Abstract
In brief
Bisphenol A (BPA) is a widely produced chemical, mostly used in the production of polycarbonate plastics, and can act as an endocrine disruptor. This paper focuses on the different effects of BPA on ovarian granulosa cells.
Abstract
Bisphenol A (BPA) is an endocrine disruptor (ED), widely used as a comonomer or an additive in the plastics industry. It can be found in food and beverage plastic packaging, epoxy resins, thermal paper and other common products. To date, there have only been several experimental studies to have examined how BPA exposure affects human and mammalian follicular granulosa cells (GCs) in vitro and in vivo; the collected evidence data show that BPA negatively affects the GCs by altering steroidogenesis and gene expression, inducing autophagy, apoptosis and cellular oxidative stress through reactive oxygen species production. Exposure to BPA can also lead to abnormally constrained or elevated cellular proliferation and can even reduce cell viability. Therefore, research on EDs such as BPA is important as it provides some important insights into the causes and development of infertility, ovarian cancer and other conditions related to impaired ovarian and GC function. Folic acid, a biologic form of vitamin B9, is a methyl donor that can neutralize the toxic effects of the BPA exposure and is, as a common food supplement, an interesting option for researching its protective role against ubiquitous harmful EDs such as BPA.
Introduction
Infertility, defined as the inability to conceive naturally, is an ongoing problem among many young couples worldwide (10–15% globally), and the causes of infertility often remain unknown (Pivonello et al. 2020). Problems with fertility can affect either partner, that is, the male, the female or both. The most common causes of female infertility are endometriosis, problems with the fallopian tubes, ovarian infertility such as polycystic ovary syndrome (PCOS), low ovarian reserve, poor ovarian response to hormonal stimulation and premature ovarian insufficiency (POI), also called early menopause (Tamrakar & Bastakoti 2019). In some cases, where the cause of infertility has not been identified, it is known as idiopathic infertility. The causes and risks of infertility are often endogenous, but there are also several exogenous factors that can affect fertility. These include sexually transmitted infections, such as chlamydia, smoking, alcohol and other environmental factors, such as continuous exposure to harmful chemicals or metals (Gallo 2022, Sharpe & Franks 2002). It is also possible that endocrine disruptors play a role.
Endocrine-disrupting chemicals or endocrine disruptors (EDs) are a special class of chemicals that can disrupt and affect the endocrine system and can thus be harmful to humans and animals (Kabir et al. 2015). With all the evidence collected, EDs show the ability to disrupt the endocrine system on many levels and dysregulate the expression of hormones and their respective receptors, which can lead to different pathological outcomes, including female infertility (Jozkowiak et al. 2022, Land et al. 2022 , Bellavia et al. 2023).
Bisphenol A (BPA) is one of the most common and ubiquitous EDs and is widely used in the making of many consumer products, including food packaging and bottles (Konieczna et al. 2015, Chen et al. 2016). The most important sources of BPA are food and beverages, as BPA leaches from food and beverage packaging, especially after heating (Acconcia et al. 2015, Konieczna et al. 2015). BPA in humans is metabolized in the liver and later excreted by urine (Pivonello et al. 2020). Several studies have concluded that BPA negatively affects the reproductive system largely through binding to estrogen receptors (ERs) and thus stimulating estrogen function or via oxidative stress-related damage (Tsutsumi 2005). BPA can affect ovarian function, especially steroidogenesis and follicular growth, and even ovarian morphology (Meli et al. 2020).
BPA can affect various phenotypes that are regulated by the natural hormone, estrogen, which is produced in the ovaries and is responsible for the development and regulation of the female reproductive system and secondary sex characteristics. BPA shows a high affinity for ERs because it expresses the phenol group similar to estradiol (E2) and exhibits estrogen-mimicking behavior (Ma et al. 2019, Stavridis et al. 2022, Tarafdar et al. 2022). BPA has been found to interact with ERs (Wehbe et al. 2020) located both in the cell membrane (G-protein-coupled receptor GPR30) and in the cytoplasm/nucleus (ERα (ESR1) and ERβ (ESR2)), which are encoded by genes ESR1 and ESR2 and distributed in different cells and tissues: ERα in hypothalamus, endometrium and breast cancer cells and ERβ in ovarian granulosa cells (GCs), brain, kidney, bone, heart, lungs, intestinal mucosa and endothelial cells. The BPA activity includes changes of hypothalamus ERα and ERβ expression at relatively low doses (260 μg/kg body weight (BW)/day and lower), thus indicating that even concentrations, lower than the estimated daily intake doses, can affect the organism. Additionally, more studies have evidenced the ability of BPA to bind G-protein-coupled estrogen receptors (GPR30) and interfere with estrogen-activated signaling pathways (Nadal et al. 2018, Stavridis et al. 2022, Chevalier & Fénichel 2015). BPA binds to estrogen and GPCRs with affinity, which is approximately 10,000 times lower than that of the steroid hormone E2; however, its actions are not always dose dependent and as such hard to predict (Wetherill et al. 2007).
All this suggests that BPA could potentially arouse estrogen function and contribute to the pathogenesis of various diseases of the female reproductive system by disrupting the hypothalamic–pituitary–gonadal (HPG) axis (Rebuli et al. 2014). This way, BPA exposure may lead to alterations in female reproductive hormone levels (Miao et al. 2015) and cause steroid hormone imbalance, PCOS, menstrual cycle disorder, POI, and lowered ovarian reserve (Souter et al. 2013, Li et al. 2021, Shi et al. 2021).
The ED BPA has the capacity to bind androgen receptors (ARs) and inhibit androgen function, and, knowing the principle of HPG axis (Fig. 1), it may interfere with gonadotropin-releasing hormone (GnRH) released from hypothalamus (Xu et al. 2018), gonadotropins such as follicle-stimulating hormone (FSH) and luteinizing hormone (LH) released from pituitary, and progesterone synthesized by the adrenal cortex as well as the ovaries (also ovarian corpus luteum during pregnancy) which are all important for different aspects of reproductive health and function in women (Miao et al. 2015, Vahedi et al. 2016 , Chen et al. 2018, Lazúrová et al. 2021, Liet al. 2021). The pulsatile release of GnRH from the hypothalamus induces the release of LH and FSH from the pituitary which act on ovarian follicles to release progesterone and estrogen; E2 is one of three estrogens produced that regulates the release of LH and GnRH by a negative feedback action (Adachi et al. 2007). Because BPA possesses the ability to mimic the action of E2, it can bind to ERα receptor (Fang et al. 2000) and in this way disrupt the functioning of the hypothalamus (kisspeptin KISS1) and pituitary gland; moreover, BPA can also target LH and FSH receptors (LHCGR and FSHR), several growth factors and proteins/enzymes involved in the process of steroidogenesis in the female reproductive tract (Mukhopadhyay et al. 2022).
The effect of BPA on the hypothalamus–pituitary–gonadal axis. BPA may bind to hypothalamic and ovarian granulosa cell estrogen receptors (ERα and ERβ, respectively) and disrupt the release of GnRH from hypothalamus and gonadotropins (FSH and LH) from the pituitary. Consequently, it affects the steroid hormone feedback at all levels which can cause alterations within the female reproductive cycle. BPA, bisphenol A; GnRH, gonadotropin-releasing hormone; FSH, follicle-stimulating hormone; LH, luteinizing hormone.
