Final Thesis PDF

Possible Impacts of PCBs on Female Reproductive
Health
By
Nimra Khan
CIIT/FA19-BSO-054/ISB
Sammar
BS Thesis
In
Biosciences
COMSATS University Islamabad, Pakistan
SPRING 2023
i
COMSATS University Islamabad
Possible Impacts of PCBs on Female Reproductive Health
A Thesis Presented to
In Partial Fulfilment
Of the requirement for the Degree of
BS Biosciences
By
Nimra Khan
Sammar
SPRING 2023
ii
An Undergraduate Thesis submitted to the Department of Bioscience as partial fulfilment of

the requirement for the award of Degree of BS Biosciences.
Name Registration Number
Nimra Khan CIIT/FA19-BSO-054/ISB
Sammar CIIT/FA19-BSO-070/ISB
Supervisor
Dr. Syed Tahir Abbas Shah
Associate Professor
Department of Biosciences
Co-Supervisor
Dr. Saira Amir
Assistant Professor
iii
Final Approval
This thesis titled
By
Nimra Khan
Sammar
Has been approved.

For the COMSATS University Islamabad
External Examiner:
Supervisor:

Co-supervisor:
Dr. Saira Amir
Head of the Department:
Dr. Muhammad Qaiser Fatmi
iv
DECLARATION
We, Nimra Khan (CIIT/FA19-BSO-054/ISB) and Sammar (CIIT/FA19-BSO-070/ISB)

hereby declare that I have produced the work presented in this thesis, during the
scheduled period of study.I also declare that I have not taken any material from any
source except referred to wherever due that amount of plagiarism is within acceptable
range. If a violation of HEC rules on research has occurred in this thesis. I shall be liable
to punishable actionunder the plagiarism rule of HEC.
Date:
Nimra Khan
(CIIT/FA19-BSO-054/ISB)
______________________
Sammar
(CIIT/FA19-BSO-070/ISB)
v
Certificate
It is certified that Nimra Khan CIIT/FA19-BSO-054/ISB and Sammar CIIT/FA19-BSO-070/ISB have
carried out all the work related to this thesis under my supervision at the Department of Biosciences,
COMSATS University, Islamabad.
Date:
Supervisor:

Associate Professor
Department of
Biosciences
Head of the Department:
Dr. Muhammad Qaiser Fatmi

COMSATS University
Islamabad
vi
DEDICATION
Dedicated To My Beloved Parents
Whose prayers, affection and encouragement

made meable to get success.
vii
ACKNOWLEDGEMENT
First of all, we would like to thank Almighty Allah for awarding us strength to meet
our research goals.
With profound gratitude, we would like to thank our worthy supervisor Dr Syed Tahir
Abbas Shah Associate Professor, COMSATS University Islamabad for all the help,
encouragement, and guidance.
We are obligated to our Co-Supervisor Dr Saira Amir Assistant Professor, COMSATS

University Islamabad for providing all the support and help to perform our lab work
and accomplishing research goals in time.
We are highly indebted to our beloved parents, who always sacrificed their own needs
for us. No words are enough to thank them for exceptional support, love,
encouragement and assistance throughout the completion of my degree. We are
thankful to our sisters for their support and encouragement throughout our degree.
Last, we would like to thank our friends Shandana Qasim, Shermeen Syed, Wajiha
Khalil and Ma'am Nazia, a constant source of energy and love.
viii
Abstract
PCBs are organochlorine pesticides that despite being banned are continued to be
detected in organisms and the environment till today. The aim of this study was to
determine the effects that exposure to PCBs has on female reproductive health and how
it influences female infertility. A study was conducted on 52 women (22 fertile and 30
infertile) belonging from urban and rural areas of Punjab (67.3%) and KPK (32.7%),
retrieving hair and urine samples to trace the concentration of the various PCB
congeners to determine their correlation with female sterility. A meta-analysis was
performed prior to the study including 40 research articles from the years 2008-2023
that contained in-vitro as well as epidemiological studies. The analysis revealed that
exposure to low levels of PCBs affected normal endocrine functioning leading to
infertility issues while high exposure to PCBs resulted in decreased production of
estradiol in the body causing reproductive impairment. PCBs were also found to be
associated with ovarian, endometrium, cervical, and vaginal cancer. The research study
found that PCB101 was in the highest concentration in the hair of control and infertile
women while PCB180 was concentrated in elevated amounts in the urine samples of
the participants. Solid- phase microextraction gas chromatography-gas spectrometry
(SPME/GC-MS) was used to monitor the analytes. Results revealed that the diagnosis
namely miscarriages, primary, and secondary infertility had a statistically significant
association with PCB concentrations with P value 0.001. Likewise, the conditions
normal, endometriosis, infertile, no eggs, and PCOS also showed to have a positive
correlation with PCB concentrations indicating that PCBs might be the cause for these
disorders with P value 0.000. Due to the small samples sizes and because most of the
participants were patients suffering from reproductive disorders, the results of the study
cannot be stated to be conclusive. More research is required in this area of study to find
more convincing evidence.
ix
Table of Contents
1. Introduction: ........................................................................................................... 1
1.1. Female Reproductive System: ......................................................................... 6
1.2. Female Infertility:............................................................................................ 8
1.3. PCBS Role in Female Infertility: .................................................................... 9
1.3.1. Endometriosis: ......................................................................................... 9
1.3.2. Female Fecundability: ............................................................................ 10
1.3.3. Implantation Failure: .............................................................................. 11
1.3.4. Intergenerational transmission: .............................................................. 11
1.3.5. Polycystic ovary syndrome: ................................................................... 12
1.3.6. Primary ovarian inefficiency: ................................................................ 12
1.4. PCBs and reproductive track cancers: ........................................................... 13
1.4.1. Ovarian cancer: .................................................................................. 13
1.4.2. Endometrial cancer:............................................................................ 13
1.4.3. Cervical cancer: .................................................................................. 14
1.4.4. Vaginal cancer: ................................................................................... 14
2. Methodology:........................................................................................................ 17
2.1. Meta Analysis:.................................................................................... 18
2.2. Exposure Analysis: ............................................................................. 20
2.2.1. Population ...................................................................................... 20
2.2.2. Reagent and materials .................................................................... 20
2.2.3. Urine sample treatment .................................................................. 20
2.2.4. Hair samples treatment .................................................................. 21
2.2.5. Data analysis .................................................................................. 21
3. Results: ................................................................................................................. 22
3.1. META ANALYSIS ...................................................................... 23
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3.1.1. PCBs and female infertility: ................................................... 23
3.1.1.1. Endometriosis: ................................................................. 23
3.1.1.2. Female Fecundability:...................................................... 25
3.1.1.3. Implantation Failure:........................................................ 26
3.1.1.4. Polycystic ovary syndrome: ............................................. 26
3.1.1.5. Primary ovarian inefficiency ........................................... 27
3.1.1.6. Intergenerational Transfer................................................ 27
3.1.2. PCBs and Reproductive Track Cancers: ................................ 30
3.2. Exposure Analysis: ....................................................................... 36
4. Discussion ............................................................................................................. 55
5. Conclusion: ........................................................................................................... 65
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Table of Figures
Figure 1 structure of PCB (Hens & Hens, 2017) .......................................................... 4

Figure 2 Female Reproductive System (Australia, 2022) .............................................. 8
Figure 3 PRISMA Flowchart ....................................................................................... 19
Figure 4 PCBs and Infertility.. .................................................................................... 29
Figure 5 PCB and Reproductive Tract Cancers. ........................................................ 34
Figure 6 PCBs concentration in Hair samples ............................................................. 44
Figure 7 concentration of PCBs in Hair samples ......................................................... 45
Figure 8 Concentration of PCB 28 in control and infertile .......................................... 45
Figure 9 Concentration of PCB 52 in control and infertile .......................................... 46
Figure 10 Concentration of PCB 138 in control and infertile ...................................... 46
Figure 15 PCBs concentration in Urine samples ......................................................... 50
Figure 16 Concentration of PCBs in urine................................................................... 50
Figure 17 Concentration of PCB 28 in control and infertile ........................................ 51
Figure 18 Concentration of PCB 52 in control and infertile ........................................ 51
xii
List of Tables
Table 1 PCBs involved in Infertility and Reproductive Cancers ................................. 15

Table 2 PCB Affected Genes ...................................................................................... 35
Table 3 Demographic Data .......................................................................................... 37
Table 4 Demographic Data of control and infertile patients........................................ 38
Table 5 Lifestyle Factor ............................................................................................... 39
Table 6 Menstrual Conditions...................................................................................... 41
Table 7 Analyte exposure in Hair sample .................................................................... 43
Table 8 Analyte exposure in Urine samples ................................................................ 49
xiii
Chapter 1
Introduction
1
Endocrine disrupting chemicals (EDCs) are exogeneous agents that disturb the
synthesis, metabolism, storage, release, binding action, elimination and
transport of hormones and interfere with the development, homeostasis and
reproduction. EDCs are present in toys, detergents, air pollution, pesticides
plastic food containers, plastic bottles, toothpastes and cosmetics. We are
exposed to EDCs by food, inhalation and dermal contact. After exposure EDCs
are metabolized by liver, intestine and kidney. After exposure some of EDCs
get eliminated out of body by bile and urine (Aydemir & Ulusu, 2023). EDCs
can be natural or synthetic chemicals like bisphenols, phthalates, aromatic
hydrocarbons (PHAs), perfluorochemicals (PFCs), polychlorinated biphenyls
(PCBs), polybrominated diphenyl ethers, pesticides and dioxins (Aydemir &
Ulusu, 2023). EDCs bind binds with thyroid receptors and endocrine receptors
and alter hormone synthesis and binds with nuclear receptors like estrogen and
androgen receptors and affects reproductive system (A. Ghosh, Tripathy, &
Ghosh, 2022). EDCs are associated with infertility, polycystic ovarian
syndrome (PCOS), primary ovarian inefficiency (POI), endometriosis, obesity,
impaired follicle formation, premature birth, diabetes and uterine fibrosis
(Smith, Pfeifer, & Collins, 2003). Dysregulation of female reproductive system
leads to early menopause, dementia, reproductive dysfunction, cardiovascular
disease and mortality(Aydemir & Ulusu, 2023).
Polychlorinated biphenyl ethers (PCBs) are organochlorine aromatic

