We hypothesized that in dairy cattle maternal energy restriction applied during two gestational windows (up to day 80 or 120 of gestation) impairs ovarian and cardiovascular development in juvenile female offspring. We also investigated the role of maternal leptin and testosterone in developmental programming in calves. Holstein-Friesian heifers were randomly assigned to one of three experimental groups; starting 10 days before artificial insemination, they were individually fed at: (i) 0.6 of their maintenance energy requirements (M) up to day 80 (Nutrient Restricted, NR80) or (ii) day 120 of gestation (NR120); (iii) 1.8 M until day 120 of pregnancy (Control, CTR). Plasma leptin concentrations increased transiently in nutritionally restricted heifers pregnant with a single female calf, but maternal testosterone concentrations were not influenced by diet. Calves had similar body growth, but daughters of NR80 and NR120 had impaired ovarian development, as assessed by reduced gonadal weight, fewer surface antral & primary follicles and recovered COCs, as well as lower circulating AMH concentrations. Cardiovascular morphology and function in the offspring were not influenced by maternal diet, as determined by peripheral arterial blood pressure, echocardiography, post-mortem heart weight and aortic circumference. Regardless of its duration (until day 80 or 120 of gestation), nutritional restriction resulted in a similar alteration of ovarian development in juvenile progeny, but cardiovascular development was unaltered. Evidence suggests that the window of development that encompasses the peri-ovulatory period to the first 2.6 months of gestation is critical in ovarian programming and that maternal leptin may be involved.

In this work, we aimed to determine the role of activin A during crucial events of mouse embryogenesis and distinguish the function of the protein of zygotic origin and the one secreted by the maternal reproductive tract. To this end, we recorded the progression of development and phenotype of Inhba knockout embryos and compared them with the heterozygotes and wild-type embryos using time-lapse imaging and detection of lineage-specific markers. We revealed that the zygotic activin A deficiency does not impair the course and rate of development of embryos to the blastocyst stage. Inhba knockout embryos form functional epiblast, as evidenced by their ability to give rise to embryonic stem cells. Our study is the first to show that derivation, maintenance in culture, and pluripotency of embryo-derived embryonic stem cells are exogenous and endogenous activin A-independent. However, the implantation competence of activin A-deficient embryos may be compromised as indicated in the outgrowth assay.

The female reproductive system ages before any other physiological system, making it a sensitive indicator of aging. Early reproductive aging is associated with the early onset of infertility and an increased risk of several diseases. During aging, systemic and reproductive oxidative stress and inflammation levels increase through inflammasome activation, leading to ovarian follicle loss. Other markers of reproductive aging include increased fibrosis and shortening of telomeres in ovarian cells. The factors that accelerate reproductive aging are unclear, but likely involve exposure to endocrine-disrupting chemicals such as phthalates. Di(2-ethylhexyl) phthalate (DEHP) is a widely used phthalate and humans are exposed to it daily. Several studies show that DEHP induces reproductive toxicity by affecting estrous cyclicity, follicle numbers, and hormone levels. However, little is known about the mechanisms underlying DEHP-induced early onset of reproductive aging. Thus, this study tested the hypothesis that dietary exposure to DEHP induces early reproductive aging by affecting inflammation, fibrosis, and the expression of telomere regulators and antioxidant enzymes. Adult CD-1 female mice were exposed to vehicle (corn oil) or DEHP (0.5, 1.5, or 1500 ppm) via the chow for six months. Exposure to DEHP increased the expression of antioxidant enzymes and Casp3, increased expression of telomere-associated genes, and increased fibrosis levels in the ovary. In addition, DEHP exposure for 6 months altered ovarian and systemic inflammatory status. Collectively, our novel data suggest that 6-month dietary exposure to DEHP may accelerate reproductive aging by affecting several reproductive aging markers in female mice.

To investigate the follicle microenvironment of individuals with premature ovarian insufficiency (POI), normal ovarian reserve (normal) and advanced maternal age (AMA), and identify potential therapeutic targets.

