Comprehensive analysis of genomic complexity in the 5' end coding region of the DMD gene in patients of exons 1-2 duplications based on long-read sequencing.

BACKGROUND: Dystrophinopathies are the most common X-linked inherited muscle diseases, and the disease-causing gene is DMD. Exonic duplications are a common type of pathogenic variants in the DMD gene, however, 5' end exonic duplications containing exon 1 are less common. When assessing the pathogenicity of exonic duplications in the DMD gene, consideration must be given to their impact on the reading frame. Traditional molecular methods, such as multiplex ligation-dependent probe amplification (MLPA) and next-generation sequencing (NGS), are commonly used in clinics. However, they cannot discriminate the precise physical locations of breakpoints and structural features of genomic rearrangement. Long-read sequencing (LRS) can effectively overcome this limitation. RESULTS: We used LRS technology to perform whole genome sequencing on three families and analyze the structural variations of the DMD gene, which involves the duplications of exon 1 and/or exon 2. Two distinct variant types encompassing exon 1 in the DMD Dp427m isoform and/or Dp427c isoform are identified, which have been infrequently reported previously. In pedigree 1, the male individuals harboring duplication variant of consecutive exons 1-2 in the DMD canonical transcript (Dp427m) and exon 1 in the Dp427c transcript are normal, indicating the variant is likely benign. In pedigree 3, the patient carries complex SVs involving exon 1 of the DMD Dp427c transcript showing an obvious phenotype. The locations of the breakpoints and the characteristics of structural variants (SVs) are identified by LRS, enabling the classification of the variants' pathogenicity. CONCLUSIONS: Our research sheds light on the complexity of DMD variants encompassing Dp427c/Dp427m promoter regions and emphasizes the importance of cautious interpretation when assessing the pathogenicity of DMD 5' end exonic duplications, particularly in carrier screening scenarios without an affected proband.

Single-Cell Quantitative Proteomic Analysis of Human Oocyte Maturation Revealed High Heterogeneity in In Vitro-Matured Oocytes.

Oocyte maturation is pertinent to the success of in vitro maturation (IVM), which is used to overcome female infertility, and produced over 5000 live births worldwide. However, the quality of human IVM oocytes has not been investigated at single-cell proteome level. Here, we quantified 2094 proteins in human oocytes during in vitro and in vivo maturation (IVO) by single-cell proteomic analysis and identified 176 differential proteins between IVO and germinal vesicle oocytes and 45 between IVM and IVO oocytes including maternal effect proteins, with potential contribution to the clinically observed decreased fertilization, implantation, and birth rates using human IVM oocytes. IVM and IVO oocytes showed separate clusters in principal component analysis, with higher inter-cell variability among IVM oocytes, and have little correlation between mRNA and protein changes during maturation. The patients with the most aberrantly expressed proteins in IVM oocytes had the lowest level of estradiol per mature follicle on trigger day. Our data provide a rich resource to evaluate effect of IVM on oocyte quality and study mechanism of oocyte maturation.

Developing a Predictive Model for Minimal or Mild Endometriosis as a Clinical Screening Tool in Infertile Women: Uterosacral Tenderness as a Key Predictor.

