Vaginal and endometrial microbiome dysbiosis associated with adverse embryo transfer outcomes.

BACKGROUND: Assisted reproductive technology (ART) is the most effective method to treat infertility and the pathogenesis of implantation failure after in vitro fertilization-embryo transfer (IVF-ET) is a challenging filed in infertility. Microbes in the female reproductive tract are considered to be associated with gynecological and obstetric diseases. However, its effects on embryo implantation failure are unsured. PURPOSE: This study aimed to investigate reproductive tract dysbiosis, identify different bacteria in reproductive tract as potential biomarkers of embryo implantation failure and demonstrate the pathogenesis through metabolites analysis. METHODS: We compared the data from 16S rRNA gene and metagenome in reproductive tracts through QIIME2 and HUMAnN2 by the times of embryo implantation failure on 239 infertile patients and 17 healthy women. RESULTS: Our study revealed a strong positive correlation between Lactobacillus abundance and embryo implantation success (IS) after IVF-ET. The microbial community composition and structure in reproductive tract showed substantially difference between the embryo implantation failure (IF) and healthy control. Moreover, we established a diagnostic model through receiver operating characteristic (ROC) with 0.913 area under curve (AUC) in IS and multiple implantation failures (MIF), verified its effectiveness with an AUC = 0.784 demonstrating microbial community alterations could efficiently discriminate MIF patients. Metagenome functional analyses of vaginal samples from another independent infertile patients after IVF-ET revealed the L-lysine synthesis pathway enriched in IF patients, along with ascended vaginal pH and decreased Lactobacillus abundance. CONCLUSIONS: This study clarifies several independent relationships of bacteria in vagina and endometrial fluid on embryo implantation failure and undoubtedly broadens the understanding about female reproductive health.

Effects of organochlorine pesticides on human and rat 17ß-hydroxysteroid dehydrogenase 1 activity: Structure-activity relationship and in silico docking analysis.

The objective of this study was to examine the effect of 11 organochlorine pesticides on human and rat 17ß-Hydroxysteroid dehydrogenase 1 (17ß-HSD1) in human placental and rat ovarian microsome and on estradiol production in BeWo cells. The results showed that the IC50 values for endosulfan, fenhexamid, chlordecone, and rhothane on human 17ß-HSD1 were 21.37, 73.25, 92.80, and 117.69 µM. Kinetic analysis revealed that endosulfan acts as a competitive inhibitor, fenhexamid as a mixed/competitive inhibitor, chlordecone and rhothane as a mixed/uncompetitive inhibitor. In BeWo cells, all insecticides except endosulfan significantly decreased estradiol production at 100 µM. For rats, the IC50 values for dimethomorph, fenhexamid, and chlordecone were 11.98, 36.92, and 109.14 µM. Dimethomorph acts as a mixed inhibitor, while fenhexamid acts as a mixed/competitive inhibitor. Docking analysis revealed that endosulfan and fenhexamid bind to the steroid-binding site of human 17ß-HSD1. On the other hand, chlordecone and rhothane binds to a different site other than the steroid and NADPH-binding site. Dimethomorph binds to the steroid/NADPH binding site, and fenhexamid binds to the steroid binding site of rat 17ß-HSD1. Bivariate correlation analysis showed a positive correlation between IC50 values and LogP for human 17ß-HSD1, while a slight negative correlation was observed between IC50 values and the number of HBA. ADMET analysis provided insights into the toxicokinetics and toxicity of organochlorine pesticides. In conclusion, this study identified the inhibitory effects of 3-4 organochlorine pesticides and binding mechanisms on human and rat 17ß-HSD1, as well as their impact on hormone production.

Embryo selection through non-invasive preimplantation genetic testing with cell-free DNA in spent culture media: a protocol for a multicentre, double-blind, randomised controlled trial.

