Increased levels of villus-derived exosomal miR-29a-3p in normal pregnancy than uRPL patients suppresses decidual NK cell production of interferon-γ and exerts a therapeutic effect in abortion-prone mice.

OBJECTIVE: Recurrent pregnancy loss (RPL) patients have higher absolute numbers of decidual natural killer (dNK) cells with elevated intracellular IFN-γ levels leading to a pro-inflammatory cytokine milieu, which contributes to RPL pathogenesis. The main objective of this study was twofold: first to explore the regulatory effects and mechanisms of villus-derived exosomes (vEXOs) from induced abortion patients or RPL patients at the level of intracellular IFN-γ in dNK cells; second to determine the validity of application of vEXOs in the treatment of unexplained RPL (uRPL) through in vitro experiments and mouse models. METHODS: Exosomes were isolated from villus explants by ultracentrifugation, co-cultured with dNK cells, and purified by enzymatic digestion and magnetically activated cell sorting. Flow cytometry, enzyme-linked immunosorbent assays, and RT-qPCR were used to determine IFN-γ levels. Comparative miRNA analysis of vEXOs from induced abortion (IA) and uRPL patients was used to screen potential candidates involved in dNK regulation, which was further confirmed by luciferase reporter assays. IA-vEXOs were electroporated with therapeutic miRNAs and encapsulated in a China Food and Drug Administration (CFDA)-approved hyaluronate gel (HA-Gel), which has been used as a clinical biomaterial in cell therapy for > 30 years. In vivo tracking was performed using 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyaine iodide (DiR) labelling. Tail-vein and uterine horn injections were used to evaluate therapeutic effects of the engineered exosomes in an abortion-prone mouse model (CBA/J × DBA/2 J). Placental growth was evaluated based on placental weight. IFN-γ mRNA levels in mouse placentas were measured by RT-qPCR. RESULTS: IFN-γ levels were significantly higher in dNK cells of uRPL patients than in IA patients. Both uRPL-vEXOs and IA-vEXOs could be efficiently internalized by dNK cells, whereas uRPL-vEXOs could not reduce the expression of IFN-γ by dNK cells as much as IA-vEXOs. Mechanistically, miR-29a-3p was delivered by vEXOs to inhibit IFN-γ production by binding to the 3' UTR of IFN-γ mRNA in dNK cells. For in vivo treatment, application of the HA-Gel effectively prolonged the residence time of vEXOs in the uterine cavity via sustained release. Engineered vEXOs loaded with miR-29a-3p reduced the embryo resorption rate in RPL mice with no signs of systemic toxicity. CONCLUSION: Our study provides the first evidence that villi can regulate dNK cell production of IFN-γ via exosome-mediated transfer of miR-29a-3p, which deepens our understanding of maternal-fetal immune tolerance for pregnancy maintenance. Based on this, we developed a new strategy to mix engineered vEXOs with HA-Gel, which exhibited good therapeutic effects in mice with uRPL and could be used for potential clinical applications in uRPL treatment.

TSG6-Exo@CS/GP Attenuates Endometrium Fibrosis by Inhibiting Macrophage Activation in a Murine IUA Model.

Intrauterine adhesion (IUA) is characterized by the formation of fibrous scar tissue within the uterine cavity, which significantly impacts female reproductive health and even leads to infertility. Unfortunately, severe cases of IUA currently lack effective treatments. This study presents a novel approach that utilizes tumor necrosis factor-(TNF) stimulated gene 6 (TSG6)-modified exosomes (Exos) in conjunction with an injectable thermosensitive hydrogel (CS/GP) to mitigate the occurrence of IUA by reducing endometrium fibrosis in a mouse IUA model. This study demonstrate that TSG6-modified Exos effectively inhibits the activation of inflammatory M1-like macrophages during the initial stages of inflammation and maintains the balance of macrophage phenotypes (M1/M2) during the repair phase. Moreover, TSG6 inhibits the interaction between macrophages and endometrial stromal fibroblasts, thereby preventing the activation of stromal fibroblasts into myofibroblasts. Furthermore, this research indicates that CS/GP facilitates the sustained release of TSG6-modified Exos, leading to a significant reduction in both the manifestations of IUA and the extent of endometrium fibrosis. Collectively, through the successful construction of CS/GP loaded with TSG6-modified Exos, a reduction in the occurrence and progression of IUA is achieved by mitigating endometrium fibrosis. Consequently, this approach holds promise for the treatment of IUA.

