The effects of plasticizers on the ovary

Abstract

Endocrine disrupting chemicals pose a threat to health and reproduction. Plasticizers such as phthalates and bisphenols are particularly problematic because they are present in many consumer products and exposure can begin in utero and continue throughout the lifetime of the individual. Evidence suggests that these chemicals can have ancestral and transgenerational effects, making them a huge public health concern for the reproductive health of current and future generations. Studies performed in rodents or using rodent- or human-derived tissues have been critical for understanding the toxic effects of plasticizers on the ovary and their mechanisms of action. This review addresses current in vitro and rodent-based in vivo studies investigating the effects of bisphenols and phthalates on ovarian health, female reproduction, and correlations between human exposure and reproductive pathologies.

Introduction

Plasticizers are chemicals used in manufacturing to make materials more flexible and elastic. These chemicals are ubiquitous in the environment and can be found in medical supplies and devices, clothing, toys, food packaging, and building materials. Recently, plasticizers such as bisphenol A (BPA) and phthalates have emerged as endocrine disrupting chemicals (EDC). EDCs are chemicals or chemical mixtures that interfere with the body’s normal hormone response, often through disrupting endogenous hormone production, interfering with hormone signaling, or increasing metabolism of target hormones. Reproductive tissues, including the ovary, are particularly sensitive to the endocrine disrupting effects of these chemicals. The ovary is the female gonad and serves as the site of follicle development (folliculogenesis) and steroid hormone production (steroidogenesis). These processes are hormonally regulated and highly susceptible to EDC exposure. This review describes recent studies performed using rodent models and isolated human tissues that identified the endocrine disrupting actions of common plasticizers such as bisphenols and phthalates on the ovary and highlight human epidemiological studies exploring their links to reproductive pathologies.

Effects of plasticizers on folliculogenesis

One of the primary functions of the ovary is folliculogenesis. During this process, ovarian follicles undergo a maturation process to prepare for ovulation and release of the oocyte for fertilization [1]. The stages of folliculogenesis are depicted in Figure 1. Females are born with a finite number of immature primordial follicles that mature throughout their reproductive life. Either inhibition or acceleration of folliculogenesis can have profound effects on reproduction. Inhibition of folliculogenesis can prevent the development of pre-ovulatory follicles that will release a viable oocyte upon ovulation [2]. Conversely, accelerated folliculogenesis can lead to faster depletion of ovarian follicles and premature onset of reproductive senescence [2]. Exposure to plasticizers has been linked to shifts in ovarian follicle populations, indicating disruption of folliculogenesis (Table 1). Recent rodent studies demonstrated that perinatal exposure to BPA can disrupt germ cell nest breakdown and decrease primary, secondary, and antral follicle populations, and decrease corpus luteum formation, suggesting that exposure may be preventing immature follicles from undergoing maturation [3–5]. In manufacturing, BPA has been replaced with analogues like bisphenol E (BPE) and bisphenol S (BPS). However, exposure to these new analogues in rodents is associated with shifts in follicle distribution towards early stages of folliculogenesis, similar to BPA, calling into question their viability as safer alternatives to BPA [3, 4, 6]. Other studies have shown that adult bisphenol exposure decreased primordial, primary, and antral follicle populations and decreased the formation of corpora lutea in mice [7, 8]. Further, both perinatal and adult exposures to bisphenols increased atresia and the breakdown and resorption of ovarian follicles in both mice and rats [4, 8, 9].

Figure 1. Effects of bisphenols and phthalates on folliculogenesis.

Current studies demonstrate bisphenols and phthalates can impair folliculogenesis by altering follicle populations. Major findings from these studies are summarized here. ↓ indicate decreased and ↑ indicate increased follicle populations relative to vehicle controls. PMM refers to studies performed with phthalate metabolite mixtures.

Table 1:

