The study is aimed to screen the differential expressed genes (DEGs) related to sperm DNA fragmentation in men and provide reference basis of the sperm selection in assisted reproduction based on DNA fragmentation. We evaluated 60 semen samples from patients with high, medium or low sperm DNA fragmentation index (DFI). Using multicolor flow cytometry, we measured the content of reactive oxygen species (ROS), phosphatidylserine (PS) externalization and mitochondrial membrane potential (MMP) in these sperm samples. The results revealed that the more ROS content and PS externalization were detected in the sperm with higher DFI, but there was lower MMP level in the high DFI sperm. Next, we conducted RNA sequencing (RNA-seq) on 3 groups of sperm samples with high, medium and low DFI. Then, Gene Ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were performed on DEGs. Furthermore, we utilized qRT-PCR to validated the significantly DEGs from the RNA-seq assay. The transcriptome results showed a total of 5334 DEGs were found in the sperm sample with high, medium and low DFI. According to GO and KEGG analysis 421 down-regulated genes in the high DFI group were related to oxidative stress and spermatogenesis. Thirteen novel genes were also identified that most likely were involved in sperm DNA fragmentation, which were further validated by qRT-PCR. In conclusion, our study suggested that the sperms with highly fragmented DNA were accompanied by down-regulation of a series of genes related to antioxidant and spermatogenesis.

Male infertility and impaired spermatogenesis are significant concerns in reproductive health, often linked to disruptions in the cellular and molecular processes within the testis. The cellular composition and transcriptional dynamics of human testicular tissue are crucial for understanding these issues. Previous studies have largely relied on bulk tissue analysis, which obscures the distinct roles and interactions of specific cell types. Here, through a comprehensive single-cell transcriptomic analysis of human testes across various developmental stages and pathological conditions, we reveal the intricate cellular heterogeneity and the molecular mechanisms underlying testicular function. Our study identifies significant disruptions in the differentiation trajectories of Germ cells in conditions such as Klinefelter syndrome (KS), AZFa deletion, and Sertoli-cell-only syndrome (SCOS). We further uncover key transcription factors and regulatory networks governing Leydig cell function, particularly those related to steroidogenesis and hormonal regulation. These findings highlight the organized yet complex cellular and molecular landscape of the testis and uncover critical pathways altered in male infertility. Collectively, our data suggest that targeted therapeutic strategies could be developed to address specific disruptions in testicular cell populations and their associated regulatory networks.

This study evaluated diosgenin effects on methotrexate-induced testicular injury in the rats. A single dose of methotrexate (MTX) (20 mg/kg, i.p) was administered, followed by two weeks of diosgenin treatment via gavage starting one day before methotrexate injection. Testicular damage was evaluated through histological examination of seminiferous tubules, as well as analysis of serum testosterone level, oxidative stress and inflammation biomarkers, and antioxidant levels. The results of this study showed that in the MTX-exposed group, oxidative stress indices of malondialdehyde (MDA), reactive oxygen species (ROS), nitrite and indices of inflammation consisting of tumor necrosis factor α (TNFα), and interleukin 6 (IL-6) have a significant increase compared to the control group. Additionally, reductions were observed in antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH). In addition, testosterone level decreased and signs of testicular damage were observed in the MTX group. Conversely, in the group treated with diosgenin alongside MTX at a dosage of 50 mg/kg, there was a significant decrease in oxidative stress markers (MDA, ROS, nitrite) and inflammatory markers (TNFα and IL-6). Moreover, there was a significant increase in the levels of antioxidant enzymes (SOD, CAT, and GSH). Diosgenin appears to have the potential to protect testicular tissue from damage caused by the toxic effects of MTX through the reduction of oxidative stress and inflammation.

