original article: the effect of high gravidity on the carcinogenesis of mammary gland in ta2 mice

The Effect of High Gravidity on the Carcinogenesis of MammaryGland in TA2 MiceXuan Wang1,2, Chun Huang3, Baocun Sun1,4, Yanjun Gu1, Yanfen Cui1, Xiulan Zhao4, Yan Li1, ShiwuZhang1

1Department of Pathology, Tianjin Cancer Institute and Hospital, Tianjin Medical University, Tianjin, China;2Department of Public Health, Tianjin Medical University, Tianjin, China;3Department of Medical Oncology, Tianjin Cancer Institute and Hospital, Tianjin Medical University, Tianjin, China;4Department of Pathology, Tianjin Medical University, Tianjin, China

Introduction

Breast cancer, a major global problem, is the leading

cause of death affecting women worldwide according

to mortality estimation and incidence,1,2 but the

mechanism of carcinogenesis is not yet been defini-

tively proven. Pregnancy has been regarded as a

protective factor since 1970s.3 The reason is that the

mammary epithelial cells are completely differenti-

ated during this period, which can inhibit the initia-

tion of the neoplastic process.4 It is commonly

accepted that nulliparity or low parity and increasing

age of the mother are risk factors for breast cancer,

while higher parity and earlier age at first pregnancy

may have a protective effect. However, recent

research has shown that pregnancy does not result

Keywords

Estradiol, progesterone, spontaneous breast

cancer, T lymphocyte subsets, TA2 mice

Correspondence

Baocun Sun, Department of Pathology, Tianjin

Cancer Institute and Hospital, Ti Yuan Bei,

Huan Hu Xi Road, He Xi District, Tianjin

300060, China.

E-mail: [email protected]

Submitted August 31, 2009;

accepted December 14, 2009.

Citation

Wang X, Huang C, Sun B, Gu Y, Cui Y, Zhao X,

Li Y, Zhang S. The effect of high gravidity on

the carcinogenesis of mammary gland in TA2

mice. Am J Reprod Immunol 2010; 63:

396–409

doi:10.1111/j.1600-0897.2009.00807.x

Problem

Spontaneous breast cancer in Tientsin Albinao 2 (TA2) mice, like

human pregnancy-associated breast cancer (PABC), often occurs in

pregnancy and puerperium, especially in mice with high gravidity. We

hypothesized that the dysfunction of cellular immunity caused by the

increase of 17b-estradiol (E2) and progesterone (P) might be one of the

reasons for carcinogenesis of mammary gland.

Method of study

We investigated the T lymphocyte subsets and the concentration of

serum hormone and cytokines in cancer-bearing, pregnant or postpar-

tum TA2 mice using flow cytometry, chemiluminescent immunoassay,

and enzyme-linked immunosorbent assay (ELISA), respectively.

Results

The number of T lymphocytes and the concentration of E2, P, interleu-

kin-2 (IL-2), IL-4, and interferon-gamma (IFN-c) changed with the

increase of pregnancy and puerperium. During four pregnancies, ele-

vated E2 and P resulted in a decrease in the number of CD3+, CD4+ T

lymphocytes, CD4+ ⁄ CD8+ ratio, and the concentration of IL-2, IL-4, and

IFN-c. Data in the fourth pregnancy were the closest to those of cancer-

bearing mice.

Conclusion

T lymphocyte subsets and concentration of IL-2, IL-4, and IFN-c are

affected by E2 and P during multiple pregnancy and delivery to some

degree, which may contribute to the genesis of spontaneous breast can-

cer in TA2 mice.

ORIGINAL ARTICLE

American Journal of Reproductive Immunology 63 (2010) 396–409

396 ª 2010 John Wiley & Sons A/S

in an immediate protective effect.5,6 Indeed, breast

cancer still can be detected during pregnancy or in

the first year after delivery no matter whether a

woman lactates, which has been defined as preg-

nancy-associated breast cancer (PABC). PABC has a

higher invasive and metastatic potential compared

with those occurring in women older than 40, and

its regional nodal and hematogenous metastasis can

be detected at an early stage.6 Furthermore, PABC

has a higher recurrence rate and often leads to worse

clinical outcome. Thus, more studies are focusing on

the carcinogenesis of PABC, and the results suggest

that hormonal factors associated with pregnancy and

lactation might play a role in PABC occurrence,

together with a specific genetic background.7–11

Hence, a specific animal model may conducive to

further elucidation of the mechanism of PABC.

