g/l per

expression/breastfeed) and nutritive protein (~0.073g/24hr per expression/breastfeed).

The concentration of lactose was steady from day 10 to 20 postpartum for all

mothers (Fig 5.23). In fact even the mother who had 2x fat content and 2x

concentration of total protein in her milk had similar concentration of lactose

compared to the rest of mothers. The overall median, IQR and range of lactose for all

days were 66.2, 59-74, 34.7-115g/l (n=1027) and 65.7, 58.3-73.7, 29.3-109g/l

(n=999), left and right breasts respectively. Although, variations of lactose were

slight, the concentrations of lactose on day 10 were statistically different (p<0.05) to

day 15-18, 20, 30, 50 and 60 postpartum for left breast and 17-20, 30, 40, 60 for right

breast (Table 5.9, 5.10, 5.15 & 5.16). Lactose concentration per expression/breastfeed

was strongly related to day (p =0.002) and duration of expression/breastfeed (p =

0.0253). The lactose concentration in milk increased from day 10 to 60 postpartum.

116

All days showed a slight but significant increase in lactose concentration per

expression relative to day 10 postpartum. Longer duration of expression/breastfeed

was associated with a lower concentration of lactose in milk (-0.083g/l per

expression/breastfeed).

The trend for energy of milk from both breast during the first two months of

lactation were similar to that for the concentration of fat (Fig 5.24). The overall

median, IQR and range of energy for all days were 22.1, 19.8-24.8, 1.45-

63.3Cal/30ml and 21.9, 19.5-24.7Cal/oz, left and right breasts respectively. The

energy of milk was different (p<0.05) between day 15 and 10, 15 and 16, 15 and 30,

15 and 40, 15 and 50, 17 and 40, 20-40 for left breast and day 15 and 30, 15 and 40,

15 and 50, 20 and 30, 20 and 40, 20 and 50 for right breast (Table 5.9, 5.10, 5.15 &

5.16). Energy per expression/breastfeed was strongly related to day (p < 0.0001),

milk volume (p <0.0001), duration of expression (p = 0.0054) and interval since last

expression (p <0.0001). Energy per expression varied from day 10 to 60 postpartum.

There was a significant increase in energy per expression on day 15 postpartum, but

on day 30 and 40 postpartum, there were significant decreases in energy per

expression/breastfeed. During the day, the trend of energy per expression gradually

decreased from mid-night. Higher milk volume obtained in an expression or

breastfeed was associated with higher milk energy. Lower energy per

expression/breastfeed was observed with longer interval since last expression and

duration of expression.

The sIgA and lactoferrin concentrations of milk from both breasts of the

individual mothers declined from day 10 to 60 postpartum. The most rapid change in

the concentrations of these proteins was during the first 20 days, then the

concentrations of these proteins reached plateau (Fig 5.25, 5.26). The median, IQR

and range of overall sIgA concentration per expression/breastfeed for all days were

0.82, 0.59-1.11, 0.05-2.89g/l (n= 1026) and 0.81, 0.58-1.14, 0.07-2.93g/l (n=1005)

and lactoferrin concentration for all days were 2.41, 1.53-3.53, 0.07-7.93g/l (n=1026)

and 2.40, 1.51-3.49, 0.04-8.81g/l (n=1005), left and right breast respectively. The

concentration of sIgA on day 10 were significantly different (p<0.05) to day 15-20, 30,

40, 50 and 60 postpartum for both breasts (Table 5.11, 5.12, 5.15 & 5.16). The

statistical difference in the concentration of lactoferrin between days of postpartum

was summarized in Table 5.19 & 5.20. The sIgA concentration in milk per breast per

expression was related to the day of expression/breastfeed (p <0.0001), milk volume

117

(p=0.012) and breast (p=0.033). The concentration of sIgA in milk per

expression/breastfeed decreased as higher milk volume obtained as well as lactation

progressed. Left breasts had 0.03/gl more sIgA per expression/breastfeed than the

right breast. The concentration of lactoferrin was also found to decrease from day 10

postpartum (p <0.0001). Low volume of milk per expression/breastfeed was

associated with low concentration of lactoferrin (p<0.034). In addition, the

concentration of lactoferrin was lower at subsequent expression/breastfeed after

morning (p=0.0248), with longer duration of expression/breastfeed occurred

(p=0.0005) and 0.12g/l of lactoferrin more from left breast per expression/breastfeed

(p=0.0104).

There were significant correlations between the concentrations for sIgA and

lactoferrin from each breast per expression/breastfeed. sIgA was positively correlated

with lactoferrin (p <0.0001, n= 2031) (Fig 5.27).

