the islamic university of gaza serum leptin was significantly higher in women with rpl compared to...
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The Islamic University of Gaza Deanship of Post-graduate Studies
Biological Sciences Master Program
Serum Leptin Level in Women with Recurrent Pregnancy Loss in Mid-Zone Governorate,
Gaza strip
Submitted in Partial Fulfillment to the Requirements for the Degree of
Master of Biological Sciences / Medical Technology
BY:
Nabil A. Alhour
M.B.BCH
Supervisor:
Prof. Dr. Maged M. Yassin Professor of Physiology
Faculty of Medicine The Islamic University of Gaza
March, 2015
I
الهذينآمنوامنكموالهذينأوتواالعلمدرجات } (المجادلة11){يرفعللاه
Nabil A. Alhour Serum Leptin Level in Women with
Recurrent Pregnancy Loss in Mid-Zone
Governorate, Gaza strip
II
Declaration
I hereby declare that this submission is my own work and that, to the best of
my knowledge and belief, it contains no material previously published or
written by another person nor material which to a substantial extent has been
accepted for the award of any other degree of the university or other institute,
except where due acknowledgement has been made in the text.
Signature Name Date
Nabil Nabil A. Alhour February, 2015
Copy Right
All rights reserved: No part of this work can be copied, translated or stored
in any retrieval system, without prior permission of the author.
III
Dedication
I dedicate this work to:
The soul of my beloved mother and to my father and his wife
My wife who encourage me to accomplish this thesis
The dearest to me; my sons and daughters Ali, Ekrima, Safia,
Aysha and Nusaiba
To my brothers and sisters
All researchers who are working to improve the quality of life
Dedication is almost expressed to the Palestinian people who
have suffered and will be struggling with the persistence to
have a free Palestine.
Nabil A. Alhour
IV
Acknowledgment
I would like to express my deepest gratitude and appreciation to my
supervisor Prof. Dr Maged M. Yassin, Professor of Physiology, Faculty of
Medicine, The Islamic University of Gaza for his planning and initiating of this
work and for his continuous support, encouragement and kind of supervision
that leads to the emergence of this work in its current form.
Special thanks for Dr. Basin Ayesh for his continuous support and
encouragements and my friend Mohamed Talal Al Hour for his help.
I would like to thank the patients for their fruitful cooperation and the staff of
Al-Nusirat Central Laboratory for their assistant in sample analysis.
My special thanks to Mr. Abdul Rahman Hamad for his help in statistical
analysis.
V
Abstract
Serum Leptin Level in Women with Recurrent Pregnancy Loss in Mid-Zone Governorate,
Gaza strip
Background: Leptin hormone is involved in supporting normal pregnancy
and its disturbance is recently implicated in the pathogenesis of various
disorders during pregnancy that might lead to recurrent pregnancy loss (RPL).
Objective: To assess serum leptin level in women with RPL in Gaza strip.
Materials and methods: This case-control study comprised 42 women with
RPL and 42 control women with no history of RPL. Questionnaire interview
was applied. Serum leptin, thyroid stimulating hormone (TSH), follicular
stimulating hormone (FSH) and Luteinizing hormone (LH) were determined.
Data were computer analyzed using SPSS version 18.0.
Results: The mean ages of cases and controls were 31.6±7.3 and 31.1±6.4
years, respectively. Menstrual data showed that the mean duration of
menstruation was significantly longer in cases compared to controls and the
number of cases who reported irregular menstruation was also significantly
higher than controls. The number of cases who have children was significantly
lower than controls. Higher number of those cases reported cesarean
compared to their counterparts of controls. Concerning abortion, 57.1% of
cases have 3 abortions and 42.9% have more than 3 abortions. When related
to frequency of abortion, the number of cases who had 3 abortions was found
to have significantly higher number of children than those who had >3
abortions. The mean gestational age of fetus at abortion was 10.2±3.9 weeks
indicating that most abortions took place in the first trimester. Toxoplasmosis
was significantly higher than controls. Toxoplasmosis was found to be
associated with abortion. The mean level of serum leptin was significantly
elevated in cases compared to controls (17.3±13.1 vs. 9.0±7.1 ng/ml, %
difference=63.1, P=0.007). However, there were no significant differences in
the mean levels of TSH, FSH and LH between cases and controls (P>0.05).
The mean level serum leptin was significantly higher in cases reported
VI
irregular menstruation that those reported regular menstruation (21.8±13.7
versus 15.1±9.3, P=0.044). Similarly, serum leptin was significantly higher in
cases that had more than three abortions compared to those who had three
abortions (22.3±12.9 versus 14.5±10.2, P=0.046). Cases reported
toxoplasmosis showed significantly higher serum leptin levels (24.8±9.5
versus 16.5±8.3, P=0.034). Finally, serum leptin exhibited positive significant
correlations with serum TSH and LH levels (r=0.359, P=0.021 and r=0.767,
P=0.000, respectively).
Conclusions: Serum leptin was significantly higher in women with RPL
compared to controls. Leptin level was higher in patients reported irregular
menstruation, had more than three abortions and those reported
toxoplasmosis. Serum leptin exhibited positive significant correlations with
serum TSH and LH levels.
Keywords: Recurrent Pregnancy Loss, Leptin, Gaza strip.
VII
الملخص
مستوىمصلهرموناللبتينعندالسيداتاللواتييعانينمناإلجهاضالمتكرر فيالمحافظةالوسطىفيقطاعغزة
شارك هرمون اللبتن ف عملة دعم الحمل الطبع وإحداث بعض االضطرابات الت تؤدي الى الخلفية:
اإلجهاض المتكرر ف بعض األحان.
مستوى اللبتن عند السدات اللوات عانن من اإلجهاض المتكرر ف المحافظة الوسطى ف قاس الهدف:
قطاع غزة
المستعملة: والطرق (. تم تنفذ 24حالة( مع الحاالت السلمة ) 24مقارنة الحاالت المرضة ) المواد
( و ثم TSH( وهرمون )LH( وهرمون )FSHاالستبان بتحدد الهرمونات التالة: هرمون اللبتن وهرمون )
.spssتحلل العالقة بنهما عبر برنامج
سنة تقربا. المعطات عن 3.2 ± ...6 و 3.6± 3..6متوسط عمر الحاالت المستهدفة كان النتائج:
الدورة الشهرة أشارت إلى ان عدد أام الدورة الشهرة زد بصورة واضحة ف الحاالت المرضة مقارنة
ت السلمة وأن الحاالت الت فها الدورة الشهرة غر منتظمة ه أضا أعلى , وعدد األطفال عند بالحاال
الحاالت المرضة أقل بصورة واضحة من الحاالت السلمة ,والعدد الكبر من الحاالت المرضة الت تنجب
% من الحاالت المرضة ..13عبر العملات القصرة كانت أكثر مقارنة مع نظائرهم ف الحاالت السلمة.
اجهاضات متكررة. عند 6% من الحاالت حدث معهم أكثر من 24.4إجهاضات متكررة و 6حدث معهم
من إجهاضات أنجبت عدد أكبر من األطفال 6 لدهم الت النظر ف العالقة بن الحاالت وجدنا أن الحاالت
± 4... اإلجهاضات عند أغلب الحمل عمر متوسط وكان. اإلجهاضات المتكررة لدهم أكثر من الذن أولئك
األولى أو بمعنى آخر ف الثلث الثالثة األشهر ف وقعت اإلجهاض حاالت معظم أن إلى مشرا أسابع 6.4
زد عند حاالت اإلجهاض المتكرر ووجد أضا أن ارتفاع نسبة العدوى بالتوكسوبالزموزس .األول من الحمل
بصورة واضحة مقارنة بالحاالت السلمة. وكان متوسط هرمون اللبتن مرتفع اللبتن مع ارتفاع نسبة هرمون
..3 ± ..4مقارنة 3.6. ± ...6بصورة واضحة ف الحاالت المرضة مقارنة مع الحاالت السلمة )
( وهرمون FSHأنه ال توجد عالقة واضحة بن الهرمونات التالة : )نانوجرام لكل مللتر %( . ومع ذلك وجد
(LH( وهرمون )TSH ف الحاالت المرضة والحاالت السلمة وأن مستوى هرمون اللبتن ف الحاالت )
(. ووجد 4.6 ± ..1.مقارنة 2..4 ±3..6المرضة فما خض الدورة الشهرة الغر منتظمة كان أعلى )
± 44.6ن أعلى بصورة واضحة ف الحاالت الت تعان من ثالث اجهاضا متكررة أو اكثر )أن مستوى اللبت
± 42.2( , ووجد أن مستوى اللبتن رتفع ف الحاالت الت عانت من عدوى بالتكسوبالزموزس )4..4
( وهرمون LH( ووجد أضا وجود عالقة قوة بن هرمون اللبتن وهرمون )2.6 ± 3.1.مقارنة 4.1
(TSH)( )
مستوى اللبتن رتفع بصورة واضحة عند السدات اللوات عانن من اإلجهاض المتكرر :االستنتاجات
مقارنة بالحاالت السلمة. رتفع مستوى هرمون اللبتن عند الحاالت اللوات عانن من الدورة الشهرة الغر
وقد اصبن بعدوى التوكسوبالزموزس. تبن ان منتظمة واللوات عانن من أكثر من ثالث إجهاضات متكررة
(.TSH( وهرمون )LHهرمون اللبتن رتبط بعالقة قوة مع وهرمون )
إجهاض متكرر , لبتن , قطاع غزة.: كلماتمفتاحية
VIII
Table of contents
Page Contents
I Declaration
II Dedication
III Acknowledgement
IV Abstract (English)
VI Abstract (Arabic)
VII Table of Contents
X List of tables
XI List of figures
XI List of Annex
Chapter 1: Introduction
1 Overview 1.1
2 General objective 1.2
2 Specific objectives 1.3
Chapter 2: Literature Review
3 Definition of recurrent pregnancy loss 2.1
3 Prevalence of recurrent pregnancy loss 2.2
4 Etiology of recurrent pregnancy loss 2.3
4 Endocrine etiologies 2.3.1
5 Autoimmune etiologies 2.3.2
6 Anatomic etiologies 2.3.3
6 Genetic etiologies 2.3.4
7 Infectious etiologies 2.3.5
7 Thrombotic etiologies 2.3.6
8 Unexplained etiologies 2.3.7
8 Environmental etiologies 2.3.8
9 Leptin 2.4
9 Definition and site of secretion 2.4.1
10 Mechanism of leptin action 2.4.2
IX
11 Leptin and female reproduction 2.4.3
11 Leptin as an ovarian and placental hormone 2.4.3.1
11 Hypothalamic-pituitary-gonadal (HPG) axis 2.4.3.2
13 Leptin and recurrent pregnancy loss 2.4.3.3
Chapter 3: Materials and Methods
14 Study design 3.1
14 Study population 3.2
14 Sampling and sample size 3.3
15 Ethical Consideration 3.4
15 Exclusion criteria 3.5
15 Data collection 3.6
15 Questionnaire interview 3.6.1
16 Specimen collection and processing 3.6.2
16 Hormonal analysis
3.7
