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Background Blood coagulation has been associated with ovulation and female infertility. In this study, the expression of the tissue factor system was examined during ovulation in immature rats; the correlation between tissue factor and ovarian hyperstimulation syndrome (OHSS) was evaluated both in rats and human follicular fluids. Methods Ovaries were obtained at various times after human chorionic gonadotropin (hCG) injection to investigate the expression of tissue factor system. Expression levels of ovarian tissue factor, tissue factor pathway inhibitor (Tfpi)-1 and Tfpi-2 genes and proteins were determined by real-time quantitative polymerase chain reaction (qPCR), and Western blot and immunofluorescence analyses, respectively. Expression levels of tissue factor system were also investigated in ovaries of OHSS-induced rats and in follicular fluid of infertile women. Results The expression of tissue factor in the preovulatory follicles was stimulated by hCG, reaching a maximum at 6 h. Tissue factor was expressed in the oocytes and the preovulatory follicles. Tfpi-2 mRNA levels were mainly increased by hCG in the granulosa cells whereas the mRNA levels of Tfpi-1 were decreased by hCG. Human CG-stimulated tissue factor expression was inhibited by the progesterone receptor antagonist. The increase in Tfpi-2 expression by hCG was decreased by the proliferator-activated receptor γ (PPARγ) antagonist. Decreased expression of the tissue factor was detected in OHSS-induced rats. Interestingly, the tissue factor concentrations in the follicular fluids of women undergoing in vitro fertilization were correlated with pregnancy but not with OHSS. Conclusions Collectively, the results indicate that tissue factor and Tfpi-2 expression is stimulated during the ovulatory process in rats; moreover, a correlation exists between the levels of tissue factor and OHSS in rats but not in humans.

Background Proper differentiation of trophoblasts in the human placenta is essential for a successful pregnancy, whereas abnormal regulation of this process may lead to adverse pregnancy outcomes, especially preeclampsia (PE). However, the underlying mechanism of trophoblast differentiation remains unclear. Previous studies have reported the involvement of alpha-actinin-4 (ACTN4) in the actin cytoskeleton dynamics and motility. Hence, we hypothesized that ACTN4 may act as an important regulator in the normal proliferation and differentiation of trophoblasts during early pregnancy. Method: To test this hypothesis, we collected villous tissues from women undergoing a legal pregnancy termination during 6–10 weeks of gestation and explanted them for cell culture and siRNA transfection. We also obtained placental tissues from PE patients and healthy pregnant women and isolated the primary cytotrophoblast (CTB) cells. The expression of ACTN4 in the CTBs of placental villi and during the differentiation of CTBs into STBs was detected by immunofluorescence, immunohistochemistry (IHC), and EdU proliferation assays. Besides, villous explant, Matrigel invasion, transwell migration assay, and Wound-healing assay were performed to identify the possible role of ACTN4 in the outgrowth of explants and the invasion, migration, and proliferation of cell column trophoblasts (CCTs). Western blot analysis was carried out to compare the protein expression level of AKT, Snail activities, and epithelial-to-mesenchymal transition (EMT) in the villi or HTR8/SVneo cells with ACTN4 knockdown. Results ACTN4 was highly expressed in CTB cells and interstitial extravillous trophoblast (iEVT) cells but not found in the syncytiotrophoblast (STB) cells in the first trimester villi. Downregulation of ACTN4 led to reduced trophoblast proliferation and explant outgrowth ex vivo, as well as iEVT invasion and migration in vitro due to disrupt of actin filaments organization. Such ACTN4 inhibition also decreased AKT and Snail activities and further impeded the EMT process. In addition, ACTN4 expression was found to be downregulated in the iEVTs from preeclamptic placentas. Conclusions Our findings suggest that ACTN4 may act as an important regulator of trophoblast proliferation and differentiation during early pregnancy, and dysregulation of this protein may contribute to preeclampsia pathogenesis.

Background Spaceflights-induced microgravity can alter various physiological processes in human’s body including the functional status of the reproductive system. Rodent model of tail-suspension hindlimb unloading is extensively used to stimulate the organs responses to the microgravity condition. This study explores the potential effects of hindlimb unloading on testicular functions and spermatogenesis in adult male rats and the underlying mechanism/s. Methods Twenty Sprague-Dawley rats were allotted into two groups: normally loaded group (control; all arms were in touch with the grid floor) and hindlimb unloaded group (HU; only the forearms were in contact with the grid floor). Results Following 30 days of exposure, the HU group saw a decline in body weight, testicular and epidydimal weights, and all semen parameters. The circulating concentrations of gonadotropin-releasing hormone (GnRH), follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone significantly decreased, while levels of kisspeptin, corticosterone, inhibin, prolactin and estradiol (E2) increased in the HU group. Intratesticular levels of 5α-reductase enzyme and dihydrotestosterone (DHT) were suppressed, while the levels of aromatase and kisspeptin were significantly elevated in the HU group. Hypothalamic kisspeptin (Kiss1) mRNA expression levels were downregulated while its receptors (Kiss1R) were upregulated in the HU group. On the contrary, the mRNA expression levels of testicular Kiss1 were upregulated while Kiss1R were downregulated. The pituitary mRNA expression levels of FSHβ and LHβ decreased in the HU group. The levels of the antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and nitric oxide (NO) concentrations, and total antioxidant capacity (TAC) were elevated while malondialdehyde (MDA) concentrations declined in the testes of HU group. The testes of the HU rats showed positive immunostaining of caspase-3, heat shock protein 70 (HSP70) and Bcl2. Conclusions Altogether, these results revealed an inhibitory effect of hindlimb unloading on kisspeptin signaling in the hypothalamic-pituitary-testicular axis with impaired spermatogenesis and steroidogenesis.

Background Spent culture medium (SCM) as a source of DNA for preimplantation genetic tests aneuploidy (PGT-A) has been widely discussed. Methods Seventy-five blastocysts that were donated for research provided a unique possibility in which multiple specimens, including trophectoderm (TE) biopsy, SCM, and paired corresponding whole blastocyst (WB) specimens from the same blastocyst source, could be utilized for the purpose of this preclinical validation. Results To conduct a validation ploidy concordance assessment, we evaluated the full chromosomal concordance rates between SCM and WB (SCM-to-WB), and between TE and WB (TE-to-WB) as well as sensitivity, specificity and overall diagnostic accuracy. 78.67% (59/75) of NGS results in the SCM group were interpretable, a significantly lower percentage than their corresponding TE and WB groups. This discrepancy manifests itself in intrinsically low quantity and poor integrity DNA from SCM. Subsequently, remarkable differences in full concordance rates (including mosaicism, and segmental aneuploidies) are seen as follows: 32.2% (SCM-to-WB, 19/59) and 69.33% (TE-to-WB, 52/75), (p . In such cases, full concordance rates were 27.27% (15/55) in SCM-to-WB, and, 76% (57/75) in TE-to-WB (p . Collectively, the NGS data from SCM also translated into lower sensitivities, Positive Predictive Value (PPV), Negative Predictive Value (NPV), overall diagnostic accuracies, and higher Negative Likelihood Ratio (NLR). Conclusions Our study reveals that DNA is detectable in the majority of SCM samples. Individual chromosomal aberration, such as segmental aneuploidy and mosaicism, can be quantitatively and qualitatively measured. However, TE still provides a more accurate and reliable high-throughput methodology for PGT-A. Meanwhile, cell-free DNA in SCM reporting lacks uniform diagnostic interpretations. Considering that this test is meant to determine which embryos are relegated to be discarded, PGT-A with cell-free DNA in SCM should not be permitted to be applied in routine clinical settings for diagnosis purpose.

Background Intra-cytoplasmic sperm injection (ICSI) is used in assisted reproductive technology (ART) laboratories. However, there is no consensus regarding the precise time intervals within ICSI cycles [oocyte pick up (OPU), oocyte denudation (DN), and ICSI], and results are inconsistent and contradictory. Thus, we aim to evaluate whether there is a concordance regarding the time intervals used in different laboratories and a concrete time that gives better laboratory and reproductive results. Methods A systematic review of the literature until July 25, 2020, was performed with the keywords “Oocyte Denudation/Denudation/Oocyte,” “Intra-cytoplasmic Sperm Injection/ICSI,” “Oocyte/Oocyte maturation/ cumulus,” and “Cumulus removal/ removal.” Articles and abstracts in English and involving human subjects referring to the effects of oocyte DN time on embryo development and clinical outcomes were included. Results Of the 294 evaluated articles, 24 (including 20 full articles and 4 abstracts) were included in this review. Eighteen studies analysed the effect of OPU-DN time on embryo development and clinical outcomes. Most of these studies concluded that OPU-DN time did not influence ICSI outcomes, but some suggested that oocytes should be incubated for a short time before DN to improve oocyte maturity and enhance ICSI outcomes. In addition to reports on positive or negligible effects, adverse effects were reported in 12 studies on DN-ICSI timing. Neither OPU-DN nor DN-ICSI time could improve live birth rate. Conclusions Oocytes should be pre-incubated for a short duration (preferably

Background Placenta previa describes a placenta that extends partially or completely over the internal cervical oss. Placenta previa is one of the leading causes of widespread postpartum hemorrhage and maternal mortality worldwide. Another cause of bleeding in pregnant women is Placenta accreta spectrum. Therefore, the aim of the present systematic review and meta-analysis is to determine the effect of prophylactic balloon occlusion of the internal iliac arteries in patients with placenta previa or placental accreta spectrum (PAS). Methods In this systematic review and meta-analysis, to identify and select relevant studies, the SID, MagIran, ScienceDirect, Embase, Scopus, PubMed, Web of Science, and Google Scholar databases were searched, using the keywords of internal iliac artery balloon, placenta, previa, balloon, accreta, increta and percreta, without a lower time limit and until 2020. The heterogeneity of the studies was examined using the I2 index, and subsequently a random effects model was applied. Data analysis was performed within the Comprehensive Meta-Analysis software (version 2). Results In the review of 29 articles with a total sample size of 1140 in the control group, and 1225 in the balloon occlusion group, the mean difference between the two groups was calculated in terms of Intraoperative blood loss index (mL) and it was derived as 3.21 ± 0.38; moreover, in 15 studies with a sample size of 887 in the control group, and 760 in the balloon occlusion group, the mean difference between the two groups in terms of gestation index (weeks) was found as 2.84 ± 0.49; and also with regards to hysterectomy balloon occlusion after prophylactic closure of the iliac artery, hysterectomy (%) balloon occlusion was calculated as 8.9 %, and this, in the hysterectomy control group (%) was obtained as 31.2 %; these differences were statistically significant and showed a positive effect of the intervention (P Conclusion The results of this study show that the use of prophylactic internal iliac artery balloon occlusion in patients with placenta previa or Placenta accreta spectrum has benefits such as reduced intraoperative blood loss, reduced hysterectomy and increased gestation (weeks), which can be considered by midwives and obstetricians.