compounds. PCBs were discovered over 100 years ago. Their use in industrial
and commercial products began in 1929. Because of their exceptional properties
they get widespread use in a short period of time. Schools, offices, factories
replace other insulating fluids with PCBs to reduce the risk of fires. Many cities,
insurance companies and industries demanded capacitors and transformers that
are only made up of PCBs. One of the reasons for their high demand was also
their low cost (Ross, 2004). For many years PCBs were widely used in industrial
and commercial applications (Ermler & Kortenkamp, 2022) such as such as in
capacitors and transformers in the form of dielectric fluids, plastics, adhesives,
varnishes, newsprints, fluorescents lights, carbon papers, printing inks, paints,
insulators, and hydraulic fluids. They have flame-retardant properties and low
electric conductivity(Montano et al., 2022). About 1.1 billion pounds of PCBs
2
were manufactured in united states between 1929 and 1977. In early 1938 health
effects were reported due to exposure to PCBs. Animal studies also began to
show an association between different congeners of PCBs and health effects. In
1968, ‘Yusho’ a massive food poisoning tragedy occurred in western Japan
including 1291 people by consuming rice oil contaminated with PCBs
(Erickson, 1997). So, in 1979 the production of PCB was banned by US EPA.
PCBs are classified as Persistent Organic Pollutants (POPs). POPs are
chemicals with long half-life in environment, long transport range, toxicity
persistence and bioaccumulation (Ermler & Kortenkamp, 2022). PCBs are
composed of 209 different compounds consisting of two connected benzene
rings and 1–10 chlorine atoms (Figure 1 structure of PCB) called congeners.
Each PCB congeners/metabolites nomenclature is based on their structure
according to the location and number of chlorine atoms in the biphenyl ring.
Originally, they were numbered by Ballschmiter and coworkers. Ballschmiter
system assign named to the congeners in ascending order starting
monochlorinated metabolites and ending at PCB 209 that is dechlorinated
congener (Grimm et al., 2015). Later, each congener is assigned number
between 1 to 209 according to International Union of Pure and Applied
Chemistry (IUPAC) nomenclature (Mills Iii, Thal, & Barney, 2007). Different
congeners have different properties, toxicity level, behaviour in different
environment (Montano et al., 2022). PCBs have different physical nature they
can range from waxy solid to oily liquids. They have a long half-life so are
difficult to degrade from the environment, they are resistant to oxidation, to
different temperatures and hydrolysis, that why considered as stable molecules.
Physical and chemical properties of PCBs depend upon the number of chlorine
atom present (Hens & Hens, 2017). Congeners are grouped into two forms;
high-chlorinated PCBs (HC-PCB) are more persistent and have more chlorine
atom present in their structure, they are more lipophilic and have high melting
point and less soluble in water. Because of their lipophilic property they are
more soluble in lipids, oils and fats. Whereas, low-chlorinated PCBs (LC-PCB)
which are less persistent and more volatile and have no or less number of
chlorine atoms in their structure. (Casey, Bush, & Carpenter, 2022). The 209
congeners of PCBs are classified as “coplanar and non-coplanar” based on the
position of chorine atom on the ring that is non-ortho(coplanar) or ortho position
3
(non-coplanar). This position decides the toxicity of the PCB due to their
difference in interaction with the aryl hydrocarbon receptor (AhR). Out of them
12 (PCB 77,81,105,114,118,123,126,156,157,167,169 and 189) have somewhat
similar chemical and physical properties like dioxins and furans and are
therefore called dioxin like PCBs (DL-PCBs) or coplanar PCBs (Jin et al.,
2020). Dioxin like PCBs are define as PCB congeners that consist of 4 or more
with only one or none substitution in their ortho position (Reddy,
Moniruzzaman, & Aminabhavi, 2019).
Figure 1 structure of PCB (Hens & Hens, 2017)
Exposure
Most of the PCBs are believed to be present in the environment because of their
long half-life and lipophilic nature. Most believe that they are present in the
environment because of the leakage that occurred from the capacitors and
transformers in which they were used (Reddy et al., 2019). Their presence in
the environment is the source of exposure to humans and animals. As they are
stable molecules, resistant to oxidation, biodegradation and reduction they can
enter particularly in any ecosystem i.e., air, water, land rivers and reside there
for many years (Montano et al., 2022). Their high solubility in lipids, oils and
fats allow them to enter animal and human tissues and become part of the food
chain. Hence the major source of exposure to PCBs is through diet. e.g. fish,
milk and dairy products (Ermler & Kortenkamp, 2022). In 1978, the daily intake
of PCB was estimated in industrial countries which was more than daily intake
estimated in 1991. Humans are also exposed to PCBs through inhalation, dermal
contact, breast milk and skin absorption. City air and the air in the buildings that
4
were constructed using PCB is another source of exposure that is usually
overlooked. PCBs involved in food products are highly chlorinated and more
persistent while PCB in air are less chlorinated and more volatile. As LC-PCB
has low potential of resistance to biodegradation, humans are more exposed to
HC-PCB through food, especially fish. Many studies are performed that
explained the association between high level of PCB in serum and consumption
of contaminated fish (Grimm et al., 2015).Lipophilic nature of PCBs allow them
to accumulate in adipose tissues and fats so PCBs accumulated in breast milks
fat of contaminated mother can be the source of exposure to the infants
(Mustieles & Arrebola, 2020). They have ability to cross placental barrier thus
causing neurodevelopmental toxicity, difficulty in learning, intellectual and
behavioural impairment in children when exposed during prenatal and perinatal
periods (Wang et al., 2019). Some longitudinal studies also demonstrated that
exposure to PCBs can cause mutagenic, teratogenic, and carcinogenic disorders
(Hens & Hens, 2017). Some studies found that PCBs exposure are associated
with hormonal imbalance that’s why are endocrine disruptors (Grimm et al.,
2015).
Effects
PCBs have negative and estrogenic effects on humans and animals (Sifakis,
Androutsopoulos, Tsatsakis, & Spandidos, 2017). Normal body endocrine
functions and normal body pathways are disrupted at epigenetic levels by
PCBs(Del Pup et al., 2015), as they can accumulate in body and bind with
various receptors (He, Zhang, & Liu, 2021). High chlorinated PCBs can cause
obesity, hyperlipidaemia, cardiovascular diseases (Aminov & Carpenter, 2020)
and low chlorinated PCBs can cause diabetes, infertility, reproductive cancers,
abnormal ovulation and menstruation (Aminov, Haase, & Carpenter, 2016) as
shown in (Table 1). Some studies also show that PCBs exposure during prenatal
and perinatal period can also affects the normal development of children (Wang
et al., 2019). In males’ negative correlation has been reported between semen
parameters and PCBs (Amir et al., 2021). Studies also showed that high level
of PCBs in women can affect menstrual cycle, longer time to pregnancy (Mrema
et al., 2013), low biomarker of ovarian reserves, abortions, sex ratio changes in
offspring, low fecundability (Han et al., 2016a). Diseases such as uterine
5
fibroids, endometriosis (Vassilopoulou et al., 2019) and polycystic ovarian
syndrome (PCOS) (Del Pup et al., 2016) have been reported to be caused by
PCBs (De Coster & Van Larebeke, 2012).
Mode of action
PCBs are persistent organic pollutants that remain in the body for longer period
of time. PCBs having one or no chlorine at their ortho position are called
coplanar PCBs and PCBs with two or more chlorines at ortho position are
termed as non-coplanar PCBs (Carpenter, 2006). AhRs are transcriptional
regulatory proteins that regulate the expression of genes. PCBs can mimic aryl
hydrocarbons and binds to AhRs and leads to adverse biological outcomes
including fetotoxicity, cancer, immune deficiencies and reproductive toxicities
(Abbott et al., 1999). Non-coplanar PCBs resemble estradiol 17- β (E2) hence
binding with estrogen receptors causes anti-estrogenic effects and multiple
reproductive abnormalities. They can also activates other receptor such as
constitutive androstane receptor, a xenobiotic receptor(Jin et al., 2020). It also
causes reduction in cytosolic estrogenic levels by regulating the transcription of
large number of dioxin responsive genes (Petrakis et al., 2017). In various
studies and experiments PCBs and their metabolites have been reported to cause
immunotoxin, hepatotoxic, cytotoxic, neurotoxic and nephrotoxic effects
(Yilmaz, Terekeci, Sandal, & Kelestimur, 2020).Toxicity of PCB is totally
dependent on its metabolism process. Arenes are electrophilic metabolic
intermediates that react with proteins, lipids and DNA and harm them. OH-
PCBs are oxidized to quinones, highly reactive species that may form covalent
bonds with DNA, proteins and other endogenous compounds. OH-PCBs, PCB
sulfates, and PCB methyl sulfones might equally persist as parent congeners
and has their own toxicities(Grimm et al., 2015)
1.1. Female Reproductive System:
Female Reproductive system: female reproductive system consists of vagina,

cervix, uterus, ovaries and fallopian tubes as shown in (Figure 2). The vagina is
muscular canal that connects uterus to the outside of body. lining mucous
membrane keeps it moist. During delivery, the vagina expands to accommodate
6
a baby. Cervix is the lowest part of uterus. it is opening that allows entry of
sperm and menstrual blood to exit. During vaginal birth, cervix dilates and
allows the baby to come out. The uterus is a hollow, pear-shaped organ for
holding a baby during pregnancy. The uterus has two parts: the corpus and the
cervix. Corpus expands during pregnancy. Ovaries are oval-shaped, small
glands located on either side of uterus and responsible for estrogen,
progesterone and egg production. Fallopian tubes are narrow tubes that allow
the attachment of ovaries and uterus. it allows egg to travel from ovaries to your
uterus. Fertilization occurs in fallopian tubes and egg implants in uterine lining
(Hayden, Barlow, & pharmacology, 1972). Eggs are produced in ovaries. These
eggs are transferred to fallopian tube where fertilization may take place.
Fertilized egg is moved to uterus. uterine lining is thickened by hormones of
menstrual cycle. Fertilized egg implants in uterine lining. If implantation
doesn’t occur, uterine lining is shed and leads to menstrual bleeding. Moreover,
female reproductive system produces sex hormones. Inhibition of female sex
hormones leads to menstrual pause or menopause (S. Yao, Lopez-Tello, &
Sferruzzi-Perri, 2021)
7
Figure 2 Female Reproductive System (Australia, 2022)
1.2. Female Infertility
Failure to conceive, after one year of regular intercourse with no contraception

