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Epigenetics impacts male fertility and reproductive disorders. RNA modifications, like m6A, influence RNA metabolism. Varicocele contributes to male infertility, and oxidative stress affects sperm function. This study investigates the expression of key RNA modification enzymes in a rat varicocele model, aiming to elucidate the relationship between varicocele, oxidative stress, and fertility. Fifteen male Wistar rats were divided into Control, Sham, and Varicocele induction groups. Varicocele was induced in the rats surgically. After 8 weeks, testicular tissues and sperm were collected for analysis, including histopathological assessment and evaluation of sperm parameters, functional tests, and gene expression of key RNA modification enzymes: METTL3 as a writer, ALKBH5 and FTO as erasers, and YTHDF2 as a reader involved in recognizing m6A-modified RNA using qRT-PCR. One-way ANOVA with post-hoc Tukey HSD was used for comparing tests within groups. Varicocele induction resulted in histological changes in testicular tissues, including irregularly variable-sized seminiferous tubules. Sperm parameters were significantly affected, with lower concentration, motility, and higher percentage of abnormal sperm in the varicocele group. Increased levels of oxidative stress markers (Sperm lipid peroxidation, and intracytoplasmic ROS) and sperm DNA damage were observed, indicating the presence of oxidative stress in varicocele. Moreover, the expression of key enzymes involved in RNA metabolism was downregulated in the varicocele group. These findings highlight the detrimental impact of varicocele on testicular health, sperm quality, and gene expression, providing insights into the underlying mechanisms of male infertility associated with varicocele.

The effective combination of semen cryopreservation and artificial insemination has a positive effect on the conservation of germplasm resources, production and breeding, etc. However, during the process of semen cryopreservation, the sperm cells are very susceptible to different degrees of physical, chemical, and oxidative stress damage. Oxidative damage is the most important factor that reduces semen quality, which is affected by factors such as dilution equilibrium, change of osmotic pressure, cold shock, and enzyme action during the freezing–thawing process, which results in the aggregation of a large amount of reactive oxygen species (ROS) in sperm cells and affects the quality of semen after thawing. Therefore, the method of adding antioxidants to semen cryoprotective diluent is usually used to improve the effect of semen cryopreservation. The aim of this experiment was to investigate the effects of adding five antioxidants (GLP, Mito Q, NAC, SLS, and SDS) to semen cryoprotection diluent on the cryopreservation effect of semen from Saanen dairy goats. The optimal preservation concentrations were screened by detecting sperm viability, plasma membrane integrity, antioxidant capacity, and acrosomal enzyme activities after thawing, and the experimental results were as follows: the optimal concentrations of GLP, Mito Q, NAC, SLS, and SDS added to semen cryopreservation diluent at different concentrations were 0.8 mg/mL, 150 nmol/L, 0.6 mg/mL, 0.15 mg/ mL, 0.6 mg/mL, and 0.15 mg/mL. The optimal concentrations of the five antioxidants were added to the diluent and analyzed after 1 week of cryopreservation, and it was found that sperm viability, plasma membrane integrity, and mitochondrial activity were significantly enhanced after thawing compared with the control group ( P  < 0.05), and their antioxidant capacity was significantly enhanced ( P  < 0.05). Therefore, the addition of the above five antioxidants to goat sperm cryodilution solution had a better enhancement of sperm cryopreservation. This study provides a useful reference for exploring the improvement of goat semen cryoprotection effect.

Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-specific liver disease, which can lead to adverse fetal outcomes, including preterm labor and intrauterine death. The pathogenesis of ICP is still unclear. We hypothesized that pathological index leads to abnormal placenta changes in ICP. Investigation of these differences in protein expression in parallel profiling is essential to understand the comprehensive pathophysiological mechanism underlying ICP. The present study screened differentially expressed proteins (DEPs) as novel diagnostic markers for ICP. Proteomic profiles of placental tissues from 32 ICP patients and 24 healthy volunteers (controls) were analyzed. Our founding was valid by following western blotting and immunohistochemistry staining, respectively. The association of the key protein expression with clinicopathological features of ICP was further analyzed. A total of 178 DEPs were identified between the ICP and control groups. Functional enrichment analysis showed these proteins were significantly enriched in the PPAR singling pathway by KEGG and PPARα/RXRα activation by IPA. Apolipoprotein A2 (APOA2) was the only upregulated protein, which uniquely identified in ICP groups and related to both pathways. Validation of western blotting and immunohistochemical staining analysis showed significantly higher APOA2 expression in the ICP group than in the control group. Furthermore, the expression of APOA2 is associated with clinicopathological features in ICP groups. Receiver operating characteristic (ROC) curve analyses showed that the AUC of APOA2 was 0.8984 (95% confidence interval (CI): 0.772–1.000). This study has identified up-regulated APOA2 associated with PPAR singling pathway and PPARα/RXRα activation in ICP. Thus, APOA2 may be involved in ICP pathogenesis, serving as a novel biomarker for its diagnosis.