Anti-Müllerian hormone (AMH) is widely used in the clinic as a biomarker for ovarian reserve and to predict ovarian response to gonadotropin stimulation. Patients with higher AMH levels tend to yield more oocytes and have better outcomes from assisted reproductive technology (ART) procedures. The goal of this study is to determine if AMH can be used to predict the outcome of controlled ovarian stimulation in rhesus macaques, which are commonly used in biomedical research, to refine animal use while maximizing oocyte yield. We hypothesized that pre-stimulation AMH values can be used to predict oocyte yield and quality. Regularly cycling adult macaques underwent controlled ovarian stimulation and baseline (pre-stimulation) plasma AMH levels were determined using an AMH-specific enzyme-linked immunoassay. Oocytes were collected by laparoscopic or ultrasound-guided aspiration, then counted and evaluated for quality and stage of meiosis. Sperm from established fertile males were used to inseminate the oocytes in vitro with fertilization success checked 14 - 16 hours later. Females were grouped by oocyte yield: low ≤ 17; mid = 18 - 41; high ≥ 42. We found that high and mid yielders had significantly higher AMH than low yielders (p<0.0001) and the percent of mature oocytes was greater in the high and mid yielders. There were no significant differences in oocyte quality or ova fertilization rate. These data suggest that AMH is a useful measure for controlled ovarian stimulation success in rhesus macaques and can be used to identify suitable animals for oocyte donation before entering them into a stimulation protocol.

Bovine viral diarrhea virus (BVDV) infection during pregnancy is a significant contributor to reproductive failures in cattle. The bovine receptor for BVDV (CD46) was previously edited with a six amino acid substitution (G82QVLAL to A82LPTFS) and shown to have significantly reduced BVDV susceptibility in a Gir heifer calf. Since a role for CD46 has been proposed in mammalian fertilization, our objective was to assess the edited heifer's fertilization rates, early embryonic development, and germline transmission conformation of the edit. Cumulus oocyte complexes were collected from the edited heifer and unedited females, fertilized with semen from an unedited bull and cultured until the blastocyst stage. Ultrasound examinations and serum progesterone concentration were also monitored to confirm estrous cyclicity in the CD46-edited heifer. Estrous cyclicity was normal with visualization of a corpus luteum and elevated progesterone concentrations. Fertilization rates and blastocyst development were not different in oocytes from edited and unedited controls. Genome sequence analysis of blastocysts confirmed germline transmission of either edited allele from the heifer. Subsequently, the CD46-edited heifer was artificially inseminated with semen from an unedited Gir bull and fertility status was confirmed with a diagnosed conception at day 35 of gestation. Thus, a six amino acid substitution in CD46 did not negatively affect fertilization of edited oocytes or early embryonic development when fertilized with semen from an unedited bull. An edited bull is still needed to similarly evaluate reproductive function of sperm cells carrying this CD46 edit.

Chronic inflammation is a key characteristic of polycystic ovary syndrome (PCOS) and is associated with follicular dysplasia in PCOS. PCOS patients treated with 1000 mg resveratrol (RES) daily for 3 months showed significant improvement in menstrual cycle regularity compared to the placebo group. This investigation explores potential impact of RES on a rat model of PCOS. Sprague-Dawley (SD) rats were subjected to a 30-day letrozole/high-fat diet interventions for PCOS model establishment, followed by RES intervention (20 mg/kg/d) for an additional 30 days. RES intervention mitigated obesity, estrous cycle irregularities, and ovulation disorders while decreasing serum testosterone and lipopolysaccharide (LPS) levels in PCOS rats. Concurrently, inflammatory markers (TNF-α, NLPR3, IL-6,) and pyroptosis-related markers (GSDMD, cleaved-Caspase-1, IL-1β, IL-18) were downregulated. Additionally, KGN cells (a human granulosa-like cell line) were treated with LPS and RES for in vitro assays. It was observed that RES (15 μM) significantly reduced ROS production and downregulated inflammatory cytokine expression in LPS-intervened KGN cells. Additionally, RES downregulated the expression levels of pyroptosis-related factors (GSDMD and cleaved-Caspase-1) and attenuated IL-18 and IL-1β secretion in LPS-induced KGN cells. Furthermore, RES intervention improved the pyroptosis-associated morphology of KGN cells after LPS treatment. In conclusion, RES may restore follicular development in PCOS rats by inhibiting inflammation and NLRP3/GSDMD/Caspase-1-mediated pyroptosis of ovarian granulosa cells, providing new insights into potential therapeutic approaches for PCOS.

