Effects of SARS-COV-2 infection during the frozen-thawed embryo transfer cycle on embryo implantation and pregnancy outcomes.

STUDY QUESTION: Does severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection during the frozen-thawed embryo transfer (FET) cycle affect embryo implantation and pregnancy rates? SUMMARY ANSWER: There is no evidence that SARS-CoV-2 infection of women during the FET cycle negatively affects embryo implantation and pregnancy rates. WHAT IS KNOWN ALREADY: Coronavirus disease 2019 (COVID-19), as a multi-systemic disease, poses a threat to reproductive health. However, the effects of SARS-CoV-2 infection on embryo implantation and pregnancy following fertility treatments, particularly FET, remain largely unknown. STUDY DESIGN, SIZE, DURATION: This retrospective cohort study, included women who underwent FET cycles between 1 November 2022 and 31 December 2022 at an academic fertility centre. PARTICIPANTS/MATERIALS, SETTING, METHODS: Women who tested positive for SARS-CoV-2 during their FET cycles were included in the COVID-19 group, while those who tested negative during the same study period were included in the non-COVID-19 group. The primary outcome was ongoing pregnancy rate. Secondary outcomes included rates of implantation, biochemical pregnancy, clinical pregnancy, early pregnancy loss, and ongoing pregnancy. Multivariate logistic regression models were applied to adjust for potential confounders including age, body mass index, gravidity, vaccination status, and endometrial preparation regimen. Subgroup analyses were conducted by time of infection with respect to transfer (prior to transfer, 1-7 days after transfer, or 8-14 days after transfer) and by level of fever (no fever, fever <39°C, or fever ≥39°C). MAIN RESULTS AND THE ROLE OF CHANCE: A total of 243 and 305 women were included in the COVID-19 and non-COVID-19 group, respectively. The rates of biochemical pregnancy (58.8% vs 62.0%, P = 0.46), clinical pregnancy (53.1% vs 54.4%, P = 0.76), implantation (46.4% vs 46.2%, P = 0.95), early pregnancy loss (24.5% vs 26.5%, P = 0.68), and ongoing pregnancy (44.4% vs 45.6%, P = 0.79) were all comparable between groups with or without infection. Results of logistic regression models, both before and after adjustment, revealed no associations between SARS-CoV-2 infection and rates of biochemical pregnancy, clinical pregnancy, early pregnancy loss, or ongoing pregnancy. Moreover, neither the time of infection with respect to transfer (prior to transfer, 1-7 days after transfer, or 8-14 days after transfer) nor the level of fever (no fever, fever <39°C, or fever ≥39°C) was found to be related to pregnancy rates. LIMITATIONS, REASONS FOR CAUTION: The retrospective nature of the study is subject to possible selection bias. Additionally, although the sample size was relatively large for the COVID-19 group, the sample sizes for certain subgroups were relatively small and lacked adequate power, so these results should be interpreted with caution. WIDER IMPLICATIONS OF THE FINDINGS: The study findings suggest that SARS-CoV-2 infection during the FET cycle in females does not affect embryo implantation and pregnancy rates including biochemical pregnancy, clinical pregnancy, early pregnancy loss, and ongoing pregnancy, indicating that cycle cancellation due to SARS-CoV-2 infection may not be necessary. Further studies are warranted to verify these findings. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the National Key Research and Development Program of China (2023YFC2705500, 2019YFA0802604), National Natural Science Foundation of China (82130046, 82101747), Shanghai leading talent program, Innovative research team of high-level local universities in Shanghai (SHSMU-ZLCX20210201, SHSMU-ZLCX20210200, SSMU-ZLCX20180401), Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital Clinical Research Innovation Cultivation Fund Program (RJPY-DZX-003), Science and Technology Commission of Shanghai Municipality (23Y11901400), Shanghai Sailing Program (21YF1425000), Shanghai's Top Priority Research Center Construction Project (2023ZZ02002), Three-Year Action Plan for Strengthening the Construction of the Public Health System in Shanghai (GWVI-11.1-36), and Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant Support (20161413). The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Ovarian ferroptosis induced by androgen is involved in pathogenesis of PCOS.