is reproductive disorder called female infertility (Deliamiezade, 2019). PCBs
are endocrine disruptors that interfere with normal body functioning and cause
multiple health effects including infertility (Kuriyama & Chahoud, 2004).
Female exposed to PCBs has shorter menstrual cycle, reduced fecundability,
PCOS, POI and long time to pregnancy. Female exposed to PCBS during
pregnancy can transfer these chemicals through placenta and by breastfeeding
to foetus and cause multiple reproductive abnormalities. (Kuriyama &
Chahoud, 2004). One out of every six or 8-12% of reproductive aged women
are suffering from infertility. 2-6% of couples are using Assisted Reproductive
Technology for conceiving.
8
1.3.PCBS Role in Female Infertility
PCBs are Persistent Organic Pollutants (POPs) that have a very long half-life.
Humans are exposed to PCBs by contaminated food, environment, and indoor
exposure. Despite being banned some PCBs are still in use. PCBs
bioaccumulate in animal fats leading to biomagnification (Fujimoto & Bloom,
2015). PCBs are present in hair, placenta, amnion, breastmilk, follicular fluid,
fetus, embryo and uterus. PCBs are associated by infertility, endometriosis,
fecundability, POI, PCOS and implantation failure (Meeker et al., 2011).
Other studies revealed that PCBs causes reduced oocytes maturation,
decreased proliferation of embryo cells, blastocyst formation and
development, decreased IVF rates, increased embryo degeneration,
implantation failure and decreased litter production (Meeker et al., 2011) (Han
et al., 2016a). PCBs effect menstrual cycle phase length, stillbirth,
spontaneous abortion rates and ability to conceive by variety of cellular
mechanisms (Han et al., 2016a). Lower concentrations of PCBs affect normal
endocrine functioning and cause infertility problems (Smith et al., 2003)
1.3.1. Endometriosis
Endometriosis is multifactorial gynaecological disorder defined as the ectopic

presence of stromal cells and functioning endometrial glands (Vichi et al.,
2012). Endometriosis is associated with infertility, internal bleeding, scarring,
dyspareunia, and pelvic pain. Endometriosis is multifactorial disorder that may
contribute to alter growth and immune response and imbalance of sex
hormones. Endometriosis causes inflammatory response in peritoneal cavity
with production of reactive oxygen species (ROS) which increases the
adhesion of endometrial cells in peritoneum. Endometrial progression leads to
infertility, hormonal imbalance, failure of immune response and genetic
predisposition (Vichi et al., 2012). 8-10% of every reproductive aged woman
is suffering from endometriosis. Endometriosis develops in reproductive-age
women and slip back after menopause, suggesting that it has estrogen
dependent growth (Trabert et al., 2010a). Many genetic and environmental
toxins like PCBs, polychlorinated-dibenzo-furans (PCDFs), polychlorinated-
9
dibenzo-dioxins (PCDDs) and tetrachlorodibenzo-p-dioxin (TCDD) are
associated with endometriosis (M. Yao et al., 2017). Non-co-planer PCB
congeners has two or more chlorine atoms at ortho position of biphenyl ring
and has strong affinity to Aryl Hydrocarbon Receptors (AhRs). Exposure to
non-coplanar PCBs causes neurological, neurotoxic, endocrine and
carcinogenic changes in body by binding to AhRs (Pauwels et al., 2001)
1.3.2. Female Fecundability
Female Fecundability is probability of conceiving during a menstrual cycle,

for the couple engaging in regular unprotected intercourse (Cohn et al., 2011).
It is also known as Time-To-Pregnancy (TTP) and depends on both male and
female by (Yland et al., 2020). A study reveals that women with high exposure
to PCBs has higher PCB serum level and increased TTP compared to the
women with lowest exposure. Two epidemiological studies conducted on
Swedish Women and New York state women showed contrasting results.
Swedish women consumed PCB enriched fish from Baltic Sea showed no
significant association between PCBs and TTP. While the study conducted on
New York state women consuming Ontario fish showed positive and dose
dependent association between PCBs and TTP. Hence TTP increases with
increase in PCBs exposure. In 1979, Taiwan mass poisoning of PCB
contaminated cooking oil incident leads to gum hyperpigmentation, chloracne,
hyperkeratosis, meibomian gland inflammation and nail discoloration. The
exposed women have irregular menstrual cycle, increased stillbirth, and higher
prevalence of anemia. Intergenerational transmission by lactational and in
utero exposure causes irregular and short menstrual cycle, elevated estradiol
level in serum and elevated level of follicular stimulating hormone in progeny
(C.-Y. Yang, Wang, Chen, Tsai, & Guo, 2008). Another study reveals that
dioxin like anti-estrogenic PCBs are associated with longer TTP and non-
dioxin like anti estrogenic PCBs is associated with shorted TTP (Han et al.,
2016a).
10
1.3.3. Implantation Failure
A competent embryo must attach to a receptive endometrial lining for

successful pregnancy. When blastocyst finally infiltrating the decidua,
implantation is considered to occur(Qu, Ming-Zhang, Yuan-Fang, Wang, &
Zhang, 2018). Embryo implantation is a hormonally regulated process, and it
requires receptive endometrium and good quality blastocyst. Inadequate
endometrium receptivity leads to two thirds of implantation failure. Pinopodes
are bleb like protrusions which grow in the surface of endometrium for limited
period, synchronized with embryo implantation. Pinopodes are markers of
implantation and absence of pinopodes indicates implantation failure.
Moreover, estradiol and progesterone are responsible for cell proliferation and
endometrium development. Deficiency of estrogen is associated with PCOS,
higher abortion rate and low chance of pregnancy (Qu, Ming-Zhang, et al.,
2018). No statistically significant association was found between PCBs
exposure and spontaneous abortion (Meeker et al., 2011).
1.3.4. Intergenerational transmission
PCBs are highly persistent organic pollutants that accumulate in human fats,
adipose tissue and breast milk and can cross placenta. Thus, mammalian
offspring are exposed to high concentration of PCBs during prenatal
development and breast feeding. A fetus is highly sensitive and can result in
permanent functional and structural abnormalities(Pocar et al., 2012).
PCB exposure in perinatal stage causes many developmental, reproductive,

and physiological effects in daughters. AhR are transcriptional regulatory
proteins. PCBs can bind with AhR and cause adverse biological and
reproductive outcomes like cancer, reproductive toxicity, immune deficiencies
and fetotoxicity. on eight-year-old children estimated that high levels of PCBs
can decrease the concentration of oestradiol E2 in children than exposure to
low level of PCBs and impaired reproductive development. Low fundus and
uteri length were observed in girls with high level exposure.
11
1.3.5. Polycystic ovary syndrome
Polycystic ovary syndrome is hyperandrogenism and insulin resistance. It is

an endocrine metabolic disorder affects 6-10% of women in childbearing age
(Vagi et al., 2014). Hyperandrogenism causes fertility dysregulations and
leads to acne, chronic ovulation alopecia and hirsutism PCOS is metabolic
disorder associated with obesity, dyslipidemia, insulin resistance and
increased risk of Type II diabetes mellitus and cardiovascular abnormalities.
PCOS is caused by both environmental and genetic factors (Q. Yang et al.,
2015). PCOS has a strong impact on quality of life and fertility (Tan,
Ignatenko, Wagner, Dokras, Seufert, Zwanziger, Dunschen, Zakaria,
Huseinovic, & Basson, 2021).
1.3.6. Primary ovarian inefficiency
Primary ovarian inefficiency is a gynaecological disorder characterized by loss

of ovarian activity and loss of primordial ovarian follicles before the age of
40. 1-4% of every woman is suffering from POI. POI is also referred to as
early menopause. POI is associated with increased level of follicle-
stimulating hormone (FSH) and luteinizing hormone (LH) and decreased level
of estrogen and anti-Mullerian hormone (AMH). POI patients are recognized
to be at increased risk for osteoporosis, mental impairment, cardiovascular
disease and mortality. POI is caused by genetic and environmental agents,
POPs like PCBs are positively associated with POI and these endocrine
disruptors resist ovarian follicular development, reduced oocyte formation,
depletion or primordial follicle pool, alteration in reproductive hormonal
levels and induce symptoms like POI in women (Pan et al., 2019). Anti-
Müllerian hormone (AMH) is involved in regulation of primordial follicle
growth. AMH is produced in granulosa cells of ovary. In the absence of AMH
follicles are recruited rapidly and are more sensitive to FSH. Thus, the absence
of AMH causes early cessation of menstrual cycle and prematurely exhausted
follicle pool (Qin et al., 2014).
12
1.4.PCBs and reproductive track cancers
PCB is one of the endocrine disrupting chemicals (EDC) and associated with
reproductive cancers (Table 1). Each year around 35,000 women between the
age of 15 and 39 are detected with cancer (Coccia et al., 2014). Reproductive
track cancers include endometrial, ovarian, vaginal, fallopian, cervical cancers.
They are also known as gynecological cancers (Gibson & Saunders, 2014).
Gynecological malignancies are the most common cancers in women and girls
worldwide and rated as the top 4 to occur in women. Endometrial cancer is the
most common cancer of all, following ovarian, then cervical and vaginal cancer,
the least common is fallopian cancer (Gibson & Saunders, 2014). However in
case of deaths, ovarian cancer causes more deaths than endometrial, vaginal and
cervical cancers (Morgan, Deoraj, Felty, Yoo, & Roy, 2016). About 15% to
50% of the association is found between endometriosis and endometrioid
ovarian tumors. Females having endometriosis have two-to-three-fold increased
risk of ovarian cancer (Herreros-Villanueva, Chen, Tsai, & Er, 2019).
1.4.1. Ovarian cancer
Most lethal of all gynecological cancers is ovarian cancer (OvCa). Due to our
failure or inability to detect the onset of ovarian cancer in females the mortality
rate is quite high. So, when females are detected with ovarian cancer they are at
advance stage of disease. Treatment for OvCa cancer includes cytoreductive
surgery and adjuvant platinum-based chemotherapy. In some studies, it is also
suggested that if adjuvant platinum-based chemotherapy is done before
cytoreductive surgery the rate of reoccurrence of the disease can be reduced (Al-
Alem et al., 2019).
1.4.2. Endometrial cancer
Endometrial cancer (EC) is defined as a tumor in endometrium (Morice, Leary,

Creutzberg, Abu-Rustum, & Darai, 2016). It is the sixth most common cancer
of all the gynecological cancers(Lu & Broaddus, 2020). Endometrial cancer
occurs in women mostly after menopause. Each year, about 42000 women dies
from this cancer and 142000 women develop endometrial cancer (Ryan, Susil,
13
Jobling, Oehler, & research, 2005). PCBs are one of the causes of EC. Many
studies showed that PCBs are associated with increased risk of EC. A case
control study was performed by Reich et al that PCB and other EDCs were
found in abdominal adipose tissue of two cases of endometrial stromal sarcoma
(Reich, Regauer, & Scharf, 2010)
1.4.3. Cervical cancer
The fourth most common of all gynecological cancer is cervical cancer.