INTRODUCTION: Morphological evaluation is used to select embryos for in vitro fertilisation. However, it does not fully reflect the implantation potential. Preimplantation genetic testing for aneuploidies (PGT-A) can detect embryonic aneuploidy, but biopsy procedure is invasive. Currently, a non-invasive PGT (ni-PGT) approach using spent medium is being evaluated. However, the clinical benefit of ni-PGT has not been clearly demonstrated. A multicentre randomised trial is needed to verify whether ni-PGT can be an new effective tool for evaluating embryos. METHODS AND ANALYSIS: Overall, 1148 couples aged 35~42 (women) receiving in vitro fertilization-intracytoplasmic sperm injection are planned to be enrolled. Couples will be digitally randomised to (1) ni-PGT and (2) conventional morphology groups at a 1:1 treatment ratio. The primary outcome will be the ongoing pregnancy rate related to the first transfer cycle within 6 months after oocyte retrieval. ETHICS AND DISSEMINATION: The study protocol is approved by the Ethics Committee of Peking University Third Hospital and the participating hospitals. The results will be disseminated through international conferences and scientific journals. TRIAL REGISTRATION NUMBER: NCT04339166.

Estradiol (E 2) Reduction Adversely Affect the Embryo Quality and Clinical Outcomes of In Vitro Fertilization and Embryo transfer (IVF-ET).

Objective: The purpose of this study was to explore the influence of decreased serum estradiol (E 2) levels during controlled ovarian hyperstimulation (COH) on in vitro fertilization and embryo transfer (IVF). Methods: The clinical data of 300 IVF-ET cycles with patients were analyzed retrospectively. According to the presence of falling E 2 level during the COH, we divided all subjects into two groups: the E 2 levels fall group (n = 120, group A) and the control group (n = 180, group B). In group A, there were 57 patients with falling E 2 with drug dosage reduction. The other 63 patients experienced the decreased E 2 level spontaneously. The clinical and laboratory variables in the groups were compared. Receiver operator characteristic (ROC) curve analyses were carried out in order to evaluate the predict value of E 2 level on the day of human chorionic gonadotropin (hCG) administration on IVF outcomes. Results: Duration and total dosage of gonadotropin (Gn) used were statistically more in group A than in group B (P < 0.001). The high-quality embryo rate was significantly lower in group A (P = 0.048). Women in group A had lower clinical pregnancy rate (P = 0.029), live birth rate (P < 0.001), ongoing pregnancy rate (P = 0.001), and higher early abortion rates (P = 0.008) than group B. Women with spontaneously falling E 2 group had a higher BMI index than those in the drug dosage reduction group (P = 0.001). More dosage and longer duration of Gn in spontaneously falling E 2 group than in the drug dosage reduction group (P < 0.01). There were no differences in clinical outcomes between the two types of E 2 decreased groups. Results from ROC showed an E 2 level <1987.5 pg/ml on the hCG day might predict early abortion in this study. The sensitivity was 58.4% and the specificity was 78.9%. In addition, an E 2 level >2020 pg/ml on the hCG day might be an index to predict live birth. The sensitivity was 57.0% and the specificity was 61.7%. Conclusions: Reduction of E 2 during COH might adversely affect the clinical pregnancy, early abortion, and ongoing pregnancy of IVF-ET.

Comparative study on risk of birth defects in singleton ART birth under high levels of estrogen after fresh embryo transfer and frozen embryo transfer.

OBJECTIVE: To investigate whether high estrogen (E2) levels caused by controlled ovarian hyperstimulation affect the birth defect rate in singleton assisted reproductive technology (ART) birth after conceived by fresh embryo transfer and frozen embryo transfer (FET). METHODS: This was a retrospective cohort study. A total of 581 women with singletons, as well as those who have become pregnant and have had an unwanted abortion under high E2 levels on trigger day were divided into three groups. Group A received FET and the E2 levels on trigger day were higher than 5000 pg/ml. Group B received fresh embryo transfer and the E2 levels were between 3000 and 5000 pg/ml. Group C received FET and the E2 levels were between 3000 and 5000 pg/ml. RESULTS: There were no significant differences in birth weight, delivery mode, preterm birth rate, and fetal sex between the three groups (p > .05). Birth defect rate in Group B was higher than that in Group A and C, and the rate between Group B and C had significant differences (p < .05). After adjusting for maternal age, BMI, and type of infertility, only a FET cycle is significantly associated with decreased birth defect rate. CONCLUSION: Fresh embryo transfer under supraphysiological level of estrogen exposure may increase the birth defect rate of ART singletons. Even after prenatal screening and diagnosis, a part of birth defect could not be detected during pregnancy. When the estrogen levels on trigger day were no lower than 3000 pg/ml, FET should be advocated to reduce the occurrence of such risk.

