The impact and inflammatory characteristics of SARS-CoV-2 infection during ovarian stimulation on the outcomes of assisted reproductive treatment.

Introduction: Despite the global prevalence of coronavirus disease 2019 (COVID-19), limited research has been conducted on the effects of SARS-CoV-2 infection on human reproduction. The aims of this study were to investigate the impact of SARS-CoV-2 infection during controlled ovarian stimulation (COS) on the outcomes of assisted reproductive treatment (ART) and the cytokine status of patients. Methods: This retrospective cohort study included 202 couples who received ART treatment, 101 couples infected with SARS-CoV-2 during COS and 101 matched uninfected couples. The parameters of ovarian stimulation and pregnancy outcomes were compared between the two groups. The All-Human Inflammation Array Q3 kit was utilized to measure cytokine levels in both blood and follicular fluid. Results: No difference was found in the number of good-quality embryos (3.3 ± 3.1 vs. 3.0 ± 2.2, P = 0.553) between the infected and uninfected groups. Among couples who received fresh embryo transfers, no difference was observed in clinical pregnancy rate (53.3% vs. 51.5%, P = 0.907). The rates of fertilization, implantation, miscarriage, ectopic pregnancy and live birth were also comparable between the two groups. After adjustments were made for confounders, regression models indicated that the quality of embryos (B = 0.16, P = 0.605) and clinical pregnancy rate (P = 0.206) remained unaffected by SARS-CoV-2 infection. The serum levels of MCP-1, TIMP-1, I-309, TNF-RI and TNF-RII were increased, while that of eotaxin-2 was decreased in COVID-19 patients. No significant difference was found in the levels of cytokines in follicular fluid between the two groups. Conclusion: Asymptomatic or mild COVID-19 during COS had no adverse effects on ART outcomes. Although mild inflammation was present in the serum, it was not detected in the follicular fluid of these patients. The subsequent immune response needs further investigation.

Downregulation of homeobox A1 in human granulosa cells is involved in diminished ovarian reserve through promoting cell apoptosis and mitochondrial dysfunction.

Granulosa cell apoptosis contributes to the occurrence of diminished ovarian reserve (DOR). HOXA1, belonging to the HOX gene family, is involved in regulating cancer cell apoptosis. However, whether HOXA1 participates in the granulosa cell apoptosis in DOR patients remains to be elucidated. In the current study, we demonstrated the differential transcriptomic landscape of granulosa cells in DOR patients compared to that in the controls and identified decreased expression of the HOXA1 gene. Meanwhile, we found that HOXA1 was a gonadotropin-response gene, in which FSH could promote its expression, whereas LH inhibited HOXA1 expression in human granulosa cells. CCK-8 assay, flow cytometry and TUNEL staining results showed that inhibition of endogenous HOXA1 expression promoted human granulosa cell apoptosis. Moreover, knockdown of HOXA1 increased Bax while reducing Bcl2 protein expression. Furthermore, we found a total of 947 differentially expressed genes (DEGs), including 426 upregulated genes and 521 downregulated genes using transcriptome sequencing technology. Enrichment analysis results showed that the DEGs were involved in apoptosis and mitochondrial function-related signaling pathways. Knockdown of HOXA1 impaired mitochondrial functions, exhibiting increased reactive oxygen species (ROS) and cytoplasmic Ca2+ levels, decreased mitochondrial membrane potential, ATP production and mitochondrial DNA (mtDNA) copy number, and abnormal mitochondrial cristae. Our findings demonstrated that aberrantly reduced HOXA1 expression induced granulosa cell apoptosis in DOR patients and impaired mitochondrial function, which highlighted the potential role of HOXA1 in the occurrence of DOR and provided new insight for the treatment of DOR.

ALKBH5 controls the meiosis-coupled mRNA clearance in oocytes by removing the N 6-methyladenosine methylation.

N6-methyladenosine (m6A) maintains maternal RNA stability in oocytes. One regulator of m6A, ALKBH5, reverses m6A deposition and is essential in RNA metabolism. However, the specific role of ALKBH5 in oocyte maturation remains elusive. Here, we show that Alkbh5 depletion causes a wide range of defects in oocyte meiosis and results in female infertility. Temporal profiling of the maternal transcriptomes revealed striking RNA accumulation in Alkbh5-/- oocytes during meiotic maturation. Analysis of m6A dynamics demonstrated that ALKBH5-mediated m6A demethylation ensures the timely degradation of maternal RNAs, which is severely disrupted following Alkbh5-/- depletion. A distinct subset of transcripts with persistent m6A peaks are recognized by the m6A reader IGF2BP2 and thus remain stabilized, resulting in impaired RNA clearance. Additionally, reducing IGF2BP2 in Alkbh5-depleted oocytes partially rescued these defects. Overall, this work identifies ALKBH5 as a key determinant of oocyte quality and unveil the facilitating role of ALKBH5-mediated m6A removal in maternal RNA decay.

