Molecular human reproduction最新文献

In women with endometriosis, monocyte chemoattractant protein 1 (MCP-1) or chemokine (C-C motif) ligand 2 (CCL2) is elevated in serum, peritoneal fluid, and endometriotic lesions, though its exact role in endometriosis is still unknown. The MCP-1 downstream molecule integrin-linked kinase (ILK) is involved in several cellular events. Our recent findings suggest that MCP-1 promotes an inflammatory response via ILK in a mouse endometriosis model. MCP-1 also favors human endometriotic cell aggregation, colonization, migration, and invasion, which are reversed by the ILK inhibitor compound (CPD) 22 (600 nM). Furthermore, the inflammatory response to MCP-1 is reduced by ILK inhibition (CPD22, 20 mg/kg body weight) in a mouse model. We studied MCP-1/chemokine (C-C motif) receptor type (CCR)2-mediated ILK signaling in endometriosis and observed a positive association of ILK and CCR2 with endometriosis in patients. Our immunoprecipitation and molecular docking studies confirmed ILK interaction with CCR2 under a high MCP-1 level in Hs832(C).TCs (human endometriotic cells). MCP-1 promotes ILK-Ser246 phosphorylation in endometriotic cells in human and mouse models. The mouse model shows the same inflammatory markers as seen in human endometriosis and mimics some of the aspects of the inflammatory reaction. Targeting ILK by CDP22 (20 mg/kg) suppresses endometriosis progression in the mouse model. Altered MCP-1-ILK signaling leads to poor pregnancy outcomes in the mouse model. Further, our in silico results suggest that CPD22 stabilizes the interaction with Asp234 and His318 residues of ILK and inhibits the Ser246 phosphorylation. In conclusion, MCP-1 activates ILK at the Ser246 residue and leads to lesion development/progression, reflecting the therapeutic importance of ILK for endometriosis management through the mouse model.

Aneuploidy in embryos poses a major barrier to successful human reproduction, contributing to nearly 50% of early miscarriages. Despite its high prevalence in human embryos, the molecular mechanisms regulating aneuploid cell fate during development remain poorly understood. This knowledge gap persists due to ethical constraints in human embryo research and the limitations of existing animal models. In this study, we identified the New World primate marmoset (Callithrix jacchus) as a suitable model for investigating aneuploidy. By calling copy number variants from single-cell RNA-sequencing data of marmoset embryonic cells, we identified heterogeneous aneuploidy, indicating chromosomal instability (CIN) in marmoset preimplantation embryos. Furthermore, marmoset aneuploidy displayed lineage-specific behavior during gastruloid differentiation, similar to humans, suggesting a conserved regulatory mechanism in lineage specification. To develop a more pluripotent cell line to study early specification, we established an efficient approach for generating naïve-like marmoset pluripotent stem cells (cjPSCs). These cells resemble preimplantation epiblast-like cells and exhibit inherent CIN. Transcriptome analysis identified potential pathways contributing to aneuploidy during early embryogenesis, including the downregulation of cell cycle checkpoint signaling and the upregulation of autophagy pathways. Additionally, we found no significant effect of spontaneously occurring aneuploidy in cjPSCs on blastoid formation, suggesting that the consequences of aneuploidy become evident only after gastrulation, with preimplantation lineages exhibiting a higher tolerance for genomic instability. Unexpectedly, aneuploidy enhanced cavity formation during blastoid development, suggesting a potential role in facilitating efficient trophectoderm differentiation. Our findings validate the marmoset as a valuable model for studying CIN during early primate development and provide insight into the mechanisms underlying the prevalence of aneuploidy in primates. Naïve-like cjPSCs recapitulate key phenotypic traits of early embryonic cells, providing a robust system for studying post-implantation aneuploid cell fates in vivo and serving as a foundation for future research in this field.

Placental DNA methylation varies across gestation and is associated with obstetrical complications. Cell-free DNA (cfDNA) from maternal plasma could provide a noninvasive approach to study placental DNA methylation in ongoing pregnancies. However, research on maternal cfDNA methylation is limited and technologically challenging. Therefore, we aimed to investigate DNA methylation in maternal cfDNA and placental tissues across gestation using the innovative methylation DNA sequencing (MeD-seq) technology. Secondly, we explored the origins of methylation differences in maternal cfDNA across gestation, and aimed to identify gestational age-associated placental DNA methylation markers directly in cfDNA. We longitudinally collected maternal cfDNA in all three trimesters and at birth (n = 10), alongside placental tissues from first trimester, second trimester, and term pregnancies (all n = 10), and used previously collected maternal blood buffy coat samples (n = 20). Different placental cell types, including syncytiotrophoblasts/cytotrophoblasts (SCTs/CTBs) (n = 10), extravillous trophoblasts (n = 7), and syncytial knotting (n = 3), and maternal cell types including spiral arteries (n = 3) and endometrial epithelium (n = 3), were isolated using laser capture microdissection. Differentially methylated regions (DMRs) identified in cfDNA from pregnant compared to non-pregnant women (n = 6) ranged from 798 to 2163 in first and third trimesters, respectively. Gradual DNA methylation changes were observed across gestation in cfDNA, placental tissues, and trophoblasts. We showed an increase in DMRs in cfDNA, that overlap with DNA methylation in placental tissues and especially trophoblasts, and in DNA methylation of placenta-specific markers across gestation, reflecting an increased placental-originated cfDNA fraction. Among 110 DMRs between first trimester and term placental tissues, those related to NXPH4, EPS8L2, AMOTL1, and IRX2 had the strongest association with gestational age in cfDNA, for which comparable associations were found in SCTs/CTBs. These DMRs were all hypomethylated in maternal buffy coat samples. This study indicates the feasibility of identifying gestational age-dependent placental DNA methylation marks in maternal cfDNA and can serve as a reference for future studies.