Tientsin Albinao 2 (TA2) is a mouse strain that

has a very high incidence of spontaneous breast can-

cer. It was bred successfully in 1969 and was certifi-

cated by the Committee on Standardized

Nomenclature for Mice in 1984. The characteristics

of TA2 include a high incidence of spontaneous

breast cancer during pregnancy or after delivery,

multiplets, and the probability of developing breast

cancer in each mammary.12–14 Our previous study

showed that the mobidity in parous females was

85% with an average parity of 2.7, while it was

41% in virgin female mice. There were less apparent

abnormalities in TA2 mammary glands during the

first and second pregnancy and puerperium. On the

contrary, atypical hyperplasia or carcinomatous

changes proved by H&E staining were found in some

of TA2 mice after the third pregnancy.15,16 The mo-

bidity in the offspring of female TA2 and male TA1

(a mouse strain with a very low incidence of sponta-

neous breast cancer, which was certificated by the

same committee in the same year) was 88.89% with

an average parity of 7.67.17 These results indicate

that gravidity and parity affect the genesis and devel-

opment of breast cancer and that TA2 mouse is a

good model to investigate PABC.

The human immune response is a key physiologi-

cal system upon which mammalian survival depends

and would be expected to be a crucial target for the

action of natural selection. T cells are a fundamental

component of the adaptive arm of this response and

are involved in the defense against pathogens. The

degree of natural variation in the levels and ratios of

the various T lymphocyte subsets may contribute to

part of the genetic risk for the development of auto-

immune disease, infectious disease, and cancer.18,19

The antitumor immune response is predominantly

sustained by CD4+ and CD8+ T cells.20 CD4+ T helper

lymphocytes have been divided into T helper 1

(Th1) and Th2 subpopulations, based on their func-

tional properties and on the profile of secreted cyto-

kines. Th1 cells are responsible for the production of

interferon-gamma (IFN-c), interleukin-2 (IL-2), and

tumor necrosis factor-alpha (TNF-a), while Th2 cells

secrete IL-4, IL-5, IL-6, IL-10, and IL-13.21 Once the

balance of Th1 and Th2 is destroyed, infectious dis-

eases, autoimmune diseases, and cancer can

occur.21–24 Gestation is associated with a transient

depression of maternal cell-mediated immunity to

protect the semi-allogeneic embryo from rejection.

The hallmark of this immune tolerance is a profound

modulation of T cell response.25 The effect of gesta-

tion and T cell responses on spontaneous breast can-

cer is not yet been studied.

In order to confirm the effect of T lymphocytes on

the genesis of PABC, TA2 mice model was utilized in

this study. We correlated the changes in quantity and

function of T lymphocyte subsets with the different

level of estrogen and progestogen during different

stages of pregnancy and puerperium, and analyzed

the tumorigenesis of mammary gland of TA2 mice.

Materials and methods

Animals

The female TA2 mice used in this study (weighing 18–

22 g, 6–8 weeks) were obtained from the Experimen-

tal Animal Center at Tianjin Medical University,

China. All mice were fed ad libitum with standard

rodent food and water in a climate-controlled envi-

ronment with a light ⁄ dark photoperiod of 12:12.

Three to four female mice were housed together with

male TA2 mouse, also from the Experimental Animal

Center at Tianjin Medical University. Female mice

were inspected for the presence of vaginal plugs every

morning. The day a vaginal plug was first observed

was defined as the first day of pregnancy (day 1 of

pregnancy). The animals were maintained in accor-

dance with the NIH Guide for the Care and Use of

Laboratory Animals. The animals were grouped by

the days of pregnancy and puerperium (Table I).

Sera and spleens were collected at the following

time points in the first pregnancy: on days 5, 10,

and 20 of pregnancy and days 1, 7, 14, 21, and 42

of puerperium. And P10, P20, PP42 in four gravidi-

GRAVIDITY ON THE CARCINOGENESIS OF MAMMARY GLAND


ª 2010 John Wiley & Sons A/S 397

ties were chosen as being the most likely based on

the first gravidity, to provide information about

when the different level of index showed signifi-

cance. Mice in different gravidities were compared

with 8-month-old virgin mice and mice with sponta-

neous breast cancer (Table II).

Carmine and H&E Staining of Mammary Gland

Inguinal mammary glands were dissected, mounted

on glass slides, fixed in Carnoy’s solution and stained

in carmine alum overnight as described for whole-

mount analysis.26

Tissues were fixed overnight in 10% neutralized

buffered formalin, processed, embedded in paraffin

and cut into 5-lm thick-section, and sections were

initially stained with hematoxylin–eosin method.

H&E staining was performed according to standard

procedures by the department of Pathology of Tian-

jin cancer institute and hospital.

Detection of Concentration of 17b-Estradiol (E2)

and Progesterone (P)

The serum concentration of E2 and P was detected

by automated chemiluminescent immunoassay sys-

tem ACS�-180 (Chiron Diagnostics Corp., Medfield,

MA, USA), according to the manufacturer’s protocol.