Effects of bisphenols and phthalates on folliculogenesis

Chemical	Model	Exposure	Main Findings	Ref.
BPA	Mouse	Perinatal Exposure
		0, 0.5, 20, or 50μg/kg/d administered orally to pregnant females on gestational day (GD) 11-birth, female pups euthanized at PND4	Increased germ cells in nests (0.5 and 5μg/kg/d) Decreased primary (0.5μg/kg/d) and secondary (0.5, 20, and 50μg/kg/d) follicles	[3]
		Adult Exposure
		0, 12.5, 25, or 50mg/kg intraperitoneal injections for 10 days, beginning at 6 weeks of age, euthanized immediately	Decreased primordial follicles, primary follicles, and corpora lutea (25 and 50mg/kg) Increased atresia (50mg/kg)	[8]
	Rat	Perinatal Exposure
		25ng/kg/d or 5mg/kg/d subcutaneously PND1–15, euthanized PND105	Decreased primordial follicles at (25ng/kg and 5mg/kg) Increased follicle atresia (5mg/kg)	[9]
		0, 2.5, 25, 250, 2500, or 25000μg/kg/day administered by gavage from GD6-PND21, euthanized immediately	Decreased primordial (250μg/kg/day), primary (2.5μg/kg/day), and pre-antral (2.5μg/kg/day) follicles	[5]
		0, 0.5, 5, or 50mg/kg o subcutaneously on post-natal day (PND) 1–10, euthanized PND75	Decreased corpora lutea and antral follicles Increased atresia (5 and 50mg/kg)	[4]
		Adult Exposure
		25ng/kg/d or 5mg/kg/d subcutaneously PND90-PND105, euthanized either PND106 or PND133	Decreased antral follicles (25ng/kg and 5mg/kg) and corpora lutea (25ng/kg) on PND 106 No change in follicle populations at either dose on PND133	[9]
BPE	Mouse	Perinatal Exposure
		0, 0.5, 20, or 50μg/kg/d administered orally to pregnant females on gestational day (GD) 11-birth, female pups euthanized at PND4	Increased germ cells in nests (0.5 and 50μg/kg/d) Decreased primary follicles (0.5, 20, and 50μg/kg/d)	[3]
BPS	Mouse	Perinatal Exposure
		0, 0.5, 20, or 50μg/kg/d of either administered orally to pregnant females on gestational day (GD) 11-birth, female pups euthanized at PND4	Increased germ cells in nests (0.5 and 20μg/kg/d) Decreased primary follicles (0.5, 20, and 50μg/kg/d)	[3]
		0, 2, 10, 50, 100, or 200μg/kg administered orally to pregnant mice 12.5–15.5 days post-coitus F1 and F2 generation females were euthanized PND21	F1 Increased primordial follicles (2 and 10μg/kg) Decreased secondary (10μg/kg) and antral (2 and 10μg/kg) follicles	[6]
			F2 No significant differences observed
		Adult Exposure
		0, 0.004, 0.375, 37.5, or 375ng/ml in drinking water for 4 weeks, beginning at 5 weeks of age, euthanized immediately	Decreased primary (37.5 and 375ng/ml), preantral (0.004, 0.375, 37.5, and 375ng/ml), and antral (0.375, 37.5, or 375ng/ml) follicles	[7]
	Rat	Perinatal Exposure
		0, 0.5, 5, or 50mg/kg subcutaneously on post-natal day (PND) 1–10, euthanized PND75	Decreased corpora lutea and antral follicles (5 and 50mg/kg) Increased atresia (50mg/kg)	[4]
DBP	Mouse	Adult Exposure
		0, 10, 100, or 1000μg/kg/d DBP administered orally to 80 day old mice for 30 days, euthanized immediately	Increased atresia (1000μg/kg/d) No change in follicle populations	[13]
DEHP	Mouse	Perinatal Exposure	F1 Decreased primordial follicles (750mg/kg/d)	[12]
		0, 0.02, 0.2, 500, and 750mg/kg/d administered orally to pregnant dams GD11-birth F1, F2, and F3 generation females were euthanized at 12 months	F2 No significant differences observed
			F3 Increased primordial follicles (0.2mg/kg/d)
		0 or 0.6mg/kg/d intraperitoneal injections PND0–4, euthanized PND5	Decreased primordial follicles	[10]
		Adult Exposure
		0, 0.02, 0.2, 20, or 200mg/kg/d administered orally to 40 day old females for 10 days, euthanized either immediately or 3, 6, or 9 months post-dosing	3 months post-dosing No significant differences observed	[14]
			6 months post-dosing No significant differences observed
			9 months post-dosing Increased preantral follicles (20 and 200mg/kg/d)
		0, 0.02, 0.2, 20, or 200mg/kg/d administered orally to 40 day old females for 10 days, euthanized 12, 15, or 18 months post-dosing	12 months post-dosing Decreased primary follicles (20mg/kg)	[15]
			15 months post-dosing Increased preantral follicles (0.2mg/kg) Decreased antral follicles (20mg/kg)
			18 months post-dosing No significant differences observed
DiNP	Mouse	Adult Exposure	3 months post-dosing Decreased primordial follicles (0.1mg/kg/d)	[14]
		0, 0.02, 0.1, 20, or 200mg/kg/d administered orally to 40 day old females for 10 days, euthanized either immediately or 3, 6, or 9 months post-dosing	6 months post-dosing Increased primary follicles (200mg/kg/d) Decreased antral follicles (0.02 and 0.1mg/kg/d)
			9 months post-dosing Decreased primary follicles (0.02mg/kg/d)
		0, 0.02, 0.1, 20, or 200mg/kg/d administered orally to 40 day old females for 10 days, euthanized 12, 15, or 18 months post-dosing	12 months post-dosing Decreased primordial follicles (20 and 200mg/kg)	[15]
			15 months post-dosing Increased preantral follicles (0.1mg/kg) Decreased antral follicles (0.1mg/kg)
			18 months post-dosing Increased primordial follicles (0.1 and 200mg/kg) Decreased antral follicles (0.02mg/kg)
Phthalate Metabolite Mixture	Mouse	Perinatal Exposure
		0, 10, 100, or 500x of phthalate metabolite mixture (SELMA study; 33% MBP, 16% MBzP, 21% MEHP, 30% MINP) administered orally to pregnant dams from GD0.5 to birth pups euthanized PND21 and PND90	PND21 Decreased secondary follicles (10, 100, and 500x) Increased follicle atresia (10, 100, and 500x)	[11]
			PND90 Decreased primary (100 and 500x) and secondary (10, 100, and 500x) follicles Increased follicle atresia (100 and 500x)