The efficacy of in vitro embryo production (IVEP) in equines is relatively limited compared to other species due to the lack of a reliable superovulation technique, limited availability of cumulus oocyte complexes (COCs), low in vitro oocyte maturation (IVM) and fertilization rates. Extracellular vesicles (EVs), which are nanoparticles involved in intercellular signaling in the ovarian environment, have shown potential as supplements to improve oocyte development during IVM. This study tested the hypothesis that EVs from small (< 20 mm) ovarian follicles could enhance fertilization rates in mares. Follicular fluid was collected postmortem, and EVs were isolated and characterized. The IVM process was conducted with or without EVs (200 µg EV protein/ml). EV internalization during IVM was examined using fluorescent labeling and confocal microscopy. Following intracytoplasmic sperm injection (ICSI), presumptive zygotes were cultured in a time-lapse system. Confocal microscopy confirmed EV internalization by COCs. Nanoparticle tracking analysis showed that obtained EVs were submicron-sized, and flow cytometry identified surface markers CD81 and CD63 on a subpopulation of EVs. Transmission electron microscopy revealed the characteristic disk shape of EV isolates. After culture, 196 oocytes (36.84 %) exhibited a first polar body and were subjected to ICSI. The EV-treated group showed a significantly higher fertilization rate (34.7 % vs. 20.2 %; P < 0.05), reduced degeneration, and increased cleavage efficiency (P < 0.1). Despite early embryonic arrest in both groups, these results suggest that follicular fluid-derived EVs could play a supportive role in equine IVF procedures.

Breast cancer (BC) is the most prevalent invasive malignant tumor. Cisplatin (DDP) is a prototype of platinum-based chemotherapy drugs, its resistance severely hinders its clinical application. This project intended to figure out the exact mechanism of KIFC1 in the DDP resistance of BC.

Polycystic Ovary Syndrome (PCOS) is a complex endocrine disorder with elusive molecular mechanisms. This study explores the competitive endogenous RNA (ceRNA) regulatory network in the cumulus cells of PCOS patients. ceRNAs are transcripts like mRNAs, miRNAs, and lncRNAs that competitively bind shared miRNAs, regulating gene expression post-transcriptionally. We analyzed mRNA, microRNA (miRNA), and long non-coding RNA (lncRNA) from two cohorts: 12 PCOS patients and 11 healthy controls (dataset GSE10946), and 5 PCOS patients and 5 healthy controls (dataset GSE72274). These microarray datasets, obtained from the Gene Expression Omnibus (GEO), helped us identify differentially expressed mRNAs, miRNAs, and lncRNAs. Our analysis revealed a significant ceRNA network, which may play a crucial role in the pathophysiology of PCOS. In this network, 5 lncRNAs, 3 miRNAs, and 36 mRNAs were identified as differentially expressed. These elements form a complex regulatory schema influencing key cellular processes related to the disease, such as cell cycle regulation and response to estrogen. The HOXA11-AS-hsa-miR-454-3p-CCND2 network emerged as a potentially valuable biomarker for PCOS diagnosis, supported by Receiver Operating Characteristic (ROC) curve analysis indicating strong predictive power. Our findings suggest that the ceRNA interactions in PCOS cumulus cells provide a deeper understanding of the disease's molecular basis and offer new avenues for therapeutic intervention. This in silico study lays the groundwork for further experimental validation of these ceRNA networks as targets for PCOS treatment.