High estrogen during ovarian stimulation induced loss of maternal imprinted methylation that is essential for placental development via overexpression of TET2 in mouse oocytes.

BACKGROUND: Ovarian stimulation (OS) during assisted reproductive technology (ART) appears to be an independent factor influencing the risk of low birth weight (LBW). Previous studies identified the association between LBW and placenta deterioration, potentially resulting from disturbed genomic DNA methylation in oocytes caused by OS. However, the mechanisms by which OS leads to aberrant DNA methylation patterns in oocytes remains unclear. METHODS: Mouse oocytes and mouse parthenogenetic embryonic stem cells (pESCs) were used to investigate the roles of OS in oocyte DNA methylation. Global 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) levels were evaluated using immunofluorescence or colorimetry. Genome-wide DNA methylation was quantified using an Agilent SureSelectXT mouse Methyl-Seq. The DNA methylation status of mesoderm-specific transcript homologue (Mest) promoter region was analyzed using bisulfite sequencing polymerase chain reaction (BSP). The regulatory network between estrogen receptor alpha (ERα, ESR1) and DNA methylation status of Mest promoter region was further detected following the knockdown of ERα or ten-eleven translocation 2 (Tet2). RESULTS: OS resulted in a significant decrease in global 5mC levels and an increase in global 5hmC levels in oocytes. Further investigation revealed that supraphysiological ß-estradiol (E2) during OS induced a notable decrease in DNA 5mC and an increase in 5hmC in both oocytes and pESCs of mice, whereas inhibition of estrogen signaling abolished such induction. Moreover, Tet2 may be a direct transcriptional target gene of ERα, and through the ERα-TET2 axis, supraphysiological E2 resulted in the reduced global levels of DNA 5mC. Furthermore, we identified that MEST, a maternal imprinted gene essential for placental development, lost its imprinted methylation in parthenogenetic placentas originating from OS, and ERα and TET2 combined together to form a protein complex that may promote Mest demethylation. CONCLUSIONS: In this study, a possible mechanism of loss of DNA methylation in oocyte caused by OS was revealed, which may help increase safety and reduce epigenetic abnormalities in ART procedures.

Identifying a panel of nine genes as novel specific model in endometriosis noninvasive diagnosis.

OBJECTIVE: To study biomarkers to develop a novel diagnosis model for endometriosis and validate it using clinical samples. DESIGN: We used publicly available data sets and weighted gene coexpression network analysis to identify differentially expressed genes. Ten machine learning algorithms were used to develop an integrative model for predicting endometriosis. The accuracy and robustness of the model were validated using data sets and clinical samples. SETTING: Department of Obstetrics and Gynecology, Tangdu Hospital, Air Force Medical University, Xi'an, Shaanxi, China. PATIENT(S): The study included clinical patients between the ages of 20 and 40 years who required laparoscopic surgery and who had not undergone hormone therapy within the previous 3 months. All the healthy individuals had given birth to a child at least once in their lives. Patients with inflammatory conditions, malignant diseases, immune diseases, myoma, or adenomyosis were excluded. Paraffin blocks of the samples were collected (case, n = 5; control, n = 5). Blood samples of 58 individuals were collected (case, n = 28; control, n = 30). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The areas under the receiver operator characteristic curve of our diagnostic model were measured for data sets and clinical samples. Multiplex immunohistochemical staining and real-time quantitative polymerase chain reaction assays were used for the validation of the model from tissue slides and peripheral blood samples. RESULT(S): A nine-gene panel endometriosis messenger RNA score (EMScore), was constructed to distinguish the patients with endometriosis from healthy individuals using algorithms. The EMScore accurately predicted endometriosis, and the areas under the receiver operator characteristic curve of our diagnostic model were 0.920, and 0.942 for tissue and blood samples, respectively. Moreover, the EMScore outperformed other acknowledged signatures for predicting endometriosis across seven clinical cohorts. Overall, the EMScore constitutes a sensitive and specific noninvasive diagnostic method for endometriosis. CONCLUSION(S): We developed the EMScore, a novel model that can aid in the diagnosis of endometriosis using peripheral blood samples. This study will contribute to the development of improved clinical noninvasive and sensitive diagnostic tools for endometriosis. These nine genes might be potential target molecules for treating endometriosis.