Approximately half million women are diagnosed with cervical cancer each
year. Main cause of cervical cancer is high risk subtype of human papilloma
virus (HPV) (Cohen, Jhingran, Oaknin, & Denny, 2019). Studies showed that
PCB 118, 126 and 153 are associated with cervical cancer (Zhang, Song, Li,
& methods, 2019)
1.4.4. Vaginal cancer
Vaginal cancer is a rare gynecological cancer, approximately 3% of all cancers.

Mostly this cancer occurs in postmenopausal women but infection with human
papillomavirus (HPV) can increase the risk of vaginal cancer in young women
(Rajaram, Maheshwari, Srivastava, obstetrics, & gynaecology, 2015).
14
Table 1 PCBs involved in Infertility and Reproductive Cancers
Sr.no PCBs Reproductive References PCBS Infertility References

Track
Cancers
1 PCB 78 Ovarian (Morgan et PCB180, Endometriosis (Vichi et

PCB 118 cancer al., 2016) PCB118, al., 2012)
PCB 188 PCB153, (Trabert et
PCB138, al., 2010a)
PCB170
2 PCB 153 Uterine (Morgan et PCB138/ Primary (Murati et

PCB 118 cancer al., 2016) 158, Ovarian al., 2015)
PCB 138 PCB153, Inefficiency
PCB180 (POI)
3 PCB 138 Cervical (Zhang, PCB153 Implantation

PCB cancer Song, & Li, Failure
118, 2019)
PCB 126
PCB 153
15
4 PCB 188 Endometri (Chen et PCB 153 Uterine (Yoshizaw
PCB 126 al cancer al., 2015) inflammation a et al.,
PCB 135 (Caserta, 2009)
De Marco,
Besharat, &
Costanzi,
2022)
1.3. Aims and Objectives
The aim of the study was to determine the impact of PCBs on female infertility
living in agriculture areas of Punjab and KPK and association between
demographic factors, lifeastyle factors and menstrutaion status with female
infertility.
16
Chapter 2
Methodology
17
2.1. Meta Analysis
We systematically reviewed the experimental and epidemiological studies of
past 15 years (2008-2023) on humans, mice, turtle, and other animals to study
the associated between PCBs, female reproductive track cancers and female
infertility on multiple electronic databases like Google Scholar and PubMed.
The search terms like “Polychlorinated Biphenyls/PCBs”, “PCBs and Female
infertility” and “PCBs and female reproductive track cancers” were used to
retrieve the relevant publications. We primarily focused on the research articles
containing epidemiological information on human female and experimental
animal data, including mechanistic and molecular information. After careful
screening about 1830 duplicate articles were excluded. All the articles before
the timeline were discarded. Approximately 163 irrelevant, insufficient, non-
original research, lack of direct evidence and different language publications
were discarded. After full-text assessment only 232 articles remained. We didn’t
omit any article based on the negative or positive findings. Nearly 40 articles
were reviewed following the 2020 Preferred Reporting Items for Systematic
Review and Meta Analysis Protocols (PRISMA) statement (Figure 3).
18
Figure 3 PRISMA Flowchart
19
2.2. Exposure Analysis
The present work was carried out in accordance with The Code of Ethics of the
World Medical Association (Declaration of Helsinki) for experiments involving
humans http://www.wma.net/e/policy/b3.htm and the Uniform Requirements
for manuscripts submitted to Biomedical journals
http://www.nejm.org/general/text/requirements/1.htm. The study was approved
by the Ethical review board, COMSATS University Islamabad and written
informed consent was obtained by all participants.
2.2.1. Population
Infertile females (who were unable to conceive for over 12 months), inhabitant
of two main areas of Pakistan including Chakwal (Punjab) and Dera Ismail
Khan (KPK) participated in the research study. Predesigned questionnaires
were used to collect complete information of family history and personal health
after taking informed consent of all the participants. Hair and urine samples
were collected for POPs monitoring. Females were categorized into cycle
irregularities, diagnosed infertility.
2.2.2. Reagent and materials

Seven Congeners of PCB mentioned in Stockholm convention including PCBs
(28, 52, 101, 118, 138, 153 and 180) were used as an indicator to monitor the
exposure of PCB and association between PCBs and infertility. The PCB
congeners were obtained from Fluke Analytical-Sigma-Aldrich (Sigma-
Aldrich, St. Louis, USA).
2.2.3. Urine sample treatment

Pooled urine samples were used which contain no detectable PCBs. For
analysis, urine samples were transferred to Toxicology laboratory, University
of Crete, Greece after samples were stored at -20 Celsius. To remove all the
solid particles samples were thawed at room temperature and centrifuged at
4000 rmp for 5 minutes. Approximately, 3ml of urine in addition with 0.3 g of
NaCl and 10 ng of TCN were added to SPME vials and placed in GC-MS tray.
The spiking levels of PCBs were 0, 0.0025, 0.005, 0.01, 0.025, 0.05 ng/Ml.
20
2.2.4. Hair samples treatment
For head hair sample collection sterilized scissors were used to cut the near
roots. Samples were stored in paper envelopes. Until analysis they were kept in
a dark room at room temperature. N- hexane was used at concentration of 100
ppm to prepare all the stock solution. Mixed solution of PCBs at different
concentrations were prepared by using stock solutions for working standard
solutions and spiked samples. All stocks and working solutions were stored at
Pooled human hair samples with detected levels below the limit of
quantification values were spiked with all target analytes at concentration levels
0, 1, 2.5, 5 and 20 ng/mg for PCBs. Hair weight 100 mg was cut into small
pieces. Distilled water was used to wash hair for 3-4 minutes. Then was rinsed
with n-hexane/dichloromethane. Sample was transferred to SPME vials after
drying at 40 Celsius where 1ml of nanopure H2O, 0.3 g of NaCl and 10 ng of
TCN (internal control) and 2 mL of 10N NaOH were added (Barmpas et al.,
2020; Tzatzarakis et al., 2014). The peaks were observed with respective
retention time and area was calculated for each observed peak after the analysis
was completed. Before all the analysis standards and spiked samples were run
five times with blank samples to check the normal functioning of the system.
2.2.5. Data analysis

The area of the observed peaks was calculated for each ion peak against
retention time. Microsoft Excel (Microsoft Corporation, USA) was used to
calculate the concentration of all ions using the line formula y = ax + b. For
further analysis, IBM Corp. Released 2017. IBM SPSS Statistics for Windows.
Version 25.0. Armnonk, NY: IBM Corp. was used. ANOVA, bivariate
correlation, linear regression, and Pearson’s chi square was run to analyze the
relationship between various factors. Graphs were drawn using IBM Corp.
Released 2017. IBM SPSS Statistics for Windows. Version 25.0. Armnonk,
NY: IBM Corp. most recent version of SPSS; Microsoft Excel (Microsoft
Corporation, USA); and Origin (Pro), Version 2019. Origin Lab Corp.,
Northampton, MA, USA. P- value below 0.050 was considered statistically
significant.
21
Chapter 3
Results
22
3.1. Meta analysis
3.1.1. PCBs and female infertility
Table , Failure to conceive, after one year of regular intercourse with no

contraception is reproductive disorder called female infertility (Deliamiezade,
2019). PCBs are endocrine disruptors that interfere with normal body
functioning and gene regulations (Table 2) causing multiple health effects
including infertility (Kuriyama & Chahoud, 2004). Polychlorinated biphenyls
(PCBS) are synthetic chemicals and flame retardant used in electric equipment.
Production of PCBS was banned in 1977 but they still exist in the environment
due to their persistent nature and accumulate in human body due to their
lipophilicity and long half-life(Williams et al., 2021). Polychlorinated
biphenyls are endocrine disruptors that interfere with normal body functioning
and cause multiple health effects including infertility. Female exposed to PCBs
has shorter menstrual cycle, reduced fecundability, PCOS, POI and long time
to pregnancy as shown in (Figure 4). Female exposed to PCBS during
pregnancy can transfer these chemicals through placenta and by breastfeeding
to foetus and cause multiple reproductive abnormalities. (Kuriyama &
Chahoud, 2004)
3.1.1.1. Endometriosis
A multifactorial gynaecological disorder defined as the ectopic presence of