The objective of this study was to examine the impact of freeze-thawing on the levels of oxidative stress, fatty acids, vitamins A, D, E, and K, cholesterol, and amino acids, as well as on spermatological parameters, in ram semen. Semen was collected and pooled from each of the seven rams twice a week for three weeks. The mixed semen was diluted with tris + egg yolk diluent at 38 °C (Group 38 °C) and the temperature was reduced to 5 °C (Group 5 °C). Following the glycerolization-equilibration process (Group G-E), the samples were automatically frozen in liquid nitrogen vapor at -140 °C. The semen samples were thawed 24 h after freezing (Group Frozen-Thawed, F-T). A comparison of Group 38 °C with Group F-T revealed significant differences in several parameters. Motility rates, kinematic values, percentage of membrane integrity (HOS), some PUFA levels, ∑SFA and amino acid levels were significantly lower in Group F-T. Conversely, the ratio of dead and static spermatozoa, lipid peroxidation level, some PUFA levels, ∑MUFA, vitamins A, E and cholesterol levels were significantly higher in Group F-T. The majority of these alterations were also evident in semen samples subjected to G-E treatment. In conclusion, exposure of ram semen to G-E and F-T treatments results in modifications to semen, fatty acid, vitamin, and amino acid profiles, accompanied by elevated levels of lipid peroxidation. Moreover, this study demonstrated, for the first time, that oxidative stress was induced, some amino acid levels were altered, vitamin A and E levels were increased, vitamin D and K levels were not affected, and β-sitosterol levels were decreased after freeze-thawing in ram semen.

The fertility of women is crucial for the well-being of individuals and families. However, various factors such as chemotherapy, lifestyle changes, among others, may lead to a decline in female fertility, thus emphasizing the significance of preserving and restoring fertility. Stem cells, with their unique capacity for self-renewal and pluripotent differentiation, have made significant strides in areas such as ovarian tissue cryopreservation, in vitro culture of frozen-thawed ovarian tissue, and construction of ovarian-like organs. This review aims to summarize the latest findings in these fields, highlighting the pivotal role, mechanisms, and future prospects of stem cell technology in preserving and restoring female fertility. Additionally, the importance of interdisciplinary collaboration is underscored, as personalized stem cell therapy regimens tailored through interdisciplinary cooperation between reproductive medicine and stem cell fields hold promise in providing reliable solutions for the preservation and restoration of female fertility.

Chemotherapy-induced ovarian toxicity in patients with cancer significantly affects future fertility depending on the age of initiation of treatment. However, the mechanisms underlying the age-related depletion of the ovarian reserve are not well understood. We investigated the effects of chemotherapy on pre- and postpubertal ovarian reserves in a mouse model. Juvenile (3-week-old) and adult (8-week-old) mice were injected with vehicle or cyclophosphamide (CPA;100 mg/kg). We assessed the short-term effects at 24 h and 72 h after injection and the long-term effects at 10 and 12 weeks of age by counting the follicles. The number of primordial follicles in the juvenile group was significantly reduced by CPA treatment compared with that in the adult group. To elucidate the mechanisms of this depletion, we performed immunostaining for γH2AX, cleaved PARP1, and FOXO3 at 24 h post-treatment. CPA-treated juvenile mice had a significantly higher proportion of γH2AX-positive primordial follicles, indicating double-strand DNA breaks. By contrast, 4-hydroperoxy CPA, an activated analog of CPA, induced γH2AX-positive primordial follicles in both groups in vitro, suggesting age-dependent differences in humoral ovarian microenvironment. Moreover, the level of cleaved PARP1 was specifically elevated in CPA-treated juvenile mice. However, primordial follicle activation was unaffected in the CPA-treated groups, as assessed by FOXO3 translocation. In conclusion, our findings suggest that ovaries in juveniles are more susceptible to DNA damage and subsequent apoptosis, leading to a higher rate of primordial follicle depletion. Therefore, it is crucial to recognize that cancer treatment, especially in children, can exert a substantial influence on future fertility.

