ORIGINAL�ARTICLE

ABSTRACT
Objective: To study the teratogenic effects of Bisphenol A on the testicular histogenesis of developing rat pups 
during prenatal and early postnatal life. 
Study Design: Laboratory-Based Randomized Control Trial. 
Place and Duration of Study: The present study was carried out at Anatomy Department of Islamic 
International Medical College Rawalpindi in association with National Institute of Health (NIH) Islamabad from 
September 2018 to September 2019.
Materials and Methods: Eight weeks old 10 pregnant female rats were divided into 2 groups each containing 5 
pregnant female rats. Pups were delivered by spontaneous vaginal delivery. Control group A was composed of 
15 male rat pups from 5 pregnant female rats fed on the standard diet during pregnancy and lactation till day 
21. Experimental group B was composed of 15 male rat pups from 5 pregnant female rats which were given 
250μg/kg/day Bisphenol A subcutaneously during pregnancy and lactation till day 21.
Results: Deterioration of histological parameters was significantly seen in group B. Tubule differentiation 
index(TDI) in group B was 69.22%; significantly reduced as compared to control group A. The modified 
Johnsen's criteria in group B was score 3 (in 33.3% rats ) and score 1 (in 26.6% rats) as compared to score 7 (in 
100% rats) in control group A. 66.6% rats in group B showed severe (grade 4) and 26.6% rats showed moderate 
(grade 3) epithelial degeneration as compared to control group A. 46.6% rats in group B showed severe (grade 
4) and 40% had moderate (grade3) congestion as compared to control group A.46.6% rats in group B showed 
severe (grade 4) vacuolization and 33.3% had moderate (grade3) vacuolization as compared to control group.
Conclusion: The teratogenic effect of BPA on developing rat testes is evident when given during prenatal and 
early postnatal life. The teratogenicity of BPA may adversely affect spermatogenesis and male fertility.

Key Words: Bisphenol A, Developing Rats, Early Postnatal Testicular Teratogen, Prenatal.
5

bisphenol A.  The European Food Safety Authority 
(EFSA) has determined the temporary tolerable daily 

6
intake of BPA which is 4μg/kg/day.  According to the 
data published by EFSA in 2015; BPA exposure for 

7
men and women is 1.01 to 1.06μg/kg/day.
Bisphenol A is a ubiquitous endocrine disrupting 
chemicals (EDC) which is used in synthesis of epoxy 
resins and polycarbonate plastics, food cans, water 
pipes, receipt papers, baby feeding bottles and 

3
currency notes.  Reproductive system is one of the 

8
main systems targeted by this endocrine disruptor.  
Exposure to such chemicals in humans is a probable 

9
cause of male infertility.  Developing testis is 
fundamental component of male reproductive 
system which is susceptible to teratogens. BPA has 
deteriorating effects in pregnancy when exposed 
during critical period of embryonic testicular 

10
development.  Animal studies revealed that BPA has 

11
a potent teratogenic potential.  In rats during 
prenatal testicular development (gestational days: 

Introduction
Bisphenol A was manufactured first by A. P. Dianin in 
1891 and investigated in 1930s during the             
hunt for synthetic estrogens and is thus an important 

1 2
endocrine disrupting chemical. '  BPA is a 
xenoestrogen which mimics estrogens and is able to 

3
bind estrogen receptors.  Edward Charles Dodd first 
identified the estrogenic characteristics of BPA when 

4
he was in search of an estrogen.  A study on BPA 
reported that humans are exposed to 10μg/day of 

Bisphenol A: A Testicular Teratogen in Developing Rats During Prenatal and 
Early Postnatal Life
Tooba Khurshid, Rehana Rana, Shabana Ali, Huma Beenish, Ali Ahmed, Syeda Sara Bano

Correspondence:
Dr. Tooba Khurshid
Senior lecturer
Department of Anatomy
Islamic International Medical College
Riphah International University, Islamabad
E-mail: khursheedtooba@gmail.com

Department of Anatomy
Islamic International Medical College
Riphah International University, Islamabad

Funding Source: NIL; Conflict of Interest: NIL
Received: October 30, 2019; Revised: January 20, 2021
Accepted: January 28, 2021

Bisphenol A: A Testicular Teratogen in RatsJIIMC 2021 Vol. 16, No.1

19



13.5-16.5), there is migration of primordial germ 
cells towards genital ridge along with sertoli cells 
differentiation, formation of seminiferous cords and 
leydig cells differentiation. Postnatal testicular 
development includes neonatal period (3–7 post 
natal days), early infantile period (8–14 days), late 
infantile period (15–20 days), juvenile period (21–32 

12
days) and peri-pubertal period (32–55 days).  
Oxidative stress induced by BPA during the testes 
development is the key factor in aetiology of male 

11
infertility.  Exposure of pre- implantation embryos 
to BPA impairs testes development and disrupts 

