In vivo gamete toxicology in the context of in vitro fertilization: a narrative review.

Success of ART can be measured by both fertilization and pregnancy rates, as well as health and longevity of ART-conceived offspring. Outcomes are highly dependent on the inherent fertility of the parents. As infertility rates continue to rise, optimized ART will only become more crucial. Thus, it is essential that we continue to deepen our understanding of toxicants that may impact gamete quality. Although clinicians are generally aware of potential environmental risks, in vivo gamete exposures are too often excluded during an infertility work-up and treatment. Substantial and growing evidence that gamete toxicology impacts natural fertility and ART outcomes supports change. Assessment of risk should become an integral part of every fertility evaluation. In short, studies of gamete toxicology are more important than ever, and understanding individual exposures and mechanisms could drastically improve future ART success.

The success of ART, as measured by rates of development of euploid embryos, implantation after embryo transfer, and ultimately the full-term birth of a healthy child, is dependent on many factors, including the fertilization capacity of the gametes and the genetic integrity and developmental potential of the resulting embryo. Gamete quality can be compromised when an individual is exposed to environmental pollutants via inhalation, dermal absorption, and ingestion ( 1 – 4 ). This review will highlight in vivo exposures that may affect human gamete quality and, ultimately, ART success, focusing on heavy metals, polyfluoroalkyl substances (PFAS), persistent organic pollutants (POPs), and airborne contaminants (a summary of clinical associations can be observed in Table 1 ). Although much of the existing human data is epidemiological or correlative, valuable in vitro and animal studies can be used to supplement our somewhat limited understanding of human gamete toxicology in terms of mechanism and causation. Our goal is to provide a comprehensive summary of known environmental risks and make a case for individual toxicity assessment to fertility care providers, fertility patients, and the general ART community.

Heavy metals are naturally occurring metallic elements that possess roughly five times the density of water. Some heavy metals, such as iron (Fe), magnesium (Mg), and zinc (Zn), are essential in dietary moderation, whereas others, including mercury (Hg), cadmium (Cd), and lead (Pb), are non-essential, toxic, and tend to both bioaccumulate and biomagnify ( 7 , 8 ). These elements are generally acquired through a balanced diet. Humans are ubiquitously exposed to non-essential heavy metals through their environment via natural (e.g., corrosion and atmospheric deposition) and anthropogenic (e.g., mining, industrial use, and agricultural) sources. Non-essential heavy metals can disrupt cellular structure and function and accumulating within cells and intercellular spaces, including reproductive cells and tissues ( 7 , 9 ). The heavy metals Hg, Cd, and Pb are considered endocrine-disrupting chemicals (EDCs), binding to estrogen receptors and other hormone receptors and disrupting intra-cellular signaling cascades ( 10 – 12 ). Heavy metals may also bind to sulfhydryl enzyme groups, which generally disrupts a variety of energy production and signal transduction pathways ( 13 , 14 ). Heavy metal exposures have been linked with human infertility. In women, blood levels of Pb and Cd are positively associated with ovarian intrafollicular heavy metal levels and ultrastructural alterations in both the oocyte and surrounding cumulus cells ( 15 ). Ultrastructural effects may result from metabolic stress (endoplasmic reticulum and mitochondrial disruption), zona pellucida alterations, and increased exposure to reactive oxygen species (ROS) ( 15 ). Overall disruption to oocyte maturation and quality were significant and cumulus cells became steroidogenically inactive ( 15 ); both could contribute to poor oocyte competence in vivo and in vitro . Additional studies have linked heavy metal exposures to negative reproductive clinical outcomes. In women undergoing IVF, hair concentrations of Hg, lithium (Li), and arsenic (As) are elevated over women who conceived naturally ( 16 , 17 ), and high Hg concentrations in hair resulted in lower ovarian follicle numbers and oocyte yields following ovarian stimulation ( 18 ), with the vast majority of recovered oocytes being immature ( 19 ). High Hg levels in FF are negatively correlated with pregnancy and live birth rates ( 20 ), and high Hg levels in blood are associated with an increased frequency of recovering immature oocytes but not with impaired fertilization ( 21 ). A conflicting study reported no differences in follicle number following ovarian stimulation, fertilization rates, embryo quality, and clinical pregnancy and live birth rates in patients with elevated hair Hg levels when compared to those