Several rodent-based studies suggest phthalates can disrupt folliculogenesis. Perinatal exposure to phthalates such as di(2-ethylhexyl) phthalate (DEHP) or a mixture of phthalate metabolites decreased primordial, primary, and secondary follicle populations in mice [10–12]. Brehm et al. (2018) demonstrated that exposing pregnant dams to DEHP affected follicle populations in the resulting pups (filial generation 1, F1) and altered follicle populations in the subsequent F2 and F3 generations, indicating that DEHP can have transgenerational effects on folliculogenesis [12]. In adult mice, subchronic exposure to DEHP or diisononyl phthalate (DiNP), a DEHP replacement chemical, altered follicle populations throughout the lifetime of the aging mouse, whereas exposure to dibutyl phthalate (DBP) did not appear to impact folliculogenesis [13–15]. Similar to bisphenols, some phthalates have been linked to increased follicular atresia in mice [11, 13].

In addition to changes in follicle populations, plasticizers target the oocyte and disrupt its maturation and communication with other cell types present in the follicle. In mice, exposure to BPA in vitro or BPS exposure in vivo can promote germinal vesicle breakdown and increase the meiotic progression of oocytes, respectively [6, 16]. Additionally, bisphenols can impair communication with granulosa cells through downregulation of the oocyte derived paracrine factors bone morphogenetic protein 15 (Bmp15) and growth/differentiation factor 9 (Gdf9), as well as reducing gap junctional intercellular communication between the cumulus cells and the oocyte [6, 16, 17]. In contrast to bisphenols, exposure to phthalates such as DBP reduces germinal vesicle breakdown and disrupts meiosis in mouse oocytes [18, 19]. Use of estrogen receptor (ESR) antagonists indicates that DBP-induced disruption of meiosis is dependent on ESR1 and ESR2 activation, although it is unclear how DBP is modifying ESR signaling [18]. Altogether, these studies demonstrate that bisphenols and phthalates disrupt the normal progression of folliculogenesis and impair oocyte maturation. These effects can have serious repercussions for fertility.

Effects of plasticizers on follicle health and potential mechanisms of action

In vitro, both bisphenols and phthalates can reduce follicle growth and viability in mouse ovarian follicles, suggesting that the health of the follicle is adversely affected with exposure to these chemicals [17, 20, 21]. However, the mechanisms by which plasticizers affect follicle health and development are not fully understood. Recent studies have identified several cellular processes and molecular targets that are altered with plasticizer exposure. These are discussed below.

Oxidative Stress

Several studies have demonstrated that phthalates and bisphenols induce oxidative stress in mouse ovarian follicles, mouse oocytes, and human granulosa cells [18, 22–24]. Liu et al. (2019) demonstrated that the oxidative stress induced by DEHP exposure was dependent on the induction and activity of an oxidative stress related factor xanthine dehydrogenase (XDH) in the neonatal mouse ovary in vitro. Inhibition of XDH with RNAi rescued the ovary from DEHP-induced oxidative stress [24]. Phthalate- or bisphenol-mediated increases in oxidative stress are often accompanied by altered expression/activity of the antioxidant enzymes superoxide dismutase (SOD1), glutathione peroxidase (GPX1), and catalase (CAT) that work to break down reactive oxygen species (ROS) [23, 24]. This altered expression/activity of antioxidant enzymes often leads to accumulation of ROS and results in DNA damage [18, 22, 24]. Additionally, exposure to phthalates such as DBP can decrease ovarian expression of DNA damage repair genes, including breast cancer 1/2 (Brca1/2), ATM serine/threonine kinase (Atm), and RAD50 double strand break repair protein (Rad50) in mouse antral follicles [13]. These data suggest that phthalate or bisphenol exposed ovaries may not be equipped with the machinery to repair ROS-induced DNA damage.