16 Determination of serum leptin
3.7.1
18 Determination of serum thyroid stimulating hormone 3.7.2
20 Determination of serum follicle stimulating hormone 3.7.3
22 Determination of serum luteinizing hormone 3.7.4
25 3.8 Statistical analysis 3.8
Chapter 4: Results
26 Socio-demographic data of the study population
4.1
28 Premenstrual symptoms among the study population 4.2
29 Duration of menstruation and menstrual symptoms of the study
population
4.3
31 Pregnancy and delivery information of the study population 4.4
34 Recurrent abortion among cases 4.5
35 Frequency of abortion in relation to have children among cases 4.6
36 Chronic diseases among the study population 4.7
37 Family history of recurrent abortion and various disorders among 4.8
X
the study population
39 Serum levels of leptin, TSH and gonadotropins of the study
population
4.9
41 Relations of leptin 4.10
41 Serum leptin in relation to regularity of menstruation, having
children and type of delivery among cases
4.10.1
43 Serum leptin in relation to frequency of abortion and toxoplasmosis
among cases
4.10.2
46 Serum leptin in relation to TSH, FSH and LH levels of cases 4.10.3
Chapter 5:Discussion
49 Sociodemoghraphic data and menstrual aspects of the study
population
5.1
50 Pregnancy, delivery and recurrent abortion among the study
population
5.2
51 Family history of recurrent abortion and various disorders among
the study population
5.3
51 Serum levels of leptin, TSH and gonadotropins (FSH and LH) of
the study population
5.4
53 Relations of serum leptin to the studied parameters among cases 5.5
Chapter 6:Conclusions and Recommendations
55 Conclusions 6.1
56 Recommendations 6.2
57 CHAPTER 7:REFERENCES
Page List of tables 49 Personal profile of the study population
Table 4.1
51 Socioeconomic data of the study population
Table 4.2
52 Duration of coronary artery disease among cases Table 4.3
53 Serum vitamin D levels of the study population Table 4.4
54 Different categories of serum vitamin D levels of the study Table 4.5
XI
population
56 Cardiac enzymes activities of the study population Table 4.6
57 Serum alkaline phosphatase (ALP) activity of the study
population
Table 4.7
58 lipid profile of the study population Table 4.8
59 Serum calcium and phosphorus of the Study population Table 4.9
60 Duration of coronary artery disease in relation to serum
vitamin D levels of the cases.
Table4.10
61 Vitamin D levels in relation to cardiac enzyme of the study
population
Table 4.11
63 Vitamin D levels in relation to lipid profile activities of the study
population
Table 4.12
65 Vitamin D levels in relation to alkaline phosphatase (ALP) of
the study population
Table 4.13
66 Vitamin D levels in relation to calcium and phosphorus of the
study population
Table 4.14
Page List of figures
4 Coronary arteries of the heart Figure 2.1
7 Pathophysiology of coronary artery disease Figure 2.2
15 Structure of vitamin D Figure 2.3
16 Synthesis of vitamin D Figure 4.4
17 Action of vitamin D Figure 2.5
53 Serum vitamin D levels in cases and controls Figure 4.1
XII
55 Different categories of serum vitamin D levels Figure 4.2
62 Vitamin D level in relation to cardiac enzymes activities of the study population
Figure 4.3
64 Vitamin D level in relation to lipid profile of the study population
Figure 4.4
67 Vitamin D level in relation to calcium and phosphorus concentration of the study population .
Figure 4.5
List of Appendices
Page Description Appendix
94 Ministry of Health permission letter Annex1
95 Public Aid Society permission letter Annex2
96 Interview questionnaire Annex3
1
Chapter 1
Introduction
1.1 Overview
Recurrent pregnancy loss (RPL) is one of the most frustrating and difficult
areas in reproductive medicine because the etiology is often unknown and
there are few evidence-based diagnostic and treatment strategies. It differs
from infertility and classically refers to the occurrence of three or more
consecutive losses of clinically recognized pregnancies prior to the 20th week
of gestation (Loss, 2010).
The occurrence of RPL affects about 1-5% of couples (Baek et al., 2007).
Clinical miscarriages (those occurring after the sixth week of gestation) occur
in 8% of pregnancies (Wang et al., 2003). Most clinically apparent
miscarriages (two thirds to three-quarters in various studies) occur during the
first trimester (Rosenthal, 2006; Allison et al., 2011 and Gonçalves et al.,
2014). About two-thirds of pregnancies in the United States of America ended
in live birth (Ventura et al., 2012).
Recurrent pregnancy loss is a heterogeneous condition that has many
possible causes including genetic, hormonal, metabolic, uterine anatomical,
infectious, environmental, occupational and personal habits, thrombophilia, or
immune disorders (Ford and Schust, 2009; Pluchino et al., 2014 and
Gaboon et al., 2015). The risk of miscarriage is increased with maternal age,
and influenced by history and number of previous cesarean delivery (Abou-
Gabal et al., 2013 and Nankali et al., 2014). However, up to 50% of cases of
RPL have a clearly defined etiology (The Practice Committee of the
American Society for Reproductive Medicine, 2012).
Originally, leptin is one of the metabolic hormones that is identified as an
adipocyte-derived protein. The circulating leptin concentration therefore
directly reflects the amount of body fat (Wolf et al., 2001). For years, leptin
2
hormone was regarded as an exclusive regulator of satiety and energy
homeostasis. Its role in pregnancy was later suggested by the findings that is
synthesized within the fetoplacental unit (Forhead and Fowden 2009 and
Pérez-Pérez et al., 2015). Compelling evidence also has implicated leptin in
reproductive functions such as the regulation of ovarian function, oocyte
maturation, embryo development and implantation (Herrid et al., 2014).
Dysregulation of leptin metabolism and/or function may be implicated in the
pathogenesis of various disorders during pregnancy such as recurrent
miscarriage (Grattan et al., 2007 and Baban et al., 2010).
Although spontaneous pregnancy loss can be physically and emotionally
taxing for couples, especially when faced with recurrent losses, there is
underreporting and lack of research on this condition in the Gaza Strip. Few
recent studies have been focused on autoimmunity and genetic aspects of
RPL in the Gaza Strip (AL Derawi, 2009; Sharif, 2012 and Jaber and
Sharif, 2014). However, there was no previous study investigated endocrine
and clinical features of RPL in Gaza Strip. Therefore, the present study is the
first to assess serum leptin level in RPL which may open a new avenue in the
management of this reproductive disorder.
1.2 General objective
The General objective of the present study is to assess serum leptin level in
women with RPL in Gaza Strip.
1.3 Specific objectives
1. To identify socio-demographic and clinical data of the study population.
2. To determine serum leptin level in cases compared to controls.
3. To measure thyroid stimulating hormone (TSH), follicular stimulating
hormone (FSH) and Luteinizing hormone (LH) in cases and controls.
4. To verify the relationship between serum leptin and the various studied
parameters.
3
Chapter 2
Literature Review
2.1 Definition of recurrent pregnancy loss
Recurrent pregnancy loss, recurrent miscarriage, or habitual abortion is
defined as repeated occurrence of 3 or more miscarriages before 24th week
of gestation. The modern definition, however, is the spontaneous loss of 2 or
more consecutive pregnancies before 20 weeks of gestation (Aruna et al.,
2010). The American Society for Reproductive Medicine defines RPL as two
or more failed pregnancies (documented by ultrasound or histopathological
examination) and suggests some assessment after each loss with a thorough
evaluation after three or more losses (American Society for Reproductive
Medicine, 2008). While some physicians recommend evaluation after the loss
of two pregnancies, and others will not begin a workup until the loss of at least
three pregnancies (Grattan et al., 2007). In addition, Zhang et al. (2012)
defined RPL as the occurrence of three or more clinically detectable
pregnancy losses before the 20th week of gestation with the same partner.
2.2 Prevalence of recurrent pregnancy loss
About one third of all clinically recognized pregnancies end in miscarriage and
two thirds in live birth (Ventura et al., 2012). The accurate prevalence of RPL
is not available, but it has been estimated that 2%-5% of women experience
RPL with the majority of these cases occurring in the first trimester (van
Niekerk et al., 2013 and Gonçalves et al., 2014). The pathophysiological
mechanisms of RPL are as yet poorly understood and the aetiologies remain
unexplained in up to 50% of affected couples (The Practice Committee of
the American Society for Reproductive Medicine, 2012). However,
endocrine disorders play a major role in approximately 8% to 20% of RPL
(Ford and Schust, 2009 and Pluchino et al., 2014).
4
2.3 Etiology of recurrent pregnancy loss
The etiologies of RPL include endocrine disorders, immunologic
abnormalities, genetic factors, anatomic abnormalities, infections, heritable
and/or acquired thrombophilias, environmental factors and unexplained
etiologies (Figure 2.1).
Figure 2.1 Etiology of recurrent pregnancy loss (Ford and Schust, 2009).
APS: Antiphospholipid antibody syndrome.