stromal cells and functioning endometrial glands (Vichi et al., 2012). Most
common symptoms include heavy internal bleeding, scarring, inflammation,
painful cramps, painful intercourse and infertility. endometriosis develops in
reproductive-age women and slip back after menopause, suggesting that it has
estrogen dependent growth (Trabert et al., 2010b). Endometriosis develops in
reproductive-age women and slip back after menopause, suggesting that it has
estrogen dependent growth (Trabert et al., 2010a). There are two in vivo studies
conducted on mice to identify the impact of PCBs on endometriosis. Exposure
to PCB126, a congener of PCB both in-vivo and in vitro effects the estrogenic
biosynthesis and inflammatory response in mice. Dioxin like PCBs are more
associated with endometriosis than non-dioxin like PCBs according to studies.
23
PCB126 not only stimulates the E2 biogenesis but also increases interleukin- 6
and interleukin-8 secretions. In some studies, it is also reported that CB126
stimulates the transition of testosterone in oestradiol. LXA4 is an endogenous
anti-inflammatory mediator in the endometrium. It can inhibit the development
of endometriosis but PCB126 expose decreases levels of LXA4 means
development of inflammation. Both E2 biogenesis and inflammation leads to
the development of endometriosis as reported by Huang et al (Huang et al.,
2017). PCB-118 results in open uterine lumen, abnormal endometriosis, poorly
developed pinopodes and compact stromal cells. PCB-118 exposure causes
hypermethylation in promoter regions and impairs endometrial receptivity and
causes the failure of embryo implantation by interfering with the expressions of
HOXA10 and ITGB3 reported by Qu et al (Qu, Wang, & Zhang, 2018).
Multiple epidemiological studies were conducted to identify the association
between PCBs and endometriosis while few epidemiological studies showed
contrasting results. A case-control study conducted on 343 Italian women, 181
cases and 162 controls to study the impact of polychlorinated biphenyls on
glutathione transferase polymorphisms (GSTM1, GSTT1, GSTP1, and GSTA1)
associated with the risk of endometriosis. This study reveals that GSTT1 is
negatively associated with endometriosis while the GSTP1 wild-type genotype,
medium-high blood level of PCB-153 and high level of PCB-180 increases the
risk of endometriosis. (Vichi et al., 2012).
While another epidemiological case-control study conducted on western
Washington state women. In this study 251 newly diagnosed cases between
1996-2001 were recruited and this study reveals no significant association
between endometriosis, PCBs, and estrogenic PCBs. Negative-association was
observed between endometriosis and PCB-170, PCB-201 and PCB-196. The
differences in the results of two studies might be due to the geographic
variability in PCBs concentration (Trabert et al., 2010a). PCBs, phthalates and
dioxins were the main suspected EDCs in endometriosis and uterine fibroids
leading towards female infertility reported by Le Moal et al (Le Moal et al.,
2016). In 2013 French Institute for Public Health Surveillance (InVS) organized
a workshop in which human reproductive health and general environment
network (HURGENT) was founded for forming a multi-country monitoring
system for reproductive health indicators. Out of many indicators’ PCBs,
24
phthalates and dioxins were the main suspected EDCs in endometriosis and
uterine fibroids leading towards female infertility reported by Le Moal et al (Le
Moal et al., 2016).
3.1.1.2. Female Fecundability

Female fecundability is probability of conceiving during a menstrual cycle, for
the couple engaging in regular unprotected intercourse(Cohn et al., 2011). It is
also known as time-to-pregnancy and depends on both male and female by
(Yland et al., 2020). Four epidemiological studies were conducted to analyze
the association between maternal serum PCBs concentration and daughter’s
fecundability. Polychlorinated biphenyls are endocrine disruptors that affect
oocyte and reproductive development with reduced female fecundability in
offspring of PCB exposed mothers (Han et al., 2016a). An epidemiological
study was conducted to analyze the association between maternal serum PCBs
concentration and time to pregnancy in daughters reveals that PCB exposure
before or during pregnancy causes short and long time to pregnancies. PCB-66,
PCB-74, PCB105, PCB118, PCB156, PCB-167 are anti-estrogenic PCBs
associated with long time to pregnancies. While PCB-56 was associated with
shorter time to pregnancies. PCB-136, PCB-118, PCB-167 and PCB-170 were
associated with reduced fecundability in daughters. (Gennings et al., 2013).
Chevrier et al (Chevrier et al., 2013) conducted epidemiological study on 3421
pregnant women to investigate the time to pregnancy. The amount of 12 PCBs,
10 PBDE and 14 organo-chlorine was measured in cord blood. Results showed
that shellfish consumption increases the time to pregnancy. PCB-153, PCB-187
and DDE are associated with reduced fecundability. Time to pregnancy
increases with increase in concentrations of PCBs and organochlorines
concentrations in blood. Another epidemiological study was conducted to
investigate the relationship between persistent environment pollutants and
couple fecundity reveals that PCB-167 and PCB-209 were consistently
associated with reduced fecundability in female. The strongest associations
were observed for PCB-167 in females and PCB-138 in males. (Louis et al.,
2013). A study by Yang et al (C.-Y. Yang et al., 2008) reveals that women
exposed to PCBs in Taiwan cooking oil incident has time-to-pregnancy of 4
months while control has 3 months. An epidemiological study was conducted
25
to reveal the association between maternal in utero exposure to PCBs and
Michigan female fecundability in offspring. There was negative association
between DDE and female fecundability. While polychlorinated biphenyls are
endocrine disruptor that effect oocyte and reproductive development has
reduced female fecundability in offspring of PCB exposed mothers (Han et al.,
2016b)
3.1.1.3. Implantation Failure
A competent embryo must attach to a receptive endometrial lining for

successful pregnancy. When blastocyst is finally infiltrating the decidua.
implantation is considered to occur. PCBs have been linked to increased
pregnancy failure and failed embryo implantation in many epidemiological and
in vivo studies reported by Barrett et al (Barrett, 2011). Endocrine disruptors
such as PCBs cause infertility problems in women. High level of PCBs in
women blood, low chance to get pregnant. There is 50% chance that high PCBs
level decreases the chance to get pregnant and if become pregnant implantation
problems and miscarriages are most likely to occur as reported by Pizzorno et
al (Pizzorno, 2018). Highest level of PCBs was observed in resting female who
do not off load their PCBs then comes sexually immature, lactating and pregnant
females (Murphy et al., 2015). Meeker et al (Meeker et al., 2011) conducted an
epidemiological two phase study from 1994-1998 and 1999-2003. 2350 couples
were enrolled in study. reported that PCB-153 is associated with failed
implantation and follows dose-dependent trend while PCB-118 reduces live
births rate and doesn’t follow dose-dependent pattern.
3.1.1.4. Polycystic ovary syndrome
Polycystic ovary syndrome is hyperandrogenism and insulin resistance.