The feasibility of switching from IVF to IVM combined with all-blastocyst-culture and transfer for patients with ovarian hyperstimulation syndrome tendency.

OBJECTIVE: To investigate the feasibility of switching from in vitro fertilization (IVF) to in vitro maturation (IVM) combined with all-blastocyst-culture and transfer as a supplementary infertility treatment in patients with ovarian hyperstimulation syndrome (OHSS) tendency METHODS: Retrospective cohort study including 184 patients who switched from IVF and underwent 192 IVM cycles between January 2016 and December 2020. The outcomes were compared between cleavage-stage embryo transfer (group A, n = 74) and blastocyst-stage transfer (group B, n = 52) groups. RESULTS: The OHSS rate is 0%. 66 cycles were canceled for transfer. Among the 126 transfer cycles, number of retrieved oocytes, proportion of metaphase II oocytes, cleavage rate, and proportion of high-quality embryos on day 3 post-fertilization are significantly lower in group A than that in group B. On the contrary, number of transferred embryos is significantly lower in group B than that in group A, whereas the rates of implantation, clinical pregnancy, and live births are significantly higher in group B than that in group A. CONCLUSION: Timely switching to IVM combined with all-blastocyst-culture and transfer for patients undergoing controlled ovarian hyperstimulation and exhibiting characteristics of OHSS tendency is feasible as a supplementary infertility treatment.

Two high-fidelity variants: efSaCas9 and SaCas9-HF, which one is better?

CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated endonuclease Cas9) nucleases have been widely applied for genome engineering. Staphylococcus aureus Cas9 (SaCas9) is compact, which can be packaged in AAV (adeno-associated virus) vector for in vivo gene editing. While, wild-type SaCas9 can induce unwanted off-target mutations and substantially limits the applications. So far, there are two reported SaCas9 variants with high-fidelity, including efSaCas9 from our previous study and SaCas9-HF. However, it remains unknown which one possessing the better fidelity and higher activity. Here, we performed a parallel comparison of efSaCas9 and SaCas9-HF in human cells through fluorescent reporter system and target deep sequencing, respectively. The results demonstrated that efSaCas9 possesses higher cleavage activity and fidelity than SaCas9-HF at the most endogenous sites in human cells. Collectively, our study provides insights for the rational selection of suitable SaCas9 for human genome editing.

A patient with severe congenital neutropenia harbors a missense ELANE mutation due to paternal germline mosaicism.

BACKGROUND: Clinical and genetic characteristics of ELANE mutation of a 3-year-old male who had a severe congenital neutropenia (SCN) were examined. We then investigated whether CRISPR/Cas9-mediated gene editing could correct the mutation. PROCEDURE: The proband underwent extensive clinical assessments, such as exome sequencing and bioinformatics analysis, so that pathogenic genes could be identified. Sanger sequencing was also utilized for confirmation. The cell line, 293-ELANE, harboring ELANE mutation was generated, and the mutation was then corrected by CRISPR/Cas9-mediated homology-directed repair (HDR). RESULTS: The ELANE gene test in the proband unveiled a heterozygous de novo missense mutation: c. 248T > A (p.V83D), which was not detected in his asymptomatic parents who had provided peripheral blood samples. We found that 46.01% of his father's sperm cells had the same mutation. These results demonstrate that the proband inherited the ELANE mutation from his father, who had an average neutrophil count but had a germline mosaicism. The highest repair efficiency of CRISPR/Cas9-mediated HDR for 293-ELANE is 4.43%. CONCLUSIONS: We identified a missense mutation (p.V83D) in ELANE that causes SCN. This is the first report on paternal semen mosaicism of an ELANE mutation. Our study paves the way for preimplantation genetic diagnosis (PGD) based on ELANE mutation prevention and clinical treatment of congenital disabilities.