STUDY QUESTION: Does ovarian ferroptosis play an active role in the development of polycystic ovary syndrome (PCOS)? SUMMARY ANSWER: Increased ovarian ferroptosis was present in PCOS ovaries and the inhibition of ferroptosis with ferrostatin-1 (Fer-1) ameliorated polycystic ovary morphology and anovulation. WHAT IS KNOWN ALREADY: Programmed cell death plays a fundamental role in ovarian follicle development. However, the types and mechanisms of cell death involved in the ovary are yet to be elucidated. Ferroptosis is a recently discovered iron-dependent programmed cell death. Impaired iron metabolism and cell death have been observed in women with PCOS, the main cause of anovulatory infertility. Additionally, previous studies reported that an abnormal expression of noncoding RNA may promote ferroptosis in immortalized ovarian granulosa cell lines. However, little is known about whether ovarian ferroptosis is increased in PCOS, and there is insufficient direct evidence for a role of ferroptosis in PCOS, and the underlying mechanism. Moreover, the effect of the inhibition of ferroptosis with Fer-1 in PCOS remains unclear. STUDY DESIGN SIZE DURATION: Ferroptosis was evaluated in human granulosa cells (hGCs) from non-PCOS (n = 6-16) and PCOS (n = 7-18) patients. The experimental study was completed in vitro using primary hGCs from women undergoing IVF. Improvements in PCOS indicators following ferroptosis inhibition with Fer-1 were investigated in a dehydroepiandrosterone (DHEA)-induced PCOS rat model (n = 8 per group). PARTICIPANTS/MATERIALS SETTING METHODS: Ovarian ferroptosis was evaluated in the following ways: by detecting iron concentrations via ELISA and fluorescent probes; measuring malondialdehyde (MDA) concentrations via ELISA; assessing ferroptosis-related protein abundance with western blotting; observing mitochondrial morphology with transmission electron microscopy; and determining cell viability. Primary hGCs were collected from women undergoing IVF. They were treated with dihydrotestosterone (DHT) for 24 h. The effect of DHT on ferroptosis was examined in the presence or absence of small interfering RNA-mediated knockdown of the putative receptor coregulator for signaling molecules. The role of ovarian ferroptosis in PCOS progression was explored in vivo in rats. The DHEA-induced PCOS rat model was treated with the ferroptosis inhibitor, Fer-1, and the oocytes and metaphase II oocytes were counted after ovarian stimulation. Additionally, rats were treated with the ferroptosis inducer, RSL3, to further explore the effect of ferroptosis. The concentrations of testosterone, FSH, and LH were assessed. MAIN RESULTS AND THE ROLE OF CHANCE: Increased ferroptosis was detected in the ovaries of patients with PCOS and in rats with DHEA-induced PCOS. Increased concentrations of Fe2+ (P < 0.05) and MDA (P < 0.05), and upregulated nuclear receptor coactivator 4 protein levels, and downregulated ferritin heavy chain 1 (FTH1) and glutathione peroxidase 4 (GPX4) proteins were observed in the hGCs in patients with PCOS and ovaries of PCOS rats (P < 0.05 versus control). DHT was shown to induce ferroptosis via activation of NOCA4-dependent ferritinophagy. The inhibition of ferroptosis with Fer-1 in rats ameliorated a cluster of PCOS traits including impaired glucose tolerance, irregular estrous cycles, reproductive hormone dysfunction, hyperandrogenism, polycystic ovaries, anovulation, and oocyte quality (P < 0.05). Treating rats with RSL3 resulted in polycystic ovaries and hyperandrogenism (P < 0.05). LARGE-SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: Although ovarian-targeted ferroptosis inhibition may be a more targeted treatment for PCOS, the underlying mechanisms in the cycle between ferroptosis and hyperandrogenism require further exploration. Additionally, since PCOS shows high heterogeneity, it is important to investigate whether ferroptosis increases are present in all patients with PCOS. WIDER IMPLICATIONS OF THE FINDINGS: Androgen-induced ovarian ferroptosis appears to play a role in the pathogenesis of PCOS, which potentially makes it a promising treatment target in PCOS. STUDY FUNDING/COMPETING INTERESTS: This study was supported by the National Key R&D Program of China (2023YFC2705500, 2023YFC2705505, 2019YFA0802604), National Natural Science Foundation of China (No. 82130046, 82320108009, 82101708, 82101747, and 82001517), Shanghai leading talent program, Innovative research team of high-level local universities in Shanghai (No. SHSMU-ZLCX20210201, No. SSMU-ZLCX20180401), Shanghai Jiaotong University School of Medicine, Affiliated Renji Hospital Clinical Research Innovation Cultivation Fund Program (RJPY-DZX-003) and Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant Support (No. 20161413), Shanghai's Top Priority Research Center Construction Project (2023ZZ02002), and Three-Year Action Plan for Strengthening the Construction of the Public Health System in Shanghai (GWVI-11.1-36). The authors report no competing interests.

Perfluorobutane (Sonazoid) Contrast-enhanced ultrasound to diagnose hepatocellular carcinoma: A systematic review and meta-analysis.