Citation: Reproduction 165, 5; 10.1530/REP-22-0257
In women with PCOS, urinary BPA levels negatively associate with ovarian steroid hormones such as E2, testosterone, free androgen index, and testosterone, thus suggesting a possible suppressive effect of BPA on ovarian steroidogenesis (Lazúrová et al. 2021). In another study, increased urinary BPA levels in PCOS patients tended to be related to serum anti-Müllerian hormone (AMH) and day-3 FSH levels, while there was no association with inhibin (Zhou et al. 2016). It was evidenced that BPA shows the ability to dysregulate AMH and AMHRII expression in different cells and tissues, including oocytes and cumulus–oocyte complex, which indicates its damaging influence on female reproduction within highly hormone-sensitive follicular environment and ovarian reserve (Zhou et al. 2016, Czubacka et al. 2021, Saleh et al. 2021).
In addition, BPA can affect and disrupt the thyroid hormone system, where due to similar structure, BPA can bind to thyroid hormone receptor (TR; particularly TRα and TRβ) positioned in proximity to the cell nucleus, acting as a transcription repressor, AR, pregnane X receptor and peroxisome proliferator-activated receptor (Marino et al. 2012, Acconcia et al. 2015). So, BPA acts antagonistically by inhibiting TR-induced transcription of thyroxine and triiodothyronine-responsive genes (Kim & Park 2019). In addition, BPA can also affect transcription of factors, involved in thyroid hormone synthesis (TPO, NIS, Tg and PAX8) and thyroid development (Slc5a5) (Gorini et al. 2020).
EDs can be found in almost every industry field, agriculture and retail. Exposure can happen through ingestion, inhalation of air particles and/or gases and dermal absorption (Gore et al. 2014, Rashid et al. 2020). EDs can accumulate in fat tissues and remain there for a long time. The effect that EDs have on human health can often remain hidden and is not evident until later in life (Rattan et al. 2017). There are many different animal studies, epidemiological studies and clinical reports that demonstrate how EDs affect male and female reproduction, thyroid functioning, nervous system, metabolism and related comorbidities such as obesity and diabetes (Crain et al. 2008, Kabir et al. 2015, Bonde et al. 2016, Kiess et al. 2021, Sang et al. 2021). It is worrying that research on animal models (e.g. rats and mice) has shown that perinatal exposure to low doses of BPA during the critical period of hypothalamic sexual differentiation modifies the activity of the HPG axis in the offspring, with advanced puberty and consequences for later life in adult animals (Sadowski et al. 2014, Moustafa & Ahmed 2016, Ma et al. 2017, Oliveira et al. 2017 , Ahsan et al. 2018, Xu et al. 2018 ). Also in humans, some findings suggest that BPA exposure (possibly during pregnancy) appears to be related to an earlier age at onset of puberty (precocious puberty) in girls, especially in those who are obese (Supornsilchai et al. 2016 ).
The harmful effects and toxicity of BPA are connected with both oxidative stress and stress markers (Gassman 2017) and through activating nuclear, orphan (aryl hydrocarbon receptor) and noncanonical steroid hormone receptors, most notably two estrogen-type receptors, ERα and ERβ, which, upon binding, change conformation and travel into the nucleus or activate extranuclear, nongenomic pathways; they bind to DNA to regulate the activity of different genes (as a DNA-binding transcription factors). However, they also have additional functions independent of DNA binding. Different studies have shown that the increased production of reactive oxygen species (ROS) majorly contributes to BPA toxicity by disrupting cellular redox balance, depleting antioxidant reserves and thus causing mitochondrial distress and changes in cellular signaling pathways, which can lead to inflammation or apoptosis (Wetherill et al. 2007, Moon et al. 2012). According to the European Food Safety Authority (EFSA) report (2015), the current daily dose of BPA exposure (tolerable daily intake (TDI): 4 μg/kg BW/day and specific migration limit: 0.05 mg/kg food) does not present a threat to human health (Almeida et al. 2018).
This review aims to offer some new connections and insights regarding the consequences of damage caused by BPA to ovarian tissue and function, with special emphasis on follicular GCs which are significantly involved in steroidogenesis. This review will focus on studies describing how BPA affects human healthy and malignant (immortalized) and animal GCs at their molecular level since they are closely associated with the development and function of the ovaries, follicles and oocytes and consequently with female (in)fertility.
Methods
This review is based on studies that were retrieved by systematic search in PubMed (MEDLINE) using the following keyword combinations: ‘'(BPA OR Bisphenol A AND Granulosa)’' with 67 results, ‘'(BPA OR Bisphenol A AND Fertility)’' with 354 results, ‘'(BPA OR Bisphenol A AND Cumulus)’ with 19 results, ‘'(BPA OR Bisphenol A AND folic*)’ with 27 results and ‘'(BPA OR Bisphenol A AND Oxidative stress)’' with 634 results. Only experimental studies and existing review articles published in English from 2000 to 2022 were selected. Other criteria included experimental studies in humans and mammals and studies that only analyzed the stand-alone effects of BPA. We tried to determine the possible negative effects of BPA on ovarian function, especially follicular GCs.
Findings
We found a number of negative effects of BPA on ovarian function, including GCs, in both humans and animal models. To better understand the negative effects of BPA on the ovaries, we provide some basic knowledge on the ovary, from folliculogenesis/oogenesis, follicles, oocytes and follicular GCs to steroidogenesis.
Folliculogenesis, follicles, oocytes, GCs and steroidogenesis
The most important ovarian unit is the ovarian follicle, a structure of tightly packed somatic cells, mostly granulosa and theca cells, with an oocyte inside (Baerwald et al. 2012). Each follicle has the potential to mature and someday release a mature oocyte. Ovarian follicles exist at several developmental stages, starting with small primordial follicles, which consist of an immature oocyte, surrounded by a single layer of flattened GCs (Yamada & Satoh 1997). Primordial follicles are formed during fetal development, and, as the reserve of immature oocytes, they are the most abundant follicular stage in the ovary. Located in the cortical periphery, most of them never develop beyond the primordial phase (Wallace & Kelsey 2010, Monniaux et al. 2019). The exact mechanism that dictates the transition to the next follicular stage is still unknown (Monniaux et al. 2019). The primordial follicle stage is followed by transformation into primary follicles that are 10 times larger. An increase in size is a consequence of a growing oocyte inside the follicle and a change in the shape of the surrounding GCs that are becoming metabolically and secretorily active in this stage. They communicate with each other through special tight and gap junctions that allow them to exchange small molecules (Baerwald et al. 2012). In this phase, a thick glycoprotein-rich layer, called the zona pellucida, develops around the oocyte and borders the GC layer. During the growth of the follicle, GCs start to multiply, and the previously single-cell granulosa layer becomes thicker, which depicts a transformation into the secondary follicle. In this stage, theca cells from the surrounding environment merge with the outer layer of a secondary follicle and create a new peripheral layer called the thecal layer (Fortune et al. 2000). The thecal layer is, in contrast to the avascular granulosa layer, intertwined with blood vessels, enabling the delivery of nutrients into the follicle. Some secondary follicles develop further into tertiary follicles with a newly formed fluid-filled cavity called the antrum. Antral fluid contains many different protein and steroid hormones, enzymes and ions (Fortune et al. 2000). The granulosa layer of tertiary follicles consists of several layers of GCs, and the thecal layer has now developed into two different layers, the theca interna and theca externa. The oocyte is now protruding into the antral cavity, surrounded by a special layer of GCs called the cumulus oophorus (Monniaux et al. 2019). GCs are very important as they feed the oocyte and direct it in its growth and development inside the follicle. Tertiary follicles can be divided into three subgroups, depending on their stage of maturity: early tertiary follicles, late tertiary follicles and the most mature late or Graafian follicles. During each menstrual cycle, only one Graafian follicle achieves final maturation and evolves into the preovulatory follicle, while the other follicles die (atresia) (Monniaux et al. 2019).
