Trace exposures to the toxic metals mercury (Hg), cadmium (Cd) and lead (Pb) may threaten human reproductive health. The aim of this study is to generate biologically-plausible hypotheses concerning associations between Hg, Cd, and Pb and in vitro fertilization (IVF) endpoints. For 15 female IVF patients, a multivariable log-binomial model suggests a 75% reduction in the probability for a retrieved oocyte to be in metaphase-II arrest for each microg/dL increase in blood Pb concentration (relative risk (RR)=0.25, 95% confidence interval (CI) 0.03-2.50, P=0.240). For 15 male IVF partners, each microg/L increase in urine Cd concentration is associated with an 81% decrease in the probability for oocyte fertilization (RR=0.19, 95% CI 0.03-1.35, P=0.097). Because of the magnitude of the effects, these results warrant a comprehensive study with sufficient statistical power to further evaluate these hypotheses.

Low-level environmental exposure to Hg, Pb and Cd may interfere with pregnancy during in vitro fertilization (IVF). The aim of this study was to generate hypotheses concerning associations between background exposures and pregnancy. In modified Poisson regression models including 24 women and adjusted for urine Cd and creatinine, blood Pb, age, race and smoking, 1 μg/L increases in blood Hg are associated with decreases of 35% (P=0.03) and 33% (P=0.01) in clinical and biochemical pregnancies, respectively. In alternate Poisson models including 26 women and adjusted for blood Pb, blood Hg, age, race and smoking, 1 μg/L increases in blood Cd are associated with decreases of 94% (P=0.01) and 82% (P=0.04) in clinical and biochemical pregnancies, respectively. No effects are detected in 15 men, although inverse associations are suggested for urine cadmium and pregnancy. These data suggest that low-level, background exposures to Hg and Cd may interfere with pregnancy following IVF.

Persistent organic pollutants (POPs) are ubiquitously distributed among the U.S. population and adversely impact human reproduction. These compounds have been detected in human ovarian follicular fluid (FF), where they directly contact a developing oocyte. As a pilot investigation, we measured 43 polychlorinated biphenyl (PCB) congeners, p,p'-dichlorodiphenyltrichloroethane (DDT), and its persistent metabolite p,p'-dichlorodiphenyldichloroethylene (DDE) in residual FF collected from 32 women undergoing in vitro fertilization (IVF). We identified significant inverse associations between higher levels of PCB congeners and indicators of ovarian reserve (e.g., antral follicle count), follicular response to administered gonadotropins (e.g., peak estradiol, number of oocytes retrieved, endometrial thickness), intermediate IVF endpoints (e.g., oocyte fertilization and embryo quality), and clinical IVF outcomes (e.g., embryo implantation and live birth), after adjusting for body mass index, cigarette smoking, race, and age. Our results suggest that ongoing exposure to POPs impacts IVF and merit confirmation in a larger and more definitive future study.

Litter size and other conventional measures in rodents are common end-points in the assessment of xenobiotics for reprotoxic effects. However, since litter size may be normal despite reduced semen quality, we established and tested a mouse in vitro fertilization/in vitro culture (IVF/IVC) system to assess other aspects of reprotoxicity of xenobiotic exposure. Two pesticides, vinclozolin (V) and chlormequat (C), were added to feed in low (40 and 900 ppm, respectively) and high (300 and 2700 ppm, respectively) doses and compared to control (nil pesticide). Exposed males were used for natural mating to evaluate litter size and then used for IVF/IVC and sperm evaluation. The IVF/IVC system detected significant adverse effect of high dose of vinclozolin on blastocyst formation, which was not detected by conventional measures such as litter size or sperm motility and viability. We conclude that assessment based on IVF/IVC measures may complement litter size and other conventional end-points.

