Epigenetic changes caused by methylphenidate hydrochloride on paternal inheritance have been suggested in fish, yet a subject to be determined in mammals. In rats, we showed increased sperm DNA fragmentation and reduced embryonic viability. In the present report, male Wistar rats (n = 21) were divided into two groups: control and methylphenidate. The control group received 1 mL/kg of distilled water, while the methylphenidate group received 5 mg/kg by gavage from 38 to 68 days of age on a single daily dose. After this period, there was an interval before exposed rats started a mating schedule with untreated/normally cycling females. Morphological quality and key epigenetic marks in the blastocysts were assessed. Immunocytochemistry was performed in fresh blastocysts to quantify the trimethylated histones H3K4, H3K9, and H4K20. Treatment with methylphenidate reduced the mean quality of blastocysts by 43.57% (p = 0.02), as well as increased those classified as "poor" by more than 150% (p < 0.001). Epigenetic marks were also altered, with an increase in the intensity of H3K9me3 (p = 0.01), a reduction of H4K20me3 (p = 0.05) and a nonsignificant increase of H3K4me3 (p = 0.34). The results suggest that the decline in blastocyst quality is highly associated with subchronic use of this psychostimulant by adolescent males. This is the first report showing the risks posed by methylphenidate to the epigenetic signature of a mammalian blastocyst following paternal exposure.

Hypoxia-inducible factor 1-alpha (HIF-1α) is essential for glycolysis regulation. Its expression in the endometrium is significantly reduced in recurrent implantation failure (RIF), indicating that lower levels of HIF-1α may contribute to embryo implantation failure. O-GlcNAcylation is a dynamic posttranslational modification mediated by O-GlcNAc transferase (OGT), known to regulate HIF-1α in cancer cells. However, it remains unclear whether OGT affects glycolytic processes in uterine endometrial stromal cells (ESCs) and its potential role in embryo implantation. This study utilized In Vitro and In Vivo experiments to investigate the role of OGT in decidualization and embryo implantation, along with its underlying mechanisms. Our findings show that OGT expression is significantly reduced in the endometrium of patients with RIF. Additionally, OGT knockdown led to failed embryo implantation in mice. Further analysis revealed that OGT promotes decidualization by stabilizing HIF-1α, which enhances glycolytic activity. Inhibiting OGT resulted in insufficient decidualization among human ESCs. Moreover, our results indicate that OGT partially regulates CCL2 secretion by maintaining HIF-1α levels within human ESCs, which is essential for successful embryo implantation. Based on these findings, we propose that OGT represents a novel and promising therapeutic target for both the diagnosis and treatment of RIF.

Clusterin (CLU), one of the main glycoproteins in mammalian semen and the male reproductive tract, plays a role in spermatogenesis and sperm maturation. Given the poor reliability of classic seminal studies in determining male-fertilizing capacity and the differences in CLU abundance between normal and abnormal spermatozoa, we investigated the potential value of mRNA-CLU levels and protein distribution in spermatozoa as markers of sperm quality and predictors of male fertility. This multicenter study included 90 patients undergoing in vitro fertilization (IVF) treatment with their partners, and a control group of 36 fertile males with normal seminograms. We assessed the relationship between IVF treatment outcomes, seminogram variables, mRNA-CLU levels byquantitative real-time-PCR and CLU distribution by immunostaining in spermatozoa. Our study reveals CLU staining in the acrosome (p = 0.002, OR 14.8, 95% CI: 2.7-79.3) and mRNA-CLU levels (p = 0.005, OR 10.85, 95% CI: 2.0-57.4) as independent risk factors for pregnancy failure, irrespective of traditional seminogram variables. Additionally, our results suggest that CLU, and specially its secreted isoform, constitutes a component of the protein pool that human spermatozoa can produce during its maturation process, exhibiting a variable abundance and distribution in spermatozoa from fertile men compared to those in patients with altered seminograms and infertile patients with normal seminograms. Our study is the first to identify mRNA-CLU levels and CLU immunostaining in the spermatozoa acrosome as independent risk factors for pregnancy failure, with distribution patterns correlating with sperm maturity and seminogram alterations.

Premature ovarian insufficiency (POI) patients experience a decline in ovarian function and a reduction in serum reproductive hormones, leading to a significant impact on the outcomes of assisted reproductive technology. Despite the absence of an effective clinical treatment to restore fertility in POI patients, recent research has indicated that cord blood plasma (CBP) derived from human umbilical cord blood (hUCB) may offer therapeutic benefits for various degenerative diseases. The primary aim of this study is to explore approaches for enhancing ovarian function and serum reproductive hormones through the administration of CBP in a murine model. Initially, hUCB was utilized to obtain CBP (CBP), which was subsequently analyzed for cytokine and growth factor profiles in comparison to adult blood plasma (ABP) by use of flow cytometry. Subsequently, POI mouse models were established through the induction of 4-vinylcyclohexene diepoxide, followed by the injection of CBP into the tail. At 7, 14, and 21 days posttreatment, mouse ovaries and blood were collected, and their estrus cycle, body weight, and ovarian weights were evaluated using precise electronic balance. Finally, ovarian morphology and follicle number were assessed through HE staining, while serum levels of anti-Müllerian hormone (AMH), estradiol (E2) and follicle-stimulating hormone (FSH) were determined by ELISA. Our study revealed that individuals with CBP exhibited significantly lower concentrations of proinflammatory cytokines, including IL-β (p < 0.01) and IL-2 (p < 0.05), while displaying elevated levels of anti-inflammatory cytokines and chemokines, such as IL-2, IL-4, IL-6, IL-8, IL-12P70, IL-17A, IP-10, interferon-γ, and tumor necrosis factor-α (p < 0.01). Furthermore, CBP demonstrated remarkably higher levels of growth factors, including transforming growth factor-β1, vascular endothelial growth factor, and insulin-like growth factor-1 (p < 0.01) than ABP. Notably, our investigation also revealed that CBP restored the content of serum reproductive hormones, such as AMH, E2, and FSH (p < 0.05), and increased the number of primordial and primary follicles (p < 0.01) and decreased the number of luteal and atretic follicles (p < 0.01) in vivo. Our findings suggested that CBP-secreted cytokines and growth factors could be restored POI ovarian function, enhanced serum reproductive hormones and rescued follicular development in vivo. These findings further support the potential of CBP as a promising strategy in clinical applications for POI related infertility.

Cystic ovary disease (COD) is a common cause of subfertility in dairy cattle. Therefore, the aim of this study was to provide novel concepts for cyst classification and to investigate the effects of COD on tubal microarchitecture, oviductal metabolic function, and the formation of the sperm reservoir. Bovine Fallopian tubes affected by follicular cysts, follicular cysts with luteinization and luteal cysts were investigated by a variety of microscopic and histological techniques and compared to control cows in metestrus and diestrus. We defined three types of cysts involved in COD, each of which had a characteristic wall thickness, inner wall appearance and cellular pattern within the cyst aspirate. Regarding the Fallopian tube, each cyst type was associated with a characteristic morphology, specifically the microarchitecture of the folds in ampulla, epithelial cell ratios, and ciliated/secretory cell size and form. Furthermore, each cyst type showed different patterns of tubal glycoprotein and acidic mucopolysaccharide synthesis, which was highly variable as compared to the controls. Our studies are the first to characterize the effects of COD on the Fallopian tube, which promotes the establishment of novel, cyst-specific therapeutic concepts in cattle and helps gain a holistic view of the causes of subfertility in cows with COD.