118

Table 5.13 Median, IQR and range of milk production of left breast




Table 5.14 Median, IQR and range of milk production of right


Right breast


Frequency 10 127 7 6-7 5-8

20 135 6 6-7 3-8

30 128 6 5-7 3-9

40 114 6 5-7 2-8

50 99 6 5-7 4-9

60 89 6 5-7 4-8

Vol/exp 10 127 38 23-61 5-160

or bf 20 135 56 37-81 12-199

30 128 55 42-80 5-177

40 114 56 34-73 5-263

50 99 51 34-75 12-187

60 89 51 36-70 1-189



Left breast


Frequency 10 127 7 6-7 5-8

20 136 6 6-7 3-8

30 135 6 5-7 3-9

40 118 6 6-7 1-9

50 107 6 5-7 4-9

60 93 6 5-7 3-8

Vol/exp 10 127 45 30-65 2-142

or bf 20 136 51 35-85 9-226

30 135 51 35-86 8-171

40 118 48 32-75 1-145

50 107 49 35-80 7-175

60 93 49 38-62 1-163

119

Table 5.15 Median, IQR and range of milk component of left



Left breast


Fat 10 98 45.1 37.0-51.6 18.7-68.9

(g/l) 20 102 43.8 38.4-51.4 22.2-182.7

30 90 44.0 36.2-52.5 16.7-118.6

40 69 39.2 30.0-50.5 14.5-88.3

50 51 41.2 36.8-54.2 21.2-75.6

60 45 46.8 37.9-55.1 17.4-72.9

Total 10 98 20.8 17.3-24.6 9.6-38.1

Protein 20 102 18.1 14.2-23.0 7.9-53.3

(g/l) 30 90 13.9 11.6-17.7 7.9-36.1

40 70 13.4 10.9-16.4 6.0-28.3

50 52 12.5 10.1-15.7 6.9-22.7

60 45 11.4 9.1-14.9 5.0-22.6

Nutritive 10 98 0.95 0.12- 3.40 0.60- 1.48

Protein 20 102 1.04 0.20- 6.54 0.69- 1.79

(g/24hr) 30 89 0.89 0.24- 4.73 0.60- 1.36

40 70 0.78 0.34- 3.02 0.56- 1.13

50 52 0.73 0.20- 2.69 0.51- 1.03

60 45 0.61 0.02- 2.48 0.39- 0.83

Lactose 10 98 62.5 55.6-69.7 41.3-85.6

(g/l) 20 103 65.7 59.0-74.6 38.8-94.2

30 90 65.9 59.6-72.7 34.7-91.5

40 71 67.7 58.5-75.5 40.1-92.2

50 52 71.4 61.5-77.7 48.0-115.9

60 45 68.4 58.6-78.8 47.0-90.7

Energy 10 98 22.4 19.7-24.0 13.5-29.3

(Cal/30ml) 20 103 21.9 20.1-24.4 8.7-63.3

30 90 21.3 19.7-24.1 12.5-43.3

40 71 19.8 17.6-23.2 7.9-34.0

50 52 21.3 19.7-24.3 9.9-31.7

60 45 21.6 19.7-24.0 15.0-31.1

120

Table 5.16 Median, IQR and range of milk component of right


Right breast


Fat 10 95 45.2 38.1-53.1 15.4-64.2

(g/l) 20 101 44.7 37.7-53.5 23.2-167.9

30 87 40.1 32.8-48.8 21.9-123.5

40 67 38.7 30.3-49.2 13.8-80.8

50 48 40.3 34.5-48.3 22.0-78.0

60 46 47.3 36.2-54.2 18.2-72.3

Total 10 95 20.6 16.9-26.0 9.9-37.1

Protein 20 101 18.2 15.4-22.5 7.3-47.8

(g/l) 30 88 14.2 11.4-17.5 6.9-29.2

40 69 13.7 9.6-15.6 6.3-28.6

50 45 12.2 9.6-14.5 5.3-23.2

60 46 12.3 9.3-14.1 3.5-19.0

Nutritive 10 95 0.85 0.06- 3.50 0.57- 1.36

Protein 20 101 1.05 0.23- 4.20 0.70- 1.96

(g/24hr) 30 88 0.90 0.14- 5.12 0.53- 1.37

40 69 0.71 0.07- 3.37 0.48- 1.12

50 45 0.54 0.17- 3.38 0.39- 0.78

60 46 0.47 0.02- 2.87 0.31- 0.80

Lactose 10 95 61.5 53.7-68.2 31.8-86.2

(g/l) 20 102 65.3 59.1-74.1 44.5-91.0

30 87 67.2 59.2-75.9 41.2-94.6

40 68 68.3 62.0-73.1 38.7-96.0

50 46 66.6 57.6-72.4 48.2-89.2

60 46 72.6 56.4-80.5 46.8-109.7

Energy 10 95 22.9 20.1-24.5 10.5-29.3

(Cal/30ml) 20 102 22.1 20.1-25.3 8.7-57.6

30 88 20.5 18.9-22.6 10.2-45.2

40 69 20.5 17.1-22.9 9.0-33.8

50 48 19.3 17.5-22.0 9.2-32.6

60 46 22.0 19.7-25.2 15.8-29.1


121

Table 5.17 Median, IQR and range of sIgA and lactoferrin of left


Left Breast Day N Median IQR Range

sIgA 10 98 1.17 0.70-1.62 0.34-2.60

(g/l) 20 103 0.78 0.57-1.08 0.10-1.96

30 90 0.75 0.55-1.00 0.08-2.01

40 71 0.80 0.57-1.13 0.07-2.10

50 52 0.81 0.65-1.02 0.05-2.02

60 45 0.75 0.35-1.04 0.05-2.21

Lactoferrin 10 98 3.20 2.26-4.63 0.69-7.93

(g/l) 20 103 2.14 1.31-3.33 0.34-5.99

30 90 1.96 1.19-2.92 0.12-5.13

40 71 1.60 1.06-2.93 0.11-4.62

50 52 2.13 1.30-2.69 0.07-4.86

60 45 1.60 1.16-1.98 0.20-3.12


Table 5.18 Median, IQR and range of sIgA and lactoferrin of right


Right Breast Day N Median IQR Range

sIgA 10 95 1.02 0.64-1.49 0.35-2.93

(g/l) 20 102 0.76 0.50-1.11 0.11-1.63

30 88 0.73 0.55-1.06 0.24-1.92

40 70 0.80 0.58-1.15 0.14-2.14

50 47 0.79 0.65-1.12 0.10-2.02

60 46 0.80 0.40-1.16 0.08-1.43

Lactoferrin 10 95 3.34 2.05-4.77 0.91-8.82

(g/l) 20 102 2.13 1.14-3.10 0.06-4.87

30 88 1.82 1.25-2.92 0.12-6.58

40 70 1.59 1.02-2.88 0.04-4.66

50 47 2.09 1.23-2.78 0.43-4.69

60 46 1.45 0.93-2.53 0.04-3.60


122

Table 5.19 Statistical difference in the concentration of lactoferrin between days 10,

15-20, 30, 40, 50 and 60 postpartum (left breast).

Day 10 15 16 17 18 19 20 30 40 50 60

10 ----- ND ND * * * * * * * *

15 ----- ND ND ND * * * * * *

16 ----- ND ND ND ND * * * *

17 ----- ND ND ND * * ND *

18 ----- ND ND * * * *

19 ----- * ND ND ND ND

20 ----- ND ND ND *

30 ----- ND ND ND

40 ----- ND ND

50 ----- ND

60 -----

* - p<0.05, ND – no statistical different

Table 5.20 Statistical difference in the concentration of lactoferrin between days 10,

15-20, 30, 40, 50 and 60 postpartum (right breast).

Day 10 15 16 17 18 19 20 30 40 50 60

10 ----- ND ND * * * * * * * *

15 ----- ND ND ND * * * * * *

16 ----- ND ND ND * * * * *

17 ----- ND ND ND ND * ND *

18 ----- ND ND ND * ND *

19 ----- * ND ND ND ND

20 ----- ND ND ND ND

30 ----- ND ND ND

40 ----- ND ND

50 ----- ND

60 -----

* - p<0.05, ND – no statistical different

123

Figure 5.18 Frequency of expression/breasfeed per breast per day per mother from

day 10 to 60 postpartum. Each colour represents one mother.

Postpartum (days)

Fre

qu

en

cy

of

ex

pre

ss

ion

/bre

as

tfe

ed

Left

Right

10 20 30 40 50 60

6

0

3

9

6

0

3

9

124

Figure 5.19 Milk volume per breast per day per mother from day 10 to 60 postpartum.

Each point was the mean of milk volume per day per mother.

Postpartum (days)

Left

Right

10 20 30 40 50 60

100

0

200

100

0

200 M

ilk

vo

lum

e (

g/e

xp

res

sio

n/b

rea

stf

ee

d)

125

Figure 5.20 Fat content per breast per day per mother from day 10 to 60 postpartum.

Each point was the mean of milk fat content per day per mother.

Mil

k F

at

(g/l)

Left

Right

80

0

40

120

80

120

Postpartum (days)

10 20 30 40 50 60

40

0

126

Figure 5.21 Total protein concentration per breast per day per mother from day 10 to

60 postpartum. Each point was the mean of total protein concentration per day per

mother.

Postpartum (days)

Left

Right

10 20 30 40 50 60

40

0

20

60

40

20

60

Mil

k T

ota

l p

rote

in (

g/l)

0

127

Figure 5.22 Nutritive protein per breast per day per mother from day 10 to 60

postpartum. Each point was the mean amount of other milk protein per day per

mother.

Postpartum (days)

10 20 30 40 50 60

40

0

20

60

40

20

60

Nu

trit

ive

pro

tein

(g

/24

hr)

0

Left

Right

128

Figure 5.23 Lactose concentration per breast per day per mother from day 10 to 60

postpartum. Each point was the mean of lactose concentration per day per mother.

Left

Right

Postpartum (days)

Mil

k L

ac

tose

(g

/l)

Left

Right

10 20 30 40 50 60

60

0

100

60

0

100

20

20

129

Figure 5.24 Energy per breast per day per mother from day 10 to 60 postpartum. Each

point was the mean energy per day per mother.