6
testosterone synthesis.  A study of BPA exposed rats 
reve a l e d  a b n o r m a l  s p e r m at i d s ,  i nte rst i t i a l  
congestion, scanty cellular components and empty 
tubular lumina with significant decrease in Leydig 
cell count, germinal epithelium height and tubular 

5
diameter.  
Numerous phenol derivatives were detected in 
water bodies of river Indus in Pakistan. All samples 
we re  a d u l te rate d  w i t h  P h e n o l , 2 , 4 - b i s ( 1 , 1 -

13
dimethylethyl).  To the best of researcher's 
knowledge, in Pakistan where significant quantity of 
BPA is leached into water, such a study was needed to 
be conducted in pregnant rats to see teratogenic role 
of BPA during prenatal and early postnatal life. With 
exposure to BPA, this study could highlight the 
probable reason of increasing male infertility in 
Pakistan by identifying the problem in progression of 
spermatogenesis in rat pups with the help of 
seminiferous tubule differentiation and histological 
scoring.

Materials and Methods 
The experimental study was carried out by 
randomized control trial in animal house of National 
institute of health, Islamabad (NIH) from September 
2018 to September 2019 after approval of Ethics 
Review Committee. Randomization was by simple 
random sampling. Eight weeks old 10 female and 5 
male albino Sprague Dawley rats were kept under 
standard temperature at 22 ± 0.5˚C in stainless steel 
cages in 12-hour light dark cycle with 50% humidity. 
Adult female rats were caged with adult male rats (2 
females/ male). They were given food and water ad 
libidum for 7-days to acclimatize. They mated under 
normal conditions in cage. Female rats with presence 
of vaginal plugs were considered at day 0 of 
pregnancy. Pregnant rats were divided into 2 groups. 

Each group comprised of 5 pregnant rats. One group 
was fed on normal diet and other group was given 
BPA (Table I). Pups were delivered by spontaneous 
vaginal delivery at time of birth. Male rat pups which 
were 21 days old were included in the study. Male 
pups with obvious pathology or male pups older than 
21 days old were rejected. Delivered male pups were 
divided into two groups as in Table I. This study 
constituted a sample size of 30 male rat pups.

Table I: Grouping of Animals (N=30)

After 42 days of experiment, 21 days old male rat 
pups were euthanized and dissected. Testes were 
fixed in Bouin's fixative, stained with Eosin and 
Hematoxylin and examined under X10 and X40 
power of light microscope and parametric data was 
collected by the principal researcher. Microscopic 
parameters included tubule differentiation index (% 
of tubules with more than 3 layers of differentiated 
germinal epithelial cells) and assessment of 
spermatogenesis with modified Johnsen's crirteria 
from 1-7 with 1 being the lowest (absence of 
germinal epithelium and cells) and 7 being the 
highest (presence of rounded spermatids). In 
between 7 and 1 spermatogonia, spermatocytes and 

14
sertoli cells were seen . These two parameters were 
assessed by independent samples T test. The 
parameters of epithelial degeneration, congestion 
and vacuolization were classified according to 
International Harmonization of Toxicologic 

15
Pathology Nomenclature.  These three parameters 
were assessed by Chi square test. The data was 
analysed by SPSS version 21. A p-value of less than 
0.05 was considered significant. 

Results
Tubule differentiation index (TDI) and modified 
Johnsen's criteria were reduced in group B 
significantly as compared to control group A (Figure 
1, Table II). Severe and moderate grades of epithelial 

Bisphenol A: A Testicular Teratogen in RatsJIIMC 2021 Vol. 16, No.1

20



d e g e n e ra t i o n ,  i n t e r s t i t i a l  c o n g e s t i o n  a n d  
vacuolization were seen in rats of group B as 
compared to control group A. (Figure 2, Table III, 
Figure3, Table III, Figure 4, Table III respectively).

worldwide. Almost half of these cases are attributed 
to male partner. A study by Majid et al (2019) 
depicted that oxidative stress due to EDCs is one of 
major mechanisms involved in causing male 

16
infertility.  Programming of masculinization occurs 
during the gestation days 15–18 in laboratory albino 

Discussion
Infertility is defined as failure to conceive after 
unprotected and frequent sexual intercourse for 
more than a year which affects 15% couples 

Fig 1: Figure 19  TDI and Assessment of Spermatogenesis
with Modified Johnsen's Criteria in Testes of Albino Rat 
Showing Score 7 in Control Group A and Score 3 in 
Experimental Group B. (A: Control Group A, B: 
Experimental Group B) (H&E, X40)

Table II: Group Wise Distribu�on of Tubule Differen�a�on

Index (ANOVA) and Assessment of Spermatogenesis with

Modified Johnsen's Criteria (Chi square test) of 

Seminiferous Tubules of Testes among the Control and 

the Experimental Groups in Albino Rats (N=30).