In males, 95% of testosterone is synthesized by Leydig cells, and a deficiency in this synthesis will cause metabolic disorders and multiple organ dysfunction. Testosterone deficiency is not only affected by aged or diseased Leydig cells, which have been studied extensively, but is also closely related to the development of the testis. At present, the focus on the mechanism of testis development includes epigenetic and hormone regulation. However, testicular development is constrained by the external tough tunica albuginea, suggesting that mechanical signals may also play an important role in the regulation of testis development; however, this is not yet well understood. In this in vitro study, we found that a gradual increase in extracellular substrate stiffness for testis development leads to the activation of mechanical signals to promote cytoskeleton remodeling. Eventually, the mechanical signal mediates changes in the mitochondrial-endoplasmic reticulum and affects the synthesis of testosterone in Leydig cells. Through organoid and animal experiments, we found that targeting mechanical signaling pathways that regulate testosterone biosynthesis is feasible. This provides a new angle for further exploration of testis development and new insights into how substrate stiffness affects the testis, raising new clues for clinical applications.

Protamine 2 (Prm2/PRM2), together with Protamine 1 (Prm1/PRM1), constitute the two protamines found in both murine and human sperm. During spermiogenesis in haploid male germ cells, chromatin undergoes significant condensation, a phase in which most histones are replaced by a species-specific ratio of these two protamines. Altered PRM1/PRM2 ratios are associated with subfertility and infertility in both male mice and men. Notably, during histone-to-protamine exchange, a small fraction of histones remains (ranging from 1% to 15%) bound to DNA. The regulatory roles of these residual histones, governed by post-translational modifications (PTMs), play a pivotal role in spermatogenesis, particularly in chromatin remodelling and epigenetic regulation of genes during sperm differentiation or even in early embryogenesis. In this study, utilizing a Prm2-deficient mouse model and conducting an analysis of sperm samples from men exhibiting either normozoospermia or atypical spermiograms, we observed alterations in the methylation and acetylation profiles of histones H3 and H4. Subsequent in-depth analysis revealed that discrepancies in protamine ratios do not significantly influence the PTMs of histones in testicular sperm. In murine epididymal sperm, altered protamine ratios are associated with reduced acetylation of histone H4 (H4ac), a phenomenon similarly observed in ejaculated sperm from men. In particular, H4K5ac and H4K12ac were identified as the two modifications that appear to decrease as a result of reduced Prm2/PRM2 levels. Our findings reveal that Protamine 2 is necessary for the maintenance of specific histone PTMs, such as acetylation, which is essential for proper spermatogenesis and particularly for chromatin remodelling.

In patients with testicular germ cell tumours (TGCT), sperm cryopreservation prior to anti-cancer treatment represents the main fertility preservation approach. However, it is associated with a low sperm recovery rate after thawing. Since sperm is a high-energy demanding cell, which is supplied by glycolysis and oxidative phosphorylation (OXPHOS), mitochondrial dysfunctionality can directly result in sperm anomalies. In this study, we investigated the bioenergetic pattern of cryopreserved sperm of TGCT patients in comparison with normozoospermic samples using two state-of-the-art methods: the Extracellular Flux Analyzer (XF Analyzer) and two-photon fluorescence lifetime imaging microscopy (2P-FLIM), in order to assess the contributions of OXPHOS and glycolysis to energy provision. A novel protocol for the combined measurement of OXPHOS (oxygen consumption rate: OCR) and glycolysis (extracellular acidification rate: ECAR) using the XF Analyzer was developed together with a unique customized AI-based approach for semiautomated processing of 2P-FLIM images. Our study delivers optimized low-HEPES modified human tubal fluid media (mHTF) for sperm handling during pre-analytical and analytical phases, to maintain sperm physiological parameters and optimal OCR, equivalent to OXPHOS. The negative effect of cryopreservation was signified by the deterioration of both bioenergetic pathways represented by modified OCR and ECAR curves and the derived parameters. This was true for normozoospermic as well as samples from TGCT patients, which showed even stronger damage within the respiratory chain compared to the level of glycolytic activity impairment. The impact of cryopreservation and pathology are supported by 2P-FLIM analysis, showing a significant decrease in bound NADH in contrast to unbound NAD(P)H, which reflects decreased metabolic activity in samples from TGCT patients. Our study provides novel insights into the impact of TGCT on sperm bioenergetics and delivers a verified protocol to be used for the assessment of human sperm metabolic activity, which can be a valuable tool for further research and clinical andrology.

