Endocrine disruption and female reproductive health: Implications on cross-talk between endocrine and autocrine/paracrine axes in the ovary

INTRODUCTION

Reproduction is the sole process in any species that is responsible to sustain its progeny on earth. A female who starts her journey with 2 million oocytes ends with just 400 follicles capable of ovulating during her reproductive life.^[1] This marks the immense importance of every single follicle for the nature that has taken all considerations intelligently in designing and making it of utmost quality for positive successful fertilization. In today’s urbanized globe, females are exposed to a myriad of anthropogenic substances, drugs, and exogenous compounds in the environment, popularly termed as endocrine disrupting chemicals (EDCs), which might lead to an overabundance of infertility related disorders. In addition to altered lifestyle, early and recurrent pregnancy; multitudes of environmental anthropogenic contaminants, poor nutritional status, sanitation facility, and ill-developed infrastructure at rural areas are posing great risks on female reproductive health specially in developing nations. The majority of EDCs interfere with neuroendocrine and endocrine functions thereby distressing reproduction and embryonic development.^[2] Moreover, females are constantly undergoing steroidal/nonsteroidal drug regimens during various ovarian disorders, for example, for ovulation induction in women with polycystic ovary syndrome (PCOS), aromatase inhibition therapy for advanced breast cancer or even drugs used as contraceptive pills that might pose side effects on female reproductive health.^[3,4] In this context, active and systematic research initiatives may help to better understand the complex dialogs between neuroendocrine, endocrine and juxtacrine modulators, their potential targets, and extent of damage inflicted by various harmful agents [Figure 1]. Based on these major amendments in existing policies, long-term planning and implementation of child and women health-care measures at the grass-root level may help in empowering women of this country to gain access in the globe of sound reproductive health.

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Figure 1:

Risks of drugs/environmental toxicants on female reproduction.

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ENDOCRINE DISRUPTORS: AN UPDATE

A functional endocrine system is a balanced network of chemical messengers in the form of hormones that regulate and coordinate bodily functions. In the present milieu of 21^st century with growing urbanization and profuse use of medications, a plethora of chemicals exist that possess the ability to interfere with the natural course of hormone action. They are potent enough to disrupt the normal homeostatic processes of hormone activity and have been termed as endocrine-disrupting chemicals (EDCs). Endocrine disruptors are known to (a) mimic the effects of endogenous hormones; (b) antagonize the effects of endogenous hormones; (c) alter the pattern of synthesis and metabolism of natural hormones; and (d) modify hormone receptor levels.^[5] Considering the pervasive nature and risks involved from exposure to EDCs, many of which are estrogenic in nature, United States Environmental Protection Agency developed a two-tier screening and testing program to detect xenobiotics. While tier I screening protocol included battery of assays to identify chemicals that interact with estrogen, androgen, and thyroid systems, tier two screening was executed for those chemicals that are positive in first screening and to further determine whether the chemical substance actually could interfere with endocrine-related processes.^[6]

IMPACT OF EDC EXPOSURE ON FEMALE REPRODUCTIVE HEALTH: A FOCUS ON XENOESTROGENS

A major subset of EDCs capable of eliciting estrogenic effects is the “xenoestrogens” (XEs) that have received a global concern for putting female reproduction at stake. XEs can exert their influence at both genomic and non-genomic level thereby activating a wide variety of signaling pathways and downstream kinases.^[7] Exposure to XEs has been reported to dysregulate the entire hypothalamic-pituitary-gonadal system through increased GnRH production resulting in an increased endogenous estrogen production and premature puberty.^[8] Estrogen, the female steroid hormone, regulates a wide array of female reproductive events, including endometrial growth, menstrual cycle regulation, and breast development. Interestingly, ovaries and uteri are most sensitive to XEs leading to a surfeit of female reproductive disorders such as early puberty, premature ovarian failure, impaired fertility as well as breast, ovarian, and uterine cancers.^[9] Given the host of endocrine abnormalities associated with multitude of ovarian diseases (obesity, PCOS, endometriosis, ovarian cancer, etc.), EDCs in the environment are worth considering to have plausible association with such pathophysiologic conditions. Over 70% of the women have been diagnosed with sporadic breast cancer and this risk has increased at an exponential rate with the increased exposure to estrogenic chemicals.^[10] Endometriosis, another estrogen-dependent gynecological disease, wherein endometrium-like tissue grows outside uterine cavity is also one of the most common causes of infertility in women of reproductive age.^[11] Surveys have revealed that women who have used estrogen replacement therapy are more likely to develop endometrial cancer than those who never underwent estrogen replacement therapy.^[12] Moreover, estrogen also stimulates the growth of ovarian tumor cell lines expressing the estrogen receptor (ER), making ovarian cancer preponderant among women undergoing various hormone therapy regimens.^[13] Collectively, these reports suggest that EDCs can disrupt a wide range of reproductive functions through hormone-driven processes leading to elevated risks for a spectrum of diseases.

