Molecular human reproduction最新文献

Cyclic AMP (cAMP) regulates multiple aspects of sperm function essential for attaining fertilizing ability; therefore, its intracellular levels must be tightly controlled to ensure proper signaling dynamics. In recent years, multidrug resistance-associated protein 4 (MRP4) has emerged as a novel regulator of cAMP homeostasis by facilitating its efflux. Previous studies from our laboratory have identified MRP4 in mammalian sperm and demonstrated its role in capacitation-associated events in murine and bovine species. However, its relevance in human sperm remains unknown. Our study demonstrates a functional MRP4 in human sperm, as its pharmacological inhibition led to a rapid (5 min) intracellular cAMP accumulation and a subsequent decrease in extracellular levels (30 min) under capacitating conditions, as assessed by radio binding protein assay. At the biological level, MRP4 inhibition resulted in a decrease in tyrosine phosphorylation at 360 min of capacitation. Membrane hyperpolarization was also affected, diminishing the induced acrosome reaction. Moreover, a significant reduction in intracellular Ca2+ levels was observed, leading to a significant decrease in progressive and total motility, as well as an inhibition of hyperactivation. In addition, our results indicated that the decrease in Ca2+ levels was due to impaired CatSper channel activity upon MRP4 inhibition. These findings suggest that MRP4-mediated cAMP efflux is essential for proper sperm function, playing a critical role in maintaining nucleotide homeostasis, which is a key determinant in the regulation of human sperm capacitation.

Fertilization is the process by which sperm and oocyte recognize each other and fuse to form a zygote. Fertilization failure is a common issue encountered in ART. However, clinically, there is a lack of identification of the specific stage at which fertilization is arrested and the development of personalized improvement strategies. In this study, we conducted a fertilization failure assay (FFA) in a cohort of 445 couples undergoing IVF (n = 394) and ICSI (n = 51) who experienced total fertilization failure (TFF). In subsequent cycles, comparative analysis was performed to assess both fertilization rates and good-quality embryo rates between couples at different arrest stages undergoing either conventional ICSI or ICSI with assisted oocyte activation (ICSI-AOA). Additionally, whole-exome sequencing (WES, n = 20) was implemented to investigate potential genetic mutations associated with specific fertilization arrest stages. It found that unfertilized oocytes were categorized into four fertilization arrest phenotypes: (i) sperm penetration/fusion deficiency (SPD), (ii) histone incorporation deficiency (HID), (iii) pronuclear-formation deficiency (PFD), and (iv) mixed deficiency. SPD was the primary cause of TFF after IVF (67.8%), and this deficiency was circumvented in ICSI cycles. HID and PFD were the causes of TFF after ICSI. Couples with ≥50.0% oocytes arrested at the histone incorporation stage or ≥75.7% oocytes arrested at the pronuclear formation stage were at high risk of low fertilization rate following ICSI. Couples with ≥50.0% of oocytes arrested at the histone incorporation stage may achieve improved fertilization and embryo outcomes through ICSI-AOA. Genetic mutations associated with male-factor etiologies of fertilization failure have been identified in this patient population. In contrast, for couples with arrest at the PFD, ICSI-AOA shows no beneficial effects on fertilization or embryo development. Genetic mutations associated with female-factor etiologies of fertilization failure have been identified in this patient population. In conclusion, FFA delineates the arrest stages during fertilization failure, facilitates analysis of insemination progression, and provides clinical reference for subsequent treatment cycles.