2.3.1 Endocrine etiologies
Endocrine disorders play a major role (up to 20%) in RPL (Ford and Schust,
2009 and Pluchino et al., 2014). Luteal phase defect (LPD), polycystic
ovarian syndrome (PCOS), insulin resistance, thyroid disease,
hyperprolactinemia and recently hyperleptinaemia are among the
endocrinologic disorders implicated in RPL (Fox-Lee and Schust, 2007 and
Nanda et al., 2012). Traditionally, LPD has been proposed to result from
inadequate production of progesterone by the corpus luteum and endometrial
5
maturation insufficient for proper placentation and then more likely RPL (Shah
and Nagarajan, 2013 and Pluchino et al., 2014). Polycystic ovarian
syndrome is the most commonly identified ultrasound abnormality amongst
women with RPL (Larsen ET AL., 2013 and Jeve and Davies, 2014). Insulin
resistance and the resultant hyperinsulinemia that is often present in cases of
PCOS may play a role in RPL (Chakraborty et al., 2013). Autoimmune
thyroid disease is by far the most frequent cause of hypothyroidism in women
of reproductive age (Lata et al., 2013). Hypothyroidism may be a frequent
cause in RPL. Severe maternal hypothyroidism early in gestation is strongly
associated with fetal distress and abortion (Barapatre and Vaidya, 2013).
Therefore, thyroid autoantibodies were employed as a marker for at-risk
pregnancies (Mehran et al., 2013). In addition, evaluation of endocrine
disorders includes measurement of TSH level. Prolactin is commonly
measured in women with RPL, as elevated prolactin levels are associated
with ovulatory dysfunction. Hyperprolactinemia may be associated with
recurrent pregnancy loss through alterations in the hypothalamic-pituitary-
ovarian axis, resulting in impaired folliculogenesis and oocyte maturation,
and/or a short luteal phase (The Practice Committee of the American
Society for Reproductive Medicine, 2012 and Pluchino et al., 2014).
Finally, several studies reported that dysregulation of leptin metabolism and/or
function is implicated in the pathogenesis of various disorders during
pregnancy that might lead to recurrent miscarriage (Baban et al., 2010 and
Nanda et al., 2012).
2.3.2 Autoimmune etiologies
Autoimmune disorders contribute to about 20% of the cases of RPL (Ford
and Schust, 2009). As previously mentioned thyroid autoimmunity is linked to
RPL and in many instances thyroid autoantibodies were employed as a
marker for at-risk pregnancies (Mehran et al., 2013). Antiphospholipid
syndrome (APS) is another autoimmune disorder that has been clearly linked
with many poor obstetric outcomes, including RPL (Derksen and de Groot,
2008). The most widely accepted tests are for lupus anticoagulant,
anticardiolipin antibody and anti-2 glycoprotein I (Ford and Schust, 2009
6
and Keeling et al., 2012). Antiphospholipid antibodies have a variety of
effects on the trophoblast, including inhibition of villous cytotrophoblast
differentiation and extravillous cytotrophoblast invasion into the deciduas,
induction of syncytiotrophoblast apoptosis, and initiation of maternal
inflamatory pathways on the syncytiotrophoblast surface (Marchetti et al.,
2013 and Tong et al., 2015).
2.3.3 Anatomic etiologies
Anatomic abnormalities account for 10% to 15% of cases of RPL and are
generally thought to cause miscarriage by interrupting the vasculature of the
endometrium, prompting abnormal and inadequate placentation (Ford and
Schust, 2009). Anatomic abnormalities may include congenital uterine
anomalies, intrauterine adhesions, and uterine fibroids or polyps (Cholkeri-
Singh, 2014 and Sugiura-Ogasawara et al., 2015). Diagnostic evaluation for
uterine anatomic anomalies should include office hysteroscopy or
hysterosalpingography (Cholkeri-Singh, 2014). Myomectomy should be
considered in cases of submucosal fibroids or any type fibroids larger than 5
cm. Myomectomy can be performed via open laparotomy, laparoscopy, or
hysteroscopy (Saravelos et al., 2011 and Kim et al., 2013).
2.3.4 Genetic etiologies
Approximately 2% to 5% of RPL is associated with a parental balanced
structural chromosome rearrangement, most commonly balanced reciprocal
or Robertsonian translocations (Ford and Schust, 2009; Sharif, 2012 and
Gaboon et al., 2015). Additional structural abnormalities associated with RPL
include chromosomal inversions, insertions, and mosaicism (Chaithra et al.,
2011 and Gonçalves et al., 2014). Single gene defects, such as those
associated with cystic fibrosis or sickle cell anemia, are seldom associated
with RPL (Goddard and Bourke, 2009 and Silva-Pinto et al., 2014).
Appropriate evaluation of RPL should include parental karyotyping. Genetic
counseling is indicated in all cases of RPL associated with parental
chromosomal abnormalities (van Niekerk et al., 2013 and Hyde and Schust,
2015).
7
2.3.5 Infectious etiologies
Certain infections, including Listeria monocytogenes, Toxoplasma gondii,
rubella, herpes simplex virus, measles, cytomegalovirus and coxsackieviruses
are known or suspected to play a role in RPL with a proposed incidence of
0.5-5% (Fox-Lee and Schust 2007; Ford and Schust, 2009; Hussan, 2013
and Ariani and Chaich, 2014). Most of these infectious agents, particularly
Toxoplasma gondii, were identified more frequently in vaginal and cervical
cultures and serum from women with recurrent miscarriages (Penta et al.,
2003 and AL-Ani, 2011). The proposed mechanisms for infectious causes of
pregnancy loss may include direct infection of the uterus, fetus or placenta;
placental insufficiency; chronic endometritis or endocervicitis; amnionitis and
infected intrauterine device (Ford and Schust, 2009 and Robbins et al.,
2012).
2.3.6 Thrombotic etiologies
Both inherited and combined inherited/acquired thrombophilias are common,
with more than 15% of the white population carrying an inherited
thrombophilic mutation (Greer, 2003). The potential association between RPL
and heritable thrombophilias is based on the theory that impaired placental
development and function secondary to venous and/or arterial thrombosis
could lead to miscarriage (Bogdanova and Markoff, 2010 and Hansda and
Roychowdhury, 2012). The heritable thrombophilias most often linked to
RPL include hyperhomocysteinemia resulting from methylene tetrahydrofolate
reductase mutations, activated protein C resistance associated with factor V
Leiden mutations, protein C and protein S deficiencies, prothrombin promoter
mutations and antithrombin mutations (Bogdanova and Markoff, 2010 and
Gawish and Al-Khamees, 2013). Acquired thrombophilias associated with
RPL include hyperhomocysteinemia and activated protein C resistance
(Bozikova et al., 2015).
8
2.3.7 Unexplained etiologies
Almost half of RPL patients remain without a definitive diagnosis i.e with
unexplained RPL (Ford and Schust, 2009). The most effective therapy for
patients with unexplained RPL is often the most simple: antenatal counseling
and psychological support. These measures have been shown to have
subsequent pregnancy success rates reaching to 50-60% (Nakano et al.,
2011 and Lund et al., 2012).
2.3.8 Environmental etiologies
Links between RPL and occupational and environmental factors have been
suggested, although the studies performed are difficult to draw strong
conclusions (Fox-Lee and Schust, 2007 and van Niekerk et al., 2013).
Three particular exposures; smoking, alcohol and caffeine have gained
particular attention because of their widespread use and modifiable nature.
However, their association with RPL was relatively weak (Mohamed et al.,
2014).
9
2.4 Leptin
2.4.1 Definition and site of secretion
Leptin was identified through positional cloning of the obese (ob) gene, which
is mutated in the massively obese ob/ob mouse, and it has a pivotal role in
regulating food intake and energy expenditure (Zhang, 1994 and Stanley et
al., 2005 and Oswal and Yeo, 2010). Leptin is a small peptide hormone (146
amino acids, 16-kDa Protein) which adopts a typical four-helical bundle
cytokine structure with four anti-parallel helices in an up-up-down-down
arrangement (Figure 2.1, Peelman et al., 2014). Leptin is mainly produced in
adipose tissue (Coelho et al., 2013). The expression of leptin receptors has
also been demonstrated in ovaries of different species (Zendron et al., 2014).
In addition, leptin is synthesized within the fetoplacental unit (Maymó et al.,
2009; Tessier et al., 2013 and Vázquez et al., 2015).
)Peelman et al., 2014( leptinhuman Structure of Figure 2.1
11
2.4.2 Mechanism of leptin action
Leptin binds to the leptin receptor (Ob-Rb) and activates Janus-family tyrosine
kinase 2 (JAK2) by auto- or cross-phosphorylation. JAK2 then phosphorylates
the other tyrosine residues (Tyr985, Tyr1077, Tyr1138), which act as sites for the
binding of intracellular signaling molecules. Signal Transducer and Activator
of Transcription 3 (STAT-3) is a substrate of JAK 2 and subsequently binds to
the phosphorylated Tyr1138. STAT-3 then undergoes dimerization and
translocates into the cell nucleus to function as a promoter for gene
transcription. Members of the suppressor of cytokine signaling proteins
(SOCS) family (SOCS1 and SOCS3) suppress the action of leptin by binding
to JAK 2 and tyrosine residues (Paz-Filho et al., 2012 and Adya et al.,
2015).
11
Figure 2.2 Mechanism of action of leptin (Paz-Filho et al., 2012)
2.4.3 Leptin and female reproduction
Leptin is an indispensable factor in the metabolic control of female puberty
and fertility. It is involved in all stages of normal pregnancy as well as in
reproductive disorders such as RPL.
2.4.3.1 Leptin as an ovarian and placental hormone
Leptin can be produced by human ovarian follicles, both in granulosa and
cumulus cells. It was confirmed that preovulatory follicles express leptin gene
at the levels of mRNA and protein (Joo et al., 2010 and Smolinska et al.,
2013). Although the expression of leptin receptors has been demonstrated in
ovaries, the mechanism through which leptin controls ovulation is not fully
understood (Trisolini et al., 2013). In addition, it has been accepted that
leptin may play a role in the regulation of the menstrual cycle. It is well
recognized also that menstrual irregularities and infertility are common in
women with abnormal expression of the ob gene product (Elias and Purohit,
2013). Furthermore, leptin hormone was shown to be produced by both
maternal and fetal adipose tissues, as well as by the placental trophoblast.
