Molecular human reproduction最新文献

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.

Among rare cases of teratozoospermia, MMAF (multiple morphological abnormalities of the flagellum) syndrome is a complex genetic disorder involving at least 70 different genes. As the name suggests, patients with MMAF syndrome produce spermatozoa with multiple flagellar defects, rendering them immobile and non-fertilizing, leading to complete infertility in affected men. The only viable treatment option is ICSI. What is less understood is the presence of the various types of head defects in the spermatozoa, which are consistently present. Due to the involvement of numerous genes and the limited number of patients with MMAF syndrome, research on head defects and their impact on embryonic development remains insufficiently explored. To address these questions, a comparative study was conducted under controlled experimental conditions using four knockout (KO) mouse lines targeting Cfap43, Cfap44, Armc2, and Ccdc146 genes, all associated with MMAF syndrome in humans and mice. Each KO line underwent a detailed examination of nuclear defects, including morphology, DNA compaction, chromosomal architecture, and ploidy. The study revealed significant heterogeneity among the four lineages, with the extent of defects varying depending on the lineage, ranked as Ccdc146-/- > Cfap43-/- > Armc2-/- ≈ Cfap44-/-. The developmental potential of sperm from males in each lineage was assessed by injecting them into wild-type oocytes, and embryo development was monitored up to the blastocyst stage. Sperm from all KO lines exhibited a marked decrease in supporting embryo development compared to the wild-type, with developmental failure rates ranked as follows: Ccdc146 > Cfap43 > Armc2 > Cfap44-deficient sperm. The degree of developmental failure thus correlated with the severity of nuclear defects, and zygotes produced with sperm from Ccdc146-/- and Cfap43-/- mice showed the highest rates of developmental impairment. These findings from preclinical models highlight the heterogeneous nature of MMAF syndrome, both in terms of sperm nuclear defects and developmental potentials. Genetic characterization in humans is therefore crucial for improving therapeutic counselling in affected individuals.

Biphasic IVM can be offered as a patient-friendly alternative to conventional ovarian stimulation in IVF patients predicted to be hyper-responsive to ovarian stimulation. However, cumulative live birth rates after IVM per cycle are lower than after conventional ovarian stimulation for IVF. In different animal species, supplementation of IVM media with oocyte-secreted factors (OSFs) improves oocyte developmental competence through the expression of pro-ovulatory genes in cumulus cells. Whether the addition of OSFs in human biphasic IVM culture impacts the transcriptome of oocytes and cumulus cells retrieved from small antral follicles in minimally stimulated non-hCG-triggered IVM cycles remains to be elucidated. To answer this, human cumulus-oocyte complexes (COCs) that were fully surrounded by cumulus cells or partially denuded at the time of retrieval were cultured in a biphasic IVM system either without or with the addition of pro-cumulin, a GDF9:BMP15 heterodimer. Oocytes and their accompanying cumulus cells were collected separately, and single-cell RNA-seq libraries were generated. The transcriptomic profile of cumulus cells revealed that pro-cumulin upregulated the expression of genes involved in cumulus cell expansion and proliferation while downregulating steroidogenesis, luteinization, and apoptosis pathways. Moreover, pro-cumulin modulated the immature oocyte transcriptome during the pre-maturation step, including regulating translation, apoptosis, and mitochondria remodeling pathways in the growing germinal vesicle oocytes. The addition of pro-cumulin also restored the transcriptomic profile of matured metaphase II oocytes that were partially denuded at collection. These results suggest that cumulus cell and oocyte transcriptome regulation by pro-cumulin may increase the number of developmentally competent oocytes after biphasic IVM treatment. Future studies should assess the effects of pro-cumulin addition in human biphasic IVM at the proteomic level and the embryological outcomes, particularly its potential to enhance outcomes of oocytes that are partially denuded at COC collection.

In patients with mosaic Turner syndrome, the ovarian somatic cells (granulosa and stromal cells) display a high level of aneuploidy with a 45,X karyotype, which may affect gene expression in the ovary and contribute to their reduced fertility. The aim of the current research is to study the effect of aneuploidy of somatic ovarian cells on gene expression in ovarian cortex stromal cells and small ovarian follicles from mosaic (45,X/46,XX) Turner syndrome patients. To this end, ovarian cortical tissue was obtained by laparoscopic surgery from eight mosaic Turner syndrome patients (aged 5-19 years) and eight controls (aged 6-18 years). The tissue was fractionated to obtain purified follicles and stromal cells. Part of the purified fractions was used to determine the X chromosomal content of ovarian cells of Turner syndrome patients by interphase FISH, while the remaining part was used to compare the gene expression profile of these cells to controls. The results demonstrated that high level 45,X haploidy in cortical stromal cells of Turner syndrome patients had no effect on gene expression, gross morphology of the ovary, or histological appearance of the cortex compared to controls. Gene expression analysis of purified small follicles of Turner syndrome patients with mainly 45,X granulosa cells revealed aberrant expression of 11 genes. Of these, six were upregulated (CD24, TLR1, EPHA2, PLXND1, ST6GALNAC5, and NOX4) while five genes (CRYAB, DLX1, PCYT2, TNFRSF8, and CA12) were downregulated compared to follicles of controls. Interestingly, the overexpressed genes in these small follicles were all associated with more advanced stages of follicular development. The consequences of this abnormal gene expression in follicles for Turner syndrome patients remain to be investigated, but they are likely to affect fertility.