Although EDCs might enter into living organisms throughout their life, a particularly vulnerable time for exposure is the prenatal period, covering the entire voyage from gamete production, and fertilization through to intrauterine and post-natal development of progeny.^[14] Developmental exposure to EDCs may lead to serious alterations that will persist into adult life, marking the field of endocrine disruption and female reproductive health a major area of concern. Bisphenol-A (BPA), a widely used plasticizer having xenoestrogenic properties, has been reported to be higher in circulation of women with PCOS. BPA reportedly potentially displaces a proportion of bound androgens or interferes with androgen catabolism leading to androgen excess in PCOS.^[15] BPA can be transferred from the mother to child both through lactational and transplacental routes. Moreover, women undergoing infertility treatment has been demonstrated to have an inverse correlation between BPA levels in the urine with the number of quality eggs recovered in in vitro fertilization.^[16] Recently, BPA per os (p.o.) has been shown to impact uterine morphology and androgen synthesis by interfering with the RNA methylation-related genes.^[17] Notably, bisphenol S and fluorine-9-bisphenol, supposedly more environment friendly alternatives of BPA, have been shown to promote aberrant spindle formation in mature mouse and porcine oocytes.^[18,19] Moreover, women exposed to di(2-ethylhexyl) phthalate (DEHP), a phthalate ester used in medical bags and tubing, may promote shorter pregnancy duration and increased incidences of miscarriage.^[20] Numerous uterine defects that include precocious menstruation, irregular estrous cycle, and even short reproductive lifespan are well documented in mice treated with genistein-an isoflavone phytoestrogen, acting as a potent angiogenesis inhibitor.^[21] Surprisingly, diethylstilbestrol, a nonsteroidal synthetic estrogen prescribed to pregnant women earlier, has been implicated in multi-generational effects in addition to reproductive tract dysfunction and poor pregnancy outcomes.^[22] These evidences suggest a close association between developmental EDC exposure and adverse reproductive outcomes.

OOGENESIS: AN UPDATE ON AUTOCRINE/ PARACRINE ENTITIES

Oogenesis, the mother process of continuation of life, is under the tight surveillance of intra-ovarian factors aiming to develop fertilizable female gametes of utmost quality, a pre-requisite for perpetuation of the species. Ovary, the supreme player of this long run, relies on amalgamation of neuroendocrine, endocrine, and autocrine/paracrine factors.^[23] Regulation of ovarian development and function is primarily controlled by the concert of hypothalamus-pituitary-gonadal axis, FSH, and LH being the sole players to stimulate synthesis of major gonadal sex steroids.^[24] Binding of FSH to its cognate receptor (FSHR) at follicular cell surface promotes growth, while luteinizing hormone receptor (LHR) drives the oocyte toward final maturation.^[24] In addition to gonadotropins and gonadal steroids, an increasing body of evidence in recent times have suggested the participation of a wide spectrum of locally produced non-steroidal peptide factors from the follicular theca-granulosa cell layer and the oocyte itself, thus forming a bi-directional regulatory network within the ovarian follicles. The notion of the oocyte as a passive partner receiving cues from its surrounding counterpart, the somatic follicular cells, has been replaced with the current perception of a complex yet regulated crosstalk between the granulosa- and oocyte-derived factors to orchestrate follicle development.^[25-27] Recent few years have seen remarkable parallel contribution of a variety of growth factors, namely, insulin-like growth factors (IGFs),^[28-30] epidermal growth factor (EGF) family members,^[31-33] TGFβ superfamily members^[34-36] in the ovary of mammals as well as fish. While IGFs role in modulation of steroidogenesis, oocyte maturation, follicular growth, and survival of growing oocytes has been reported widely, recent evidences recognized EGF family as yet another essential mediator of LH action on oocyte maturation in mammals. Moreover, oocyte-derived factors belonging to the TGFβ super family, primarily GDF9 and BMP15, are expressed during early stages of follicle development in the oocytes of mammalian species.^[27,37,38]

BIDIRECTIONAL COMMUNICATION IN THE OVARY: A TARGET FOR EDC ACTION?