STUDY OBJECTIVE: To develop a noninvasive predictive model based on patients with infertility for identifying minimal or mild endometriosis. DESIGN: A retrospective cohort study. SETTING: This study was conducted at a tertiary referral center. PATIENTS: A total of consecutive 1365 patients with infertility who underwent laparoscopy between January 2013 and August 2020 were divided into a training set (n = 910) for developing the predictive model and a validation set (n = 455) to confirm the model's prediction efficiency. The patients were randomly assigned in a 2:1 ratio. INTERVENTIONS: Sensitivities, specificities, area under the curve, the Hosmer-Lemeshow goodness of fit test, Net Reclassification Improvement index, and Integrated Discrimination Improvement index were evaluated in the training set to select the optimum model. In the validation set, the model's discriminations, calibrations, and clinical use were tested for validation. MEASUREMENTS AND MAIN RESULTS: In the training set, there were 587 patients with minimal or mild endometriosis and 323 patients without endometriosis. The combination of clinical parameters in the model was evaluated for both statistical and clinical significance. The best-performing model ultimately included body mass index, dysmenorrhea, dyspareunia, uterosacral tenderness, and serum cancer antigen 125 (CA-125). The nomogram based on this model demonstrated sensitivities of 87.7% and 93.3%, specificities of 68.6% and 66.4%, and area under the curve of 0.84 (95% confidence interval 0.81-0.87) and 0.85 (95% confidence interval 0.80-0.89) for the training and validation sets, respectively. Calibration curves and decision curve analyses also indicated that the model had good calibration and clinical value. Uterosacral tenderness emerged as the most valuable predictor. CONCLUSION: This study successfully developed a predictive model with high accuracy in identifying infertile women with minimal or mild endometriosis based on clinical characteristics, signs, and cost-effective blood tests. This model would assist clinicians in screening infertile women for minimal or mild endometriosis, thereby facilitating early diagnosis and treatment.

Bi-allelic Missense Pathogenic Variants in TRIP13 Cause Female Infertility Characterized by Oocyte Maturation Arrest.

Normal oocyte meiosis is a prerequisite for successful human reproduction, and abnormalities in the process will result in infertility. In 2016, we identified mutations in TUBB8 as responsible for human oocyte meiotic arrest. However, the underlying genetic factors for most affected individuals remain unknown. TRIP13, encoding an AAA-ATPase, is a key component of the spindle assembly checkpoint, and recurrent homozygous nonsense variants and a splicing variant in TRIP13 are reported to cause Wilms tumors in children. In this study, we identified homozygous and compound heterozygous missense pathogenic variants in TRIP13 responsible for female infertility mainly characterized by oocyte meiotic arrest in five individuals from four independent families. Individuals from three families suffered from oocyte maturation arrest, whereas the individual from the fourth family had abnormal zygote cleavage. All displayed only the infertility phenotype without Wilms tumors or any other abnormalities. In vitro and in vivo studies showed that the identified variants reduced the protein abundance of TRIP13 and caused its downstream molecule, HORMAD2, to accumulate in HeLa cells and in proband-derived lymphoblastoid cells. The chromosome mis-segregation assay showed that variants did not have any effects on mitosis. Injecting TRIP13 cRNA into oocytes from one affected individual was able to rescue the phenotype, which has implications for future therapeutic treatments. This study reports pathogenic variants in TRIP13 responsible for oocyte meiotic arrest, and it highlights the pivotal but different roles of TRIP13 in meiosis and mitosis. These findings also indicate that different dosage effects of mutant TRIP13 might result in two distinct human diseases.

Mutations in PLCZ1 induce male infertility associated with polyspermy and fertilization failure.

PURPOSE: To investigate the genetic causes of polyspermy and total fertilization failure (TFF) in two independent male patients suffering from male infertility. METHODS: Immunofluorescence (IF) staining was used to detect the localization of the PLCζ protein in sperm and the maternal pronucleus in the zygote. Genomic DNA samples were extracted from the peripheral blood of patients and their families. The ExAC database was used to identify the frequency of corresponding mutations. The PLCZ1 mutations were validated by Sanger sequencing. The pathogenicity of the identified mutations and their possible effects on the protein were assessed using in silico tools and molecular modeling. RESULTS: We identified a reported homozygous mutation c.588C > A (p.Cys196Ter) and a compound heterozygous mutation c.2 T > C(p.Met1Thr)/c.590G > A (p.Arg197His) with one novel mutation in PLCZ1. The IF results showed that these multipronuclear zygotes formed as a result of polyspermy. In silico analysis predicted that the mutations result in disease-causing proteins. IF staining revealed that PLCζ is abnormally localized in the sperm samples from the two affected patients. Assisted oocyte activation (AOA) successfully rescued polyspermy and TFF and achieved pregnancy in two patients with the PLCZ1 mutation. CONCLUSION: We identified a homozygous mutation in PLCZ1 (c.588C > A [p.Cys196Ter]) in a male patient with polyspermy after in vitro fertilization (IVF) as well as a compound heterozygous mutation c.2 T > C(p.Met1Thr)/c.590G > A (p.Arg197His) with one novel mutation in a male patient with fertilization failure after intracytoplasmic sperm injection (ICSI), and we provide evidence that the homozygous mutation can cause polyspermy and the compound heterozygous mutation can cause fertilization failure.