Postpartum (days)

Mil

k e

nerg

y (

Cal/3

0m

l)

Left

Right

10 20 30 40 50 60

30

0

10

50

30

0

10

50

130

Figure 5.25 sIgA concentration per breast per day per mother from day 10 to 60

postpartum. Each point was the mean sIgA concentration per day per mother.

Postpartum (days)

sIg

A (

g/l

) Left

Right

10 20 30 40 50 60

1

0

2

1

0

2

131

Figure 5.26 Lactoferrin concentration per breast per day from day 10 to 60 postpartum.

Each point was the mean lactoferrin concentration per day. Each colour represents

one mother.

Postpartum (days)

Lac

tofe

rrin

(g

/l)

Left

Right

10 20 30 40 50 60

2

4

0

6

2

4

0

6

132

Figure 5.27 The regression analysis between sIgA (g/l) and lactoferrin (g/l).

Blue- left breast, pink- right breast

sIg

A

(g/l

)

0 2 6 10

Left

Right

Lactoferrin (g/l)

1

2

3

0

1

2

3

0

sIgA = 1.07(lactoferrin) + 1.63

R2= 0.2

sIgA = 1.10(lactoferrin) + 1.61

R2= 0.12

133

5.4 Discussion

The mean daily milk production for the preterm mothers (n=25) was 681±

367g/24hr for days 10 and 15 postpartum, and 727± 269g/24hr for day 30 postpartum.

This was greater than milk productions previously reported for similar times

postpartum of 493± 330ml/24hr and 606± 369ml/24hr (Hopkinson et al., 1988), 520±

390ml/24hr and 690± 350ml/24hr (Hopkinson et al., 1992), 507± 78ml/24hr and 571±

93ml/24hr (Hill et al., 1999) and 552± 429ml/24hr and 556± 444ml/24hr (Hill &

Aldag, 2005d), respectively.

It is likely that the higher milk production observed in my study was due to the

slightly later recruitment of mothers as discussed in Chapter 4.

Except for one mother, who attempted to breastfeed twice, once on day 10 and

once on day 17 postpartum, all other mothers were exclusively expressing milk from

day 10 to 20 postpartum. From day 30 onwards, mothers began to introduce

breastfeeds and transitioned to various degrees from feeding expressed breastmilk to a

combination of breastfeeding and feeding expressed breastmilk to their babies. Term

babies require at least 400ml/24h from 1-6 months postpartum to maintain growth and

development (Kent et al., 2006). Assuming a similar minimal level of milk

production would be required when pre-term babies have fully transitioned to

breastfeeding, 19 (83%) of the 23 mothers who remained in the study at 60 days

postpartum would have been able to meet this minimal requirement for milk

production.

In previous studies, breastfeeding mothers were found to produce more milk

from their right breast (Kent et al., 2006). However, in my study there was no

significant difference in the daily milk production between the left (376 192g/24hr)

and right (376 195g/24hr) breasts at either day 10-20 postpartum (when the mothers

were fully expressing) or left (345 152g/24hr) and right (357 142g/24hr) breasts at

day 30-60 postpartum (when the mothers were transitioning to breastfeeding) found in

this study. On the other hand, Engstrom et al. (2007) found that the right breasts of

preterm mothers with high pumping frequency often produced more milk than their

left breasts. However, this study was based on one expression session on particular

days and my analysis was for 24hr milk productions. They also based their analysis

on the proportional differences between left and right breasts to control for the large

differences in milk production between pre-term mothers. Expressing my 24hr milk

134

productions as proportional differences between left and right breasts did not result in

significant differences between breasts (Fig 5.10). Since I did not find a difference

between left and right breasts, it is tempting to suggest that the differences between

left and right breasts in breastfeeding mothers was due to a baby preference. However,

the conflicting findings of these two studies for pump dependent mothers does not

permit any firm conclusions to be made in relation to the influence of the

breastfeeding baby versus breast expression on differences in milk production that

have been reported between left and right breasts.

Applying the regression analysis describe in chapter 4 to the entire data set (i.e.

from 10 to 60 days of lactation) resulted in 40 of the 46 breasts having the same

significant correlation between the percentage of milk yield and the interval since

previous expressions as the analysis of the data for day 15 to 20 postpartum.

Therefore it is possible to extrapolate the result from the data analysis for day 15 to 20

postpartum to the entire dataset. However, two of the breasts changed from non-

significant to significant correlations and four had the reverse change. These switches

might be due to the fact that the babies began to transit to fully breastfed, therefore,

the baby’s appetite may have influenced milk yield at a breastfeeding session adding

to the observed variation. Nevertheless, the relationship held for 87% of the breasts

from 10 to 60 days of lactation confirming the importance of interval between breast

expressions as a determinant of the variation in milk yield at a breast expression.

Previous studies on the fat content of preterm milk in established lactation

have been carried out at approximately two, four and eight weeks postpartum and

these times compare with my results for 15, 30 and 60 days postpartum. The mean

content of fat in milk samples at day 15, 30 and 60 postpartum from my study (Table

5.19) were slightly higher than those reported by Gross et al. (1980), Anderson et al.

(1981), Lemons et al. (1982) and Butte et al. (1984). Whereas the fat content of milk

at 30 days postpartum reported by Lepage et al. (1984) was similar, those reported by

Sann et al. (1981) were about half my findings (Table 5.21). The fat content of

breastmilk is subject to two major sources of error. First, sampling error is a

significant problem because the fat content of milk can increase several fold from the

beginning to the end of either a breastfeed (mean increase from 31 to 83g/l) (Daly et

al., 1993); (mean increase from 29 to 53g/l) (Kent et al., 2006) or breast expression

(mean increase in my study was from 37 to 67g/l). Thus it is important to obtain a

mixed sample of the total volume of milk expressed by either measuring each

135

expression over the 24 hr period or collecting all the milk expressed over the 24 hr

period. Whereas Sann et al. (1981), Anderson et al. (1981), Lemons et al. (1982) and

Lepage et al. (1984) took a sample from the pooled milk collected over a 24hr period

for each subject, Gross et al. (1980) and Butte et al. (1984) took a single morning

sample for each subject. This latter sampling protocol also would be expected to give

a reasonable estimate of the average fat content of breast milk because my results

showed that there was no significant difference in the fat content of milk over the day,

that is, from morning, day, evening and night (Fig 5.3). Secondly, a variety of assay

methods have been used to measure the fat content of breastmilk and therefore some

variation can occur depending on the specificity of the assay. I measured the

esterified fatty acid content of the milk fat assuming that almost all the milk fat was

triacylglycerols and that the average molecular weight of breastmilk fatty acids was

843.1Da (Jensen, 1989). Gross et al. (1980) and Butte et al. (1984) used the Rose-

Gottlieb method which measured the weight of the dried lipid extract whereas Sann et

al. (1981) extracted the lipid using chloroform-methanol according to Folch et al.

(1957). Anderson et al. (1981) and Lemons et al. (1982) used a colorimetric method

which utilized the sulfuric acid charring technique to determine the content of fat.