Cell Death Pathways

Mouse-based studies demonstrate that bisphenol or phthalate exposure can increase apoptosis, or programmed cell death, in vivo and in vitro [8, 18, 19, 22, 24, 25]. Further, exposure to BPA or an environmentally relevant mixture of phthalate metabolites is associated with increases in caspase expression/cleavage and/or shifts in expression of pro- and anti-apoptotic factors in mouse ovaries [8, 20, 22]. Additionally, bisphenols and phthalates can induce autophagy in mouse ovarian tissues [10, 22]. Autophagy is a process by which damaged cellular components are degraded, often leading to cell death [26]. DEHP induces autophagy in the mouse ovary through activation of 5’ AMP-activated protein kinase (AMPK) and downstream factors S-phase kinase-associated protein 2 (SKP2) and coactivator associated arginine methyltransferase 1 (CARM1) [10].

Epigenetic Modifications

Plasticizer exposure has been shown to cause epigenetic modifications. In pregnant mouse dams dosed with BPS, analysis of oocytes isolated from the F1 generation showed decreased histone H3 lysine K4 (H3K4) methylation and increased histone H3 lysine K9 (H3K9) methylation compared to controls [6]. H3K4 methylation is considered a mark of active transcription, whereas H3K9 methylation is associated with transcriptional repression [27]. Another study demonstrated that BPA decreased expression of histone deacetylase 7 (HDAC7) in mouse ovarian follicles and increased global acetylation of H3K9 and histone H4 lysine K12 (H4K12) in isolated oocytes [28]. Rattan et al. (2019) showed that exposing pregnant mouse dams to DEHP increases global DNA methylation in the resulting F1 generation and decreases methylation in the F3 generation, suggesting that plasticizer-induced epigenetic changes are transgenerational [29]. Collectively, these studies suggest that phthalate/bisphenol induced changes in methylation or acetylation could be altering chromatin conformation, resulting in aberrant gene expression.

Effects of plasticizers on steroidogenesis

The ovary is the primary source of progestins, estrogens, and androgens that are critical for maintaining reproductive health and fertility. These sex steroid hormones are produced by the concerted actions of steroidogenic enzymes located in the theca and granulosa cells of the ovarian follicle [1]. These steroidogenic enzymes and the hormones they produce are depicted in Figure 2. The process of steroidogenesis is highly susceptible to the endocrine disrupting actions of plasticizers (Table 2). In rodents, gestational/neonatal exposure to BPA, as well as its replacement chemicals BPE and BPS can disrupt steroidogenesis by increasing testosterone production and dysregulating expression of steroidogenic enzymes, including steroidogenic acute regulatory protein (Star) and cytochrome P450 family 17 subfamily A member 1 (Cyp17a1) [3, 4]. Furthermore, gestational exposure to bisphenols can have transgenerational effects, leading to increased circulating estradiol levels and altered expression of steroidogenic enzymes cytochrome P450 family 11 subfamily A member 1 (Cyp11a1), and Cyp17a1 in F3 female mice [30]. Similarly, adult exposure to BPA decreases estradiol production in mice [31].

Figure 2. Effects of bisphenols and phthalates on steroidogenesis.

Current studies demonstrate bisphenols and phthalates can alter steroidogenesis by dysregulating expression of steroidogenic enzymes and altering steroid hormone production. Major findings from these studies are summarized here. ↓ indicate decreased and ↑ indicate increased gene/protein expression or hormone production relative to vehicle controls. PMM refers to studies performed with phthalate metabolite mixtures.