[Effect of complete Y chromosome AZFc microdeletion on embryo euploidy].

OBJECTIVE: Preimplantation genetic testing (PGT) was performed to analyze the embryo euploidy in patients with complete Y chromosome AZFc microdeletion. METHODS: The clinical data of complete AZFc microdeletion underwent PGT from January 2013 to December 2021 in Reproductive Medicine Center of the First Affiliated Hospital of Nanjing Medical University were retrospectively analyzed. The patients with monogenic disease who underwent PGT during the same period were set as the control group. The basic characteristics, fertilization rate, Day 3 high quality embryo rate, blastocyst formation rate, embryo euploid rate, 45, X embryo ratio was compared between the two groups. RESULTS: A total of 220 patients were included, including 91 patients with complete AZFc microdeletion and 129 patients with monogenic disease. There was no significant difference in age between the two groups. In semen parameters, the sperm concentration, total sperm count and progressive motility in AZFc microdeletion group were lower than those in monogenic disease group, and the differences were statistically significant (P=0.001). The fertilization rate of AZFc microdeletion group was lower than that in monogenic disease group (P=0.012), and there was no significant difference in the number of MII oocytes, Day 3 high-quality embryo rate and blastocyst formation rate. A total of 933 blastocysts were successfully tested, including 496 blastocysts in AZFc deletion group and 437 blastocysts in monogenic disease group. The euploid, aneuploid and mosaic rates of the AZFc microdeletion group were 57.26%, 24.60% and 18.14%, respectively, while those of the monogenic disease group were 66.13%, 23.80% and 10.07%, with statistically significant differences between the two groups (P=0.001). Further analysis of the two groups of aneuploid embryos showed that aberrations occurred most commonly in chromosome16 (3.87%), X (3.46%), 13 (2.44%), 22 (2.24%) and 19 (2.03%) in AZFc microdeletion group, respectively, while the monogenic disease group was 22 (4.35%), 16 (2.97%), 7 (2.74%), 15(1.60%) and 2(1.60%), respectively. The proportion of sex chromosome abnormality in AZFc microdeletion group was higher than that in monogenic disease group (P=0.039), and there was no significant difference in the proportion of 45,X between the two groups. CONCLUSION: Compared with monogenic disease group, The embryo euploid rate in AZFc microdeletion patients decreased and the proportion of 45, X embryos did not increase significantly. It is recommended to select euploid female embryos by PGT, which not only avoids vertical transmission of AZFc microdeletion, but also reduces the risk of miscarriage due to aneuploid embryos.

Peroxiredoxin 4 secreted by cumulus cells ameliorates the maturation of oocytes in vitro.