Background Asthenozoospermia is one of the most common causes of male infertility, and its genetic etiology is poorly understood. DNAH9 is a core component of outer dynein arms in cilia and flagellum. It was reported that variants of DNAH9 (OMIM: 603330) might cause primary ciliary dyskinesia (PCD). However, variants in DNAH9 lead to nonsyndromic severe asthenozoospermia have yet to be reported. Methods Whole exome sequencing (WES) was performed for two individuals with nonsyndromic severe asthenozoospermia from two non-consanguineous families, and Sanger sequencing was performed to verify the identified variants and parental origins. Sperm routine analysis, sperm vitality rate and sperm morphology analysis were performed according the WHO guidelines 2010 (5th edition). Transmission electron microscopy (TEM, TECNAI-10, 80 kV, Philips, Holland) was used to observe ultrastructures of sperm tail. Quantitative realtime-PCR and immunofluorescence staining were performed to detect the expression of DNAH9-mRNA and location of DNAH9-protein. Furthermore, assisted reproductive procedures were applied. Results By WES and Sanger sequencing, compound heterozygous DNAH9 (NM_001372.4) variants were identified in the two individuals with nonsyndromic severe asthenozoospermia (F1 II-1: c.302dupT, p.Leu101fs*47 / c.6956A > G, p.Asp2319Gly; F2 II-1: c.6294 T > A, p.Phe2098Leu / c.10571 T > A, p.Leu3524Gln). Progressive rates less than 1% with normal sperm morphology rates and normal vitality rates were found in both of the two subjects. No respiratory phenotypes, situs inversus or other malformations were found by detailed medical history, physical examination and lung CT scans etc. Moreover, the expression of DNAH9-mRNA was significantly decreased in sperm from F1 II-1. And expression of DNAH9 is lower in sperm tail by immunofluorescence staining in F1 II-1 compared with normal control. Notably, by intracytoplasmic sperm injection (ICSI), F1 II-1 and his partner successfully achieved clinical pregnancy. Conclusions We identified DNAH9 as a novel pathogenic gene for nonsyndromic severe asthenospermia, and ICSI can contribute to favorable pregnancy outcomes for these patients.

Background TTF1 is a transcription factor that is expressed in the hypothalamus after birth and plays crucial roles in pubertal development. TTF1 may regulate the expression of the Kiss1 gene, which may drive puberty onset in the hypothalamic arcuate (ARC) and anterior ventral paraventricular (AVPV) nuclei. Methods A dual-luciferase reporter assay was used to detect binding between TTF1 and the Kiss1 gene promoter. To investigate the effects of TTF1, we modified TTF1 expression in cell lines and in the ARC or AVPV nucleus of 21-day-old female rats via lentivirus infection. TTF1 and other puberty onset-related genes were detected by qRT-PCR and western blot analyses. Results The in vitro data indicated that TTF1 knockdown (KD) significantly reduced Kiss1 and GnRH expression. Overexpression (OE) of TTF1 promoted Kiss1 expression. In vivo, the expression of Kiss1 and GnRH decreased significantly in the rats with hypothalamic ARC- or AVPV-specific TTF1 KD. The TTF1-KD rats showed vaginal opening delay. H&E staining revealed that the corpus luteum was obviously reduced at the early puberty and adult stages in the rats with ARC- or AVPV-specific TTF1 KD. Conclusion TTF1 bound to the promoter of the Kiss1 gene and enhanced its expression. For 21-day-old female rats, decreased TTF1 in the hypothalamic ARC or AVPV nucleus resulted in delayed vaginal opening and ovarian abnormalities. These observations suggested that TTF1 regulates puberty onset by promoting the expression of Kiss1 and plays an important role in gonad development.

Background Irisin, which is cleaved from fibronectin type III domain-containing protein 5 (Fndc5), plays an important role in energy homeostasis. The link between energy metabolism and reproduction is well known. However, the biological actions of irisin in reproduction remain largely unexplored. Methods In this study, we generated Fndc5 gene mutation to create irisin deficient mice. Female wild-type (WT) and Fndc5 mutant mice were fed with standard chow for 48 weeks. Firstly, the survival rate, body weight and fertility were described in mice. Secondly, the levels of steroid hormones in serum were measured by ELISA, and the estrus cycle and the appearance of follicles were determined by vaginal smears and ovarian continuous sections. Thirdly, mRNA-sequencing analysis was used to compare gene expression between the ovaries of Fndc5 mutant mice and those of WT mice. Finally, the effects of exogenous irisin on steroid hormone production was investigated in KGN cells. Results The mice lacking irisin presented increased mortality, reduced body weight and poor fertility. Analysis of sex hormones showed decreased levels of estradiol, follicle-stimulating hormone and luteinizing hormone, and elevated progesterone levels in Fndc5 mutant mice. Irisin deficiency in mice was associated with irregular estrus, reduced ratio of antral follicles. The expressions of Akr1c18, Mamld1, and Cyp19a1, which are involved in the synthesis of steroid hormones, were reduced in the ovaries of mutant mice. Exogenous irisin could promote the expression of Akr1c18, Mamld1, and Cyp19a1 in KGN cells, stimulating estradiol production and inhibiting progesterone secretion. Conclusions Irisin deficiency was related to disordered endocrinology metabolism in mice. The irisin deficient mice showed poor growth and development, and decreased fertility. Irisin likely have effects on the expressions of Akr1c18, Mamld1 and Cyp19a1 in ovary, regulating the steroid hormone production. This study provides novel insights into the potential role of irisin in mammalian growth and reproduction.

Background The uterine cervix is a mechanical and immunological barrier against ascending infection during pregnancy. Cervical insufficiency (CI), a painless cervical dilation that occurs in the mid-trimester, is an important cause of extremely preterm birth. We hypothesized that women with CI have a differential transcriptomic profile. Therefore, we compared the transcriptomic profile of peripheral blood in women with CI and that of controls. Methods RNA sequencing was used to generate the global gene expression profiles of 11 women with CI and 4 controls, and differential expression analysis was performed to identify genes showing significant expression changes between the CI (n = 11) and control (n = 4) groups as well as between the CI-preterm (n = 7) and CI-term (n = 4) groups. Gene set enrichment was assessed in terms of Gene Ontology processes, and a subset of differentially expressed genes in CI was validated in a different sample-set by qRT-PCR and ELISA. Results Thirty genes were differentially expressed between the CI and control groups. Differentially upregulated genes in the CI group included neutrophil-mediated immunity-associated (DEFA3 and ELANE) and bicarbonate transport-related genes. The serum concentration of alpha defensin 3 was significantly higher in women with CI than in controls (P = 0.014). Analysis of differential gene expression according to pregnancy outcomes revealed 338 differentially expressed genes between the CI-term and CI-preterm groups. Immune and defense response to organism-associated genes and influenza A and NOD-like receptor signaling pathways were upregulated in the CI-term group. Conclusions Our results revealed significant differences in the whole blood transcriptomic profiles of women with CI compared to those of controls. Different immune responses in women with CI may affect pregnancy outcomes.

Background There has been an interest in the relationship between ABO blood groups and infertility. Many studies have investigated the association of ABO blood groups with diminished ovarian reserve (DOR), ovarian hyperstimulation syndrome (OHSS), and outcomes of assisted reproductive technology (ART), with controversial results. Methods A systematic review and meta-analysis was conducted to evaluating the association of ABO blood groups with DOR, OHSS, and outcomes of ART. Results Thirteen studies performed between 2010 and 2018 were included in this meta-analysis. DOR, OHSS, live birth rate (LBR), clinical pregnancy rate (CPR), miscarriage rate (MR) were reported in 9, 2, 4, 3, 2 studies, respectively. The combined results showed similar risk of DOR among individuals with blood group A (RR, 0.98; 95% confidence interval [CI], 0.85, 1.13), B (RR, 0.96; 95% CI, 0.76, 1.20), AB (RR, 1.00; 95% CI, 0.76, 1.30), and non-O (RR, 0.94; 95% CI, 0.79, 1.11) as compared to those with blood group O. Meta-analysis showed that the incidences of OHSS were similar in women with blood group A (RR, 1.05; 95% CI, 0.66, 1.66), B (RR, 1.04; 95% CI, 0.46, 2.35), AB (RR, 0.51; 95% CI, 0.10, 2.56), non-O (RR, 1.02; 95% CI, 0.65, 1.57) with blood group O. As to the clinical outcomes, meta-analysis showed no difference in LBR among individuals with blood group A (RR, 1.27; 95% CI, 0.74, 2.17), B (RR, 1.47; 95% CI, 0.95, 2.29), AB (RR, 1.48; 95% CI, 0.76, 2.90), non-O (RR, 1.28; 95% CI, 0.83, 1.98) when compared to those with blood group O. Similarly, the results also found that there were no difference in CPR and MR between women with blood A (CPR: RR, 1.12), B (CPR: RR, 1.08), AB (CPR: RR, 1.05), non-O (CPR: RR, 1.05; MR: RR, 0.94) and blood group O. Conclusions ABO blood groups may not be associated with DOR, OHSS, LBR, CPR, and MR of ART. Infertility and ART outcomes are influenced by multiple factors. Blood groups should not be taken into account excessively during diagnosis and treatment of infertile women.

