Cryptorchidism, among the most frequent congenital urogenital anomalies in male infants, has a global prevalence ranging from 1% to 9%. Characterized by failure of testicular descent into the scrotum, such a condition exposes the testes to elevated temperature, oxidative stress, and impaired spermatogenesis, significantly increasing risks of infertility and testicular cancer. Although orchiopexy is the standard treatment, long-term spermatogenic defects often persist, highlighting the need to elucidate underlying molecular mechanisms. Although the main mechanistic experiments were performed in GC-1 spg cells through genetic manipulation of cPKCγ, an additional heat-stress experiment was included to simulate the high-temperature condition associated with cryptorchidism. In the present study, the role of conventional protein kinase C gamma (cPKCγ) in regulating mitophagy and apoptosis was investigated using mouse spermatogonial GC-1 cells. Through siRNA-mediated knockdown and adenovirus-induced overexpression, cPKCγ upregulation was demonstrated to significantly induce cellular apoptosis and disrupt mitochondrial function, indicated by elevated malondialdehyde (MDA) levels, reduced superoxide dismutase (SOD) activity, decreased ATP production, increased 8-hydroxy-2'-deoxyguanosine (8-OHdG) accumulation, increased mitochondrial reactive oxygen species (ROS), and loss of mitochondrial membrane potential. Mechanistically, cPKCγ was found to inhibit the UCHL1/HIF-1α signaling pathway, suppressing mitophagy, as evidenced by a reduced LC3-II/LC3-I ratio and decreased PINK1 and Parkin expression. Importantly, adverse effects induced by cPKCγ overexpression were substantially rescued through UCHL1 or HIF-1α overexpression, or rapamycin treatment. Conversely, knockdown of UCHL1 or HIF-1α partially counteracted the protective effects of cPKCγ silencing on cell viability, oxidative stress, and mitophagy activation. Furthermore, heat stress upregulated endogenous cPKCγ and suppressed UCHL1/HIF-1α signaling and mitophagy-related proteins in GC-1 spg cells. Collectively, cPKCγ exacerbates spermatogonial apoptosis by repressing UCHL1/HIF-1α-dependent mitophagy, suggesting a potential molecular mechanism for spermatogonial vulnerability under cryptorchidism-related heat stress.
The development of computer-assisted sperm analysis (CASA) systems has expanded the number of analyzable seminal parameters. The aim of this study was to define the optimal frame rate analysis using CASA-Mot system, and to evaluate the effect of different dog breeds on the sperm kinematic variables measured. Ninety-seven ejaculates from 49 adult males of 11 dog breeds enrolled in an artificial insemination program were collected, extended and analyzed at 37 °C. Samples were obtained via manual stimulation at the Sangüeso Veterinary Clinic in Valencia, Spain, and were immediately analyzed using the ISAS®v1 system. Videos were acquired at 500 frames per second (fps) and computationally down sampled to 25-400 fps. Sperm kinematic variables were modeled using an exponential regression to estimate the frame rate required to reach 95% of the asymptotic value. Curvilinear velocity (VCL) was the most frame-rate sensitive parameter. The optimal frame rate calculated from the kinematic variable VCL varied among breeds, ranging from 433.72 fps for the Miniature Schnauzer to 939.67 fps for the Golden Retriever. Furthermore, significant differences were found in all analyzed sperm kinematic parameters between breeds. Marked breed effects were detected in motility, concentration and kinematic patterns at 500 fps, with Jack Russell, French Bulldog, Giant Schnauzer and Golden Retriever showing the highest total and fast motility, and Dachshund and Miniature Schnauzer the lowest. Overall, dogs require higher frame rates than most domestic species for reliable CASA-based kinematic assessment, and breed effects should be considered in future research when interpreting canine semen data.