BACKGROUND: Peroxiredoxin 4 (Prdx4) in the endoplasmic reticulum (ER) is the only secretory member of the antioxidant Prdx family. Our previous studies demonstrated that Prdx4 in cumulus cells (CCs) ameliorated the maturation of oocytes in vitro and enhanced oocyte developmental competence by preventing CCs apoptosis caused by oxidative stress (OS) through gap junctions. In this study, we aimed to determine whether Prdx4 released by CCs can repair meiotic defects in mouse oocytes by co-culturing immature (germinal vesicle) oocytes with CCs from mature oocytes in the absence of gap junctions. RESULTS: The OS-induced meiotic defects in mouse oocytes were impeded by co-culture with CCs, as evidenced by the increased first polar body (PB1) extrusion rate and decreased ROS level. CCs increased Prdx4 expression and lowered IRE1α, Bip expression in H2O2-treated oocytes. After knockdown of Prdx4 expression in CCs, the rate of PB1 extrusion in the oocytes was significantly reduced to the level detected in H2O2 group, and ER stress was not alleviated. CO-IP and immunofluorescence co-localization experiments demonstrated that Prdx4 interacted with PDIA6 in the oocytes and the Pearson's R value was 0.69 calculated using ImageJ. CONCLUSIONS: Cumulus cells can promote the maturation of oocytes in vitro by secreting Prdx4 in a paracrine manner and serve as a promising therapeutic antioxidant for improving the quality of oocytes, especially aging oocytes, in clinical in vitro maturation (IVM).

Nucleoporin37 may play a role in early embryo development in human and mice.

Maternal-effect genes (MEGs) play an important role in maintaining the survival and development of mammalian embryos at the cleavage stage after fertilization. Despite long-term efforts, the MEGs that regulate preimplantation embryo development remain largely unknown. Here, using whole-exome sequencing and homozygosity mapping, we identified a potential candidate gene associated with early embryo development: nucleoporin37 (NUP37), a nucleoporin gene that encodes a member of the nuclear pore complexes and regulates nuclear pore permeability and nucleocytoplasmic transport. Moreover, we determined the temporal and spatial expression patterns of Nup37 in mouse oocytes and early embryos, and explored the role of NUP37 in oocyte maturation and preimplantation embryo development. Immunoprecipitation assays confirmed that yes-associated protein-1 (YAP1) binds to TEA domain transcription factor 4 (TEAD4) and NUP37. Furthermore, Nup37 gene knockdown reduced the nuclear import of YAP1 and down-regulated the expression of YAP1-TEAD pathway downstream genes Rrm2 and Rpl13 in early embryos. Our study provides evidence that maternal NUP37 contributes to the nuclear import of YAP1 and then activates the YAP1-TEAD pathway, a signalling pathway essential for zygotic genome activation. Nup37 may be a key gene involved in preimplantation embryo development in mammals.

Novel biallelic mutations in PADI6 in patients with early embryonic arrest.

Peptidyl arginine deiminase, type VI (PADI6) is a member of the subcortical maternal complex (SCMC), which plays vital roles in mammalian embryogenesis. Most mutations in SCMC members have been reported to cause human embryonic arrest, and a total of 15 mutations in PADI6 have been shown to be responsible for early embryonic arrest according to previous studies. However, the genetic factors behind this phenotype remain to be understood in further detail. Here, we identified 13 novel mutations and 4 previously reported mutations of PADI6 in 14 patients who were diagnosed with abnormal embryonic development caused by early arrest, embryonic fragmentation, and recurrent implantation failure. Most of the mutations were predicted by in silico analysis to be deleterious or damaging to the function of PADI6. In addition, the total and East Asian population frequencies of the mutations were low or absent in the gnomAD database. Our study expands the mutational spectrum in PADI6 and will provide precise targets for genetic counseling in the future.

