The complex anatomical, biochemical and immunological architecture of the ovine cervix presents a substantial barrier to frozen-thawed (FT) ram spermatozoa migration following cervical artificial insemination (AI). A key component of the immunological barriers involves interactions between FT sperm and polymorphonuclear neutrophils (PMN), which are known to infiltrate the female reproductive tract (FRT) following semen deposition and engage in effector mechanisms, specifically degranulation, release of neutrophil extracellular traps (NETs) and phagocytosis. Seminal plasma (SP) has been shown to play a critical immunomodulatory role in regulating sperm-PMN interactions across several species, including sheep, reducing sperm-PMN binding in vitro. During in vitro processing and cryopreservation, SP is inherently diluted, potentially exacerbating immune-mediated clearance of FT sperm. Identifying the specific components of SP responsible for this immunoprotective effect may inform targeted supplementation strategies. Extracellular vesicles (EVs), a heterogenous class of SP-containing proteins, lipids, metabolites and nucleic acids, are also emerging as key mediators of immunological communication. Their potential role in modulating sperm-PMN interactions warrants further investigation to help understand sperm-PMN interactions and implications for improving survival of FT ram sperm in the cervix following AI.

The initial barrier encountered by the embryo during the pivotal phase of implantation and subsequent pregnancy maintenance is the maternal endometrial epithelial barrier. To elucidate the interaction between the forkhead Box O1 (FoxO1) molecule, which is present in the endometrial luminal epithelium during mouse embryo implantation, and the embryonic heparin binding epidermal growth factor (HBEGF) and phosphoinositide 3-kinase(PI3K) signaling pathways, along with their potential roles in modulating the epithelial barrier. A normal pregnancy group and a pseudopregnancy model established through uterine bead transfer using bovine serum albumin (BSA)- or HBEGF-coated beads with or without the PI3K inhibitor (Wortmannin) were analyzed by comprehensive immunofluorescence, providing insights into the distinct expression patterns and probable functions of HBEGF and FoxO1 within the endometrial milieu. Pharmacological inhibition of PI3K by Wortmannin suppresses HBEGF expression and disrupts FoxO1 signaling in the luminal epithelium, leading to nuclear accumulation of FoxO1 in the crypt epithelium and ultimately preventing luminal closure, required for implantation. This research underscores the critical role of PI3K, HBEGF, and FoxO1 signaling in facilitating the embryo's ability to breach the epithelial barrier during implantation.

In 2024, the reproductive biology research community in Australia and New Zealand reunited in Adelaide for the Society for Reproductive Biology (SRB) Annual Meeting. The conference showcased major advances made in key areas of reproductive biology, with symposia dedicated to five key themes: (1) exploring comparative reproductive biology across species; (2) the multifaceted impact of climate change on reproduction; (3) innovations in drug development and repurposing for reproductive health; (4) revolutionising fertility and assisted reproductive technologies; and (5) cultivating inclusion in the scientific community. This review summarises these symposia and discusses the relevant emerging research questions and the future research directions. Main advances from each theme include: (1) the utility of non-traditional model organisms and archival samples in research; (2) the importance of further understanding the effects of heat stress on male and female fertility during pregnancy in humans and broader species; (3) collaboration between researchers, industry and policymakers to overcome barriers in contraceptive development; (4) ensuring robust and ethical use of artificial intelligence for clinical and fundamental research; and (5) the significance of maintaining diversity and inclusion in science to promote discoveries and ensure health data is available for diverse population groups.

Context The induction of oocytes from embryonic stem cells (ESCs) in vitro provides a promising tool for the treatment of female infertility. Various molecules are involved in this complex process, which requires further elucidation. Aims This study aims to screen for factors that induce the differentiation of ESCs into oocytes in vitro by constructing transcription factor (TF)-mediated gene regulatory networks (GRNs) during the formation of oocytes. Methods Based on publicly available multi-omics data, the weighted gene co-expression network analysis (WGCNA) method identified oocyte-specific TFs and key oocyte-specific genes. Additionally, chromatin immunoprecipitation (ChIP) sequencing data and ChIP-qPCR analysis were used to examine GRNs mediated by oocyte-specific TFs. Key results First, by analyzing assay for transposase-accessible chromatin sequencing (ATAC-seq) and DNase I hypersensitive site sequencing (DNase-seq) data from human and mouse ESCs, primordial germ cells (PGCs), and oocytes, we identified five and three oocyte-specific TFs, respectively. RNA sequencing and WGCNA further revealed 38 key oocyte-specific genes. Subsequently, when comparing cell-specific TFs in mouse and human oocytes, we identified three overlapping oocyte-specific TFs (NFYA, NFYB, and NFYC). Notably, NFYA exhibited significantly elevated expression levels in oocytes compared to ESCs and PGCs. Additionally, ChIP-qPCR results demonstrated that NFYA was relatively enriched at the promoter region of the key oocyte-specific gene, m6 A demethylase Alkbh5 . Conclusions This study provides preliminary insights into the role of cell-specific TFs and TF-mediated GRNs in oocyte formation by identifying oocyte-specific genes and key oocyte-specific TFs. Implications The findings indicate that their intricate regulatory mechanisms may significantly contribute to enhancing the efficiency of differentiating ESCs into oocytes.

