Molecular human reproduction最新文献

Mammalian oocyte maturation is a tightly regulated process essential for successful fertilization and embryonic development. Meiotic resumption in mammalian oocytes is controlled by various regulatory factors, including the tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein (YWHA/14-3-3). However, the specific functions of individual YWHA isoforms in oocyte meiosis remain poorly understood. In this study, we revealed that knockdown of Ywhaz, one of the isoforms of YWHA, using short interfering RNA (siRNA) or morpholino oligomers (MOs), accelerates meiotic resumption in mouse oocytes. To elucidate the mechanism underlying YWHAZ-mediated meiotic resumption, we thus explored its interactions with potential target proteins. Co-immunoprecipitation and immunofluorescence analyses demonstrated a physical interaction between YWHAZ and phosphorylated CDC25B. Additionally, we identified the protein kinases responsible for YWHAZ phosphorylation at distinct residues. Specifically, JNK1, CSNK1A1/CKIα, and protein kinase B (PKB/Akt) were found to phosphorylate YWHAZ at Serine 184/186, Threonine 232, and Serine 58, respectively. Notably, phosphorylation of YWHAZ at serine 58 by PKB/Akt promoted meiotic resumption in mouse oocytes. Furthermore, we found the formation of a heterodimer between YWHAZ and YWHAQ. Our results provide insights into the PKB/Akt-YWHAZ-CDC25B signalling pathway and illuminate the functional influence of YWHAZ phosphorylation in meiotic regulation.

We have previously shown that ligustilide (LIG) treatment can restore endometrial receptivity and promote implantation in pregnant rats with low endometrial receptivity (LER). Using mRNA sequencing technology, we found that desmoglein-2 (Dsg2) is one of the differentially expressed genes, which is downregulated in rats with LER but elevated after LIG treatment. DSG2 is an adhesion protein that exists in epithelial tissues and serves as a crucial component of the junction complex for intercellular adhesion. The ability to adhere between cells is considered essential for successful embryo implantation. Hence, we speculated that DSG2 might be involved in the mechanism by which LIG treats LER. Immunofluorescence staining, western blots, and immunohistochemical staining were performed to confirm the restored expression of DSG2 by LIG treatment in the endometrial tissues of LER rats. Then, we investigated the effect of DSG2 on endometrial receptivity by injecting small interfering (si)Dsg2 (for silencing Dsg2 expression) into one uterine horn of a pregnant rat and siNC (for a normal control) into the other. It was found that the side of the endometrium injected with siDsg2 showed significant changes in the expression of multiple receptivity-related molecules and obvious embryo loss. In addition, we found that these receptivity-related molecules were affected by siDsg2 in Ishikawa (an endometrial cell line) cells. Dsg2 knockdown reduced the ability of Ishikawa cells to adhere to trophoblastic spheroids. Furthermore, the effect of LIG on endometrial cell-trophoblast adhesion was partially abolished by Dsg2 silencing. In conclusion, our findings suggest that the protective effect of LIG against LER might be achieved by increasing DSG2 expression. DSG2 enhancement might become a novel strategy for the development of LIG-inspired treatments to improve endometrial receptivity.

This study aimed to investigate the benefits of melatonin supplementation during the blastocyst vitrification and thawing process and explore underlying mechanisms to prevent apoptotic events. We evaluated blastocysts in three groups: fresh blastocysts (Control), non-melatonin-treated vitrification (VT), and melatonin-treated vitrification (MVT). We compared their developmental potential and oxidative stress levels to analyse effects of optimized melatonin supplementation. Additionally, transcriptome analysis in blastocysts by Smart-seq2 was performed to investigate the underlying transcriptional mechanism. Antioxidant enzyme and mitochondrial function protein expression were investigated by immunofluorescence staining. Our results showed that supplementation of melatonin to the vitrification and warming solution significantly reduced the apoptotic cell proportion (P < 0.001) while increasing the numbers of inner cell mass (P < 0.001), trophectoderm (P < 0.001), and total cell counts (P < 0.001). Melatonin protected against oxidative stress and restored mitochondrial dysfunction in blastocysts, as evident from increased mitochondrial activity (P < 0.05) and reduced levels of Ca2+ (P < 0.05) and reactive oxygen species (P < 0.05). Importantly, melatonin alleviated cryodamage and preserved blastocyst ultrastructure, and rebalanced altered gene expression induced by the vitrification and warming. These results suggest that adding 10-10 M melatonin to vitrification and warming solutions protects mouse blastocysts against the detrimental effects of oxidative stress and enhances the efficiency of cryopreservation.