Androgen Excess Increases Food Intake in a Rat Polycystic Ovary Syndrome Model by Downregulating Hypothalamus Insulin and Leptin Signaling Pathways Preceding Weight Gain.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a common reproductive and metabolic disorder characterized by high androgen levels. The aim of this study was to evaluate the effects of hyperandrogenism on the hypothalamus and subsequently on the food intake and obesity in females. METHODS: A dihydroxy testosterone (DHT)-induced rat model was established to recapitulate the hyperandrogenism features of PCOS patients. Body weight and food intake of the rats were recorded. The food intake of DHT-induced rats was restricted by pair feeding to exclude possible effects of weight gain on the hypothalamus. The expression levels of relevant proteins and mRNAs in the hypothalamus and primary hypothalamic neurons exposed to DHT were analyzed by Western blotting and RT-PCR, respectively. The leptin levels in the serum and cerebrospinal fluid (CSF) were measured, and leptin was injected via the intracerebroventricular (ICV) route to test the leptin sensitivity of the hypothalamus. RESULTS: The excessive prepuberty androgen levels in the DHT-induced rats markedly elevated food intake prior to weight gain. Consistent with this, the expression of neuropeptide Y and agouti-related peptide mRNAs was upregulated, which occurred prior to obesity and even with restricted food intake. In addition, the hypothalamic sensitivity to insulin and leptin was also impaired in the DHT-induced rats before obesity and with restricted food intake. DHT significantly reduced the leptin levels in the CSF, and ICV injection of leptin inhibited the DHT-induced increase in food intake. CONCLUSIONS: Androgen excess increased food intake in rats and promoted obesity by downregulating insulin and leptin signaling in the hypothalamus, most likely by suppressing leptin levels in the CSF.

Associations between antenatal corticosteroid exposure and neurodevelopment in infants.

BACKGROUND: It has been well recognized that antenatal administration of dexamethasone to pregnant women at risk of preterm delivery may markedly accelerate fetal maturation and reduce the risk of adverse perinatal outcomes in their preterm infants, particularly for births before 34 weeks of gestation. Since 2015, antenatal corticosteroid administration has been extended beyond 34 weeks of gestation by clinical guidelines, as it might have beneficial effects on fetal maturation and perinatal outcomes. However, concerns regarding the potential influence of antenatal corticosteroid treatment on offspring neurodevelopment have been raised. OBJECTIVE: This study aimed to investigate whether maternal antenatal corticosteroid administration was associated with neurodevelopment in infants at 1 year of age. STUDY DESIGN: In this prospective and longitudinal birth cohort study, women were followed up throughout gestation, and their infants underwent a Bayley Scales of Infant and Toddler Development, Third Edition, screening test at 1 year of age between December 2018 and September 2020. Finally, 1609 pregnant women and 1759 infants were included in the current study. Using a generalized linear mixed model, we examined the association between antenatal corticosteroid exposure and infant neurodevelopment in cognitive, receptive communication, expressive communication, fine motor, and gross motor functions. RESULTS: Of the 1759 infants eligible for this study, 1453 (82.6%) were singletons. A total of 710 infants were exposed to antenatal corticosteroids, among whom 415 were dexamethasone exposed and 483 were prednisone exposed. Dexamethasone was prescribed most often in late pregnancy, whereas prednisone was often used before 8 weeks of gestation among women who conceived through assisted reproductive technology. Compared with those who had no exposure, antenatal corticosteroid exposure was associated with an increased risk of infants being noncompetent in the cognitive development domain after adjusting for conventional risk factors (adjusted risk ratio, 1.53; 95% confidence interval, 1.08-2.18; P=.017). For medication-specific exposure, those exposed vs not exposed to antenatal dexamethasone were 1.62-fold (95% confidence interval, 1.10-2.38; P=.014) more likely to be noncompetent in the cognitive development domain at 1 year. The association did not vary markedly between preterm and term infants, singletons and twins, or assisted reproductive technology-conceived and spontaneously conceived infants (all P>.05 for heterogeneity). In contrast, a null association was observed for the risk of being noncompetent in any domain of neurodevelopment with antenatal prednisone exposure at early pregnancy. CONCLUSION: Here, antenatal corticosteroid, particularly dexamethasone exposure, was markedly associated with an increased risk of infants being noncompetent in the cognitive development domain at 1 year of age. These findings may provide new information when weighing the benefits and potential risks of maternal antenatal corticosteroid administration.