Context The increasing use of nanoparticles (NPs) in various consumer, agricultural, and pharmaceutical applications has raised considerable concern about their potential risks to human health and the environment. Aims This study investigated the progressive toxic effects of silver nanoparticles (AgNPs) in mouse testes after single and repeated exposure. Methods Prepubertal male mice were exposed to AgNPs by gavage at 50, 200, and 500mg/kg body weight. Testis, epididymis, and serum were collected and subjected to histopathological analysis. Key results Daily exposure to AgNPs for 7 and 15days (n =8) decreased sperm count, while increasing abnormal sperm count and testicular atrophy in a dose- and exposure-time-dependent manner. A single exposure to AgNPs at a dose of 200mg/kg body weight (n =8) resulted in testicular histopathological changes and spermatogenic cell apoptosis in a time-dependent manner. The highest number of apoptotic cells was detected 24h after exposure, whereas testicular testosterone (TT) concentrations decreased at 12 and 24h. To explore whether AgNPs suppress TT concentrations by affecting the hypothalamus-pituitary-testicular (HPT) axis, we analyzed serum LH concentrations; however, no significant changes in LH levels were found. Conclusion This study showed that AgNPs cause potential adverse effects on the testis, specifically, spermatogenic cell apoptosis, and impaired spermatogenesis in an exposure time- and dose-dependent manner. The testicular toxicity was not associated with suppression of the HPT axis, possibly involving other mechanisms. Implications These findings contribute to the broader discussion on NP safety and regulatory considerations, particularly regarding their reproductive toxicity.

Context In the past decade, flow cytometry has become a useful tool for evaluating cellular viability characteristics for non-domestic animals such as non-human primates, marine animals, and birds. This technology has the potential to vastly improve sperm-quality assessments, concentration counts and cell sorting in a more time-efficient and reliable manner. Aims The study aimed to validate the efficacy of using flow cytometry for amphibian sperm by comparing its results with those obtained through traditional means of sperm-quality assessment. Methods Sperm samples were collected from testes macerates of the African clawed frog (Xenopus laevis ) and subjected to both flow cytometry and microscopy analyses. Flow cytometry allowed for the simultaneous assessment of sperm viability and concentration by using fluorescent probes, whereas microscopy provided a traditional means of assessing sperm characteristics. Key results Sperm concentrations measured by flow cytometry and fluorescent microscopy were highly correlated, although flow cytometry methods estimated higher concentrations. Sperm viability measured by flow cytometry and that measured by fluorescent microscopy were not significantly correlated and were significantly different, varying by only ~8% in viability, on average. Conclusions Although flow cytometry overestimated concentration and live/dead assessments, the discrepancies were slight enough to indicate that flow cytometry can still be a valuable method for assessing amphibian sperm. Implications These results validated the utility of flow cytometry as a reliable tool for assessing amphibian sperm viability and concentration, offering a promising alternative to traditional, time-consuming methods.

Pre-implantation genetic testing for aneuploidy (PGT-A) via embryo biopsy helps in embryo selection by assessing embryo ploidy. However, clinical practice needs to consider the invasive nature of embryo biopsy, potential mosaicism, and inaccurate representation of the entire embryo. This creates a significant clinical need for improved diagnostic practices that do not harm embryos or raise treatment costs. Consequently, there has been an increasing focus on developing non-invasive technologies to enhance embryo selection. Such innovations include non-invasive PGT-A, artificial intelligence (AI) algorithms, and non-invasive metabolic imaging. The latter measures cellular metabolism through autofluorescence of metabolic cofactors. Notably, hyperspectral microscopy and fluorescence lifetime imaging microscopy (FLIM) have revealed unique metabolic activity signatures in aneuploid embryos and human fibroblasts. These methods have demonstrated high accuracy in distinguishing between euploid and aneuploid embryos. Thus, this review discusses the clinical challenges associated with PGT-A and emphasizes the need for novel solutions such as metabolic imaging. Additionally, it explores how aneuploidy affects cell behaviour and metabolism, offering an opinion perspective on future research directions in this field of research.