AIM: This meta-analysis aimed to assess the precision of Sonazoid contrast-enhanced ultrasound (CEUS) to distinguish hepatocellular carcinoma (HCC) from focal liver lesions (FLLs). MATERIAL AND METHODS: The Cochrane Library, Embase, PubMed, and Web of Science databases were systematically searched and checked for studies using Sonazoid CEUS to characterize HCC. A comprehensive meta-analysis was conducted, involving data pooling, subgroup analyses, meta-regression, and investigation of publication bias. RESULTS: The meta-analysis included fourteen studies. The overall diagnostic accuracy for characterizing HCC was as follows (all ranges show the 95% confidence interval): pooled sensitivity of 0.87 (0.80-0.92), pooled specificity of 0.95 (0.91-0.97), and a diagnostic odds ratio of 121 (61-241). The overall weighted area under the curve was 0.97 (0.95-0.98). The pooled sensitivity, specificity, and diagnostic odds ratio for Sonazoid and Sonovue were 0.75 (0.63- 0.84), 0.97 (0.86-0.99), 82 (15-445); and 0.64 (0.51-0.76), 0.98 (0.91-0.99), 72 (17-311), respectively. The sources of heterogeneity were identified as the study location, prevailing risk factor, reference diagnosis standard, criteria of Sonazoid CUES, and the proportion of cases of HCC. We observed no potential publication bias. CONCLUSION: Sonazoid CEUS is efficient to distinguish HCC from FLLs, with good sensitivity and specificity. It is comparable to Sonovue CEUS to diagnose HCC.

The effect of peak serum estradiol level during ovarian stimulation on cumulative live birth and obstetric outcomes in freeze-all cycles.

Objective: To determine whether the peak serum estradiol (E2) level during ovarian stimulation affects the cumulative live birth rate (CLBR) and obstetric outcomes in freeze-all cycles. Methods: This retrospective cohort study involved patients who underwent their first cycle of in vitro fertilization followed by a freeze-all strategy and frozen embryo transfer cycles between January 2014 and June 2019 at a tertiary care center. Patients were categorized into four groups according to quartiles of peak serum E2 levels during ovarian stimulation (Q1-Q4). The primary outcome was CLBR. Secondary outcomes included obstetric and neonatal outcomes of singleton and twin pregnancies. Poisson or logistic regression was applied to control for potential confounders for outcome measures, as appropriate. Generalized estimating equations were used to account for multiple cycles from the same patient for the outcome of CLBR. Results: A total of 11237 patients were included in the analysis. Cumulatively, live births occurred in 8410 women (74.8%). The live birth rate (LBR) and CLBR improved as quartiles of peak E2 levels increased (49.7%, 52.1%, 54.9%, and 56.4% for LBR; 65.1%, 74.3%, 78.4%, and 81.6% for CLBR, from the lowest to the highest quartile of estradiol levels, respectively, P<0.001). Such association remained significant for CLBR after accounting for potential confounders in multivariable regression models, whereas the relationship between LBR and peak E2 levels did not reach statistical significance. In addition, no significant differences were noticed in adverse obstetric and neonatal outcomes (gestational diabetes mellitus, pregnancy-induced hypertension, preeclampsia, placental disorders, preterm birth, low birthweight, and small for gestational age) amongst E2 quartiles for either singleton or twin live births, both before and after adjustment. Conclusion: In freeze-all cycles, higher peak serum E2 levels during ovarian stimulation were associated with increased CLBR, without increasing the risks of adverse obstetric and neonatal outcomes.

Novel mutations in PLCZ1 lead to early embryonic arrest as a male factor.

Early embryonic arrest is one of the causes of assist reproduction technology (ART) failure. We have previously reported that the first sperm-derived genetic factor, ACTL7a mutations, could lead to early embryonic arrest. However, whether there are other male genetic factors associated with early embryonic arrest remains elusive. Here, we reported bi-allelic mutations in PLCZ1, a well-known causal gene of total fertilization failure, in four infertile males. Among these mutations, p.403_404del, p.I489S, and p.W536X were newly reported in this study. Histological and Western blotting analysis of the patients' sperm indicated these variants as loss-of-function mutations. These patients manifested normal conventional semen parameters and ultra-structures in sperm heads. However, among four in vitro fertilization (IVF) cycles, 81.8% (18/22) of the oocytes were polyspermic fertilized, which was rarely reported in PLCZ1-related male patients. In the following six ICSI cycles, artificial oocyte activation (AOA) was applied and successfully rescued the fertilization failure and polyspermy phenotypes, with 31.3% (15/48) of the MII oocytes normally fertilized. However, 60.0% (9/15) of these normally fertilized zygotes were arrested at 2-5-cell stage, with one failing to cleave, indicating that PLCZ1 was not only necessary for fertilization, but also crucial for early embryonic development. However, these rescued zygotes showed a lower potential in developing into blastocysts when cultured in vitro. Thus, fresh cleavage transfer was tried and two live births were successfully achieved thereafter. In conclusion, this study provided novel mutations in PLCZ1 gene to expand the pathogenic mutational spectrum in male infertility and demonstrated that PLCZ1 was a crucial sperm-related genetic factor for early embryonic arrest. We also proposed that cleavage transfer after ICSI and AOA treatment could be a potential treatment method for male patients carrying bi-allelic mutations in PLCZ1.

Local glucose elevation activates pyroptosis via NLRP3 inflammasome in ovarian granulosa cells of overweight patients.