[Analysis of three Chinese pedigrees affected with recurrent hydatidiform mole due to variants of NLRP7 gene].

OBJECTIVE: To explore the genetic etiology of recurrent hydatidiform mole (RHM) and provide accurate guidance for reproduction. METHODS: Peripheral venous blood samples of the probands with RHM and members from 5 unrelated pedigrees were collected. Genomic DNA was extracted by using routine method, and whole exome sequencing was carried out to detect variants of RHM-associated genes including NLRP7 and KHDC3L. Sanger sequencing and real-time quantitative PCR (RT-qPCR) were used to validate the candidate variants and delineate their parental origin. RESULTS: Homozygous or compound heterozygous variants of the NLRP7 gene were identified in four patients from three pedigrees, which included a homozygous deletion of exon 1 to 4 of NLRP7 in patient P1 and her elder sister, compound heterozygous variants of NLRP7 c.939delG (p.Q314Sfs*6) pat and c.1533delG (p.N512Tfs*4) mat in patient P2, and compound heterozygous variants of NLRP7 c.2389_2390delTC (p.A798Qfs*6) pat and c.2165A>G (p.D722G) mat in patient P4. All variants were interpreted as pathogenic or likely pathogenic according to the American College of Medical and Genomics (ACMG) guidelines. Among these, NLRP7 exons 1 to 4 deletion, c.939delG (p.Q314Sfs*6), c.1533delG (p.N512Tfs*4) and c.2389_2390delTC (p.A798Qfs*6) were unreported previously. CONCLUSION: Variants of the NLRP7 gene probably underlay autosomal recessive RHM in the three pedigrees, and definitive molecular diagnosis is beneficial for accurate genetic counseling. Above finding has also enriched the spectrum of the NLRP7 variants underlying RHM.

Prediction of live birth probability after in vitro fertilization and intracytoplasmic sperm injection treatment: A multi-center retrospective study in Chinese population.

AIM: To develop a prediction model to estimate the chances of live birth over multiple cycles of in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) treatment. METHODS: A retrospective cohort study was launched in three reproductive centers including 10 824 couples who received 14 106 treatment cycles with known pregnancy outcomes by the end of 2016. Discrete time logistic regression was used to establish the model and a nomogram was developed to predict the chance of live birth on plain paper-based final predictors. RESULTS: Among 10 824 couples, 5809 (53.7%) ended up with a live birth with several successive transplant cycles. What's more, we found that younger female age (p < 0.001), smaller cycle number (p < 0.001), female body mass index (p < 0.001), male factor (p < 0.001), ovulation disorder (p = 0.006), and higher endometrial thickness (p < 0.001) were significantly associated with increased live birth rate. Discrimination of the model expressed by area under the curve (AUC) was 0.66. CONCLUSION: Our study will help shape couples' expectations of their ART outcome, allowing them to plan their treatments more efficiently and prepare emotionally and financially.

Genetic etiologic analysis in 74 Chinese Han women with idiopathic premature ovarian insufficiency by combined molecular genetic testing.

PURPOSE: To identify the disease-causing genes of Chinese Han women with idiopathic premature ovarian insufficiency (POI). METHODS: Seventy-four Chinese Han women with idiopathic POI were collected to analyze the genetic etiology. Triplet repeat-primed polymerase chain reaction (TP-PCR) was performed to screen the FMR1 (CGG)n premutation, and then 60 POI-related genes were sequenced by targeted next-generation sequencing (NGS) in POI patients with normal FMR1. RESULTS: A total of one patient (1/74) with FMR1 premutation was identified. Targeted NGS revealed that 15.07% (11/73) patients had pathogenic or likely pathogenic variants of Mendelian genes (FOXL2, EIF2B2, CYP17A1, CLPP, MCM9, GDF9, MSH5, ERCC6, POLG). Ten novel variants in six Mendelian genes were identified, such as CLPP c.355A>C (p.I119L) and c.688A>C (p.M230L), MCM9 c.1157C>T (p.T386M) and c.1291A>G (p.M431V), GDF9 c. 238C>T (p.Q80X), MSH5 c.604G>C (p.G202R) and c.2063T>C (p.I688T), ERCC6 c.C1769C>T (p.P590L), POLG c.2832G>C (p.E944D), and c.2821A>G (p.I941V). CONCLUSION: This study suggested targeted NGS was an efficient etiologic test for idiopathic POI patients without FMR1 premutation and enriched the variant spectrum of POI-related genes.