Lipids play numerous pivotal physiological roles in mammalian reproduction, being indispensable for oocyte competence acquisition and post-fertilization embryonic development. Profiling lipids in minute samples, such as oocytes, presents challenges but has been accomplished through mass spectrometry technologies like Multiple Reaction Monitoring (MRM) profiling. With the dual objectives of simplifying workflow and examining the influence of preanalytical conditions, we assessed whether transportation at room temperature affects the lipid profile of bovine oocytes. To this end, samples were prepared using either monophasic (methanol only) or biphasic liquid extraction protocols (Bligh & Dyer method) and transported either on dry ice or at room temperature inside sealed-vacuum packages to prevent lipid oxidation. Subsequently, employing a comprehensive method, we screened a list of 316 MRMs from 10 different lipid subclasses in oocyte lipid extracts. Principal Component Analysis (PCA) revealed similar lipid profiles concerning temperature during transportation, whereas clear differentiation among samples was observed based on the lipid extraction method. Univariate analysis indicated that the one-phase methanol extraction resulted in higher relative abundances of phospholipids, except for phosphatidylserines. Conversely, the Bligh & Dyer extraction favored the detection of neutral intracellular lipids (triacylglycerols, free fatty acids, cholesteryl esters, and acyl-carnitines). Consequently, lipid recovery was directly correlated with the polarity of lipid class and the extraction method. Regarding transportation temperature, phosphatidylethanolamine, triacylglycerol, and free fatty acids exhibited lower abundances when samples were transported at room temperature. Based on multivariate and univariate analyses, we conclude that if samples undergo the same lipid extraction protocol and are transported in the same batch at room temperature inside vacuum-sealed bags, it is feasible to analyze lipid extracts of bovine oocytes and still obtain informative lipid profiling results.

Cryopreservation of testicular tissue holds an important role in the field of fertility preservation, particularly for prepubertal boys diagnosed with cancer. However, prepubertal testicular tissue cryopreservation is still considered to be in the experimental stage necessitating the refinement of cryopreservation protocol. Considering the fact that loss of membrane lipids is the primary cause of freeze–thaw-induced loss of testicular cell functions, in this study, we explored the beneficial properties of exogenous supplementation of membrane lipids in the form of liposomes in enhancing the cryosurvival of prepubertal testicular tissue. The freezing medium supplemented with liposomes (prepared from soy lecithin, phosphatidylethanolamine, phosphatidylserine, and cholesterol) was used for the experiments. Prepubertal testicular tissues from Swiss albino mice were cryopreserved in a liposome-containing freezing medium (LFM) composed of 0.25 mg/mL liposomes, 5% DMSO, and 30% FCS in the DMEM/F12 medium using a slow freezing protocol. The tissues were thawed and assessed for various testicular cell functions. Freezing in LFM mitigated the loss of viability, decreased malondialdehyde level ( p  < 0.05), and reduced apoptosis ( p  < 0.05) in the testicular cells compared to the testicular tissue cryopreserved in the control freezing medium (CFM). Further, DMSO (5%) appears to be the ideal penetrating cryoprotectant for prepubertal testicular tissue cryopreservation with liposome-based freezing medium. Similar enhancement in cryosurvival of cells was observed in adult human testicular tissue frozen with LFM. These findings highlight the translational value of liposome-based freezing medium in the cryopreservation of testicular tissue of prepubertal boys undergoing chemotherapy.

Endometrial carcinoma (EC) is a common malignant tumor in women with high mortality and relapse rates. Mitochondrial permeability transition (MPT)-driven necrosis is a novel form of programmed cell death. The MPT-driven necrosis related lncRNAs (MRLs) involved in EC development remain unclear. We aimed to predict the outcomes of patients with EC by constructing a novel prognostic model based on MRLs and explore potential molecular functions. A risk prognostic model was developed utilizing multi-Cox regression in conjunction with the Least Absolute Shrinkage and Selection Operator (LASSO) regression algorithm, which was based on MRLs. The predictive efficacy of the model was evaluated through receiver operating characteristic (ROC) curve analysis, as well as nomogram and concordance index (C-index) assessments. Patients were categorized into high- and low-risk groups based on their median risk scores. Notably, the high-risk group exhibited significantly poorer overall survival (OS) outcomes. Gene ontology (GO) and Gene set enrichment analysis (GSEA) demonstrated that Hedgehog and cell cycle pathways were enriched in the high-risk group. Tumor Immune Dysfunction and Exclusion (TIDE) displayed that patients in the high-risk group showed a high likelihood of immune evasion and less effective immunotherapy. A significant disparity in immune function was also observed between two groups. Based on the nine-MRLs, drug sensitivity analysis identified several anticancer drugs with potential efficacy in prognosis. Meanwhile, the results demonstrated that OGFRP1 plays a carcinogenic role by affecting mitochondrial membrane permeability in EC. Therefore, the risk model constructed by nine MRLs could be used to predict the clinical outcomes and therapeutic responses in patients with EC effectively.

