Comparing GDF9 in mature follicles and clinical outcomes across different PCOS phenotype.

Background: Polycystic ovary syndrome (PCOS) is main cause of anovulatory infertility in women with gestational age. There are currently four distinct phenotypes associated with individualized endocrinology and metabolism. Growth differentiation factor 9 (GDF9) is a candidate as potential biomarker for the assessment of oocyte competence. The effect on oocyte capacity has not been evaluated and analyzed in PCOS phenotypes. Objective: We aimed to screen the expression levels of GDF9 in mature follicles of women with controlled ovarian hyperstimulation (COS) with different PCOS phenotypes. To determine the correlation between the expression level of GDF9 and oocyte development ability. Methods: In Part 1, we conducted a retrospective study comparing the clinical outcomes and endocrine characteristics of patients with PCOS according to different subgroups (depending on the presence or absence of the main features of polycystic ovarian morphology (PCOM), hyperandrogenism (HA), and oligo-anovulation (OA)) and non-PCOS control group. We stratified PCOS as phenotype A (n = 29), phenotype B (n = 18) and phenotype D (n = 24). In Part 2, the expression of GDF9 in follicular fluid (FF) and cumulus cells (CCs) were detected by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry, respectively. Results: In Part 1, the baseline clinical, hormonal, and ultrasonographic characteristics of the study population were matched with the presence or absence of the cardinal features of each PCOS phenotypes showed a clear difference. Phenotypes A and D had statistically significant associations with blastocyst formation and clinical pregnancy compared with phenotypes B (p < 0.001). In Part 2, the levels of GDF9 in FF and CCs for phenotype A and B were significantly were higher than those of phenotype D (P = 0.019, P = 0.0015, respectively). Multivariate logistic regression analysis showed that GDF9 was an important independent predictor of blastocyst formation (P＜0.001). The blastocyst formation rate of phenotype A was higher than that of phenotype B and D (P＜0.001). Combining the results of the two parts, GDF9 appears to play a powerful role in the development of embryos into blastocysts. Conclusions: GDF9 expression varies with different PCOS phenotypes. Phenotype A had higher GDF9 levels and blastocyst formation ability.

Inhibition of FoxO1 alleviates polycystic ovarian syndrome by reducing inflammation and the immune response.

The aim of this study was to explore the role of forkhead box transcription Factor O1 (FoxO1) in chronic inflammation in polycystic ovary syndrome (PCOS). A PCOS rat model was constructed as an in vivo model by letrozole induction, and granulosa cells (GCs) from PCOS rats were isolated and cultured as an in vitro cellular model. FoxO1 was knocked down by shRNA and siRNA in the PCOS rat model and GCs model, respectively. H&E staining was conducted to evaluate the effect of FoxO1 inhibition on ovarian pathology and dysfunction in PCOS rats. The levels of inflammatory cytokines in the ovaries and uterus of PCOS rats and in GCs were assessed by ELISA. Flow cytometry was used to evaluate the changes in the contents of neutrophils and macrophages in the peripheral blood and spleen of PCOS rats. CCK-8 assays and Annexin V-FITC/PI staining were performed to evaluate the proliferation and apoptosis of GCs. The expression of genes and proteins related to the TLR4/NF-κB/NLRP3 pathway in GCs was determined by RT-qPCR and Western blotting. The results indicated that FoxO1 was highly expressed in PCOS rat model. Inhibition of FoxO1 significantly mitigated the pathological changes and dysfunction in the ovaries of PCOS rats while also suppressing inflammation and fibrosis in the ovaries and uterus. Moreover, knocking down FoxO1 facilitated the restoration of the normal ratio of neutrophils and macrophages in the peripheral blood and spleen of PCOS rats and promoted M2 polarization of macrophages. Additionally, inhibition of FoxO1 promoted the proliferation of GCs and inhibited the inflammatory response in GCs. Furthermore, FoxO1 knockdown inhibited the activation of the NF-κB pathway and the formation of the NLRP3 inflammasome in GCs. In conclusion, inhibition of FoxO1 can alleviate PCOS by inhibiting the TLR4/NF-κB/NLRP3 pathway to reduce inflammation and the immune response.