Lepage et al. (1984) used the sum of the fatty acids determined from gas liquid

chromatography. These different methods of fat content measurement may account

for the wide variations of fat content between studies

The content of fat in milk was not statistically different between the left and

right breasts of individual women. However, it is important to investigate each breast

independently, not only to get a valid estimate of milk fat production each day but

also because changes in the volume of milk available in the breast before and after

each breastfeed over a 24hr period explained 68% of the variation in the fat content of

the milk in the breasts of term mothers (Daly et al., 1993). Conversely, the changes in

the fat content of the milk can be used to estimate the degree of fullness of the breast

at any point in time (Cox et al., 1996). Statistical analysis also showed that the

content of fat in preterm milk was positively associated with milk volume per

expression/breastfeed but negatively associated with interval since last expression.

This finding was similar to term mothers who were exclusively breastfeeding their

babies. Their breastmilk, as measured in hind milk samples, had a high content of fat

after larger volumes of milk were consumed by their babies (Kent et al., 2006).

However, lower fat contents were observed at longer intervals between expressions

136

and this is consistent with the observations in Chapter 3 that after a longer interval the

breast is not completely drained of milk until the second hourly expression. Thus, it

would be expected that the hind milk obtained after a longer interval would have a

lower fat content.

The variation in the content of fat in fore- and hind- milk at day 15 and day 20

was investigated to determine if variation in the degree of milk removal during each

breast expression could explain part of the variation in milk yield. Analysis of the fat

content of the milk according to the groups in the regression analysis (see Chapter 4)

showed that there were significant differences in the fore-, mid- and hind- milk fat

content between the four groups. Relative to the non significant group in the

regression analysis, the groups 1 and 2 with R square (>0.25) in the regression

analysis had significant higher volume of milk per expression/breastfeed estimated

from the content of fat in the fore-, mid- and hind- milk. The estimated mean volume

of the group (R2= 0.25 to 0.50) for fore- and mid- milk were 11g and 109g per

expression/breastfeed, while the estimated mean volume of the group (R2

> 0.50) for

fore-, mid-, hind- milk were 23g, 127g and 8g per expression/breastfeed. This

suggested that in addition to the interval between breast expressions, the efficacy of

expression was also responsible for some of the observed variation in milk

composition.

However, a higher (p <0.0026) content of fat in the hind milk was associated

with higher milk yields in all four groups. This implied that more of the available milk

was removed when higher milk yields were obtained, suggesting that in addition to

interval between breast expressions the efficacy of expression was responsible for

some of the observed variation in milk yield. It was of interest that the fat content of

mixed milk was only significantly higher with higher yields in groups of breasts that

either did not show a significant response to interval or showed that interval

accounted for >50% of the variation. These two groups also had significantly higher

milk production (Table 4.2). However the explanation for this relationship for these

two groups is not apparent. Nevertheless, more research is required to identify the

sources of variation (other than interval between expressions) in the yield of milk at a

breast expression so that advice can be provided to pump dependent mothers to

minimize their effort and optimize their milk production.

137

Table 5.21 Fat content of preterm milk at day 15, 30 and

60 postpartum by current and other studies

Fat (g/l)

Day 15 Day 30 Day 60

My study L 53± 21 46± 16 46± 13

R 51± 20 43± 15 46± 12

Gross et al. (1980) 44± 14 40± 12

Sann et al. (1981) 25± 8

Anderson et al. (1981) 43± 9.5 41± 8

Lemons et al. (1982) 34± 7.6 32± 6.4 35± 9.6

Butte et al. (1984) 35± 9 40± 12 42± 13

Lepage et al. (1984) 47± 5

mean ± SD, L- left breast, R- right breast

As with milk fat, previous studies on the concentration of protein in preterm

milk in established lactation have been carried out at approximately two, four and

eight weeks postpartum and these times compare with my results for 15, 30 and 60

days postpartum (Table 5.22). The mean concentration of total protein was slightly

higher than previous reports on day 15 of lactation but similar at days 30 and 60

postpartum (Table 5.22)

All studies found that the concentration of protein in milk was higher on day

15 and declined gradually to 11 to 12 g/l by day 60 postpartum. A similar decline has

been observed by other workers (Table 5.22). Although, the method of protein

analysis in this study was different to previous studies, the standard curve for the

BioRad dye binding assay was prepared from preterm breastmilk with a known

concentration of protein that had been determined using the Kjeldhal method (see

chapter 2). There was no difference in the concentration of total protein between left

and right breasts and in agreement with the finding of Atkinson et al. (1978) the

concentration of total protein in milk was not related to volume of milk per

expression/breastfeeding. The statistical analysis also found that there was a

significant positive relationship between the concentration of protein and frequency of

expression with an increase of 1.41g/l for every additional expression per day.

Although the type of changes in the concentration of total protein in the milk of

preterm mothers with stage of lactation were similar to those reported for term

138

mothers (Gross et al., 1982), the concentration of total protein was consistently higher

in the milk of the preterm mothers. The reason for this higher concentration of total

protein is not readily apparent because in the past these babies did not survive

therefore evolutionary pressure could not have been responsible for the favorable

higher concentrations of total protein in preterm mother’s milk. Therefore, it is likely

that the premature initiation of lactation may have influenced the concentration of

total protein in the preterm milk.

Table 5.22 Protein concentration of preterm milk at day

15, 30 and 60 postpartum by other studies

Protein (g/l)


My study L 20± 7 15± 5 12± 4

R 20± 7 15± 5 12± 3

Gross et al. (1980) 14 4 12± 2

Sann et al. (1981) 18± 6

Anderson et al. (1981) 17 3 14± 3

Lemons et al. (1982) 15 3 12± 2 12± 2

Butte et al. (1984) 15 1 14± 1 11± 3

Lepage et al. (1984) 19± 3


While the milk proteins lactalbumin and casein have high biological value,

evidence suggests that the protective glycoproteins, sIgA and lactoferrin, are not

readily digested (Davidson & Lonnerdal, 1987). Therefore, the absolute amount of

nutritive protein was calculated by a formula (section 5.6.7). The median of nutritive

protein, in Fig5.22, represented approximately 98% of the absolute amount of total

protein in preterm milk. Nevertheless, the concentration of total protein in preterm

milk was higher than in term milk. Furthermore sIgA plus lactoferrin constitute a

higher proportion (approximately 32%) of the concentration of total protein in term

milk (Lonnerdal & Atkinson, 1995) and concentrations of sIgA and lactoferrin in

preterm milk were similar to that in term milk (Montagne et al., 1999). Thus the

concentration of proteins other than sIgA and lactoferrin must be even higher in

preterm milk than suggested by the total protein concentration. Studies are required to

139

identify the relative composition of the nutritive proteins in preterm milk as the amino

acid composition of these proteins could be of significance in maintaining nitrogen

balance in preterm babies. Furthermore, understanding the composition of the

nutritive protein may provide the key for improving current methods of fortification

mother’s own milk with appropriate amino acid supplements for preterm babies.

The mean concentrations of lactose in milk at day 15, 30 and 60 postpartum

were within the range of values reported by previous studies (Table 5.23). The

change in the concentration of lactose in milk from both breasts was very consistent

between days. Lactose is the most osmotically active component in the milk. As its

concentration increases, it draws water into the lumen thus raising the overall volume

of milk in the breasts (Neville & Allen, 1983) and therefore it was expected that the

concentration of lactose in preterm milk would show less variation than that of the

other macro-nutrients. The concentration of lactose was similar both between left and

right breasts and from day 15 to 60 of lactation and this finding is in agreement with

previous preterm studies (Table 5.23). The concentration of lactose in the preterm

milk is also similar to that reported for term milk at a similar stage of lactation (Gross

et al., 1980; Lemons et al., 1982) suggesting that lactose synthesis and osmotic

balance was not influenced by the premature initiation of lactation in preterm mothers.