Integrated bioinformatics analysis elucidates granulosa cell whole-transcriptome landscape of PCOS in China.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a common reproductive, neuroendocrine, and metabolic disorder in women of reproductive age that affects up to 5-10% of women of reproductive age. The aetiology of follicle development arrest and critical issues regarding the abnormal follicular development in PCOS remain unclear. The present study aims to systematically evaluate granulosa cell whole-transcriptome sequencing data to gain more insights into the transcriptomic landscape and molecular mechanism of PCOS in China. METHODS: In the present study, the microarray datasets GSE138518, GSE168404, GSE193123, GSE138572, GSE95728, and GSE145296 were downloaded from the Gene Expression Omnibus (GEO) database. Subsequently, differential expression analysis was performed on the PCOS and control groups, followed by functional interaction prediction analysis to investigate gene-regulatory circuits in PCOS. Finally, hub genes and their associated ncRNAs were validated by qPCR in human-luteinized granulosa (hGL) cells and were correlated with the clinical characteristics of the patients. RESULTS: A total of 200 differentially expressed mRNAs, 3 differentially expressed miRNAs, 52 differentially expressed lncRNAs, and 66 differentially expressed circRNAs were found in PCOS samples compared with controls. GO and KEGG enrichment analyses indicated that the DEGs were mostly enriched in phospholipid metabolic processes, steroid biosynthesis and inflammation related pathways. In addition, the upregulated miRNA hsa-miR-205-5p was significantly enriched in the ceRNA network, and two hub genes, MVD and PNPLA3, were regulated by hsa-miR-205-5p, which means that hsa-miR-205-5p may play a fundamental role in the pathogenesis of PCOS. We also found that MVD and PNPLA3 were related to metabolic processes and ovarian steroidogenesis, which may be the cause of the follicle development arrest in PCOS patients. CONCLUSIONS: In summary, we systematically constructed a ceRNA network depicting the interactions between the ncRNAs and the hub genes in PCOS and control subjects and correlated the hub genes with the clinical characteristics of the patients, which provides valuable insights into the granulosa cell whole-transcriptome landscape of PCOS in China.

Serum steroid metabolome on the day of oocyte retrieval in women with polycystic ovarian syndrome and its association with pregnancy outcome of in vitro fertilization.

Steroid hormone level is a crucial factor affecting the outcomes of in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI). The purpose of this study was to evaluate serum steroid metabolome on the day of oocyte retrieval in women with polycystic ovarian syndrome (PCOS) and explore whether specific steroids can be potential indicators to improve the prediction of pregnancy outcomes in PCOS patients undergoing IVF/ICSI. In this study, the serum levels of 21 steroids in 89 women with PCOS and 73 control women without PCOS on the day of oocyte retrieval of the first IVF/ICSI treatment cycle were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). All patients subsequently received good-quality embryo transfer, and the correlation between their steroid profiles and pregnancy outcomes of the first embryo transfer (ET) was retrospectively analyzed. We found PCOS patients had aberrant levels of 11 out of 21 steroid hormones compared to control individuals, with androgen steroid hormones being considerably enhanced. Enzyme activity evaluation indicated that PCOS women might have abnormal activity of CYP17A1, CYP21A2, CYP11B2, CYP19A1, HSD3B, HSD11B, and HSD17B. Additionally, the level of 18-hydroxycorticosterone (p = 0.014), corticosterone (p = 0.035), and 17-hydroxypregnenolone (p = 0.005) were markedly higher in live birth group than in non- live birth group for PCOS women following frozen embryo transfer (FET). Multiple logistic regressions indicated that 18-hydrocorticosterone and 17-hydroxypregnenolone were independently associated with live birth outcomes of PCOS women following FET. Receiver operating characteristic (ROC) curve analysis revealed that 0.595 ng/mL for 18-hydrocorticosterone level (AUC: 0.6936, p = 0.014).and 2.829 ng/mL for 17-hydroxypregnenolone level (AUC: 0.7215, p = 0.005) were the best cutoff values to predict live birth outcomes of PCOS. In conclusion, the blood steroid metabolome was closely related to the IVF/ICSI outcomes of PCOS patients. 18-hydroxycorticosterone and 17-hydroxypregnenolone might be potential indicators to predict pregnancy outcomes of PCOS undergoing IVF/ICSI treatment.