Serum samples were stored at )80�C and measured

under the same conditions. All the assays were

carried out at the department of Clinical Laboratory

in Tianjin General Hospital by using ACS estradiol-6

and progesterone from Simmens medical solutions

Diagnostics (Tarrytown, NY, USA). All samples were

analyzed in duplicate.

Assay of T Lymphocyte Subsets

Splenic cells from mice were isolated, and the cell

suspensions were then washed three times with an

ice-cold phosphate buffered solution (PBS) buffer. A

total of 105 splenic cells diluted in 0.1 mL PBS were

stained at 4�C, in the dark, for 30 min, using the

following monoclonal antibodies purchased from

Biolegend (San Diego, CA, USA): Phycoerythrin

(PE)-labeled antibody to the mouse CD3+ clone

(17A2), the fluorescein isothiocyanate (FITC)-labeled

antibody to the mouse CD4+ clone (GK1.5), and the

PE ⁄ Cy5-labeled antibody to the mouse CD8+ clone

(53–6.7) at the dilution recommended by the manu-

facturer. The fluorescence was analyzed using a flow

cytometer (FACS Aria; BD Company, Franklin Lakes,

NJ, USA). Total CD3+ T lymphocytes and the

percentage of CD4+ or CD8+ T lymphocytes were

analyzed using the cell quest program.

Detection of Concentration of IL-2, IL-4, and IFN-c

Mice were bled at the indicated days, and sera were

aliquoted and stored at )80�C until use. The concen-

tration of IL-2 and IL-4 in sera was analyzed by

enzyme-linked immunosorbent assay (ELISA). The

Table I The Groups of Animals in First Pregnancy (n = 5)

Groups

Normal

controlaDay 5 of

pregnancy

Day 10 of

pregnancy

Day 20 of

pregnancy

Day 1 of

puerperium

Day 7 of

puerperium

Day 14 of

puerperium

Day 21 of

puerperium

Day 42 of

puerperium

Abv. NC P5 P10 P20 PP1 PP7 PP14 PP21 PP42

aNon-pregnant and non-tumor adult mice (6–8 weeks) were taken as normal control.

Table II The Groups of Animals in Different Gravidities (n = 5)

Groups

Control

First pregnancy Second pregnancy Third pregnancy Forth pregnancy8 months old virgin Spontaneous breast cancer

Abv. 8MV SBC 1P 2P 3P 4P

*Non-pregnant and non-tumor adult 8 months old virgin mice were taken as negative control.

Mice with spontaneous breast cancer were taken as positive control.

WANG ET AL.



mouse IL-2 ELISA kit (Cat. No. 431007), mouse IL-4

ELISA kit (Cat. No. 431107), and mouse IFN-c ELISA

kit (Cat. No. 430807) from Biolegend were used to

quantify each cytokine following the manufacturer’s

instructions. Each multiplex assay was performed in

duplicate on two different occasions according to the

manufacturers’ specifications. OD means were used

to determine the concentrations of cytokines in the

samples according to the standard curves which

were generated by using the reference cytokine

concentrations supplied by the manufacturers.

Statistical Analysis

All data were analyzed using spss statistical software

(version 13.0 for Windows; SPSS, Chicago, IL, USA;

www.spss.com). The data were analyzed using a

one-way anova. The differences were considered

significant at P value <0.05. Correlation was

determined by linear regression.

Results

The Morphology of Mammary Gland of TA2 Mice

in Different Gravidities

Carmine-stained whole mounts of mammary glands

and H&E staining from TA2 mice showed that ductal

growth occured extensively, and the ducts under-

went further branching following the onset of

pregnancy compared with normal control (NC). Epi-

thelial cells multiplied to form the lobuloalveolar

structure. The interstitial adipose tissue disappeared

progressively, and proliferating epithelial cells filled

the interductal spaces. Lobulus were obviously

extended and the number of lobulus increased on

P20 with the increase of gravidity, the gland became

larger and adipose tissue decreased in size. We could

not find the ductal structure and distinguish the duct

and lobulus in 3P and 4P, they extruded each other.

The number of alveolus increased, while the struc-

ture became more disordered and the interstitial tis-

sue between lobules became less especially in 3P and

4P. On PP42, the developed alveoli regressed and

were replaced by a large amount of fatty tissue and

connective tissue during involution. The mammary

gland in 1P almost returned to antepartum shape.

The number of glandular lobule was lower, and the

gland was small in 1P. The regressed alveoli in 2P

were more than 1P, but the adipose tissue was less.

The mammary glands during involution in 1P and

2P were similar to 8MV. The mammary gland could

not return to the antepartum shape completely in 3P

and hyperplasia glandular lobule mass were found.

Atypical hyperplasia was also seen in H&E sections

in some places. The detail of morphology of mam-

mary glands is shown in Fig. 1.