Effect of endometrial thickness and embryo quality on live-birth rate of fresh IVF/ICSI cycles: a retrospective cohort study.

BACKGROUND: Successful implantation and delivery require both the functional embryo and receptive endometrium in assisted reproductive technology (ART) cycles. However, little is known about embryo-endometrial interaction on live-birth. We aimed to investigate the independent effect and interaction of endometrial thickness (EMT) and embryo quality on live-birth in fresh embryo transfer (ET) cycles. METHODS: We conducted a retrospective cohort study including 15,012 ART cycles between 2013 and 2016 in three centers in China. Poisson regression with generalized estimating equations was employed to calculate relative risks (RRs) and 95% confidence intervals (CIs). We estimated the interaction of embryo quality and EMT on live-birth rate (LBR). RESULTS: The LBR per cycle was 42.8% overall. LBR increased with increasing EMT and reached a plateau (50.6 to 54.2%) when EMT was 11 mm or thicker. Embryo quality represented by cumulative score was associated with LBR independently of number of embryos transferred and EMT. LBR was not increased with thicker EMT when only Q1 cleavage-stage embryo transferred (aRR 0.95, 95%CI 0.61-1.46). LBR was not increased significantly with thicker EMT with transfer of two good-quality cleavage-stage embryos and any blastocyst combination except Q1 group. There was significant interaction between EMT and embryo quality on LBR for cleavage-stage ETs (P=0.023). CONCLUSIONS: This study demonstrated the nonlinear EMT-LBR association and the EMT cut-off value of 11 mm which may be of more clinical significance for predicting live-birth. Embryo quality is an independent prognostic tool for LBR. Our finding of significant embryo-endometrial interaction indicates combination of EMT and embryos quality might improve the prognostic value in clinical practice for live-birth in patients undergoing transfer of 1-2 fresh cleavage-stage embryos.

Novel mutations in LHCGR (luteinizing hormone/choriogonadotropin receptor): expanding the spectrum of mutations responsible for human empty follicle syndrome.

PURPOSE: To screen novel mutations in LHCGR responsible for empty follicle syndrome and explore the pathological mechanism of mutations. METHODS: Four affected individuals diagnosed with infertility-associated anovulation or oligo-ovulation from three independent families were recruited. Sanger sequencing was used to identify the LHCGR mutations in affected individuals. Western blot was performed to evaluate the effects of mutations on LHCGR protein levels. Immunofluorescence was done to explore the effects of mutations on LHCGR subcellular localization. The ATP levels were measured to infer the functional effects of the mutations on LHCGR. RESULTS: In the present study, three novel biallelic mutations in LHCGR were identified in four affected individuals from three independent families with empty follicle syndrome or oligo-ovulation. All biallelic mutations were inherited from the proband of their parents. The western blot showed that the identified mutations decreased LHCGR protein level and altered the glycosylation pattern. The immunofluorescence showed an ectopic subcellular localization of LHCGR in cultured HeLa cells. Besides, the mutations in LHCGR also reduced the cellular ATP consumption. CONCLUSION: These findings confirm previous studies and expand the mutational spectrum of LHCGR, which will provide genetic diagnostic marker for patients with empty follicle syndrome.

Histone methyltransferase SETD2 is required for meiotic maturation in mouse oocyte.