Numerous reports from mammalian models have depicted EDC modulation of follicular dynamics at the level of gonadotropin receptors. HPTE (bis-hydroxy methoxychlor), the active metabolite of the organochlorine pesticide methoxychlor, is a potent suppressor of progesterone and LH receptor in rat granulosa cells.^[39] Further, dioxins such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can negatively influence both expression and stability of LH receptor transcripts in the cultured rat granulosa cells.^[40] Not limited to mammals, BPA and diisononyl phthalate can modulate follicle-stimulating hormone receptor and LHR expression in zebrafish ovary.^[41,42] Besides, adverse impact of EDC exposure on ovarian steroidogenesis and local (juxtacrine) factors has been reported earlier. Previously, serum estradiol and progesterone levels have been shown to undergo significant alteration in rats following DDT exposure.^[43] Besides, negative impact of TCDD, a halogenated hydrocarbon, and on aryl hydrocarbon receptors has earlier been implicated in proliferation of granulosa cells, gonadotropin receptor expression, folliculogenesis, and intra-ovarian estrogen signaling.^[44] Besides, EDCs such as polycyclic aromatic hydrocarbons, cadmium, and isoflavones impair ovarian steroidogenesis.^[45-47] Not limited to mammals, the negative impact of BPA on transcript abundance of ovarian steroidogenic enzyme genes has been elucidated in rare minnow.^[48] Recently, the ability of BPA to deregulate epigenetic mechanism leading to negative impact on female reproductive system in zebrafish has also been reported.^[41] Besides, the deleterious impact of EDCs on ovarian growth factor expression and function has also been studied in recent past.^[49,50] TCDD at environmentally relevant concentration can negatively modulate the interaction between IGF1 and FSH leading to decreased LH receptor mRNA abundance in human granulosa cells.^[40] Moreover, studies from human luteinized granulosa cells have shown the up regulation of VEGF and IGF1 due to 1,1-dichloro-2,2-bis(p-chlorophenyl) ethylene, a metabolite of DDT, eventually leading to impaired ovarian steroidogenesis and infertility.^[51] Notwithstanding, BPA attenuation of FSH-induced aromatase expression in human granulosa-like tumor cell line has been shown to involve peroxisome proliferator-activated receptor-gamma signaling and reduced expression of IGF1 and IGF receptors (IGFR).^[50] Although, a group of researchers has demonstrated the altered mRNA expressions of IGF-1/IGFR on BPA exposure in a piscine model;^[49] existing literature and research on fish ovarian growth factors are far from adequate and requires more research initiatives to unveil the molecular mechanism underlying EDC modulation of ovarian functions.

Although EDC-induced ovotoxicity has been demonstrated in several mammalian and non-mammalian models, our understanding of the relative importance of free radicals-induced stress, inflammatory or apoptotic response in promotion of deleterious biological changes in the ovary is limited. For gaining an in-depth knowledge about the extent of damage inflicted by various EDCs and their probable interference with diverse intra-ovarian signaling events, we need thorough research initiatives not only on complex dialogues between neuroendocrine, endocrine, and juxtacrine modulators but also on the redox balance, inflammatory, and apoptotic status of the ovarian follicles.