Pelvic organ prolapse (POP) is a condition that significantly affects women's quality of life. The pathological mechanism of POP is not yet fully understood, and its pathogenesis is often caused by multiple factors, including the metabolic imbalance of the extracellular matrix (ECM). This study aims to investigate the role of miR-5195-3p, a microRNA, in the pathology of POP and its regulatory mechanism. Using various molecular biology techniques such as qRT-PCR, FISH, immunohistochemistry, and western blot, miR-5195-3p expression was examined in vaginal wall tissues obtained from POP patients. Results revealed an up-regulation of miR-5195-3p expression in these tissues, showing a negative correlation with the expression of ECM-related proteins. Further analysis using bioinformatics tools identified LOX as a potential target in POP. Dual luciferase reporter gene experiments confirmed LOX as a direct target of miR-5195-3p. Interestingly, regulating the expression of LOX also influenced the TGF-β1 signaling pathway and had an impact on ECM metabolism. This finding suggests that miR-5195-3p controls ECM metabolism by targeting LOX and modulating the TGF-β1 signaling pathway. In conclusion, this study unveils the involvement of miR-5195-3p in the pathological mechanism of POP by regulating ECM metabolism through the LOX/TGF-β1 axis. These findings reveal new mechanisms in the pathogenesis of pelvic POP, providing a theoretical foundation and therapeutic targets for further research on POP treatment.

Developmental exposure to environmental chemicals (ECs) perturbs establishment and maintenance of the ovarian reserve across the reproductive lifetime, leading to premature follicle depletion and ovarian aging. Considering humans are exposed to a complex mixture of ECs, real-life models assessing their cumulative impact on the ovarian reserve are needed. Biosolids is a source of real-life mixture of ECs. While earlier studies demonstrated that grazing pregnant sheep on biosolids-treated pastures (BTP) did not influence establishment of the ovarian reserve in fetal life, its impact on subsequent depletion of ovarian reserve during reproductive life of offspring is unknown. We hypothesized that developmental exposure to biosolids accelerates depletion of ovarian reserve. Ovaries were collected from F1 juveniles (9.5 weeks) and adults (2.5 years) born to F0 ewes grazed on control inorganic fertilizer pastures or BTP from before conception and throughout gestation. The impact on follicular density, activation rate, and Anti-Müllerian hormone (AMH; mediator of activation) expression by immunohistochemistry was determined. Activation rate was increased in F1 BTP juveniles with a corresponding reduction in primordial follicle density. In contrast, activation rate and ovarian reserve were similar between control and F1 BTP adults. The density of AMH-positive antral follicles was lower in BTP juveniles, whereas AMH expression tended to be higher in antral follicles of BTP adults, consistent with the changes in the ovarian reserve. These findings of detrimental effects of developmental exposure to biosolids during juvenile life that normalizes in adults is supportive of a shift in activation rate likely related to peripubertal hormonal changes.

Extracellular vesicles, particularly exosomes, play a pivotal role in the cellular mechanisms underlying cancer. This review explores the various functions of exosomes in the progression, growth, and metastasis of cancers affecting the male and female reproductive systems. Exosomes are identified as key mediators in intercellular communication, capable of transferring bioactive molecules such as miRNAs, proteins, and other nucleic acids that influence cancer cell behavior and tumor microenvironment interactions. It has been shown that nc-RNAs transported by exosomes play an important role in tumor growth processes. Significant molecules that may serve as biomarkers in the development and progression of male reproductive cancers include miR-125a-5p, miR-21, miR-375, the miR-371 ~ 373 cluster, and miR-145-5p. For female reproductive cancers, significant miRNAs include miR-26a-5p, miR-148b, miR-205, and miRNA-423-3p. This review highlights the potential of these ncRNAs as biomarkers and prognostics in tumor diagnostics. Understanding the diverse roles of exosomes may hold promise for developing new therapeutic strategies and improving treatment outcomes for cancer patients.