Overweight, with an increasing prevalence worldwide, significantly impairs the clinical outcomes following in vitro fertilization (IVF). Hyperglycemia, hyperlipidemia, and metabolic disorders are always accompanied by the majority of overweight patients. The association between granulosa cell function and metabolic alterations in follicular fluid including lipids, proteins, and growth factors has been extensively documented. However, the effects of higher glucose level on ovarian granulosa cells (GCs), remain largely unknown. In this study, we identified that overweight women had elevated follicular glucose level which profoundly activated NLRP3 inflammasome and pyroptosis. An in vitro correlation between follicular high glucose, NLRP3 inflammasome and pyroptosis was also established. More importantly, in granulosa cells of overweight patients, the activation of the NLRP3 inflammasome and pyroptosis induced by high glucose was involved in the dysregulation of estradiol synthesis. Our study may provide new options to interpretate and improve IVF outcomes in overweight women.

Follicular fluid lipidomic profiling reveals potential biomarkers of polycystic ovary syndrome: A pilot study.

Background: Polycystic ovary syndrome (PCOS) is a heterogeneous endocrine disorder associated with multiple metabolic conditions including obesity, insulin resistance, and dyslipidemia. PCOS is the most common cause of anovulatory infertility; however, the molecular diversity of the ovarian follicle microenvironment is not fully understood. This study aimed to investigate the follicular fluid (FF) lipidomic profiles in different phenotypes of PCOS and to explore novel lipid biomarkers. Methods: A total of 25 women with PCOS and 12 women without PCOS who underwent in vitro fertilization and embryo transfer were recruited, and their FF samples were collected for the lipidomic study. Liquid chromatography-tandem mass spectrometry was used to compare the differential abundance of FF lipids between patients with different PCOS phenotypes and controls. Subsequently, correlations between specific lipid concentrations in FF and high-quality embryo rate (HQER) were analyzed to further evaluate the potential interferences of lipid levels with oocyte quality in PCOS. Candidate biomarkers were then compared via receiver operating characteristic (ROC) curve analysis. Results: In total, 19 lipids were identified in ovarian FF. Of these, the concentrations of ceramide (Cer) and free fatty acids (FFA) in FF were significantly increased, whereas those of lysophosphatidylglycerol (LPG) were reduced in women with PCOS compared to controls, especially in obese and insulin-resistant groups. In addition, six subclasses of ceramide, FFA, and LPG were correlated with oocyte quality. Twenty-three lipid subclasses were identified as potential biomarkers of PCOS, and ROC analysis indicated the prognostic value of Cer,36:1;2, FFA C14:1, and LPG,18:0 on HQER in patients with PCOS. Conclusions: Our study showed the unique lipidomic profiles in FF from women with PCOS. Moreover, it provided metabolic signatures as well as candidate biomarkers that help to better understand the pathogenesis of PCOS.

Increased expression of HOXA11-AS attenuates endometrial decidualization in recurrent implantation failure patients.

Endometrial decidualization is a prerequisite for implantation, and impaired decidualization is associated with recurrent implantation failure (RIF). Coding genes of the HOX family have been clarified as critical regulators in endometrial decidualization, but the role of long non-coding RNAs (lncRNAs) in the HOX gene family has yet to be determined. The aim of this study was to clarify the possible roles of lncRNAs in the HOX gene family in decidualization. In this study, we identified that HOXA11-AS was the most reduced lncRNA in the HOX family in the human endometrium during the window of implantation, and it was elevated in RIF patients. Mechanistically, HOXA11-AS negatively regulated decidualization through competitive interaction with PTBP1, an RNA-binding protein. Binding of PTBP1 to HOXA11-AS limited PTBP1 availability to regulate PKM1/2 alternative splicing, resulting in enhanced PKM1 and diminished PKM2 expression, thus attenuating decidualization. The pattern of high HOXA11-AS expression and impaired PKM2 splicing was consistently observed in RIF patients. Collectively, our study indicates that the increase of HOXA11-AS is detrimental to endometrial decidualization, likely contributing to RIF. Our study may shed light on the pathogenesis and treatment of RIF.

Elevated SAA1 promotes the development of insulin resistance in ovarian granulosa cells in polycystic ovary syndrome.