Steroidogenesis in follicular cells
Ovarian follicles produce and secrete three groups of steroid hormones that are essential for successful reproduction (Bremer & Miller 2014). These three groups of hormones are estrogens, androgens and progestins, and all of them have the same precursor, cholesterol. Cholesterol enters theca cells through receptor-mediated endocytosis or is de novo synthesized in theca cells (Miller & Auchus 2011). Steroidogenic acute regulatory (StAR) protein then manages cholesterol transport to the mitochondrial matrix, where the production of steroid hormones occurs (Soccio & Breslow 2003). The production and synthesis of steroid hormones is called steroidogenesis and consists of multiple steps of specific enzymatic modifications of steroid molecules. Each type of ovarian follicular cell has its own role in steroidogenesis, and each type of cell synthesizes specific steroidogenic enzymes. Glandular cells of the theca interna use cholesterol with several intermediate precursor enzymes, such as cytochrome P450 family 17 subfamily A member 1 (CYP17A1), 3β-hydroxysteroid dehydrogenase (HSD3b), 17a-hydroxylase-17,20-desmolase (CYP45017a) and 17β-hydroxysteroid dehydrogenase (HSD17b), to produce androstenedione, which then passes into the GC layer (Marti et al. 2017). GCs convert androstenedione to testosterone, and the latter is then converted to E2 by aromatase (CYP450arom; CYP19A1). E2 later diffuses into the thecal layer and enters the bloodstream from there. This is called the two-cell model of ovarian steroid hormone production (Jones & Lopez 2006). GCs of preantral follicles also produce AMH, which regulates follicular development by preventing the maturation of early follicles, recruiting and selecting dominant follicles and inhibiting others by preventing their access to FSH (Bedenk et al. 2020). As a biomarker, AMH is crucial for assessing the relative size of ovarian reserve and even for measuring some aspects of ovarian function, such as PCOS and POI (Bedenk et al. 2020).
Sources of human GCs and immortalized GC lines for research
An interesting source of GCs is the follicular fluid, obtained by ultrasound-guided aspiration of ovarian follicles to obtain oocytes for in vitro fertilization (IVF) after hormonal stimulation of the ovaries (Beschta et al. 2021). In assisted conception in clinical practice, follicular fluid is usually discarded after oocyte removal but can be a valuable source of GCs for research. GCs can be isolated from follicular fluid on the basis of the expression of various markers, such as FSHR, cytochrome P450 family subfamily A member 1 (CYP19A1), AMH, nuclear receptor subfamily 5 group A member 2 (NR5A2) and mitochondria localized glutamic acid-rich protein (MGARP) (Kossowska-Tomaszczuk et al. 2009, Hatzirodos et al. 2015).
More studies have been performed on the GC line COV434, an immortalized human GC line with the majority of the essential functions preserved; the addition of FSH stimulates cellular growth, proliferation and synthesis and secretion of estrogens (Zhang et al. 2000). It was established from a primary human granulosa tumor from a 27-year-old woman with metastatic GC carcinoma. GCs possess three specific cellular functions: the production of estrogen in response to FSH, the expression of specific biomarkers of apoptosis necessary for inducing follicular atresia and the ability to communicate with other follicular cells. Possessing them all, the COV434 cell line is crucial for ovarian and follicular research in vitro (Zhang et al. 2000).
The KGN cell line is another human ovarian granulosa-like tumor cell line established from a patient with invasive ovarian GC carcinoma. The KGN cell line maintains most of the distinct GC functions, most notably steroidogenesis, normal apoptosis and FSH receptor expression. This cell line thus allows the study of different aspects of human GCs in vitro (Nishi et al. 2001).
Animal models, especially mammalian models, are essential for biological research because of extraordinary physiological and anatomical similarity to human (Barré-Sinoussi & Montagutelli 2015). Most mammals and humans show very similar folliculogenesis, oogenesis and embryonic development. Animal GCs (and other cells of the reproductive system) are very important for research because they provide reliable and normally functioning primary cellular models, which offer, in comparison to hormone-treated luteinized human GCs and immortalized GCs, the closest approximation to in vivo conditions (Sabry et al. 2022). Most researches used bovine, ovine, porcine and murine GCs.
Bisphenol A
One of the most extensively studied EDs is BPA. BPA, 4,40-dihydroxy-2,2-diphenylpropane, is a commonly used organic compound and was first synthesized as a synthetic estrogen (Acconcia et al. 2015). BPA has two phenol groups and is mostly used as a comonomer in the production of polycarbonate plastic and epoxy resins as a flame retardant and antioxidant. It can be found in many different consumer products, mostly food packaging, plastic bottles, nursing bottles, lenses, dental materials, thermal paper and so on. BPA-containing polycarbonate plastic materials are strong, tough, transparent and highly resistant to heat- and cold-induced degradation and acidic environments (Vasiljevic & Harner 2021).
Because of its hormone-mimicking features, BPA can be placed in a group of chemicals called xenoestrogens, more specifically EDs, as they imitate the body’s own 17β-E2 actions by binding to ERs. Although its effect is not proven to be very strong, its wide prevalence raises many concerns. Most people are frequently, even daily, exposed to low concentrations of BPA. Most ingested BPA is through food and drink, as BPA is used in epoxy coatings and plastics. Other sources of human exposure are clothing, thermal paper (e.g. purchase invoices), dental fillings, other devices, air and dust particles from which BPA is absorbed through skin contact (Geens et al. 2012). BPA can be detected in human serum, plasma, saliva, hair, amniotic fluid and cord blood; after being converted to more water-soluble forms through glucuronidation, BPA leaves the body through the urine, with a half-life of approximately 6 h (Huang et al. 2018). BPA can also accumulate in different human and animal tissues, affecting and altering their functions (Cimmino et al. 2020).
An assortment of several independent studies have reported an estimated dietary daily BPA intake of approximately 0.4–4.2 μg/kg BW/day (Ribeiro et al. 2017); this assessment puts human daily exposure in the range of ‘low-dose’ exposure with no documented side effects (the lowest no observed adverse effect level concentration is 20 mg/kg BW/day in rats) (Ribeiro et al. 2017). The EFSA estimates the maximum TDI of BPA to be approximately 50 μg/kg BW/day with possible reevaluations in the future (Ćwiek-Ludwicka 2015). Daily exposure variates between different countries, with Italy being the country with maximal estimated BPA intake and Tunisia the country with the lowest BPA daily intake (Huang et al. 2017). BPA intake also depends on dietary customs such as higher consumption of canned foods, which were related to higher urinary levels of BPA (Liu et al. 2018, Hartle et al. 2022) and occupational groups including thermal paper factory, plastic factory and epoxy resin manufacturing factory workers with higher urinal BPA concentrations than in control groups (Hines et al. 2017, Kouidhi et al. 2017, Ribeiro et al. 2017, Bousoumah et al. 2021).