Level of Serum E2 and P of TA2 Mice

Changes during first pregnancy

The concentration of serum E2 and P in pregnancy

was significantly different from NC (E2: F = 5.491,

P = 0.000; P: F = 17.953, P = 0.000). They increased

once the female mice were in gestation and reached

the summit on P20. The concentration of E2

decreased gradually after delivery. However, the

level of P decreased sharply on PP1 and fluctuated

during different days after parturition (Fig. 2a). The

level of P went up significantly on P5 (P = 0.002),

P10 (P = 0.000), and P20 (P = 0.000) compared with

NC. The level of E2 was higher on P10 (P = 0.005),

P20 (P = 0.000), and PP1 (P = 0.041) compared with

NC. No significant changes were observed between

NC and other postpartum groups.

Changes in different gravidities

The concentration of serum E2 and P went up with

the increase of gravidity (Fig. 2b,c). E2 increased sig-

nificantly on P20 (F = 6.585, P = 0.004) among four

gravidities. Notably, E2 in 4P was approximately

twice as high as that in 1P (P = 0.001) and 2P

(P = 0.002). The same tendency was observed in the

level of P (F = 3.314, P = 0.042), and 4P was signifi-

cantly higher than 1P (P = 0.007). No significant dif-

ferences were found in E2 (F = 1.866, P = 0.176)

and P (F = 2.198, P = 0.128) on PP42 in different

gravidities and 8MV.

Lymphocyte Subsets

Changes during first pregnancy

Compared with NC, a significant decrease in CD3+ T

lymphocytes was found during pregnancy and puer-

perium (F = 18.094, P = 0.000). Suppressor ⁄ cyto-

toxic (CD8+) T lymphocytes on P10 (P = 0.025) and

P20 (P = 0.012) increased while helper ⁄ inducer

(CD4+) T lymphocytes went down from the begin-

ning of pregnancy and significantly decreased on

P10 (P = 0.004) and P20 (P = 0.005), which resulted

in the marked reduction in CD4+ ⁄ CD8+ ratio

(Fig. 2a,b). No significant changes were found in




CD4+ T, CD8+ T lymphocytes or in CD4+ ⁄ CD8+ ratio

at any stage of puerperium.

Changes in different gravidities, 8MV and SBC

The percentage of CD3+, CD4+, CD8+ T lymphocytes

and CD4+ ⁄ CD8+ ratio were different in four gravidi-

ties (Figs 3c–4f). They were compared with those in

8MV and SBC (Table III and IV). All indexes signifi-

cantly decreased with the increase of gravidity

(P < 0.001) except that CD8+ T lymphocytes

increased gradually. The most remarkable changes

occurred in 4P. Data in 4P were the closest to those

in SBC and were the farthest from those in 8MV. No

differences were observed in the data between every

time point of 4P and SBC. CD4+ T lymphocytes on

P10 in 1–3P, on P20 in 1–4P were the same as those

in SBC, while CD4+ T lymphocytes on P20 in 4P

were unexpectedly lower than SBC (Table III).

Concentration of Serum IL-2, IL-4, and IFN-c

Changes in the first pregnancy

Significantly lower concentration of serum IL-2,

IL-4, IFN-c and IFN-c ⁄ IL-4 ratio were observed

compared with NC (Fig. 4a,b). The concentration of

IL-2 reduced significantly on P10, P20 and PP7,

PP21 (P < 0.05). IL-2 tended to increase again on

PP1 but decreased gradually on PP7, PP14 and PP21

at last, and then increased again on PP42. The con-

centration of IL-4 on P5 was slightly higher than

NC, but decreased immediately, especially on P20,

PP1, PP7, and PP21 (P < 0.05). The concentration of

IFN-c reduced significantly on every stage during

pregnancy and puerperium (P < 0.05). The IFN-c ⁄ IL-

4 ratio decreased significantly on P5 and P10

(P < 0.05), while increased on PP1, PP7, and PP42.

The concentration of IL-2, IL-4, IFN-c and the IFN-

c ⁄ IL-4 ratio recovered on PP42.

Changes in different gravidities, 8MV and SBC

The concentration of serum IL-2, IL-4, and IFN-cdecreased significantly with the increase of gravidity

(P < 0.05), while the IFN-c ⁄ IL-4 ratio decreased

slightly. The differences in 1P–4P were showed in

Fig. 4c–f. The differences on different time point

among gravidities, 8MV, and SBC are shown in

Table V and Table VI. The data in 4P were close to

SBC, and the levels of IL-2, IL-4, and IFN-c on P10

Fig. 1 Carmine-stained wholemounts of ingu-

inal mammary glands and H&E staining tissues

of NC, P20, PP42 and 8MV. NC&8MV: only

duct were seen. No atypical hyperplasia was

found. 1&2P: ducts connected with lobulus in

terminal were seen clearly. More stromal

tissue and less lobulus and no hyperplasia in

epithelia were observed on P20. Less glandu-

lar lobule was found on PP42. 3&4P: intersec-

tion among lobulus proliferated like mass

appeared on P20. Atypical hyperplasia was

seen on PP42. The mammary glands were

stained with carmine red in i and were

sectioned and stained with H&E in ii.