Evaluation of non-invasive gene detection in preimplantation embryos: a systematic review and meta-analysis.

BACKGROUND: Genetic abnormalities in embryos are responsible for most miscarriages and repeated embryo implantation failures, so a reliable preimplantation genetic screening method is urgently needed. Non-invasive preimplantation genetic testing (niPGT) is a potential method for embryo genetic diagnosis. However, the value of its application is controversial. This meta-analysis aimed to investigate and validate the diagnostic value of niPGT in patients undergoing in vitro fertilization (IVF). METHODS: This review used the "Preferred Reporting Items" as a systematic review and meta-analysis of the diagnostic test accuracy (PRISMA-DTA) statement. We searched PubMed, Embase, Web of Science Core Collection, and Cochrane Library up to May 2022 to retrieve non-invasive preimplantation gene detection studies. The eligible research quality was evaluated following the quality assessment study-2 system for diagnostic accuracy. The pooled receiver operator characteristic curve (SROC) and the area under SROC (AUC) were used to evaluate diagnostic performance quantitatively. Threshold effect, subgroup analysis, and meta-regression analysis were used to explore the source of heterogeneity. Deeks' funnel plots and sensitivity analyses were used to test the publication bias and stability of the meta-analysis, respectively. FINDINGS: Twenty studies met the inclusion criteria. The pooled sensitivity, specificity, and AUC were 0.84 (95% CI 0.72-0.91), 0.85 (95% CI 0.74-0.92), and 0.91 (95% CI 0.88-0.93), respectively. Subgroup analysis showed that the spent culture medium (SCM) subgroup had higher sensitivity and lower specificity than the SCM combined with the blastocoel fluid (BF) subgroup. Subgroup analysis showed that the study sensitivity and specificity of < 100 cases were higher than those of ≥ 100. Heterogeneity (chi-square) analysis revealed that sample size might be a potential source of heterogeneity. Sensitivity analysis and Deeks' funnel plots indicated that our results were relatively robust and free from publication bias. INTERPRETATION: The present meta-analysis indicated that the pooled sensitivity, specificity, and AUC of niPGT in preimplantation genetic testing were 0.84, 0.85, and 0.91, respectively. niPGT may have high detection accuracy and may serve as an alternative model for embryonic analysis. Additionally, by subgroup analysis, we found that BF did not improve the accuracy of niPGT in embryos. In the future, large-scale studies are needed to determine the detection value of niPGT.

Fertility Outcome and Safety of Ethiodized Poppy Seed Oil for Hysterosalpingography in 1,053 Infertile Patients: A Real-World Study.

Objective: Ethiodized poppy seed oil for hysterosalpingography (HSG) is reported to display some therapeutic effect on infertility, but big a sample-size study under real clinical settings is still lacking to verify the speculation. Thus, this real-world study enrolled 1,053 infertile patients who underwent ethiodized poppy seed oil-based HSG to explore its fertility enhancement value. Method: A total of 1,053 infertile patients who underwent HSG using ethiodized poppy seed oil as the contrast medium were retrospectively analyzed. The live birth rate and 3-, 6-, 12-month and total pregnancy rate were retrieved. Besides, adverse events during and after HSG were recorded. Results: The 3-, 6-, 12-month and total pregnancy rate was 22, 36.8, 50, and 53.8%, respectively. The total live birth rate was 42.7%. Sub-group analyses showed that pregnancy rate was 53.7, 53.8, 54.1, and 62.4% in subgroups of primary infertility patients, secondary infertility patients, infertility patients with fallopian tube disease, and infertility patients with unknown cause, respectively. Meanwhile the live birth rate was 44.3, 41.3, 41.5, and 59.2% in these subgroups, separately. Multivariate logistic regression analysis disclosed that BMI ≥ 24 kg/m2, history of dysmenorrhea, and abnormity of sperm count or motility-related infertility were independently correlated with reduced pregnancy rate and livebirth rate (All Ps < 0.05). Adverse events mainly included pain (20.6%) and interstitial reflux (7.9%), which were mild and tolerable. Conclusion: Ethiodized poppy seed oil for HSG discloses a satisfying fertility outcome with a tolerable safety profile in infertile patients; meanwhile, this effect might be influenced by BMI, history of dysmenorrhea, and paternal abnormity of sperm.