However, trends of exposure are changing, mostly due to the general transition to BPA analogues in the production of baby bottles and food packaging, but the temporal trends of exposure vary globally (e.g. comparison of temporal trends of BPA exposure between Los Angeles and Beijing showed steady decline of exposure in Los Angeles through the years, while no significant temporal trend was observed in Beijing (Lin et al. 2020)).
Other BPA analogs, such as bisphenol F (BPF) and bisphenol S (BPS), are also known and increasingly used as ‘safer alternatives’ but are much less researched. While BPS is usually used in epoxy glues and thermal receipt papers, BPF has a broader spectrum of use; it can be used in the dental industry, prosthetics, food packaging, plastic industry and so on. (Chen et al. 2016). Human exposure to bisphenol analogs is similar to BPA exposure, that is, oral and dermal, mostly through food and skin contact. Although data and studies on BPA analogs are still limited, reported results show a wide range of different toxic effects, such as cytotoxicity, genotoxicity, endocrine disruption and impaired reproductive health, suggesting that bisphenol analogs might not be as safe as previously thought (Rochester & Bolden 2015). However, more research is still needed to prove the estimated harmful effects of bisphenol analogs and classify them as xenoestrogens.
General health effects of BPA
In 2017, the European Chemical Agency decided that BPA should be classified as a chemical of high concern due to its role as an ED. Many studies on animal models have found strong connections between EDs and different metabolic diseases or altered body functions, as they imitate human estrogen 17β-E2 by binding to ERs and triggering a cellular response (Alonso-Magdalena et al. 2012). BPA works through genomic and nongenomic pathways, which can be triggered by nonspecific dose responses (Alonso-Magdalena et al. 2012). Nonmonotonic dose responses (NMDRs) are doses where the slope of the dose–response curve pattern is not just increasing or decreasing with dose but changes direction within the range of examined doses (Vandenberg et al. 2012). There are few examples how different doses of BPA trigger different cell responses: (1) through binding to ERβ, low doses of BPA alter glucose-induced insulin secretion and decrease the expression of genes encoding calcium channels, decreasing calcium influx in mouse pancreatic β cells, yet activating said genes at high doses through ERβ and ERα; (2) at very low doses, BPA binds to GPR30/G protein-coupled estrogen receptor 1 (GPER), thereby inducing fast-acting intracellular signaling but with doses increasing, BPA first binds to nuclear ERs and later, at really high concentrations, to ARs and TRs, altering their transcriptional activity (vom Saal & Vandenberg 2021). By binding to different receptors, BPA can affect many normal body functions, including causing obesity via thyroid dysfunction (Rezg et al. 2014, Heindel et al. 2017), altering thyroid function by binding to TRs (Andrianou et al. 2016) and many more actions, even affecting male and female fertility (Maffini et al. 2006, Radwan et al. 2018).
BPA can also affect some other diseases, such as breast and ovarian cancer (Guo et al. 2020, Segovia-Mendoza et al. 2020, Sang et al. 2021, Wan et al. 2021), colon cancer (Chen et al. 2015, Jun et al. 2021), respiratory diseases (Gascon et al. 2015, Wu et al. 2021), cardiovascular diseases (Moreno-Gómez-Toledano et al. 2021, Naomi et al. 2022), insulin resistance and type 2 diabetes mellitus (Soundararajan et al. 2019, Wade et al. 2020, Farrugia et al. 2021), obesity (Wu et al. 2020) and depression (Perera et al. 2016, Wiersielis et al. 2020).
Negative impacts of BPA on ovarian function and infertility
More research groups have analyzed the possible effects of BPA on female fertility and the correlation between serum and urine BPA levels and infertility (Ziv-Gal & Flaws 2016). The results from these studies show that women who struggle with infertility have higher serum BPA levels than those of fertile women (Ziv-Gal & Flaws 2016, Pivonello et al. 2020). Furthermore, BPA levels are also associated with poorer oocyte maturation rates, fertilization outcomes and embryo quality and can thus interfere with IVF treatments in infertile patients (Mok-Lin et al. 2010, Bloom et al. 2011). The exact reason for this is still unknown, and not all studies even agree on this association (Mínguez-Alarcón et al. 2015). The resulting mismatch might be due to differences in the sample size and health status among participating women. A study on mice has shown that BPA interrupts the normal cell cycle and upregulates atresia genes in mouse antral follicles, which may cause inhibited follicular growth and even follicular atresia (Peretz et al. 2012), which could be a major factor in decreased fertility. Some studies have also suggested a possible connection between BPA toxicity and PCOS; BPA is believed to worsen PCOS pathogenesis through various mechanisms within the HPO axis (Rutkowska & Rachoń 2014, Kechagias et al. 2020).
BPA toxicity on human follicular GCs
One of the most likely targets of BPA action in the ovaries are GCs in the follicles. This has been demonstrated in both immortalized GC lines and GCs derived from follicular fluid from the IVF process (Fig. 2). Let us summarize some of these findings.
The flowchart of in vivo and in vitro experiments of BPA effects on ovarian granulosa cells. In vitro studies mostly use animal GCs, human immortalized GC cell lines and human GCs, obtained by the follicle aspiration in the IVF procedure. GCs are isolated from the ovaries and then exposed to BPA in vitro and later analyzed on toxic effects. In vivo studies are performed on different animal models, such as rats, mice, pigs and cows, treated with BPA. GCs from treated animals are later isolated and analyzed on their properties.
Citation: Reproduction 165, 5; 10.1530/REP-22-0257
Immortalized GC lines COV434 and KGN
Many studies have shown that BPA adversely affects immortalized GCs in a great variety of ways. We will describe some of them in the following section.
Cell viability and proliferation
BPA affects GC viability in a concentration- and time-dependent manner. Studies have used different methods and BPA concentrations to assess the effect of BPA exposure on immortalized cell line viability and have obtained similar results. Exposure to higher concentrations of BPA can significantly decrease cell viability in a time-dependent manner, especially concentrations of 100 µmol/L and higher (Qi et al. 2020, Bujnakova Mlynarcikova & Scsukova 2021, Huang et al. 2021, Liu et al. 2021a). Some previous studies from the same authors used lower concentrations (up to 10 µmol/L) to evaluate how BPA exposure influences cell viability and obtained no significant results (Mlynarcikova & Scsukova 2020). One study concentrated on assessing environmental concentrations of BPA (0.5, 5, 50 and 500 μg/L) and shorter times of exposure (6 h) and concluded that environmental levels of BPA do not decrease GC viability (Shi et al. 2021a). However, human exposure to BPA is constant, and future studies should focus on how environmental doses of BPA affect GCs over longer periods of time. It is also important to add that GC lines represent malignant cells from granulosa tumors and may act differently in response to BPA exposure.
It is not exactly certain how and if BPA treatment affects GC proliferation, as studies present different results. Bujnakova Mlynarcikova & Scsukova (2021) and Kwintkiewicz and colleagues both examined how standard concentrations (up to 100 µmol/L) affect cell proliferation and obtained opposing results. One study showed no significant effect (Bujnakova Mlynarcikova & Scsukova 2021), while another found that BPA inhibits GC proliferation in a dose-dependent fashion, with a maximum inhibition of 92 ± 2% at 100 μM BPA (Kwintkiewicz et al. 2010). Interestingly, lower, environmental doses of BPA (0.5 and 5 µg/L; 10 and 50 nM) sometimes even promote GC proliferation (Hoffmann et al. 2017, Shi et al. 2021). BPA exposure can affect cell morphology and function, as it significantly increases lipid droplet concentrations and their volumes (Rajkumar et al. 2021) or promotes nuclear and endoplasmic reticulum enlargement due to increased proliferation (Shi et al. 2021a). The increase in the lipid droplet number might be due to changes in cholesterol transport or to dysfunction in the fusion of autophagosomes and lysosomes (Rajkumar et al. 2021).