with normal levels ( 22 ), emphasizing the need for further in vitro and animal studies of Hg toxicity. In men, Hg can cross the blood-testis barrier and accumulate in Sertoli and Leydig cells ( 23 ). Elevated seminal plasma Hg has been associated with both poor sperm morphology and motility ( 24 ). In IVF, a 1 ug/L increase in male urinary Hg has been associated with an 81% decrease in oocyte fertilization rates ( 21 ). Increased FF Pb has been shown to significantly impair fertilization in IVF ( 25 ), whilst elevated Pb levels in blood worsen overall IVF outcomes and are associated with reduced oocyte recovery, decreased implantation and pregnancy rates, and the induction of CpG methylation changes in whole blood ( 26 – 28 ). A study by Kumar et al. found no negative correlation between IVF outcomes and elevated serum Pb, Cd, or Zn ( 29 ), again meriting further research. In men, increased Pb in seminal plasma has been associated with up to a 47% loss in sperm motility, decreased fertilization rates, and reduced biomarkers of sperm function (mannose receptor expression and mannose-induced acrosome reaction) ( 30 , 31 ). Although not correlated with IVF directly, elevated blood Pb in men has also been repeatedly associated with reduced sperm motility and morphology, which could easily translate to impaired IVF outcomes ( 32 – 34 ). Other heavy metals influencing IVF outcomes include chromium (Cr), manganese (Mn), As, Cd, and Zn. Elevated FF Cr and Mn have been associated with reduced oocyte retrieval numbers and an increased percentage of immature oocytes among those recovered ( 35 , 36 ). High As in FF has been associated with fewer high-quality cleavage-stage embryos, whereas high FF Cd has been linked to reduced pregnancy and live birth rates ( 36 ). In serum, elevated Mg and Cd have been shown to reduce IVF success by as much as 83.9% and 89.4%, respectively ( 28 ). In contrast, elevated serum copper (Cu) in women has been associated with increased oocyte recovery, and FF Zn was elevated in women who had undergone ET when compared to those who did not due to issues with uterine receptivity, ovarian swelling, or failed fertilization, indicating that some heavy metals could be beneficial to the IVF process ( 28 ). For men, one study observed no differences in either serum (Zn, Cu), seminal plasma (Zn), or blood (Cd, Pb) heavy metals and IVF outcomes ( 29 ). However, other studies have reported detrimental effects on sperm number, viability, and motility under whole body exposure to As, Cd, Pb, and Hg, possibly a result of epididymal damage ( 37 ). Persistent Organic Pollutants (POPs) are generally lipophilic and get their name from their lengthy environmental half-lives and resistance to various forms of environmental degradation. Both characteristics lead to bioaccumulation and widespread exposures ( 38 , 39 ). POPs include flame retardants [polybrominated diphenyl ethers (PBDEs)], industrial chemicals and their byproducts [polychlorinated biphenyls (PCBs) and per- and polyfluoroalkyl substances (PFAS)], and pesticides [organochloride pesticides (OCPs)] ( 38 , 39 ). This section will discuss general attributes of POPs, specifically PCBs, PBDEs, and OCPs. Further information on PFAS can be found in section 3c . A non-targeted screening of FF in women undergoing IVF revealed complex mixtures of POPs, which affect the microenvironment within the follicle and could alter follicle development, oocyte competence, and embryo quality ( 40 ). Generally, blood concentrations can be used as an approximation of FF concentrations ( 41 ). However, different POPs have different rates of transfer from blood into FF, with the rate of OCB uptake exceeding that of PCBs, and PCBs exceeding PFAS ( 41 ). PCBs and PFAS levels are positively associated with biomarkers of ovarian reserve and responsiveness, however they are negatively associated with oocyte and embryo quality ( 41 – 44 ). PCBs, used in plasticizers, carbonless copy paper, adhesives, inks, and other products, are a chemical class used in the United States between the late 1920s until their eventual ban in 1979 ( 45 ). PCBs are composed of a benzene ring with various degrees of chlorination that generally correlate with toxicity ( 46 ). Many PCBs are also endocrine disrupting and possess estrogenic, thyroidogenic, or androgenic properties ( 45 , 47 , 48 ). Stimulatory or inhibitory effects vary based on chemical structure. Although each PCB congener harbors unique mechanisms of toxicity, they are generally carcinogenic, can cause thyroid conditions, are disruptive to liver, cardiovascular, immune, and neurologic function, and may cause reproductive harm and infertility ( 46 ). Many PCBs are aryl hydrocarbon receptor (AHR) ligands in their nonplanar conformations ( 49 ) The AHR plays an integral role in adaptive immunity and response to environmental stimuli ( 50 ) In the male reproductive system, AHR expression is positively associated with better sperm quality parameters and fertility