EDCS ON OVARIAN REDOX BALANCE, INFLAMMATORY, AND APOPTOTIC RESPONSE

Reactive oxygen species (ROS) are the by-products of oxidative metabolism and play crucial role in different cell signaling pathways. An imbalance between the production and clearance of ROS concomitant with attenuated antioxidant enzyme activity leads to the induction of elevated oxidative stress. Altered redox balance may lead to increased lipid peroxidation, heightened apoptosis, inflammation, and mitochondrial dysfunction at various target tissues.^[52] Our recent data demonstrate disruption of metabolic homeostasis congruent with compromised inflammation, insulin signaling, and steatosis in BPA-exposed model freshwater teleost (Labeo bata)^[53] indicating potential threat to aquatic species favored for human consumption. Importantly, EDC-mediated stress response may have malefic impact on biological macromolecules and intracellular machinery causing reproductive dysfunction. Moreover, EDCs may modulate immune cell dysfunction leading to acute or chronic inflammation. Importantly, EDC exposure may induce polycystic ovarian disease (PCOD), an inflammatory ovarian disorder. EDC induced oxidative stress can produce advanced glycation end products (AGEs), pro-inflammatory glycotoxin, that promote activation of pro-inflammatory condition and contributes significantly in ageing and female fertility. While it has been reported earlier that woman with PCOS have high serum AGEs,^[54] exposure to persistent organic pollutants (POPs) such as polychlorinated biphenyls (PCBs), hexachlorobenzene, BPA, and phthalates leads to the formation of AGEs and inflammatory disorders including PCOD.^[54] Moreover, congruent with altered GnRH pulsatality, high dose of BPA has been shown to elicit PCOD-like critical phenotypes characterized through increased plasma testosterone and 17β estradiol (E2) levels in rodents.^[55] Apparently, EDC modulation of sex steroid receptors and steroidogenesis with hyperandrogenism may alter redox balance, inflammatory response, insulin sensitivity leading to PCOS, or inflammatory reproductive disorders. Furthermore, organophosphate and carbamate pesticides have potent role in endocrine disruption and reproductive disorders such as pelvic inflammatory disease that results into infertility, birth defects, and adverse pregnancy outcomes.^[56] Another important inflammatory disorder, commonly referred as endometriosis, reveals growth of endometrial tissue and stroma outside the endometrial cavity and has been considered as one of the main reasons behind infertility in females. Certain EDCs such as persistent chemicals (metals, dioxin like compounds, polybrominated diphenyl ethers, and PCBs) and some non-persistent chemicals (BPA and phthalates) can exacerbate endometriosis such as lesions potentially through altered ER functions and elevated inflammatory signaling.^[57] Interestingly, low-dose BPA exposure in human trophoblastic cell in vitro induces apoptosis and inflammation that leads to the etiopathology of preeclampsia,^[58] an immune system dysfunction related disorder observed at certain stages of gestation due to hypertension where chronic immune activation takes place due to imbalance between CD4⁺ T cells and regulatory T cells as well as pro-inflammatory and anti-inflammatory cytokines.

While enrolment and survival of primordial follicles, selection or loss of follicles through apoptotic screening, luteogenesis, and atresia are of paramount importance for the regulation of well-orchestrated reproductive fitness, elevated oxidative stress has significant negative influence on apoptotic signaling, death of follicular cells, disrupted steroidogenesis, and premature reproductive aging. Congruent with increase in B-cell lymphoma 2 (BCL2)-associated X protein (Bax), transformation-related protein 53 expression but decreased level of Cyclin D2, BPA exposure may prevent follicular cell proliferation, growth of follicles leading to follicular atresia.^[59] BPA-induced apoptosis in granulosa cells involves elevated Bax expression, caspase 3 activation, and attenuated G2-M1 transition promoting follicular atresia and luteal regression.^[60,61] Besides, methylmercury may act as a potent EDC by lowering E2 level in circulation and promoting ovarian primary growth arrest as well as follicular apoptosis.^[62] Moreover, dioxin and DEHP-induced granulosa cell apoptosis in mammals has been reported earlier.^[63,64] Recently, Wang et al.^[65] have shown the effect of another potent EDC soy isoflavones on ovarian apoptosis in rats marked by chromatin marginalization, condensation of the nucleus with increased of apoptotic bodies and vacuolation in granulosa cells. Congruent with reduced Bcl2 expression, elevated expression of Fas, caspase 3, caspase 8, and Bax has also been reported. More recently, BPA modulation of gonadotropin receptors, markers associated with steroidogenesis and growth factor expression along with altered ERα/ERβ homeostasis has been reported from our laboratory as a fallout of elevated oxidative/nitrosative stress, elicited inflammatory, and cell death modulators in the zebrafish ovary.^[66] Clearly, the tight link between EDC mediated deregulated redox balance, inflammation, induction of apoptosis, and loss of ovarian homeostasis has been less investigated topic and need a steadfast approach in future.