Steroidogenesis
Along with inducing oxidative stress and cellular apoptosis, BPA exposure may alter steroid hormone synthesis and metabolism in vivo and in vitro and cause steroid hormone imbalance, PCOS and disorders of the estrus cycle in animals. Based on the findings in GC lines thus far, the results can vary. KGN cells exposed to different BPA concentrations (0.5, 5, 50 and 500 μg/L) showed a significant decrease in the production of progesterone and E2 and the E2/testosterone ratio in a dose-independent manner; lower concentrations attenuated E2 levels, while higher concentrations had no effect (Qi et al. 2020). Lin and colleagues reported a dose-dependent effect of BPA exposure on impaired E2 production (Lin et al. 2021a), while another study described how only the lowest and highest BPA doses significantly decreased the levels of progesterone and the E2/testosterone ratio, whereas the middle two concentrations had no significant effect (Shi et al. 2021a). NMDRs go against typical dose–response relationships and are frequently associated with EDs. NMDRs can be explained by opposing effects, influenced by multiple receptors with different affinity or negative feedback loops with an increasing dose (Lagarde et al. 2015). Different experimental results can be a method- and dose-dependent consequence, with low conclusive value; more studies on GC cell lines should be executed in the future, with emphasis on method and protocol standardization.
It is not well understood how BPA influences steroid synthesis; some of the mechanisms are connected with BPA upregulating or downregulating the expression of steroidogenesis-related genes. Studies show opposing results regarding the influence of BPA on specific gene expression; Liu and colleagues (2021) discovered that in KGN cells, low concentrations of BPA (10–11 to 10–8 M) increase the expression of forkhead box L2 (FOXL2), a transcription factor involved in ovarian function and development. BPA also elevates aromatase (CYP19A1) expression and consequently E2 production (Kwintkiewicz et al. 2010) while inducing β-catenin (CTNNB1) expression in the so-called BPA-CTNNB1-FOXL2-CYP19A1-E2 signaling cascade (Liu et al. 2021b). Different outcomes of BPA exposure are presented in an older study from 2012 in which higher concentrations of BPA (50 μM) downregulated CYP19 gene transcription and lowered CYP19 activities (Watanabe et al. 2012) or had no significant effects on CYP19 at all (Shi et al. 2021a). The differences in the results might be a consequence of different exposure concentrations and methods or materials used to analyze the BPA influence. BPA exposure can decrease ferredoxin and ferredoxin reductase expression in a dose-dependent fashion. Both of these genes regulate progesterone synthesis in GCs. Exposure to BPA can also inhibit the expression of the testosterone-related genes hydroxysteroid 17-beta dehydrogenase 2 and 3 (HSD17B2 and HSD17B3) and the E2 production-related gene hydroxysteroid 17-beta dehydrogenase 1 (HSD17B1) while simultaneously upregulating p450 oxidoreductase expression (Shi et al. 2021a). It has been shown that BPA does not affectGPR30 mRNA expression but stimulates KGN cell proliferation and activity by binding to it (Hoffmann et al. 2017). BPA does not appear to affect the expression of nuclear receptors, which might be important targets of BPA (Mlynarcikova & Scsukova 2020).
Gene expression
Vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) are important regulators of blood vessel development and angiogenesis produced by GCs. These factors are important for normal ovarian functioning and physiology, and many pathological conditions are related to VEGF and PDGF dysregulation. BPA significantly decreases the VEGF and PDGF expression in the COV434 cell line, although the mechanism of action has not yet been researched (Bujnakova Mlynarcikova & Scsukova 2021). Exposure to BPA also decreases the expression of the autocrine and paracrine protein chemerin and its receptor G protein-coupled chemokine-like receptor 1 (CLKMR1) in these cells (Hoffmann et al. 2018) and causes overexpression of peroxisome proliferator-activated receptor gamma (PPARG) (Kwintkiewicz et al. 2010).
Oxidative stress
As an ED, BPA impairs cellular redox homeostasis by inducing oxidative stress by decreasing antioxidant enzyme activity and altering the oxidation balance in mitochondria and cells. BPA exposure at higher concentrations significantly increased cellular ROS levels, causing a decrease in the antioxidant activity of the selected antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione synthase (GSH) and mitochondrial SODs (P < 0.05) in the KGN and COV434 cell lines (Huang et al. 2020, Mlynarcikova & Scsukova 2020). Exposure to BPA thus increases the levels of ROS in KGN cells by reducing intracellular antioxidant capacity and increasing cellular Ca2+ concentrations, which induce apoptosis. Huang et al. went even further and expounded the mechanism of BPA-related apoptosis. They studied whether and how inhibitors of associated ERs restrict apoptosis induced by BPA exposure. They discovered that treatment with G15, a GPER inhibitor, significantly prevented KGN cell apoptosis and influenced ROS and Ca2+ levels, while the ERα and ERβ inhibitors methylpiperidinopyrazole (MPP) and PHTPP had no effect. These studies show that BPA-induced oxidative stress causes KGN cell apoptosis through a GPER-dependent pathway and through increasing Ca2+ accumulation. BPA in high concentrations also increases markers of nucleic acid damage, such as 8-OHdG, malondialdehyde (MDA), an indicator of lipid damage, and protein carbonyls, which indicate protein damage (Huang et al. 2020). Macromolecular damage caused by BPA-induced oxidative stress is one of the triggers of cellular apoptosis, and GC apoptosis is one of the main factors of follicular dysfunction and atresia. Further studies with lower exposure concentrations are needed to better assess how even environmental BPA concentrations affect GCs and follicles.
Autophagy
Lin et al. studied how BPA affects KGN cells, an ovarian GC line, with respect to autophagy, apoptosis and endocrine disruption. KGN cells were treated with different concentrations of BPA (1, 10 or 100 nM) for different times (24, 48 or 72 h) and later analyzed with various methods to assess apoptosis and autophagy. BPA-induced cell apoptosis was measured by flow cytometry and TUNEL assays. GC autophagy was examined by immunofluorescence staining and western blotting after 48 h of culture. The 10 and 100 nM BPA exposure groups showed significant increases in the autophagic markers microtubule-associated protein 1A/1B light chain 3B and Beclin 1. Western blot analysis also showed significantly increased levels of 5’ AMP-activated protein kinase (AMPK) and unc-51-like autophagy activating kinase (ULK1) along with decreased mammalian target of rapamycin (mTOR) levels. A study also investigated BPA levels in IVF patients and revealed that the oocyte retirement rate, oocyte maturation rate and embryo implantation rate significantly decreased with increasing urinary BPA concentrations. The researchers also analyzed how BPA impacted mouse folliculogenesis and discovered that BPA exposure leads to a decrease in GC dilation of the follicles and decreased GC-related hormone concentrations (Lin et al. 2021b).