WANG ET AL.



were closer to SBC than those on the other two time

points. There was no difference in IFN-c ⁄ IL-4 ratio

between each group of 4P and SBC.

Association between hormone and the indexes of immune

function

Negative correlations between CD4+ T lymphocytes,

CD4+ ⁄ CD8+ ratio, and E2 ⁄ P were confirmed during

pregnancy and puerperium (Fig. 5a,b,e,f). Positive

correlations between CD8+ T lymphocytes and E2 ⁄ Pwere observed (Fig. 5c,d). There was no correlation

between IL-2 ⁄ IL-4 ⁄ IFN-c and E2 ⁄ P (P > 0.05).

Discussion

TA2 mouse, an inbred strain originating from

Kunming mouse, has about 200 serial passages and

stable hereditary characters. It has a very high

incidence of spontaneous breast cancer, but the

mechanism of its carcinogenesis is not known. Our

previous studies showed that the carcinogenesis of

mammary gland might be closely related to preg-

nancy and gravidity.15,17 Usually, immune systems

can response to cancer cells with immune modula-

tion and immune surveillance in normal body. But

in pregnant TA2 mice, immune suppressive effects of

elevated estradiol and progesterone will induce

proliferation and differentiation of the ductal and

lobular–alveolar epithelium.27–29 The decrease of

immune surveillance will make the proliferation of

mutational breast epithelium out of control, and

thus will lead to an increased risk of breast cancer if

tumor cells exist and their proliferation is trig-

gered.30 Therefore, we hypothesized that multiple

Fig. 2 Serum estradiol and progesterone concentration. (a) Serum estradiol and progesterone concentration in pregnancy and puerperium dif-

fered from control *P < 0.05, **P < 0.01. (b) 4P differed from 1P and 2P in estradiol and differed from 1P in progesterone on P20 **P < 0.01. (c)

No significant changes were found in different gravidities compared with the 8MV.




pregnancy and delivery and the impairment of

immune function might be associated to the carcino-

genesis of mammary gland in TA2 mice.

Once the mice became pregnant, their mammary

gland went through a ‘hyperplasia-involution’ pro-

cess. Experiencing multiple pregnancy and delivery

Fig. 3 The changes of T lymphocytes subsets. (a and b) T lymphocytes subsets in pregnancy and puerperium differed from control *P < 0.05, **P

< 0.01. (c) CD3+ T lymphocytes in 4P were significant lower than 1P~3P on P20 and PP42. (d) CD4+ T lymphocytes in 4P were significant lower than

1P~2P on every time point. (e) CD8+ T lymphocytes in 4P were significant higher than 1P~2P on P20 and PP42. (f) CD4+/CD8+ ratio in 4P was signif-

icant lower than 1P on P10, 1P~2P on P20 and PP42. Significant difference was marked by *.

WANG ET AL.



can make their mammary glands go into a ‘hyper-

plasia-involution-hyperplasia-involution’ cycle. With

strong reproductive capacity and short and frequent

estrus cycle, female mice usually enter the estrus

period quickly after delivery and copulate with the

male successfully. They usually become pregnant

again during lactation. Thus, they skip the period of

involution resulting in exposure of their mammary

glands to high level of estrogen and progestogen for

a prolonged period.31 Therefore, we can imagine that

there will be two possible outcomes. One is that

some of the mammary epithelia with stronger capac-

ity for proliferation and invasion will grow more

quickly because of multiple pregnancies. On the

contrary, those epithelia with a lower capacity for

proliferation and invasion will disappear through the

apoptotic pathway. Both of these result in incom-

plete involution or partial proliferation. The contri-

bution of the unique involution process of the breast

to PABC susceptibility warrants further investiga-

tion.11

Breast cancer is a type of hormone-dependent

tumor.32 Some evidence has shown that the increase

level of estradiol and progesterone will promote the

proliferation and growth of mammary glands, and

the concentration of serum estradiol and progester-

one is closely associated with the risk of breast can-

cer.27,33 Once estrogen is combined with a specific

receptor, it will activate the target gene downstream,

and then stimulate the proliferation of mammary

ductal epithelium. Progesterone has a synergistic

effect on stimulating mammary alveolus epithelium

cells with estradiol.34 In the full term of pregnancy,

the mammary gland is exposed to a physiological

high level of estradiol and progesterone. In this

research, we have found that the concentration of

estradiol and progesterone significantly increased

during pregnancy and slightly increased after deliv-

Table III The Percentage of CD4+ and CD8+ T Lymphocytes on P10, P20, and PP42 in Four Gravidities (mean ± S.D., n = 5)