BACKGROUND: Insulin resistance (IR) contributes to ovarian dysfunctions in polycystic ovarian syndrome (PCOS) patients. Serum amyloid A1 (SAA1) is an acute phase protein produced primarily by the liver in response to inflammation. In addition to its role in inflammation, SAA1 may participate in IR development in peripheral tissues. Yet, expressional regulation of SAA1 in the ovary and its role in the pathogenesis of ovarian IR in PCOS remain elusive. METHODS: Follicular fluid, granulosa cells and peripheral venous blood were collected from PCOS and non-PCOS patients with and without IR to measure SAA1 abundance for analysis of its correlation with IR status. The effects of SAA1 on its own expression and insulin signaling pathway were investigated in cultured primary granulosa cells. RESULTS: Ovarian granulosa cells were capable of producing SAA1, which could be induced by SAA1 per se. Moreover, the abundance of SAA1 significantly increased in granulosa cells and follicular fluid in PCOS patients with IR. SAA1 treatment significantly attenuated insulin-stimulated membrane translocation of glucose transporter 4 and glucose uptake in granulosa cells through induction of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression with subsequent inhibition of Akt phosphorylation. These effects of SAA1 could be blocked by inhibitors for toll-like receptors 2/4 (TLR 2/4) and nuclear factor kappa light chain enhancer of activated B (NF-κB). CONCLUSIONS: Human granulosa cells are capable of feedforward production of SAA1, which significantly increased in PCOS patients with IR. Excessive SAA1 reduces insulin sensitivity in granulosa cells via induction of PTEN and subsequent inhibition of Akt phosphorylation upon activation of TLR2/4 and NF-κB pathway. These findings highlight that elevation of SAA1 in the ovary promotes the development of IR in granulosa cells of PCOS patients.

Addressing the role of 11ß-hydroxysteroid dehydrogenase type 1 in the development of polycystic ovary syndrome and the putative therapeutic effects of its selective inhibition in a preclinical model.

BACKGROUND: Polycystic ovary syndrome (PCOS) is the most common metabolic and endocrine disorder among reproductive-age women, and the leading cause of anovulatory infertility. 11ß-hydroxysteroid dehydrogenases-1 (11ß-HSD1) catalysing the conversion of inactive cortisone to active cortisol plays a crucial role in various metabolic diseases. However, whether 11ß-HSD1 is associated with the pathogenesis of PCOS and whether 11ß-HSD1 can be a treating target of PCOS remain unknown. METHODS: This study was first designed to explore the role of 11ß-HSD1 in PCOS development and the effect of selective 11ß-HSD1 inhibitor administration on PCOS treatment. Follicular fluid and granulosa cells (GCs) were collected from 32 non-PCOS patients and 37 patients with PCOS to measure cortisol and 11ß-HSDs levels. Female Sprague-Dawley rats (3-week-old) were injected with dehydroepiandrosterone (DHEA) to induce PCOS and their ovaries were collected to measure the abundance of corticosterone (CORT) and 11ß-HSDs. To determine the role of 11ß-HSD1 in PCOS development, we overexpressed 11ß-HSD1 in the ovaries of female rats (5-week-old) or knocked down the expression of 11ß-HSD1 in the ovaries from PCOS rats via lentivirus injection. After lentivirus infection, the body weights, ovarian weights, estrous cycles, reproductive hormones and morphology of the ovary were analysed in rats from different experimental groups. Then to figure out the translational potential of the selective 11ß-HSD1 inhibitor in treating PCOS, PCOS rats were treated with BVT.2733, a selective 11ß-HSD1 inhibitor and a cluster of PCOS-like traits were analysed, including insulin sensitivity, ovulatory function and fertility of rats from the Control, PCOS and PCOS+BVT groups. Rat ovarian explants and human GCs were used to explore the effect of CORT or cortisol on ovarian extracellular matrix remodelling. RESULTS: The elevated expression of 11ß-HSD1 contributed to the increased cortisol and corticosterone (CORT) concentrations observed in the ovaries of PCOS patients and PCOS rats respectively. Our results showed that ovarian overexpression of 11ß-HSD1 induced a cluster of PCOS phenotypes in rats including irregular estrous cycles, reproductive hormone dysfunction and polycystic ovaries. While knockdown of ovarian 11ß-HSD1 of PCOS rats reversed these PCOS-like changes. Additionally, the selective 11ß-HSD1 inhibitor BVT.2733 alleviated PCOS symptoms such as insulin resistance (IR), irregular estrous cycles, reproductive hormone dysfunction, polycystic ovaries, ovulatory dysfunction and subfertility. Moreover, we showed that cortisol target ovarian insulin signalling pathway and ovarian extracellular matrix (ECM) remodelling in vivo, in ovarian explants and in GCs. CONCLUSION: Elevated 11ß-HSD1 abundance in ovarian is involved in the pathogenesis of PCOS by impairing insulin signalling pathway and ECM remodelling. Selective inhibition of 11ß-HSD1 ameliorates a cluster of PCOS phenotypes. Our study demonstrates the selective 11ß-HSD1 inhibitor as a novel and promising strategy for the treatment of PCOS.

Deficiency of Sirtuin 1 Impedes Endometrial Decidualization in Recurrent Implantation Failure Patients.