Table 2:

Effects of bisphenols and phthalates on steroidogenesis

Chemical	Model	Exposure	Main Findings	Ref.
BPA	Mouse	Perinatal Exposure
		0, 0.5, 20, or 5μg/kg/d administered orally to pregnant females GD11-birth; euthanized at 3, 6, or 9 months	No changes in hormone levels or steroidogenic enzyme expression at any dose at any time point	[3]
		0, 0.5, 20, or 50μg/kg/d administered orally to pregnant females GD11-birth F3 females were euthanized at 3, 6, or 9 months of age	3 month No change in hormone levels Increased expression of Star (0.5μg/kg)	[30]
			6 months Increased estradiol (0.5μg/kg) Increased expression of Cyp11a1 (50μg/kg) Decreased expression of Cyp17a1 (0.5 and 50μg/kg)
			9 months No change in hormone levels or steroidogenic enzyme expression
		Adult Exposure
		0, 5, 50, or 500μg/kg/d administered to 6 week old mice for 28 days; euthanized immediately	Decreased estradiol (5 and 500μg/kg/d)	[31]
		In Vitro Exposure
		Preantral follicles cultured in vitro with 0, 4.5, or 45μM for 8, or 10 days	Decreased estradiol following 8 and 10 days (45μM)	[17]
	Rat	Perinatal Exposure
		0, 2.5, 25, 250, 2500, or 25000μg/kg/day administered by gavage from GD6-1 year, euthanized immediately	Decreased estradiol (2500 and 25000μg/kg)	[5]
		0, 0.5, 5, or 50mg/kg subcutaneously PND 1–10, euthanized PND75	Decreased progesterone (50mg/kg) Increased testosterone (50mg/kg) and estradiol (50mg/kg)	[4]
		In Vitro Exposure
		Granulosa cells cultured in vitro with 0, 0.1, 1, 10, 50, or 100μM for 24–48 hours	Decreased progesterone (100μM); rescued by co-treatment with 22-hydroxycholesterol or pregnenolone Increased expression of Star, Cyp11a1, and Hsd3b1 (100μM); Star expression restored to control level by co-treatment with cholesterol	[32]
	Human	In Vitro Exposure
		Cumulus granulosa cells cultured in vitro with 0, 0.000001, 0.001, 1, or 100μM BPA for 48 hours	Increased progesterone (100μM) Decreased estradiol (100μM) Increased expression of Star (100μM); expression level is restored to control levels with pharmacological inhibition of either ERK1/2, PPARG, or EGFR signaling Decreased expression of Cyp19a1 (0.000001, 0.001, 1, and 100μM)	[33]
BPE	Mouse	Perinatal Exposure
		0, 0.5, 20, or 50μg/kg/d administered orally to pregnant females GD11-birth; euthanized at 3, 6, or 9 months	3 months No changes in hormone levels Increased expression of Cyp11a1(0.5μg/kg) and Hsd3b1(0.5μg/kg)	[3]
			6 months No changes in hormone levels Increased expression of Star (0.5μg/kg), Cyp11a1 (0.5 and 20μg/kg), Cyp19a1 (50μg/kg)
			9 months Increased testosterone (0.5μg/kg) Increased Star (20μg/kg) and Cyp17a1 (0.5μg/kg)
		0, 0.5, 20, or 50μg/kg/d administered orally to pregnant females GD11-birth F3 females were euthanized at 3, 6, or 9 months of age	3 month No change in hormone levels Increased expression of Cyp11a1 (50μg/kg)	[30]
			6 months No changes in hormone levels Increased expression of Cyp11a1 (0.5μg/kg) Decreased expression of Cyp17a1 (0.5 and 50μg/kg)
			9 months No change in hormone levels No change in steroidogenic enzyme expression
BPS	Mouse	Perinatal Exposure
		0, 0.5, 20, or 50μg/kg/d administered orally to pregnant females GD11-birth; euthanized at 3, 6, or 9 months	3 months No changes in hormone levels Increased expression of Cyp11a1(0.5μg/kg)	[3]
			6 months No changes in hormone levels No change in steroidogenic enzyme expression
			9 months Increased testosterone (50μg/kg) No change in steroidogenic enzyme expression
		0, 0.5, 20, or 50μg/kg/d administered orally to pregnant females GD11-birth F3 females were euthanized at 3, 6, or 9 months of age	3 month No change in hormone levels Increased expression of Cyp11a1 (0.5μg/kg)	[30]
			6 months Increased estradiol (50μg/kg) Decreased expression of Cyp17a1 (0.5 and 50μg/kg)
			9 months No change in hormone levels No change in expression of steroidogenic enzymes
	Rat	Perinatal Exposure
		0, 0.5, 5, or 50mg/kg subcutaneously PND1–10, euthanized PND75	Decreased progesterone (50mg/kg) Increased testosterone (50mg/kg)	[4]