Testicular development and spermatogenesis in mice involve complex and dynamic gene regulation and chromatin remodelling. In this study, Real-time fluorescence quantitative PCR (RT-qPCR), Western Blot (WB), Immunofluorescence (IF), transfection and other techniques were used to analyse the expression of Ino80d mRNA and its encoded proteins in mouse testicular tissue and mouse spermatogonial cells, and to further analyse the possible target-regulatory relationship and function of miR-92a-3p and Ino80d. We found that Ino80d mRNA and protein expression was up-regulated in adult mouse testis tissue relative to juvenile mouse testis tissue, whereas miR-92a-3p expression was down-regulated in adult mouse testis tissue. Immunofluorescence results showed that the Ino80d protein was mainly localized in the nucleus of male germ cells. Ino80d protein expression is higher in spermatogonia, spermatid and lower in primary spermatocytes, secondary spermatocytes and sperm. There is a decreasing trend in development from spermatogonia to secondary spermatocytes. The transfection results showed that the expression levels of Ino80d mRNA and protein were down-regulated after overexpression of miR-92a-3p in mouse spermatogonia. Increased miR-92a-3p may be a key factor in inhibiting the expression of Ino80d mRNA and proteins in the miR-92a-3p mimics group of mouse spermatogonial cells, whereas differential expression may be a result of the negative regulation of miR-92a-3p, which regulates testicular development and spermatogenesis in mice.

Ketoprofen and meloxicam, non-steroidal anti-inflammatory drugs widely used in clinical practice, lack comprehensive investigation regarding their impact on male reproductive health, particularly on epididymal duct contractions and sperm parameters. Therefore, this study investigated the negative effects of ketoprofen or meloxicam on the contractions of the epididymal duct, sperm parameters, and serum testosterone levels in rats. Firstly, we assessed the in vitro effects of ketoprofen or meloxicam (1-100 μM) on the contractions of the epididymal duct elicited by noradrenaline. Rats were also orally treated with 5 mg/kg ketoprofen or 1 mg/kg meloxicam for 15 days following evaluation of epididymal duct contractions, sperm parameters, and serum testosterone levels. In vitro exposure to meloxicam (100 μM), but not ketoprofen, significantly reduced the maximum effect of noradrenaline in epididymal duct. Moreover, in vivo administration of ketoprofen and meloxicam decreased testosterone levels, sperm production, and sperm count in the caput/corpus region of the rat epididymis. Conversely, the sperm count in the cauda epididymis remained unchanged in animals treated with both ketoprofen and meloxicam. Meloxicam, but not ketoprofen, caused a delay in sperm transit time in the cauda region of the epididymis. In vivo treatment with both ketoprofen or meloxicam hindered the noradrenaline-induced contractions in the epididymal duct. In conclusion, ketoprofen and meloxicam can modify sperm parameters by decreasing testosterone levels and the contractions of the epididymal duct isolated from the distal cauda region of the rat epididymis.

A reduction in myometrial contractile activity can lead to inadequate cleaning of the uterine lumen, resulting in persistent endometritis and potentially endometrosis in mares. Oxytocin (OXT) is a key hormonal regulator of myometrial contraction. While epigenetic regulation of myometrial gene expression has been studied in humans, there is limited information on the expression of DNA methyltransferases (DNMTs) and ten-eleven translocation enzymes (TETs) in the myometrium of mares. This study aimed to evaluate the mRNA transcription of these enzymes and the potential role of DNA methylation in the expression of the OXT receptor (OXTR) gene in the myometrium of mares with endometrosis. Myometrial samples were collected post-mortem during the mid-luteal (n = 23) and follicular (n = 20) phases of the estrous cycle and assessed according to Kenney and Doig endometrial category (I, IIA, IIB, III). mRNA transcription of OXTR, DNMT1, -3A, -3B and TET1, -2, -3 were determined using qPCR. DNA methylation analysis at CpG islands of OXTR exons 1 and 2 was performed using bisulfite pyrosequencing. Myometrial OXTR mRNA transcription and DNA methylation in its promoter region showed no significant differences between categories, although increased methylation was observed at CpG island position 6 in exon 2. DNMT1, TET2, and TET3 mRNA transcription was altered in the equine myometrium depending on the phase of the estrous cycle and the severity of endometrosis (P < 0.05). These findings indicate that DNMTs and TETs were expressed in myometrium in a manner specific to the severity of endometrosis and phases of the estrous cycle, suggesting a potential regulatory role in DNA methylation of myometrial gene expression.