Table 5.23 Lactose concentration of preterm milk at day

15, 30 and 60 postpartum by other studies

Lacotse (g/l)


My study L 67± 11 67± 11 68± 12

R 66± 10 67±11 70± 15

Gross et al. (1980) 62 8 69± 10

Sann et al. (1981) 65± 18

Anderson et al. (1981) 56 2 60± 3

Lemons et al. (1982) 69 5 68± 7 67± 6


The energy content of the preterm milk was similar in the left and right breasts

and was similar to that reported in previous studies (Table 5.24). Energy content of

milk is either calculated from macronutrient concentration (fat, protein and lactose)

140

(used in this study, Gross et al. (1980) and Anderson et al. (1981)) or determined by

bomb calorimeter (used by Lemons et al. (1982), Butte et al. (1984) and Lepage et al.

(1984)). In this study, energy was calculated based on the equation recommended by

NHMRC (2005) (see section 5.2.8). However, different factors were used by Gross et

al. (1980) (energy = fat x 8.87 + protein x 4.27 +lactose x 3.87, that is, metabolizable

energy) and Anderson et al. (1981) (energy = fat x 9.25 + protein x 5.65 +lactose x

3.95, that is, gross energy). Furthermore, Hosol et al. (2005) demonstrated that there

was a difference between calculated value for energy from the energy formula (energy

= fat x 9.16 + protein x 4.22 +lactose x 3.87) and measured value for energy by bomb

calorimeter. Nevertheless, despite the differences in milk composition and the

methods employed to determine the energy content of the preterm milk there is

remarkably close agreement in the reported values for the energy content of preterm

milk from day 15 to 60 postpartum (Table 5.24).

Table 5.24 Energy content of preterm milk at day 15, 30

and 60 postpartum by current and other studies

Energy (Cal/30ml)


My study L 24± 6 22± 5 22± 4

R 24± 6 21± 5 22± 4

Gross et al. (1980) 22 4 21± 4

Anderson et al. (1981) 22 2 21± 2

Lemons et al. (1982) 23 3 22± 3 23± 2

Butte et al. (1984) 19 3 20± 3 19± 4

Lepage et al. (1984) 23± 2


The mean concentrations of sIgA in milk at day 15, 30 and 60 postpartum

were 0.9± 0.5g/l, 0.8± 0.4g/l, 0.8± 0.5g/l, for both breasts. They were within the

range of the values reported by Montagne et al. (1999) of 1.7± 0.9g/l (day 14), but

slightly lower than the value reported by Lonnerdal et al. (1979) of 1.0± 0.3g/l (term

mothers, day 14-40). Furthermore they were similar to the concentration of sIgA in

milk from term mothers who had history of atopic symptoms (0.7± 0.4g/l and 0.6±

0.4g/l, left and right breasts respectively) (Lai, 2002). It is of interest that there was

141

small variation within mothers between breast but large variation in the concentration

of sIgA between mothers (Fig 5.25). Furthermore, mothers with low concentration of

sIgA in milk at day 10 of lactation tended to continue to have low concentration up to

day 60 postpartum and those with higher concentrations of sIgA had higher

concentration up to day 60. Statistical analysis suggested that this large variation was

not due to the difference in the expression regime between mothers. Sepsis and

necrotizing enterocolitis frequently occur in preterm babies. Feeding human milk to

these babies has been found to reduce the onset of both sepsis and necrotizing

enterocolitis (Lucas & Cole, 1990b). The large variations in the concentration of sIgA

between mothers suggested that some babies receive considerably less sIgA

protection then other babies. It would be important to determine if the babies of

mothers with lower concentration of sIgA in their milk are more prone to necrotizing

enterocolitis and/or sepsis.

The mean concentrations of lactoferrin in milk at day 15, 30 and 60

postpartum were 3.0± 1.3g/l and 3.0±1.3g/l, 2.1± 1.2g/l and 2.2±1.4g/l, 1.6± 0.7g/l

and 1.7± 0.9g/l, left and right breasts respectively. They were within the range of the

values reported by Montagne et al. (1999) of 2.7 0.7g/l (day 14), but slightly higher

concentration than the value reported by Lonnerdal et al. (1979) of 1.9± 0.4g/l (term

mothers, day 14-40). Furthermore they were similar to the concentration of

lactoferrin in milk from term mothers who had history of atopic symptoms (2.7±

2.0g/l and 2.5± 1.9g/l, left and right breasts respectively) (Lai, 2002). There was a

weak positive correlation between sIgA and lactoferrin (Fig 5.27). Thus if a low

concentration of sIgA was observed in the milk of a preterm mother, there would be a

tendency for the mother to have a low concentration of lactoferrin in her milk.

Therefore milk of these mothers would provide much less innate immune protection

and may expose their babies to infection and disease. In this connection Lai (2002)

observed that the babies of mothers with lower concentrations of lactoferrin in their

milk had a greater risk of respiratory tract infection. However further research is

needed to confirm this finding.

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Chapter 6

General discussion and clinical implications

6.1 General discussion

Most mothers who deliver at term have the physiological capacity to provide

milk for their babies. Once lactation is established, the appetite of the baby regulates

milk production (Dewey & Lonnerdal, 1986). The regulation is through the local

inhibition of milk synthesis that acts independently in each breast as milk accumulates

between breastfeeds. Therefore increased milk removal from the breast by either

expressing the breast after each breastfeed (Dewey & Lonnerdal, 1986) or increasing

breastfeeding frequency (Egli et al., 1961) will increase milk production. Many

mothers who deliver prematurely have a delayed initiation of lactation (Cregan et al.,

2002) and most mothers have a low supply of milk. Furthermore, the inability of

preterm babies to feed directly from the breasts due to either sickness or immature co-

ordination of the suck, swallow, breathe reflex, results in their mothers being

dependent on breast expression (usually with an electric breast pump) to provide

breast milk for their babies.

Current expression recommendations for preterm mothers are consistent with

the finding from the dairy industry that increased milking frequency results in an

increase in daily milk production. DeCarvalho et al. (1985) first reported a strong

correlation between daily milk production and frequency of expression of preterm

mothers. Hopkinson et al. (1988) and Hill et al. (2001) also found that preterm

mothers who were expressing milk greater than 6 times per day had a greater increase

in total milk volume at the first six weeks postpartum. These consistent findings have

lead to an optimal expression regime that is now widely practiced. That is, preterm

mothers should express their breasts frequently (~6 times per day) and at least 100

minutes per day. In addition, the high frequency of breastmilk expression was shown

to improve the success of the initiation of lactation for preterm mothers (Hill et al.

2001). Therefore, increased expression frequency is perceived as the way to increase

daily milk production. Obviously such a recommendation is very demanding for

mothers who are very concerned about their fragile sick babies. In addition the studies

on which these recommendations were derived do not take into account potential

differences between breasts within mothers. My study is the first comprehensive

143

study that has collected every volume and sample of milk per expressions per breast

per day on day 10, 15-20, 30, 40, 50 and 60 postpartum. This intensive sample

collection is able to determine the variation in the yield and composition of breastmilk

from each breast of preterm mothers per day as well as during established lactation

(10 to 60 days postpartum). Furthermore, I also investigated the short time influence

of a high frequency of breast expression (hourly expression up to 8 hr) on milk yield

of term mothers in established lactation.