To add a enhance understanding of polycystic ovary syndrome (PCOS) at the molecular level, this investigation aimed to find the genes and crucial pathways linked with PCOS by using integrated bioinformatics analysis. Based on the expression profiling by high throughput sequencing data GSE84958 derived from the Gene Expression Omnibus, the differentially expressed genes (DEGs) between PCOS samples and normal controls were identified. With DEGs, we performed a series of functional enrichment analyses. Then, a protein–protein interaction (PPI) network, miRNA - target genes and TF - target gene networks were constructed and visualized, with which the hub gene nodes were screened out. Finally, validation of hub genes was performed by using receiver operating characteristic (ROC) and RT-PCR. Molecular docking studies performed. A total of 739 DEGs were screened out, among which 360 genes were up regulated and 379 genes were down regulated. GO enrichment analysis indicated that up regulated genes were mainly involved in peptide metabolic process, organelle envelope and RNA binding and the down regulated genes were significantly enriched in plasma membrane bounded cell projection organization, neuron projection and DNA-binding transcription factor activity, RNA polymerase II-specific. REACTOME pathway enrichment analysis showed that the up regulated genes were mainly enriched in translation and respiratory electron transport and the down regulated genes were mainly enriched in generic transcription pathway and transmembrane transport of small molecules. The top 10 hub genes in the constructed PPI network, miRNA - target gene network and TF - target gene network were SAA1, ADCY6, POLR2K, RPS15, RPS15A, CTNND1, ESR1, NEDD4L, KNTC1 and NGFR. The modules analysis showed that genes in the top 2 significant modules of PPI network were mainly associated with respiratory electron transport and signalling by NGF, respectively. We find a series of crucial genes along with the pathways that were most closely related with PCOS initiation and advancement. Our investigations provide a more detailed molecular mechanism for the progression of PCOS, detail information on the potential biomarkers and therapeutic targets.

Background Energy balance is closely related to reproductive function, wherein hypothalamic kisspeptin mediates regulation of the energy balance. However, the central mechanism of kisspeptin in the regulation of male reproductive function under different energy balance states is unclear. Here, high-fat diet (HFD) and exercise were used to change the energy balance to explore the role of leptin and inflammation in the regulation of kisspeptin and the hypothalamic-pituitary-testis (HPT) axis. Methods Four-week-old male C57BL/6 J mice were randomly assigned to a normal control group (n = 16) or an HFD (n = 49) group. After 10 weeks of HFD feeding, obese mice were randomly divided into obesity control (n = 16), obesity moderate-load exercise (n = 16), or obesity high-load exercise (n = 17) groups. The obesity moderate-load exercise and obesity high-load exercise groups performed exercise (swimming) for 120 min/day and 120 min × 2 times/day (6 h interval), 5 days/week for 8 weeks, respectively. Results Compared to the mice in the normal group, in obese mice, the mRNA and protein expression of the leptin receptor, kiss, interleukin-10 (IL-10), and gonadotropin-releasing hormone (GnRH) decreased in the hypothalamus; serum luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone levels and sperm quality decreased; and serum leptin, estradiol, and tumor necrosis factor-α (TNF-α) levels and sperm apoptosis increased. Moderate- and high-load exercise effectively reduced body fat and serum leptin levels but had the opposite effects on the hypothalamus and serum IL-10 and TNF-α levels. Moderate-load exercise had anti-inflammatory effects accompanied by increased mRNA and protein expression of kiss and GnRH in the hypothalamus and increased serum FSH, LH, and testosterone levels and improved sperm quality. High-load exercise also promoted inflammation, with no significant effect on the mRNA and protein expression of kiss and GnRH in the hypothalamus, serum sex hormone level, or sperm quality. Moderate-load exercise improved leptin resistance and inflammation and reduced the inhibition of kisspeptin and the HPT axis in obese mice. The inflammatory response induced by high-load exercise may counteract the positive effect of improving leptin resistance on kisspeptin and HPT. Conclusion During changes in energy balance, leptin and inflammation jointly regulate kisspeptin expression on the HPT axis.

Caloric restriction (CR), an energy-restricted intervention with undernutrition instead of malnutrition, is widely known to prolong lifespan and protect against the age-related deteriorations. Recently it is found that CR significantly affects female reproduction via hypothalamic (corticotropin releasing hormone, neuropeptide Y, agouti-related peptide) and peripheral (leptin, ghrelin, insulin, insulin-like growth factor) mediators, which can regulate the energy homeostasis. Although CR reduces the fertility in female mammals, it exerts positive effects like preserving reproductive capacity. In this review, we aim to discuss the comprehensive effects of CR on the central hypothalamus-pituitary-gonad axis and peripheral ovary and uterus. In addition, we emphasize the influence of CR during pregnancy and highlight the relationship between CR and reproductive-associated diseases. Fully understanding and analyzing the effects of CR on the female reproduction could provide better strategies for the management and prevention of female reproductive dysfunctions.

Background Ovarian teratoma-associated anti-N-methyl-D-aspartate receptor encephalitis (NMDAR-E) is a severe autoimmune neurological disorder, and the influence of teratoma-induced autoantibodies on the pathogenesis remains unclear. Methods Ovarian teratoma tissues were collected from teratoma patients with and without NMDAR-E. Proteins were extracted and then analyzed using iTRAQ-coupled LC–MS/MS, which was followed by bioinformatics analysis. Candidate proteins were verified by Western blotting and immunohistochemistry. Results In total, 36 differentially expressed proteins (DEPs) were identified between the control group and NMDAR-E group, and the bioinformatics analysis revealed that the DEPs were mainly involved in immune-related pathways, especially HLA-A and HLA-DRB1. The western blotting results for HLA-A and HLA-DRB1 were consistent with the results of the iTRAQ analysis. Additionally, the immunohistochemical data revealed that the aggregation of HLA-A (+) and HLA-DRB1 (+) cells was more apparent in the teratoma tissues of NMDAR-E patients compared with that in the tissues of controls. Conclusion Our investigation indicated that HLA-A and HLA-DRB1 might be involved in mediating ovarian teratoma-associated NMDAR-E. These findings provide new insights into the pathophysiological mechanisms and provide information for the functional exploration of proteins in the future.

Many factors, including postponement of marriage, increased life expectancy, and improved success with assisted reproductive technologies have been contributing to increased paternal age in developed nations. This increased average paternal age has led to concerns about adverse effects of advanced paternal age on sperm quality, assisted reproductive outcomes, and the health of the offspring conceived by older fathers. This review discusses the association between advanced paternal age and sperm parameters, assisted reproduction success rates, and offspring health.

Background High anti-Müllerian hormone (AMH) levels and 25-hydroxyvitamin D [25(OH)D] deficiency have been associated with polycystic ovarian syndrome (PCOS) in adult women, and implicated in its pathogenesis. Herein we determined if the level of both AMH and 25(OH)D are altered in adolescent females with clinical features of PCOS. Methods This is a cross-sectional study utilizing a retrospective chart review of 128 patients aged 12–20 referred to an academic adolescent gynecology and endocrinology clinic for an evaluation of suspected PCOS. Unadjusted comparisons of AMH and 25(OH)D distributions between subjects with and without PCOS were performed using the Wilcoxon Rank Sum test. Quantile regression was used to compare the median AMH and 25(OH)D between subject groups; adjusting for race, ethnicity, BMI, insurance type, age, and season when bloodwork was performed. Results Seventy-four subjects were classified as having PCOS by meeting ≥2 of the three Rotterdam diagnostic criteria, and 47 subjects met only one Rotterdam diagnostic criteria, and were used as the comparative non-PCOS group. There were statistically significant unadjusted differences in median levels of AMH and 25(OH)D. In the adjusted analyses, median AMH was significantly higher in the PCOS group compared to the non-PCOS group (+ 2.39 ng/mL, 95% CI 0.43, 4.35, p = 0.018); 25(OH)D was significantly lower in the PCOS group (− 9.01 ng/mL, 95% CI -14.49, − 3.53 p = 0.001). In our sample, adolescents in both groups had insufficient 25(OH)D level (22 ng/mL) and elevated BMI (32.2 kg/m2). Conclusions Adolescents with PCOS display high levels of AMH and low 25(OH)D levels. Since traditional clinical markers of PCOS may be physiologic in adolescents, AMH and 25(OH)D may be used as surrogate markers of PCOS risk in adolescents.

Human embryogenesis frequently coinciding with cell division mistakes contributing to pervasive embryonic aneuploidy/mosaicism. While embryo self-correction was elegantly demonstrated in mouse models, human studies are lacking. Here we are witness to human embryos ability to eliminate/expel abnormal blastomeres as cell debris/fragments. Each blastocyst and its corresponding debris were separated and underwent whole genome amplification. Seven of the 11 pairs of blastocysts and their corresponding cell debris/fragments revealed discordant results. Of the 9 euploid blastocysts, four showed euploid debris, while in the others, the debris were aneuploid. In the remaining pairs, the debris showed additional aneuploidy to those presented by their corresponding blastocyst. The observed ability of human embryos to self-correction doubts many invasive and non-invasive preimplantation testing for aneuploidy at the blastocyst stage, rendering high rate of false positive (discarding “good” embryos) by identifying the cell-free DNA originated from the expelled cell debris, as aneuploidy/mosaic blastocyst.