Miscarriage and preterm birth (PB) remain major challenges in obstetric care and are often associated with excessive inflammation at the feto-maternal interface. Although the role of progesterone (P4) in maintaining pregnancy is well known, its anti-inflammatory effects in immune-mediated pregnancy complications remain poorly understood. In this study, we investigated the impact of prophylactic P4 administration on miscarriage using a mouse model induced by α-galactosylceramide (αGC), a potent activator of invariant natural killer T (iNKT) cells. Prophylactic, but not therapeutic, P4 administration significantly reduced miscarriage rates. Flow cytometry analysis revealed that P4 suppressed the activity of iNKT cells and the production of inflammatory cytokines by these cells in the myometrium. Moreover, P4 reduced the immunostimulatory activity of antigen-presenting cells, particularly macrophages, by downregulating co-stimulatory molecules and interleukin (IL)-12 production. Immunohistochemistry and flow cytometry results demonstrated that the progesterone receptor (PR) was predominantly expressed on myometrial macrophages. Ex vivo experiments further confirmed that P4 directly modulates macrophage function, decreasing IL-12 and increasing IL-10 production. These findings suggest that prophylactic P4 administration mitigates immune activation at the feto-maternal interface by targeting macrophages, thereby suppressing iNKT cell-mediated inflammation and preventing miscarriage. This study highlights the importance of innate immune modulation in reproductive immunology and the potential of P4 as a prophylactic agent for preventing inflammation-associated miscarriage and PB.

Endometriosis, defined as the growth of endometrial-like tissues outside the uterus, is a common disease among women. Numerous in vivo rodent models of endometriosis have been developed to explore multiple aspects of this poorly understood disease. Heterologous models utilize human endometrial tissues engrafted into immunocompromized mice, while homologous models engraft rodent endometrium into immunocompetent mice or rats. Heterologous models of endometriosis more closely replicate the human disease; however, the murine humoral immune response must be suppressed to prevent rejection of the xenograft tissue. Although the innate immune system remains intact, suppression of the humoral response leads to a markedly different local and systemic immune environments compared to humans. Despite this limitation, experiments using heterologous models have contributed significantly to our understanding of endometriosis establishment and progression, the pre-clinical effectiveness of various therapeutic strategies, and genetically modifiable host factors that contribute to disease. Unfortunately, a lack of harmonization of the models used by different laboratories has impeded the reproducibility and comparability of results between groups. Therefore, the World Endometriosis Research Foundation (WERF) formed an international working group of experts in heterologous models of endometriosis to develop guidelines and protocols that could contribute to unifying experimental approaches across laboratories. Nine critical variables were identified: (i) mouse strain; (ii) human tissue type; (iii) hormonal status of the human tissue donor; (iv) human tissue preparation; (v) method and location of tissue placement; (vi) hormonal status of the recipient animal; (vii) whether or not mice were engrafted with human immune cells; (viii) endpoint assessments; and (ix) number and type of replicates. Herein, we outline important considerations for each major variable and make recommendations for unification of approaches. Widespread adoption of harmonized protocols and implementation of standardized documentation and reporting should further improve the reproducibility and translation of experimental findings both within and between laboratories.