Genetic factors are one of the basic determinants affecting ontogenesis in mammals. Nevertheless, on the one hand, epigenetic factors have been found to exert the preponderant and insightful impact on the intracellular mechanistic networks related to not only initiation and suppression, but also up- and downregulation of gene expression in all the phases of ontogenetic development in a variety of mammalian species. On the other hand, impairments in the epigenetic mechanisms underlying reprogramming of transcriptional activity of genes (termed epimutations) not only give rise to a broad spectrum of acute and chronic developmental abnormalities in mammalian embryos, foetuses and neonates, but also contribute to premature/expedited senescence or neoplastic transformation of cells and even neurodegenerative and mental disorders. The current article is focused on the unveiling the present knowledge aimed at the identification, classification and characterization of epigenetic agents as well as multifaceted interpretation of current and coming trends targeted at recognizing the epigenetic background of proper ontogenesis in mammals. Moreover, the next objective of this paper is to unravel the mechanistic insights into a wide array of disturbances leading to molecular imbalance taking place during epigenetic reprogramming of genomic DNA. The above-indicated imbalance seems to play a predominant role in the initiation and progression of anatomo-, histo-, and physiopathological processes throughout ontogenetic development. Conclusively, different modalities of epigenetically assisted therapeutic procedures that have been exemplified in the current article, might be the powerful and promiseful tools reliable and feasible in the medical treatments of several diseases triggered by dysfunctions in the epigenetic landscapes, e.g., myelodysplastic syndromes or epilepsy.

The neuroendocrine regulation of the stress-reproductive axis in reptiles is complex due to the diverse reproductive strategies adopted by these animals. Consequently, the underlying mechanisms by which stress can affect the reproductive axis remain opaque in reptiles. In the present study, we examined the effect of stress on the seasonal and FSH-induced ovarian recrudescence during the breeding and non-breeding phases of the cycle in the tropical and subtropical house gecko Hemidactylus frenatus. During the recrudescence phase of the ovarian cycle, exposure of lizards to various stressors (handling, confinement, chasing, and noise) caused a significant increase in the percentage of corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone (ACTH)-immunoreactive (ir) content in the median eminence (ME) and/or pars distalis of the pituitary gland (PD), concomitant with a significant decrease in the release of gonadotropin-releasing hormone (GnRH)-ir content into the ME and PD, and number of oogonia in the germinal bed and absence of the stage IV and V (vitellogenic) follicles in the ovary compared to experimental controls. During the non-breeding phase, treatment of stressed lizards with FSH did not stimulate the development of stage IV and V follicles, in contrast to their appearance in FSH-only-treated lizards. Collectively, these findings suggest that exposure to stressors prevents the seasonal ovarian recrudescence, possibly mediated through the suppression of hypothalamic GnRH release into the ME and PD and/or directly at the level of the ovary.

Pregnancy-associated glycoproteins (PAGs) are synthesized in the placental cells of ruminants and are detectable in blood, milk, and urine. Many of these proteins have been obtained and characterized from placental extracts by precipitation with 80 % ammonium sulfate. The possibility of purifying PAGs by precipitation with other concentrations of ammonium sulfate remains unexplored. We aimed to study PAG proteins obtained from extracts of ovine placenta at 100 days of gestation through precipitation with 40 % ammonium sulfate (Extract 40). The main protein complex (130 kDa) was obtained after Extract 40 precipitation. Under reducing SDS-PAGE conditions, the 130 kDa complex dissociated in two PAG proteins with apparent molecular weights of 52.1 kDa and 26.1 kDa. The 130 kDa protein appeared to be a molecular complex consisting of two copies of the 52.1 kDa protein linked to one copy of the 26.1 kDa protein, presumably by disulfide bonds. Furthermore, the 52.1 kDa protein consisted of at least three isoforms with distinct isoelectric points. Amino acid microsequencing of the 52.1 kDa protein revealed a chimeric structure containing amino acid sequences of PAG1, PAG4, PAG6, and PAG1-like proteins. This procedure recovered a novel 130 kDa protein complex composed of 26.1 kDa and two 52.1 kDa PAGs. To the best of our knowledge, this has not been previously reported as heterologous polymeric molecules.