Specific receptors in the uterine endometrium, trophoblast, and fetus facilitate
direct effects of leptin on implantation, placental endocrine function, and
conceptus development (Maymó et al., 2009 and Tessier et al., 2013).
2.4.3.2 Hypothalamic-pituitary-gonadal (HPG) axis
Leptin acts on the HPG axis and interplays with key elements of the female
reproductive system (Figure 2.3). The positive/permissive effects of leptin,
produced by the white adipose tissue, are primarily conducted at central,
hypothalamic levels, where leptin can modulate gonadotropin-releasing
hormone secretion by modulating the activity of stimulatory afferents. Among
these, a role of Kiss1 neurons has been suggested; yet, compelling evidence
suggests that leptin effects on Kiss1 neurons are mostly indirect. In addition,
stimulatory actions of leptin at the pituitary level to increase gonadotropin
secretion have been described. In contrast, the reported effects of leptin on
12
the gonads are mostly inhibitory; e.g., high leptin levels have been shown to
directly inhibit ovarian and testicular steroidogenesis. The positive interplay
between leptin and insulin, produced by the pancreas, is also depicted
(Vázquez et al., 2015).
Figure 2.3 Graphical summary of the major sites of action of leptin in the
control of the hypothalamic-pituitary-gonadal axis (Vázquez et al., 2015).
WAT: White adipose tissue, GnRH: Gonadotropin-releasing hormone, LH:
Luteinizing hormone; FSH: Follicle stimulating hormone.
13
2.4.3.3 Leptin and recurrent pregnancy loss
Leptin hormone is involved in supporting all stages of normal pregnancy and
its disturbance may be implicated in the pathogenesis of various disorders
during pregnancy that might lead to RPL (Grattan et al., 2007). The role of
leptin in RPL is not well known. However, elevated levels of serum leptin were
detected in women with RPL (Baban et al., 2010; Saeed et al., 2010). In
addition, Nanda et al. (2012) demonstrated increased maternal serum leptin
concentration at 11-13 weeks’ gestation in pathological pregnancies that may
subsequently leads to abortion. In this context, Žaneta et al. (2004)
suggested that elevation of leptin level could be an indicator for the cessation
of pregnancy naturally either at term or due to pathology at any time during
gestation. It is accepted that hyperleptinaemia induces maternal leptin
resistance which may be associated with impaired placentation and embryo
implantation (Herrid et al., 2014 and Wang et al., 2014).
14
Chapter 3
Materials and Methods
3.1 Study design
The present study is a case control investigation. Case-control studies are
often used to identify factors that may contribute to a medical condition by
comparing subjects who have that condition/disease (the "cases") with
patients who do not have the condition/disease but are otherwise similar (the
"controls"). Case-control studies are quick, widely used, relatively inexpensive
to implement, require comparatively fewer subjects, and allow for multiple
exposures or risk factors to be assessed for one outcome (Mann, 2003 and
Song and Chung, 2010).
3.2 Study population
The study population comprised 42 women with recurrent abortion attending
Al-Aqsa hospital in Gaza strip (cases) and 42 `women with no history of
recurrent abortion who served as controls. Cases and controls were age
matched.
3.3 Sampling and sample size
Women with recurrent abortion aged 18-40 years were selected randomly
from Al-Aqsa hospital in Gaza strip. Control women with no history of
recurrent abortion were selected from the general population. The sample
size calculations were based on the formula for case-control studies. EPI-
INFO statistical package version 3.5.1 was used with 95% CI, 80% power and
50% proportion as conservative and OR >2. The sample size in case of 1:1
ratio of case control was found to be 40:40. For a no-response expectation,
the sample size was increased to 42 patients. The controls also consisted of
42 women.
15
3.4 Ethical consideration
The participants were given a full explanation about the purpose of the study
and assurance about the confidentiality of the information and that the
participation was optional.
3.5 Exclusion criteria
* Cases and controls aged < 18 years and > 40 years old.
* Women with progesterone deficiencies (serum progesterone not less than
0.15 ng/ml) which support early pregnancies.
* Women with uterine fibroid.
* Women on medication e.g. aspirin and hormone therapy.
3.6 Data collection
3.6.1 Questionnaire interview
A meeting interview was used for filling in the questionnaire which designated
for matching the study needs (Annex 1). All interviews were conducted face to
face by the researcher herself. The questionnaire was based on recent
studies conducted on recurrent abortion with some modifications (AL Derawi,
2009 and Ahamed et al., 2014). During the study the interviewer explained to
the participants any of the confused questions that will not clear to them. Most
questions were the yes/no question which offers a dichotomous choices
(Backestrom and Hursh-Cesar, 2012). The validity of the questionnaire was
tested by 5 specialists in the fields of obstetrics and gynecology,
endocrinology and public health. The questionnaire was piloted with 5 women
not included in the study, and modified as necessary. The questionnaire
included questions on socio-demographic data (Age, residence, education,
employment and family income/month); premenstrual symptoms (back pain,
breast pain and psychological changes); duration of menstruation and
menstrual symptoms (menstruation regularity, heavy menstruation and
dysmenorrheal); pregnancy and delivery information (age at first pregnancy,
have children and type of delivery); data on recurrent abortion (frequency of
abortion, gestational age of fetus at abortion and time interval between two
16
abortions); chronic diseases (thyroid gland disease, hypertension and
diabetes); and family history of recurrent abortion and various disorders
(abdominal surgery, ectopic pregnancy, vesicular mole and toxoplasmosis).
3.6.2 Specimen collection and processing
Blood samples were collected from 42 women with recurrent abortion and 42
control women with no history of recurrent abortion. Twelve hours fasting
overnight venous blood samples (6 ml) were drawn by the researcher himself
into plastic tubes from each control and case. The blood samples were left for
a while without anticoagulant to allow blood to clot. Serum samples were
obtained by centrifugation at 3000 rpm/10 minutes for hormonal analysis.
3.7 Hormonal analysis
3.7.1 Determination of serum leptin
Determination of human serum leptin level was carried out by competitive
enzyme immunoassay Diagnostic System Laboratories (DSL), USA
Principle of the assay
The DSL-10-23100 ACTIVE Human Leptin ELISA is an enzymatically
amplified "twostep" sandwich-type immunoassay. In the assay, standards,
controls and unknown serum or plasma samples were incubated in
microtitration wells, which have been coated with anti-human leptin antibody.
After incubation and washing, the wells were treated with another anti-human
leptin detection antibody labeled with the enzyme horseradish peroxidase
(HRP). After a second incubation and washing step, the wells were incubated
with the substrate tetramethylbenzidine (TMB). An acidic stopping solution
was then added and the degree of enzymatic turnover of the substrate was
determined by dual wavelength absorbance measurement at 450. The
absorbance measured was directly proportional to the concentration of human
leptin present. A set of human leptin standards was used to plot a standard
curve of absorbance versus human leptin concentration from which the
human leptin concentrations in the sample can be calculated. The assay
procedure sheets were available with the kit, the application of assay
procedure mentioned below.
17
Assay procedure
Annabel all specimens and reagents to reach room temperature (~25°C) and
mix
thoroughly by gentle inversion before use. Standards, Controls and samples
should be assayed in duplicate.
1. The microtitration strips were marked to be used.
2. Twenty five microlitters of the standards, controls and samples were
pipeted into the appropriate wells.
3. One hundred microlitters of the assay buffer E were added to each well
using a semi-automatic dispenser.
4. Incubate the wells with shaking at a fast speed (500-700 rpm) on an orbital
microplate shaker, at room temperature (~25 °C) for 2 hours.
5. Aspirate and wash each well 5 times with the wash solution using an
automatic microplate washer. Blot dry by inverting plate on porous material.
6. The antibody-enzyme conjugate solution was prepared by diluting the
antibody-enzyme conjugate concentrate in the assay buffer.
7. One hundred microlitters of the antibody-enzyme conjugate solution was
added to each well using a semi-automatic dispenser.
8. The wells were incubated, shaked at a fast speed (500-700 rpm) on an
orbital
microplate shaker, at room temperature (~25 °C) for 1 hour.
9. Each well aspirate and wash 5 times with the wash solution using an
automatic
microplate washer and Blot dry by inverting plate on absorbent material.
10. One hundred microlitters of the TMB Chromogen solution was added to
each well using a semi-automatic dispenser.
11. Incubate the wells, shaking at a fast speed (500-700 rpm) on an orbital
microplate shaker, at room temperature (~25°C) for 10 minutes. Avoid
exposure
to direct sunlight.
12. One hundred Microlitters of the stopping solution (0.2M sulfuric acid) was
added to each well using a semi-automatic dispenser.
18
13. The absorbance of the solution in the wells was read within 30 minutes,
using a microplate reader set to 450 nm.
Calculation
A. The mean absorbance for each standard, control and samples were
calculated.
B. Plot the log of the human leptin concentrations in ng/mL along the x-axis
versus the mean absorbance readings for each of the standards along the y-
axis versus, using a linear curve-fit. (Alternatively, the data can be plotted
linear vs. linear and a smoothed spine curve-fit can be used).
C. Determine the human leptin concentrations of the controls and samples
from the standard curve by matching their mean absorbance readings with the
corresponding human leptin concentrations.
3.7.2 Determination of serum thyroid stimulating hormone
The Teco Thyroid Stimulating Hormone ELISA test kit was used for the
quantitative determination of thyroid stimulating hormone (TSH) concentration
in serum.
Principle of the assay
The essential reagents required for an immunoenzymometric assay include
excess amount of antibodies (both enzyme conjugated and immobilized) with
high affinity, high specificity and contain different epitopes with distinct
recognition and native antigen. In this assay procedure, the immobilization
takes place at the surface of a microplate well through the interaction of
streptavidin coated on the well and exogenously added biotinylated
monoclonal anti-TSH antibody. Upon mixing, a reaction results between the
native antigen contained in serum, the monoclonal biotinylated antibody and
the enzyme-labeled antibody, without competition or steric hindrance, to form
a soluble sandwich complex.