Infertility impacts up to 17.5% of reproductive-aged couples worldwide. To aid in conception, many couples turn to ART, such as IVF. IVF can introduce both physical and environmental stressors that may alter DNA methylation regulation, an important and dynamic process during early fetal development. This meta-analysis aims to assess the differences in the placental DNA methylome between spontaneous and IVF pregnancies. Potential datasets were identified by searching the NCBI Gene Expression Omnibus (GEO) using keywords related to IVF in human participant studies published before November 2023. In our combined fetal sex population (N = 575) from three eligible GEO datasets, 127 autosomal cytosine guanine dinucleotides (CpGs) were significant (False Discovery Rate (FDR) <0.05) between IVF (n = 96) and spontaneous (n = 479) placentae, with 47 CpGs considered differentially methylated (FDR < 0.05 and |Δβ| > 0.05). Stratification by fetal sex revealed no significant autosomal CpGs in fetal female placentae (N = 281); however, in the fetal male placentae (N = 294), we identified nine autosomal CpGs that reached statistical significance between IVF (n = 56) and spontaneous (n = 238) placentae, with three CpGs considered differentially methylated. Fetal male placentae had lower proportions of trophoblasts (P < 0.0001) and stromal cells (P = 0.007) and higher proportions of syncytiotrophoblasts (P = 0.0001) compared to fetal female placentae, regardless of conception type. IVF placentae had higher proportions of stromal cells (P = 0.01) and lower proportions of syncytiotrophoblasts (P = 0.01) compared to spontaneous placentae, regardless of sex. Controlling for cell-type proportions in linear models reduced test statistic inflation and identified new significant CpGs that may previously have been masked by cell-type heterogeneity. The results of this meta-analysis are critical to further understand the impact of IVF on tissue epigenetics, which may help with understanding the connections between IVF and negative pregnancy outcomes. Additionally, our study suggests that sex-specific differences in placental DNA methylation and cell composition should be considered as factors for future placental DNA methylation analyses.

Premature ovarian insufficiency (POI) is a major cause of female infertility, for which effective therapies remain limited. S14G-Humanin (HNG), a potent analogue of Humanin, exhibits strong antioxidant and anti-apoptotic properties and has demonstrated cytoprotective effects in various tissues, including the ovary. In this study, a cyclophosphamide (CP)-induced POI mouse model was established to evaluate both the ovarian damage induced by chemotherapy and the protective effects of HNG. HNG administration significantly increased the number of primordial follicles (P = 0.044) and growing follicles (all P > 0.05), as well as corpora lutea (P = 0.09). Moreover, HNG markedly improved oocyte quality (P = 0.009), significantly lowering the proportion of abnormal ovulated oocytes (P = 0.002). Fertility outcomes were also enhanced: CP treatment significantly reduced litter size compared to controls (4.6 ± 1.1 vs 8.0 ± 1.0; P < 0.001), whereas HNG treatment significantly mitigated this reduction (6.2 ± 0.8 vs 4.6 ± 1.1; P = 0.029). Mechanistically, HNG alleviated oxidative stress and apoptosis in ovarian tissues (all P < 0.05), reduced ROS levels (P = 0.034), and restored mitochondrial membrane potential (P = 0.004) in a human granulosa cell line. Furthermore, HNG significantly upregulated PGC-1α expression and enhanced AMPK phosphorylation in both in vivo and in vitro models (both P < 0.05). Collectively, these findings demonstrate that HNG confers significant protection against chemotherapy-induced ovarian damage and highlight its potential as a novel therapeutic agent for chemotherapy-induced ovarian damage.