Decidualization is driven by differentiation of human endometrial stromal cells (ESCs), and is a prerequisite for successful implantation and establishment of pregnancy. The critical role of impaired decidualization in women suffered recurrent implantation failure (RIF) has been established, while the underlying mechanism is poorly understood. In the present study, we verified the essential role of Sirtuin1 (SIRT1) in regulating differentiation and maintaining reactive oxygen species (ROS) homeostasis of human ESCs during decidualization. The abundance of SIRT1 was decreased in RIF patients both in the endometria during window of implantation phase and in the decidualized ESCs. Downregulation of SIRT1 disrupted the intracellular ROS homeostasis during decidualization of ESC, manifested as the accumulation of intracellular ROS level and the reduction of antioxidant stress molecules. Elimination of ROS with N-acetyl-L-cysteine (NAC) could rescued the decidualization inhibition caused by SIRT1 knockdown. Further, we explored the insufficient expression of SIRT1 in ESC affected the deacetylation of forkhead box O1 (FOXO1), and thus inhibited the transcriptional activity of FOXO1. This could account for the dysregulation of intracellular ROS homeostasis during decidualization and decreased expression of decidual markers. Collectively, our findings provided insight into the role of down-regulated SIRT1 in the poor decidual response of ESCs in RIF patients.

Induction of autophagy by Beclin-1 in granulosa cells contributes to follicular progesterone elevation in ovarian endometriosis.

Endometriosis is a common gynecological disease in which ovarian dysfunction can be an important cause of infertility. Elevated progesterone (P4) levels during the follicular phase is possibly associated with impaired oocyte quality and pregnancy outcome in endometriosis. Beclin-1 (BECN1), an essential mediator of autophagy, has been shown to be related to the development and progression of endometriosis. This study aimed to investigate the autophagic activity in ovarian granulosa cells (GCs) of patients with endometriosis and to clarify the role of BECN1 in preovulatory P4 elevation. Our results demonstrated that serum P4/estradiol (E2) ratio and P4-to-follicle index (the average P4 secretion per follicle) on the day of human chorionic gonadotropin administration were elevated in women with ovarian endometriosis. Increased expression of BECN1 and enhanced autophagy were observed in GCs of patients with ovarian endometriomas. In cultured GCs, BECN1 knockdown reduced P4 secretion and the expression of key steroidogenic enzymes; whereas overexpression of BECN1 resulted in induced P4 production with activated biosynthesis pathway. Moreover, inhibition of autophagy by BECN1 knockdown significantly attenuated low-density lipoprotein (LDL)-induced P4 synthesis. These findings provide new insights into the role of BECN1 in late follicular P4 elevation in patients with endometriosis by promoting the degradation pathway of LDL for P4 biosynthesis via lysosome activation in GCs, and have potential therapeutic implications for the improvement of oocyte quality in women affected by endometriosis.

Novel role of CXCL14 in modulating STAR expression in luteinized granulosa cells: implication for progesterone synthesis in PCOS patients.

Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in reproductive-age women. Reduced progesterone levels are associated with luteal phase deficiency in women with PCOS. The levels of C-X-C motif chemokine ligand-14 (CXCL14) were previously reported to be decreased in human-luteinized granulosa (hGL) cells derived from PCOS patients. However, the function of CXCL14 in hGL cells and whether CXCL14 affects the synthesis of progesterone in hGL cells remain unclear. In the present study, the levels of CXCL14 were reduced in follicular fluid and hGL cells in PCOS patients, accompanied by decreased progesterone levels in follicular fluid and decreased steroidogenic acute regulatory (STAR) expression in hGL cells. CXCL14 administration partially reversed the low progesterone production and STAR expression in hGL cells obtained from PCOS patients. In primary hGL cells, CXCL14 upregulated STAR expression and progesterone production. CXCL14 activated the phosphorylation of cyclic adenosine monophosphate response element-binding protein (CREB) and CREB inhibitor attenuated the modulation of StAR expression by CXCL14. P38 and Jun N-terminal kinase (JNK) pathways were also activated by CXCL14 and inhibition of p38 and JNK attenuated the increase of phosphorylation of CREB, STAR expression and progesterone production caused by CXCL14. Our findings revealed the novel role of CXCL14 in upregulation of STAR expression and progesterone synthesis through CREB phosphorylation via activation of p38 and JNK pathways in hGL cells. This is likely contributing to the dysfunction in steroidogenesis in granulosa cells from PCOS patients.

A Novel Molecule in Human Cyclic Endometrium: LncRNA TUNAR Is Involved in Embryo Implantation.