DEHP	Mouse	Perinatal Exposure
		0, 0.02, 0.2, 200, 500, or 750mg/kg/day administered orally to pregnant mice GD10.5-birth F1, F2, and F3 females were euthanized on PND8, 21, or 60	F1 Increased estradiol on PND8 (0.02 and 750mg/kg) and PND60 (750mg/kg) Decreased testosterone on PND60 (0.2mg/kg)	[34]
			F2 Increased progesterone on PND21 (500mg/kg) Decreased progesterone on PND60 (500 and 750mg/kg)
			F3 No change in hormone levels
		0, 0.02, 0.2, 500, or 750mg/kg/day administered orally to pregnant mice GD10.5-birth F1, F2, and F3 females were euthanized on PND21	F1 Increased Hsd3b1 (750mg/kg)	[29]
			F2 Decreased Hsd3b1 (0.02mg/kg), Hsd17b1 (0.02, 500, and 750mg/kg), and Cyp19a1 (0.02, 500, 750mg/kg)
			F3 Decreased Hsd17b1 (0.02 and 750mg/kg) and Cyp1b1 (0.2mg/kg)
		0, 0.02, 0.2, 500, and 750mg/kg/d administered orally to pregnant dams GD11-birth F1, F2, and F3 generation females were euthanized at 12 months	F1 Decreased testosterone (500mg/kg) Increased estradiol (500 and 750mg/kg)	[12]
			F2 Decreased progesterone (0.2mg/kg) and testosterone (0.02mg/kg)
			F3 Decreased testosterone (0.02 and 500mg/kg) Increased estradiol (0.02mg/kg)
		Adult Exposure		[14]
		0, 0.02, 0.2, 20, or 200mg/kg/d administered orally to 40 day old females for 10 days, euthanized either immediately or 3, 6, or 9 months post-dosing	Immediate Increased progesterone (200mg/kg) Decreased estradiol (20mg/kg)
			3 months post-dosing No changes in hormone levels
			6 months post-dosing Increased estradiol (0.02 and 0.2mg/kg)
			9 months post-dosing No changes in hormone levels
	Rat	Adult Exposure
		0, 300, 1000, or 3000mg/kg/d administered orally to adult rats for 4 weeks; euthanized immediately	Decreased progesterone (1000 and 3000mg/kg), testosterone (3000mg/kg), and estradiol (1000 and 3000mg/kg)	[25]
DiNP	Mouse	Adult Exposure
		0, 0.02, 0.1, 20, or 200mg/kg/d administered orally to 40 day old females for 10 days, euthanized either immediately or 3, 6, or 9 months post-dosing	Immediate Decreased testosterone (0.02, 0.2, and 200mg/kg DiNP) and estradiol (200mg/kg DiNP)	[14]
			3 months post-dosing Decreased progesterone and testosterone (0.1mg/kg DiNP)
			6 months post-dosing Decreased testosterone (0.1mg/kg DiNP) Increased estradiol (0.1 and 200mg/kg DiNP)
			9 months post-dosing No changes in hormone levels
MEHP	Rat	In Vitro Exposure
		Granulosa cells cultured in vitro with 0, 25, 50, 100, or 200μM for 24 hours	Increased progesterone and estradiol (100 and 200 μM)	[35]
Phthalate Metabolite Mixture	Mouse	Perinatal Exposure
		0, 10, 100, or 500x of phthalate metabolite mixture (SELMA study; 33% MBP, 16% MBzP, 21% MEHP, 30% MINP) administered orally to pregnant dams from GD0.5 to birth pups euthanized PND21 and PND90	PND21 Decreased Cyp19a1 (10 and 100x)	[11]
			PND90 Increased Star (500x) Decreased Cyp17a1 (100 and 500x)
		In Vitro Exposure
		Antral follicles cultured in vitro with 0, 0.065, 0.65, 6.5, 65, or 325μg/ml of a phthalate metabolite mixture (36.7% MEP, 19.4% MEHP, 15.3% MBP, 10.2% MiBP, 10.2% MiNP, and 8.2% MBzP) for 24 or 96 hours	24 hours hormone levels Increased pregnenolone (325μg/ml), progesterone (65μg/ml), androstenedione 65 and 325μg/ml), testosterone (6.5, 65, and 325μg/ml), and estrogen (65μg/ml)	[20]
			24 hours steroidogenic enzyme expression Increased Star (65 and 325μg/ml), Cyp1a1 (325μg/ml), and Cyp1b1 (0.65μg/ml) Decreased Cyp11a1 (325μg/ml), Hsd3b1 (0.65, 6.5, 65, and 325μg/ml), Cyp17a1 (0.65, 65, and 325μg/ml), Hsd17b1 (65 and 325μg/ml), Cyp19a1 (65 and 325μg/ml), Cyp1a1 (0.65μg/ml), and Cyp1b1 (325μg/ml)
			96 hours hormone levels Increased progesterone (6.5, 65, and 325μg/ml), dehydroepiandrosterone (DHEA) (65μg/ml), and testosterone (65 and 325μg/ml) Decreased estradiol (325μg/ml)
			96 hours steroidogenic enzyme expression Increased Star (65 and 325μg/ml), and Cyp1a1 (325μg/ml) Decreased Cyp11a1 (325μg/ml), Hsd3b1 (325μg/ml), Cyp17a1 (65 and 325μg/ml), Hsd17b1 (65 and 325μg/ml), Cyp19a1 (6.5, 65, and 325μg/ml), and Cyp1b1 (65 and 325μg/ml)