Hyperandrogenism causes fertility dysregulations and leads to acne, chronic
ovulation alopecia and hirsutism (Tan, Ignatenko, Wagner, Dokras, Seufert,
Zwanziger, Dunschen, Zakaria, Huseinovic, & Basson, 2021). A case control
study was conducted on Han females in north China to observe the association
between PCB and PCOS suggested that PCBs are risk factors for female
reproductive health as reported by Yang et al (Q. Yang et al., 2015).
26
3.1.1.5. Primary ovarian inefficiency
Primary ovarian inefficiency is a gynaecological disorder characterized by loss
of ovarian activity and loss of primordial ovarian follicles. POI is loss of
ovarian activity before the age of 40. POI is associated with abnormal level of
reproductive hormones LH, FSH and AMH and early menopause in female.
Pan et al (Pan et al., 2019) conducted case-control study on Chinese female
population wit 157 cases and 217 controls. This study reported that PCB- 77,
PCB-105, PCB-81, PCB-126, PCB-138, PCB-153 and luteinizing hormone
showed positive correlation with POI and recurrent miscarriages. Chinese
Hamster ovary cells were exposed to different concentrations 10, 25, 50, 75,100
micro moles of PCB-77 for 4, 24, 48 and 72-hours’ period. PCB77 exposure
causes loss of cell viability due to apoptotic cell death and 4-folds increase in
catapase-3 activity. 48 hours exposure to PCB-77 disturbs normal cell division
by increasing Go/G1 phase, increase in S phase and progression of sub-G1
phases. Thus PCB-77 exposure can cause reproductive disorders and interfere
with ovarian cell viability Murati et al (Murati et al., 2015)
3.1.1.6.Intergenerational Transfer
PCB exposure in perinatal stage causes many developmental, reproductive, and
physiological effects in daughters. AhR are transcriptional regulatory proteins.
PCBs can bind with AhR and cause adverse biological and reproductive
outcomes like cancer, reproductive toxicity, immune deficiencies and
fetotoxicity reported by Abbott et al (Abbott et al., 1999). A study conducted
by SU et al (Su et al., 2012) on eight-year-old children estimated that high levels
of PCBs can decrease the concentration of estradiol E2 in children than
exposure to low level of PCBs and impaired reproductive development. Low
fundus and uteri length were observed in girls with high level exposure. When
pregnant Sprague-Dawley rats were exposed to Aroclor 1221 (A1221) many
effects were observed by Steinberg et al (Steinberg, Walker, Juenger, Woller,
& Gore, 2008) such as deviation of sex ratio toward females, alteration in
luteinizing hormone and progesterone levels. So low levels of PCBs are
associated with increased risk of infertility. Walker et al (Walker, Kermath,
Woller, & Gore, 2013) reported that in females A1221 exposure has sexually
27
dimorphic effects when exposed in gestation period and alters reproductive
aging in female rats. A genome wide transcriptome study conducted on zebra
fish with different groups and exposed to 3 different concentrations of POPs
low, medium and high. Parental exposure to persistent organic pollutants in
zebrafish induces epigenetic changes in HNF4A, insulin, LH, Follicle
stimulating hormone (FSH) and NF-κB producing genes, regulators of
endocrine signaling, metabolic homeostasis, reproduction, immune functions
and cancer development reported by Lyche et al (Lyche et al., 2013) . A
postnatal follow up study conducted on pregnant rats exposed to different
concentrations (0, 10, 30, 100, 300, 1000mg/kg) on gestational day 7-10. In
utero and lactational exposure to non-dioxin like PCB-180 induces changes in
RAP-RXR heterodimer. Retinoic acid signalling pathway and retinoid X
receptors (RAP-RXR) have an essential role in reproductive development and
fertility by Alarcon et al (Alarcón et al., 2021). Gallo et al (Gallo, Ravenscroft,
Carpenter, Schell, & Environment, 2018) reported in study that every unit
increase in the level of PCBs 5 folds greater risk of FSH: LH. PCB 105,
52,95,101 and 47 are the cause of the increase ratio of FSH: LH means less
ovarian responsivity. A study conducted on rats reveals that PCB-126 exposure
causes an increase in FSH and decrease in progesterone, anti-mullerian
hormone (AMH) and 17B- estradiol. It causes reduction in uterine area, number
of uterine glands and follicle corpora lutea reported by Klenov et al (Klenov et
al., 2021). The processes through which PCB causes infertility is demonstrated
in (Figure 2).
28
Figure 4 PCBs and Infertility. PCBs exposure causes epigenetic modification in genes
CYP1A1, CYP1B1 and HOXA10 and leads to endometriosis. Primary ovarian
inefficiency is caused by PCB-138/158, PCB-153 and PCB-180. PCB exposure causes
polycystic ovaries. Intergenerational transmission of PCB by NF-KB signalling
pathway, Retinoic acid signalling pathway and Aryl hydrocarbon signalling pathway
leads to impaired reproduction, infertility, cancers, and epigenetic changes.
29
3.1.2.PCBs and Reproductive Track Cancers
PCB is one of the endocrine disrupting chemicals (EDC). Some epidemiological
studies support that high levels of EDCs are associated with increased risk of
gynecological cancers such as endometrial, cervical and ovarian cancers (Den
Boon et al., 2015) (Roy et al., 2015) while other studies observe no
association(Ruder, Hein, Hopf, & Waters, 2014) (Donat-Vargas et al., 2016). A
cohort study that was performed on large population of 36777 middle age and
elderly Swedish women in order to find the association between dietary PCBs
and ovarian, breast and endometrial cancer but Donat-vargas et al (Donat-
Vargas et al., 2016) in a 14 year follow up observed that dietary PCBs exposure
does not play role in development of ovarian, breast and endometrial cancers.
Similarly, a cohort study performed on 24,865 capacitor manufacture workers
by Ruder et al (Ruder et al., 2014) , workers were exposed to PCBs at plants in
Indiana, Massachusetts and New York. These workers were followed to
evaluate the mortality, but no association was observed between PCBs and risk
of mortality by ovarian and endometrial cancer. PCBs also affect the normal
estrogenic signaling pathways that leads towards the increased risk of
gynecological cancers. Congeners of PCBs are reported to be higher in women
with ovarian, and uterine cancers. Morgan et al (Morgan et al., 2016) in his
cross sectional study including women of age 20 to 85 years observed a possible
association between increase risk of ovarian and uterine cancers and exposure
to xenoestrogen PCBs. He reported that women who were diagnosed with
ovarian and cervical cancers have high PCBs levels. He further observed that
dioxin like PCBs were more associated with ovarian cancer and non-dioxin like
were associated with ovarian and uterine cancers to be more specific sum of
non-dioxin like PCBS and PCB 138 were associated with cervical and uterine
cancer and sum of dioxin like PCB and PCB 74 and 118 were associated with
increased risk of ovarian cancer. As we know, PCB can be exposed through
food intake and has been associated with reproductive problems including
uterine leiomyomata (UL). High consumption of PCB contaminated food can
increase the risk of cancer. As reported by Lambertino et al (Lambertino,
Turyk, Anderson, Freels, & Persky, 2011) in his cohort study including over
4200 participants that high consumption of Great lake sport is associated with
30
self-reported uterine Leiomyomata and is a source of exposure to PCBs. High
level of PCBs in sport fish can increase risk of UL in women. But in
breastfeeding women lower association was found, as breast feeding can lower
the risk of UL as it reduces the burden of PCB in lactating women. Another
Faroese birth cohort study recruited between 1986-1987 and followed until
adulthood observed the association that was reported by Leung et al (Leung et
al., 2018). He studied the exposure of PCB-105, PCB-118 and NDL-PCBS on
72 participants. Faroese consume seafood contaminated with PCBS which
trigger DNA methylation changes in genes in umbilical cord white blood cells
(UCWBCS). Umbilical cord blood samples and hair samples from mother were
taken. This study reveals that PCB-105 is associated with methylation at
significant numbers of sites. PCB-105 cause methylation changes at 73 CPG
sites and 33 genes, out of which 13 genes are involved in cancers, reproductive
system disorders, organismal injuries and abnormalities. Embryonic Lethal
Abnormal Vision Drosophila, Homolog-like 1 (ELAVL1) play an important
role in cancer progression and PCB-105 effect ELAVLI associated cancer
network in females. Thus, PCB-105 is most epigenetically active toxicant in
females. Likewise in 1972 a large quantity of PCB contaminated oil spilled into
the river. To find the association between PCBs and different health effects a
study was conducted. Helmfrid et al (Helmfrid, Berglund, Löfman, & Wingren,
2012) observed that community contaminated with PCB are associated with
many reproductive problems and cancers. High association was observed
between total cancers and high consumption of perch fish contaminated with
PCBs. Mothers that were exposed to PCBs before the age of 5 had more preterm
child deliveries and risk of breast cancer.
Experimental studies also observed an association between PCBs exposure and

gynecological cancers. PCB effects are not only observed in humans but are
also involved in reproductive problems in animals. A study was performed to
observe the association between PCB and DDT and incidence of uterine
leiomyomata in 257 Baltic gray seal females examined during 1973–2007 gray
seal. Bredhult et al (Bredhult, Bäcklin, Bignert, & Olovsson, 2008)
demonstrated a significant association between PCB exposure and development
of uterine leiomyomata in Baltic gray seal females.Similarly another study
31
performed by Yoshizawa et al (Yoshizawa et al., 2009)on Harlan Sprague-
Dawley female rats , he reported that when rats were exposed to different dioxin
and non-dioxin like compound. PCB 153 and binary mixture of PCB 126 and
153 were associated with inflammation in the ovary and cystic endometrial
hyperplasia, PCB 153 exposure is associated with inflammation in uterus and
PCB118 is associated with uterine carcinoma. All these effects were caused by
PCBs by disrupting normal endocrine pathways of the reproductive organs.
Inhibition of catechol-O methyltransferase (COMT) contributes to estrogen
mediated tumorigenesis. To confirm the hypothesis that PCB catechol
metabolites effects the expression of COMT Ho et al (Ho et al., 2008)
performed the experiment on MCF-7 cells results showed that PCB were
involved in the PCB mediated carcinogens by down regulating the expression
of COMT and inhibiting its activity. PCBs also effects expression of many
genes, aberrant expression of genes can cause many severe problems such as
cancers. Ghosh et al (S. Ghosh et al., 2015a) in his study exposed Human
primary Blood Mono-nuclear cells (PBMC) to the mixture of PCBS (PCB-118,
PCB-138, PCB-153, PCB170, PCB-180) for 48 hours. Blood samples were
extracted form 6 participants, 3 male and 3 females for epigenetic studies and
their association with endocrine system disorders, developmental disorders,
genetic disorders and cancers. 100 genes were differentially expressed in
exposed individuals, 16% were upregulated and 84% were downregulated.
PRAD genes associated with insulin resistance in type II diabetes were
upregulated. Aryl hydrocarbon receptor nuclear translocator ARNT gene
involved in xenobiotic metabolism was downregulated. LEPR gene involved in
lipid metabolism was downregulated and leads to obesity. CYP2D6 gene
involved in oxidation of wide range of substrates was downregulated and cause
poor neuro-behavioral development in exposed children. Cancer related gene
P53 that also act as tumor suppressor and prevents cancer was downregulated
in exposed individuals resulting in development of multiple cancer such as
ovarian and breast cancer. Similarly Qu et al (Qu, Wang, et al., 2018) in his
study observed that PCB 188 is involved in the down regulation of HOXA10
gene, which resulted in the development of endometriosis and it also
progression of several cancers such as endometrial carcinoma, ovarian cancer,
and breast cancer. Experiments performed on cell lines also showed that PCB
32
can cause reproduction track cancers. Guo et al (Guo et al., 2020) in his recent
study cultured ovarian stem cells OVCAR-3 cells and treated them different
concentrations (10-6-10-10M) of Bisphenol A and polychlorinated biphenyls for
24 hours. OVCAR-3 stem cells are pluripotent, immortal and can differentiate
into different tumor cells. Exposure to 10-7M BPA and PCB for 24 hours
increased the numbers and size of cells. It caused activation of WNT signaling
pathway which leads to ovarian cancer. CD44+ and CD24- were increased with
treatment to PCBS and BPA which indicated tumor growth. Exposure also
changed messenger RNA expression, downregulation of ALDH1A1 and
CD133 and upregulation of NANOG, OCT4 and SOX2. Indicating PCBs and
PBA has oncogenic effect that can cause ovarian cancer. Human cervical
carcinoma Hela cells require aerobic glycolysis for cancer cells survival.
Pyruvate kinase M2 (PKM2) pathway is mediator of aerobic glycolysis leads to
progression of cancer cells and causes cervical cancer. Zhang et al (Zhang,
Song, & Li, 2019) in his study also observed that when human cervical
carcinoma cells were exposed to different concentrations (0.01, 0.1, 1, 10, 100
nM) of PCB 118, PCB126 and PCB 153 for 24 to 48 hours results in the
enhancement of cell viability, promotes aerobic glycolysis, increases glucose
consumption and lactose production. Exposure upregulates the expressions of
GLUT1, PDK, LDHA and increases protein level of PKM2. Indicating that
PCB 126, 118 and 153 exposures are associated with cervical cancers in human.
Ishikawa cells are ideal models to study the impact of estrogen on endometrial
epithelium. Ishikawa cell lines were exposed to different concentrations (o.3, 3
and 30 Micro mole) of PCB-126 most toxic environmental congener and PCB-
153 most prominent. This study reveals that exposure to PCB-126 increases
Ishikawa cell viability while PCB-153 has no significant cytotoxic effect. PCB-
126 and PCB-153 upregulate the activity of Superoxide Dismutase SOD1
activity. Interleukin 8 and its receptors are involved in endometrial cancer are
downregulated by PCB exposure and cause cell death at high dose. PCB-126
causes proliferation of Ishikawa cells and effect endometrial tissues. PCB-153
exposure inhibits the expression of TNF-alpha. PCB-126 binds with aryl
hydrocarbon receptors and estrogen receptors and abolish the expression of
inflammatory factors reported by Chen et al (Chen et al., 2015). (Figure 5)
describes the whole association between PCBs and reproductive tract cancers.
33
Figure 5 PCB and Reproductive Tract Cancers. Different congeners of PCB causes
reproductive cancers following WNT, PKM2 pathways and downregulation of
P54 and COMT when exposed to MFC-7 cells, ovarian stem cell and human
cervical carcinoma Hella cells
34
Table 2 PCB Affec8ted Genes
Sr Genes PCBs References