This retrospective study aims to compare laboratory and clinical outcomes of a microfluidic sperm sorting device (ZyMōt) versus a standard density gradient centrifugation (DGC) technique in ICSI cycles using autologous or donor oocytes. A total of 1,091 cycles utilizing ZyMōt and 1,176 cycles employing DGC method were included to investigate fertilization rates, blastocyst developmental rates, embryo euploidy, clinical pregnancy/live birth rates (CPR/LBR) after single embryo transfer. The ZyMōt group yielded lower DNA fragmentation and higher motility in spermatozoa compared to DGC method. Fertilization was increased (81.0% vs.78.7%; P =0.003), and the proportion of day 5 blastocysts was significantly greater using ZyMōt than the DGC method (25.6 % vs. 18.3.1%; P <0.001). The hierarchical multivariate regression models revealed that ZyMōt sperm processing contributed significantly to increased blastulation rates than the DGC method ß = 0.11, P<0.001), after adjusting for female age, male age, and infertility diagnosis. Sperm processed by ZyMōt did not enhance blastocyst euploidy rates in all stratified ages (20-40 years old). However, the cumulative CPR per retrieval using euploid embryos were noticeably higher in ZyMōt versus DGC cohorts in both oocyte donor (82.0% and 59.6%; P= 0.0042) and non-donor (73.6% and 64.5%; P = 0.0152) groups. Similarly, LBR in ZyMōt were improved significantly in both donor and non-donor groups (68.0% versus 49.7%; P= 0.0252, and 62.6% versus 52.3%; P = 0.0099, respectively). Our findings demonstrate that ZyMōt offers several advantages over the conventional DGC approach, resulting in enhanced fertilization rates, significantly increased blastocyst developmental potential and improved clinical outcomes.
Folliculogenesis is a complex, multi-stage process crucial for the establishment and maintenance of female fertility through the production of a developmentally competent oocyte. Folliculogenesis, including follicular formation, activation, growth and maturation, relies on a finely tuned spatiotemporal crosstalk between germ cells, somatic cells, and the hypothalamic-pituitary-ovarian axis. This work provides a comprehensive overview of the cellular dynamics and molecular mechanisms underlying each stage of follicular development. A particular emphasis is placed on the interaction of growth factors, transcriptional networks, signaling pathways and endocrine cues that collectively govern follicular fate and oocyte quality. Disruptions in these interactions lead to emergence of pathological conditions such as premature ovarian insufficiency and age-related infertility. We further highlight the dual aspects of oocyte maturation, nuclear and cytoplasmic, as major determinants of developmental competence, and explore the role of spindle dynamics, organelle redistribution and epigenetic reprograming in this process. The bidirectional communication between oocytes and cumulus cells, mediated by paracrine signaling and jap junctions, is underscored as a pivotal regulator of oocyte metabolic activity, redox homeostasis, and meiotic competence. A better understanding of the oocyte-cumulus cell interaction offers new approaches for refining the in vitro maturation systems and improving assisted reproductive technologies. A special attention is given to the emerging use of cumulus cell-derived biomarkers for noninvasive assessment of oocyte quality and prediction of preimplantation embryo development. Taken together, this article presents an integrated framework to guide future research in reproductive biology, regenerative medicine, and fertility preservation.
Male infertility remains an understudied yet significant contributor to global reproductive health challenges, with up to 50% of infertility cases involving a male factor and a large proportion still classified as idiopathic. Recent advances in generative artificial intelligence (AI), particularly large language models (LLMs), offer a transformative opportunity to tackle persistent gaps in understanding the genetic, epigenetic, and environmental determinants of male infertility. This chapter explores the scientific potential of LLMs and related AI technologies in accelerating discov-eries across the male reproductive research continuum - from interpreting complex genomic data and identifying novel gene - environment interactions to enhancing sperm quality assessment and predicting an unborn child's long-term health risks stemming from paternal factors. Real-world examples and emerging case studies illustrate how generative AI can help fertility researchers learn rapidly, synthesize massive volumes of literature, generate hypotheses, design experiments, and reveal patterns that conventional analyses may miss. The narrative further reflects on the implications of using AI to forecast offspring health via polygenic risk scoring and in silico developmental simulations, highlighting both technical promise and ethical considerations. Written from the perspective of a computational scientist collaborating with fertility experts, this chapter demonstrates how interdisciplinary approaches, amplified by LLMs, can lower barriers between computer science and reproductive biology. By embracing generative AI responsibly - with attention to data quality, interpretability, and social responsibility - male infertility researchers stand poised to unlock novel insights that will benefit not only current patients but also future generations.