Prolonged pituitary downregulation with gonadotropin-releasing hormone agonist improves the live-birth rate: a retrospective cohort study comparing 3 different protocols.

BACKGROUND: The long protocol has been recognized as the gold standard in controlled ovarian hyperstimulation (COH). However, the full dose of gonadotropin-releasing hormone agonist (GnRH-a) under the prolonged protocol has become increasingly popular in China. This study sought to compare pregnancy outcomes among the following 3 groups: a long protocol group, and 2 types of improved prolonged protocol groups. METHODS: A retrospective cohort study was conducted of 550 patients undergoing fresh embryo transfer (ET). Patients were treated either with the improved prolonged protocol in the follicular phase (Group 1; n=288) or the mid-luteal phase (Group 2; n=143), or the long protocol (Group 3; n=119). The clinical and laboratory outcomes of the 3 groups were compared. RESULTS: The general characteristics of the women in the 3 groups were comparable. On the day on which gonadotropin (Gn) was first administered and on the day on which human chorionic gonadotropin (hCG) was administered, the luteinizing hormone (LH) levels of patients in both Groups 1 and 2 were lower than those of patients in Group 3. The number of oocytes retrieved, fertilized, and cleaved, and the number of high-quality embryos in the 3 procedures were similar. However, the number of transferred embryos, the rate of blastocyst progression, and the rate of implantation differed. The clinical pregnancy rates (CPRs)were significantly higher in the prolonged protocol groups (62.5% and 61.5%) than the long protocol group (48.7%). Further, statistically significant differences in the live-birth rates (LBRs) (56.9% vs. 57.3% vs. 42.9%) were observed. However, no differences in early abortion rates were found. CONCLUSIONS: As a result of pituitary downregulation with GnRH-a, the prolonged groups had better CPRs and LBRs than the long protocol group. The prolonged protocol in the mid-luteal phase was equally effective as that in the early follicular phase in fresh in-vitro fertilization (IVF)/intracytoplasmic sperm injection-embryo transfer (ICSI-ET) cycles. High LH levels on the day of hCG may be a predictor of adverse clinical outcomes.

Prednisone combined with letrozole reduced risk of ovarian hyperstimulation syndrome (OHSS) in women undergoing long-term gonadotropin-releasing hormone analog treatment.

BACKGROUND: Ovarian hyperstimulation syndrome (OHSS) is a severe disease that can lead to serious complication. Letrozole has been applied during controlled ovarian hyperstimulation (COH) to reduce the rate of OHSS in women undergoing long-term Gonadotropin-releasing Hormone Analog (GnRHa) treatment for assisted fertility. Prednisone can prevent vasodilatation and increased vascular permeability, which is common during OHSS. However, few studies have evaluated the combined effect of letrozole and prednisone in preventing severe OHSS and is the aim of our retrospective study of patients receiving GnRHa treatment. METHODS: A total of 296 women who accepted autologous in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) treatments were included in this retrospective study. There were three groups: 146 women had letrozole, including letrozole alone (LE group, n=60) and letrozole with prednisone (LE + Pre group, n=86), and 150 women had no treatment (C group). Severe OHSS was diagnosed according to clinical evidence of hydrothorax, severe dyspnea, oliguria/anuria, and intractable nausea/vomiting. RESULTS: The addition of prednisone to letrozole successfully reduced the occurrence rate of severe OHSS than those women administered letrozole alone (55.0% vs. 70.6%, P=0.022). However, the ongoing pregnancy rate was lower in the LE + Pre group than that in the LE-alone group (64.3% vs. 87.0%, P=0.025). Surprisingly, progesterone level on the trigger day (>0.895 ng/mL) is a strong predictor for pregnancy failure with a specificity of 68.3% and sensitivity of 65.7% in the LE-alone group. CONCLUSIONS: Treatment with a combination of letrozole and prednisone may lower the rate of severe OHSS in women with prolonged gonadotropin-releasing hormone agonist protocol during assisted fertility treatment. When the progesterone level on trigger day is over 0.895 ng/mL, letrozole treatment may negatively affect clinical pregnancy.