p-value ≤ 0.05

Fig 2: Germinal Epithelial Degenera�on (Severe) in Testes
of Albino Rat (Arrow) Shown in Experimental Group B. 
Negligible Degenera�on is Seen in Control Group A (A: 
Control Group A, B: Experimental Group B) (H&E, X40)

Fig 3: Inters��al Conges�on (Severe) in Testes of Albino 
Rat (Arrow) Shown in Experimental Group B. Negligible 
Conges�on is Seen in Control Group A (A: Control Group 
A, B: Experimental Group B) (H&E, X40)

Fig 4: Vacuoliza�on of Germinal Epithelium (Severe) in 
Testes of Albino Rat (Arrows) Shown in Experimental 
Group B. Negligible Vacuoliza�on is Seen in Control 
Group A (A: Control Group A, B: Experimental Group B) 
(H&E, X40)

Table III: Group wise Distribu�on of Tubular 
Degenera�on, Inters��al Conges�on and Germinal 
Epithelial Vacuoliza�on (Chi Square Test) In Testes of 
Albino Rats among the Control and the Experimental 
Groups

p-value ≤ 0.05

Bisphenol A: A Testicular Teratogen in RatsJIIMC 2021 Vol. 16, No.1

21



rats and in humans between weeks 8–14 of 
17

gestation.  Animal studies have shown that 
pregnant females are at risk from BPA exposure since 

18 19
BPA is a known teratogen as well. '  In humans, BPA 
has been detected in maternal and fetal plasma, 
amniotic fluid, placental tissue and in milk of 

20
lactating mothers.  
In present study, BPA was administered during 
pregnancy and 21 days after delivery during 
lactation. Testicular histogenesis was assessed by 
quantitative and qualitative parameters. BPA 
adversely affected its development owing to its 
teratogenicity. TDI and modified Johnsen's criteria 
have been evaluated in this study both prenatally 
and in early postnatal period to see teratogenic 
potential of BPA and its role in development of 
subsequent male infertility.
Differentiation of spermatogenic epithelium is 
indicated by tubule differentiation index. In present 
study, TDI in group B was significantly reduced as 
compared to control group which was due to 
disruption of germinal epithelial cells lipid 
membranes which occurred by generation of ROS by 
BPA. A study on diabetic rat has shown reduction in 

21
TDI in testes showing tubular damage.
Modified Johnsen's criteria manifests progression of 
stages of spermatogenesis. The reduced score in this 
study with BPA indicated spermatogenic arrest due 
to Leydig cells disruption which caused testosterone 
levels to fall. Since testosterone is principally 
implicated in progression of spermatogenesis so the 
score fell. Along with tubular degeneration there was 
loss of cells of spermatogenic lineage. This has been 
supported by a study in which BPA with dose of 

22
100mg/kg/day was used.  
In a study, BPA has induced mitochondria-mediated 
apoptosis due to activation of caspases in cytosol of 

23
hepatic cells.  In present study group B, significant 
degeneration of germinal epithelium was seen. Loss 
of normal cellular array and architecture of 
spermatogenic lineage was clearly evident in group 
B. As a result, sloughing of germinal epithelium from 
basement membranes occurred due to increased 
movement of smooth muscle cells as a result of 
ongoing inflammation by the released ROS. This 
result is in line with a study in which BPA-induced 
oxidative damage is seen in kidney, testis and liver of 

24 25
rats with degenerative changes. '  

In present study, testicular interstitium showed 
significant congestion in BPA group. This was due to 
ongoing inflammation by free radicals which led to 
recruitment of inflammatory mediators in testicular 
blood vessels. This caused increased blood flow and 
hyperemia by focal dilation of blood vessels evident 

26
as congestion.  This result has been supported by 

27
various studies on rat testes when exposed to BPA.
Vacuolization is due to disturbance of fluid balance in 
spermatogenic and Sertoli cells. Uncharged 
lipophilic bases and phospholipids enter cells by 
diffusion which become positively charged in 
organelles. Accumulation of charged form of      
weak bases increases intraorganellar osmotic 
pressure which attracts water from extracellular 

28
compartment and thus vacuoles appear.  In present 
study, severe vacuolization of spermatogenic cells 
and Sertoli cells was seen in group B rats as compared 
to control group. This result is in accordance with 

29
other studies.
Being a rodent based developmental study it has 
offered constraints in inducing pregnancy in all the 
rats at the same time. Lactation was an important 
event during the current study in which mothers and 
newborn pups showed individual variations. 
This study recommends future investigation           
into the imperative male infertility markers like 
s e m e n  a n a l y s i s ,  s p e r m  m o r p h o l o g y  a n d  
immunohistochemistry. 

Conclusion
The present study indicates that BPA has detrimental 
effects on histology of developing testes owing to its 
teratogenic potential. Thus, spermatogenesis and 
male fertility may be adversely affected by BPA 
especially when exposed to pregnant and nursing 
mothers.

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