SET-domain-containing 2 (SETD2), a member of the histone lysine methyltransferase family, has been reported to be involved in multiple biological processes. However, the function of SETD2 during oocyte maturation has not been addressed. In this study, we find that mouse oocytes are incapable of progressing through meiosis completely once SETD2 is specifically depleted. These oocytes present an abnormal spindle morphology and deficient chromosome movement, with disrupted kinetochore-microtubule attachments, consequently producing aneuploidy eggs. In line with this, the BubR1 signal is markedly elevated in metaphase kinetochores of oocytes with SETD2 depletion, indicative of the activation of spindle assembly checkpoint. In addition, we note that loss of SETD2 results in a drastic decrease in the trimethylation level of H3K36 in oocytes. Collectively, our data demonstrate that SETD2 is required for oocyte maturation and indicate a novel mechanism controlling the meiotic apparatus.

SET protein up-regulated testosterone production in the cultured preantral follicles.

BACKGROUND: We found previously that the expression of SET gene was up-regulated in polycystic ovaries. Evidences suggested that SET protein was essential for regulating both the promoter activity of CYP17A1 and the biological activity of P450c17. In this study, we explored whether SET regulated androgen production in preantral follicles. METHODS: The mouse preantral follicles were cultured in vitro. Testosterone secretion and expression of steroidogenic enzymes were observed in the preantral follicles treated in vitro by SET overexpression and knockdown. RESULTS: Testosterone levels in the media of the AdCMV-SET infected follicles significantly increased, and the CYP17A1 and HSD3B2 expression also significantly increased (P < 0.05). Testosterone levels in AdSiRNA-SET infected group decreased, and so did CYP17A1 and HSD3B2 expression (P < 0.05). CONCLUSIONS: SET played a positive role in regulating ovarian androgen biosynthesis by enhancing the transcription of steroidogenic enzymes CYP17A1 and HSD3B2, which maybe contribute to the hyperandrogenism in PCOS.

The association between polycystic ovary syndrome and ectopic pregnancy after in vitro fertilization and embryo transfer.

OBJECTIVE: We sought to assess the association between polycystic ovary syndrome (PCOS) and ectopic pregnancy after in vitro fertilization-embryo transfer (ET). STUDY DESIGN: In this retrospective cohort study, we included 5339 women who had clinical pregnancies after in vitro fertilization treatment (PCOS, 205 women; non-PCOS, 5134 women) at Nanjing Medical University (China) between 2007 and 2011. Fresh and cryo-thawed ET cycles were analyzed respectively. The primary outcome measure was the occurrence of ectopic pregnancy. Multivariate logistic regression analysis was used to adjust for important confounders. RESULTS: In fresh ET cycles of women who were undergoing controlled ovarian hyperstimulation (COH; n = 3303), women with PCOS had 3.06 times higher risk of ectopic pregnancy compared with those without PCOS (7.0% vs 2.4%; adjusted odds ratio [aOR], 3.06; 95% confidence interval [CI], 1.34-6.96). In the stratified analysis, for women without PCOS, the high estradiol group (>4085 pg/mL) had higher ectopic pregnancy rates compared with the low estradiol group (≤4085 pg/mL; 3.4% vs 2.0%; aOR, 1.99; 95% CI, 1.19-3.35); however, for women with PCOS, both high and low estradiol groups had high ectopic pregnancy rates (5.6% vs 7.7%; aOR, 0.92; 95% CI, 0.15-5.67). In cryo-thawed ET cycles without COH (n = 2036), the ectopic rates between women with and without PCOS were similar (2.2% vs 2.0%; aOR, 0.94; 95% CI, 0.22-4.07). CONCLUSION: PCOS was associated with an increased risk of ectopic pregnancy after COH in fresh ET cycles, but not in cryo-thawed ET cycles. A possible explanation is that, compared with women without PCOS, women with PCOS appear to hold a lower threshold of hyperphysiologic estradiol level that triggers the occurrence of ectopic pregnancy after COH.