Epithelial-mesenchymal transition (EMT) is known to play a crucial role in the development of endometriosis (EMs). However, the exact mechanisms involved in EMT regulation in EMs are not well understood. In this study, we performed comprehensive research using clinical samples, single-cell sequencing, and in vivo/in vitro models to investigate the effects of advanced oxidation protein products (AOPPs) on EMT and the underlying mechanisms in EMs. Combining bioinformatics analysis with experimental validation, our results show that AOPPs accumulate in EMs tissues, and their levels positively correlate with the expression of EMT markers in fibrotic lesions of EMs patients. Stimulation with AOPPs leads to a concentration- and time-dependent alteration of EMT markers expression in both in vitro and in vivo models. These effects are mainly mediated by the generation of reactive oxygen species and nitrite, along with the activation of the ERK and P38 signaling pathways. In chronic administration studies using normal rats, AOPPs induce EMT and enhance collagen deposition. These findings significantly contribute to our understanding of the molecular mechanisms of EMs and provide a foundation for future research and therapeutic development in this field.

Dysfunction in trophoblast cells is closely associated with the development of recurrent spontaneous abortion (RSA). Previous reports have indicated that microRNA (miR)-200c was upregulated in the serum of patients who have had abortions. This study aimed to investigate the regulatory effects and mechanisms of miR-200c in trophoblast cells. The human extravillous trophoblast cell line HTR-8/SVneo was either subjected to knockdown or overexpression of miR-200c, and its levels were measured using RT-qPCR. The cell behaviors of HTR-8/SVneo were assessed using CCK-8, Transwell, wound healing assays, and flow cytometry. Western blotting was used to detect the protein levels of Ki67, Bcl-2, Bax, MMP2/9, and PI3K/Akt-related markers. The findings revealed that miR-200c levels were higher in the villous tissues of URSA patients. Depletion of miR-200c impeded HTR-8/SVneo cell apoptosis and enhanced cell migration, invasiveness, and proliferation, while overexpression of miR-200c exhibited the opposite effects. The data suggested that mechanistically, miR-200c inactivated PI3K/Akt signaling in trophoblast cells. Furthermore, rescue experiments demonstrated that blocking PI3K/Akt signaling reversed the effects of miR-200c depletion on HTR-8/SVneo cell behavior. Therefore, miR-200c depletion can potentially improve trophoblast cell function by activating PI3K/Akt signaling.

AhR knockout mice are not completely infertile; however, they do experience decreased litter sizes after repeated pregnancies. This study revealed that the decrease in the number of live births is partly due to fetal deaths leading to miscarriages. Interestingly, fetal mortality was found to be linked only to maternal AhR gene defects and not the fetal genotype. Furthermore, we observed no significant changes in litter sizes in allogenic pregnancy, where AhR-KO female mice were crossed with ICR male mice. The results indicated that the absence of AhR in the dams affected the expression of immune tolerance-related genes in both the placenta and fetus. Specifically, FoxP3 and indoleamine 2,3-dioxygenase-1 (IDO1) mRNA levels were lower in the placentas of AhR-KO dams than in those of wild-type dams. Moreover, there were elevated levels of IL-1β and IFN-γ mRNA in the placentas of the AhR-KO dams, which indicated increased inflammation. However, the mRNA expression levels of IL-6 and IDO1 were low despite the elevated mRNA levels of IL-1β and IFN-γ, which may be because AhR is directly involved in IL-6 and IDO1 transcription. These findings imply that in AhR-KO mice, fetal death may be attributed to the disturbance of fetal-maternal immune tolerance as a result of increased inflammation and reduced IDO1 and FoxP3 mRNA levels.