BackgroundTrophoblast cells are required for the establishment of pregnancy and fetal development. Apoptosis is an essential feature for trophoblast invasion. Uncontrolled trophoblast apoptosis is related to some complicate pregnancies. Oxidative stress (OS) is an important inducer of trophoblast apoptosis. Cyclosporin A (CsA) has been shown to promote the activity of trophoblast cells and reduce OS-induced oxidative injury. We investigated the role and mechanism of CsA in oxidative stress-induced trophoblast cell apoptosis.MethodsJEG-3 cells were cocultured with H2O2and CsA. Cell viability and morphology were measured by MTT assay and DAPI staining. Cell apoptosis was tested with annexin V/PI staining. The expression of Bcl-2-associated X protein (Bax), B-cell lymphoma/leukemia-2 (Bcl-2), cleaved poly (ADP-ribose) polymerase (PARP) and pro-caspase-3 was assayed by western blotting. The protein expression and phosphorylation of p53 and mitogen-activated protein kinase (MAPK) kinases (JNK, ERK1/2 and p38) were examined by western blotting.ResultsCsA increased the viability, alleviated morphological injury and reduced cell apoptosis of the H2O2-treated JEG-3 cells. CsA also attenuated the activation of p53, decreased the expression of Bax and cleavage of PARP, and increased the expression of Bcl-2 and pro-caspase-3 in the JEG-3 treated with H2O2. Furthermore, CsA reduced the activation of JNK and P38 but had no significant effect on the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) in the H2O2-treated JEG-3 cells. Promoting the activation of JNK and p38 impaired the protective effect of CsA on OS-induced trophoblast apoptosis.ConclusionsThese results suggested that CsA protected trophoblast cells from OS-induced apoptosis via the inhibition of the p53 and JNK/p38 signaling pathways.

The toxic effects of ionizing radiation on the gonads have been widely recognized. Sphingosine 1-phosphate (S1P) has a protective effect on ovarian injury, and although it is known that mitochondria are involved in this process, the specific mechanism is not fully understood. The present study analysed the changes in the serum AMH and ovarian histology in Sprague-Dawley female rats exposed to X-ray radiation only or co-administered with S1P. The mRNA expression profile of ovarian tissue was further analysed via next-generation sequencing and bioinformatics approaches to screen out candidate mitochondria-related genes. Finally, differentially expressed target genes were verified by real-time PCR. The results showed that ionizing radiation could reduce the serum AMH level, destroy ovarian structure and decrease the number of follicles in rats, while S1P administration significantly attenuated the impairment of ovarian function. Gene ontology (GO) and KEGG pathway analysis revealed that a variety of genes related to mitochondrial function were differentially expressed, and the protective effect of S1P on mitochondria was more obvious in the acute phase 24 h after radiation. The differentially expressed mitochondrial function-related genes associated with the protective effect of S1P were UQCRH, MICU2 and GPX4, which were subsequently verified by RT-PCR. Therefore, ionizing radiation has a significant effect on ovarian function, and S1P has a protective effect on radiation-induced ovarian injury, in which mitochondria may play an important role. This study sheds new light on the mechanism of radiation-induced ovarian injury and helps develop a novel potential strategy to control it.

Background Long non-coding RNAs (lncRNAs) have a size of more than 200 bp and are known to regulate a host of crucial cellular processes like proliferation, differentiation and apoptosis by regulating gene expression. While small noncoding RNAs (ncRNAs) such as miRNAs, siRNAs, Piwi-interacting RNAs have been extensively studied in male germ cell development, the role of lncRNAs in spermatogenesis remains largely unknown. Objective In this article, we have reviewed the biology and role of lncRNAs in spermatogenesis along with the tools available for data analysis. Results and conclusions Till date, three microarray and four RNA-seq studies have been undertaken to identify lncRNAs in mouse testes or germ cells. These studies were done on pre-natal, post-natal, adult testis, and different germ cells to identify lncRNAs regulating spermatogenesis. In case of humans, five RNA-seq studies on different germ cell populations, including two on sperm, were undertaken. We compared three studies on human germ cells to identify common lncRNAs and found 15 lncRNAs (LINC00635, LINC00521, LINC00174, LINC00654, LINC00710, LINC00226, LINC00326, LINC00494, LINC00535, LINC00616, LINC00662, LINC00668, LINC00467, LINC00608, and LINC00658) to show consistent differential expression across these studies. Some of the targets of these lncRNAs included CENPB, FAM98B, GOLGA6 family, RPGR, TPM2, GNB5, KCNQ10T1, TAZ, LIN28A, CDKN2B, CDKN2A, CDKN1A, CDKN1B, CDKN1C, EZH2, SUZ12, VEGFA genes. A lone study on human male infertility identified 9879 differentially expressed lncRNAs with three (lnc32058, lnc09522, and lnc98497) of them showing specific and high expression in immotile sperm in comparison to normal motile sperm. A few lncRNAs (Mrhl, Drm, Spga-lncRNAs, NLC1-C, HongrES2, Tsx, LncRNA-tcam1, Tug1, Tesra, AK015322, Gm2044, and LncRNA033862) have been functionally validated for their roles in spermatogenesis. Apart from rodents and humans, studies on sheep and bull have also identified lncRNAs potentially important for spermatogenesis. A number of these non-coding RNAs are strong candidates for further research on their roles in spermatogenesis.

Background Patients found to be poor ovarian responders (POR) are a challenging patient population for any assisted reproduction technology. Despite attempts at various controlled ovarian stimulation schemes, reproductive outcomes in this patient population have not improved. In recent years, the DuoStim protocol (both follicular and luteal phase stimulation during the same menstrual cycle) has shown a potential for use in patients with POR. Methods This retrospective study reviewed the medical records of 304 women who were diagnosed as POR and underwent the DuoStim protocol. We compared follicular phase stimulation (FPS) data and luteal phase stimulation (LPS) data of the same patients. We also compared the effects of different trigger drugs including urine human chorionic gonadotropin (uHCG; 10,000 IU), recombinant human chorionic gonadotropin (rHCG; 250 μg), and gonadotropin-releasing hormone agonist (GnRH-a; 0.2 mg) at the FPS and LPS stages. Results POR undergoing the DuoStim protocol resulted in a significantly higher number of oocytes retrieved, normal fertilised oocytes, cleaved embryos, cryopreserved embryos, and good quality embryos at the LPS stage than at the FPS stage. Trigger drugs at the FPS stage did not affect the FPS stage data. Regardless of the stage, rHCG and GnRH-a yielded significantly more cryopreserved embryos and good quality embryos than uHCG. Conclusion The use of GnRH-a or rHCG as the trigger drug may be better than uHCG in both the FPS and LPS stages for POR undergoing the DuoStim protocol. This will increase the number of good quality embryos at the LPS stage. We found that the LPS stage results in more oocytes (and therefore more embryos) than the FPS stage.

Background The current evidence on the association between obesity-associated markers and semen quality, serum reproductive hormones and lipids remains inconsistent. In this study, we tested the hypothesis that, in infertile Chinese men, body mass index (BMI) negatively correlates with sperm concentration, serum total testosterone (TT), and high-density lipoprotein cholesterol (HDL-C). The relationship between other obesity-associated markers and semen quality parameters, serum reproductive hormones, lipids and leptin were also investigated. Methods 181 Chinese infertile men were recruited from September 2018 to September 2019. Their obesity-associated markers, semen parameters, and serum reproductive hormones, lipids and leptin were detected. Statistical analysis was performed to assess the relationship between obesity-associated markers and semen quality, serum reproductive hormones, lipids and leptin. Result(s) Statistically negative correlation was found between other obesity-associated markers (e.g. waist-to-hip ratio and waist-to-height ratio) and semen parameters (e.g. sperm concentration, ratio of progressive motility and ratio of non-progressive motility), while no significant correlation was found between BMI and semen quality, serum reproductive hormones, lipids and leptin. Ratio of morphologically normal sperm was negatively correlated with serum lipids including total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C), leptin and seminal superoxide dismutase. Ratio of progressive sperm, sperm concentration and ratio of morphologically normal sperm exhibited significantly lower values in overweight group than normal group. Estradiol (E2) and E2/TT were significantly higher in obese group than normal group, while TT level was significantly lower in obese group than normal group. Univariate and multivariate analysis indicated that TC was significantly associated with BMI. Serum leptin concentration was positively correlated with seminal leptin concentration in overweight and obese groups. Conclusion(s) No significant correlation was found between BMI and sperm concentration, serum TT and HDL-C, while other obesity-associated markers were found to negatively correlate with sperm concentration, ratio of progressive motility and ratio of non-progressive motility. Statistically significant correlations between serum reproductive hormones, lipids and leptin also existed in Chinese infertile men.

Background There is a growing body of evidence on low serum vitamin-D levels and the risk of uterine leiomyomas (UL). Therefore, this systematic review and meta-analysis was conducted to investigate the association between serum vitamin D levels and UL occurrence. Methods Searches were systematically conducted of the electronic databases PubMed, Scopus, EMBASE, Web of Science (ISI), Cochrane library, Ovid, and Google Scholar to identify relevant studies from inception until February 6, 2020. Heterogeneity across the included studies was examined using Cochran’s Q and I-square (I2). Data was pooled using random effects modeling and expressed as standardized mean differences (SMDs). Results Nine eligible studies with a total of 1730 participants (835 patients with UL and 895 controls) were included in the current meta-analysis. Pooled results with random effects modeling indicated that serum vitamin D levels were significantly lower in patients with UL than in the control group (n = 9, SMD = − 0.67; 95% CI, − 0.98, − 0.35, p 2 = 89.3%, p n = 4, SMD = − 1.20; 95% CI, − 1.45, − 0.96, p 2 = 30.6%, p = 0.229) were lower than those from Europe (n = 3, SMD = − 0.34; 95% CI, − 0.49, − 0.18, p 2 = 0.0%, p = 0.602) and Africa (n = 2, SMD = − 0.13; 95% CI, − 0.29, 0.04, p = 0.128; I2 = 0.0%, p = 0.417), although the difference was not significant in Africa. Publication year was also found to be a potential contributor’s variable in the pooled SMD using the meta-regression method (t = − 3.00, p = 0.02). Conclusions To the best of our knowledge, the current meta-analysis showed for the first time that serum vitamin D levels were significantly lower in women with UL in selected populations.