While advanced maternal age is associated with significant changes in oocyte gene expression, these are not global changes but limited to a fraction of the transcriptome. However, there is little consensus on the specific genes affected, and on the transcriptomic signatures of age-related declines in oocyte quality. To characterize the effects of age on the human MII oocyte transcriptome, here we take a two-part approach. We first generated single-oocyte Smart-seq2 datasets from 10 younger (21-29 years) and 10 older (37-43 years) donors, identifying genes differentially expressed between the two groups, then cross-referenced our results with those of 12 studies (9 human, 3 mouse) performing equivalent analyses using a variety of single-cell transcriptomic or microarray platforms. Technical differences notwithstanding, we found considerable discordance between the datasets, suggesting that age-related signatures of differential gene expression are not easily reproducible. Independent corroboration of age-associated changes in expression was limited to few genes, with the vast majority only supported by one of the 13 datasets, including our own. Nevertheless, we identified 40 genes whose expression significantly altered with age in multiple studies, highlighting common processes underlying ageing, including dysregulated proteostasis. As human Smart-seq2 oocyte libraries are challenging to procure and rare in public archives, we next implemented a meta-analytic method for their re-use, combining our 20 oocytes with 130 pre-existing libraries sourced from 12 different studies and representing a continuous age range of 18-43 years. We identified 25 genes whose expression level significantly correlated with age and corroborated 14 of these genes with RT-PCR, including the proteasomal subunits PSMA1 and PSMA2, both of which were downregulated in older oocytes. Overall, our findings are consistent with both pronounced inter-oocyte heterogeneity in transcription and with oocyte ageing being a multifactorial process to which bona fide transcriptomic changes may only play a restricted role, while proteomic changes play more pronounced roles.

In vitro follicle culture (IFC) is an emerging fertility preservation alternative for women and children with cancer. Because two-dimensional (2D) IFC results in oocytes of suboptimal quality in mice and cannot support follicle growth in humans, the search for an optimal three-dimensional (3D) method that preserves the follicular structure is ongoing, and both matrix-free and hydrogel encapsulation systems are being explored. Our aim was to compare several 3D mouse IFC systems, including matrix-free and hydrogel encapsulation approaches. Secondary follicles were cultured for 12 days in a matrix-free non-attachment (NA) system, a Poly-Ethylene-Glycol (PEG) hydrogel, an extracellular-matrix-derived soft hydrogel (ES), and a 2D attachment (AT) control. We assessed follicle growth, survival, hormone secretion, theca cell localization, oocyte meiotic competence and diameter, gene expression in oocytes and cumulus cells, as well as oocyte fertilization potential. Metaphase II oocyte rates were significantly higher in the NA (75 ± 12.4%, n = 79) and AT systems (77 ± 12.6%, n = 109) compared to the ES (33.4 ± 9.5%, n = 40, P < 0.01), while low antral follicle rates from the PEG system led to its exclusion from the comparison. Similarly, following IVF, 2-cell rates were significantly higher in the NA (47.7 ± 17.6%, n = 147, P < 0.01) and AT (40.2 ± 9.7%, n = 132, P < 0.05) systems compared to the ES (23.5 ± 9.3%, n = 63). Furthermore, cumulus cells from the NA condition displayed a more in vivo-like gene expression profile than other conditions. No differences were detected in follicle survival, oocyte diameter, blastocyst rate, or quality between conditions. Lastly, we observed major differences in theca cell localization and hormone secretion levels that require further investigation. Our findings demonstrate the efficiency of the NA system over complex encapsulation methodologies, as it enhanced oocyte meiotic and developmental competence compared to the ES. However, as the study is limited by the lack of human data and the use of Fetal Bovine Serum (FBS) in the culture medium, further research is required to translate our findings to humans.

Spermatogonial stem cells (SSCs) serve as the foundational units for all adult male germ cells, playing a critical role in male fertility. These cells are characterized by their unique ability to balance self-renewal and differentiation, ensuring the maintenance of tissue homeostasis while simultaneously producing mature spermatozoa. Recent studies have highlighted the pivotal role of fibroblast growth factor (FGF) signaling in regulating SSC self-renewal, with various FGFs and their corresponding FGF receptors (FGFRs) being implicated in the intricate processes governing SSC homeostasis. This review aims to provide a comprehensive overview of the characteristic expression patterns of FGFs and FGFRs within germ cells and their surrounding somatic cells. In addition, we will conduct in-depth research on the multifaceted functions and regulatory mechanisms of FGF signaling in SSCs and elucidate its potential clinical significance. Understanding these mechanisms not only enhances our knowledge of male reproductive biology but also opens avenues for innovative clinical applications aimed at safeguarding fertility in vulnerable populations.