The concept of the autocrine inhibition of milk production as a result of the

accumulation of an inhibitory factor (FIL, Chapter 1, section 1.4.2) in the milk of a

full breast has been used to explain the observation of increased milk production

associated with more frequent breastfeeding and/or breast expression. Furthermore, it

has been shown that the inhibitory factor acts with a unique mechanism, that inhibits

the secretion of milk from the Golgi vesicles (Rennison et al., 1993) and this enables

a rapid down regulation of secretion as milk accumulates in the breast with longer

intervals between milk removal. However, Daly et al. (1996) found that the inhibition

of milk synthesis in the breasts of mothers who delivered at term occurred after

intervals of more than 6-8 hr. This suggested that the impact of autocrine inhibition

of milk production was influenced by the efficacy of the expression regime.

Daly et al. (1996) found that a large variation in milk yield from each breast of

term mothers resulted in difficulties in assessing the impact of the expression regime

on the milk production (Fig 4.7). However, by expressing the volume per expression

as a proportion of 24hr milk production (actual milk yield), it was possible to

standardize the difference in the milk yield between breasts and allowed the analysis

of the relationship between proportional milk yield and interval since previous

expression. Furthermore, by expressing the 24hr milk production as 100%, the

cumulative expected milk yield (expected milk yield) curve could be calculated for all

times from 0 to 24 hr (Fig 4.8) assuming that the rate of milk synthesis over the day

was constant.

When the milk yield per expression per breast was expressed as a proportion

of 24hr milk production (Chapter 4), similar responses were observed in mothers who

had delivered prematurely (Fig. 4.8-4.30) to that reported by Daly et al. (1996) for

term mothers. The interval since previous expression was significantly correlated with

the actual milk yield for 40 of the 46 breasts (87%). The greater number of breasts

(46) compare to the 8 in Daly’s study studied in this project allowed me to explain the

144

range in the variation in actual milk yield by the variation in the interval since

previous expression (varied from 25 to 81%, Chapter 4). In some breasts, the interval

between breast expressions explained most of the variation in actual milk yield, that

is, the actual milk yield had a similar slope to the expected milk yield regression line.

In these breasts extending the interval between breast expressions up to 6 hr did not

decrease actual milk yield even in high producing breasts (Fig 4.8- 9) suggesting that

local autocrine inhibition was not an important factor in regulating milk production in

these breasts. Whereas in other breasts the slope of the actual milk yield regression

line was less than the expected milk yield regression line and expression intervals of

greater than 4 hr resulted in a decrease in actual milk yield (Fig 4.18), suggesting that

the autocrine control mechanism responded rapidly to increasing intervals, i.e.

breastmilk accumulation, in these breasts. These observations are consistent with

those of Daly et al. (1996) who measured the change in volume of mother’s breasts

and found that the rate of milk synthesis was relatively constant in mothers who

breastfed relatively frequently and decreased in mothers when they had a long interval

between breastfeeds. In this connection it is of interest that Kent et al. (2006) found

that term babies of exclusively breastfeeding mothers breastfed from a breast

approximately 6 times per day. Therefore it is likely that autocrine inhibition does not

play an important role in regulating milk production in a significant proportion of

breast. Thus the impact of local inhibition on actual milk yield as the interval between

breast expressions/breastfeeding increases probably depends on the rate of milk

synthesis, the capacity of the breast to store milk, the breasts sensitivity to the

inhibitory factor(s) and the efficiency of milk removal at the previous breast

expression. The relative influences of these factors result in actual milk yield in some

breasts showing a high dependence on the interval between expressions while other

breasts show little (Fig 4.26) or no effect (Fig. 4.30). Furthermore, the influence on

the accumulation of milk in the breast on inhibition of milk secretion may be quite

separate to the mechanism increasing milk synthesis in response to increasing the

frequency of milk removal.

A constant yield of milk was obtained from the 3rd

expression onward when

term mothers in established lactation expressed their breasts at hourly intervals for

periods of up to eight hours in a controlled environment (Chapter 3). This constant

yield of milk suggests that only the newly synthesized milk was obtained from the 3rd

hourly expression onward. Thus it was concluded that the synthesis of breastmilk

145

occurs at a constant intrinsic rate when autocrine inhibition is negated by expressing

the breast every hour. Furthermore, it was found that the hourly milk yield from the

3rd

expression onwards was not significantly different from the hourly milk yield

calculated by dividing the daily milk production by 24. It should be noted that this

latter value equates to the expected milk yield at one hour calculated in Chapter 4.

Although more frequent milk removal has been demonstrated to increase milk

synthesis in dairy animals (Wilde et al., 1987a), sows (Knight et al., 1998) and

women (Dewey & Lonnerdal, 1986) the response time for increasing milk synthesis is

much longer (days rather than hours) than that for the autocrine inhibition of milk

secretion. Therefore, there must be another mechanisms or factors that are

influencing the milk synthesis of the breasts.

The measurement of milk production in breastfeeding mothers is calculated

over a period of at least 24 hour because milk intake at each breastfeed is usually

determined by the infant’s appetite rather than the availability of breastmilk (Kent et

al., 2006). Presumably as the ‘stored’ milk was removed at the 1st and 2

nd hourly

expressions, only the newly synthesized milk was obtained from the 3rd

hourly

expression onward (Fig 3.3). This intrinsic rate of milk synthesis was similar to the

hourly rate obtained from the 24hr milk production by test weight method. Therefore

it would be possible to obtain an estimate of daily milk production by multiplying the

volume of milk expressed at the 3rd

hourly expression with 24.

Hunting and gathering societies are thought to represent some aspects of early

human development (Konner & Worthman, 1980). In these societies infants are

always close to their mothers and breastfeeding occurs for a few minutes at a time at

least once each hour. It could be predicted from my findings that under this

breastfeeding regime, autocrine inhibition of milk secretion due to breast fullness

would be unlikely to occur and that the frequent removal of milk from the breast

would stimulate high levels of milk synthesis. This may explain why poorly

nourished women in developing countries are able to produce similar volumes of

breastmilk to well nourished women in developed countries (Prentice et al., 1986).

The intensive sampling procedure in this study provided an opportunity to

observe the changes in the composition of milk between breasts, throughout the day,

between days and during the first two months of lactation; which something that has

not been previously studied to this degree of detail. The trends for fat and lactose

were to increase during the first 20 days and reach a steady state from day 30

146

postpartum onward, while the trend for total protein, nutritive protein, sIgA and

lactoferrin was to gradually decline.

The content of fat in milk of preterm mothers in this study was higher than

that found in previous studies ((Gross et al., 1980; Anderson et al., 1981; Sann et al.,

1981; Lemons et al., 1982; Butte et al., 1984; Lepage et al., 1984)). This difference

might be related to the different methods use in the determination of fat content. My

method of fat measurement does not involve the extraction of fatty acids from

triacylglycerols and all the reactions were preformed in a single tube for each milk

sample. On the other hand, other methods from other studies required extraction and

conversion before the measurement took place. Thus, it was possible that some

content of fat was lost during the extraction phase, which resulted in lower fat content

found by those studies. Therefore, this may suggest that the content of fat in preterm

milk is higher and offers higher energy than previously thought. Future studies on the

content of fat will provide more information about the importance of the high fat

content in milk for the growth and development of babies.