Stem Cell Therapies for Human Infertility: Advantages and Challenges.

Physical and mental health and hormonal imbalance are associated with the problems related to infertility and reproductive disorders. The rate of infertility has increased globally over the years, due to various reasons. Given the psychosocial implications of infertility and its effects on the life of the affected people, there has been an increased focus on its treatment over the last several years. Assisted reproductive technology can only solve about 50% of the cases. Moreover, it contains significant risks and does not solve the fundamental problem of infertility. As pluripotent stem cells have the potential to differentiate into almost any type of cell, they have been widely regarded as a promising option in the development of stem cell-based fertility treatments, which could even correct genetic diseases in offspring. These advancements in reproductive biotechnology present both challenges and possibilities for solving infertility problems caused by various unexplainable factors. This review briefly presents the different types of infertility disorders and the potential applications of stem cells in the treatment of these reproductive diseases.

Technical note: developmental validation of a novel 41-plex Y-STR system for the direct amplification of reference samples.

The SureID® PathFinder Plus is a new 6-dye, 41-plex Y-STR system that includes the 17 loci from the Yfiler® kit (DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, and Y-GATA-H4) plus 14 rapidly mutating Y-STR loci (DYS449, DYS481, DYS518, DYS527a/b, DYS533, DYS549, DYS570, DYS576, DYS627, DYF387S1a/b, and DYF404S1), and 10 low-medium mutation loci (DYS388, DYS444, DYS447, DYS460, DYS522, DYS557, DYS593, DYS596, DYS643, and DYS645). The inclusion of the 14 rapidly mutating Y-STR loci improves the discrimination of related individuals. Conversely, the 10 low-medium mutation loci are suitable not only for familial searching but also for providing a higher refinement in the construction of Y chromosome phylogenetic relationships among lineages. The 41-plex Y-STR system is designed for direct amplification of reference samples, such as blood samples on an FTA® Card, gauze, tissue, or cotton substrates as well as hair root or buccal samples on swabs. We performed developmental validation work including accuracy, stability, stutter precision, species specificity, sensitivity, PCR inhibitors, reproducibility, parallel testing of the system, and suitability for use on DNA mixtures. In addition, mutations of the loci were analyzed by 754 DNA-confirmed father-son pairs. The results demonstrate that this kit, developed in-house, is time-efficient, accurate, reliable, and highly informative for forensic database, familial searching, and distinguishing related males.

Biallelic mutations in Sperm flagellum 2 cause human multiple morphological abnormalities of the sperm flagella (MMAF) phenotype.

Male patients with multiple morphological abnormalities of the sperm flagella (MMAF) are infertile and exhibit absent, short, coiled, bent and/or irregular sperm flagella. Mutations in the SPEF2 gene reduce sperm motility and cause sperm tail defects in animal models and humans. In the present study, we performed a genetic analysis on an MMAF patient and identified novel biallelic mutations in the SPEF2 gene. The biallelic mutations were confirmed by Sanger sequencing and in silico analysis revealed that, these variations were deleterious. The expression of truncated SPEF2 protein was reduced significantly in the patient's spermatozoa. The spermatozoa harbored biallelic mutations and showed severe ultrastructural defects in the axoneme and mitochondrial sheath. Our data suggest that biallelic mutations in SPEF2 can cause severe sperm flagellum defects, thus providing a novel candidate genetic pathogen for the human MMAF phenotype.

MiR-222 promotes the progression of polycystic ovary syndrome by targeting p27 Kip1.