no.
Upregulated Downregulated
1 CYP1A1, GPX1, GGT1, IL6 PCB-126
CYP1A2,
CYP1B1, PON1,
MPO1, (46)
CYP17A1,
HSB17B3,
TNFSF10,
BMP2, CCL24,
CLL5, CCL19,
PF4, CYP19A1
2 GLUT1, LDHA,
PDK, ALDH1A1
(60)
PCB-126,
PCB-118,
PCB-153
3 CD133, SOX2, PCB-126, PCB-
NANOG, OCT- 153 (59)
04
4 SOD1, PRAD ARNT, CYP2D6, PCB-118, PCB-
LEPR, P53, IL-8, 138, PCB-170, (58)
TNF-a PCB-180, PCB-
153, PCB-126
5 HOXA10 PCB-118 (24)
35
3.2.Exposure Analysis
1.2.1. Demographic Data
This study was conducted to observe PCBs concentration in hair and urine
samples of females to find the association between PCBs and female infertility.
A total of 52 participants from Khyber Pakhtunkhwa (KPK) and Punjab were
included in this study. 35 samples were from Punjab and 17 were from KPK. 52
urine and 52 hair samples were analyzed. Basic characteristics included (age,
residence (rural, urban), BMI, conditions (normal, infertile, endometriosis,
POCs, no egg), diagnosis (Primary infertility, secondary infertility,
miscarriages). Out of 52 participants, 12 were <=25 years (23.10%). 17
participants were between the ages of 26-30 (32.70%). 13 participants were
between 31-35 years (25%). 6 participants were between the ages of 36-40 years
(11.50%). 1 participant was in the age group of 41-45 years (1.90%). 1
participant was in the age group of 46-50 years (1.90%). Participants with BMI
less than 18.5 were considered underweight and out of 52 participants 14 were
underweight, that is 26.90%. participants having BMI between 18.5-25 were
considered normal. 17 participants fell in normal category, that is 32.70%.
Participants with BMI within 25-30 were considered overweight and 3
participants were overweight, that is 5.80%. BMI>=30 was considered obese, 3
participants which is 5.80% were obese. 42 participants (80.80%) were included
from rural areas and 10 participants (19.20) were included from urban areas.
Out of 52 participants 22 (42.30 %) were normal, 2 (3.80%) were infertile, 10
(19.20%) were suffering from endometriosis, 10 (30.80%) were PCOs patients
and 2 (3.80%) had no eggs. Diagnosis contains 40(76.90%) participants with
primary infertility, 10 (19.20%) participants with secondary infertility and 2
(3.80%) participants with miscarriage shown in (Table 3).
36
Table 3 Demographic Data
Frequency Percentage
Characteristics
Age (years)
<=25 12 23.10%
26-30 17 32.70%
31-35 13 25%
36-40 6 11.50%
41-45 1 1.90%
46-50 1 1.90%
BMI
Underweight 14 26.90%
Normal 17 32.70%
Overweight 3 5.80%
Obese 3 5.80%
Residence
Rural 42 80.80%
Urban 10 19.20%
Condition
Normal 22 42.30%
Infertile 2 3.80%
Endometriosis 10 19.20%
PCOS 16 30.80%
No Eggs 2 3.80%
Diagnosis
Primary Infertility 40 76.90%
Secondary Infertility 10 19.20%
Miscarriages 2 3.80%
37
According to (Table 4) there is no considerable difference in the means of controls and
infertile patients with regards to the demographic factors age, BMI, bleeding length,
and age at menarche. Since the p-value of none of the factors is equal to or less than
0.05, this indicates that these factors are not significantly correlated with female
infertility.
Table 4 Demographic Data of control and infertile patients.
p-
Std. value
n Mean Error 95% CI Min Max (KW)
Lower Upper
34.1 18.0
Age Control 21 30.90 1.56 27.65 6 0 50.00 0.61
31.9 22.0
Infertile 29 29.97 0.97 27.98 5 0 45.00
32.0 18.0
Total 50 30.36 0.86 28.64 8 0 50.00
24.0 16.2
BMI Control 14 21.85 0.99 19.71 0 0 31.36 0.98
25.6 16.1
Infertile 23 22.87 1.33 20.12 2 1 43.20
24.3 16.1
Total 37 22.49 0.90 20.66 1 1 43.20
Bleeding
Length Control 21 4.67 0.42 3.79 5.55 1.00 7.00 0.53
Infertile 30 4.50 0.53 3.43 5.57 0.00 15.00
Total 51 4.57 0.35 3.86 5.27 0.00 15.00
Age at
Menarch 13.6 10.0
e Control 16 12.63 0.47 11.62 3 0 17.00 0.09
14.4
Infertile 28 13.64 0.40 12.83 5 9.00 18.00
13.9
Total 44 13.27 0.31 12.65 0 9.00 18.00
38
1.2.2. Lifestyle Factors
Table 5 shows the comparison of lifestyle factors in control and infertile

patients. According to the table, none of the lifestyle factors including
residence, age group, weight, exercise, dieting status, caffeine intake, smoking
status, sexual activity, contraception usage, eating disorder, and sleeping
disorder show any significant correlation with either of the patient groups. This
is because the retrieved p-values after applying the Pearson chi-square test
exceed the 0.05 threshold.
Table 5 Lifestyle Factor
Pearson Chi-
Control Infertile Square p-value
Residence
Rural 16 26
Urban 6 4 0.208
Age Group
<=25 4 8
26-30 8 9
31-35 5 8
36-40 3 3
41-45 0 1
46-50 1 0 0.719
Weight
Underweight 5 9
Normal 7 10
Overweight 1 2
Obese 1 2 0.984
Exercise
Sedentary 14 18
Mild exercise 6 11
Occasional
exercise 1 1 0.821
39
Dieting status
Yes 2 3
No 19 27 0.955
Caffeine
Intake
None 0 1
Coffee 9 14
Tea 12 15 0.651
Smoking
Status
Smoker 1 4
Non-smoker 20 26 0.311
Sexual
Activity
Sexually active 17 24
sexually
inactive 0 3 0.155
Contraception
Usage
Yes 0 1
No 21 29 0.398
Eating
Disorders
Yes 10 18
No 11 12 0.382
Sleeping
Disorder
Yes 10 10
No 11 20 0.304
40
1.2.3. Menstrual Conditions
Table 6 displays the association between menstrual conditions such as

diagnosis, conditions, menstruation status, age at menarche, bleeding
length, and depression with the control and infertile groups. After applying
the Pearson chi-square test the p-values indicating the significance of the
menstruation conditions were obtained. The p-values of conditions and
diagnosis were 0.000 and 0.001 respectively which are statistically
significant suggesting that these factors have an interrelationship with the
control and infertile groups. While the factors menstruation status, age at
menarche, bleeding length, and depression showed no association with them
due to their statistically insignificant p-values.
Table 6 Menstrual Conditions
Condition Control Infertile p-value

Normal 22 0
Endometriosis 0 10
Infertile 0 2
No eggs 0 2
PCOS 0 16
sexually
inactive 0 3 0.000
Diagnosis
Miscarriages 2 0
Primary
Infertility 20 20
Secondary
Infertility 0 20 0.001
Menstruation
Status
Regular 14 14
Irregular 6 13 0.21
Age at
menarche 0.098
41
9-13 (years) 11 12
14-18(years) 5 16
Bleeding
Length
0-4(days) 10 15
5-15(days) 11 15
No 14 17 0.867
42
1.2.4. Hair And Urine Analysis
1.2.4.1.Hair Samples
As shown in Table 7 , total of 52 hair samples were taken from females’

participants. Out of them, 35 samples were from Punjab and 17 were from KPK.
Participants between the age group 0f 20 -29, 46% hair samples were collected
following 44% of samples from females between the age group of 30-39 and lastly
8% from females above the age of 40. The table below indicates the concentration
of PCBs detected in hair samples. PCB 101 was detected in 12
participants(23.076%) and has the maximum detected value of 8.370 pg/mg with
standard error 4.772 pg/mg. while PCB 28 was detected in 21
participants(40.384%) and has the lowest detected value, that is 0.181 pg/mg with
standard error 0.023 pg/mg. PCB 118, PCB 138, PCB153 and PCB 180 have mean
values 2.825 pg/mg, 3.76 pg/mg, 5.635 pg/mg and 3.938 pg/mg with standard error
0.432 pg/mg, 0.16 pg/mg, 0.173 pg/mg and 0.568 pg/mg.
Table 7 Analyte exposure in Hair sample
Sample Hair
Compound Mean± SE (pg/mg) Range(pg/mg)
PCB28 0.181± 0.023 0.014-0.629
PCB52 0.513± 0.016 0.008-7.352
PCB101 8.370± 4.772 0.010-227.432
PCB118 2.825± 0.432 0.015-20.112
PCB138 3.76± 0.16 0.044-4.16
PCB153 5.635± 0.173 0.168-5.97
PCB180 3.938± 0.568 0.207-32.442
The clustered bar graph in (Figure 6) indicates that the mean concentrations of the
several PCB congeners found in the hair samples of women in the control and infertile
groups. PCB101 has the highest concentration in the infertile group suggesting it might
play a role in causing reproductive infertility hindering female reproductive health
while in comparison its concentration in the control group is significantly lower which
43
further supports this presumption. PCB 153 appears to have approximately the same
concentration in both groups indicating it might not have any contribution in
obstructing fertility in women. PCB28 has the lowest concentration in both groups
which can be attributed to low exposure to the compound.
Figure 6 PCBs concentration in Hair samples
Figure 7 explains that PCB 28 is significantly different between control and