Protein in milk was not only nutritive, i.e. provides essential amino acids but

also consists of proteins, such as sIgA and lactoferrin, that carry out specific functions

(Lonnerdal & Atkinson, 1995). Since the concentrations of sIgA and lactoferrin in

the milk of preterm mothers were similar and consistent with that in the milk of term

mothers (Montagne et al., 1999), the increased concentration of total protein in

preterm milk would suggest an increase in the concentration of nutritive protein.

Alpha- lactalbumin, one of the nutritive proteins, is an important protein that forms

lactose synthetase with galactosyltransferase to catalyze the formation of lactose in

the Golgi apparatus of the lactocytes (Neville & Allen, 1983). As lactation

progresses, the breasts of the preterm mothers may undergo substantial changes in

milk synthesis and the lactocytes in the breasts may prioritize the synthesis of alpha-

lactablumin and galactosyltransferase to increase the output of lactose. Therefore the

high concentration of nutritive protein might indicate an increase in the milk synthesis

of the breasts. Future studies on the composition of the nutritive protein will provide

more information about the importance of the extra nutritive protein in milk for the

growth and development of babies.

The positive relationship between both the total and nutritive protein and the

frequency of expression suggests a higher concentration of protein in milk could be

achieved with additional breast expressions per day. Further examination of this

147

relationship may provide more information to permit the development of an

expression regime to maximize the concentration of protein in mother’s own milk for

preterm babies. This would provide a greater benefit to preterm babies who have a

higher protein requirement.

The concentrations of sIgA and lactoferrin were greatly varied between

mothers (ranging from 0.05 to 2.93g/l of sIgA and 0.04 to 8.82g/l of lactoferrin).

Since the preterm babies’ immune system is very immature, a baby who receives

lower amounts of these immune proteins may be more prone to the development of

necrotizing enterocolitis and sepsis. Lai (2002) reported that low concentrations of

sIgA (< 0.6g/l) in term milk were associated with a higher risk of contracting

respiratory tract infections by 12 months of age in term infants. Therefore further

investigation of the relationship between the concentrations of sIgA and lactoferrin,

and the frequency of onset of infection in these babies could allow better assessment

of the minimum amount of these proteins rquired to protect them against infections

and diseases.

6.2 Clinical implications

Clinically, the goal for pre-term mothers for their total daily milk production

ranges from 350g/24hr (Meier, personal communication) upto 500ml/24hr (Hill et al.,

2005c). Currently there are no universal or standard international guidelines for the

volume of milk that these mothers should produce per day. If there is a need for a

guideline then the minimum daily milk production that mothers should aim for is the

minimum volume of milk required for an exclusively breastfed babies at 1- 6 months

which is 440ml/24hr (Kent et al., 2006). In my study, 21 of 25 mothers produced

more than 450ml/24hr from 10 to 60 days of lactation. As discussed in Chapter 4, the

high proportion of mothers producing in excess of 450ml/24hr was probably due to

the recruitment of mothers at 5-10 days after birth rather than immediately after

delivery. Therefore it was likely that most recruited mothers had established their

lactation with their expression regime.

Current expression regimes place a great demand on the preterm mothers at a

time of extreme stress. Hence any evidence-based advice that can optimize milk

production and minimize the effort for the mothers would greatly enhance their

chance of having a successful lactation. Although, the short-term control of milk

148

synthesis occurs at the level of the individual breasts, practical advice for mothers

needs to be similar for both their breasts. An examination of the regression analysis

for individual breasts could be used to predict expression regimes that could be

beneficial to preterm mothers. This evidence-based analysis would be able to design

an optimized regime that removes most milk from each breast at each expression,

which could trigger a series of enzymatic events in the breast that result in an increase

in milk production.

The regression lines for the actual milk yields and the expected milk yields

had similar slopes and intercepts for both breasts for three of 25 mothers (Fig 4.8-10).

Furthermore these mothers had high daily milk productions (>900ml/24hr). Their

average frequency was 5.1 ±0.5 expressions per day per breast between days 15 and

20 postpartum. Since the proportion of 24hr milk production was highly correlated (r

= 0.74 to 0.84) with interval since previous expression for these mothers, the milk

yield from the breasts of these mothers would not respond to changes in the

expression regime. Regression analysis predicts that the interval of expression could

extend from 4.8 to 7 hr without compromising the milk production. This would

reduce the frequency of expression from 5 to 3.4 times per day and save on average

48 ± 14 minutes of expressing per day for each mother. Furthermore since the median

frequency for these mothers was 5, 50% of the time the mothers were expressing at

intervals that were shorter than 4.7hr with some intervals as short as 2-3 hours,

therefore apart from decreasing the expressing time per day, the extended interval

would enable the mothers more flexibility in planning their daily activities.

The slopes of the regression lines for the actual milk yield and expected milk

yield were different for 7 of 25 mothers (Fig 4.11-14, 4.19, 4.20 and 4.26) but similar

responses were observed form both breasts of these mothers. The daily milk

production was >450ml/24hr (mean of 818 ± 49ml/24hr). The average frequency of

expression for these mothers was 6.2 ± 0.5 and total expressing time was 112 ± 14

minutes per day between days 15 and 20 postpartum. Extending the interval from 4hr

to 5hr per expression predicts a similar daily milk production for these mothers but

the frequency of expression would be reduced from 6 to 4 times per day and save on

average 34 ± 8 minutes per day per breast. However it would be necessary to monitor

these mothers because the slope of the regression line of the actual milk yield shows

that milk production was compromised with extended intervals between breastfeeds.

In addition, the correlation between the proportion of 24hr milk production and

149

interval since last expression (r = 0.45 to 0.83) was greatly varied, therefore it is likely

that there would be a graded response between mothers in the improvement of the

daily milk production associated with optimizing the interval between breast

expressions.

Two of 25 mothers had daily milk production <450ml/24h (246ml/24hr for

mother in Fig 4.18 and 321ml/24hr for mother in Fig 4.27). The slopes of the

regression lines for the actual milk yield and expected milk yield were different but

similar responses were observed between the left and right breasts for each mother.

They expressed on average 5.3 ±0.6 times with per day between days 15 and 20

postpartum. The slope of the regression line predicts that there would be an

improvement in both mothers’ milk productions if the interval between breast

expressions did not exceed 4hr. According to the regression equation for the actual

milk yield for both breasts in Fig 4.18, 23% of 24hr milk production should be

achieved at 4hr interval. On this basis it would be predicted that 4hr intervals would

result in an increase of 38% in the 24hr milk yield per breast and therefore an increase

of 72% in total daily milk production for both breasts. That is, these findings predict

that an extra 177ml/24hr could be obtained with an expression interval of 4 hr and her

daily milk production would increase to 423ml/24hr which would meet the minimum

requirement of milk production. For the mother in Fig 4.27, 4hr intervals would

result in an increase of 16% in the 24hr milk yield per breast and therefore increase

her total daily milk production by 32%. That is, increasing her daily milk production

to 437ml/24hr. However, since the correlation between the proportion of 24hr milk

production and the interval since previous expression for each mother was not very

strong (r = 0.36 to 0.64), the predicted improvement in milk production by reducing

the longer intervals between breast expressions would be a maximum expected

response.