Effect of endometrial thickness on obstetric and neonatal outcomes in assisted reproduction: a systematic review and meta-analysis.

PURPOSE: This systematic review and meta-analysis aimed to explore the relationship of endometrial thickness (EMT) with obstetric and neonatal outcomes in assisted reproductive cycles. METHODS: PubMed, EMBASE, Cochrane Library and Web of Science were searched for eligible studies through April 2023. Obstetric outcomes include placenta previa, placental abruption, hypertensive disorders of pregnancy (HDP), gestational diabetes mellitus (GDM) and cesarean section (CS). Neonatal outcomes include birthweight, low birth weight (LBW), gestational age (GA), preterm birth (PTB), small for gestational age (SGA) and large for gestational age (LGA). The effect size was estimated as odds ratio (OR) or mean difference (MD) with 95% confidence interval (CI) using a random-effects model. Inter-study heterogeneity was assessed by the chi-square homogeneity test. One-study removal method was used to determine the sensitivity of the meta-analysis. RESULTS: Nineteen studies involving 76,404 cycles were included. The pooled results revealed significant differences between the thin endometrium group and the normal group in placental abruption (OR = 2.45, 95% CI: 1.11-5.38, P = 0.03; I2 = 0%), HDP (OR = 1.72, 95% CI: 1.44-2.05, P < 0.0001; I2 = 0%), CS (OR = 1.33, 95% CI: 1.06-1.67, P = 0.01; I2 = 77%), GA (MD = -1.27 day, 95% CI: -2.41- -1.02, P = 0.03; I2 = 73%), PTB (OR = 1.56, 95% CI: 1.34-1.81, P < 0.0001; I2 = 33%), birthweight (MD = -78.88 g, 95% CI: -115.79- -41.98, P < 0.0001; I2 = 48%), LBW (OR = 1.84, 95% CI: 1.52-2.22, P < 0.00001; I2 = 3%) and SGA (OR = 1.41, 95% CI: 1.17-1.70, P = 0.0003; I2 = 15%). No statistical differences were found in placenta previa, GDM, and LGA. CONCLUSION: Thin endometrium was associated with lower birthweight or GA and higher risks of placental abruption, HDP, CS, PTB, LBW and SGA. Therefore, these pregnancies need special attention and close follow-up by obstetricians. Due to the limited number of included studies, further studies are needed to confirm the results.

Maternal circulating exosomal miR-185-5p levels as a predictive biomarker in patients with recurrent pregnancy loss.

PURPOSE: The aim of this study was to explore the predictive role of microRNAs (miRNAs) from maternal serum exosomes in early recurrent pregnancy loss (RPL) and the related mechanism in early pregnancy. METHODS: Maternal serum was collected from pregnant women with RPL history or women with ongoing pregnancy (OP); serum exosomes were extracted and identified. Differentially expressed (DE) miRNAs in exosomes were screened by RNA sequencing and further validated by qRT-PCR. Next, the predictive value of exosomal miRNA and the clinical indicators for subsequent miscarriage in RPL patients were evaluated. Additionally, we verified the regulatory relationship between miR-185-5p and vascular endothelial growth factor (VEGF) in decidual natural killer (dNK) cells by overloading or inhibiting the exosomal miR-185-5p level in trophoblast cells. RESULTS: The miRNA sequencing revealed 43 DE miRNAs between OP and RPL patients. The five most significant DE miRNAs (miR-22-3p, miR-185-5p, miR-335-3p, miR-362-5p, and miR-378a-3p) were selected for identification, and miR-185-5p was increased in RPL patients. The area under curve (AUC) of the receiver operating characteristic was 0.925 when using miR-185-5p as a biomarker for subsequent miscarriage in RPL patients. In addition, miR-185-5p in exosomes secreted from HTR-8 cells reduces VEGF expression of dNK cells. CONCLUSIONS: The current study, for the first time, successfully constructed the correlation between maternal circulating exosomal miR-185-5p expression pattern and RPL, which may be involved in the pathogenesis of RPL by downregulating the VEGFA of dNK cells and perturbing angiogenesis at the maternal-fetal interface.