The impact of environmental chemicals like persistent organic pollutants (POPs) on reproductive health is still poorly understood, despite the high societal and economical costs. The aim of the present study was to systematically review and evaluate the human evidence on the associations between internal levels of POPs and in vitro Fertilization (IVF) outcomes among women. We applied a protocol based on the National Toxicology Program Office of Health Assessment and Translation's guidelines for the study search, selection and quality assessment. Fifteen studies were finally retained in the present work. The results showed that main families of POPs are still pervasive in follicular fluid and serum of women undergoing IVF treatments. Globally, we found inconsistent findings across studies for specific exposure-outcome dyads, suggesting that adverse effects of POPs on IVF outcomes cannot be ruled out. Specifically, there is evidence that POPs, notably some polychlorinated biphenyls and organochlorine pesticides, may impair embryo quality and pregnancy rates. Most studies have been performed in small cohorts (n<50) and focused on PCBs and OCPs, whereas major research gaps remain for emerging compounds (e.g. perfluoroalkylated substances) and the most clinically relevant outcome, live birth rate. The overall evidence presented 'serious' or 'very serious' risk of bias, mainly due to the lack of consideration of relevant confounding variables, low sample size or underreporting of methods. Globally, we judged the level of evidence being "low". Given the high economical and societal costs associated to infertility and IVF, further well-designed research is urged to fill the highlighted gaps.

To be fertilization competent, spermatozoa undergo a series of changes in the female reproductive tract collectively referred to as capacitation. In an attempt to understand, if ornidazole, a known anti-fertility drug, adversely affects sperm functions by targeting capacitation, we designed experiments to study the influence of this drug on hyperactivation (HA), capacitation-associated protein tyrosine phosphorylation (pY) and the acrosome reaction (AR). Addition of ornidazole at 0 h, inhibited the onset of HA and total pY in a dose dependent manner. However, when ornidazole was added at 3.5h, severe effects were still seen on HA and pY of high molecular weight proteins but, pY of lower M(r) proteins (50-56 kDa) was affected only marginally. Further, lower doses of ornidazole (5 and 10 mM) had greater inhibitory effect when added at 0 h, while addition of ornidazole at 3.5 h required higher doses of ornidazole (25 mM) to cause significant inhibition of acrosome reaction. Collectively, through in vitro studies, we demonstrate that ornidazole affects the onset and progression of hamster sperm hyperactivation, capacitation associated protein tyrosine phosphorylation and acrosome reaction, and the severity depends on the dose (5, 10 or 25 mM) and the time of addition (0 or 3.5 h) of the drug to the spermatozoa.

This study was conducted to investigate the applicability of an in vitro technique for maturation, fertilization, cleavage, and growth to blastocysts of bovine oocytes to investigate reproductive toxicologic effects. During maturation, the oocytes were exposed to the di-ortho-substituted PCB congener 2,2',4,4',5,5'-CB (PCB 153) in the three concentrations 0.84 ng/mL, 8.4 ng/mL, and 84 ng/mL or to the non-ortho-substituted PCB congener 3,3'4,4',5-CB (PCB 126) in the three concentrations 1.006 pg/mL, 10.06 pg/mL, and 100.6 pg/mL and compared with control groups. PCB 153 had no effect on maturation but resulted in a reduced proportion of oocytes that cleaved at the highest concentration. There were no differences in blastocyst development among groups. PCB 126 resulted in a reduction in maturation percentage at the highest concentration and in blastocyst development at all concentrations. These results demonstrated adverse effects of PCB congeners on bovine oocytes and showed that this system can be used to evaluate toxic effects on oocytes and preimplantation-stage embryos.