Polycystic ovary syndrome (PCOS) is one of the most complex and common reproductive and endocrinologic disorders in the child-bearing age of women. Recently, miR-222 were reported to be associated with the etiology of PCOS. However, the function of miR-222 during the pathogenesis of PCOS remains unclear. In the present study, we aimed to investigate the role of miR-222 in PCOS. Firstly, miR-222 expression was examined by quantitative real-time PCR (qRT-PCR) in PCOS. The effects of miR-222 on proliferation, apoptosis and cell cycle in KGN cells were analyzed by CCK-8 assay and flow cytometry analysis, respectively. In addition, bioinformatics analysis was used to predict the target genes of miR-222, and dual-luciferase reporter assay was applied to verified the interaction between miR-222 and p27 Kip1 in KGN cells. Moreover, the expressions of p27 Kip1 in KGN cells treated with miR-222 mimics or miR-222 inhibitor were evaluated by qRT-PCR and western blot assays. The results showed that the expression of miR-222 was remarkably upregulated in PCOS tissues compared with corresponding normal tissues. In the gain-of-function and loss-of-function assays, we revealed that miR-222 mimics significantly promoted cell proliferation, while miR-222 inhibitor induced cell apoptosis and cell cycle arrested. Furthermore, p27 Kip1 was identified as a target gene of miR-222, and could be negatively regulated by miR-222 mimics in KGN cells. In conclusion, our findings suggested that miR-222 may promote the progression of PCOS by targeting p27 Kip1.

Dibutyl Phthalate Inhibits the Effects of Follicle-Stimulating Hormone on Rat Granulosa Cells Through Down-Regulation of Follicle-Stimulating Hormone Receptor.

Dibutyl phthalate (DBP) is used worldwide in solvents and plasticizers. The cytotoxicity and potential tumorigenic effect of DBP have been reported. DBP has also been shown to impact reproductive function. In this study, to further evaluate the effects of DBP on granulosa cells (GCs), we treated rat GCs in vitro with DBP before evaluation of the biological alterations of these GCs. We found that DBP did not induce significant GC death at the tested concentrations. However, follicle-stimulating hormone (FSH)-induced KIT ligand (KITLG) expression in GCs was significantly reduced at both mRNA and protein levels by DBP treatment in a dose-dependent manner. The down-regulation of KITLG was due to the down-regulation of expression of FSH receptor (FSHR) in GCs. Down-regulation of FSHR impaired FSH-induced intracellular signaling in GCs, demonstrated by decreased phosphorylation of AKT and mechanistic target of rapamycin (mTOR). Furthermore, DBP treatment also reduced FSH-induced expression of hypoxia-inducible factor 1-alpha (HIF1A), which is an important signaling component for KITLG expression. Other FSH-induced biological effects, such as production of estradiol and progesterone, as well as GC proliferation, were also suppressed by DBP. Therefore, our study discovered a unique mechanism underlying the toxicity of DBP on GCs. These findings may initiate the development of novel therapeutic interventions for DBP-induced damage to GCs.

Zinc protects sperm from being damaged by reactive oxygen species in assisted reproduction techniques.

The aim of this study was to explore the effect of zinc on hydrogen peroxide-induced sperm damage in assisted reproduction techniques. First, sperms were selected from semen samples of 20 healthy men prepared by density gradient centrifugation. Selected sperm were treated with either 0.001% H(2)O(2), 12.5 nM ZnCL(2), 0.001% H(2)O(2) + 12.5 nM ZnCL(2) or 0.9% NaCl(2) (control). After this treatment, the motility, viability, membrane integrity and DNA fragmentation of sperms in each group were analysed by Goodline sperm detection system, optical microscopy and sperm DNA fragmentation assay. Poorer motility, vitality, membrane integrity and more DNA damage were found in sperms treated by H(2)O(2), compared with control. When sperms were treated with both H(2)O(2) and zinc, however, all indicators were improved compared with H(2)O(2) alone. There was a close association between oxidative stimulation and sperm injury; zinc could inhibit hydrogen peroxide-induced damage of sperm in assisted reproductive technology. However, the presence of zinc in culture medium can decrease the sperm quality without addition of peroxide.