Apoptosis
A few studies that have examined the effect of BPA on the KGN immortalized GC line have shown that BPA exposure reduces cell viability (Huang et al. 2021, Liu et al. 2021). After treatment with different concentrations (30 and 50 μM for 24 h) and analysis with different methods, KGN cell viability was significantly reduced. Exposing KGN cells to higher concentrations of BPA also significantly increased cellular ROS levels and decreased the antioxidant ability of the selected antioxidant enzymes SOD, CAT and GSH (P < 0.05). Flow cytometry was used to determine the effect of BPA on KGN cell apoptosis. A lower concentration of BPA (30 μM) had no significant effect on cellular apoptosis, while a higher concentration (50 μM) did. Western blotting was used to assess how BPA exposure influences pro- and antiapoptotic Bax and Bcl2 protein expression. BPA exposure decreased the Bcl2/Bax ratio and significantly induced apoptosis. Apoptosis-inducing caspase 3 protein activity was also measured using flow cytometry. Caspase 3 activity did not significantly increase after BPA exposure. BPA exposure also causes intracellular stress by activating apoptosis signal-regulating kinase 1 (ASK1), c-jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) signaling cascades. Phosphorylated stress protein levels were analyzed with western blotting (Liu et al. 2021). A similar study on KGN cells was conducted by Huang and colleagues (2021) but with different BPA concentrations (0, 0.1, 1, 10 and 100 μM) and times (12 h and 24 h). They discovered that BPA positively influences KGN cell apoptosis in a concentration- and time-dependent manner in comparison with that in the control group. The effects of BPA exposure on cell apoptosis were stronger after a longer period of exposure (24 h), in addition to a significant change in the protein expression levels of the pro- and anti-apoptosis regulators Bax, Bak, Bcl2 and Mcl-1, as measured with western blotting. This method was also used to test caspase 3 activity, which was significantly increased after exposure to 100 μM BPA. Moreover, BPA exposure also drastically decreased the mitochondrial membrane potential after 24 h of exposure to 100 μM BPA. Phosphorylation of stress response factors, such as extracellular signal-regulated kinases 1/2, p38, JNK, AKT and ASK1, was assessed with western blotting, and a significant increase in the phosphorylation of JNK and ASK1 was found (Huang et al. 2021).
BPA effects on human GCs isolated from follicular fluid
The results of different studies have shown that BPA has a negative effect on GCs obtained from healthy ovaries and follicular fluid retrieved by ultrasound-guided aspiration during the IVF procedure similar to that on immortalized GC lines. This is not surprising, as we mentioned before that immortalized GCs largely retain the properties of ‘healthy’ GCs (Zhang et al. 2000). However, GCs from the follicular fluid in the IVF procedure are more difficult to access for research than commercially available cell lines (e.g. COV434 and KGN) and can only be used with the permission of the Medical Ethics Committee and the written informed consent of the patient. As a result, few studies of this kind have been published but, similar to cell line studies, BPA has been shown to negatively affect steroidogenesis and gene expression in GCs. Some of these findings are as follows:
Steroidogenesis
There are few studies on human GCs to date that have examined BPA and steroidogenesis. Pogrmic-Majkic and colleagues (2019) analyzed how BPA affects STAR and CYP19A1 mRNA levels in human cumulus GCs and reported that higher BPA concentrations (100 μM) significantly increased STAR mRNA levels after a longer exposure time (48 h), whereas lower doses had no significant effect. In contrast, all examined BPA concentrations decreased the CYP19A1 mRNA expression. The observed results corresponded with later analyses on progesterone and E2 production; BPA exposure increased progesterone production and decreased E2 levels (Pogrmic-Majkic et al. 2019). Slightly contradictory outcomes were observed in a 2016 study in which even lower concentrations of BPA significantly impaired E2 production and progesterone synthesis after downregulating steroidogenesis-related proteins and enzymes (CYP11A1, 3β-HSD and CYP19A1). However, BPA exposure had no effect on StAR expression in this study (Mansur et al. 2016). Wang and colleagues compared the BPA effect at lower doses (0.01 to 1 μM for 24 h) on GCs from PCOS and non-PCOS patients with significantly different urine BPA concentrations between the two groups. Aromatase and E2 production was impaired in a concentration-dependent manner in GCs from PCOS patients with higher urine BPA levels but not in GCs from non-PCOS patients. The difference between BPA-related outcomes is probably due to the ovarian hormonal treatment of PCOS patients and the small sample sizes but could suggest that BPA exposure has a role in ovarian dysfunction (Wang et al. 2017). Ehrlich and colleagues took a slightly different approach and examined the connection between CYP19 gene expression in GCs, obtained from IVF pactents, and urinary BPA concentration but found no significant association between the two. However, it is not fully understood how well urine BPA concentration mirrors the BPA concentrations in ovarian cells, so future experiments should focus on different comparison models, perhaps follicular fluid instead of urine (Ehrlich et al. 2013).
The results show that in vitro BPA exposure can affect human GC steroidogenesis and alter steroid hormone production, mostly in a dose-dependent manner, but further studies are needed to examine how every day, chronic exposure to lower BPA concentrations deregulates human ovarian steroidogenesis. The results of the BPA effect on GCs isolated from the follicular fluid of IVF patients may differ from other studies on GCs because of the previous in vivo hormonal treatment of the ovaries, but no major discrepancies were found between studies on human and animal models. To date, there are few studies about how BPA exposure affects GCs. Most of them study BPA exposure in relation to steroid hormones and related genes and/or altered gene expression in GCs. There are no data on BPA-induced oxidative stress in GCs.
Gene expression
Some studies of GCs from follicular fluid from IVF procedures showed that higher concentrations of BPA can significantly affect and upregulate the expression of many GC genes involved in various cell mechanisms, namely, cell cycle progression, cell proliferation, lipid and steroid mechanisms, chromosomal segregation, oogenesis and apoptosis (Mansur et al. 2017). BPA dose dependently increases tribbles pseudokinase 3 (TRIB3) and insulin-like growth factorbinding protein 1 (IGFBP1) and attenuates 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1) expression, which are genes involved in ovarian physiology and folliculogenesis. Gene TRIB3 is involved in lipid metabolism through PPARG activity and insulin-signaling pathways, while IGFBP1 gene participates in glucose metabolism, follicular growth, steroidogenesis and ovulation and HMGCS1 participates in cholesterol synthesis (Prudente et al. 2012, Kessler et al. 2014). Similar to the KGN cell line, BPA downregulates FSH-induced aromatase expression and E2 secretion by stimulating the expression of PPARγ, an aromatase inhibitor (Kwintkiewicz et al. 2010, Pogrmic-Majkic et al. 2019). Interestingly, there is no linear correlation between urinary BPA levels and aromatase (CYP19) expression in vivo (Ehrlich et al. 2013). BPA also decreases the expression of other genes, such as gap junction protein alpha 1 (GJA1) and connexin 43 (Cx43), which codes for the gap junction protein connexin, through the estrogen-receptor-dependent signaling pathway and MAPK pathway (Lin et al. 2021a). BPA exposure also changed the expression of extracellular vesicle miRNAs (EV-miRNAs) and their cellular gene targets but only at high concentrations (200–20,000 ng/mL). The EV-miRNA let-7g-5p was the most deregulated EV-miRNA in a study from 2019 (Rodosthenous et al. 2019). let-7g-5p overexpression can disrupt important GC functions and induce apoptosis and autophagy. In contrast, BPA exposure downregulates miR-212-3p, which is normally present in GCs and is regulated by LH. miR-212-3p participates in cell signaling and proliferation and is important for cell viability (Rodosthenous et al. 2019).