The aim of this study was to perform a dose-response test to determine whether bovine oocytes exposed to dichlorodiphenyltrichloroethane (DDT), hexachlorocyclohexane (gammaHCH), or methoxychlor (MXC) in vitro would exhibit changes in maturation rates, cleavage rates at Day 2, or blastocyst rates at Day 7 to 8 after fertilization in vitro (IVF). All three pesticides affected maturation and degeneration rates in a dose-dependent manner, but to different extents. Higher concentrations of pesticides were associated with higher rates of chromatin degeneration. Because the maturation of bovine oocytes was depressed in a dose-dependent manner, the fertilizability and further embryonic development of in vitro matured oocytes was studied at the lowest previously tested concentration (7.25 microg/mL) only. No significant difference in fertilization rates was seen between unexposed control and treated groups. The cleavage rates did not differ among groups 48 h after IVF. The number of morulae and blastocysts on Day 7 to 8 after IVF, which is commonly used as a parameter for normal development, was significantly different between control and DDT- and gammaHCH-treated groups, but not between the control and MXC groups. The pesticides did not differ significantly among themselves. These results show that the tested pesticides decrease the rate of normal oocyte maturation in vitro in a dose-dependent manner. The effect of the lowest concentration of pesticides is seen only after Day 7 of embryo development.

Spermatozoa require the capacitation, a series of biochemical events, to perform fertilization. Many toxic compounds can interfere in this process, including perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), which belong to the perfluoroalkyl substances (PFAS). Since both substances are found in many everyday materials and are highly persistent, they accumulate in organisms where they have been associated with fertility problems. This study analyzes the effects of PFOS and PFOA on the functionality of boar spermatozoa, and changes in the plasma membrane (PM) during capacitation. The median lethal concentrations (LC50) of PFOS and PFOA were 460 and 1894 µM, respectively, while the mean inhibitory concentrations of capacitation (ICC50) were 274 µM and 1458 µM, respectively. The ICC50 of PFOA was insufficient to reduce the capacitation, but 950 µM (½ LC50) of PFOA and the ICC50 of PFOS significantly reduced the number of capacitated spermatozoa. PFOS and PFOA also impeded the progesterone (P4)-induced acrosomal reaction (iAR). These effects occur despite the accumulation of [Ca2+]i under capacitating conditions. The accumulation of [Ca2+]i produces saturation, which prevents its entry through ionophore A23187 and P4 in the presence of PFOS. Membrane potential (Emv) was deregulated. Both PFAS affected lipid membrane conductance mediated by valinomycin. The spermatozoa presented 49% and 47% of membrane dysfunction with PFOS and PFOA, respectively. By causing membrane damage, both substances prevented the release of cholesterol and altered the organization of membrane microdomains (MMDs). Data indicate that both PFAS caused alterations in PM functionality.

Exposure to di-butyl phthalate (DBP) exerts negative effects on female fertility in animal models, but human studies remain limited. Here, the effects of DBP exposure on mural granulosa cell function were investigated in primary cultures from women undergoing in vitro fertilization. Cultured cells treated with various doses of DBP (0, 0.01μg/mL, 0.1μg/mL, 1μg/mL, 10μg/mL, or 100μg/mL) for 48h were assessed using enzyme-linked immunosorbent assay and qRT-PCR. Treatment with 100μg/mL DBP resulted in significantly lower 17β-estradiol and progesterone production (p<0.01). It also resulted in altered mRNA expression of steroidogenic, angiogenic, and epidermal growth factor-like growth factor genes: CYP11A1 (p<0.001), CYP19A1 (aromatase) (p<0.001), VEGF-A (p<0.02), BTC (p=0.009), and EREG (p=0.04). StAR expression was impaired after exposure to both 10 and 100μg/mL (p<0.03 and p<0.001, respectively). Our results indicate that in vitro exposure of granulosa cells to high doses of DBP alters cell functions.