Unexplained infertility (UI) affects ∼10% of infertile couples, yet standard diagnostic protocols fail to identify a cause. Follicular fluid (FF), which supports oocyte development, contains metabolites that may reflect underlying molecular disturbances. In this exploratory study, we investigated the FF metabolome of women with UI and compared it with controls to explore metabolic alterations associated with UI. FF was collected during oocyte retrieval from 20 women undergoing IVF (ten with UI, ten with male factor infertility), matched for age, BMI, stimulation, and fertilization protocols. Metabolomic profiling was performed using hydrophilic interaction and reversed-phase liquid chromatography coupled to Q-TOF-MS/MS, followed by metabolite identification (XCMS Online and MetaboAnalyst) and KEGG pathway analysis. Approximately 2000 features were detected. Differential metabolites were identified by OPLS-DA (VIP > 2) and validated using univariate metrics such as fold change (|log2FC| > 1), statistical significance (p < 0.05), and ROC analysis (AUC > 0.8). Twelve metabolites, including diacylglycerols, phosphatidic acids, vitamin D3 derivatives (VitD3-glucosiduronate, 1α-hydroxy-2β-(5-hydroxypentoxy)-VitD3), asparaginyl-asparagine, 3α-hydroxy-6-oxo-5β-cholan-24-oic acid, Leu-Pro-Ala-Ser-Phe, triacylglycerols, phosphatidylcholine, and lactosyl-ceramide were significantly decreased, while Ile-Lys-Val-Val was increased in women with UI. Pathway analysis highlighted disruptions in glycerophospholipid, glycerolipid, steroid, and linoleic acid metabolism. Consistent with the untargeted findings, targeted analysis demonstrated significantly reduced levels of follicular 25-hydroxyvitamin D [25(OH)D] in women with UI despite uniform oral supplementation, indicating dysregulated follicular vitamin D availability. Whilst the study was limited by sample size, the metabolome analysis was performed in triplicate for each sample, thus providing preliminary insights into the metabolic disruptions in FF from women with UI.
Sperm flagellar axoneme comprises microtubules (MT) and associated machinery and is an integral determinant of sperm motility. Reports from our lab show reduced levels of acetyl α-tubulin, and HDAC6, along with compromised axoneme polymerization in sperm of asthenozoospermic men. These observations prompted us to identify the sperm repertoire of HDAC6-interacting proteins(HIPs) associated with the MTs. HIPs and Microtubule associated protein (MAP) fractions, respectively, were isolated from sperm of normozoospermic individuals, subjected to tandem mass spectrometry(MS) using a bottom-up approach and proteins in the two groups were identified. 1224 and 315 proteins were identified in the respective groups. Seven clusters of HIPs were among the top 20 significant clusters. Proteins were manually curated from these relevant clusters and overlapped with the MAPs dataset which identified 14 HDAC6 interacting proteins to be associated with MTs (HMAPs). On further analysis with MAP analyzer-LZTFL1, RAB7A, AIFM1 demonstrated low specificity toward MT whereas MYH10 and CFAP53 demonstrated high specificity. Among these HMAPs, EEF1A2, MYH10, ANXA1, TUFM, SOD1, and SRSF7 are known to interact with HDAC6 as documented in the BioGRID database. Interaction of CFAP53 with HDAC6 was validated by double immunofluorescence staining and co-immunoprecipitation in rat sperm. LFQ-DDA analysis of these HMAPs, revealed significantly lower abundance of CFAP53 and TUFM with higher abundance of MYH10 in asthenozoospermic men. Their differential expression in men with poor sperm motility as well as enrichment of acetylation on these HMAPs highlights their association with HDAC6 in maintaining axonemal stability/dynamicity and acetylation-deacetylation to the extent required for sperm motility, although interpretation is limited by the small sample size, restricted availability of human sperm for experimental validation, and reliance on in silico acetylation predictions.