EDC MEDIATED DISRUPTION OF ENERGY SENSING HOMEOSTASIS AND ULTIMATE OVARIAN MISFORTUNE

Oxidative stress mediated compromised mitochondrial functions leads to the disruption of energy sensing homeostasis mechanism. Overproduction of ROS, either through leakage of mitochondrial electron transport system or as the byproduct of metabolic pathways, and elevated oxidative stress led to activation of poly ADP (adenosine diphosphate)-ribose polymerase, a DNA repairing NAD+ consuming enzyme. Besides, ROS inactivation of NAM phosphoribosyltransferase enzyme, the rate limiting component in the mammalian NAD biosynthesis pathway, leads to downregulation of energy sensing marker Sirtuin (Silent mating type information regulation 2 homolog) (SIRT1), an NAD-dependent protein deacetylase that links transcriptional machinery to intracellular energetics and participates in the coordination of cellular functions such as cell cycle, response to DNA damage, metabolism, apoptosis, and autophagy.^[67,68] SIRT1 is the regulatory component other than energy sensors such as adenosine monophosphate-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and has crucial role in the regulation of inflammation, apoptosis, and metabolism. Although the molecular mechanism is not yet clearly understood, SIRT modulation of reproductive parameters, for example, spermatogenesis, germ cell apoptosis and premature ovarian failure, or even maintenance of follicle reserve has been reported earlier.^[69,70] Downregulation of SIRT1 results into different metabolic disorders such as disruption of lipid and glucose metabolism that results into insulin resistance, type 2 diabetes which also has negative influence on female reproductive fitness. Moreover, SIRT1 has important role in reproductive events such as folliculogenesis, steroidogenesis, spindle assembly, and chromosome alignment. SIRT1 regulation of folliculogenesis in rat ovary and FSH-mediated steroidogenesis in granulosa cells through Forkhead box O3 a and steroidogenic acute regulatory protein (StAR) have been reported earlier.^[71,72] Moreover, proliferation and secretory activity of porcine granulosa cells is regulated by SIRT1 modulation of NF-κ and p53 function.^[73] Besides, SIRT2 activation of histone H4K16 and α-tubulin may regulate metaphase II spindle assembly, chromosome alignment, and aging process in mouse oocytes.^[74] Transcriptional activation of FoxO by AMPK signaling in nutrient-depleted conditions helps in the maintenance of reproductive activities.^[75] Folliculogenesis, luteinization, progesterone secretion, and oxidative stress response are controlled by SIRT3 modulation of glutamate dehydrogenase, superoxide dismutase 1, catalase, 17beta-hydroxysteroid dehydrogenase 1, StAR, and human cytochrome P450 aromatase in human granulosa and cumulus cells.^[76] Thus, alteration in energy sensors has profound negative impact on female fertility or reproductive fitness.

CONCLUSION

A new life developing within a mother is prone to high risk factors at its various levels of journey starting from preconception, pre-implantation, and fetal period followed by its arrival on earth. Urbanization, pollution, various therapeutic regimens, etc., are proving detrimental for female fertility, requiring utmost concern and attention. To deal and combat with these issues seriously, we need continuous research initiatives to unknot the complex dialogs between neuroendocrine, endocrine and juxtacrine modulators, the basis of reproductive endocrinology [Figure 2]. Accordingly, one can envisage proper assessment of endocrine disruptors with the ultimate goal of making healthy, viable, and superior quality eggs. Restricted production and/or phasing out their use coupled with large scale monitoring are important measures to clean up the environmental burden of EDCs. While, bioaccumulation of EDCs in food chain may pose serious threat to human health and reproductive fitness, more specifically gamete quality, transgenerational adversities, and development of metabolic disorder in both mother and child, microbial degradation of POPs as a component of mitigation strategy may be of significant influence in future.

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Figure 2:

A schematic representation of endocrine-disrupting chemical-induced oxidative stress and its influence on impaired ovarian homeostasis and female infertility (red and blue arrows signify negative and promoting influence, respectively).

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