Here we report a retrospective analysis of negative effects of routine enrofloxacin treatment of recurrent diarrhea on the ovary and the developing oocytes of the common marmoset, a small New World primate. The most deleterious effect on oocytes was observed about two months post treatment suggesting that the enrofloxacin effect is on early growing follicles. Manifestations of toxicity included decreased numbers of growing follicles and recovered culturable oocytes, as well as signs of early atresia of granulosa cells. In addition, increased amounts of holed stroma after treatment strongly suggested increased death of the early growing follicles. Of the oocytes judged to be of adequate quality for culture, maturation rates were not affected but fertilization of in vitro matured MII oocytes and subsequent cleavage rates were severely reduced in the enrofloxacin treated animals. Further, the arrested oocytes, which failed to mature or fertilize, showed obvious meiotic spindle abnormalities.

There continues to be a need to develop in vivo high-throughput screening (HTS) and computational methods to screen chemicals for interaction with the estrogen, androgen, and thyroid pathways and as complements to in vitro HTS assays. This study explored the utility of an embryonic zebrafish HTS approach to identify and classify endocrine bioactivity using phenotypically-anchored transcriptome profiling. Transcriptome analysis was conducted on zebrafish embryos exposed to 25 estrogen-, androgen-, or thyroid-active chemicals at concentrations that elicited adverse malformations or mortality at 120 h post-fertilization in 80% of animals exposed. Analysis of the top 1000 significant differentially expressed transcripts and developmental toxicity profiles across all treatments identified a unique transcriptional and phenotypic signature for thyroid hormone receptor agonists. This unique signature has the potential to be used as a tiered in vivo HTS and may aid in identifying chemicals that interact with the thyroid hormone receptor.

Hepatitis B virus (HBV) can invade the male germline, and sperm-introduced HBV genes could be transcribed in embryo. This study was to explore whether viral gene transcription is regulated by host genes. Embryos were produced by in vitro fertilization of hamster oocytes with human sperm containing the HBV genome. Total RNA extracted from test and control embryos were subjected to SMART-PCR, SSH, microarray hybridization, sequencing and BLAST analysis. Twenty-nine sequences showing significant identity to five human gene families were identified, with CSH2, EIF4G2, PCBD2, PSG4 and TTN selected to represent target genes. Using qRT-PCR, when CSH2 and PCBD2 (or EIF4G2, PSG4 and TTN) were silenced by RNAi, transcriptional levels of HBV s and x genes decreased (or increased). This is the first report that host genes participate in regulation of sperm-introduced HBV gene transcription in embryo, which is critical to prevent negative impact of HBV infection on early embryonic development.

Oxidative stress and trace elements in the oocytes environment is explored in endometriosis and impact on in vitro fertilization (IVF) outcome assessed. Follicular fluid was aspirated at the time of oocyte retrieval from endometriosis (n=200) and tubal infertility (n=140) and the analytes measured using spectroscopy and HPLC. Increased concentration of reactive oxygen species (ROS), nitric oxide (NO), lipid peroxidation (LPO), iron, lead, cadmium and reduced levels of total antioxidant capacity (TAC), superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), glutathione reductase (GR), vitamins A, C, E, copper, zinc and selenium was observed compared to tubal infertility. Increased ROS and NO in endometriosis and tubal infertility associated with poor oocytes and embryo quality. Increased levels of ROS, NO, LPO, cadmium and lead were observed in women who did not become pregnant compared to women who did. Intrafollicular zinc levels were higher in women with endometriosis who subsequently became pregnant following IVF.

Although most children conceived by assisted reproductive technology (ART) are healthy, there are concerns regarding the potential long-term health implications of ART. It has been reported that alterations in insulin-induced gene (INSIG), sterol regulatory element binding protein (SREBP), and SREBP cleavage-activating protein (SCAP) are involved in cardiometabolic changes. Thus, ART mouse models were established via in vitro fertilization (IVF), intracytoplasmic injection (ICSI), and in vitro oocyte maturation (IVM). A significantly higher systolic blood pressure was identified in the IVM aged female mice. In addition, abnormalities in the blood lipids and liver function were identified in the IVM- or ICSI-conceived elderly mice. Furthermore, ICSI or IVM significantly affected the hepatic expression and methylation of INSIG-SCAP-SREBP from a young to old age. Our animal data indicated that ICSI or IVM result in a higher risk of cholesterol metabolism dysfunction in older mice, which may be associated with long-term alterations of INSIG-SCAP-SREBP.