Embryo implantation rate remains an inefficient process in in vitro fertilization and embryo transfer (IVF-ET) cycles. The role long non-coding RNA (lncRNA) plays in embryo implantation remains unclear. We aimed to investigate the expression pattern of lncRNA TCL1 upstream neural differentiation-associated RNA (TUNAR) in human cyclic endometrium and clarify the role of TUNAR in the development of endometrial receptivity. Endometrial biopsies were collected at the late proliferative phase, luteinizing hormone (LH) + 2 and LH + 7, from patients with or without recurrent implantation failure (RIF). Real-time RT PCR was performed to detect the level of lncRNAs. After pZW1-snoVector-TUNAR transfection, multiple function of TUNAR in endometrial epithelial cells (EECs) and endometrial stromal cells (ESCs) was investigated. The expression of TUNAR in endometrium was found down-regulated at LH + 7 and up-regulated in RIF patients. In proliferative phase, TUNAR was overwhelmingly more abundant in ESCs and regulated its proliferation. In LH + 7, the difference in the expression of TUNAR between ESCs and EECs was narrowed. Overexpression of TUNAR not only impaired spheroid attachment to EECs, but also inhibited decidualization of ESCs. TUNAR was found expressed in human endometrium for the first time, which might be involved in embryo implantation by modulating the blastocyst attachment to the endometrial epithelium and regulating the proliferation and decidualization of ESCs. Our study helps us to better understand the molecular mechanisms of embryo implantation and may provide a promising biomarker of endometrial receptivity.

Larimichthys crocea is a suitable bioindicator for monitoring short-term Cd discharge along the coast: An experimental study.

The present study evaluated the feasibility of using a marine cage fish Larimichthys crocea as a model for monitoring short-time Cd discharge near the sewage outlet. Fish were exposed to 0, 20, 100, 500 and 2500 µg/L for 6 h. Cd concentrations in gills, and left and right lobes of hepatopancreas were examined as well as activity levels of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathion-S-transferase (GST), glutathione reductase (GR), lipid peroxidation, glutathione (GSH) and mRNA levels of 19 genes encoding these enzymes. Cd concentrations increased at 100, 500 and 2500 µg/L Cd in gill and at 2500 µg/L Cd in hepatopancreas. Lipid peroxidation increased and GSH levels declined in gills at 2500 µg/L Cd. On the contrary, oxidative damage was not observed in hepatopancreas but GSH levels increased at all tested concentrations of Cd in the left lobe and at 20 µg/L Cd in the right lobe. The enhanced antioxidant response was confirmed in gills due to the increased activity levels of antioxidant enzymes and the up-regulated mRNA levels of most genes. However, disordered antioxidant response was observed in hepatopancreas, showing a dose- and lobe-dependent effect. RNA-seq and q-PCR analyses were performed to investigate differently expressed genes between both lobes under different concentrations of Cd. The most significantly enriched pathway term was pancreatic secretion, where the right lobe showed higher mRNA levels of 18 genes encoding pancreatic digestive enzymes than the left one under Cd stress. Interestingly, both lobes had the same mRNA levels of digestive enzyme genes and antioxidant genes in fish without Cd exposure. Overall, Larimichthys crocea is very sensitive to environmental exposure to cadmium. The present study for the first time investigates Cd-induced antioxidant response in Larimichthys crocea, also is the first to find lobe-dependent effects in fish.

The upregulation of 11ß-HSD1 in ovarian granulosa cells by cortisol and interleukin-1ß in polycystic ovary syndrome.

Our previous study have demonstrated the elevated cortisol concentration in the follicular fluid (FF) contributed to the insulin resistance of the granulosa cells (GCs) in PCOS, but the complicated cortisol generation mechanisms are still unknown. 11ß-hydroxysteroid type 1(11ß-HSD1) mainly functions as reductase in intact cells, converting cortisone to cortisol. Cortisol and IL-1ß are known to induce 11ß-HSD1 in number of tissues, but few results were obtained in ovarian GCs In this study, FF and GCs from PCOS and non-PCOS patients were collected to study the interaction of cortisol and IL-1ß in 11ß-HSD1 expression. The ELISA and qRT-PCR revealed that the cortisol and IL-1ß concentration in FF and 11ß-HSD1 abundance in GCs were elevated in PCOS patients. By using cultured GCs in vitro, we demonstrated that both cortisol and IL-1ß could stimulate 11ß-HSD1 expression. The induction of 11ß-HSD1 by IL-1ß was further inducted by cortisol, whereas the induction of IL-1ß and IL-6 expression by IL-1ß was completely inhibited by cortisol. In conclusion, cortisol and IL-1ß preformed a synergistically upregulation of 11ß-HSD1 expression in GCs, contributing to the accumulation of cortisol in FF of PCOS patients. This may lead to the metabolic disorders of the ovary.

Elevated levels of arachidonic acid metabolites in follicular fluid of PCOS patients.

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in reproductive-age women usually accompanied by lipid metabolic disorders. However, it remains unknown whether arachidonic acid (AA) and its metabolites in follicular fluid (FF) were altered in PCOS patients. This study was intended to measure the levels of AA and its metabolites in the FF of non-obese PCOS patients that underwent in vitro fertilization (IVF) and to explore the possible causes of the alterations. Thirty-nine non-obese women with PCOS and 30 non-obese women without PCOS were enrolled. AA and its metabolites were measured by liquid chromatography-mass spectrometry. The levels of AA metabolites generated via cyclooxygenase-2 (COX-2) pathway and cytochrome P450 epoxygenase pathway but not lipoxygenase (LOX) pathway were significantly higher in the FF of PCOS patients. The metabolites generated via COX-2 pathway were significantly correlated with levels of testosterone and fasting insulin in serum. The in vitro study further demonstrated that insulin but not testosterone could promote the IL-1ß and hCG-induced COX-2 expression and prostaglandin E2 (PGE2) secretion in primary human granulosa cells. In conclusion, there was an elevation in AA metabolites in FF of PCOS patients. Insulin played a pivotal role in the increased AA metabolites generated via COX-2, which could be interpreted as another novel molecular pathophysiological mechanism of PCOS.