BackgroundMicrodissection testicular sperm extraction (microTESE) in men with non-obstructive azoospermia (NOA) is the procedure that results in the highest number of sperm cells retrieved for in vitro fertilization (IVF). This study presents a novel assessment of predictors of sperm retrieval as well as downstream embryology and pregnancy outcomes in cases of men with NOA undergoing microTESE.MethodsA retrospective chart review of 72 men who underwent microTESE for predictors of fertility outcomes including sperm retrieved at microTESE, embryology progression to embryo transfer (ET), clinical pregnancy, live birth, and surplus sperm retrieved for additional IVF/intracytoplasmic injection cycles beyond one initial cycle. Statistical models for each of these outcomes were fitted, with ap-value of ResultsSeventy-two men underwent microTESE, and 51/72 (70.8%) had sperm retrieved. Of those, 29/43 (67.4%) reached ET. Of the couples who underwent ET, 21/29 (72.4%) achieved pregnancy and 18/29 (62.1%) resulted in live birth. Of the men with sperm retrieved, 38/51 (74.5%) had surplus sperm cryopreserved beyond the initial IVF cycle.Age, testicular volume, FSH, and testicular histopathology were assessed as predictors for sperm retrieved at microTESE, progression to ET, pregnancy, live birth, and surplus sperm. There were no preoperative predictors of sperm retrieval, clinical pregnancy, or live birth. Age predicted reaching ET, with older men having increased odds. FSH level had a negative relationship with surplus sperm retrieved. Men with hypospermatogenesis histology had higher rates of sperm retrieval, clinical pregnancy, live birth, and having surplus sperm.ConclusionsMen who underwent microTESE with a hypospermatogenesis histopathology had better outcomes, including higher rates of sperm retrieval, clinical pregnancy, live birth, and having surplus sperm retrieved. Increasing male partner age increased the odds of reaching ET. No other clinical factors were predictive for the outcomes considered.

Histone acetylation is a critical epigenetic modification that changes chromatin architecture and regulates gene expression by opening or closing the chromatin structure. It plays an essential role in cell cycle progression and differentiation. The human endometrium goes through cycles of regeneration, proliferation, differentiation, and degradation each month; each phase requiring strict epigenetic regulation for the proper functioning of the endometrium. Aberrant histone acetylation and alterations in levels of two acetylation modulators - histone acetylases (HATs) and histone deacetylases (HDACs) - have been associated with endometrial pathologies such as endometrial cancer, implantation failures, and endometriosis. Thus, histone acetylation is likely to have an essential role in the regulation of endometrial remodelling throughout the menstrual cycle.

Premature Ovarian Insufficiency (POI) is viewed as a type of infertility in which the menopausal status occurs before the physiological age. Several therapeutic strategies have been introduced in clinic for POI treatment, although the outputs are not fully convincing. Platelet-rich plasma (PRP) is a unique blood product widely applied in regenerative medicine, which is based on the releasing of the growth factors present in platelets α-granules. In the current investigation, we examined the effectiveness of PRP as a therapeutic alternative for POI animals. POI in Wistar albino rats was induced by daily intraperitoneal (IP) administration of gonadotoxic chemical agent, 4-vinylcyclohexene dioxide (VCD) (160 mg/ kg) for 15 consecutive days. After POI induction, the PRP solution was directly injected intra-ovarian in two concentrations via a surgical intervention. Every two weeks post-injection, pathological changes were monitored in the ovaries using Hematoxylin-Eosin staining method, until eight weeks. Follicle Stimulating Hormone (FSH) content in serum was measured, together with the expression of the angiogenic-related transcripts ANGPT2 and KDR by real-time qPCR. Furthermore the fertility status of the treated rats was evaluated by mating trials. Histopathological examination revealed successful POI induction via the depletion of morphologically normal follicles in rats following VCD treatment compared to the control rats. The injection of PRP at two concentrations reduced the number and extent of the follicular atresia and inflammatory responses (p ANGPT2 and KDR transcripts were significantly increased in POI rats due to enhanced inflammation, while these values were modulated after PRP administration (p p > 0.05). Nevertheless, a clear improvement in litter counts was found in POI rats receiving PRP compared to the non-treated POI group, being able to consider PRP as a facile, quick, accessible, safe and relatively cheap alternative therapeutic strategy to revert POI-related pathologies.

Recently a novel method based on horizontal sperm migration in injection dishes has been introduced as an additional tool for preparation of semen sample in assisted reproductive technology (ART) procedures. In the present study, we evaluated both timing and reproductive outcomes in a randomized controlled study including 1034 intra-cytoplasmic sperm injection (ICSI) procedures followed by fresh embryo transfer. Couples enrolled were divided into two sub-groups, namely conventional swim-up method (Group A), and horizontal sperm migration in injection dishes (Group B).No significant differences were found between groups with respect to fertilization rate, implantation success, clinical pregnancy outcomes and ongoing pregnancies. On the contrary, both cleavage and blastocyst rates were statistically higher in Group B, suggesting superior efficiency and safety of this innovative technique also including time-saving and cheaper costs as compared to the classical swim-up sperm preparation.Our data support the interpretation of the horizontal sperm migration as a promising procedure for semen preparation in ART cycles.

BackgroundThrough this prospective study, we aimed to explore the change of molecular modification after the transient scrotal hyperthermia on human sperm.MethodsTen healthy subjects selected with strict screening criteria underwent testicular warming in a 43 °C water bath for 30 min a day for 10 consecutive days. Semen samples were collected 2 weeks before the first heat treatment and 6 weeks after the first heat treatment. Proteins from the samples were labeled with isobaric tags for relative and absolute quantitation and analyzed by two-dimensional liquid chromatography–tandem mass spectrometry.ResultsIn contrast to the control, of the 3446 proteins identified, 61 proteins were deregulated: 28 were up-regulated and 33 were down-regulated. Approximately 95% of the differentially expressed proteins were found to participate in spermatogenesis, fertilization, or other aspects of reproduction. In particular, the expression of sperm motility and energy metabolism-related proteins AKAP4, SPESP1, ODF1, ODF2, GAPDHS, and ACTRT2, validated by western blotting of the proteins obtained from human and mouse samples, tended to be reduced under scrotal hyperthermia.ConclusionsThe results indicated that the proteins AKAP4, ODF1, ODF2, GAPDHS, SPESP1, and ACTRT2, play an important role in the heat-induced reversible reduction in sperm concentration and motility and have the potential to be the biomarkers and clinical targets for scrotal heat treatment induced male infertility.

Background There is a need for a reference material to support the development and ensure the quality of immunoassays for human AMH. A batch of ampoules, coded 16/190, containing lyophilised recombinant AMH was evaluated in a WHO Collaborative Study. The aims of the study were to determine the AMH content in terms of the calibration of each immunoassay method, to predict long-term stability and to assess the suitability of the preparation to calibrate AMH immunoassays. Methods Study participants were asked to report the AMH content of specific dilutions of coded ampoules of 16/190 and a comparator preparation containing approximately half the AMH content. In each assay, participants also reported the AMH content of 22 patient samples to assess commutability. A robust all-laboratory geometric mean of the content estimates was determined using the laboratory geometric mean estimates. Commutability was assessed using a difference in bias approach. Stability was predicted by the measurement of thermally accelerated degradation samples. Results Seven laboratories performed twenty-one immunoassay method-platform combinations, sixteen of which provided data which met the validity criteria, giving a consensus geometric mean estimate of AMH content of 511 ng/ampoule (95% CI, 426–612, n = 16, GCV 42%) and a robust geometric mean of 489 ng/ampoule. By contrast, the GCV% for the all-laboratory geometric mean of the relative content estimates for the comparator sample to 16/190 was 12%. Commutability was assessed using 20 of the 22 representative patient samples. Of the valid assays, 16/190 was within the limits of acceptable commutability for 6 methods, partially commutable for a further 3 methods and non-commutable when measured by 7 methods. The preparation was predicted to be highly stable when stored at − 20 °C. Conclusion The majority of methods met the validity criteria. Content estimates showed a high between-method variability, yet assays exhibited a similar proportionality of response as demonstrated using the comparator sample. 16/190 was commutable in some but not all methods. On the basis of these results, it was agreed by the WHO Expert Committee on Biological Standardization to establish 16/190 as a WHO Reference Reagent for AMH with a content defined by consensus immunoassay of 489 ng/ampoule.

Background Fertility preservation is particularly challenging in young women diagnosed with hematopoietic cancers, as transplantation of cryopreserved ovarian cortex in these women carries the risk for re-introducing cancer cells. Therefore, the construction of a bioengineered ovary that can accommodate isolated small follicles was proposed as an alternative to minimize the risk of malignancy transmission. Various options for viable bioengineered scaffolds have been reported in the literature. Previously, we reported three protocols for producing mouse ovarian scaffolds with the decellularization technique. The present study examined these scaffolds further, specifically with regards to their extracellular composition, biocompatibility and ability to support recellularization with mesenchymal stem cells. Material and methods Three decellularization protocols based on 0.5% sodium dodecyl sulfate (Protocol 1; P1), or 2% sodium deoxycholate (P2), or a combination of the two detergents (P3) were applied to produce three types of scaffolds. The levels of collagen, elastin and sulfated glycosaminoglycans (sGAGs) were quantified in the remaining extracellular matrix. Detailed immunofluorescence and scanning electron microscopy imaging were conducted to assess the morphology and recellularization efficiency of the constructs after 14 days in vitro utilizing red fluorescent protein-labelled mesenchymal stem cells. Results All protocols efficiently removed the DNA while the elastin content was not significantly reduced during the procedures. The SDS-protocol (P1) reduced the sGAG and the collagen content more than the SDC-protocol (P2). All scaffolds were biocompatible and recellularization was successful, particularly in several P2-derived scaffolds. The cells were extensively distributed throughout the constructs, with a denser distribution observed towards the ovarian cortex. The cell density was not significantly different (400 to 550 cells/mm2) between scaffold types. However, there was a tendency towards a higher cell density in the SDC-derived constructs. Scanning electron microscope images showed fibrous scaffolds with a dense repopulated surface structure. Conclusions While there were differences in the key structural macromolecules between protocols, all scaffolds were biocompatible and showed effective recellularization. The results indicate that our SDC-protocol might be better than our SDS-protocol. However, additional studies are necessary to determine their suitability for attachment of small follicles and folliculogenesis.