infertile females. In the graph below you can see that in infertile and controls
the concentration of PCB 101 is the highest but this did not mean that they are
significantly different. PCB 153 has the lowest concentration among infertile
and controls.
44
Figure 7 concentration of PCBs in Hair samples
Infertile
Control
ns
ns-non-significant
Figure 8 Concentration of PCB 28 in control and infertile
45
Infertile
ns
Control
ns-non-significant
Infertile
Control
ns
ns-non-significant
46
Infertile
ns Control
ns-non- significant
Infertile
ns Control
ns-non-significant
47
Infertile
ns Control
ns-non-significant
Infertile
Control
ns
ns-non-significant
48
1.2.4.2. Urine Sample:
Table 8 displays that out of 52 participants from which urine samples were
collected, 22 (42.30%) were normal, 2 (3.80%) were infertile, 10 (19.20%)
were having endometriosis, 16 (30.80%) suffering from PCOs and 2 (3.80%)
were with no eggs. PCB 180 was detected in 44 participants (84.615 %) and
has the maximum detected mean value 0.056 ± 0.029 following PCB 153,
PCB 138, PCB101, PCB 118, PCB 52 having mean values 0.018±0.004,
0.011±0.002, 0.010±0.001, 0.009±0.001, 0.006±0.001 while PCB 28 was
detected in all the participants (100%) and has the lowest detected mean value
0.004 ± 0.001.
Table 8 Analyte exposure in Urine samples
Sample Urine
Compound Mean± SE (ng/ml) Range(ng/ml)
PCB28 0.004± 0.001 0.000-0.016
PCB52 0.006± 0.001 0.000-0.025
PCB101 0.010± 0.001 0.000-0.039
PCB118 0.009± 0.001 0.000-0.038
PCB138 0.011± 0.002 0.000-0.096
PCB153 0.018± 0.004 0.000-0.215
PCB180 0.056± 0.029 0.000-1.526
Figure 8 below shows the mean concentrations of multiple PCB congeners in

the urine samples of control and infertile groups. The mean concentration of
PCB 180 is the highest in both groups which can be linked to the reproductive
disorders experienced by women with higher concentrations of this compound.
The second compound with the highest concentrations in both groups is
PCB153 while that with the lowest concentration is PCB28.
49
Figure 15 PCBs concentration in Urine samples
There was no statistically significant difference between congeners of PCBs in
control and infertile females in urine sample. Whereas the concentration PCB
180 is highest and PCB28 is the lowest in controls and infertile as shown in
Figure 9 below.
Figure 16 Concentration of PCBs in urine
50
Infertile
Control
ns
ns-non-significant
Figure 17 concentration of PCB 28 in control and infertile
Infertile
ns
Control
ns-non-significant
51
Infertile
ns
Control
ns-non-significant
52
Infertile
ns Control
Infertile
ns Control
53
\
ns Infertile
Control
54
Chapter 4
Discussion
55
PCBs are organic halogens also known as xenobiotics. Environmental exposure
to PCBs has been associated with female infertility. This study was conducted
to reveal the association between PCBs and female sterility and how exposure
to them will affect overall female reproductive health. The chosen population
included women from the areas of KPK and Punjab. Hair and urine samples
were obtained from 52 women belonging to agriculture areas where they were
constantly exposed to the several congeners of PCBs. PCB28, PCB52, PCB101,
PCB118, PCB138, PCB153, and PCB180 were detected in most of the hair and
urine samples. These PCBs were detected in considerable amounts in the hair
samples as compared to the urine samples in which the analyte concentrations
were barely observable. PCB101 was found in statistically significant
concentrations in the hair samples of infertile groups as compared with control
groups. Although considerable difference was observed in PCB 101
concentration in controls and infertility, but results were not significant enough
to establish a strong association, but it implies that PCB 101 might have an
impact on infertility. Studies reveal that PCB101 when mixed with other PCBs
induces anti-proliferative effects(S. Ghosh et al., 2015b). Another study reveals
that PCB 101 increases the rate of cell death at high concentrations proportional
to the amount of exposure to the mixture (Ferrante et al., 2011). While in urine
samples PCB180 was in significantly higher concentration in infertile as
compared with controls. Vichi et al reported in his study that higher level of
PCB-180 increases the risk of endometriosis (Vichi et al., 2012). Alarcon et al
determined that in-utero and lactational exposure to PCB-180 induces changes
in the RAP-RXR heterodimer which plays an essential role in reproductive
development and fertility (Alarcón et al., 2021). To summarize, results in this
study show no significant association between demographic and lifestyle factors
with female infertility. There was significant difference between controls and
infertile with conditions like endometriosis, infertility, no eggs, and polycystic
ovary syndrome which indicates that these conditions contribute to female
infertility. Endometrial progression leads to infertility, hormonal imbalance,
failure of immune response and genetic predisposition as reported by (Ferrante
et al., 2011). PCB-118 exposure causes hypermethylation in promoter regions
and impairs endometrial receptivity and causes the failure of embryo
implantation (Qu, Wang, et al., 2018). PCOS has a strong impact on female
56
fertility (Tan, Ignatenko, Wagner, Dokras, Seufert, Zwanziger, Dunschen,
Zakaria, Huseinovic, Basson, et al., 2021). Yang et al reported is his study that
PCBs and PCOs are risk factors for female reproductive health (Q. Yang et al.,
2015). Significant difference was observed between the normal and participants
having reproductive disorders which indicated that exposure to relevant PCBs
could have impact on female infertility. There is 50% chance that high PCBs
levels decrease the possibility for the embryo to implant with further increased
possibility miscarriages being likely to occur (Pizzorno, 2018). Meeker et al
reported in his study that PCB-153 is associated with failed implantation while
PCB-118 reduces live births rate and doesn’t follow dose-dependent pattern
(Meeker et al., 2011). The association of PCBs with female infertility remains
unclear because of small sample size. However, it is noteworthy that PCBs
concentrations are considerably higher in infertile group as compared with
control group which means that PCBs may contribute to female infertility.
57
Chapter 5
Conclusion
58
PCBs concentration was measured in hair and urine of 52 female participants
belonging to Punjab and KPK. PCBs are persistent organic pollutants and have
a long half-life in the human body and contribute to multiple health effects in
humans. Our results showed no significant association of lifestyle factors like
dieting status, smoking status, sexual activity, eating status etc. with control and
infertile groups. Demographic factors like age, BMI, residence etc. showed no
significant correlation with controls and infertile groups. While menstruation
status is significantly correlated with infertile groups. Conditions like
endometriosis, PCOS, no eggs and sexual inactivity showed significant p-value
of 0.000 which means these factors contributes to female infertility. likewise,
miscarriages, primary infertility, secondary infertility, and other reproductive
abnormalities showed the p-value of 0.001 which means that these factors are
significantly associated with female infertility. PCBs concentration detected in
hair samples were considerably higher as compared with urine samples which
indicates that PCBs can persist for longer period in human body. PCB 101 was
detected in maximum amount in hair samples of infertile groups and least
detected PCB was PCB 28. While in urine samples PCB 180 was recorded in
maximum concentration and PCB 28 was in lowest concentration in infertile
group. Our results showed that exposure to these analytes like PCBs contribute
to female infertility but due to sample samples size and most of the female
participants already suffering from reproductive disorders like endometriosis,
PCOS, primary infertility and secondary infertility, results are still unclear.
Detail research is needed to assess real consequences of PCBs exposure on
female infertility.
59
Chapter 6
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Possible Impacts of PCBs on Female Reproductive

Possible Impacts of PCBs on Female Reproductive Health

COMSATS University Islamabad

An Undergraduate Thesis submitted to the Department of Bioscience as partial fulfilment of

Name Registration Number

Nimra Khan CIIT/FA19-BSO-054/ISB

COMSATS University Islamabad

This thesis titled

Possible Impacts of PCBs on Female Reproductive Health

Has been approved.

Dr. Syed Tahir Abbas Shah

We, Nimra Khan (CIIT/FA19-BSO-054/ISB) and Sammar (CIIT/FA19-BSO-070/ISB)

Dr. Syed Tahir Abbas Shah

Head of the Department:

Dr. Muhammad Qaiser Fatmi

Dedicated To My Beloved Parents

Whose prayers, affection and encouragement

We are obligated to our Co-Supervisor Dr Saira Amir Assistant Professor, COMSATS

1.1. Female Reproductive System: ......................................................................... 6

1.2. Female Infertility:............................................................................................ 8

1.3. PCBS Role in Female Infertility: .................................................................... 9

1.3.1. Endometriosis: ......................................................................................... 9

1.3.2. Female Fecundability: ............................................................................ 10

1.3.3. Implantation Failure: .............................................................................. 11

1.3.4. Intergenerational transmission: .............................................................. 11

1.3.5. Polycystic ovary syndrome: ................................................................... 12

1.3.6. Primary ovarian inefficiency: ................................................................ 12

1.4. PCBs and reproductive track cancers: ........................................................... 13

1.4.1. Ovarian cancer: .................................................................................. 13

1.4.2. Endometrial cancer:............................................................................ 13

1.4.3. Cervical cancer: .................................................................................. 14

1.4.4. Vaginal cancer: ................................................................................... 14

2.1. Meta Analysis:.................................................................................... 18

2.2. Exposure Analysis: ............................................................................. 20

2.2.1. Population ...................................................................................... 20

2.2.2. Reagent and materials .................................................................... 20

2.2.3. Urine sample treatment .................................................................. 20

2.2.4. Hair samples treatment .................................................................. 21

2.2.5. Data analysis .................................................................................. 21

3.1. META ANALYSIS ...................................................................... 23

3.1.1.1. Endometriosis: ................................................................. 23

3.1.1.2. Female Fecundability:...................................................... 25

3.1.1.3. Implantation Failure:........................................................ 26

3.1.1.4. Polycystic ovary syndrome: ............................................. 26

3.1.1.5. Primary ovarian inefficiency ........................................... 27

3.1.1.6. Intergenerational Transfer................................................ 27

3.1.2. PCBs and Reproductive Track Cancers: ................................ 30

3.2. Exposure Analysis: ....................................................................... 36

Figure 1 structure of PCB (Hens & Hens, 2017) .......................................................... 4

Table 1 PCBs involved in Infertility and Reproductive Cancers ................................. 15

Polychlorinated biphenyl ethers (PCBs) are organochlorine aromatic

Figure 1 structure of PCB (Hens & Hens, 2017)

1.1. Female Reproductive System:

Female Reproductive system: female reproductive system consists of vagina,

1.2. Female Infertility

Failure to conceive, after one year of regular intercourse with no contraception

Endometriosis is multifactorial gynaecological disorder defined as the ectopic

1.3.2. Female Fecundability

Female Fecundability is probability of conceiving during a menstrual cycle,

A competent embryo must attach to a receptive endometrial lining for

1.3.4. Intergenerational transmission

PCB exposure in perinatal stage causes many developmental, reproductive,