Groups

CD4 CD8

1P 2P 3P 4P 1P 2P 3P 4P

P10 58.26 ± 6.16a 55.40 ± 5.19a 53.65 ± 2.87a 51.25 ± 1.62ab 32.16 ± 3.09 35.70 ± 5.86 36.15 ± 0.59 36.00 ± 4.16a

P20 58.87 ± 2.17a 59.08 ± 2.59a 57.19 ± 5.79a 53.43 ± 1.31a 32.68 ± 2.93 32.19 ± 3.63 34.86 ± 1.94 37.08 ± 2.18a

PP42 67.55 ± 4.21b 67.30 ± 1.73b 60.65 ± 2.13 57.83 ± 2.99a 30.08 ± 3.66 29.85 ± 2.46 35.33 ± 0.49 35.45 ± 1.81a

8MV 65.21 ± 2.03b 65.21 ± 2.03b 65.21 ± 2.03b 65.21 ± 2.03b 30.69 ± 2.64 30.69 ± 2.64 30.69 ± 2.64 30.69 ± 2.64b

SBC 56.24 ± 4.51a 56.24 ± 4.51a 56.24 ± 4.51a 56.24 ± 4.51a 35.43 ± 6.66 35.43 ± 6.66 35.43 ± 6.66 35.43 ± 6.66a

F 9.221 15.614 9.427 27.163 1.879 1.805 2.278 2.651

P 0.000 0.000 0.000 0.000 0.138 0.153 0.083 0.049

aP < 0.05, compared with 8MV.bP < 0.05, compared with SBC

Table IV The Percentage of CD3+ T Lymphocytes and CD4+ ⁄ CD8+ T Ratio on P10, P20, and PP42 in Four Gravidities (mean ± S.D., n = 5)

Groups

CD3 CD4 ⁄ CD8

1P 2P 3P 4P 1P 2P 3P 4P

P10 12.78 ± 2.79a 11.36 ± 2.14ab 10.52 ± 2.38a 8.05 ± 3.17a 1.85 ± 0.34b 1.64 ± .046ab 1.48 ± 0.08a 1.44 ± 0.19a

P20 22.78 ± 3.80ab 22.14 ± 3.97ab 21.59 ± 3.04ab 12.88 ± 3.55a 1.81 ± 0.14a 1.85 ± 0.18ab 1.64 ± 0.13a 1.45 ± 0.09a

PP42 51.53 ± 20.60ab 42.88 ± 15.55b 36.72 ± 11.81b 20.00 ± 7.53ab 2.28 ± 0.38b 2.28 ± 0.26b 1.72 ± 0.08a 1.64 ± 0.16a

8MV 40.00 ± 8.50b 40.00 ± 8.50b 40.00 ± 8.50b 40.00 ± 8.50b 2.14 ± 0.25b 2.14 ± 0.25b 2.14 ± 0.25b 2.14 ± 0.25b

SBC 8.84 ± 3.95a 8.84 ± 3.95a 8.84 ± 3.95a 8.84 ± 3.95a 1.50 ± 0.30a 1.50 ± 0.30a 1.50 ± 0.30a 1.50 ± 0.30a

F 22.358 20.917 33.810 37.007 7.251 10.012 12.911 12.763

P 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

aP < 0.05, compared with 8MV.bP < 0.05, compared with SBC.




ery with the increase of gravidity. Although the level

of hormone did not elevated obviously, we observed

the marked morphology changes in mammary gland.

We thought that it was due to the long-term effect

of high estrogen and progestrogen. Meanwhile, it

was confirmed that the continuous treatment with

estradiol and progesterone would result in high

mammary tumor incidence.35 Mammary carcinogen-

esis required continuous exposure to high concentra-

tion of both estrogen and progesterone, not estrogen

or progesterone alone.36 This conclusion was coinci-

dent with ours. In addition, a negative correlation

Fig. 4 The concentration of TA2 mice serum IL-2, IL-4 and IFN-gamma. (a and b) The concentration of serum IL-2, IL-4 and IFN-gamma/IL-4 ratio

during pregnancy and puerperium were significantly different from NC. (c) IL-2 in 4P was significantly lower than 1~2P on P10, 1P on P20 or 1~3P

on PP42. (d) IL-4 in 4P was significantly lower than 1P on P10 and P20 or 1~2P on PP42. (e) IFN-gamma in 4P was significantly lower than 1~2P on

P10 and P20 or 1~3P on PP42. (f) IFN-gamma/IL-4 in 4P was significantly lower than 1P on PP42. Significant difference was marked by *.

WANG ET AL.



was found between the concentration of estra-

diol ⁄ progesterone and the parameters promoting the

immune function in this study. As the gravidity

increased, the correlation became more prominent.