Interleukin-1 increases cyclooxygenase-2 expression and prostaglandin E2 production in human granulosa-lutein cell via nuclear factor kappa B/P65 and extracellular signal-regulated kinase 1/2 signaling pathways.

A multitude of cytokines have been reported to participate in the folliculogenesis process in female. Interleukin-1 (IL-1), belonging to interleukin family, is originally identified as an important immune factor involved in inflammation response. Besides the immunity system, IL-1 is also expressed in reproductive system. However, the role of IL-1 in regulating ovarian follicle function remains to be elucidated. In the current study, using the primary human granulosa-lutein (hGL) and immortalized human granulosa-like tumor cell line (KGN) models, we demonstrated that both IL-1α and IL-1ß increased prostaglandin E2 (PGE2) production via upregulating its cyclooxygenase (COX) enzyme COX-2 expression in human granulosa cells. Mechanistically, IL-1α and IL-1ß treatment activated nuclear factor kappa B (NF-κB) signaling pathway. Using the specific siRNA to knock down endogenous gene expression, we found that the inhibition of p65 expression abolished IL-1α and IL-1ß-induced upregulation of COX-2 expression whereas knockdown of p50 and p52 had no effect. Moreover, our results also showed that IL-1α and IL-1ß promoted the nuclear translocation of p65. ChIP assay demonstrated the transcriptional regulation of p65 on COX-2 expression. Additionally, we also found that IL-1α and IL-1ß could activate the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. The inhibition of ERK1/2 signaling pathway activation reversed IL-1α and IL-1ß-induced upregulation of COX-2 expression. Our findings shed light on the cellular and molecular mechanisms by which IL-1 modulates the COX-2 expression through NF-κB/P65 and ERK1/2 signaling pathways in human granulosa cells.

The BMP2 Signaling Axis Promotes Invasive Differentiation of Human Trophoblasts.

Embryo implantation and trophoblast invasion are principal limiting factors of pregnancy establishment. Aberrant embryo development or improper trophoblast differentiation and invasion may lead to various unfavorable pregnancy-related outcomes, including early pregnancy loss (EPL). Our clinical data show that the serum BMP2 levels were significantly increased during the first trimester of pregnancy and that the serum and BMP2 expression levels were lower in women with EPL than in women with normal early pregnancies. Moreover, we observed that BMP2 was expressed in oocytes and trophoblast cells of cleaved embryos and blastocysts prior to implantation in both humans and mice. Exogenous BMP2 promoted embryonic development by enhancing blastocyst formation and hatching in mice. LncRNA NR026833.1 was upregulated by BMP2 and promoted SNAIL expression by competitively binding to miR-502-5p. SNAIL induced MMP2 expression and promoted cell invasion in primary extravillous trophoblast cells. BMP2 promotes the invasive differentiation of mouse trophoblast stem cells by downregulating the expression of TS cell marker and upregulating the expression of trophoblast giant cell marker and labyrinthine/spongiotrophoblast marker. Our findings provide significant insights into the regulatory roles of BMP2 in the development of the placenta, which may give us a framework to explore new therapeutic strategies to pregnancy-related complications.

Melatonin Ameliorates Coxsackievirus B3-Induced Myocarditis by Regulating Apoptosis and Autophagy.

Current therapeutics options for viral myocarditis are unsatisfactory. Melatonin (MLT), a hormone secreted by the pineal gland and other organs, has protective effects on ischemic heart injury. However, the potential therapeutic effect of MLT on viral myocarditis is unknown. In this study, we investigated the protective effect of MLT on viral myocarditis in a mouse model of myocarditis infected with coxsackievirus B3 (CVB3) and explored the probable mechanisms. Mice with CVB3-induced myocarditis displayed inflammatory cell infiltration and interstitial edema. MLT treatment significantly ameliorated the myocardial injuries. In addition, the rate of autophagy changed, although apoptosis was inhibited in mouse hearts following treatment with MLT. These results suggest that MLT has a strong therapeutic effect on acute viral myocarditis, which is associated with changes in autophagy and apoptosis in the heart. Thus, MLT could be a promising novel therapeutic approach against viral myocarditis.