Simultaneously, the complex is deposited to the well through the high affinity
reaction of streptavidin and biotinylated antibody. After equilibrium is attained,
the antibody-bound fraction is separated from unbound antigen by
19
decantation or aspiration. The enzyme activity in the antibody-bound fraction
is directly proportional to the native antigen concentration.
Reagents and Materials provided
1. Streptavidin coated microplate: containing 96 wells per kit
2. Enzyme Conjugate: containing TSH antibody conjugated with HRP and
biotinylated antibody in buffer, 13 ml.
3. TSH Reference Standard set: (7 1-mL vials) containing 0, 0.5, 2.5, 5.0, 10,
20 and 40 μIU/ml TSH antigen in buffer. Exact concentrations are given on
the package labels on a lot specific basis.
4. TMB Solution: containing TMB and H2O2 reagent in amber
bottle, 11 ml.
5. Stop Solution: containing diluted hydrochloric acid, 8 ml.
6. Wash Solution concentrate: 20 ml.
Reagent preparation
1. All reagents should be allowed to reach room temperature (18-25 c) before
use.
2. Working wash solution: add 20 mL of wash solution concentrate to 1000
mL of deionized water, and mix well. The amount of working wash solution
depends on the assay procedure described below. The working wash solution
is stable at room temperature for at least 7 days.
Assay procedure
1. Secure the desired number of coated wells in the holder. Prepare data
sheet with sample identification.
2. Pipette 50 μl of standards, sample and controls into each well.
3. Pipette 100 μl of conjugate reagent into each well. Mix thoroughly for 30
seconds.
4. Incubate at room temperature for 60 minutes.
5. Discard the contents of the wells by decantation or aspiration. If decanting,
blot the plate dry with absorbent paper.
21
6. Pipette 300 μl of working wash solution, decant or aspirate. Repeat 2
additional times for a total of 3 washes. An automatic ELISA washer may be
used.
7. Add 100 μl of TMB reagent into each well. Gently mix for 10 seconds.
9. Incubate at room temperature in the dark for 15 minutes without shaking.
10. Pipette 50 μl of stop solution to each well and gently mix for 10-20
seconds. It is critical to make sure that the blue color change to yellow color
completely.
11. Read the absorbance at 450 nm for each well.
Expected values and sensitivity
The TSH ELISA was performed in a study of 160 random adult samples and
yielded a normal range of (0.4 to 6.0 μIU/ml).
3.7.3 Determination of serum follicle stimulating hormone
Follicle stimulating hormone level was determined by using ELISA TECO kit
for FSH.
Principle of the assay
The essential reagents required for an immunoenzymometric assay include
excess amount of antibodies (both enzyme conjugated and immobilized) with
high affinity, high specificity and contain different epitopes with distinct
recognition and native antigen. In this assay procedure, the immobilization
takes place at the surface of a microplate well through the interaction of
streptavidin coated on the well and exogenously added biotinylated
monoclonal anti-FSH antibody. Upon mixing, a reaction results between the
native antigen contained in serum, the monoclonal biotinylated antibody and
the enzyme-labeled antibody, without competition or steric hindrance, to from
a soluble sandwich complex. Simultaneously, the complex is deposited to the
well through the high affinity reaction of streptavidin and biotinylated antibody.
After equilibrium is attained, the antibody-bound fraction is separated from
unbound antigen by decantation or aspiration. The enzyme activity in the
antibody-bound fraction is directly proportional to the native antigen
concentration. By utilizing several different serum references of known
21
antigen value, a dose response curve can be generated from which the
antigen concentration of an unknown can be ascertained.
Kit components
One stripholder containing 96 microtitration wells coated with streptavidin, six
FSH reference standards with concentrations of approximately (0, 5.0, 10, 25,
50 and 100 mIU/mL). Enzyme conjugate, TMB chromogen solution, stop
solution and wash solution concentrate.
Assay procedure
All samples and reagents were allowed to reach at room temperature
(~25°C). Reagents mixed by gentle inversion before use. Standards, controls
and samples assayed in duplicate.
1. Microtitration strip was marked to be used.
2. Fifty µL of the standards, controls and samples were added into each
appropriate well.
3. One hundred µL of conjugate reagent were added into each well using a
precision pipette and then mixed for 30 seconds.
4. The wells were incubated for 60 minutes at room temperature (~25°C).
5. Each well was aspirated and washed 3 times by added 300 µL of working
wash solution.
6. One hundred µL of TMB reagent were added into each well and gently
mixed for 10 second.
7. The wells were incubated in the dark for 15 minutes at room temperature
(~25°C) without shaking.
8. Fifty µL of stop solution were added into each well and gently mixed for 10-
20 second.
9. The absorbance for each well was read at 450 nm.
Calculation of results
The absorbance for each standard, control, or samples were obtained, and
then the standerd curve prepared by plotted the absorbance readings for each
of the standards along the Y-axis versus stander concentrations in mIU/mL
along the X-axis. The mean absorbance values for each sample were
22
determined the corresponding concentration of FSH in mIU/mL from the
standerd curve (Figure 3.1).
Figure 3.1 Follicle stimulating hormone standard curve
Normal reference value of FSH for adult female
The normal rang between 2.5-10.0 mIU/mL.
3.7.4 Determination of serum luteinizing hormone
Luteinizing hormone level was determined by using ELISA TECO kit for LH.
Principle of the assay
The essential reagents required for an immunoenzymometric assay include
excess amount of antibodies (both enzyme conjugated and immobilized) with
high affinity, high specificity and contain different epitopes with distinct
recognition and native antigen. In this assay procedure, the immobilization
takes place at the surface of a microplate well through the interaction of
streptavidin coated on the well and exogenously added biotinylated
monoclonal anti-LH antibody. Upon mixing, a reaction results between the
native antigen contained in serum, the monoclonal biotinylated antibody and
the enzyme-labeled antibody, without competition or steric hindrance, to from
FSH
00.20.40.60.8
11.21.41.61.8
22.22.42.62.8
3
0 10 20 30 40 50 60 70 80 90 100
AbsStandard concentration (mIU/mL)
Ab
sorb
an
ce a
t 4
50 n
m
23
a soluble sandwich complex. Simultaneously, the complex is deposited to the
well through the high affinity reaction of streptavidin and biotinylated antibody.
After equilibrium is attained, the antibody-bound fraction is separated from
unbound antigen by decantation or aspiration. The enzyme activity in the
antibody-bound fraction is directly proportional to the native antigen
concentration. By utilizing several different serum references of known
antigen value, a dose response curve can be generated from which the
antigen concentration of an unknown can be ascertained.
Kit components
One stripholder containing 96 microtitration wells coated with streptavidin, six
LH reference standards with concentrations of approximately (0, 5.0, 25, 50,
100 and 200 mIU/mL). Enzyme conjugate, TMB chromogen solution, stop
solution and wash solution concentrate.
Assay procedure
All samples and reagents were allowed to reach at room temperature
(~25°C). Reagents mixed by gentle inversion before use. Standards, controls
and samples assayed in duplicate.
1. Microtitration strip was marked to be used.
2. Fifty µL of the standards, controls and samples were added into each
appropriate well.
3. One hundred µL of conjugate reagent were added into each well using a
precision pipette and then mixed for 30 seconds.
4. The wells were incubated for 60 minutes at room temperature (~25°C).
5. Each well was aspirated and washed 3 times by added 300 µL of working
wash solution.
6. One hundred µL of TMB reagent were added into each well and gently
mixed for 10 second.
7. The wells were incubated in the dark for 15 minutes at room temperature
(~25°C) without shaking.
8. Fifty µL of stop solution were added into each well and gently mixed for 10-
20 second.
9. The absorbance for each well was read at 450 nm.
24
Calculation of results
The absorbance for each standard, control, or samples were obtained, and
then the stander curve prepared by plotted the absorbance readings for each
of the standards along the Y-axis versus standerd concentrations in mIU/mL
along the X-axis, the mean absorbance values for each sample were
determined the corresponding concentration of LH in mIU/mL from the
standerd curve (Figure 3.2).
Figure 3.2 Luteinizing hormone standard curve
Normal reference values of LH for adult male
The normal rang between 2.0-13.0 mIU/mL.
Abs
0
0.5
1
1.5
2
2.5
3
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105
AbsStandard concentration (mIU/mL)
Ab
sorb
an
ce a
t 4
50 n
m
25
3.8 Statistical analysis
Data were computer analyzed using SPSS/ PC (Statistical Package for the
Social Science Inc. Chicago, Illinois USA, version 18.0) statistical package.
* Simple distribution of the study variables and the cross tabulation were
applied.
* Chi-square (2) was used to identify the significance of the relations,
associations, and interactions among various variables. Yates’s continuity
correction test, 2 (corrected), was used when not more than 20% of the cells had
an expected frequency of less than five and when the expected numbers were
small.
* The independent sample t-test procedure was used to compare means of
quantitative variables by the separated cases into two qualitative groups such
as the relationship between cases and controls leptin.
* Pearson's correlation test was applied.
* The results in all the above mentioned procedures were accepted as
statistical significant when the p-value was less than 5% (p<0.05).
* Range as minimum and maximum values was used.
* The percentage difference was calculated according to the formula:
Percentage difference equals the absolute value of the change in value,
divided by the average of the 2 numbers, all multiplied by 100.
Percent difference = (| (V1 - V2) | / ((V1 + V2)/2)) X 100.
* SPSS program version 18.0 was also used for correlation graph
plotting of leptin with TSH, FSH and LH. Excel program version 11 was
used for chart graphs plotting.
26
Chapter 4
Results
4.1 Socio-demographic data of the study population
The present study is a case control design. The study population comprised
42 women with recurrent abortion attending Al-Aqsa hospital (cases) in Gaza
strip and 42 control women with no history of recurrent abortion. Table 4.1
summarizes socio-demographic data of the study population. The mean age
of cases and controls were 31.6±7.3 and 31.1±6.4 years old respectively. The
independent sample t-test showed no significant difference between mean
ages of cases and controls (t=0.297, P=0.767). The highest number of cases
and controls was from Al-Nusirat and the lowest number was from Al-Zawaida
(Figures 4.1 and 4.2). Analysis of the educational status of the study
population showed no significant difference between various educational
levels of cases and controls (2 corrected=1.545, P=0.672). Employment and
family income showed also no significant differences between cases and
controls (2=0.309, P=0.003 and 2=3.383, P=0.184, respectively).