A majority of common metabolic diseases can result in excessive lipolysis, leading to elevated levels of non-esterified fatty acids (NEFAs) in the body fluids. In females, increased NEFA levels in the follicular fluid markedly alter the functions of intrafollicular cells such as granulosa cells (GCs) and oocytes. Therefore, elevated levels of NEFAs have been suggested to be a significant player of subfertility in females of both human and economically important animal species such as cattle, buffalo, sheep, pig, chicken, and dog. However, the effects imposed by saturated and unsaturated fatty acids (SFAs and UFAs) on ovarian follicles are controversial. The present review emphasizes that SFAs induce apoptosis in granulosa and cumulus cells of ovarian follicles in different species. They further could adversely affect oocyte maturation and developmental competence. Many types of UFAs affect steroidogenesis and proliferation processes and could be detrimental for follicular cells, especially when at elevated concentrations. Interestingly, monounsaturated fatty acids (MUFAs) appear to contribute to the etiology of the polycystic ovarian syndrome (PCOS) as they were found to induce the transcription and translation of the androgenic transcription factor SOX9 while downregulating its estrogenic counterpart FOXL2 in GCs. Overall, this review presents our revised understanding of the effects of different fatty acids on the female reproductive success, which may allow other researchers and clinicians to investigate the mechanisms for treating metabolic stress-induced female infertility.

Background The purpose of this study was to assess whether increased body mass index (BMI) negatively affects assisted reproductive technology (ART) outcomes among gestational carriers. Methods A retrospective matched case-control cohort, including all gestational carrier (GC) cycles performed at CReATe Fertility Centre (Toronto, ON, Canada) between 2003 and 2016. Setting A Canadian fertility clinic, with a large surrogacy program. Patients All gestational carriers that had undergone a cycle completed to a transfer at our clinic, and had BMI and outcome data available, were matched by BMI to infertile patients treated at our clinic during the same years provided they had undergone a cycle completed to a transfer, and had outcomes data available. Interventions None. Main outcome measures Clinical pregnancies rates, miscarriage rates and live birth rates. Results BMI was not a reliable prediction factor of any of the measured outcomes. Importantly, the gestational carrier population had better outcomes and a significantly lower overall incidence of maternal, fetal and neonatal complications when compared with infertile patients, treated at our clinic during the same years. Conclusion BMI is not a reliable predictor of outcomes among gestational carriers.

BACKGROUND: A recently published Position Statement (PS) by the Preimplantation Genetics Diagnosis International Society (PGDIS) regarding utilization of preimplantation genetic testing for aneuploidy (PGT-A) in association with in vitro fertilization (IVF) contained inaccuracies and misrepresentations. Because opinions issued by the PGDIS have since 2016 determined worldwide IVF practice, corrections appear of importance.METHODS: The International Do No Harm Group in IVF (IDNHG-IVF) is a spontaneously coalesced body of international investigators, concerned with increasing utilization of add-ons to IVF. It is responsible for the presented consensus statement, which as a final document was reached after review of the pertinent literature and again revised after the recent publication of the STAR trial and related commentaries.RESULTS: In contrast to the PGDIA-PS, we recommend restrictions to the increasing, and by IVF centers now often even mandated, utilization of PGT-A in IVF cycles. While PGT-A has been proposed as a tool for achieving enhanced singleton livebirth outcomes through embryo selection, continued false-positive rates and increasing evidence for embryonic self-correction downstream from the testing stage, has led IDNHG-IVF to conclude that currently available data are insufficient to impose overreaching recommendations for PGT-A utilization.DISCUSSION: Here presented consensus offers an alternative to the 2019 PGDIS position statement regarding utilization of preimplantation genetic testing for aneuploidy (PGT-A) in association with in vitro fertilization (IVF). Mindful of what appears to offer best outcomes for patients, and in full consideration of patient autonomy, here presented opinion is based on best available evidence, with the goal of improving safety and efficacy of IVF and minimizing wastage of embryos with potential for healthy births.CONCLUSIONS: As the PGDIS never suggested restrictions on clinical utilization of PGT-A in IVF, here presented rebuttal represents an act of self-regulation by parts of the IVF community in attempts to control increasing utilization of different unproven recent add-ons to IVF.

Background Patients with severe oligospermia and nonobstructive azoospermia have very limited numbers of viable sperm in their epididymal and testicular samples. Thus, cryopreservation of their sperm is performed to avoid repeated sperm retrievals and to preserve their sperm from any side effects of any treatment regimens. Main body The development of intracytoplasmic sperm injection technology has extended the therapeutic capacity of assisted reproductive technology for men with azoospermia via the surgical or percutaneous isolation of sperm from the testis/epididymis. The conventional cryopreservation techniques are inadequate for preserving individually selected sperm. The technique for freezing single sperm was first developed in 1997 and has been explored from the perspective of frozen carriers, freezing programs, and cryoprotectant formulations. Among these methods, advances in frozen carriers have directly improved single-sperm freezing technology. In this review, we evaluate the different technologies for the cryopreservation of single sperm by discussing the advantages and disadvantages of different freezing methods, their clinical applications, and the outcomes for a range of frozen carriers. Conclusion Our review article describes the latest and current technologies implemented for the cryopreservation of single sperm that could potentially benefit patients with severe oligospermia and who rarely have any sperm in their ejaculate. This review provides a platform to understand the process and pitfalls of single-sperm cryopreservation to ensure further improvements in the cryopreservation technology in future studies.

Background The endometrial preparation during frozen embryo transfer (FET) can be performed by natural cycle (NC), hormone replacement therapy (HRT) cycle and cycle with ovulation induction (OI). Whether different FET preparation protocols can affect maternal and neonatal outcomes is still inconclusive. Methods This was a retrospective cohort study that included 6886 women who delivered singleton live birth babies after 28 weeks of pregnancy underwent FET from January, 2015 to July, 2018. Women were divided into three groups according to the protocols used for endometrial preparation during FET: NC group (N = 4727), HRT group (N = 1642) and OI group (N = 517). Results After adjusting for the effect of age, body mass index (BMI), irregular menstruation, antral follicle count (AFC), endometrial thickness, the levels of testosterone, anti-Müllerian hormone (AMH), preconceptional fasting glucose (PFG), systolic and diastolic pressure et al., the HRT group had higher risk of hypertensive disorders of pregnancy (HDP) compared with the NC group (adjusted odds ratio (aOR) 2.00, 95% confidence interval (CI) 1.54–2.60). Singletons born after HRT FET were at increased risk of low birth weight (LBW) compared to NC group (aOR 1.49, 95%CI 1.09–2.06). The risks of preterm birth (PTB) in the HRT and OI group were elevated compared with the NC group (aOR 1.78, 95%CI 1.39–2.28 and aOR 1.51, 95%CI 1.02–2.23, respectively). Conclusions The HRT protocol for endometrial preparation during frozen embryo transfer of blastocysts was associated with increased risk of maternal and neonatal complications, compared to the NC and OI protocol.

The role of prostaglandin E2 (PGE2) in the successful resumption of oocyte meiosis and cumulus expansion has been well-documented. However, there remains very little information available on the influence of PGE2 on other processes that occur during oocyte maturation. In this study, we supplemented a maturation medium with PGE2 and monitored oocyte quality markers, glucose metabolism, mitochondrial status, oxidative stress, and apoptosis in the cumulus-oocyte complexes (COCs), using a well-established in vitro model of embryo production in cattle. We found that this increased availability of PGE2 during maturation led to an increase in the expression of genes associated with oocyte competence and improved the quality of blastocysts produced. Prostaglandin E2 also appeared to stimulate glucose uptake and lactate production in the COCs, both influencing the expression of enzymes involved in glycolysis and the hexosamine biosynthetic pathway. We found that PGE2 reduced intracellular reactive oxygen species levels, and simultaneously increased glutathione concentration and stimulated antioxidant gene expression in the oocyte. These results indicate that PGE2 has an important role in the protection of oocytes against oxidative stress. Mitochondrial membrane potential was also improved in PGE2-treated oocytes, and there was a reduction in the occurrence of apoptosis in the COCs. Promotion of an anti-apoptotic balance in transcription of genes involved in apoptosis was present in both oocytes and the cumulus cells. In summary, PGE2 could represent a novel autocrine/paracrine player in the mechanisms that can facilitate successful oocyte maturation and oocyte survival in the cow.

Background The blastocyst morphology provided valuable roles for predicting pregnancy and live birth, but was still not fully understood for evaluating miscarriage. The aim of this study was to explore the association between blastocyst morphologic evaluation and first trimester miscarriage combined with karyotype of miscarried conceptus. Methods This retrospective cohort study included a total of 2873 clinical pregnancy cycles with single blastocyst transfer performed from January 2013 to April 2019. Chromosome karyotype of miscarried conceptus was analyzed via single nucleotide polymorphism array analysis. Miscarriage and karyotype of miscarried conceptus associated with blastocyst morphology were analyzed by chi-square and logistic regression analysis. Results A total of 354 (12.3%) cycles resulted in first trimester miscarriage. Miscarriage rates increased with trophectoderm (TE) grade from A to C (P = 0.012), while three morphologic parameters (blastocoele expansion degree, inner cell mass (ICM) and TE) showed no statistical significance with miscarriage after multivariable analysis. The rate of aneuploidy was 47.7% (83 of 174) in total miscarried conceptuses. For euploid miscarriages, the grade B of ICM occupied a higher proportion compared with aneuploidy, with OR of 2.474, (95% CI, 1.311–4.699), P = 0.005. Conclusions Chromosomal aberration of embryo is an important genetic factor for first trimester miscarriage, and the quality of ICM is a potential indicator for euploid miscarriage. Blastocysts with grade A of ICM should be given priority during single blastocyst transfer to reduce potential miscarriage.