In vitro, BPA exposure can affect steroidogenesis by decreasing estradiol production in cultured mouse preantral follicles [17]. In primary rat granulosa cells, BPA decreased levels of progesterone and increased expression of Star, and these effects were partially reversed by co-treatment with cholesterol, suggesting that BPA may be impairing steroidogenesis by disrupting cholesterol homeostasis [32]. In contrast, BPA exposure increased progesterone in human cumulus granulosa cells [33]. This was accompanied by increased Star expression that was dependent on peroxisome proliferator activated receptor gamma (PPARG), epidermal growth factor receptor (EGFR), and extracellular signal-regulated kinase 1/2 (ERK1/2) activity [33]. These data suggest that the effects of BPA on steroidogenesis may be species specific.

Recent studies have demonstrated endocrine disrupting effects of phthalates on steroidogenesis. Gestational exposure to DEHP in mice can alter steroidogenesis in multiple generations, leading to increased estradiol and decreased testosterone levels in the F1 generation, and decreased progesterone levels in the F2 generation [12, 34]. Additionally, gestational DEHP exposure can induce transgenerational changes in the ovarian expression of steroidogenic enzymes, including hydroxysteroid 3-beta dehydrogenase 1 (Hsd3b1), hydroxysteroid 17-beta dehydrogenase 1 (Hsd17b1), cytochrome P450 family 19 subfamily A member 1 (Cyp19a1), and cytochrome P450 family 1 subfamily B member 1 (Cyp1b1) [29]. Repouskou et al., (2019) reported that gestational exposure to a mixture of epidemiologically defined phthalate metabolites did not alter circulating estradiol, but it decreased expression of steroidogenic enzymes Cyp19a1 and Cyp17a1 at 21 and 90 days post exposure in mice, respectively [11]. Adult exposure to DEHP or the DEHP replacement chemical DiNP altered levels of estradiol, progesterone, and testosterone compared to control mice and these altered hormone levels persisted up to 9 months post exposure [14].

In vitro, mouse antral follicles exposed to an environmentally relevant mixture of phthalate metabolites containing monoethyl phthalate (MEP), mono(2-ethylhexyl) phthalate (MEHP), monobutyl phthalate (MBP), monoisobutyl phthalate (MiBP), monoisononyl phthalate (MiNP), and monobenzyl phthalate (MBzP) secreted lower concentrations of estradiol and higher concentrations of progesterone and testosterone than vehicle exposed follicles [20]. These changes in hormone levels were coupled with increased expression of Star and cytochrome P450 family 1 subfamily A member 1 (Cyp1a1) and decreased expression of Cyp19a1 compared to control [20]. In contrast, MEHP exposure in vitro increased both estradiol and progesterone production in primary rat granulosa cells compared to control [35]. This difference in estradiol response could be due to the net effect of multiple phthalates in the former study, a lack of theca-granulosa communication in the latter study, and/or potential inter-species differences existing between mice and rats [20, 35].

Effects of plasticizers on reproductive outcomes

Current data indicate that bisphenols and phthalates can affect different parameters of female reproduction and fertility. In mice, gestational exposure to BPA, BPE, or BPS accelerated the onset of puberty, disrupted estrous cyclicity, reduced pregnancy rate, and reduced the number of mouse pups born compared to controls [3]. In contrast, neonatal exposure to either BPA or BPS delayed onset of puberty in rats [4]. The differences observed in these studies could be attributed to different windows of exposure (gestational vs. neonatal) or inter-species differences between mice and rats in response to toxicants [3, 4]. Transgenerational studies show that exposing pregnant mouse dams to BPA, BPE, or BPS can impact the reproductive function of F3 generation females, by accelerating the onset of puberty, disrupting the estrous cycle, and reducing pregnancy rate [30]. Similarly, gestational exposure to DEHP can exert transgenerational effects by altering estrous cyclicity in the F1 and F3 generations in mice [12]. In adults, exposure to DEHP (in mice and rats) or DiNP (in mice) has been shown to alter estrous cyclicity [15, 25, 36]. These changes in cyclicity, along with reduced fertility, can persist long after termination of DEHP or DiNP exposure [15, 36].