27
Table 4.1 Socio-demographic data of the study population
Socio-demographic data Cases (n=42)
Controls (n=42)
Test p- value
No. % No. %
Age (Year) <25 25-35 >35
8
24 10
19.1 57.1 23.8
9 25 8
21.4 59.5 19.1
2
0.301
0.860
Mean age±SD 31.6±7.3 31.1±6.4 t 0.297 0.767
Residence Al-Zawaida Al-Nusirat Al-Maghazi Al-Bureij Der Elbalah
6
12 6 7
11
14.3 28.6 14.3 16.7 26.2
5 11 7 8 11
11.9 26.2 16.7 19.1 26.2
2
0.278
0.991
Education University Secondary school Preparatory school Primary school
14 18 7 3
33.3 42.9 16.7 7.1
21 13 6 2
50.0 31.0 14.3 4.8
2
1.545
0.672*
Employment Yes
No
7
35
16.7 83.3
9 33
21.4 78.6
2
0.309
0.578
Family income/month (NIS)**
<1000 1000-2000 >2000
21 13 8
50.0 31.0 19.0
13 16 13
31.0 38.0 31.0
2
3.383
0.184
*P-value of 2 (corrected) test.
P>0.05:Not significan. **
NIS: new Israeli Shekels.
28
Figure 4.1 Distribution of cases (n=42) by location.
Figure 4.2 Distribution of controls (n=42) by location.
6 (14.3%)
12 (28.6%)
6 (14.3%)
7 (16.7%)
11 (26.2%)
Cases
Al-Zawaida
Al-Nusirat
Al-Maghazi
Al-Burij
Der Elbalah
5 (11.9%)
11 (26.2%)
7 (16.7%)
8 (19.1%)
11 (26.2%)
Controls
Al-Zawaida
Al-Nusirat
Al-Maghazi
Al-Burij
Der Elbalah
29
4.2 Premenstrual symptoms among the study
population
Table 4.2 provides the premenstrual symptoms among the study population.
Three were no significant differences between cases and controls with regard
to back pain, breast pain and psychological changes (P>0.05).
Table 4.2 Premenstrual symptoms of the study population
Symptoms Cases (n=42)
Controls (n=42)
2
p- value
No. % No. %
Back pain Yes No Breast pain Yes No Psychological changes Yes No
31 11
10 32
20 22
73.8 26.2
23.8 76.2
47.6 52.4
29 13
15 27
19 23
69.0 31.0
35.7 64.3
45.2 54.8
0.233
1.424
0.048
0.629
0.233
0.827
P>0.05:Not significant.
4.3 Duration of menstruation and menstrual
symptoms of the study population
As indicated in Table 4.3 and Figures 4.3 and 4.4, the mean duration of
menstruation was significantly higher in cases compared to controls (6.4±1.5
versus 5.6±1.2 days, t=2.219 and P=0.032). The number of cases who
reported irregular menstruation 14 (33.3%) was higher than controls 5 (11.9
%). The difference between the two groups was significant (2=5.509 and
P=0.019). Concerning heavy menstruation and dysmenorrheal, there was no
significant differences between cases and controls (P>0.05).
31
Table 4.3 Duration of menstruation and menstrual symptoms of the study
population
Item Cases (n=42)
Controls (n=42)
Test
p- value
Mean duration of menstruation±SD (Days) Range (min-max)
6.4±1.5 (3-10)
5.6±1.2 (4-7)
t
2.219
0.032
Menstrual symptoms Regular menstruation Yes No
Heavy menstruation Yes No
Dysmenorrhea Yes No
No.
28 14
15 27
17 25
%
66.7 33.3
35.7 64.3
40.5 59.5
No.
37 5
13 29
20 22
%
88.1 11.9
31.0 69.0
47.652.4
2
2
2
5.509
0.214
0.435
0.019
0.644
0.510
P<0.05: Significant, P>0.05: Not significant.
1
2
3
4
5
6
7
Case Control
6.4
5.6
Days
Figure 4.3 Mean duration of menstruation among the study population
31
4.4 Pregnancy and delivery information of the study
population
Table 4.4 and Figures 4.5 and 4.6 illustrate pregnancy and delivery
information of the study population. The mean age of cases and controls at
first pregnancy were 21.2±4.0 and 21.1±2.6, respectively (t=0.164 and
P=0.870). The number of cases who have children 25 (59.5%) was
significantly lower than controls 40 (95.2%) with 2=13.331 and P=0.000). Out
of 25 cases who have children, 19 (76.0%) reported normal delivery and 6
(24.0%) reported cesarean compared to 39 (97.5%) and 1 (2.5%) controls
(2=5.332 and P=0.021).
0
5
10
15
20
25
30
35
40
Yes No
28
14
37
5
Num
be
r
Figure 4.4 Regularity of menstruation among the study population
Case
Control
32
Table 4.4 Pregnancy and delivery information of the study population
Item Cases (n=42)
Controls (n=42)
Test p-
value* mean±SD mean±SD
Age±SD at first pregnancy (Year)
Range (min-max
21.2±4.0
(16-32)
21.1±2.6
(17-27)
t 0.164 0.870
Have children
Yes
No
Type of delivery
Normal
Cesarean
No.
25
17
19
6
%
59.5
40.5
76.0
24.0
No.
40
2
39
1
%
95.2
4.8
97.5
2.5
2
2
13.331
5.332
0.000
0.021
*P-value of 2 (corrected) test.
P<0.05: Significant, P>0.05: Not significant.
0
5
10
15
20
25
30
35
40
Yes No
25
17
40
2
Num
be
r
Figure 4.5 Having children among the study population
Case
Control
33
4.5 Recurrent abortion among cases
Data on recurrent abortion among cases are summarized in Table 4.5. The
numbers of cases who had 3 and more than 3 consecutive abortions were 24
(57.1%) and 18 (42.9%), respectively. The mean frequency of abortion was
4.7±2.3. In addition, the mean gestational age of fetus at abortion was
10.2±3.9 weeks and the mean time interval between two abortions was
8.4±7.3 months.
0
10
20
30
40
Normal Cesarean
28
14
39
3
Num
be
r
Figure 4.6 Type of delivery among the study population who have children
Case
Control
34
Table 4.5 Recurrent abortion among cases (n=42)
Abortion No. %
Frequency of abortion 3 >3
24 18
57.1% 42.9%
Mean frequency of abortion Range (min-max)
Mean± SD 4.7±2.3 (3-13)
Gestational age of fetus at abortion (Week)
Range (min-max)
10.2±3.9
(4-20)
Time interval between two abortions (Month) Range (min-max)
8.4±7.3 (1-36)
4.6 Frequency of abortion in relation to have children
among cases
Table 4.6 points out the frequency of abortion in relation to have children
among cases. The number of cases who had 3 abortions was found to have
higher number of children 18 (42.9%) than those who had >3 abortions 7
(16.7%). The difference between the two groups was significant (2=5.567
and P=0.018).
Table 4.6 Frequency of abortion in relation to have children among cases
(n=42)
Have children Frequency of abortion 2 p- value
3
No. %
>3
No. %
Yes 18 42.9 7 16.7 5.567 0.018
No 6 14.3 11 26.2
35
4.7 Chronic diseases among the study population
As depicted from Table 4.7, there were no significant differences in the
various reported chronic diseases (Thyroid gland disease, hypertension and
diabetes) between cases and controls (P>0.05).
Table 4.7 Chronic diseases among the study population
Disease Cases (n=42)
Controls (n=42)
2 p-
value*
No. % No. %
Thyroid gland disease Yes No
2 40
4.8
95.2
0 42
0.0 100
0.512
0.474
Hypertension Yes No
5 37
11.9 88.1
2 40
4.8 95.2
0.623
0.430
Diabetes
Yes No
2 40
4.8
95.2
0 42
0.0 100
0.512
0.474
*P-value of 2 (corrected) test.
P>0.05:Not significant.
4.8 Family history of recurrent abortion and various
disorders among the study population
Table 4.8 demonstrates family history of recurrent abortion and various
disorders among the study population. There were no significant differences
between cases and controls in term of family history of recurrent abortion,
abdominal surgery, ectopic pregnancy and vesicular mole (P>0.05). However,
the number of cases reported toxoplasmosis 6 (14.3%) was significantly
higher than controls 0 (0.0%) with 2=4.487 and P=0.034 (Table 4.8 and
Figure 4.7).
36
Table 4.8 Family history of recurrent abortion and various disorders among
the study population
Item Cases (n=42)
Controls (n=42)
2 p-
value
No. % No. %
Family history of recurrent abortion
Yes No
5 37
11.9 88.1
2 40
4.8 95.2
0.623
0.430*
Abdominal surgery Yes No
9 33
21.4 78.6
6 36
14.3 85.7
0.730
0.393
Ectopic pregnancy
Yes No
4 38
9.5
90.5
2 40
4.8 95.2
0.179
0.672*
Vesicular mole Yes No
3 39
7.1
92.9
1 41
2.4 97.6
0.262
0.609*
Toxoplasmosis Yes No
6 36
14.3 85.7
0 42
0
100
4.487
0.034*
*P-value of 2 (corrected) test.
P<0.05: Significant, P>0.05: Not significant.
0
10
20
30
40
50
Yes No
6
36
0
42
Num
be
r
Figure 4.7 Toxoplasmosis among the study population
Case
Control
37
4.9 Serum levels of leptin, TSH and gonadotropins of
the study population
Table 4.9 shows serum levels of leptin, TSH and gonadotropins (FSH and LH)
of the study population. The mean level of serum leptin was significantly
elevated in cases compared to controls as illustrated in Table 4.9 and Figure
4.8 (17.3±13.1 vs. 9.0±7.1 ng/ml, % difference=63.1, t=2.813 and P=0.007).