Effects of SLIRP on Sperm Motility and Oxidative Stress.

BACKGROUND: Deficient spermatozoon motility is one of the main causes of male infertility. However, there are still no accurate and effective treatments in a clinical setting for male asthenospermia. Exploring the genes and mechanism of asthenospermia has become one of the hot topics in reproductive medicine. Our aim is to study the effect of SLRIP on human spermatozoon motility and oxidative stress. METHODS: Sperm samples were collected including a normospermia group (60 cases) and an asthenospermia group (50 cases). SLIRP protein expression in spermatozoa was examined by western blotting, and relative mRNA expression of SLIRP in spermatozoa was quantified by reverse transcription polymerase chain reaction. Levels of reactive oxygen species (ROS), adenosine triphosphate (ATP) content, and the activity of manganese superoxide dismutase (MnSOD) in spermatozoa were also measured. RESULTS: The mRNA level and protein expression of SLIRP in the asthenospermia group were significantly reduced compared with those in the normospermia group. The ROS active oxygen level in the asthenospermia group significantly increased; however, the ATP content decreased significantly as well as the activity of MnSOD. CONCLUSION: SLIRP regulates human male fertility, and SLIRP and sperm progressive motility are positively correlated. The expression of SLIRP is declined, oxidative damage is increased, and energy metabolism is decreased in spermatozoa of asthenospermia patients compared to normospermia participants.

Prolonged pituitary down-regulation with full-dose of gonadotropin-releasing hormone agonist in different menstrual cycles: a retrospective cohort study.

BACKGROUND: The efficiency of prolonged down-regulation caused by a full-dose of gonadotropin-releasing hormone agonist (GnRH-a) injected during different menstrual phases has not yet been researched. Our goal was to evaluate the effects of GnRH-a, which was used in different phases of the menstrual cycle in patients undergoing in vitro fertilization and embryo transfer. METHODS: This was a retrospective cohort study. A total of 320 patients received a prolonged pituitary down-regulated full-dose (3.75 mg) of triptorelin in the early follicular phase, and 160 patients received the same full-dose of triptorelin during the mid-luteal phase. Clinical and laboratory outcomes were compared between the two groups. RESULTS: The basic characteristics of the two groups were comparable. The mean number of retrieved oocytes, fertilized oocytes, cleavage oocytes and good quality embryos were comparable between the two groups. Although there was a higher antral follicle count, cyst formation rate, fertilization rate and cleavage rate in the follicular phase group, no statistically significant effects were seen on implantation rate (41.15% vs. 45.91%), clinical pregnancy rate (60.38% vs. 61.36%), ongoing pregnancy rate (57.74% vs. 57.58%), live birth rate (56.23% vs. 57.58%) or early abortion rate (2.64% vs. 3.79%) per fresh transfer cycle. Moreover, severe ovarian hyperstimulation syndrome rates at the early stage (1.89% vs. 2.27%) were low in both groups. CONCLUSIONS: Prolonged pituitary down-regulation achieved by utilizing a full-dose of GnRH-a administrated in either phase of the menstrual cycle can have a positive effect on ongoing pregnancy rate and live-birth rate per fresh embryo transfer cycle. Ovarian cyst formation rate was higher in the follicular phase group, but this did not have any adverse impact on clinical results.

Differentiation of human induced pluripotent stem cells into Leydig-like cells with molecular compounds.