Background This study investigates the effect of letrozole on hormone profiles, semen parameters, body mass index (BMI), degree of oxidative stress and sperm chromatin integrity in men with idiopathic oligo/astheno/teratozoospermia (iOAT) and T:E2 ratio ≤ 10. Materials and methods This study is a longitudinal, prospective, interventional and open-labelled clinical trial. Semen samples were collected from 20 iOAT men with low serum testosterone (T) to estradiol (E2) ratio (T:E2 ratio ≤ 10). The participants were treated with 2.5 mg letrozole orally per day for 3 months. Then, sperm parameters, hormone profiles, BMI, chromatin integrity and intracellular reactive oxygen species (ROS) level were assessed pre- and post- treatment. The chromatin integrity was evaluated by assessment of DNA fragmentation (with TUNEL assay) and protamine deficiency (with Chromomycin A3, CMA3). Also, the intracellular ROS levels were investigated by 2′, 7′-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. Finally, the differences between the parameters evaluated before and after letrozole treatment were analyzed with the t-test and the Wilcoxon signed-rank test. Results Sperm concentration, percentage of sperm motility and its normal morphology increased significantly after letrozole treatment. Moreover, serum testosterone level increased but estradiol level decreased significantly following treatment. The mean of T:E2 ratio improved 1600%. Also, letrozole treatment significantly reduced the percentage of sperm TUNEL positivity and sperm CMA3 positivity. While no significant difference was observed between intracellular ROS levels and BMI before and after treatment. Finally, as a notable result, four spontaneous pregnancies (20%) were achieved after treatment. Conclusions Letrozole treatment can effectively increase spontaneous pregnancies by improving sperm parameters and sperm chromatin integrity in men with iOAT and T:E2 ratio ≤ 10. Trial registration Trial registration: IRCT, IRCT20191030045283N1. Registered 16 November 2019 - Retrospectively registered, https://fa.irct.ir/user/trial/43484/view

Background Decreased endometrial thickness (EMT) has been suggested to be associated with reduced birthweight of in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) newborns. Considering the differences in ovarian stimulation degree and laboratory procedures between IVF/ICSI and IUI treatment, we aim to investigate whether EMT has any influence on IUI infant outcomes as well. Methods This was a retrospective cohort study of 1016 patients who had singleton livebirths after IUI treatment cycles from January 2008 to December 2018 at a tertiary-care academic medical center in China. Patients were categorized into three groups by the 10th and 90th percentile of peak EMT: ≤7.6, 7.7–13.0 and ≥ 13.1 mm. The primary outcomes of the study were preterm birth (PTB), low birthweight (LBW) and small-for-gestational age (SGA). Multiple regression analyses were performed after controlling for a variety of potential confounders. Results No significant differences were found among the three groups in gestational age, birthweight and birthweight Z-score. Compared with the EMT 7.7–13.0 mm group, the incidences of PTB, LBW and SGA were 5.5% (adjusted odds ratio [aOR] 0.81, 95% confidence interval [CI] 0.33–2.01), 6.4% (aOR 1.44, 95% CI 0.58–3.58) and 7.3% (aOR 1.21, 95% CI 0.53–2.76) in the EMT ≤7.6 mm group, respectively. Similarly, EMT ≥13.1 mm was not significantly associated with risks of PTB (aOR 0.63, 95% CI 0.24–1.65), LBW (aOR 0.57, 95% CI 0.17–1.95) and SGA (aOR 0.73, 95% CI 0.28–1.92). The odds of other adverse neonatal outcomes, including macrosomia, large-for-gestational age and major congenital malformations, did not show significant differences before and after adjustment in both EMT ≤7.6 and ≥ 13.1 mm groups. Conclusions EMT is not independently associated with adverse perinatal outcomes in IUI cycles. This novel finding would provide reassuring information for IUI patients with thin endometrial linings regarding their neonatal health. However, further prospective cohort studies with larger datasets are needed to confirm the conclusion.

Background Abnormal androgen receptor (AR) genes can cause androgen insensitivity syndrome (AIS), and AIS can be classified into complete androgen insensitivity syndrome (CAIS), partial androgen insensitivity syndrome (PAIS) and mild AIS. We investigated the characteristics of clinical manifestations, serum sex hormone levels and AR gene mutations of 39 AIS patients, which provided deeper insight into this disease. Methods We prospectively evaluated 39 patients with 46, XY disorders of sex development (46, XY DSD) who were diagnosed with AIS at the Department of Endocrinology of Shanghai Children’s Hospital from 2014 to 2019. We analysed clinical data from the patients including hormone levels and AR gene sequences. Furthermore, we screened the AR gene sequences of the 39 AIS patients to identify probable mutations. Results The 39 AIS patients came from 37 different families; 19 of the patients presented CAIS, and 20 of them presented PAIS. The CAIS patients exhibited a higher cryptorchidism rate than the PAIS (100 and 55%, P = 0.001). There were no significant difference between the CAIS and PAIS groups regarding the levels of inhibin B (INHB), sex hormone-binding globulin (SHBG), basal luteinizing hormone (LH), testosterone (T), or basal dihydrotestosterone (DHT), the T:DHT ratio, DHT levels after human chorionic gonadotropin (HCG) stimulation or T levels after HCG stimulation. However, the hormone levels of AMH (P = 0.010), peak LH (P = 0.033), basal FSH (P = 0.009) and peak FSH (P = 0.033) showed significant differences between the CAIS group and the PAIS group. Twenty-one reported pathogenic and 9 novel AR mutations were identified. Spontaneous AR mutations were found in 5 AIS patients, and 21 patients inherited mutations from their mothers, who carried heterozygous mutations. Conclusions Forty-six XY DSD patients with cryptorchidism and female phenotypes were highly suspected of having AIS. We demonstrated that CAIS patients could not be distinguished by their hormone levels alone. Compared with PAIS patients, CAIS patients exhibited higher basal FSH, peak FSH, and peak LH hormone levels but lower AMH expression. We identified 21 reported pathogenic AR mutations and 9 novel AR mutations that led to different types of AIS. Missense mutations were the major cause of AIS and mostly occurred in exon 7 of the AR gene. These findings provided deeper insight into the diagnosis and classification of AIS and will even contributed to its clinical assessment.

Background We have previously shown that hsa-miR-423-5p expression in ovarian granulosa cells is decreased in high ovarian response populations. The objective of the present study was to find the target gene and mechanism for miR-423-5p involved in ovarian response regulation. Methods (a) TargetScan was used to predict the target gene of hsa-miR-423-5p. (b) A model for hsa-miR-423-5p overexpression or inhibition was constructed by transfecting KGN cells with lentivirus. CSF1 mRNA and protein expression and luciferase activity were measured. (c) The cell cycles of control and lentivirus treated KGN cells were analyzed. Western blot was used to measure the expression of CDKN1A in KGN cells. (d) The concentration of E2 in KGN cell culture medium were measured. Results (a) TargetScan revealed that the 3′ un-translated region of CSF1 matched 11 bases at the 5′ end of miR-423-5p, making it a likely target gene. (b) Overexpression or inhibition of miR-423-5p were associated with respective decreases or increases in CSF1 expression (both mRNA and protein) (p p p p Conclusions Colony stimulating factor 1 is a target gene of miR-423-5p and that it may regulate ovarian response to ovulation induction by affecting granulosa cells proliferation and estrogen secretion.

Background The interface between environmental risk factors and genetic factors could contribute to the pathogenesis of hyperandrogenism and insulin resistance in polycystic ovary syndrome (PCOS); however, the underlying complex mechanism remains to be elucidated. Methods We used dehydroepiandrosterone (DHEA)-induced PCOS-like rat model to measure circadian clock genes and insulin resistance-related genes. Additionally, we performed in vitro experiments in mature adipocytes to verify the molecular mechanisms. Results DHEA-induced PCOS-like rats exhibited insulin resistance and arrhythmic expression of circadian clock genes in the liver and adipose tissues, particularly showing decreased brain and muscle ARNT-like protein 1 (BMAL1) expression. In addition, hyperandrogenism gave rise to negative regulation of BMAL1 expression to nicotinamide phosphoribosyltransferase and sirtuin 1, which further inhibited downstream glucose transporter type 4, leading to insulin resistance in mature adipocytes, which was consistent with our previous results in HepG2 cells. Conclusions Decreased BMAL1 expression in the liver and adipose played a potentially novel role in the contribution of hyperandrogenism to insulin resistance, which might be a possible mechanism accounting for the pathogenesis of PCOS.

Background The study aimed to investigate whether and how general and partial time intervals between processes, from human chorionic gonadotrophin (HCG) trigger to intracytoplasmic sperm injection (ICSI), affected the laboratory and reproductive outcomes in ICSI cycles. Methods This was a retrospective data analysis of 3602 women who underwent ICSI treatment cycles using partner or donor sperms, performed at Reproduction Medicine Center of Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology (Wuhan, China) between October 2016 and September 2018. The clinical pregnancy rate was the major outcome in the study. The fertilization and available embryo rates were secondary outcomes. Results Data from 3602 consecutive fresh ICSI cycles was analysed. Multivariate linear regression and logistic regression analysis of factors related to fertilization and clinical pregnancy rates showed that fertilization rate (P = 0.001) and clinical pregnancy rate (P = 0.037) were significantly associated with denudation (DN)-ICSI interval. Long DN-ICSI interval was associated with higher rate of fertilization than short DN-ICSI interval but significantly decreased clinical pregnancy rate when the interval is over 4 h (P Conclusions DN-ICSI time interval can act as an independent predictor for clinical outcomes in ICSI cycles. The optimal time for ICSI is within 4 h after oocyte denudation for excellent laboratory and reproductive outcomes in ICSI cycles.