On the other hand, there were no significant differences in the mean levels of
TSH, FSH and LH between cases and controls (P>0.05).
Table 4.9 Serum levels of leptin, TSH and gonadotropins (FSH and LH) of the
study population
Hormone Cases (n=42) mean±SD
Controls (n=42) mean±SD
% difference
t P-value
Leptin (ng/ml) (min-max)
17.3±13.1 (2.3-46.4)
9.0±7.1 (2.1-34.1)
63.1 2.813 0.007
TSH (μIU/mL)* (min-max)
3.0±1.8 (0.8-9.1)
2.9±1.7 (0.5-7.1)
3.4 0.087 0.931
FSH (mIU/ml)**
(min-max) 4.7±1.7 (2.7-9.2)
4.6±1.8 (1.9-7.8)
2.2 1.189 0.240
LH (mIU/ml)*** (min-max)
6.7±2.6 (2.9-14.0)
6.0±2.1 (2.3-10.1)
11.0 0.258 0.797
*TSH: Thyroid stimulating hormone, **FSH: Follicle stimulating hormone, ***LH: Luteinizing
hormone. P<0.05: Significant, P>0.05: Not significant.
38
0
5
10
15
20
Case Control
17.3
9.0
Le
ptin
leve
l (n
g/m
l)
Figure 4.8 Serum leptin of the study population
39
4.10 Relations of leptin
4.10.1 Serum leptin in relation to regularity of menstruation,
having children and type of delivery among cases
The relationships of serum leptin level with regularity of menstruation, having
children and type of delivery among cases are provided in Table 4.10. The t-
test indicated that leptin level was significantly higher in cases reported
irregular menstruation that those reported regular menstruation as shown in
Table 4.10 and Figure 4.9 (21.8±13.7 versus 15.1±9.3 ng/ml, t=2.076,
P=0.044). However, no significant differences were recorded between leptin
level and having children or type of delivery among cases (P>0.05).
Table 4.10 Serum leptin level in relation to regularity of menstruation, having
children and type of delivery among cases
Leptin level (ng/ml)
mean±SD
t-test
P-value
Regular menstruation
Yes (n=28)
No (n=14)
15.1±9.3
21.8±13.7
2.076
0.044
Have child
Yes (n=25)
No (n=17)
15.4±9.7
20.5±12.8
1.429
0.163
Type of delivery
Normal (n=19)
Cesarean (n=6)
16.0±9.1
20.9±11.2
1.095
0.285
41
4.10.2 Serum leptin in relation to frequency of abortion and
toxoplasmosis among cases
Table 4.11 and Figures 4.10 and 4.11 display the relationship of serum leptin
with frequency of abortion and toxoplasmosis among cases. The mean level
of leptin was significantly higher in cases who had more than three abortions
compared to those who had three abortions (22.3±12.9 versus 14.5±10.2
ng/ml, t=2.065, P=0.046). In addition, there was a significant increase in
serum leptin in cases who reported toxoplasmosis in comparison to those who
did not (24.8±9.5 versus 16.5±8.3 ng/ml, t=2.201, P=0.034).
Table 4.11 Serum leptin level in relation to frequency of abortion and
toxoplasmosis among cases
Leptin level (ng/ml)
mean±SD
t-test
P-value
Frequency of abortion
3 (n=24)
>3 (n=18)
14.5±10.2
22.3±12.9
2.065
0.046
Toxoplasmosis
Yes (n=6)
No (n=36)
24.8±9.5
16.5±8.3
2.201
0.034
0
5
10
15
20
25
Yes No
15.1
21.8
Le
ptin
leve
l (n
g/m
l)
Figure 4.9 Serum leptin level in relation to menestrual regularity among cases
41
0
5
10
15
20
25
3 Abortions > 3 Abortions
14.5
22.3
Le
ptin
leve
l (n
g/m
l)
Figure 4.10 Serum leptin level in relation to frequency of abortion among cases
0
5
10
15
20
25
Yes No
24.8
16.5
Le
ptin
leve
l (n
g/m
l)
Figure 4.11 Serum leptin level in relation to toxoplasmosis among cases
42
4.10.3 Serum leptin in relation to TSH, FSH and LH levels of
cases
The relationships of serum leptin with TSH, FSH and LH levels of cases are
presented in Table 4.12 and Figures 4.12, 4.13 and 4.14. The Pearson
correlation test showed positive significant correlations between the level of
leptin and the levels of TSH and LH (r=0.359, P=0.021 and r=0.767, P=0.000,
respectively). However, none significant positive correlation was found
between leptin level and FSH level (r=0.173, P=0.280).
Table 4.12 Serum leptin level in relation to TSH and gonadotropins (FSH and
LH) levels of cases
Hormone
Leptin level (ng/ml)
Pearson correlation
(r)
P-value
TSH (μIU/mL)* 0.359 0.021
FSH (mIU/ml)** 0.173 0.280
LH (mIU/ml)*** 0.767 0.000
*TSH: Thyroid stimulating hormone, **FSH: Follicle stimulating hormone, ***LH: Luteinizing
hormone. P<0.05: Significant, P>0.05: Not significant.
Figure 4.12 Serum leptin level in relation to thyroid
stimulating hormone (TSH) level of cases
43
Figure 4.13 Serum leptin level in relation to follicle
stimulating hormone (FSH) level of cases
Figure 4.14 Serum leptin level in relation to Luteinizing
hormone (LH) level of cases
44
Chapter 5
Discussion
Recurrent abortion affects up to 5% of women worldwide and endocrine
disorders play a major role in such pregnancy loss (Ford and Schust, 2009
and Pluchino et al., 2014). Pregnancy loss can be very distressing and even
devastating for couples, especially when faced with recurrent losses. Couples
rarely obtain clear-cut reasons for the repeated failure to sustain a pregnancy,
nor the prospect of a fail-safe treatment. Despite that, there is underreporting
and lack of research on this condition in the Gaza Strip. Few recent studies
have been focused on autoimmunity and genetic aspects of recurrent abortion
in the Gaza Strip (AL Derawi, 2009; Sharif, 2012 and Jaber and Sharif,
2014). However, there was no previous study investigated endocrine and
clinical features of recurrent abortion in Gaza Strip. Therefore, the present
study is the first to assess serum leptin level in recurrent abortion which may
open a new avenue in the disease management.
5.1 Sociodemoghraphic data and menstrual aspects of
the study population
The present study is a case control investigation included 42 women with
recurrent abortion (mean age 31.6±7.3 years) and 42 control women with no
history of recurrent abortion (mean age 31.1±6.4 years). Analysis of
Sociodemoghraphic data of the study population showed no significant
differences between cases and controls in terms of residence, education,
employment and family income/month implying that such factors are more
likely not to be associated with recurrent abortion. This finding is in agreement
with that obtained by Maconochie et al. (2007). Similarly, premenstrual
symptoms including back pain, breast pain and psychological changes
exhibited no significant differences between cases and controls indicating that
such symptoms are not associated with recurrent abortion. There is no high-
quality evidence that shows any relationship between RPL and occupational
factors or stress (van Niekerk et al., 2013). However, the mean duration of
45
menstruation was significantly longer in cases compared to controls. Also, the
number of cases who reported irregular menstruation was significantly higher
than controls. This means that women with longer duration or irregular
menstruation are more likely to be at a higher risk of recurrent abortion. Ford
and Schust (2009) reported that interrupting the vasculature of the
endometrium promote abnormal and inadequate placentation and
consequently leads to miscarriage. Lack of pregnancy symptoms, seeming
continuation of ―periods,‖ irregular menses was listed as one reason for
having an abortion past 12 weeks (Planned Parenthood Federation of
America, 2014). Heavy menstruation and dysmenorrheal seem to be not
associated with recurrent abortion at least in the present study.
5.2 Pregnancy, delivery and recurrent abortion among
the study population
Data presented in this study revealed that the number of cases who have
children was significantly lower than controls. Higher number of those cases
reported cesarean compared to their counterparts of controls. Such result
coincides with the idea that recurrent abortion and cesareans may reduce the
possibility of having children which may impose psychological and social
effects on couples, particularly women. Similar result was documented by The
Practice Committee of the American Society for Reproductive Medicine
(2012) who pointed out that increased rates of cesarean delivery are
associated with pregnancy loss. In addition, Nankali et al. (2014) reported
that history and number of previous cesarean delivery is important to have
abnormal placentation in subsequent pregnancies. Concerning abortion
among cases, the present results showed that 57.1% of cases have 3
abortions and 42.9% have more than 3 abortions. When related to frequency
of abortion, the number of cases who had 3 abortions was found to have
significantly higher number of children than those who had >3 abortions. This
supports the previous result that increase rate of abortion reduces the
possibility of having children. It is generally accepted that increased abortion
availability leads to lower birth rates and even higher abortion (Bitler and
Zavodny 2002 and Pazol et al., 2013). In the present study the mean
46
gestational age of fetus at abortion was found to be 10.2±3.9 weeks indicating
that most abortions took place in the first trimester i.e. 12 weeks of gestation.
Such result is in accordance with that obtained by Kiwi (2006); Allison et al.
(2011) and Gonçalves et al. (2014) who declared that most of miscarriages
occur in the first trimester.
5.3 Family history of recurrent abortion and various
disorders among the study population
As depicted from the present results, few cases reported thyroid gland
disease, hypertension and diabetes making the association between these
chronic disorders and recurrent abortion not significant. However, if the study
focused on the direct impact of one of these disorders on recurrent abortion
among larger sample size of cases, the result will more likely to be different.
Regarding family history of recurrent abortion and other disorders including
abdominal surgery, ectopic pregnancy and vesicular mole, the results
revealed no significant differences between cases and controls. However, the
number of cases reported toxoplasmosis was significantly higher than
controls. Penta et al. (2003) identified Toxoplasma gondii more frequently in
vaginal and cervical cultures and serum from women with sporadic
miscarriages. Indeed the involvement of Toxoplasma gondii infection as a
cause of recurrent abortion has been accepted (Montoya and Remington,
2008 and Chintapalli and Padmaja, 2013). Women infected during the first
trimester could be very harmful to the fetus because after maternal acquisition
of Toxoplasma gondii for the first time during gestation the parasite will enter
the fetal circulation by the infection through the placenta. This may result in
severe congenital toxoplasmosis and can result in death of the fetus and
abortion (Montoya and Liesenfeld, 2004; Ford and Schust, 2009 and
Robbins et al., 2012). However, the implication of Toxoplasma gondii in RPL
needs further investigation.