Mammalian target of rapamycin (mTOR) inhibitors have been used clinically as anticancer and immunosuppressive agents for over 20 years, demonstrating their safety after long-term administration. These inhibitors exhibit various effects, including inhibition of cell proliferation, interaction with the oestrogen and progesterone pathways, immunosuppression, regulation of angiogenesis, and control of autophagy. We evaluated the potential of mTOR inhibitors as therapeutic agents for endometriosis, examined the secondary benefits related to reproductive function, and assessed how their side effects can be managed. We conducted a thorough review of publications on the role of the mTOR pathway and the effectiveness of mTOR inhibitors in endometriosis patients. These results indicate that the mTOR pathway is activated in endometriosis. Additionally, mTOR inhibitors have shown efficacy as monotherapies for endometriosis. They may alleviate resistance to hormonal therapy in endometriosis, suggesting a potential synergistic effect when used in combination with hormonal therapy. The potential reproductive benefits of mTOR inhibitors include decreased miscarriage rates, improved implantation, and prevention of age-related follicular loss and ovarian hyperstimulation syndrome. Activation of the mTOR pathway has also been implicated in the malignant transformation of endometriosis. Preclinical studies suggest that the dosage of mTOR inhibitors needed for treating endometriosis may be lower than that required for anticancer or immunosuppressive therapy, potentially reducing dosage-dependent side effects. In conclusion, while mTOR inhibitors, which allow for pregnancy during oral administration, show potential for clinical use in all stages of endometriosis, current evidence is limited to preclinical studies, and further research is needed to confirm clinical effectiveness.

Endometrial collagen I undergoes dynamic degradation and remodelling in response to endometrial stromal cell (ESC) decidualization and embryo implantation. However, excessive collagen I deposition in the endometrium during the implantation window may impair decidualization, causing embryo implantation failure in patients with endometriosis (EMS). We found that endometrial collagen I expression during the mid-secretory phase was increased in the EMS group of patients. Collagen I stimulation resulted in decreased expression of the decidualization markers prolactin and insulin-like growth factor binding protein-1 in ESCs, impeding ESC transformation to a decidual morphology and decreasing the blastocyst-like spheroid expansion area in vitro. Treatment with extracellular vesicles (EVs) derived from the ectopic ESCs of EMS patients (EMS-EVs) increased collagen I expression in vivo and in vitro and decreased the blastocyst-like spheroid expansion area. Furthermore, EV microRNA (miRNA) sequencing revealed that there were 40 upregulated and 77 downregulated miRNAs in EMS-EVs when compared to the EVs derived from ESCs in the endometrium of control patients (CTL-EVs), including increased expression of miR-25-3p that targets phosphatase and tensin homolog (PTEN). We also found that PTEN expression was decreased and p-Akt expression was increased in the endometrium of EMS patients and EMS-EV-treated ESCs. miR-25-3p transfected ESCs exhibited increased collagen I, decreased PTEN, and increased p-Akt. Additionally, an EV uptake study further showed that EMS-EVs were preferentially taken up by ESCs rather than by endometrial epithelial cells. These results suggest that EMS-EVs encapsulating miR-25-3p might be preferentially taken up by eutopic ESCs where they may induce endometrial collagen I deposition to impair ESC decidualization in EMS.