Background Molecular analyses of vitamin D in a typical cycling endometrium has received minimal research attention in the reproductive field. This study was designed to assess how expression of the endometrial vitamin D receptor (VDR) and CYP27B1, a vitamin D metabolizing enzyme, change during the menstrual cycle in women of reproductive age. In addition, this study explores the association between expression of vitamin D-VDR system and endometrial receptivity during the implantation window. Methods Sixteen patients underwent standardized in vitro fertilization (IVF) treatment and freeze-all techniques. Before embryo transfer, total serum 25(OH) D levels were determined through blood samples and VDR, CYP27B1, HOXA10, and CYP19 expression were determined through endometrial samples. Endometrial receptivity was also assessed using an electron microscope. Results We found that VDR protein expression was significantly lower throughout the endometrial secretory phase compared to the proliferative phase, while CYP27B1 expression remained constant during the menstrual cycle. During the implantation window, ultrastructural evaluation showed that higher serum vitamin D levels were associated with more mature pinopodes; VDR and HOXA10 protein expression were substantially elevated in pregnant women compared to non-pregnant women; and VDR protein levels were positively correlated with HOXA10 levels. In addition, serum vitamin D levels were positively correlated with VDR and HOXA10 protein levels in the endometrium. Conclusions Women with increased VDR expression in the endometrium, especially during the implantation window of the menstrual cycle, were significantly more likely to be pregnant than women with decreased expression. Our results support the hypothesis that the Vitamin D-VDR system performs a role during the development of endometrial receptivity.

Background In young women with poor ovarian response, luteal-phase ovarian stimulation (LPOS) is a potential method for collecting competent oocytes. The aim of this study was to assess the efficacy of LPOS compared with follicular phase ovarian stimulation (FPOS) in young women with poor ovarian response (POR). Methods This single-center, prospective, randomized pilot study compared LPOS and FPOS in women with POR fulfilling Bologna criteria who underwent in vitro fertilization at the Instituto Bernabeu. The primary outcome was the number of metaphase II (MII) oocytes obtained by follicular puncture. Results Sixty women were included in the study, with 27 women completing LPOS and 30 undergoing FPOS. There was no statistically significant difference in the number of MII oocytes obtained between the LPOS group and the FPOS group (2.1 ± 2.0 vs. 2.6 ± 2.2, p = 0.31). Length of stimulation was also similar in both groups (8.35 ± 2.8 vs. 8.15 ± 4.1 days, p = 0.69). Similarly, there was no significant difference in the follicle-stimulating hormone total dose, number of cumulus-oocyte complexes, survival rate, fertilization rate, or cancellation rate between groups. A significantly higher Ovarian Sensitivity Index was observed in the LPOS group versus the FPOS group (0.96 vs. 0.57, p = 0.037). Conclusion LPOS was comparable with FPOS in terms of efficacy and may improve ovarian responsiveness in young women with POR. Trial registration ClinicalTrials.gov identifier: NCT02625532; EudraCT identifier: 2015–003856-31.

BRCA mutation carriers face various situations that influence their fertility potential. There is still a lack of guideline or expert consensus on Fertility Preservation (FP) in BRCA mutation carriers and the necessity and safety of FP in BRCA mutation carriers is still in dispute. This review aims to focus on the population of BRCA mutation carriers by analyzing the existing FP strategies, comprehensively comparing the pros and cons of each strategy and its applicability.FP is a suggestion for BRCA mutation carriers with birth planning. Different FP strategies have different characteristics. Considering the particularity of BRCA mutation carriers, multiple factors need to be carefully considered. This review focuses on the applicability of each FP method for carriers under various circumstances. Available FP strategies including oocyte cryopreservation, ovarian tissue cryopreservation, preimplantation genetic diagnosis, and egg/embryo donation are analyzed by comparing existing methods comprehensively. In the attempt to provide an up-to-date decision-making guidance. Conditions taking into consideration were the carrier’s age, the risk of breast and ovarian metastasis, plans for oncotherapy, FP outcome, time available for FP intervention and accessibility.Overall, FP is necessary and safe for BRCA mutation carriers. Among all available FP methods, oocyte cryopreservation is the most reliable procedure; ovarian tissue cryopreservation is the only way for preserving both fertility and endocrine function, recommended for pre-pubertal carriers and when time is limited for oocyte stimulation. A clear framework provides frontline clinical practitioners a new thought and eventually benefit thousands of BRCA mutation carriers.

Background Congenital hypogonadotropic hypogonadism (CHH) is a rare disease, triggered by defective GnRH secretion, that is usually diagnosed in late adolescence or early adulthood due to the lack of spontaneous pubertal development. To date more than 30 genes have been associated with CHH pathogenesis with X-linked recessive, autosomal dominant, autosomal recessive and oligogenic modes of inheritance. Defective sense of smell is present in about 50–60% of CHH patients and called Kallmann syndrome (KS), in contrast to patients with normal sense of smell referred to as normosmic CHH. ANOS1 and FGFR1 genes are all well established in the pathogenesis of CHH and have been extensively studied in many reported cohorts. Due to rarity and heterogenicity of the condition the mutational spectrum, even in classical CHH genes, have yet to be fully characterized. Methods To address this issue we screened for ANOS1 and FGFR1 variants in a cohort of 47 unrelated CHH subjects using targeted panel sequencing. All potentially pathogenic variants have been validated with Sanger sequencing. Results Sequencing revealed two ANOS1 and four FGFR1 mutations in six subjects, of which five are novel and one had been previously reported in CHH. Novel variants include a single base pair deletion c.313delT in exon 3 of ANOS1, three missense variants of FGFR1 predicted to result in the single amino acid substitutions c.331C > T (p.R111C), c.1964 T > C (p.L655P) and c.2167G > A (p.E723K) and a 15 bp deletion c.374_388delTGCCCGCAGACTCCG in exon 4 of FGFR1. Based on ACMG–AMP criteria reported variants were assigned to class 5, pathogenic or class 4, likely pathogenic. Protein structural predictions, the rarity of novel variants and amino acid conservation in case of missense substitutions all provide strong evidence that these mutations are highly likely to be deleterious. Conclusions Despite the fact that ANOS1 and FGFR1 are classical CHH genes and were thoroughly explored in several CHH cohorts we identified new, yet undescribed variants within their sequence. Our results support the genetic complexity of the disorder. The knowledge of the full genetic spectrum of CHH is increasingly important in order to be able to deliver the best personalised medical care to our patients.

Background Gestational diabetes mellitus (GDM) has a high prevalence in the period of pregnancy. However, the lack of gold standards in current screening and diagnostic methods posed the biggest limitation. Regulation of gene expression caused by DNA methylation plays an important role in metabolic diseases. In this study, we aimed to screen GDM diagnostic markers, and establish a diagnostic model for predicting GDM. Methods First, we acquired data of DNA methylation and gene expression in GDM samples (N = 41) and normal samples (N = 41) from the Gene Expression Omnibus (GEO) database. After pre-processing the data, linear models were used to identify differentially expressed genes (DEGs). Then we performed pathway enrichment analysis to extract relationships among genes from pathways, construct pathway networks, and further analyzed the relationship between gene expression and methylation of promoter regions. We screened for genes which are significantly negatively correlated with methylation and established mRNA-mRNA-CpGs network. The network topology was further analyzed to screen hub genes which were recognized as robust GDM biomarkers. Finally, the samples were randomly divided into training set (N = 28) and internal verification set (N = 27), and the support vector machine (SVM) ten-fold cross-validation method was used to establish a diagnostic classifier, which verified on internal and external data sets. Results In this study, we identified 465 significant DEGs. Functional enrichment analysis revealed that these genes were associated with Type I diabetes mellitus and immunization. And we constructed an interactional network including 1091 genes by using the regulatory relationships of all 30 enriched pathways. 184 epigenetics regulated genes were screened by analyzing the relationship between gene expression and promoter regions’ methylation in the network. Moreover, the accuracy rate in the training data set was increased up to 96.3, and 82.1% in the internal validation set, and 97.3% in external validation data sets after establishing diagnostic classifiers which were performed by analyzing the gene expression profiles of obtained 10 hub genes from this network, combined with SVM. Conclusions This study provided new features for the diagnosis of GDM and may contribute to the diagnosis and personalized treatment of GDM.

Background This article describes the research trends in sperm DNA fragmentation (SDF) over the past 20 years (1999–2018) using a scientometric approach. Methods A stepwise approach was adopted to retrieve scientometric data (articles per year, authors, affiliations, journals, countries) from Scopus and analyze the publication pattern of SDF with reference to key areas of research in the field of Andrology. Results A total of 2121 articles were retrieved related to SDF. Our data revealed an increasing research trend in SDF (n = 33 to n = 173) over the past 20 years (R2 = 0.894). Most productive country in publications was the USA (n = 450), while Agarwal A. (n = 129) being the most productive author. Most of the articles in SDF were primarily focused on lifestyle (n = 157), asthenozoospermia (n = 135) and varicocele (130). Mechanistic studies on SDF were published twice as much as prognostic/diagnostic studies, with significant emphasis on oxidative stress. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was the most widely used technique to evaluate SDF. Publications on SDF related to assisted reproductive techniques also showed a linear increasing trend (R2 = 0.933). Conclusions Our analysis revealed an increasing trend in SDF publications predominantly investigating lifestyle, asthenozoospermia and varicocele conditions with TUNEL being the most widely used technique. A substantial increase in research is warranted to establish SDF as prognostic/diagnostic parameter to evaluate clinical scenarios and ART outcomes.