Pregnancy can affect the maternal immune system.37

In this study, the immunosuppression by estradiol

and progesterone became more significant with

the increase of gravidity. Recent studies in vivo and

in vitro have confirmed the fact that estrogen and

progestrogen can inhibit immune function.38–40 The

higher concentration of estrogen and progestrogen

in physiological level will usually lead to stronger

inhibition of immune system. Furthermore, estradiol

plus progesterone shows much more inhibitory

action than estradiol or progesterone alone. 41–43

Hence, we suppose that the persistently high levels

of estrogen and progesterone cannot make the

immune function of TA2 mice recover, thus mak-

ing the pregnant mice vulnerable to some diseases

such as cancer. Therefore, the increase of gravidity

can indirectly promote the genesis of breast cancer

through long-term elevated estrogen and progester-

one. Recent studies indicate that parity-induced

protection against mammary carcinogenesis is not

permanent, and this protective effect can be reversed

by increasing the promotional environment, such as

long-term administration of estrogen and progester-

one.44 Hormonal changes during pregnancy are

regarded as important mediators which act as

double-edged sword45–47; however, the complex

interactions between the endocrine system and

immunological reactions in humans are still not well

understood.

Both the innate and adaptive immune responses

are believed to be involved in controlling the growth

of many cancers. T lymphocytes are a major compo-

nent of the adaptive immune system. In our study,

we found that CD3+ T lymphocytes decreased in

pregnancy and after delivery. CD4+ T lymphocytes

and CD4+ ⁄ CD8+ T ratio decreased in middle and late

Table V The Concentration of Serum IL-2 and IL-4 on P10, P20, and PP42 in Four Gravidities (mean ± S.D., n = 5)

Groups

IL-2 IL-4

1P 2P 3P 4P 1P 2P 3P 4P

P10 230.37 ± 62.89b 183.25 ± 68.02a 155.51 ± 53.77a 119.24 ± 60.97a 360.07 ± 90.23b 301.31 ± 45.60b 271.59 ± 61.71b 223.10 ± 88.96a

P20 278.91 ± 83.87b 250.99 ± 131.30b 214.41 ± 65.51ab 193.07 ± 42.23ab 340.73 ± 66.36b 289.05 ± 69.99b 269.61 ± 72.67b 239.64 ± 59.83a

PP42 387.81 ± 124.47b 367.96 ± 106.48b 263.48 ± 85.21b 139.97 ± 14.37a 395.13 ± 165.55b 361.21 ± 56.63b 335.31 ± 78.70b 292.50 ± 71.08b

8MV 310.53 ± 48.36b 310.53 ± 48.36b 310.53 ± 48.36b 310.53 ± 48.36b 325.45 ± 78.43b 325.45 ± 78.43b 325.45 ± 78.43b 325.45 ± 78.43b

SBC 120.38 ± 28.72a 120.38 ± 28.72a 120.38 ± 28.72a 120.38 ± 28.72a 169.55 ± 68.91a 169.55 ± 68.91a 169.55 ± 68.91a 169.55 ± 68.91a

F 13.743 11.813 13.035 24.436 6.337 8.892 6.361 4.207

P 0.000 0.000 0.000 0.000 0.001 0.000 0.001 0.009


Table VI The Concentration of Serum IFN-c and IFN-c ⁄ IL-4 on P10, P20, and PP42 in Four Gravidities (mean ± S.D., n = 5)

Groups

IFN-c IFN-c ⁄ IL-4

1P 2P 3P 4P 1P 2P 3P 4P

P10 315.61 ± 86.39b 245.58 ± 51.35ab 211.30 ± 27.06ab 167.72 ± 26.52a 0.82 ± 0.18a 0.78 ± 0.07a 0.77 ± 0.05a 0.74 ± 0.16a

P20 355.33 ± 58.15b 286.47 ± 36.13b 225.34 ± 29.31ab 198.99 ± 55.71ab 1.00 ± 0.10b 0.94 ± 0.19b 0.91 ± 0.27b 0.83 ± 0.10a

PP42 366.43 ± 80.80b 313.19 ± 52.17b 295.66 ± 49.08b 215.46 ± 57.35ab 1.15 ± 0.29b 0.91 ± 0.20b 0.89 ± 0.10b 0.85 ± 0.15a

8MV 337.43 ± 70.80b 337.43 ± 70.80b 337.43 ± 70.80b 337.43 ± 70.80b 1.07 ± 0.22b 1.07 ± 0.22b 1.07 ± 0.22b 1.07 ± 0.22b

SBC 128.89 ± 27.62a 128.89 ± 27.62a 128.89 ± 27.62a 128.89 ± 27.62a 0.69 ± 0.08a 0.69 ± 0.08a 0.69 ± 0.08a 0.69 ± 0.08a

F 14.389 17.814 21.217 16.199 6.596 5.634 5.236 6.592

P 0.000 0.000 0.000 0.000 0.001 0.002 0.003 0.001





pregnancy and increased after delivery. However,

CD8+ T lymphocytes showed the opposite trend. Our

results were similar to the results of the human

study by Watanabe et al.37 Meanwhile, our study

also showed that gravidity could affect T lympho-

cytes subsets and high gravidities differed from low

gravidities. To the best of our knowledge, this is the

first report of these effects.