Context Sperm immobilization factor (SIF) isolated from Staphylococcus aureus has been implicated earlier in the laboratory in infertility due to its negative impact on sperm function. Moreover, SIF was found to bind not only to human and mouse spermatozoa but also to several bacteria. Among the array of bacteria, we selected Shigella flexneri to investigate if it shares antigenic determinants with spermatozoa. Aim The study aimed to delineate receptors from S. flexneri that imitate the sperm receptor in binding SIF, to assess its therapeutic role in SIF-mediated sperm impairment. Methods Sperm immobilization binding receptor (SBR) from S. flexneri (SF-SBR) was isolated via ultrasonication and 4M NaCl treatment, purified via chromatography and its molecular weight was estimated. The ability of SF-SBR to counteract SIF-induced sperm impairment was assessed in vitro . Further analysis included isothermal titration calorimetry and fourier-transform infrared spectroscopy to quantify the interaction of SIF and SF-SBR. Key results SF-SBR ameliorated the sperm parameters adversely affected by SIF. It also interfered with the binding of fluorescein isothiocyanate-labelled SIF to spermatozoa and bacteria. The SIF and SF-SBR interaction showed favourable thermodynamics with an entropy of 28.12J/molK and free energy of -18.48kJ/mol, supporting the concept of mimicry. Conclusions The study confirmed that SF-SBR shares an epitope with the receptor on mouse spermatozoa, affirming the presence of molecular mimicry between spermatozoa and S. flexneri . Implications These findings indicate that SF-SBR can potentially be used to counteract the effects of SIF on spermatozoa, hinting at novel therapeutic approaches for treating infertility caused by microbial sperm immobilization factors.

Context Understanding of central nervous system mechanisms related to age-related infertility remains limited. Fibril α-synuclein, distinct from its monomer form, is implicated in age-related diseases and propagates among neurons akin to prions. Aims We compared α-synuclein expression in gonadotropin-releasing hormone-expressing neurons (GnRH neurons) in the pre-optic area, arcuate nucleus, and median eminence of healthy heifers and aged cows to determine its role in age-related infertility. Methods We analysed mRNA and protein expression, along with fluorescent immunohistochemistry for GnRH and α-synuclein, followed by Congo red staining to detect amyloid deposits, and confocal microscopy. Key results Both mRNA and protein expressions of α-synuclein were confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and western blots in bovine cortex, hippocampus, and anterior and posterior hypothalamus tissues. Significant differences in α-synuclein mRNA expression were observed in the cortex and hippocampus between young and old cows. Western blots showed five bands of α-synuclein, probably reflecting monomer, dimer, and oligomers, in the cortex, hippocampus, hypothalamus tissues, and there were significant differences in some bands between young and old cows. Bright-field and polarised light microscopy did not detect obvious amyloid deposition in aged hypothalami; however, higher-sensitive confocal microscopy unveiled strong positive signal of Congo red and α-synuclein in GnRH neurons in aged hypothalami. Additionally, α-synuclein expression was detected in immortalised GnRH neurons, GT1-7 cells. Conclusion Alpha-synuclein was expressed in GnRH neurons, and some differences were observed between young and old hypothalami. Implications Alpha-synuclein may play an important role in aging-related infertility.

Context The removal or supplementation of ejaculates with seminal plasma (SP) can affect cryotolerance and post-thaw survival of spermatozoa in many species. In the Asian elephant (Elephas maximus ), elucidation of the SP proteome and investigation of how it affects spermatozoa may enable improvement of cryopreservation protocols. Aims Herein, we characterise the Asian elephant SP proteome and investigate the impacts of SP on sperm cryotolerance in the presence of conspecific or heterospecific SP. Methods Proteomic analysis of Asian elephant SP was performed using mass spectrometry on nine samples from three individuals. In a separate study, SP was removed from six ejaculates and spermatozoa were resuspended in Tris extender supplemented with: no seminal plasma (NOSP), conspecific SP from ejaculates exhibiting 'good' (GSP, >60%) or mixed sperm total motility (MSP), or horse SP (HSP). Samples underwent cryopreservation, and sperm parameters were compared prior to cryopreservation and after thawing (0 and 2h). Key results Mass spectrometry identified 155 proteins from an array of families. Significant differences were observed in post-thaw sperm quality between SP treatments: high concentrations of MSP (25%, v/v) displayed greater average path and straight-line velocity immediately after thawing (P P P Conclusions and implications These preliminary findings suggest the potential of SP to enhance the cryosurvival of Asian elephant spermatozoa, with HSP showing particularly promising results compared to conspecific SP (GSP). Further research into the specific effects of Asian elephant SP proteins is warranted.