Endometriosis is a chronic gynecological disorder characterized by progressive fibrosis, which is closely associated with clinical symptoms such as dysmenorrhea and infertility. While myofibroblast activation is central to fibrogenesis, the cellular origins and regulatory mechanisms remain incompletely understood. This study demonstrates that the macrophage-myofibroblast transition (MMT) is a novel source of myofibroblasts in endometriosis and is regulated by the TGFB1/SMAD3 signaling pathway. Using single-cell RNA sequencing, we identified a distinct subpopulation of CD68+ macrophages co-expressing ACTA2 and extracellular matrix (ECM)-related genes in the human endometrium, which exhibited a myofibroblast-like transcriptional profile and were predominantly located at a fibrotic terminal state along the pseudotime trajectory. Histological and ultrastructural analyses revealed varying degrees of fibrosis and elevated TGFB1 expression in eutopic and ectopic endometrium in endometriosis patients and mouse models. Immunofluorescence confirmed that MMT-positive cells, co-expressing CD68 and α-SMA, were enriched in endometriotic tissues and primarily derived from M2 macrophages. In mouse models of endometriosis, pharmacological inhibition of TGFB1/SMAD3 signaling significantly reduced the number of MMT-positive cells and attenuated collagen deposition, particularly in the eutopic endometrium. Furthermore, reduced ectopic lesion volume and epithelial ultrastructural damage following pathway inhibition suggested impaired ectopic lesion survival. These results demonstrate that the TGFB1/SMAD3 pathway-driven MMT might be a novel contributor to endometrial fibrosis in endometriosis. Targeting the TGFB1/SMAD3 signaling axis may provide a dual antifibrotic and anti-lesion strategy, offering therapeutic potential to intervene in the progression of endometriosis.

The death of spermatogonia leads to decreased spermatogenesis and male infertility. Spermatogonia are vulnerable to various external damaging factors, which can cause cell death. However, the mechanism is still unclear. In this study, we found that the actin-related protein T2 (ACTRT2) is specifically expressed in testicular tissue and is associated with spermatogenesis. In vitro, when GC-1 cells (spermatogonial cell line) were treated with busulfan, the proportion of cell death in the low-ACTRT2 group increased significantly. Reactive oxygen species accumulation and typical mitochondrial changes associated with ferroptosis occurred. In vivo, the seminiferous tubules in ACTRT2-/- mice were significantly shrunken. In addition, after being treated with busulfan, spermatogenesis in ACTRT2+/- mice decreased significantly compared to that in wild-type mice. In ACTRT2+/- testes, the expression levels of acyl-CoA synthetase long-chain family member 4 and arachidonic acid 15-lipoxygenase-1 (ALOX15) were upregulated, while the expression levels of solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) were downregulated. Finally, we found that the expression of solute carrier family 11 member 2 (SLC11A2), iron responsive element binding protein 2 (IREB2), and transferrin receptor protein 1 (TFRC) increased significantly in the low-ACTRT2 group, which transports iron into the cell to increase the intracellular unstable iron pool. In conclusion, ACTRT2 deficiency leads to intracellular iron overload and damage to mitochondria, ultimately increasing spermatogonia vulnerability to ferroptosis.

Nlrp5 encodes a core component of the subcortical maternal complex (SCMC), a cytoplasmic protein structure unique to the mammalian oocyte and cleavage-stage embryo. NLRP5 mutations have been identified in patients presenting with early embryo arrest, recurrent molar pregnancies, and imprinting disorders. Correct patterning of DNA methylation over imprinted domains during oogenesis is necessary for faithful imprinting of genes. It was previously shown that oocytes with mutation in the human SCMC gene KHDC3L had globally impaired methylation, indicating that integrity of the SCMC is essential for correct establishment of DNA methylation at imprinted regions. Here, we present a multi-omic analysis of an Nlrp5-null mouse model, which in germinal vesicle (GV)-stage oocytes displays a misregulation of a broad range of maternal proteins, including proteins involved in several key developmental processes. This misregulation likely underlies impaired oocyte developmental competence. Amongst impacted proteins are several epigenetic modifiers, including a substantial reduction in DNMT3L; we show that de novo DNA methylation is attenuated in Nlrp5-null oocytes, including at some imprinting control regions. This provides evidence for a mechanism of epigenetic impairment in oocytes, which could contribute to downstream misregulation of imprinted genes.