Cytokines produced by activated CD4+ T lympho-

cytes play an important role in the initiation and

Fig. 5 Correlation between T lymphocyte subsets and estradiol/progesterone.

WANG ET AL.



regulation of immune responses and coordination of

the two arms of immune system. In our study, we

detected significantly reduced IL-2 levels from

mid-pregnancy and reduced IFN-c levels from early

pregnancy. The levels of IL-4 also declined from

mid-pregnancy, but this reduction was marginal and

a significant difference was only observed in late

pregnancy. Calculating the cytokine ratios revealed a

shift from a Th1 to a pronounced Th2 cytokine

response which was significant during mid-preg-

nancy (Fig. 4e). These results were in agreement

with published results in human.25,48–51 Moreover,

our study also suggested that the level of IL-2, IFN-c,

and IL-4 changed with different gravidity. Higher

gravidity was associated with lower IL-2, IFN-c, IL-4,

and IFN-c ⁄ IL-4 ratio. The tendency was obvious

especially in the fourth pregnancy. The effects of

multiple pregnancies on the change of Th1 ⁄ Th2 ratio

have not been reported. Our results did not show

significant correlation between hormones and cyto-

kines. We thought there might be two reasons. One

was that we detected T lymphocyte cytokines only

on the serum level rather than on the intracellular

level. The other was that we correlated the level of

cytokines with single E or P, while some evidence

indicated that Th1 ⁄ Th2 ratio usually resulted from

the combined effect of estrogen and progesterone.

High estradiol dose proved to significantly decrease

IFN-c level in the T cell culture supernatant. In con-

trast, progesterone increased IFN-c production by

activated T lymphocytes but proved inefficient in a

physiological combination with estradiol.52 In this

study, we found that estradiol increased much

higher than progesterone with the increases of gra-

vidity, which led to an obvious decrease in the level

of IFN-c. IL-4 also decreased with the change of

gravidity. Because the change of IFN-c was much

more than that of IL-4, Th2 response predominated

over Th1. Therefore, the reproductive hormones

could efficiently regulate synthesis of the main

Th1 (IFN-c) and Th2 (IL-4) cytokines by T lympho-

cytes and seemed to play the key role in changing the

pregnancy-specific pattern of secreted cytokines.52

In general, Th1 ⁄ Th2 cytokine unbalanced produc-

tion might predispose to different pathologies, cancer

included.19 It is believed that a Th1 response is nec-

essary for an effective immune response to be

mounted against most tumors,53 while typical Th2

shift appears in many cancer patients. In spontane-

ous breast cancer mice, the expression of Th1 cyto-

kines was inhibited with a concomitant predominant

state of Th2 cytokines, which indicated that Th1

cytokines suppressed by pregnancy could not cause

anti-tumor cellular immunity to be activated. The

phenomenon of Th2 shift was more obvious with

the increased gravidity. Shen’s research has sug-

gested that the Th2 shift is more obvious in young

breast carcinoma tissues.54 Thus, the destroyed bal-

ance of Th1 and Th2 might be one of the reasons for

immune escape of tumor cells and contributes to the

spontaneous carcinogenesis of mammary gland.

However, the relationship between the shift in the

balance of Th1 and Th2 and carcinogenesis of mam-

mary gland in TA2 needs further investigation.

Conclusion

In summary, to the best of our knowledge, this is

the first study to focus on the explanation of carci-

nogenesis of mammary gland of TA2 mice through

the investigation of hormone levels and analysis of

immune function. Repeated pregnancy and delivery

result in continuous high level of estrogen and

progestogen, which stimulate the proliferation of

mammary ductal and lobular–alveolar epithelium.

On the other hand, the immune function is also

inhibited during the course of frequent pregnancy

and delivery. Both of these factors contribute to the

presence of abnormally proliferated epithelia and the

escape of existing tumor cells from immune sur-

veillance. These observations are important to our

understanding of the mechanisms of PABC in

human.

Acknowledgments

This work was supported by the Key Program of the

Natural Science Foundation of Tianjin (grant

043115211- 1).We thank Department of Immunol-

ogy, Tianjin Cancer Institute for their technical assis-

tance. We also thank the Experimental Animal

Center at Tianjin Medical University for providing

experimental animals.

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original article: the effect of high gravidity on the carcinogenesis of mammary gland in ta2 mice

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