Leydig cells (LCs) play crucial roles in producing testosterone, which is critical in the regulation of male reproduction and development. Low levels of testosterone will lead to male hypogonadism. LC transplantation is a promising alternative therapy for male hypogonadism. However, the source of LCs limits this strategy for clinical applications. Thus far, others have reported that LCs can be derived from stem cells by gene transfection, but the safe and effective induction method has not yet been reported. Here, we report that Leydig-like cells can be derived from human induced pluripotent stem cells (iPSCs) using a novel differentiation protocol based on molecular compounds. The iPSCs-derived Leydig-like cells (iPSC-LCs) acquired testosterone synthesis capabilities, had the similar gene expression profiles with LCs, and positively expressed Leydig cell lineage-specific protein markers LHCGR, STAR, SCARB1, SF-1, CYP11A1, HSD3B1, and HSD17B3 as well as negatively expressed iPSC-specific markers NANOG, OCT4, and SOX2. When iPSC-LCs labeled with lipophilic red dye (PKH26) were transplanted into rat testes that were selectively eliminated endogenous LCs using EDS (75 mg/kg), the transplanted iPSC-LCs could survive and function in the interstitium of testes, and accelerate the recovery of serum testosterone levels and testis weights. Collectively, these findings demonstrated that the iPSCs were able to be differentiated into Leydig-like cells by few defined molecular compounds, which may lay the safer groundwork for further clinical application of iPSC-LCs for hypogonadism.

Identification of chromosomal abnormalities and genomic features in near-triploidy/tetraploidy-acute leukemia by fluorescence in situ hybridization.

BACKGROUND: Near-triploidy/tetraploidy is rarely found in acute leukemia. Only limited data are available to characterize this condition, and it remains largely unknown. PATIENTS AND METHODS: In our study, we performed karyotype analysis on 1,031 patients diagnosed with acute leukemia from 2006 to 2018. A total of 10 patients of near-triploidy/tetraploidy karyotype were enrolled. Two cases of near-triploidy (66-79 chromosomes) and eight cases of near-tetraploidy (84-100 chromosomes) were identified. Bone marrow samples of these 10 patients were analyzed by fluorescence in situ hybridization with 19 commercially available probes that detected a small portion of gene alterations and large regions of chromosome amplifications. RESULTS: Of the six patients with acute myelocytic leukemia, we detected three cases of double t(8;21)(q22;q22) that have not been previously reported, and one of them demonstrated ins(21;8) (q22;q24q22). We also describe a novel pediatric case carrying double t(15;17)(q22;q21) and receiving targeted treatment with all-trans retinoic acid therapy. To date, this case has responded well to the regimen and has shown continuous complete remission. All patients received chemotherapy. One of them received allogeneic hematopoietic stem cell transplant (HSCT) and survived for 22 months. Eight of the 10 patients died, and the median overall survival was 11 months. CONCLUSION: Using fluorescence in situ hybridization, we identified the distinct complex karyotype of near-triploidy/tetraploidy and provided further prognostic information. Tetraploidy acute promyelocytic leukemia had favorable prognosis; thus, HSCT was not necessary. The case of insertion t(21;8)(q22;q24q22) in tetraploidy responded poorly to chemotherapy and achieved molecular remission with difficultly. Data from patients in this group indicated that near-triploidy/tetraploidy acute leukemia has poor prognosis and new therapy is urgently needed.

Comparative Effectiveness of Three Ovarian Hyperstimulation Protocol in In Vitro Fertilization (IVF) Cycles for Women with Polycystic Ovary Syndrome.

BACKGROUND The aim of this study was to evaluate the efficacy of specific in vitro fertilization (IVF) protocols for patients with polycystic ovary syndrome (PCOS), and therefore, analyze the first-rank intention IVF protocol. MATERIAL AND METHODS In this study, 408 PCOS patients (464 treatment cycles) were enrolled and assigned to one of 3 groups: group 1 [oral contraceptive long-term regimen group (OC-L protocol group, n=91)], group 2 (GnRH antagonist protocol, n=80), and group 3 [follicular phase long-term regimen group, C1-L protocol group n=293]. The endpoints are the number of eggs, oocyte maturation rate, high-quality embryo rate and clinical pregnancy rate after fresh embryos transfer, the incidence of ovarian hyperstimulation syndrome and abortion rate. RESULTS The number of eggs, oocyte maturation rate, and high-quality embryo rate in the C1-L protocol group were significantly higher than those in the other 2 groups. The fertilization rate and cleavage rate of the 3 groups were not significantly different. After fresh embryo transplantation, the pregnancy rate of C1-L protocol group was significantly higher than that of the other groups. CONCLUSIONS This study showed that the super-long downregulation in follicular phase regimen has advantages of simple treatment process, high oocyte maturation rate, high quality embryo rate, and pregnancy rate. It is a good choice for PCOS patients to promote ovulation during IVF.