Currently, there are no placenta-targeted treatments to alter the in utero environment for administration to pregnant women who receive a diagnosis of fetal growth restriction (FGR). Water-soluble polymers have a distinguished record of clinical relevance outside of pregnancy. We have demonstrated the effective delivery of polymer-based nanoparticles containing a non-viral human insulin-like growth factor 1 (IGF1) transgene to correct placental insufficiency in small animal models of FGR. Our goals were to extend these studies to a proof-of-concept study in the pregnant macaque, establish feasibility of nanoparticle-mediated gene therapy delivery to trophoblasts, and investigate the acute maternal, placental, and fetal responses to treatment. Pregnant macaques underwent ultrasound-guided intraplacental injections of nanoparticles (GFP- or IGF1-expressing plasmid under the control of the trophoblast-specific PLAC1 promoter complexed with a HPMA-DMEAMA co-polymer) at approximately gestational day 100 (term = 165 days). Fetectomy was performed 24 h (GFP; n = 1), 48 h (IGF1; n = 3) or 10 days (IGF1; n = 3) after nanoparticle delivery. Routine pathological assessment was performed on biopsied maternal tissues and placental and fetal tissues. Maternal blood was analyzed for complete blood count (CBC), immunomodulatory proteins and growth factors, progesterone (P4), and estradiol (E2). Placental ERK/AKT/mTOR signaling was assessed using Western blot and qPCR. Fluorescent microscopy and in situ hybridization confirmed placental uptake and transient transgene expression in villous syncytiotrophoblast. No off-target expression was observed in either maternal or fetal tissues. Histopathological assessment of the placenta recorded observations not necessarily related to the IGF1 nanoparticle treatment. In maternal blood, CBCs, P4, and E2 remained within the normal range for pregnant macaques across the treatment period. Changes to placental ERK and AKT signaling at 48 h and 10 days after IGF1 nanoparticle treatment indicated an upregulation in placental homeostatic mechanisms to prevent overactivity in the normal pregnancy environment. The lack of adverse maternal reaction to nanoparticle-mediated IGF1 treatment, combined with changes in placental signaling to maintain homeostasis, indicates no deleterious impact of treatment during the acute phase of study.

Endometrial receptivity is crucial for successful embryo implantation during early pregnancy. The human endometrium undergoes remodeling within each menstrual cycle to prepare or become receptive to an implanting blastocyst in the mid-secretory phase. However, the mechanisms behind these changes are not fully understood. Recently, using hormone-treated endometrial organoids to model receptivity, we identified that the transcriptional regulator WD-repeat-containing protein-61 (WDR61) was reduced in organoids derived from infertile women. In this study, we aimed to determine the role of WDR61 in endometrial receptivity. Here, we demonstrated that WDR61 immunolocalizes in the nuclei and cytosol of endometrial glandular epithelium, luminal epithelium, and stroma. The staining intensity of WDR61 was significantly higher during the receptive mid-secretory phase compared to the non-receptive proliferative phase in fertile women. In a functional experiment to model blastocyst adhesion to the endometrial epithelium, we found that adhesion of cytotrophoblast progenitor spheroids was blocked when siRNA was used to knockdown WDR61 in primary endometrial epithelial cells. Similarly, in Ishikawa cells (a receptive human endometrial epithelial cell line), siRNA knockdown of WDR61 significantly reduced the cell adhesive and proliferative capacities. qPCR revealed that WDR61 knockdown reduced expression of key genes involved in receptivity including HOXD10, MMP2, and CD44. Chromatin immunoprecipitation sequencing demonstrated that WDR61 directly targeted 2022 genes in Ishikawa cells, with functions including focal adhesion, intracellular signaling and epithelial-mesenchymal transition. Overall, these findings suggest that WDR61 promotes endometrial receptivity by modulating epithelial cell focal adhesions, proliferation, and epithelial-mesenchymal transition.

Natural killer (NK) cells are the most abundant leukocytes located at the maternal-fetal interface; they respond to pregnancy-related hormones and play a pivotal role in maintaining the homeostatic micro-environment during pregnancy. However, due to the high heterogeneity of NK cell subsets, their categorization has been controversial. Here, we review previous studies on uterine NK cell subsets, including the classic categorization based on surface markers, functional molecules, and developmental stages, as well as single-cell RNA sequencing-based clustering approaches. In addition, we summarize the potential pathways by which endometrial NK cells differentiate into decidual NK (dNK) cells, as well as the differentiation pathways of various dNK subsets. Finally, we compared the alterations in the NK cell subsets in various pregnancy-associated diseases, emphasizing the possible contribution of specific subsets to the development of the disease.

Polycystic ovary syndrome (PCOS) is a common reproductive endocrine disease, which leads to serious impairment of reproductive health in women of child-bearing age. Anovulation or oligo-ovulation is a common clinical manifestation of PCOS patients. A disturbance of the ovarian immune microenvironment contributes to the disorders of follicle development and ovulation; however, the underlying mechanism remains unclear. Here we demonstrated the protective effect of immune factor interleukin-22 (IL-22) on PCOS follicle development and ovulation. Follicular IL-22 levels were significantly lower in PCOS patients than in the control group and were positively correlated with oocyte fertilization rate and high-quality embryo rate. Additionally, IL-22 evidently improved follicle development in vitro and promoted ovulation-related gene expression, which was disrupted by the depletion of interleukin-22 receptor 1 (IL-22R1) or inhibition of STAT3 in granulosa cells. This indicates that IL-22 acts through IL-22R1 and the STAT3 signaling pathway to promote follicle development and ovulation in PCOS. In summary, this study has elucidated the vital role of the ovarian immune microenvironment in follicle development and ovulation. Application of IL-22 may provide new insights into the treatment of PCOS patients.