Intraflagellar transport 25 (IFT25) is a component of the IFT-B complex. In mice, even though this IFT component is not required for cilia formation in somatic cells, it is essential for sperm formation. However, the intracellular localization of this protein in male germ cells is not known given no reliable antibodies are available for histologic studies, and the dynamic trafficking in the developing sperm flagella is not clear. To examine localization of the protein in male germ cells and further investigate the mechanism of IFT in sperm formation, particularly to look into the dynamic trafficking of the protein, we generated a mouse IFT25-GFP knock-in (KI) mouse model using the CRISPR/cas9 system, with the mouse IFT25 protein fused with a GFP tag in the C-terminus. Three independent lines were analyzed. Western blotting using both anti-IFT25 and anti-GFP antibodies showed that the IFT25-GFP fusion protein was highly abundant only in the testis, which is consistent with the endogenous IFT25 protein. Examination of localization of the IFT25-GFP in isolated germ cells revealed that the fusion protein was present in the cytoplasm of spermatocytes and round spermatids and a strong signal was present in the developing sperm flagellar. The homozygous KI mice had normal spermatogenesis, fertility and sperm parameters. Diffusion analysis of IFT25 within the developing flagellar revealed the presence of both mobile and immobile fractions as revealed by fluorescence recovery after photobleaching (FRAP). Kymograph and FRAP analyses demonstrate the transport of IFT25-GFP within the developing tail demonstrate no apparent preference for trafficking towards and away from the cell body. The speed of trafficking depends on the stage of sperm development, ranging from highly mobile unrestricted diffusion initially, mobile punctate structures in developing sperm, and immobile punctate structures in mature sperm. Our studies demonstrate that mouse IFT25 travels along the developing sperm flagella in two directions that might be essential for functional sperm formation.

Visfatin regulates energy homeostasis, metabolism, inflammation, and reproduction via the hypothalamus-pituitary-ovary axis. Our previous study showed the visfatin gene and protein expression in the human placenta. This study aimed to investigate the in vitro effect of visfatin on the proliferation and apoptosis of placental JEG-3 and BeWo cells but also in villous explants collected from normal pregnancies and complicated by intrauterine growth restriction (IUGR), preeclampsia (PE), and gestational diabetes mellitus (GDM). We studied placenta cells viability, proliferation, cell cycle, proliferation/apoptotic factors and insulin receptor (INSR) expression, DNA fragmentation, CASP3/7 activity, and phosphorylation of ERK1/2, AKT, AMPKα, STAT3 with their involvement after pharmacological inhibition in visfatin action on proliferation and apoptosis. Visfatin (1, 10, 100 ng/mL) decreased the viability and proliferation of JEG-3 after 48 h, and a similar effect was observed via co-administration of visfatin (10 ng/mL) and insulin (10 ng/mL) in JEG-3 and BeWo after 48 h and 72 h, respectively. Visfatin reduced the transition from the G2/M phase, and expression of PCNA or cyclins D, E, A, and B in JEG-3 and PCNA in normal, IUGR, PE, and GDM placentas. It increased DNA fragmentation, CASP3/7 activity, P53, BAX/BCL2, CASP9, CASP 8, CASP3 levels in BeWo, and CASP3 expression in tested placentas. Furthermore, visfatin modulated INSR, ERK1/2, AKT, AMPKα, and STAT3 expression in JEG-3 and BeWo, and its anti-proliferative and pro-apoptotic effects occurred via mentioned factors. In conclusion, visfatin, by affecting the proliferation and apoptosis of human placenta cells, may be an important factor in the development and function of the organ.

The placenta, serving as the crucial link between maternal and infant, plays a pivotal role in maintaining a healthy pregnancy. Placental dysplasia can lead to various complications, underscoring the importance of understanding trophoblast lineage development. During peri-implantation, the trophectoderm (TE) undergoes differentiation into cytotrophoblast (CTB), syncytiotrophoblast (STB), and extravillous trophoblast (EVT). However, the specification and regulation of human trophoblast lineage during embryo implantation, particularly in the peri-implantation phase, remain to be explored. In this study, we employed a co-culture model of human endometrial cells and native embryos and analyzed the single-cell transcriptomic data of 491 human embryonic trophoblasts during E6 to E10 to identify the key regulatory factors and the lineage differentiation process during peri-implantation. Our data identified four cell subpopulations during the implantation, including a specific transitional state toward the differentiation in which the CTNND1, one crucial component of Wnt signaling pathway activated by cadherins, acted as a crucial factor. Knockdown of CTNND1 impacted the proliferative capacity of trophoblast stem cells (hTSCs), leading to early EVT-like differentiation. Intriguingly, ablation of CTNND1 compromised the terminal differentiation of hTSCs toward both STB or EVT in vitro. Those observations identified the role of cell adhesion-mediated Wnt signaling in hTSC self-renewal, as well as suggest that this signaling pathway controls a transitional state that is crucial for trophoblast lineage specification. These findings contribute valuable insights into trophoblast lineage dynamics and offer a reference for research on placental-related diseases.