Delayed Puberty by Ziram Is Associated with Down Regulation of Testicular Phosphorylated AKT1 and SIRT1/PGC-1α Signaling.

Ziram is a dimethyldithiocarbamate fungicide, which may influence the male reproductive system as a potential endocrine disruptor. We interrogated the disruption of ziram on rat progenitor Leydig cell development. Prepubertal male Sprague-Dawley rats were orally treated with 0, 2, 4, or 8 mg/kg ziram for 2 weeks. We investigated the effects of ziram on serum testosterone levels, Leydig cell number, and Leydig and Sertoli cell gene and protein expression, SIRT1/PGC-1α levels, and phosphorylation of AKT1, ERK1/2, and AMPK in vivo. We also interrogated the effects of ziram on reactive oxidative species (ROS) level, apoptosis rate, and mitochondrial membrane potential of progenitor Leydig cells in vitro. Ziram decreased serum testosterone and follicle-stimulating hormone levels, the down-regulated Leydig cell-specific gene ( Lhcgr, Scarb1, Star, Cyp17a1, and Hsd17b3), and their protein expression. However, ziram stimulated anti-Müllerian hormone production. Ziram lowered SIRT1/PGC-1α and phosphorylated protein levels of AKT1. Ziram induced ROS and apoptosis and lowered the mitochondrial membrane potential of progenitor Leydig cells in vitro. In conclusion, ziram disrupts Leydig cell development during the prepubertal period potentially through the SIRT1/PGC-1α and phosphorylated AKT1 signaling.

Identification of a novel idiopathic congenital nystagmus­causing missense mutation, p.G296C, in the FRMD7 gene.

Exploring the genetic basis for idiopathic congenital nystagmus is critical for improving our understanding of its molecular pathogenesis. In the present study, direct sequencing using gene specific primers was performed in order to identify the causative mutations in two brothers from a Chinese family who had been diagnosed with idiopathic congenital nystagmus. A comprehensive ophthalmological examination, including eye movement recordings, fundus examination, and retinal optical coherence tomography imaging was also conducted, to characterize the disease phenotype. The results revealed that the two brothers exhibited clear signs of nystagmus without any other ocular anomalies. Direct sequencing revealed a G to T transition (c.886G>T) in exon 9 of the four­point­one, ezrin, radixin, moesin domain­containing 7 (FRMD7) gene, which resulted in a conservative substitution of glycine to cysteine at codon 296 (p.G296C), leading to idiopathic congenital nystagmus in the two affected brothers. c.886G>T is a novel idiopathic congenital nystagmus­inducing mutation in the FRMD7 gene. This finding expands the spectrum of known gene mutations in idiopathic congenital nystagmus, and may be useful for faster gene diagnosis, prenatal testing, the development of potential gene therapies, and for improving the understanding of the molecular pathogenesis of idiopathic congenital nystagmus.

A Retrospective Study of Letrozole Treatment Prior to Human Chorionic Gonadotropin in Women with Polycystic Ovary Syndrome Undergoing In Vitro Fertilization at Risk of Ovarian Hyperstimulation Syndrome.