and has been shown to be integral to testicular structure and function, and fertility ( 51 , 52 ). The AHR in females regulates ovarian, oviduct, and uterine physiology, ovulation, menstrual cyclicity, and overall fertility ( 53 ). Thus, disruption by PCBs or its sister chemical group, the dioxins, can lead to dysregulation of reproductive development and function in both males and females. In a study of wildtype mice, PCB treatments caused significant alterations in uterine morphology, circulating estrogen and progesterone levels, and reduced secondary and antral follicle numbers when compared to an AHR knockout, which showed reduced sensitivity to PCB treatments ( 54 ). In a rat model, performing IVF in the presence of an AHR-agonist resulted in higher rates of polyspermy, early embryo senescence, and disruption of mitosis and nuclear positioning ( 55 ). Simultaneous addition of an AHR antagonist induced recovery of normal embryo development ( 55 ). This strongly suggests that PCBs, dioxins, and other AHR-agonizing chemicals that can accumulate within a follicle and expose a developing oocyte are capable of disrupting downstream embryo development and IVF outcomes. Many PCB compounds alter estrogen receptor (ER) activity, likely due to specific binding with the ER ( 47 , 56 , 57 ). This is supported by clinical evidence, in which exposure to PCBs is associated with lower pregnancy rates ( 58 ), longer time to pregnancy ( 59 – 62 ), abnormal hormonal patterns ( 63 ), and higher rates of teratogenesis ( 64 ), miscarriage ( 65 ), and other reproductive diseases ( 66 ). Elevated maternal PCBs have been linked to methylation changes in genes regulating the conversion of estrone to estradiol ( 66 – 70 ), and serum PCBs in postmenopausal women are negatively associated with serum LH levels, suggesting that the estrogenic effects of PCBs could disrupt ovulation in pre-menopausal women ( 63 ). In 1978, the accidental ingestion of PCB-contaminated rice oil by mothers in Taiwan resulted in reduced birth weights, disfigurement, delayed cognitive development, and behavioral problems in their children, implying a significant teratogenic effect of PCBs ( 64 ). A follow up study of the same cohort noted a 2.16 fold increase of stillbirths in the contaminated population ( 65 ). The chronic treatment of male rats with PCBs is anti-androgenic, resulting in reduced seminal vesicle, epididymal, and prostate weights ( 71 , 72 ). The effects of PCBs in human males have not been studied. PCBs have been identified within the FF and blood of women undergoing IVF ( 41 ). In FF, PCBs were associated with higher antral follicle numbers and lower embryo quality, whereas PCBs in blood were negatively associated with ovarian responsiveness to stimulation ( 41 ). The presence of heavier PCB congeners in FF has also been associated with decreases in ovarian reserve and follicular response to hormone stimulation ( 42 ). When added directly to murine oocyte maturation and IVF culture mediums, PCBs dramatically reduced fertilization rates, even at low concentrations, and were shown to disrupt embryo development ( 73 ). Further, exposure of female mice to dietary PCBs prior to copulation resulted in significant increases in implantation failure and alterations in the expression of implantation-associated genes ( 74 ). The same study observed abnormal uterine endometrial morphology in exposed animals, indicating dual effects on both embryonic and maternal tissues ( 74 ). Mouse perinatal exposure to PCB mixtures exerts a mild estrogenic effect, specifically the downregulation of the hormone response regulator, Wnt7a , and permanent alterations in uterine morphology, including disrupted gland formation and increased myometrial thickness ( 75 ). Changes in uterine morphology during development could alter embryo receptivity, influencing fertility and IVF success during adulthood. This is supported by Meeker et al., who observed that elevated serum PCB was associated with implantation failure in women undergoing IVF ( 76 ). PBDEs are flame retardants that slow ignition and combustion processes via interactions between bromine and free radicals in the gas phase ( 77 ). Despite the EPA ban in 2009, PBDEs were widely used in household products and are still found in the environment and in human samples today ( 77 ). In mammals, PBDEs are known to be endocrine disruptors and specifically detrimental to immune and reproductive health ( 78 , 79 ). PBDEs have been measured in serum and the FF of women undergoing IVF ( 41 ). PBDEs within the FF have been shown to have adverse effects on an immortalized human granulosa cell line, causing oxidative stress and a loss of viability ( 80 ). Exposure of mural and cumulus granulosa cells to PBDEs from contaminated FF has been shown to dysregulate gene expression ( 79 ). This dysregulation has been hypothesized to affect inflammatory responses within the follicle, which may, in turn, affect oocyte maturation and competency for downstream IVF ( 79 ). Epidemiological studies have demonstrated that women with elevated serum PBDEs have longer times to pregnancy and reduced fecundability overall ( 81 ). Certain PBDE congeners found in serum are associated with increased risk of implantation failure and adverse IVF outcomes (pregnancy and live birth) for non-white women, when data were stratified by race ( 82 ). Women with detectable FF PBDEs had a 10X greater chance of implantation failure when compared to women with non-detectable levels ( 83 ). In female rats, dietary PBDE exposure did not affect pregnancy rates, litter size, or fetal weights; however, there was a dose-dependent increase in the number of preantral and antral follicles through increased granulosa cell stimulation ( 80 , 84 ). The female offspring of treated mice had a notable increase in abnormal ovarian follicle development, early onset of puberty, and altered ovarian gene expression, demonstrating epigenetic effects of PBDEs ( 85 ). The effects of PBDEs on male fertility are not well understood. However, two human pilot studies observed that sperm motility, sperm concentration, and testis size decreased with increased serum PBDEs ( 86 , 87 ). OCPs are toxic synthetic pesticides that are highly persistent and capable of bioaccumulation ( 88 ). OCPs are structurally defined by one or more covalently bonded chlorine atoms, making them highly dense molecules, but otherwise exhibit significant diversity ( 88 , 89 ). OCPs include hexachlorocyclohexane (HCH), DDT (1,1,1-trichloro-2,2-bis[4-chlorophenyl]ethane), methoxyclor (MXC), and related compounds. Despite being banned in the United States since the 1970s and 1980s, these chemicals persist in the environment ( 89 – 91 ). OCPs are neurotoxic, and extreme prolonged exposure in non-targeted species, such as humans, can cause headaches, nausea, dizziness, vomiting, tremors, loss of coordination, and confusion ( 89 ). OCPs are generally endocrine disrupting. However, variations in chemical structure result in variable effects on the endocrine system, with some OCPs being estrogenic, some antiestrogenic, and some neither ( 91 ). In animal models, treatment with OCPs can be detrimental to reproductive health and fertility ( 91 – 93 ). MXC stimulates anti-mullerian hormone production in the rat ovary and inhibits follicular development ( 93 ), potentially due to disrupted gap junction formation between the oocyte and cumulus cells, which would prevent oocyte maturation and acquisition of competence ( 91 ). OCPs have been shown to impair ovulation and fertilization events after prenatal and neonatal exposure in rats ( 94 ), and after chronic exposure in adult rabbits ( 91 , 95 ). This phenomena can be partially attributed to altered granulosa cell production of cAMP, which subsequently impairs progesterone synthesis ( 96 ). In addition, elevated concentrations of blood and FF DDT metabolite, DDE, have been shown to enhance the physiological effect of FSH in in vitro- cultured human granulosa cells ( 97 ). Together, FSH and DDE stimulate aromatase production by granulosa cells and estradiol is produced ( 97 ). This cascade can lead to an overproduction of estradiol during early folliculogenesis, which can cause excessive proliferation of granulosa cells and desynchronize oocyte maturation ( 91 , 97 ). In vitro exposure of porcine follicular tissue, with a monolayer of theca interna and granulosa cells, to DDT and its metabolites revealed a time-dependent action of these toxicants to induce either estrogenic or antiestrogenic effects after a single or repeated dose, respectively ( 98 ). In humans, exposure to hexachlorobenzene is negatively associated with ovarian reserve, pregnancy and birth rates ( 41 ). Overall, normal ovarian steroidogenesis and function are drastically altered by OCP toxicity, which clearly affects downstream fertility. A 1985 study found OCPs in the FF of women undergoing oocyte retrieval, and there was a tendency for diminished oocyte recovery and success after embryo transfer in women with elevated FF OCPs ( 99 ). In another study, more than 50% of tested FF samples contained a combination of OCPs, and 33% of participants contained the specific OCP, mirex ( 100 ). Seminal plasma contained fewer OCPs than FF and seminal OCP concentration was not associated with fertilization. However, increased FF DDE was associated with reduced fertilization rates ( 100 ). In contrast, a study by Jarrell et al. reported that the presence of OCPs in FF and serum did not affect time to cleavage or cleavage rates in human IVF embryos ( 101 ). More studies of OCPs in human IVF are needed. The in vitro maturation of porcine oocytes is adversely affected by environmentally relevant concentrations of OCPs, with exposed oocytes yielding embryos with reduced developmental competency ( 102 ). In murine and bovine embryo models, in vitro exposures to DDT and MXC significantly reduced blastocyst formation rates and embryo cell numbers, and increased embryo degeneration rates