Successful pregnancy is dependent on a number of essential events, including embryo implantation, decidualization and placentation. Failure of the above process may lead to pregnancy-related complications, including preeclampsia (PE), gestational diabetes mellitus (GDM), preterm birth, fetal growth restriction (FGR), etc., may affect 15% of pregnancies, and lead to increased mortality and morbidity of pregnant women and perinatal infants, as well as the occurrence of short-term and long-term diseases. These complications have distinct etiology and pathogenesis, and the present comprehension is still lacking. Post-translational modifications (PTMs) are important events in epigenetics, altering the properties of proteins through protein hydrolysis or the addition of modification groups to one or more amino acids, with different modification states regulating subcellular localization, protein degradation, protein-protein interaction, signal transduction and gene transcription. In this review, we focus on the impact of various PTMs on the progress of embryo and placenta development and pregnancy-related complications, which will provide important experimental bases for exploring new insights into the physiology of pregnancy and pathogenesis associated with pregnancy complications.

Bisphenols are a family of chemicals used in the manufacture of consumer products containing polycarbonate plastics and epoxy resins. Studies have shown that exposure to bisphenol A (BPA) may disrupt steroidogenesis and induce adverse effects on male and female reproduction, but little is known about BPA replacements. We determined the effects of six bisphenols on the steroidogenic function of MA-10 Leydig cells and KGN granulosa cells by measuring the levels of progesterone and estradiol produced by these cells as well as the expression of transcripts involved in steroid and cholesterol biosynthesis. MA-10 and KGN cells were exposed for 48 hours to one of six bisphenols (0.01-50 μM): BPA, bisphenol F, bisphenol S, bisphenol AF, bisphenol M, or bisphenol TMC under both basal and dibutyryl cAMP (Bu2cAMP)-stimulated conditions. In MA-10 cells, most bisphenols increased the Bu2cAMP-stimulated production of progesterone. In KGN cells, there was a general decrease in progesterone production, while estradiol levels were increased following exposure to many bisphenols. qRT-PCR analyses revealed that all six bisphenols (≥1 μM) upregulated the expression of STAR, a cholesterol transporter, in both cell lines after stimulation. Key transcripts directly involved in steroid and cholesterol biosynthesis were significantly altered in a cell line, chemical, and concentration-dependent manner. The expression of upstream genes was also differentially affected. Thus, BPA and five of its analogs can disrupt steroid production in two steroidogenic cell lines and alter the levels of transcripts involved in this process. Importantly, BPA replacements do not appear to have fewer effects than BPA.

Preeclampsia (PE) is a condition of pregnancy in which symptoms of hypertension develop after 20 weeks of gestation. it can lead to placental dysfunction, maternal and perinatal mortality and morbidity. The incidence of PE is increasing, posing a serious threat to the lives of pregnant women and their unborn children. Currently, most of the research on the pathogenesis of PE has focused on placenta, However, maternal decidualization is the basis for placental formation and growth. CSPG4 (Chondroitin sulfate proteoglycan 4) is a transmembrane protein that plays a role in cell proliferation, invasion, and migration. However, its function during decidualization is not yet understood. In this study, we investigated the role of CSPG4 and found that its expression was significantly down-regulated in the decidual tissue of patients with severe PE compared to normal pregnant women. During artificially induced decidualization, CSPG4 expression was significantly increased. Knockdown of CSPG4 by siRNA inhibited decidualization, which, in turn, inhibited the invasion of trophoblast cells. In both pseudopregnant and pregnant mice, endometrial stromal cells proliferated rapidly and Cspg4 expression increased during decidualization. Therefore, we believe that CSPG4 plays a crucial role in the process of decidualization. The defect in decidualization caused by abnormal CSPG4 expression could lead to insufficient trophoblast invasion, ultimately contributing to the occurrence of PE.

Isthmus is the region of the oviduct considered a reservoir for spermatozoa, where they are retained and then released synchronously with ovulation. Integrins mediate this interaction, and it is suggested that they regulate the viability and capacitation of spermatozoa. Spermatozoa retained in the oviductal epithelial cells show specific characteristics: normal morphology, intact acrosome and plasma membrane, no DNA fragmentation, and low levels of intracellular Ca2+ and protein phosphorylation at Tyr. This work aimed to define spermatozoa's ability to adhere to an immobilized fibronectin matrix and its effects on their viability and capacitation. We found that guinea pig spermatozoa showed a high affinity for adhering to an immobilized fibronectin matrix but not to those made up of type 1 collagen or laminin. This interaction was mediated by integrins that recognize the RGD domain. Spermatozoa adhered to an immobilized fibronectin matrix were maintained in a state of low capacitation: low levels of intracellular concentration of Ca2+, protein phosphorylation in Tyr and F-actin. Also, spermatozoa kept their plasma membrane and acrosome intact, flagellum beating, and showed low activation of caspases 3/7. The spermatozoa adhered to the immobilized fibronectin matrix, gradually detached, forming rosettes and did not undergo a spontaneous acrosomal reaction but were capable of experiencing a progesterone-induced acrosomal reaction. In conclusion, the adhesion of spermatozoa to an immobilized fibronectin matrix alters the physiology of the spermatozoa, keeping them in a steady state of capacitation, increasing their viability in a similar way to what was reported for spermatozoa adhered to oviductal epithelial cells.