Endometrial polyps (EPs) are benign overgrowths of the endometrium causing abnormal uterine bleeding and infertility. Despite their clinical significance, the molecular mechanisms underlying their development and recurrence remain poorly understood, warranting comprehensive transcriptomic investigation. We hypothesized that transcriptomic differences, particularly at the single-cell level as revealed through cellular trajectory analysis, distinguish EPs from adjacent endometrium. To investigate this, paired EP and adjacent endometrium (adEN) samples were collected from 12 women undergoing hysteroscopic polypectomy (proliferative phase, n = 9; secretory phase, n = 3) and analyzed using bulk and single-cell RNA sequencing (scRNA-seq). Bulk RNA-seq revealed high transcriptional similarity between EPs and adENs, with only a few differentially expressed genes (FDR < 0.05) in proliferative-phase EPs, including upregulation of KMT2B and DLEC1 and downregulation of COL9A1 and RAB3C, potentially reflecting epigenetic regulation and protective mechanisms against tumorigenesis. scRNA-seq identified eight major cell clusters namely stromal, epithelial, endothelial, immune, perivascular, macrophage, B cell, and ciliated populations in both tissues. Pseudotime analysis revealed a mid-transcriptional arrest and enrichment of MECOM/EYA2-positive intermediate epithelial states in EPs, in contrast to the late, mature epithelial stage seen in the adENs. This aberrant epithelial maturation may be associated with impaired perivascular and endothelial differentiation, potentially contributing to defective vascular remodeling and polyp persistence. In conclusion, while EPs exhibit global transcriptomic similarity to adENs, single-cell and pseudotime analyses suggest subtle but significant disruptions in epithelial differentiation and vascular remodeling that might be involved in EPs development. Study limitations include scRNA-seq restricted to the proliferative phase, which may limit generalizability. Nevertheless, future functional studies using primary epithelial organoids derived from EPs may provide a physiologically relevant model to evaluate targeted therapeutic strategies including hormonal interventions with potential applications in infertility management.
Asthenoteratozoospermia is a major contributor to male infertility, with multiple morphological abnormalities of the flagellum (MMAF) representing a genetically heterogeneous disorder characterized by structural defects in sperm flagella. To identify the genetic determinants underlying MMAF-associated infertility, we conducted a comprehensive and systematic investigation involving Chinese infertile couples exhibiting the MMAF phenotype and undergoing assisted reproductive technology (ART). Our integrated approach combined whole-exome sequencing (WES) with Sanger sequencing for variant validation, complemented by scanning and transmission electron microscopy (SEM/TEM) to elucidate ultrastructural features. Molecular analyses included quantitative real-time PCR (qRT-PCR) and immunofluorescence (IF) to evaluate both transcriptional and translational alterations. We identified novel variants in six loci of dynein axonemal heavy chain 1 (DNAH1), including both missense and frameshift variants, across three unrelated families. Affected spermatozoa demonstrated characteristic morphological and ultrastructural abnormalities, while qRT-PCR and IF analyses revealed altered expression patterns of DNAH1. Personalized ART strategies enabled successful pregnancies in individuals harboring DNAH1 variants. While the limited sample size reflects the rarity of this genetic disorder, functional validation beyond expression analysis and structural prediction remains limited. Larger cohorts and in-depth biochemical assays will be required to generalize the findings. Nonetheless, our findings provide important insights into the genetic mechanisms of MMAF and its clinical management.
The use of assisted reproductive therapies, advances in embryo research, and developments in scientific fields such as gene editing and in vitro gametogenesis have attracted the attention of bioethicists for years. On one side, the 14-day rule has faced criticism from embryo researchers advocating for its extension. On the other side, the increasing number of cryopreserved embryos worldwide has raised practical and ethical concerns about their fate. While advancements in scientific research, especially gene editing and in-vitro gametogenesis (IVG), are not yet fully applicable, their potential future use appears to pose significant bioethical questions. In this review, we examine the evolving bioethics of embryo research, focusing on the 14-day rule, the challenges surrounding surplus cryopreserved embryos, and the future dilemmas posed by CRISPR-based gene editing, IVG, and preimplantation genetic testing for polygenic risk (PGT-P). We also highlight the critical role of multidisciplinary, patient-centered counseling in ART practice, to foster informed consent, realistic expectations, and psychosocial well-being. Finally, we underscore the need for anticipatory ethical frameworks and open-society engagement that integrate public deliberation with scientific progress to ensure that reproductive innovation proceeds responsibly, preserving both human dignity and social justice.