and the number of blastomeres undergoing apoptosis ( 103 , 104 ). It is likely that in vivo oocyte maturation occurring in the presence of elevated FF OCPs could affect human IVF outcomes following oocyte retrieval. Other environmental chemicals that may be relevant to fertility and IVF outcomes include organophosphates (OPs), phthalates, and bisphenols, including bisphenol A (BPA). OPs are commonly used as insecticides and are potent neurotoxicants, in addition to being endocrine disruptors ( 105 , 106 ). OPs have been shown to induce infertility, pregnancy losses, and teratogenesis in both humans and animals ( 107 – 109 ). In pigs, IVF and subsequent embryo culture were performed in the presence of the organophosphates malathion and diazinon ( 105 ). While diazinon did not affect oocyte viability, it significantly reduced fertilization and morula development rates ( 105 ). Malathion affected embryo viability, overall embryo development, fertilization and morula development rates ( 105 ). OPs can also impact male fertility by damaging germinal and sertoli cells, impairing spermatogenesis, negatively influencing sperm counts, and altering sperm fertilizing capacity ( 105 , 110 , 111 ). These impairments in fertilizing capacity of the sperm and failed embryo development induced by OPs would be predicted to have a major effect on IVF outcomes. The plastic industry has historically used harmful compounds in their production, including PCBs, phthalates, and BPA ( 112 ). Phthalates and BPA are widely used plasticizers that contribute to plastic softening, but concerns over reproductive and developmental toxicity have been raised ( 112 – 114 ). Chronic exposure to phthalates is associated with infertility, decreased egg and sperm quality, and increased incidence of pregnancy loss ( 115 – 119 ). In women undergoing IVF, phthalate metabolites have been detected in FF, and are not only correlated with urinary phthalate concentrations, but also with reduced antral follicle counts, lower pregnancy and live birth rates, and early pregnancy loss ( 118 – 121 ). However, few associations have been established with fertilization and embryo development rates, indicating that phthalates may be disruptive after embryo transfer. Studies of BPA have generally observed male-dominant effects. For example, when zebrafish were exposed to low levels of BPA over multiple generations, a female sex-bias was observed in the F1 and F2 generations, sperm counts and quality were reduced, and decreases in offspring survival were observed ( 114 ). Gene expression analysis in the male gonads revealed alterations in pathways involved in mitochondrial biogenesis and Wnt signaling ( 114 ). Analysis of whole larvae from exposed parents showed abnormal DNA methyltransferase expression ( 114 ). A study in rat Leydig cells observed that BPA exposure increases aromatase expression and thereby reduces testosterone synthesis ( 122 ). Human studies are lacking in this area, however increases in male urinary BPA are associated with reduced sperm concentration and motility, and worsened morphology ( 123 ). Male, but not female, serum BPA has been associated with reduced embryo quality after IVF, further implicating male factor effects in BPA reproductive toxicity ( 124 ). Per- and polyfluoroalkyl substances (PFAS), or “forever chemicals”, are a diverse group of over 15,000 synthetic chemicals that are commonly found in plastics, electronics, fire-fighting foams, non-stick pans and other surfactants ( 125 ). PFAS are a subcategory of POPs that can be further divided into three subcategories: perfluoroalkyl acids (PFAAs), perfluorooctane sulfonic acid (PFOS), and perfluorooctanoic acid (PFOA) ( 126 ). PFAS structures make them highly lipophilic and include a carbon chain in which hydrogens are replaced with fluorine molecules, creating a strong polarized bond that allows for unparalleled environmental persistence ( 126 , 127 ). Humans are ubiquitously exposed to PFAS through consumption of contaminated food (primarily fish, dairy, and meat) and water; however, the use of PFAS-containing products and inhalation are important sources of exposure ( 128 , 129 ). Concern over the effects of environmental PFAS on human health have led many governments to mandate restrictions on PFAS use and application ( 130 ). Unfortunately, PFAS have already accumulated in the environment, as well as human blood and serum, and restricted compounds have subsequently been replaced with equally bad or worse compounds ( 131 , 132 ), liver ( 133 , 134 ), brain, lung, bone, kidney ( 134 ), and other human tissues ( 133 , 135 ). PFAS are capable of acute toxicity when ingested, and their exceedingly long half-lives and ability to interfere with cellular structures and processes play a role in liver disease ( 136 ), benign and cancerous tumors ( 137 – 139 ), metabolic disruption ( 140 – 142 ), immune health ( 143 , 144 ), endocrine disruption ( 145 ), neurobehavioral defects ( 146 ), pre- and neonatal toxicity ( 