47
5.4 Serum levels of leptin, TSH and gonadotropins
(FSH and LH) of the study population
The mean level of serum leptin was found to be significantly elevated in cases
compared to controls (nearly 2 folds higher in cases), whereas no significant
differences in the mean levels of TSH, FSH and LH between cases and
controls were registered. This result leaves no dout that serum leptin is
implicated in the pathogenesis of recurrent miscarriage. However, the exact
role of leptin in pregnancy and abortion is not fully understood. Nevertheless,
elevated levels of serum leptin were detected in women with recurrent
abortion (Baban et al., 2010; Saeed et al., 2010). In addition, Nanda et al.
(2012) demonstrated increased maternal serum leptin concentration at 11-13
weeks’ gestation in pathological pregnancies that may subsequently leads to
abortion. In this context, Žaneta et al. (2004) suggested that elevation of
leptin level could be an indicator for the cessation of pregnancy naturally
either at term or due to pathology at any time during gestation. This may draw
us to a conclusion that hyperleptinaemia could be a compensatory response
predicted in patients with threatened abortion (Ali, 2011). Therefore, several
authors related serum leptin to pregnancy outcome (Kasraeian and
Jamshid, 2009 and Llaneza-Suarez et al., 2014). Literatures showed that
leptin hormone is produced by both maternal and fetal adipose tissues, as
well as by the placental trophoblast. Specific receptors in the uterine
endometrium, trophoblast, and fetus facilitate direct effects of the polypeptide
hormone on implantation, placental endocrine function, and conceptus
development (Henson and Castracane, 2006; Maymó et al., 2009 and
Tessier et al., 2013). High levels of serum leptin may induce maternal leptin
resistance possibly by the soluble isoform of the leptin receptor (Henson and
Castracane, 2006). Altered serum leptin levels and the leptin-resistant state
have been shown to be associated with 1) impaired trophoblastic invasion
(Wang et al., 2014), 2) disturbance of endometrial angiogenesis and thus
embryo implantation (Herrid et al., 2014), 3) restriction of intrauterine growth
(Kyriakakou et al., 2008) and 4) stimulation of proinflammatory cytokines
release (Lappas et al., 2005 and Saeed et al., 2010). In addition,
hyperleptinaemia was suggested to be associated with insulin resistance
48
which may be involved in miscarriage through several mechanisms such as
diminished endometrial production of the adhesion factors, insulin-like growth
factor-binding protein-1 and uterine v3 integrin (Giudice, 2006; Tian et al.,
2007; Baban et al., 2010). Therefore and finally, one can say that
dysregulation of leptin metabolism and/or function is implicated in the
pathogenesis of various disorders during pregnancy that might lead to
recurrent miscarriage.
5.5 Relations of serum leptin to the studied
parameters among cases
The present results indicated that the mean level serum leptin was
significantly higher in cases reported irregular menstruation that those
reported regular menstruation. Increased serum leptin levels were recorded in
premenstrual syndrome and polycystic ovary syndrome which are
characterized by menstrual abnormalities (Zehra et al., 2011 and Nomaira et
al., 2014). This coincides with the idea that elevated serum leptin levels and
the leptin-resistant state interrupt the vasculature of the endometrium
promoting irregular menses which was listed as one reason for having an
abortion past 12 weeks (Ford and Schust, 2009 and Planned Parenthood
Federation of America, 2014). Similarly, serum leptin was significantly higher
in cases that had more than three abortions compared to those who had three
abortions. Such positive relationship of serum leptin with frequency of abortion
do confirm the involvement of leptin in the pathophysiology of recurrent
abortion. However, this point needs further investigation. Regarding parasitic
infection, cases reported toxoplasmosis showed significantly higher serum
leptin levels in comparison to those who did not. Baltaci and Mogulkoc (2012)
concluded that Toxoplasma gondii infection caused an increase in leptin
secretion. Therefore, Toxoplasma gondii infection in women with recurrent
abortion may exacerbate the level of leptin that is already elevated and thus
supporting its role as a candidate for such abortion (Montoya and
Remington, 2008 and Chintapalli and Padmaja, 2013). As discussed
previously, Toxoplasma gondii can enter the fetal circulation by the infection
through the placenta and may result in severe congenital toxoplasmosis that
49
can result in death of the fetus and abortion (Ford and Schust, 2009 and
Robbins et al., 2012). Finally, serum leptin exhibited positive significant
correlations with serum TSH and LH levels. These findings are in agreement
with that reported by Ajala et al. (2013) and Bétry et al. (2014). The positive
correlation of leptin with TSH is convenient in terms of their effects on energy
expenditure and body weight. It has been suggested that leptin stimulates
thyrotropin releasing hormone release from the hypothalamus, directly or
indirectly, which in turn stimulates TSH secretion (Sajid et al., 2013).
Similarly, leptin was proposed to activate the release of LH through its action
on hypothalamus-pituitary axis. Bellefontaine et al. (2014) pointed out that
leptin promotes sexual maturation, which requires the pulsatile, coordinated
delivery of gonadotropin-releasing hormone to the pituitary and the resulting
surge of LH. However, the neural circuits that control the leptin-mediated
induction of the reproductive axis are not fully understood. In addition, it has
been reported that hypersecretion of basal LH with or without polycystic
ovaries is a risk factor for miscarriage (Jeve and Davies, 2014).
Nevertheless, the present results indicated that there is a link between serum
leptin, TSH and fertility hormones involved in physiological and pathological
pregnancy including recurrent abortion.
51
Chapter 6
Conclusions and Recommendations
6.1 Conclusions
* The mean duration of menstruation and the frequency of irregular
menstruation were significantly higher in cases compared to controls.
* The number of cases who have children was significantly lower than
controls. Higher number of those cases reported cesarean compared to their
counterparts of controls.
* The mean gestational age of fetus at abortion was 10.2±3.9 weeks and
increasing abortion frequency reduces the possibility of having children.
* The number of cases reported toxoplasmosis was significantly higher than
controls.
* The mean level of serum leptin was significantly elevated in cases compared
to controls.
* Serum leptin level was significantly higher in cases who reported irregular
menstruation and those who had more than three abortions.
* Serum leptin exhibited positive significant correlations with serum TSH and
LH levels.
51
6.2 Recommendations
* Frequent monitoring of serum leptin levels particularly throughout the first
trimester of pregnancy.
* Development of treatment strategy depending on leptin hormone
manipulation.
* Further research is highly recommended 1) to clarify in depth the
relationship of leptin with fertility hormones especially in obese women and to
2) assess the role of leptin in other pregnancy disorders.
52
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عالقت هريى انهبخي بحاالث اإلجهاض انخكرر /اسخبيا حىل
(Serum Leptin in Relation to Recurrent Pregnancy Loss)
(Personal Dataانبيااث انشخصيت )أوال:
انعر
يكا انسك
(Socio - Economic Dataثايا: انبيااث االجخاعيت )
انسخىي انخعهيي
)عذد سىاث انذراست(
ثاىيت عايت ⧵ دبهىو ⧵ جايعي ⧵
ابخذائي ⧵ أقم ي ثاىيت عايت ⧵
ال ⧵ عى ⧵ انسوج يعم
ال ⧵ عى ⧵ انسيذة حعم
انذخم انشهري نألسرة
)بانشيقم( 0111أقم ي ⧵
- 0111ي ⧵
0111
0111أكثر ي ⧵
(Data of Menstrual cycleثانثاك بيااث انذورة انشهريت )
هم انذورة انشهريت
يخظت؟ ال ⧵ عى ⧵
ال ⧵ عى ⧵ هم انذورة انشهريت غسيرة؟
ال ⧵ عى ⧵ هم انذورة انشهريت يؤنت؟
يىو أياو انذورة؟ كى عذد ........................
أعراض حسبق انذورة
ال ⧵ عى ⧵ آالو في انظهر
ال ⧵ عى ⧵ آالو في انثذيي
ال ⧵ عى ⧵ حغيراث فسيت
أعراض أخري
.............................................................................
(Data of Pregnancy and Abortionانحم واإلجهاض )رابعا: بيااث
ال ⧵ عى ⧵ هم هاك عقى؟
ال ⧵ عى ⧵ هم هاك إجهاض يخكرر؟
إجهاض كى عذد اإلجهاضاث؟ ......................
69
شهر عر انحم عذ اإلجهاض ..................
شهر كى انىقج بي اإلجهاضي؟ ..................
ال ⧵ عى ⧵ هم يىجذ أطفال قبم اإلجهاض
قيصريت ⧵ طبيعت ⧵ ىع انىالدة قبم اإلجهاض
ست انعر عذ انحم األول ..................
(Related Diseases with Recurrent Pregnancy lossخايسا: بيااث األيراض راث انعالقت وانخاريخ انرضي )
هم هاك حاالث عقى وإجهاض يخكرر في
انعائهت؟
ال ⧵ عى ⧵
ال ⧵ عى ⧵ هم هاك عهياث جراحيت في انبطى؟
ال ⧵ عى ⧵ هم حعاي ي شعر زائذ في انجسى؟
ال ⧵ عى ⧵ ي اعخالل في انغذة انذرقيت؟هم حعاي
ال ⧵ عى ⧵ ها حعاي ي ارحفاع في ضغط انذو؟
ال ⧵ عى ⧵ هم حعاي ي يرض انسكري؟
ال ⧵ عى ⧵ هم هاك حاريخ يرضي بحم خارج انرحى؟
ال ⧵ عى ⧵ هم هاك حاريخ يرضي بحم عقىدي؟
جرثىيت هعذوي برض انهم هاك حاريخ يرضي ن((Toxoplasmosis
ال ⧵ عى ⧵