Spontaneous abortion (SA) is a challenging and frustrating obstetric complication. Immune dysregulation at the mother-fetal interface has long been recognized as a threat to pregnancy maintenance. Decidual macrophages are key gatekeepers for immune homeostasis at the mother-fetal interface, characterized by their heterogeneity and high plasticity. Abnormalities in their number, function, and phenotype are strongly associated with pregnancy loss. However, the specific regulation mechanisms remain elusive. Here, we outline the origin and identity of the endometrial macrophages and review their diverse changes in phenotypes and functions to pregnancy initiation. More importantly, we highlight the underlying mechanisms mediating aberrant changes in macrophage polarization and functions in the context of SA, involving epigenetic landscape dysregulation, metabolic reprogramming, and aberrant communication between macrophages and other component cells at the maternal-fetal interface. Altogether, these provide a clear framework for understanding the crucial roles and prospective therapeutic targets of macrophages in SA.

Adenomyosis-the ectopic presence of endometrial glands and stroma within the myometrium-affects reproductive-age women and is associated with pain, bleeding, and subfertility, yet the immune events that precede pregnancy remain poorly defined. To address this, we investigated how the uterine immune microenvironment evolves before conception in a tamoxifen-induced murine model of adenomyosis. At 1- and 3-months post-induction, we analyzed uterine tissue by immunofluorescence, flow cytometry, and quantitative polymerase chain reaction. We found that adenomyotic uteri exhibited a sustained elevation of interleukin-6 messenger RNA, a transient interleukin-10 rise at 1 month, and stable cyclooxygenase-2 levels. Interleukin-6 receptor messenger RNA and signal transducer and activator of transcription 3 messenger RNA were both transiently downregulated at 1 month and returned to control levels by 3 months. Early in disease development, total macrophage numbers declined and displayed an alternative (M2) activation phenotype, followed by a selective loss of classically activated (M1) macrophages at later stages. B lymphocytes were consistently enriched, indicating enhanced humoral activity. Although overall T-cell counts remained stable, the CD3+ compartment underwent a marked shift from double-negative T lymphocytes toward T-helper and cytotoxic subsets, coinciding with the transient signaling changes. Limitations of our study include reliance on a single animal model, analysis at only two timepoints, and a lack of functional assessment of regulatory T cells. Future work should incorporate finer temporal profiling, single-cell transcriptomics, and validation in human tissues. These findings highlight dynamic innate-adaptive crosstalk as an early driver of adenomyosis pathology and suggest that targeting interleukin-6-mediated pathways may inform biomarker development and novel immunomodulatory interventions.

Spermatogenesis is a highly complex cellular differentiation process. Recent advances employing knockout or knock-in mouse models have functionally characterized more than 700 genes as essential for male fertility maintenance. Paradoxically, emerging evidence reveals that a substantial proportion of the continuously expanding catalog of testis-enriched genes exhibits biological dispensability for normal sperm production. To systematically catalog non-essential testis-enriched genes in murine spermatogenesis, we performed a comprehensive PubMed literature review encompassing studies published up to 1 August 2025. Through stringent inclusion criteria, this analysis consolidates data from 83 publications that identified 261 testis-enriched genes demonstrated to be non-essential for spermatogenesis. We further categorize these genes by their familial relationships and explore potential explanations for the fertile phenotype observed in these knockout models, including genetic redundancy, compensatory mechanisms, differences in knockout strategies, and environmental influences. This review provides a valuable resource to avoid unnecessary expenditures and effort by research teams.

Human fertilization is a coordinated process involving interaction of sperm with the oocyte. As the sperm pass through the female reproductive tract (FRT), they are presented with numerous challenges. These include navigating through highly viscous cervical mucus while evading immune responses to successfully fertilize the oocyte. The female immune system facilitates sperm selection while providing protection against pathogens. Neutrophils, a major component of the innate immune system, use mechanisms such as phagocytosis, neutrophil extracellular trap formation, and trogocytosis to play a crucial role in this process. With the help of sialic acid residues and N glycans present on its glycocalyx as well as certain proteins in the seminal plasma, the sperm modulate the immune responses in the FRT to their advantage. This review examines the various interactions which take place between the sperm and the FRT, the neutrophil-mediated immune reaction occurring in the FRT, as well as the adaptations the sperm employ to overcome the immune challenges. Understanding these mechanisms provides critical insights into fertility and potential therapeutic targets for infertility.