The multifaceted process of fertilization encompasses a precisely orchestrated cascade of molecular and morphological modifications of the participating male and female gametes. Fertilization culminates in the union of the maternal and paternal genomes and ultimately spark the development of a new individual. During the last decade, extensive new knowledge has been gained concerning the candidate cellular and molecular mechanisms that guide each step of the process from the sperm journey through the female reproductive tract to the gamete fusion and oocyte activation. In this review, we aim at summarizing the main molecular mechanisms of sperm and oocyte activation and fertilization from the moment sperm enters the female reproductive system up to zygote formation. Focusing on molecular determinants including but not limited to ligands, receptors, and signal transducers, we describe well established and novel molecular candidates that pave the way throughout this complex process and highlight the need for further investigation toward clinical application in assisted reproductive therapy.
Refractile bodies (RBs) in human primordial oocytes may represent a lysosomal mechanism of cellular waste management independent of hormonal stimulation or age. This observational study investigated the ultrastructural features and molecular characteristics of refractile bodies (RBs) in human primordial oocytes, focusing on the involvement of lysosomes, autophagy, and mitochondria. Ovarian tissues were obtained from 34 individuals undergoing oophorectomy as part of female-to-male gender-affirming surgery, with no clinical interventions applied. Using fluorescence microscopy, immunocytochemistry, and transmission electron microscopy (TEM), we found large RBs (>5 μm) in all individuals, with no correlation to age. RBs exhibited strong LysoTracker fluorescence, indicating acidic content. LC3, but not RAB7, colocalized with RBs, suggesting incomplete autophagic processing. TEM revealed lysosomal vesicles, mitochondrial remnants, and lipid-rich structures within RBs, some partially enclosed by isolation membranes. These features support a model in which RBs transition from passive lipid accumulation to autophagy-driven remodeling in a size-dependent manner. RBs displayed lipofuscin-like characteristics and are likely formed through lysosomal and mitophagic pathways. Their formation appears to involve both canonical and non-canonical autophagic mechanisms, independent of age or hormonal stimulation. A limitation of this study is its observational nature without functional validation.
Macrozoospermia is a rare cause of male infertility characterized by a high proportion of sperm with large, irregular heads. In this study, we identified a novel homozygous Aurora kinase C (AURKC) missense variant (c.253G > A (p.Glu85Lys)) in a Chinese infertile man using whole-exome sequencing. Segregation of the AURKC c.253G > A variant within the family was confirmed by Sanger sequencing, and In silico prediction tools suggested that the variant is pathogenic. Sperm ultrastructural abnormalities in the patient were further examined using transmission electron microscopy. Our findings identify a novel pathogenic AURKC variant associated with macrozoospermia, providing potential value for genetic diagnosis and clinical management.
Female infertility is a multifactorial condition with complex biological and clinical underpinnings. Biologically, female-related infertility may stem from disruptions in the hypothalamic-pituitary-ovarian (HPO) axis, impaired folliculogenesis, oocyte maturation defects, uterine abnormalities, endometrial dysfunction, and fallopian tube abnormalities. This review highlights key genetic mechanisms contributing to reproductive dysfunction and their relevance to diagnosis and treatment. Chromosomal abnormalities, including Turner syndrome and X-autosome translocations, also contribute to infertility and recurrent pregnancy loss (RPL). Age-related declines in oocyte quality and quantity-due to increased aneuploidy significantly impact fertility after the mid-30s. Clinical causes such as polycystic ovary syndrome (PCOS), luteal phase defects, and endometriosis contribute to infertility through hormonal imbalance, inflammation, and impaired implantation. Environmental and lifestyle factors-like endocrine-disrupting chemicals, obesity, smoking, and stress-further influence reproductive function. Evaluation requires a multidisciplinary approach combining hormonal profiling, imaging, and genetic diagnostics. Ovarian reserve assessment using anti-Müllerian hormone (AMH) and antral follicle count (AFC), hormonal evaluation along with ultrasound and hysterosalpingography, are central to clinical workups. Next-generation sequencing is enhancing the role of genetic screening in unexplained infertility and specific conditions like POI and endometriosis. Treatment options-ranging from ovulation induction to surgery and assisted reproductive technologies (ART)-are increasingly personalized based on underlying causes and patient profiles. Despite advances, many cases remain idiopathic, highlighting the need for deeper molecular research and refined phenotyping. This review emphasizes the importance of precision medicine and an evidence-based, patient-centered approach to improve fertility outcomes across a broad spectrum of infertility etiologies.