The integrated stress response (ISR) is implicated in age-related diseases, while the molecular chaperone heat shock protein 70 (HSP70) can facilitate proper protein folding. However, the regulatory mechanism of ISR in insufficient testosterone synthesis of aging Leydig cells (LCs) remains unclear. This study aims to elucidate the regulatory role of ISR in inadequate testosterone synthesis of aging LCs. We observed a positive correlation between testosterone and HSP70 levels, which were found to be decreased in elderly men. ISR was detected in testicular tissue from old mice. The expression of testosterone synthesis related protein and the content of testosterone decreased in testicular tissue of old mice. Conversely, inhibition of the integrated stress response in testicular tissue led to an increase in steroid synthase expression among old mice. Furthermore, inhibiting ISR specifically within aging LCs resulted in enhanced protein translation efficiency and increased expression levels of new HSP70 and steroidogenic acute regulatory protein (StAR). These findings suggest that ISR occurrence within aging LCs affects StAR protein expression through regulation of HSP70-mediated translation, consequently impairing testosterone synthesis.

Visfatin is expressed in the testis of chicken, humans and rodents; however, direct role of visfatin in the adult testis has not been studied. We investigated testicular responses after intra-testicular injection of FK866. The effects of visfatin inhibition were accessed at 24 hrs and 1 week post FK866 treatment. The testicular histoarchitecture were degenerated after 24 hrs of FK866 treatment along with supressed testosterone and proliferating markers and resumption in these parameters showed after 1 week. The expression of AR and ERα were down-regulated after 1 week of FK866 treatment. The expression of BCl2 was down-regulated along with a slight elevation of caspase3 after 24 hrs; however, both proteins still showed suppressed expression after 1 week. Furthermore, ERβ expression, 3βHSD, and 17βHSD were down-regulated in both groups compared to the control. Despite the down-regulation of some factors, the testicular proliferation and histoarchitecture showed resumption in the testis after 1 week of FK866 treatment. This could be due to increased testosterone secretion by suppressing aromatase expression. In conclusion, our result is the first report on the direct role of visfatin in the adult testis. Visfatin has a stimulatory role in testosterone synthesis and proliferation in the testis. Moreover, some deregulated factors in the testis after 1 week of FK866 treatment, despite normal histoarchitecture treatment, could be a compensatory mechanism after visfatin inhibitions.

Polycystic ovary syndrome (PCOS) represents a significant cause of infertility among women of reproductive age. Studies have established a close association between granulosa cells (GCs) and the abnormal follicle formation and ovulation processes characteristic of PCOS. The interactions among hsa_circ_0043533, miR-409-3p, and BCL2 were verified through luciferase activity assays. In PCOS patients, granulosa cells exhibit notably reduced apoptosis but enhanced growth, leading to their accumulation and the development of polycystic ovaries. The involvement of non-coding RNAs in PCOS has been documented, with elevated levels of hsa_circ_0043533 observed in this condition. A comprehensive series of experiments were conducted to explore the role of hsa_circ_0043533 in PCOS and elucidate its underlying mechanisms. Silencing hsa_circ_0043533 was found to promote apoptosis and hinder the migration, proliferation, and viability of KGN cells. Furthermore, we uncovered the regulatory effects of hsa_circ_0043533 on the miR-409-3p/BCL2 axis and key markers of Epithelial-Mesenchymal Transition (EMT). Additionally, it was observed that metformin modulates the hsa_circ_0043533/miR-409-3p/BCL2 axis. Overall, this study provides novel insights into the molecular mechanisms regulating granulosa cell proliferation and apoptosis in PCOS, further elucidating the molecular pathogenesis of this condition.