147 ), and decreased birth weights ( 135 , 148 ). PFAS-associated endocrine disruption, as well as their known accumulation in the umbilical cord ( 149 ), breast milk ( 133 ), and seminal plasma ( 150 ), raise concerns over reproductive toxicities ( 135 ). In a recent metanalysis of PFAS exposures and female fertility, maternal exposures to PFOA and PFOS were negatively associated with fecundability, and increased maternal blood PFOA was associated with an increased risk of infertility ( 151 ). When subanalyzed based on parity, the fertility of nulliparous women was not affected by PFOA and PFOS. Non-PFOS/PFOA PFAS were also not associated with fertility or fecundability outcomes ( 151 ). Perfluorononanoate (PFNA) accumulates in both male and female zebrafish gonads, and PFAS have been shown to cross the follicular barrier and accumulate within FF ( 152 ). Additionally, both PFNA and other PFAAs are known to disrupt the hypothalamic-pituitary-gonadal axis, reducing both male and female fertility ( 153 , 154 ). In women, epidemiological studies have found relationships between PFAS exposure and irregular menses, altered cycle length, earlier age of menopause, and reduced levels of circulating reproductive hormones ( 154 ), all of which are regulated by the hypothalamus-pituitary-ovarian axis. Perfluorooctanesulphonic acid (PFOS) exposure in zebrafish is notably detrimental, not only causing a female sex-bias and dramatically impairing sperm quality, but also causing estrogen and sex hormone-related gene expression changes in the male gonad and structural alterations in both the male and female gonads of exposed juveniles ( 155 , 156 ). Endocrine disruption has also been observed in a rat model treated with an alternate perfluoroalkyl acid, perfluorododecanonic acid, including decreases in serum estradiol ( 157 ). Alterations in ovarian gene expression related to pathways involved in cholesterol transport, steroidogenesis, and estrogen receptor activity were also found ( 157 ). In human epidemiological studies, PFAS exposures have been correlated with decreased ovarian reserve, impaired syncytiotrophoblast steroidogenesis and induced apoptosis, and decreased serum estradiol and progesterone levels ( 158 – 160 ). Ultimately, these alterations could compromise both oocyte availability and quality for downstream IVF. In males, most studies do not show that PFAS exposure alters plasma hormone concentrations ( 161 – 163 ). However, some have reported negative relationships between PFAS levels and sperm counts ( 163 , 164 ), sperm concentration ( 164 ), sperm morphology ( 165 , 166 ), and sperm DNA fragmentation and chromosomal aneuploidies (spermatozoal disomy and diploidy) ( 166 ). One in vitro study acutely exposed murine sperm directly to a PFAS cocktail and observed no evidence of cytotoxicity, and no changes in capacitation, fertilization, or DNA integrity ( 167 ). They did notice a significant developmental delay in day four pre-implantation embryos that had been created from exposed sperm ( 167 ). The mechanisms underlying this stress response have not been specifically identified ( 167 ). Other studies have reported no such changes ( 162 , 168 – 170 ), making the precise effects of PFAS on male fertility unclear and suggesting that effects may depend on the unique mechanisms exerted by specific PFAS molecules and/or their combinations. In a study by Zeng et al. (2023), PFAA was not only detected in over 85% of FF samples from women undergoing IVF, but increased PFAA levels (including n-PFOS, Br-PFOS, n-PFOA, PFHxS, PFDA and PFUnDA) were associated with reduced oocyte maturity rates and the production of fewer high-quality IVF embryos ( 152 ). Another study identified 11 PFAS types from the FF of their cohort ( 40 ). PFAS mixtures containing higher concentrations of branching PFOS isomers show greater correlations with adverse fertility parameters ( 152 ). Mechanistically, PFAAs have been hypothesized to disrupt intracellular signaling, steroidogenesis, and overall viability of granulosa and theca cells in the ovary, affecting both folliculogenesis and oocyte development ( 171 , 172 ). PFAA may also bind to peroxisome proliferator–activated receptors (PPARs), which play an important role in gamete function and oocyte development, although further research is needed ( 152 ). Other studies on FF PFAS levels and IVF found associations between high maternal and paternal plasma PFOA concentrations and negative IVF outcomes ( 43 ), high FF PFAS and decreased fertilization and embryo transfer rates ( 173 ), and high blood and FF PFAS and reduced blastocyst conversion rates ( 174 ). Others have reported no association between PFAS and fertilization ( 175 ) or IVF outcomes ( 176 ), and unexpected increases in fertilization rates after adjustment for other EDCs ( 177 ). PFOS-53-exposed in vitro- matured bovine oocytes exhibited delayed cleavage and hatched-blastocyst rates, as well as reduced blastomere numbers on day 8 post fertilization ( 178 ). Blastocyst quality was not affected ( 178 ). Gene expression analyses reveal that PFOS-treated embryos exhibit altered cell death and survival, increased metabolic stress, and changes in proliferation and differentiation pathways ( 178 ). In porcine embryos, blastocyst rates trended higher after PFHxS exposure, but these blastocysts had higher apoptosis rates ( 179 ). Cell counts were significantly elevated in PFHxS-exposed embryos ( 179 ). A study utilizing PFNA as a toxicant during bovine oocyte maturation saw impaired blastocyst formation with high concentrations of PFNA, and lipid droplet distribution was significantly altered at lower treatment concentrations, indicating disruptions in lipid metabolism at sublethal concentrations ( 180 ). Physiologically-relevant concentrations of PFOS have also been shown to inhibit porcine oocyte viability and maturation by disrupting gap junction-mediated intercellular communication within the cumulus oocyte complex ( 181 ). In the mouse, PFHxS and PFOS exposure resulted in delayed germinal vesicle breakdown and polar body extrusion, induction of ROS, decreased mitochondrial membrane potential, and perturbed meiotic capacity in the oocyte ( 182 ). Although the data have some inconsistencies, the trends strongly support a need for further investigation of the effects of specific PFAS molecules and their mixtures on fertility and IVF outcome in mammalian models and humans. Air pollution is a significant factor in multiple areas of human health, having associations with cancer ( 183 ), cardiovascular ( 184 ), and respiratory conditions ( 185 – 187 ). Considering both background natural air pollution, including volcanoes, forest fires, and biogenic sources, and anthropogenic sources, including vehicular traffic, industrial processes, and fuel combustion, the severity of air pollution continues to increase ( 187 ). Herein, air pollutants will be classified into five main groups: gaseous pollutants (including sulfur dioxide [SO 2 ], nitrate oxide [NO 2 ], carbon monoxide [CO], and ozone [O 3 ], ( 188 )), heavy metals, microorganisms, organic compounds [volatile organic compounds (VOCs)], and particulate matter (PM; PM 2.5 , PM 10 , and constituent black carbon) ( 187 , 189 , 190 ). Although some correlations have been established between individual pollutants and fertility outcomes, humans and animals are generally exposed to complex mixtures that vary widely and depend on location, time, anthropogenic activity, and atmospheric conditions ( 190 ). Thus, both total air pollution as well as the role of individual air pollutants will be discussed in relation to fertility and IVF outcomes. Particulate matter (PM) is a mixture of suspended solid and liquid droplets that are generally less than 100 microns in size ( 191 ). PM is either categorized as primary particulate matter, which is emitted directly into the atmosphere, or secondary particulate matter, which is formed from the reaction of airborne gaseous emissions. Common sources of PM include mining activities, industrial material abrasion and crushing, construction and site work activities, atmospheric formation of secondary organic aerosols, incomplete combustion forming soot, ash from combustion processes, various industrial emissions, and pollen and other biogenic sources, among others ( 191 ). One unique aspect of PM is its ability to harbor microorganisms, including viruses, spores, and bacteria, and other chemicals, such as VOCs, which enables dissemination through transport by PM in ambient air ( 191 ). The largest outdoor sources of gaseous pollutants and airborne heavy metals are from combustion processes, which include fossil fuel-derived energy production, transportation, and other industrial combustion processes ( 7 , 192 ). Among the products of combustion are VOCs, which are carbon-based molecules, either solid or liquid, that readily aerosolize at room temperature ( 193 ). In the indoor environment, VOCs originate from a variety of sources, including building materials and furniture, cosmetics, plastics, and the use of solvents and other common household and laboratory products ( 191 , 193 – 201 ). Associations between air pollution and fertility rates have been established, particularly when exposures occur during adolescence or just prior to conception ( 187 , 202 – 205 ). A 10 ug/m 3 increase in PM 2.5 was associated with a 22% decrease in fecundability ( 203 ). NO 2 levels were also inversely associated with fecundability ( 203 ). Another study reported an 11% decrease in fecundability and a 20% greater chance of infertility with each 10 ug/m 3 increase in PM 2.5 ( 206 ). Mechanistically, air pollutants are thought to access the gonads and impair gametogenesis ( 204 ). There are well-established relationships between ambient air pollution (generally focusing on commonly surveyed pollutants such as NO 2 , SO 2 , CO, and particulate matter) and adverse pregnancy outcomes, including stillbirth ( 207 ), premature birth ( 208 – 211 ), low birthweight ( 208 , 210 , 212 ), and intrauterine growth restriction ( 208 , 213 ), particularly when exposure occurs during the first trimester. In males, endocrine, gonadal, and gametal perturbations in response to air pollution exposures have all been identified. For example, postnatal exposure of male rats to diesel exhaust has been shown to exert negative effects on spermatogenesis ( 214 ). Not only were serum testosterone and estradiol significantly elevated after exposure, but FSH and LH were significantly decreased, potentially due to altered adrenal cortex hormone signaling restricting GnRH production and inhibiting spermatogenesis ( 214 ). Another study found that male rat fetuses exposed to diesel exhaust during development had significantly reduced sertoli cell numbers and overall reduced sperm production as adults ( 215 ). In humans, there is evidence that excess exposure to air traffic pollutants and ozone reduces sperm quality and sperm concentration, and increases sperm DNA fragmentation ( 216 – 218 ). Exposure to NO 2 at the patient’s home address during IVF treatment has been associated with lower pregnancy and live birth rates after embryo transfer, however oocyte recovery and embryo development rates were not affected ( 219 ). Increased O 3 exposures at a patient’s address during IVF treatment was associated with increased live birth rates if O 3 levels were elevated during the period of ovulation induction, but decreased birth rates when O 3 levels were elevated between embryo transfer and parturition ( 219 ). Similar poor outcomes have been reported in females exposed to air pollution. Female mice exposed to ambient, unfiltered air prior to reaching sexual maturity did not exhibit compromised litter size or birth weights when compared to a group exposed to filtered air, but when IVF was performed, embryos from mothers exposed to ambient air had greater inner cell mass:trophectoderm cell ratios than those exposed to filtered air, indicating disturbances during blastocyst lineage specification ( 220 ). Ultimately, this could compromise blastocyst competency and implantation rates ( 220 , 221 ). In a study of women undergoing IVF, black carbon was identified in 100% of FF samples and was also intercalated within ovarian tissues ( 190 ). Metabolomic analyses of FF samples have elucidated mechanistic relationships between exposures to NO 2 during ovarian stimulation in women undergoing IVF and the retrieval of immature oocytes ( 222 ). Elevated NO 2 exposures were also associated with changes in several key metabolic pathways, including hormone synthesis and oxidative stress pathways, which likely play a role in oocyte maturation ( 222 ). Finally, there are associations between elevated ambient PM during conception and early pregnancy loss after ET; however, specific effects on IVF laboratory outcomes were not reported ( 223 ). Although risk of exposure to VOCs is generally more relevant for gametes and embryos in the ART lab, new associations between blood and FF VOCs and reproductive diseases (including PCOS, premature ovarian failure, and endometriosis) have been established (224, 225). These recent studies have aimed to establish volatilomic and/or metabolomic signatures of both endogenous and exogenous VOCs in easily attainable biofluids in order to diagnose impaired fertility and elucidate specific pathophysiologic underpinnings of reproductive disease (224, 225). Ultimately, further research is necessary in this area to strengthen associations and identify specific mechanisms that explain the adverse reproductive effects of exogenous VOCs.

Most agree that a woman is born possessing all the oocytes she will have for her entire life, with all germ cell mitoses occurring during fetal life. As all oocytes arrest at prophase I within an unrecruited ovarian follicle, prophase I oocytes endure prolonged exposure and possible uptake of chemicals encountered throughout childhood, adolescence and adulthood prior to recruitment and ovulation ( 5 ). At the onset of puberty, serial cohorts of follicles mature and swell with follicular fluid (FF), which has also been shown to accrue toxicants, until ovulation, when typically, one of these oocytes will be exposed to the oviductal environment ( 5 , 6 ). Unlike women, male primordial germ cells (PGCs) maintain stem cell-like properties that allow for self-renewal throughout the lifetime, drastically decreasing overall exposure of mature male gametes to environmental toxicants. A post-pubertal male experiences continuous spermatogenesis throughout his reproductive life, with relatively short cycles of development and ejaculation or attrition. Thus, sperm quality is thought to be semi-resistant to environmental exposures and quality may be recoverable after a full spermatogenic cycle. Environmental contaminants come in many forms; however, this review will focus on several chemicals that may impact ART and fertility, including heavy metals, POPs, PFAS, and air pollution.