This review comprehensively discusses the innovative value and scientific mechanism of sequential embryo transfer (SET) technology in assisted reproduction. SET, by transferring embryos in stages, offers a novel approach to address clinical challenges in patients with recurrent implantation failure (RIF) and poor ovarian response (POR). The core mechanism involves bidirectional signal regulation between the embryo and endometrium. Extracellular vesicles (EVs) released by embryos during the initial transfer contain bioactive molecules like proteins, microRNA (miRNA), and mRNA. Through bidirectional molecular communication, these EVs interact with the maternal-fetal interface, promoting endometrial decidualization and establishing a molecular memory for 'embryo pre-adaptation,' thereby enhancing endometrial receptivity. Subsequent embryo transfers further optimize the pregnancy microenvironment through cumulative signaling effects, boosting embryo development and implantation success rates. Additionally, the synergistic impact of mechanical injury plays a role. Local micro-injuries induced by the transfer catheter operation trigger an inflammatory response, recruit immune cells, activate tissue repair pathways, stimulate regenerative cell and signaling molecule secretion, and enhance angiogenesis and endometrial remodeling. This 'microtrauma pre-activation' fosters favorable conditions for subsequent embryo implantation. Clinical evidence demonstrates that SET significantly elevates the clinical pregnancy and live birth rates in RIF patients without heightening the risk of multiple pregnancies. In POR patients, SET can reduce cycle cancelation rates. This review not only supports the clinical application of SET but also advances assisted reproduction from empirical to precision medicine guided by molecular mechanisms.
The male gamete, the spermatozoon, is the carrier of paternal inheritance. Besides paternal genome, the spermatozoon contributes essential factors for successful fertilization, embryonic development, and continuation of species. A dynamic and extraordinarily complex process called spermatogenesis produces spermatozoa capable of maturing and acquiring full fertilizing capacity. Spermatogenesis proceeds through a series of events that involve cell division (proliferative phase assuring continuous restocking of spermatogenic stem cells), meiosis (recombination and haploidization of paternal genome), post-meiotic differentiation (spermiogenesis; hypercondensation of sperm DNA and generation of accessory sperm structures), and post-testicular maturation (including epididymal maturation, acquisition of seminal plasma components at ejaculation and spermatozoa capacitation within the female oviduct). In this chapter, we provide a concise overview of testis organization and spermatogenesis in different mammalian species and the details of the intricate structure of human/mammalian spermatozoa. Additionally, we explore the fascinating process of spermatogenesis and its regulation. We thus aim to offer insights into the fundamental process that drives male fertility and provides the foundation of reproductive success.
Freshly ejaculated mammalian sperm have poor fertilizing ability, with fertility only gained after sperm undergo capacitation and the acrosome reaction. To visualize exposed Fc receptors (FcRs), which occur during the acrosome reaction and whose absence has been related to infertility, a novel sperm FcR binding assay (FcR assay) was developed to assess fertilizing potential of sperm in proof-of-concept studies. A competition binding assay between sperm FcR and exogenously added FcR was used to assess whether the FcR was a functioning ligand in bull sperm. Once FcR was confirmed as a functional ligand, time-based expression of FcR was then evaluated in bull and human sperm using the FcR assay. This FcR assay was then used to evaluate fertility outcomes in cattle with cryopreserved intrauterine insemination (IUI) sperm, and to evaluate sperm FcR expression in patients undergoing IUI treatment in a prospective observational study. Time-based analyses of ejaculates from bull and human sperm demonstrated characteristic, reproducible sinusoidal patterns of FcR expression that corresponded to high and low periods of fertility potential in each species. The pregnancy rate in cattle approached statistical significance using the FcR assay results to inform optimum insemination timing windows versus conventional untimed methods (73.0% vs. 68.4%, respectively; p = 0.06; 95% confidence interval [CI]: 0.98, 1.57) with a 4.4% increase in the overall pregnancy rate. In patients undergoing IUI treatment, FcR expression patterns were identified where sperm were at their optimal fertilizing state, with overall pregnancy rates increasing from 21% to 42% (p = 0.01) when inseminations occurred during the windows where the fertilizing potential of the sperm was deemed optimal. These results suggest that sperm fertility potential is quantifiable in semen samples using our novel sperm FcR assay. Importantly, the FcR assay has the ability to identify optimal fertility windows in real-time, and also in the procedure ejaculates.