1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p'-DDE, DDE), a metabolite of DDT is a persistent hormonally active environmental toxicant present in human serum and follicular fluid. The objective of this study was to investigate the effects of DDE on the expression of the ovarian vascular endothelial growth factor (VEGF) and insulin-like growth factor (IGF-1) in primary cultures of human granulosa cells and in the rat ovary. Granulosa cells were obtained at the time of oocyte retrieval for in vitro fertilization and cultured with environmentally relevant concentrations of DDE. Immature female rats were treated with 100 microg DDE/kg body weight or vehicle at 28 and 31 days of age and then euthanized at 50 days of age for collection of ovarian tissue. Expression of VEGF, the VEGF receptor fetal liver kinase (Flk-1) and IGF-1 were determined by Western blotting analysis of protein lysates from granulosa cell cultures and by immunohistochemistry in the rat ovary. DDE at concentrations of 100-1000 ng/mL increased the expression of VEGF, Flk-1 and IGF-1 in vitro in primary cultures of human granulosa cells, with the highest expression occurring at 1000 ng/mL. Similarly, acute administration of DDE resulted in a significant increase in immunoreactive VEGF, Flk-1 and IGF-1 in the rat ovary. We conclude that DDE, at levels, which have been detected in humans, alters the expression of the ovarian growth factors VEGF and IGF-1 both in vivo and in vitro. This alteration in expression of growth factors may lead to altered ovarian function as seen in polycystic ovaries and impaired fertility.

This study reviews the scientific literature on the noxious effects of cigarette smoke on the ovarian follicle, and the cumulative data on the impact of smoking on in vitro fertilization (IVF) cycle outcome. There is a close association between tobacco smoke and accelerated follicle loss, abnormal follicle growth and impairment of oocyte morphology and maturation. There is an increasing amount of evidence indicating that smoke can directly derange folliculogenesis. Increased cellular apoptosis or autophagy, DNA damage and abnormal crosstalk between oocyte and granulosa cells have been implicated in the demise of ovarian follicles. It becomes increasingly clear that maternal smoking can exert multigenerational effects on the ovarian function of the progeny. Growing evidence suggests that cigarette smoke is associated with decreased results after IVF. Further research is needed to better define the molecular mechanisms behind smoking-induced ovarian disruption.

The mammalian oviduct is a central organ for female reproduction as it is the site of fertilization and it actively transports the embryo to the uterus. The oviduct is responsive to ovarian steroids and thus, it is a potential target of endocrine disrupting chemicals. Parabens are antimicrobial compounds that are prevalently found in daily-used products. However, recent studies suggest that some parabens can impact female reproductive health. Yet, their effects on the oviduct are unknown. Here, we hypothesized that in vitro exposure of immortalized murine oviductal secretory epithelial (MOE) cells to methylparaben or propylparaben will result in disrupted cell cycle progression and increased cell death by dysregulation of molecular mechanisms that involve the cell cycle and apoptosis. Thus, we examined the effects of exposure to parabens on cell proliferation, cell cycle progression by flow cytometry, and mRNA levels of major cell cycle regulators and apoptotic factors, in MOE cells. Protein levels of estrogen and progesterone receptors were also quantified. Differences between treatments and controls were analyzed by linear mixed model followed by Dunnett post-hoc tests. The results indicate that methylparaben and propylparaben selectively reduce MOE cellular proliferation and colony numbers, compared to controls. Additionally, paraben exposure selectively dysregulates the progression through the cell cycle and decreases the levels of cell cycle regulators, compared to controls. Last, paraben selectively alters the levels of progesterone receptor. Overall, these findings suggest that parabens can affect mouse oviductal secretory epithelial cell proliferation and survival.