Polycystic ovary syndrome (PCOS) is a metabolic and endocrine disorder affecting women of reproductive age. This study examined the efficacy of etanercept (ETA), an anti-TNF-α drug, in alleviating endocrine, metabolic, and vascular dysfunction in a rat model of PCOS. Prepubertal female Wistar rats were divided into three groups: control, PCOS, and PCOS+ETA. The PCOS groups received dehydroepiandrosterone (DHEA) treatment, whereas the PCOS+ETA group received both DHEA and ETA. After 35 days, various biomarkers were evaluated, including systemic blood pressure, endothelial function, and eNOS and TNF-α expression levels in the thoracic aorta and ovaries. The PCOS group exhibited ovarian morphological changes, increased body weight, and hormonal imbalances, whereas the PCOS+ETA group showed restored levels of these parameters. Systemic blood pressure, urinary albumin levels, and protein excretion did not differ significantly differ among the groups. Endothelium-dependent relaxation, eNOS expression, TNF-α expression in the thoracic aorta, and TNF-α expression in the ovaries were restored to normal levels in the PCOS+ETA group. Furthermore, ovarian morphology was improved in the PCOS+ETA group. In conclusion, etanercept treatment shows promise in mitigating hormonal disturbances and vascular dysfunction in patients with PCOS, suggesting potential therapeutic advantages. Graphical Abstract

CatSper affects sperm function and male fertilization capacity markers, including sperm motility and egg penetration. The study has aimed to evaluate the mRNA expression of CatSper1, and CatSper3 in the spermatozoa of men with normozoospermia and Asthenoteratozoospermia, and to assess the correlation between genes expression and sperm parameters, fertilization rate, and embryo quality in intracytoplasmic sperm injection (ICSI). Reverse transcription-polymerase chain reaction was utilized to evaluate the mRNA expression of CatSper1 and CatSper3 in sperm in two patient groups: Normozoospermia (NOR; n  = 32), and Asthenoteratozoospermia (AT; n  = 22). In all patients receiving intracytoplasmic sperm injection, the fertilization rate and embryo quality were evaluated. CatSper1 , and CatSper3 mRNA expression in sperm was significantly lower in AT males than in NOR ( P  < 0.05). Levels of these genes demonstrated a significant positive correlation with sperm motility, mitochondrial membrane potential (MMP), capacitation, fertilization rate, cleavage rate, and embryo quality ( P  < 0.05) following ICSI. However, a negative correlation was found between mRNA expression of CatSper1, 3 and sperm DNA fragmentation ( P  < 0.05). Findings indicate low levels of CatSper1 and CatSper3 mRNA expression in men with Asthenoteratozoospermia, which resulted in poor sperm quality and impaired embryo development following ICSI therapy.

Early pregnancy loss (EPL) is the most common complication in assisted reproductive technology (ART). However, the precise causes for nearly 50% patients remain unexplained. In the current study, we aimed to discover the differentially expressed profiling of mRNAs and lncRNAs by RNA sequencing (RNA-seq). Human chorionic villi tissues were collected from patients with EPL and natural control (NC) group. RNA sequencing (RNA-seq) of these specimens was performed for transcriptome analysis. As a result, we identified a total of 141 mRNAs and 137 lncRNAs that were significantly differentially expressed between villi tissues from EPL and NC. Functional enrichment analyses indicated enrichment of differentially expressed genes involved in pathways were associated with growth hormone receptor binding, PI3K-Akt signaling pathway, Jak-STAT signaling pathway, transcriptional misregulation in cancer, metabolic pathways and Rap1 signaling pathway. Additionally, the co-expression networks (lncRNA–miRNA–mRNA) was constructed based on the correlation analysis between the differentially expressed RNAs.7 mRNAs and 6 lncRNAs were successfully technically validated with RT-PCR. In conclusion, our results suggest a direction for the further study of EPL-related mRNAs and lncRNAs and may ultimately assist in understanding the pathogenesis of EPL.

Our main objective was to identify the male and female parameters associated with total fertilization failure (TFF) in IVF with nonmasculine indications. The present work, IRB equivalent INS 63209, is a case–control study that evaluated all cases with TFF after conventional IVF at the Center for Human Reproduction from January 2010 to December 2019 ( n  = 154). As a control group, we analyzed all patients who did not experience fertilization failure after conventional IVF in the same period ( n  = 475). We evaluated various parameters, both male and female, assessed during infertility treatment, and only cases without masculine etiology (normal seminal parameters) were included. Ages (female and male) were not different between the groups. Moreover, AMH (anti-Müllerian hormone), semen volume, preprocessing concentration and preprocessing motility were not significantly different ( P  > 0.05). However, the number of collected oocytes (study versus control groups, median [25–75 interquartile]: 2 [1–5] and 5 [3–8]); MII (2 [1–4] and 5 [2–7]); and postprocessing motility (85 [70–90] and 90 [80–95]) were significantly different between both groups ( P  < 0.05). Furthermore, a logistic regression analysis including all significant data demonstrated that the number of collected oocytes was significantly related to IVF failure. Patients with fewer than 5 oocytes had an OR of − 1.37 (− 0.938 to − 1.827) for TFF after conventional IVF. Our results showed that a lower follicular response to controlled ovarian stimulation, evidenced by a decreased number of collected oocytes, was the most important parameter associated with IVF failure in nonmasculine infertility.