The negative impacts of BPA on GCs from immortalized cell lines and from follicular fluid from IVF procedures are summarized in Fig. 3.
Schematic representation of the GC cellular processes and related genes/proteins that BPA affects. The most researched BPA action is through binding to mER and/or GPR30 receptors, which causes intracellular oxidative stress and increases the expression of genes of oxidative stress pathways. BPA also affects steroidogenesis and alters the synthesis of steroid hormones and expression of associated enzymes. BPA also induces apoptosis by activating pro-apoptotic proteins, a response to BPA-related oxidative stress, and promotes autophagy.
Citation: Reproduction 165, 5; 10.1530/REP-22-0257
Negative effects of BPA on animal GCs in vitro and in vivo
More in vitro studies of GCs have been performed in mammalian animal species, such as mouse, rat, bovine, ovine and porcine. In animals, GCs are retrieved from the ovaries by different methods, such as needle aspiration and enzymatic degradation of ovarian tissue (Fig. 2). Most of these studies have shown that BPA adversely affects ovarian function and GCs in animals, similar to humans. The results of these papers show that animal GCs are very sensitive and respond to even very low BPA concentrations, such as 10−7 to 10−5 M (Xu et al. 2002, Liang et al. 2021). The mechanisms of the negative influence of BPA are very similar to those of human GCs and include cell viability (Bujnakova Mlynarcikova & Scsukova 2018, 2021), steroidogenesis (Xu et al. 2002, Mlynarčíková et al. 2005, Zhou et al. 2008, Samardzija et al. 2018, Wu et al. 2018, Song et al. 2019, Téteau et al. 2020, Bujnakova Mlynarcikova & Scsukova 2021), gene expression (Zhou et al. 2008, Samardzija et al. 2018), oxidative stress/ROS production (Lee et al. 2019, Sabry et al. 2022) and apoptosis (Liang et al. 2021, Xu et al. 2002) of GCs. Tables 1 and 2 show results of published in vitro studies on human and animal GCs, and it can be seen that comparable concentrations and exposure times were used for BPA exposure; moreover, similar negative effects to those in humans were observed for animal GCs. However, the results of some animal studies showed that differences in the outcome (e.g. cell viability outcomes) regarding the concentrations of BPA could possibly be due to different methods, kits and species (Bujnakova Mlynarcikova & Scsukova 2018, 2021).
Reported negative effects of BPA exposure on the ovarian GCs in human in vitro studies.
| BPA concentration | Time of exposure | Negative effects on the ovary | Reference |
|---|---|---|---|
| 20 μg/mL | 48 h | BPA altered genes and involved in cell cycle progression, mitosis, chromosomal segregation, spindle formation, lipid and steroid metabolism. | Mansur et al. (2017) |
| 100 μM | 48 h | Decreased E2 level and CYP19A1 mRNA, increased progesterone production, StAR and PPARγ mRNA expression | Pogrmic-Majkic et al. (2019) |
| 2 and 20 μg/mL | 48 h | Lowered progesterone biosynthesis, altered E2 production, reduced 3β-HSD, CYP11A1 and CYP19A1 mRNA levels and 3β-HSD, CYP11A1 and CYP19A1 protein levels | Mansur et al. (2017) |
| 10–7 M | 24 h | Reduced Cx43 mRNA levels | Lin et al. (2020) |
| 1 μM | 24 h | Reduced aromatase expression and E2 synthesis | Wang et al. (2017) |
| 20, 200, 2000 and 20,000 ng/mL | 48 h | Increased levels of let-7g-5p (20, 20,000 ng/mL), miR-191-5p (200 ng/mL) and miR-532 (2000 ng/mL). Decreased levels of miR-125b, miR-212-3p (20 ng/mL), miR-324-5p (200 ng/mL) and miR-27b-3p, miR-335 and miR-572 (20,000 ng/mL) | Rodosthenous et al. (2019) |
| 100 μM | 48 h | Increased level of PPARγ mRNA (100 μM) | Kwinktkiewicz et al. (2010) |
Reported negative effects of BPA exposure on the ovarian GCs in animal in vitro studies.
| BPA concentration | Time of exposure | Negative effects on the ovary | Reference |
|---|---|---|---|
| 10 μM | 24 h | Increased 17β-E2 concentrations, decreased apoptosis and increased proliferation | Song et al. (2019) |
| 100 μM | 48 h | Increased STAR, CYP11A1 and HSD3B1 mRNA levels and STAR protein levels and decreased basal progesterone production | Samardzija et al. (2018) |
| 100 μM | 24 and 48 h | Decreased cell viability, increased pro-apoptotic BAX protein levels and BPA-induced G2-to-M arrest. | Xu et al. (2002) |
| 10–4 M | 72 h | Decreased progesterone production | Mlynarcikova & Scsukova (2020) |
| 0, 5 and 50 µg/mL | 6, 12, 24, 48 and 72h | Reduced cell viability, increased ROS production and increased antioxidant enzymes SOD1, SOD2, CAT, GPX1 and GPX4 mRNA levels (50 μg/mL; 12 h) | Sabry et al. (2022) |
| 50 and 200 μmol/L | 24 h | Increased cell apoptosis by reducing mitochondrial membrane potential (200 μmol/L) and altered BAX, Bcl-2 and p53 protein expression | Liang et al. (2021) |
| 100 μM | 72 h | Decreased cell viability, reduced basal progesterone production and decreased StAR, CYP11A1 and HSD3B mRNA levels | Bujnakova Mlynarcikova & Scsukova (2018) |
| 10−8 M to 10−5 M | 72 h | Increased basal progesterone levels and decreased E2 production | Mlynarčíková et al. (2005) |
| 50, 100 and 200 μM | 48 h | Decreased cell viability (200 μM), reduced cell proliferation (50, 100 and 200 μM), reduced progesterone secretion (100 and 200 μM), altered E2 production and decreased AR mRNA levels. | Téteau et al. (2020) |
| 10−7 to 10−5 M | 48 h | Increased progesterone production up to 10–5 M and decreased at 10–4 M, decreased E2 levels (10–6 to 10–4 M) and altered mRNA expression of P450arom, P450scc and STAR (10–6 to 10–4 M) | Zhou et al. (2008) |
| 10 µM | 24 and 48 h | Increased E2 synthesis and elevated levels of CYP19A1 and HSD17B proteins. | Wu et al. (2018) |
The advantage of animal models is that they also allow in vivo studies of the effects of BPA, which are not possible in humans. In vivo studies on animals (mostly murine, porcine, bovine and ovine) use a different approach than experiments on isolated GCs: animals are given a specific dose of BPA and then sacrificed after a specific length of time. GCs are then isolated from ovaries/follicles and analyzed. As such research cannot be performed in humans, these findings are very important, as the effects are assumed to be comparable to those in humans. As in in vitro studies, in vivo studies have shown that BPA has a negative effect on the ovaries and GCs in mammalian species.
However, GCs from BPA-exposed animals show different responses to BPA treatment. One study reported drastically decreased E2 levels and attenuated P450arom and StAR protein levels in exposed rat GCs. Lower E2 levels were followed by enhanced follicular atresia and luteal regression, probably as a consequence of BPA-induced GC apoptosis (Lee et al. 2013). Another study on rat GCs (2019) describes opposite outcomes, with BPA significantly increasing aromatase and E2 expression. Experiments on ovine GCs reported in 2011 and 2015 found no connection between E2 levels and BPA exposure but noted elevated expression of ERα, ERβ and AR with a high cell proliferation rate in BPA-treated GCs. BPA can also alter the GC response to FSH, which can lead to weakened follicular and oocyte development (Rivera et al. 2011, 2015).