Correlation between plasticizer exposure and reproductive pathology in humans

Recent studies suggest links between plasticizer exposure and known reproductive pathologies in human populations. Indeed, epidemiological studies support a link between phthalate exposure and the occurrence of infertility. Detectable levels of phthalate metabolites (MEHP, MBP, MEP, and MBzP) were measured in the follicular fluid of women undergoing in vitro fertilization (IVF) [37, 38]. Moreover, higher concentrations of metabolites like mono-methyl phthalate (MMP), MEP, and MEHP were correlated with lower concentrations of steroid hormones in the follicular fluid of these women [38]. Recent data demonstrate that mouse ovarian follicles can metabolize parent phthalates into their monoester metabolites [39]. Elevated levels of phthalate metabolites in follicular fluid may be due to local metabolism within the ovary. Other studies have detected higher levels of BPA, MiBP, and MBP in follicular fluid, urine, and serum, respectively, from women presenting with diminished ovarian reserve compared to samples from fertile women [31, 40, 41].

Interestingly, recent rodent studies have shown increased incidence of ovarian cyst formation upon exposure to plasticizers [10, 12, 42, 43]. Cystic ovaries, along with chronic anovulation and hyperandrogenism, are classic features of polycystic ovarian syndrome (PCOS) [44]. Elevated levels of BPA in urine and serum and DEHP in follicular fluid were detected in samples from women with PCOS compared to women without PCOS [45–48]. BPA positively correlated with higher testosterone and free androgen index (measure of testosterone relative to sex hormone binding globulin) in women with PCOS [47]. Hyperinsulinemia and peripheral insulin resistance are also observed in a subset of women diagnosed with PCOS [44]. Elevated urinary BPA was positively associated with serum insulin levels and incidence of insulin resistance [45]. Together, these observations suggest that plasticizer exposure could be a contributing factor in the etiology of PCOS and subfertility/infertility.

Conclusions

It is well established that plasticizers are endocrine disrupting chemicals with deleterious effects on ovarian health. Current studies document changes in follicle populations signifying impaired folliculogenesis and dysregulation of steroid hormone production following bisphenol or phthalate exposure (Figures 1 and 2, respectively). Based on these studies, it is critical to assess the continued use of phthalates and bisphenols in consumer products because they exhibit clear ovotoxicity, their effects can persist long after termination of exposure, they can impact multiple generations with one window of exposure, and they may play a role in the development of reproductive diseases like PCOS.

Abbreviations

AMPK

5’ AMP-activated protein kinase

ATM

ATM serine/threonine kinase

BPA

bisphenol A

BPE

bisphenol E

BPS

bisphenol S

BMP15

bone morphogenetic protein 15

BRCA1/2

breast cancer 1/2

CAT

catalase

CARM1

coactivator associated arginine methyltransferase 1

CYP1A1

cytochrome P450 family 1 subfamily A member 1

CYP1B1

cytochrome P450 family 1 subfamily B member 1

CYP11A1

cytochrome P450 family 11 subfamily A member 1

CYP17A1

cytochrome P450 family 17 subfamily A member 1

CYP19A1

cytochrome P450 family 19 subfamily A member 1

DHEA

dehydroepiandrosterone

DBP

dibutyl phthalate

DEHP

di(2-ethylhexyl) phthalate

DiNP

diisononyl phthalate

EDC

endocrine disrupting chemicals

F#

filial generation

GD

gestational day

GPX1

glutathione peroxidase

GDF9

growth/differentiation factor 9

HDAC7

histone deacetylase 7

H3K4

histone H3 lysine K4

H3K9

histone H3 lysine K9

H4K12

histone H4 lysine K12

HSD3B1

hydroxysteroid 3-beta dehydrogenase 1

HSD17B1

hydroxysteroid 17-beta dehydrogenase 1

MBP

monobutyl phthalate

MBzP

monobenzyl phthalate

MEP

monoethyl phthalate

MEHP

mono(2-ethylhexyl) phthalate

MiBP

monoisobutyl phthalate

MiNP

monoisononyl phthalate

MMP

mono-methyl phthalate

PCOS

polycystic ovarian syndrome

PND

post-natal day

RAD50

RAD50 double strand break repair

ROS

reactive oxygen species

SOD1

superoxide dismutase

SKP2

S-phase kinase-associated protein 2

STAR

steroidogenic acute regulatory protein

XDH

xanthine dehydrogenase