Uterine fluid homeostasis during peri-implantation is crucial for successful embryo implantation. Taurine (Tau) plays a crucial role in regulating osmotic pressure and ion transport. However, the precise mechanisms underlying Tau-mediated regulation of uterine fluid homeostasis during peri-implantation in mice remain unclear. In this study, we generated a Tau-deficient mouse model by administering Tau-free diet to Csad knockout (Csad-/-) mice to block endogenous Tau synthesis and exogenous Tau absorption (Csad-/--Tau free). Our findings demonstrated that Csad-/-Tau free mice with diminished level of Tau exhibited decreased rates of embryo implantation and impaired fertility. Further analysis revealed that the expression of Scnn1a was down-regulated during the implantation window, while Aqp8 was upregulated in Csad-/-Tau free mice, leading to uterine luminal fluid retention and defects in luminal closure, resulting in failed embryo implantation. Additionally, it was also found that E2 inhibited uterine Csad expression and Tau synthesis, while P4 promoted them. Therefore, our findings suggest that ovarian steroid hormones regulate Csad expression and Tau synthesis, thereby affecting release and resorption of uterine luminal fluid, ultimately impacting embryo implantation success.

Endocrine disruptor chemicals (EDCs) are natural and synthetic compounds found ubiquitously in the environment that interfere with the hormonal-immune axis, potentially impacting human health and reproduction. Exposure to EDCs has been associated with numerous health risks, such as neurodevelopmental disorders, metabolic syndrome, thyroid dysfunction, infertility, and cancers. Nevertheless, the current approach to establishing causality between EDCs and disease outcomes has limitations. Epidemiological and experimental research on EDCs faces challenges in accurately assessing chemical exposure and interpreting non-monotonic dose response curves. In addition, most studies have focused on single chemicals or simple mixtures, overlooking complex real-life exposures and EDC mechanistic insights, in particular regarding their impact on the immune system. The ENDOMIX project, funded by the EU's Horizon Health Program, addresses these challenges by integrating epidemiological, risk assessment, and immunotoxicology methodologies. This systemic approach comprises the triangulation of human cohort, in vitro, and in vivo data to determine the combined effects of EDC mixtures. The present review presents and discusses current literature regarding human reproduction in the context of immunotolerance and EDC mode of action. It further underscores the ENDOMIX perspective to elucidate the impact of EDCs on immune-reproductive health.

An increase in global infertility has coincided with the accumulation of microplastics (MPs) in the environment. This trend is particularly troubling because only 10% of male infertility cases can be attributed to identifiable causes, leaving a knowledge gap in our understanding of their underlying factors. To bridge this, it is important to explore the connection between the accumulation of MPs and the observed decline in male fertility. We assessed the presence of microplastics in epididymal sperm from bulls and used it as baseline concentrations for sperm exposure. MPs were detected in all epidydimal sperm (ES) samples, with a mean concentration of 0.37 μg mL-1. Next, to investigate the effects of MPs on fertility, bovine sperm was exposed to three different concentrations of a mixture of 1.1, 0.5, and 0.3 μm polystyrene (PS) beads: (1) 0.7 μg mL-1, blood concentration of PS in cows (bPS); (2) 0.37 μg mL-1, based on the concentration of total MPs found in ES (esMP); and (3) 0.026 μg mL-1, based on the concentration of PS found in ES (esPS). All sperm samples incubated with PS exhibited reduced motility compared with the control at 0.5 h. However, PS exposure did not affect acrosome integrity or induced oxidative stress. Embryos produced from sperm exposed to PS had reduced blastocyst rates, in addition to increased ROS formation and apoptosis. By employing physiological exposure, this research provided evidence of MPs in bovine epididymal sperm and demonstrated the detrimental effect of PS on sperm functionality.