BACKGROUND Women with polycystic ovary syndrome (PCOS) undergoing in vitro fertilization (IVF) are given letrozole before a trigger injection of human chorionic gonadotropin (hCG) to lower estrogen (E2) levels, but can experience ovarian hyperstimulation syndrome (OHSS). The aim of this study was to evaluate the effect of oral letrozole, prior to administration of hCG, on the outcome of IVF and development of OHSS. MATERIAL AND METHODS Retrospective clinical review included 181 cases of women with PCOS who underwent IVF cycles with intracytoplasmic sperm injection (ICSI) and embryo transfer (ET) (IVF/ICSI-ET). The day before the use of hCG, cases were divided into a letrozole-treated group (N=78) and a non-letrozole group (N=103). An oral dose of 2.5 mg qd of letrozole was given when the peak level of E2 was ≥4000 pg/ml during ovarian stimulation and ceased before the day of egg retrieval. RESULTS The letrozole-treated group had a significant increase in the number of retrieved oocytes, viable embryos, and fresh ET rate (P>0.05); peak levels of E2, and E2 levels on the day of the egg retrieval, were significantly higher, and the fertilization rate was significantly lower (P<0.001). No significant differences were found in the rates of pregnancy, abortion, or ectopic pregnancy between the two groups (P>0.05). The incidence OHSS was lower in the letrozole-treated group, but this difference did not reach statistical significance (P>0.05). CONCLUSIONS Women with PCOS who underwent IVF, oral treatment with letrozole a day prior to treatment with hCG lowered E2 levels, but did not significantly reduce the incidence of OHSS.

Identification of a novel GJA3 mutation in a large Chinese family with congenital cataract using targeted exome sequencing.

Autosomal dominant congenital cataract (ADCC) is a clinically and genetically heterogeneous ocular disease in children that results in serious visual impairments or even blindness. Targeted exome sequencing (TES) is an efficient method used for genetic diagnoses of inherited diseases. In the present study, we used a custom-made TES panel to identify the genetic defect of a four-generation Chinese family with bilateral pulverulent nuclear cataracts. A novel heterozygous missense mutation c.443C>T (p. T148I) in GJA3 was identified. The results of the bioinformatic analysis showed that the mutation was deleterious to the structure and hemichannel function of Cx46 encoded by GJA3. Plasmids expressing wild-type and mutant human Cx46 were constructed and ectopically expressed in human lens epithelial cells (HLECs) or human embryonic kidney (HEK-293) cells. Fluorescent images indicated aggregated signals of mutant protein in the cytoplasm, and a higher protein level was also detected in T148I stable cell lines. In summary, we identified a novel mutation in GJA3 for ADCC, which provided molecular insights into the pathogenic mechanism of ADCC.

Predicting the outcome of different protocols of in vitro fertilization with anti-Muüllerian hormone levels in patients with polycystic ovary syndrome.

Objective This study evaluated associations of basal serum and follicular fluid (FF) anti-Muüllerian hormone (AMH) levels with in vitro fertilization (IVF) outcomes in polycystic ovary syndrome (PCOS) patients. Methods This prospective study included 179 consecutive women undergoing IVF, including 59 with PCOS and non-PCOS controls. Thirty PCOS cases had long gona-dotrophin-releasing hormone agonist (GnRH-a) and 29 had antagonist (GnRH-ant) protocols. Controls underwent conventional GnRH-a. Associations of basal serum and FF AMH levels with IVF outcomes were assessed. Results Median serum and FF AMH levels, antral follicle count (AFC), oestradiol human chorionic gonadotropin injection day (peak E2), and retrieved oocyte numbers were higher in PCOS patients than in controls (all P < 0.01). Oocyte maturation and high-quality embryo rates were lower in PCOS patients than in controls (P < 0.01), but both groups had similar fertilization, implantation, clinical pregnancy, and newborn rates. Peak E2 was higher in GnRH-ant than in GnRH-a protocols (16.5 nmol/L vs. 12.1 nmol/L, P < 0.05). AMH levels were correlated with AFC in PCOS patients ( P < 0.01). Peak E2 and FF AMH levels were independent predictors of oocyte number. Peak E2 predicted the fertilization rate. Conclusion Serum basal AMH levels are predictive of oocyte quantity, but not oocyte quality or IVF outcomes. Serum AMH, FF AMH, and outcomes are similar among protocols.