Effect of female coronavirus disease 2019 vaccination on assisted reproductive outcomes: a systematic review and meta-analysis.

IMPORTANCE: The effect of coronavirus disease 2019 (COVID-19) vaccination on fertility warrants clarification in women undergoing assisted reproductive treatment. OBJECTIVE: To study the association between female COVID-19 vaccination and outcomes of assisted reproductive treatment. DATA SOURCES: PubMed, Embase, the Web of Science, Cochrane Library, and medRxiv and bioRxiv were searched for eligible studies from December 1, 2019, to November 30, 2022, with no language restrictions. STUDY SELECTION AND SYNTHESIS: Observational studies comparing assisted reproductive outcomes between women with and without COVID-19 vaccination were included. The pooled estimates were calculated using the random-effects models as mean differences (MDs), standardized MDs, or odds ratios with 95% confidence intervals (CIs). Heterogeneity was evaluated using the I2 statistic. MAIN OUTCOMES: The number of oocytes retrieved and clinical pregnancy rate. RESULTS: Twenty-one cohort studies involving a total of 19,687 treatment cycles were included. In a comparison of the vaccinated vs. unvaccinated groups, the pooled MD for oocyte number was -0.06 (95% CI, -0.51 to 0.39; I2 = 0), and the pooled odds ratio for clinical pregnancy was 0.95 (95% CI, 0.85-1.05; I2 = 0). Similarly, there were no statistically significant adverse effects identified in other outcomes determined a priori, including 4 cycle characteristics, 6 laboratory parameters, and 3 pregnancy indicators. Most results were consistently unchanged in subgroup and sensitivity analyses, with no evidence of publication bias according to Egger's test. CONCLUSION AND RELEVANCE: Our work did not find significant differences in assisted reproductive outcomes between vaccinated and unvaccinated women. However, more data are warranted to confirm the safety of COVID-19 vaccination for assisted reproductive treatment and in female fertility in general.

[Villi exosomes deliver HLA-G to enhance the expression of osteoglycin and pleiotrophin in decidual NK cells].

Objective To identify the effect of HLA-G-containing exosomes on the secretory function of growth-promoting factors osteoglycin (OGN) and pleiotrophin (PTN) by decidual NK (dNK) cells. Methods dNK cells were co-cultured with HLA-G-containing exosomes from the villi of patients undergoing unexplained recurrent pregnancy loss (uRPL) and normal induced abortion, respectively. Sequentially, OGN and PTN of the dNK cells were determined using real time quantitative PCR and western blotting. Exosomes overexpressing HLA-G (HLA-GOE-EXO) were obtained by transfecting the villous trophoblast cell line HTR-8/Svneo with lentivirus LV-HLA-G. dNK cells were further co-cultured with HLA-GOE-EXO for detecting the expression of OGN and PTN, the culture supernatant of which was used to treat HTR-8/Svneo cells, and the proliferation of HTR-8/Svneo cells was detected by the CCK-8 assay. Results Exosomes derived from villi of patients receiving normal induced abortion significantly enhanced the expression of OGN and PTN in dNK cells compared with those from patients of the uRPL group. Besides, HLA-GOE-EXO markedly enhanced the expression of OGN and PTN in dNK cells. The culture supernatant of HLA-GOE-EXO treated dNK cells could promote the proliferation of HTR-8/Svneo cells. Conclusion Villi-derived HLA-G containing exosomes may enhance the secretion of growth-promoting factors in dNK cells.