The slopes of the regression lines for the actual milk yields and expected

milk yields between the left and right breast were different for eight of 25 mothers

(Fig 4.11-17 and 4.21-25). These eight mothers had average daily milk production

>450ml/24hr (mean 770 ± 52ml/24hr). Three of these mothers (Fig 4.11-17) had only

one breast that had a relatively high correlation (r = 0.76 – 0.85) between the

proportion of 24hr milk production and interval since last expression and the other

breast had a low correlation (r = 0.47 – 0.57). Extending the interval of expression up

to 5hr for these mothers would not be predicted to cause a significant decrease in milk

150

production. One breast of the remaining of 5 mothers had a low correlation (r = 0.58 -

0.66) and the other breast had either lower or no correlation (r = 0.00 – 0.45) between

the proportion of 24hr milk production and interval since previous expression.

Therefore extending the interval of expression up to 4hr for these mothers would not

be predicted to cause a significant decrease in milk production as this is still within

the range of intervals chosen by these mothers. The high milk production of these

mothers provides additional confidence in the likely success of the above prediction.

In contrast to the above 8 mothers, two mothers (Fig 4.22 & 4.28) showed the

differences in the slopes of the regression lines for the actual milk yields and expected

milk yields of the left and right breasts. But these two mothers had low milk

productions, 340 and 433 ml/24hr, respectively. However, the average interval

between breast expressions for these mothers was 3.0 ± 1.9hr and 3.5 ± 1.1hr

respectively. Furthermore, the relationship between the proportion of 24hr milk

production and interval since previous expression accounted for 19% and 45%, and

21% and 0% of the variation in milk yield for their left and right breasts respectively.

Therefore decreasing the interval between breast expressions would be predicted to

increase milk production for these mothers. Further research is needed to identify the

additional major sources of variation in milk yield, particularly in low production

expression dependent mothers, to enable evidence-based advice on increasing milk

production to be given to these mothers.

One mother had no significant correlation between the proportion of 24hr milk

production and the interval since previous expression (Fig 4.30). However, her daily

milk production was 1360 ml/24hr. Although, a prediction on expression interval

cannot be made for this mother, her high milk production could permit her to extend

her shorter intervals to see if a less demanding expression regime impacted negatively

on her daily milk production. Two other mothers were not classified because one (Fig

4.32) only collected milk yield data for two days, and the other mother (Fig 4.31)

expressed her milk strictly every four hours from day 10 to day 60 post-partum and

therefore there was no variation in the intervals between breast expressions for this

mother. Interestingly the coefficient of variation for her mean milk production over

this time was 27% suggesting that factors other than expression interval accounted for

significant variation in her milk yield at each expression. Closer examination of the

demographics and breast physiology of mothers with a strict expression interval in

151

future may provide clues for the sort of factors that contribute to the variation in the

milk production.

The use of regression analysis has provided information about the impact of

the interval since last expression on the proportion of 24hr milk yield of each breast

that can be used to optimize expression regimes for individual mothers. Interestingly,

the regression analysis has shown that for most of the mothers in this study a less

demanding expression schedule should not impair milk production. However, it

would be necessary to carry out additional research to ensure that the application of

these predictions to the fully expressing preterm mother provided the desired result of

decreasing the demand on the mother while maintaining adequate milk production.

Feeding preterm babies with fortified preterm mothers' milk is one of the

difficult challenges for modern medicine. The health status of preterm babies varies

greatly from day to day. Furthermore extreme low birth weight and very low birth

weight preterm babies have limited fluid intake, often restricted to 150ml/kg/day of

fluid. Thus the challenge is to deliver all the essential nutrients within a limited

volume of milk to meet their demand for growth and development. The fortification

of preterm mothers’ milk is commonly based on an assumed energy content of milk

of 20Cal/30ml. Energy of human milk is derived from the macro-nutrient fat, protein

and lactose. The large variation in the composition of milk between mothers found in

this study (Fig 5.13-20, 31-38) suggested that it is not appropriate to assume a

standard energy content and protein concentrations for the milk of all preterm

mothers. Measuring macro-nutrients in human milk for individual mothers is

currently labor intensive and time consuming and therefore, is not practical in a

clinical situation. However, my results show that representative samples of breastmilk

can be obtained and thereby reduce the assay demands considerably.

Since the fat content of milk increases from 36 ± 21g/l at the beginning to 67

± 27g/l at the end of an expression session, the content of fat of a mixed 24hr sample

provided the most accurate measurement. Furthermore, the variation in the fat content

of milk from 10 to 60 days postpartum in individual mothers was on average not

large, therefore measurements of the fat content at weekly intervals should provide a

reasonable estimate on which to base fortification decisions. The creamatocrit method

152

is a relatively quick and accurate method which is available to measure the content of

fat in a small sample of breastmilk (Meier et al., 2006).

The change in the concentrations of total protein and nutritive protein were

very consistent within mothers during the day, between day 10 to 20 and day 30 to 60

postpartum. Therefore, measurements of milk protein at 5-7 day intervals would give

an accurate estimate of the concentration of protein in the mothers’ milk and account

for the change in protein concentration over the course of the lactation. However due

to large variations in the concentration of protein between mothers, use of average

values of these components cannot be recommended.

The concentration of lactose in milk was very stable from day 10 to 60

postpartum and therefore a less rigorous sampling frequency would provide adequate

data for estimating the contribution that lactose makes to the energy content of

preterm milk for individual mothers.

Therefore, based on the findings of the longitudinal changes in the milk

composition of preterm mothers’ milk, it would be possible to develop a practical

plan to measure fat, protein and lactose of mothers’ own milk at specific intervals for

use as references for the process of milk fortification. Since there are instruments,

such as the creamatocrit, available for rapid measurement of fat, the determination of

fat could be done on a daily basis. Instruments such as Infra-Red Milk Analysers that

can measure multiple milk components could enable the measurement of fat, protein

and lactose. The minimum recommendation for the application of these instruments

would be weekly fat and protein measurements and fortnightly lactose measurements,

because it is much more stable in milk than the other two macronutrients. However, if

equipment and time was not permitting, then the minimum measurement of fat in

mothers’ own milk should be done every week. The minimum interval for these three

macronutrients measurements should provide a reasonable estimate of their

concentrations in the milk of individual mother. Thus they could serve as base

references for optimizing the fortification of mothers’ own milk to meet the needs of

their individual babies.

In conclusion, understanding the impact of the interval between breast

expressions on milk yield for individual breasts within mothers can be used as an

evidence base for optimizing milk production while minimizing the demands on the

mother. A greater knowledge of the variation in the concentration of macronutrients

in breastmilk would assist clinicians in the provision of optimal fortification of

153

preterm breastmilk so that it meets the demands for the growth and development for

these fragile babies. Furthermore, research is urgently required to determine if higher

concentrations of sIgA and lactoferrin in preterm mother’s milk are associated with

lower incidence of sepsis in the preterm infant. If such a relationship exists, it could

be possible to concentrate the sIgA and lactoferrrin components of human milk using

ultra-filtration techniques (Glover, 1984). Since these two proteins are quantitatively

the major innate immune proteins in human milk (comprising 30% of the total protein

in breastmilk), the concentrate could be used to fortify breastmilk that was deficient in

sIgA and lactoferrin.

154

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