Elevated chemerin induces insulin resistance in human granulosa-lutein cells from polycystic ovary syndrome patients.

The insulin resistance (IR) of ovarian granulosa cells from polycystic ovary syndrome (PCOS) aggravates the abnormalities in steroidogenesis and anovulation, and chemerin is an adipokine involved in regulating adipogenesis and glucose homeostasis. The role and underlying mechanism of chemerin in developing IR of the granulosa cells from PCOS remain unclear. Plasma, follicular fluid, and human granulosa-lutein cells (hGLs) were collected from non-PCOS and patients with PCOS with or without IR. The chemerin levels were elevated in both follicular fluid and hGL samples from patients with PCOS with IR, and the hGLs from patients with PCOS with IR showed decreased insulin sensitivity and impaired glucose uptake capacity. Moreover, treatment of chemerin attenuated insulin-stimulated glucose uptake by decreasing phosphorylation of insulin receptor substrate (IRS)1/2 Tyr612, phosphorylation of protein kinase B Ser473, and membrane translocation of glucose transporter type 4 through increasing Ser307 phosphorylation of IRS1 in cultured hGLs. These effects could be abolished by small interfering RNA-mediated knockdown of chemokine-like receptor 1. Furthermore, insulin induced the expression of chemerin in hGLs. Our findings demonstrate a novel role of chemerin in the metabolic dysfunction of PCOS, which suggested that chemerin and its receptor can be further implicated as potential therapeutic targets in the future treatment of PCOS.-Li, X., Zhu, Q., Wang, W., Qi, J., He, Y., Wang, Y., Lu, Y., Wu, H., Ding, Y., Sun, Y. Elevated chemerin induces insulin resistance in human granulosa-lutein cells from polycystic ovary syndrome patients.

High concentration of chemerin caused by ovarian hyperandrogenism may lead to poor IVF outcome in polycystic ovary syndrome: a pilot study.

A chronic low-grade inflammation state accounts for an important part of the pathogenesis of polycystic ovary syndrome (PCOS). The adipose tissue derived cytokine chemerin has recently been proven to be a proinflammatory chemokine, but its mechanism involved in the pathogenesis of PCOS remains largely unresolved. From non-obese patients with and without PCOS, follicular fluid and granulosa cells were retrieved. The effect of testosterone on the expression of chemerin and its receptors was explored in granulosa cells. IVF outcomes in different groups based on FF-chemerin (chemerin in the follicular fluid) level were further compared. The concentration of FF-chemerin, and the mRNA expression of chemerin and its receptors in granulosa cells from PCOS were significantly higher than those from non-PCOS. FF-chemerin was positively correlative to total testosterone (TT) and luteinizing hormone (LH) in the follicular fluid. Furthermore, testosterone upregulated the expression of chemerin and its receptors in vitro. The oocyte utilization rate and high-quality embryo rate were significantly decreased in the high FF-chemerin group. The upregulated chemerin levels in the ovary of PCOS patients, which may be caused by ovarian hyperandrogenism, may be a risk factor for oocyte maturation and embryo development. These findings may provide a basis for novel interventions to improve IVF outcomes.

The role of androgen in autophagy of granulosa cells from PCOS.

Hyperandrogenism is one of the most common causes for anovulation in women and increases the risk for metabolic disorder in PCOS patients. Autophagy plays an important role in dysfunction of endocrine and anovulation. However, the relationship between hyperandrogenism and autophagy in human granulosa cells of PCOS patients remains unclear. By collecting granulosa cells from PCOS patients and non-PCOS patients, we found that the abundance of autophagy-related genes ATG5, ATG7, BECN1 mRNA and the ratio of autophagy marker protein light chain 3B II/I (LC3 II/I) were significantly increased whereas the abundance of the autophagy substrate SQSTM1/p62 was decreased in ovarian granulosa cells from PCOS patients. Furthermore, we demonstrated that BECN1 mRNA abundance in human granulosa cells positively correlated with the basal level of serum total testosterone and androgen up-regulated the abundance of BECN1 mRNA and the ratio of LC3II/LC3I in a dose-dependent manner in cultured granulosa cells. These observations indicated that androgen-induced activation of autophagy may play an important role in the development of PCOS and to explore the autophagy mechanisms involved in PCOS yield new insight into the pathophysiology and therapy of the disorder.