BPA has also been proven to increase the expression of miRNA-224, a microRNA precursor that is active in mammalian ovaries, specifically GCs. BPA promotes p27 expression in ovine GCs, which results in a previously mentioned increase in cell proliferation (Rivera et al. 2011, 2015).
BPA can affect GCs through receptor binding or can cause oxidative stress via oxidative damage generated by cytotoxic ROS. Banerjee et al. studied the possible harmful effects of BPA-generated ROS on GCs isolated from treated rats. BPA-exposed GCs showed increased levels of lipid peroxidation and the inflammation markers MDA and nitric oxide along with increased levels of the proinflammatory cytokines tumor necrosis factor alpha and interleukin, which are consequences of BPA-induced oxidative stress. BPA exposure also markedly depleted antioxidant levels, namely, GSH and SOD, in treated GCs. The results showed decreased CAT activity and expression, which is clearly a response to BPA-induced oxidative damage (Banerjee et al. 2018).
Protective role of folic acid
Research on EDs is important not only for a better understanding of the causes and development of infertility, ovarian cancer and other diseases but also for reducing the toxic effects of EDs. Folic acid is an interesting option. In general, folic acid (vitamin B9) is a stable synthetic water-soluble dietary supplement that is converted into biologically active 5-methyltetrahydrofolate through several reactions in the liver. The human body’s inability to synthesize it by itself makes folic acid an essential nutrient necessary for cell division and amino acid metabolism (Gliszczynskaswiglo 2007). Folates have an important role as cofactors and coenzymes in the methylation cycle (as a methyl group donor) and DNA/RNA synthesis as donors of one-carbon groups. Folic acid and folates in general are also known for their antioxidant, cardiovascular, anticancer and neuroprotective properties. Folates have been attributed antioxidant activity because they reduce homocysteine concentrations and increase total antioxidant capacity. In this way, they protect biological molecules from oxidative damage induced by free radicals and prevent lipid peroxidation by scavenging and neutralizing highly reactive oxygen radicals. Regular folic acid supplementation (5 mg/day) increases total serum and plasma antioxidant capacity and reduces MDA levels within different researched subgroups (Asbaghi et al. 2021).
In the first few studies in women with elevated BPA levels in biological samples, it was found that when taking folic acid, the time required to conceive was shorter than in women who did not take folic acid (Philips et al. 2018). It was also found that higher urinary BPA concentrations occurred in the Netherlands in 2004–2005, which was positively associated with female obesity and the lack of folic acid use (Philips et al. 2018). Elevated BPA in the urine of patients involved in the IVF program was associated with a minor probability of embryo implantation (66%) in patients receiving <400 μg folate/day as a dietary supplement but not in patients receiving ≥400 μg folate/day (21% more likely to experience embryo implantation); a similar effect has also been identified for the possibility of clinical pregnancy and the birth of a live child (Mínguez-Alarcón et al. 2015). Patients with low serum AMH concentrations (poor ovarian reserve) included in the IVF program had higher blood AMH levels and an improved possibility of pregnancy, including naturally achieved pregnancy, after treatment with methyl donors such as folic acid (Silvestris et al. 2017). This result indicates the importance of this research, as future findings could be used to improve the chances of conceiving and giving birth in women with higher levels of EDs in biological samples who have difficulty conceiving. In established clinical practice, folic acid is prescribed to pregnant women for healthy fetal development (van Gool et al. 2018, Kancherla et al. 2022) but not in patients with high concentrations of EDs in their biological samples. In addition, folic acid has been shown to reduce the toxic effects of EDs in animal models (Bilgi et al. 2019, Lee et al. 2019).
Animal research is very important for a better understanding of the protective role of folic acid, as it can better elucidate its effects. Various animal studies, including in mammals, have shown that the diet or treatment of females with folic acid reduces the toxic effect of BPA on the ovaries, fertility and the development of pups from various aspects: it increases the activity of various antioxidant enzymes (e.g. CAT, glutathione peroxidase, and SOD) and reduces cellular oxidative stress in the blood plasma and liver of females and pups (Mou et al. 2018). Folic acid is also thought to prevent the binding of BPA to the centrosome of the dividing spindle and chromosome centromeres, thereby reducing cell division abnormalities and cell aneuploidy (Parry et al. 2002). It was suggested relatively early that treatment of female animal models with methyl donors such as folic acid nullifies the negative effect of BPA and other EDs at the epigenetic level (Dolinoy et al. 2007). In a study of pigs exposed to BPA during gestation, it was found that this treatment causes intestinal problems (reduced degradation and absorption of food) in pups; however, this negative effect was strongly reduced or eliminated in females receiving a mixture of folic acid, which was associated with methylating DNA in intestinal cells (Liu et al. 2017 ). In an animal model, it has also been found that exposure of females to BPA during gestation causes problems with the pancreas in pups due to hypermethylation of DNA for the insulin-like growth factor 2 (Igf2) gene, which did not occur in females receiving folic acid during BPA exposure (Mao et al. 2017). In mice, the toxic epigenetic effect of BPA (hypomethylation of different genes) was reduced or eliminated by treating (through feeding) females with folic acid (Singh & Li 2012). In vivo animal studies have shown that female exposure to BPA during gestation is also reflected at the epigenetic level in the offspring, so the protective role of folic acid is even more important.
Conclusions
Until now, there has not been much research investigating the effect of BPA on human granulosa cells and how it alters their functions. These studies include different models: immortal human GC lines, human GCs acquired from IVF patients, and animal GCs in vitro or isolated from BPA-treated animals (e.g. murine, porcine and ovine GCs). Most of these studies explore different, specific aspects of BPA exposure but also commonly cover basic methods, such as cell viability, streodiogenesis, gene expressions and so on. Despite this, some of the reported results in animals are contradicting and fail to offer conclusive answers combined possibly due to different species, kits and methods, but most of them confirm the findings in humans. In more articles, GCs were exposed to BPA concentrations different from those relevant in the environment. Despite the negative effects that were observed, the results are not entirely relevant and cannot be applied to the human organism. The results of both human and animal studies show that BPA negatively affects the GCs in terms of cell viability, proliferation, gene expressions, steroidogenesis, oxidative stress, ROS production, apoptosis and autophagy. Negative influences of BPA can also be expressed at the epigenetic level, which is a cause for concern, as they can also be expressed in offspring. Research on EDs such as BPA is not only important for a better understanding of the causes and development of infertility, ovarian cancer and other diseases including impaired ovarian and GC function but also from the point of view of reducing the toxic effects of EDs. An interesting option is folic acid, a methyl donor, which has already been shown to reduce the toxic effects of BPA in both human and animal models. It is important to continue this research in both human and animal models to better understand and reduce the toxic effects of BPA and other EDs in the future.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review.
Funding
The authors acknowledge the financial support from the Slovenian Public Research Agency (research grant J3-2530 to IVK, ‘young researcher’ grant to DCS).
Author contribution statement
D C S performed the literature review, wrote this article and designed the table and figures. I V K designed, revised the article, table and figures and approved the final version to be published.
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