Infertility affects a considerable number of couples at reproductive age, with an incidence of 10-15%. Approximately 25% of cases are classified as idiopathic infertility. Often, errors during the meiotic stage appear to be related to idiopathic infertility. A crucial component during the first meiotic prophase is the synaptonemal complex (SC), which plays a fundamental role in homologous chromosome pairing and meiotic recombination. In many studies with infertile patients, mutations affecting SC-coding genes have been identified. The generation of humanized models has high physiological relevance, helping to clarify the molecular bases of pathology, which in turn is essential for the development of therapeutic procedures. Here, we report the generation and characterization of genetically modified mice carrying a mutation equivalent to SYCE1 c.197-2A>G, previously found in male infertile patients, aiming to determine the actual effects of this mutation on reproductive capacity and to study the underlying molecular mechanisms. Homozygous mutants were infertile. SYCE1 protein was not detected and Syce1 transcript presented minimal levels, suggesting transcript degradation underlying the infertility mechanism. Additionally, homozygous mutants showed impaired homologous chromosome synapsis, meiotic arrest before the pachytene stage, and increased apoptosis of meiotic cells. This study validates the variant as pathogenic and causative of infertility, since the observed dramatic phenotype was attributable to this single homozygous point mutation, when compared to WT and heterozygous littermates. Moreover, although this homozygous point mutation has been only found in infertile men thus far, we anticipate that if it were present in women, it would cause infertility as well, as homozygous female mice also exhibited an infertility phenotype.

Recent advances in embryology have shown that the sister blastomeres of two-cell mouse and human embryos differ reciprocally in potency. An open question is whether the blastomeres became different as opposed to originating as different. Here we wanted to test two relevant but conflicting models: one proposing that each blastomere contains both animal and vegetal materials in balanced proportions because the plane of first cleavage runs close to the animal-vegetal axis of the fertilized oocyte (meridional cleavage); and the other model proposing that each blastomere contains variable proportions of animal and vegetal materials because the plane of the first cleavage can vary - up to an equatorial orientation - depending on the topology of fertilization. Therefore, we imposed the fertilization site in three distinct regions of mouse oocytes (animal pole, vegetal pole, equator) via ICSI. After the first zygotic cleavage, the sister blastomeres were dissociated and subjected to single-cell transcriptome analysis, keeping track of the original pair associations. Non-supervised hierarchical clustering revealed that the frequency of correct pair matches varied with the fertilization site (vegetal pole > animal pole > equator), thereby, challenging the first model of balanced partitioning. However, the inter-blastomere differences had similar signatures of gene ontology across the three groups, thereby, also challenging the competing model of variable partitioning. These conflicting observations could be reconciled if animal and vegetal materials were partitioned at the first cleavage: an event considered improbable and possibly deleterious in mammals. We tested this occurrence by keeping the fertilized oocytes immobilized from the time of ICSI until the first cleavage. Image analysis revealed that cleavage took place preferentially along the short (i.e. equatorial) diameter of the oocyte, thereby partitioning the animal and vegetal materials into the two-cell blastomeres. Our results point to a simple mechanism by which the two sister blastomeres start out as different, rather than becoming different.

Adenomyosis is a chronic, estrogen-driven disorder characterized by the presence of endometrial glands and stroma within the myometrium. Despite its significant impact on reproductive health and quality of life, the pathogenesis of the disease remains unclear. Both the glandular and stromal compartments of eutopic endometrium from women with adenomyosis show alterations compared to healthy subjects. However, the molecular mechanisms driving crosstalk between stromal cells and epithelial glands, along with paracrine signaling underlying lesion development and progression, are still poorly understood. Exosomes, small cell-derived carriers and microRNAs, namely non-coding RNA molecules, are crucial to intercellular communication within the endometrium and may elucidate interactions between the two compartments that contribute to adenomyotic lesion formation. To our knowledge, this is the first foundational study to comprehensively isolate and characterize stroma-derived exosomes from women with adenomyosis. Exosome isolation by means of differential ultracentrifugation was validated in 22 samples, including 11 healthy subjects and 11 women with adenomyosis, using nanoparticle tracking analysis, transmission electron microscopy, and flow cytometry. Profiling of microRNA in secreted exosomes revealed 10 microRNAs with significantly altered expression in adenomyosis subjects during the menstrual phase compared to controls. Thorough investigations into menstruation-specific molecular mechanisms, as well as predicted target genes and enriched pathways of exosomal microRNAs, offer promising insights into the pathogenesis of adenomyosis, shedding light on the potential mechanisms underlying stromal cell signaling and adenomyotic lesion establishment. This work does, however, have certain drawbacks, including modest sample size and limited representation due to a lack of readily available endometrial biopsies in the menstrual phase. Having done the groundwork in this study, future research should seek to validate these findings in larger cohorts and apply functional assays. Indeed, our findings can serve as a resource to elucidate the role of menstruation-specific stroma-derived microRNA-mediated signaling and its potential impact on adenomyosis development.