Improving vitrification efficiency of human in vitro matured oocytes by the addition of LEA proteins.

STUDY QUESTION: Can the addition of late embryogenesis-abundant (LEA) proteins as a cryoprotective agent during the vitrification cryopreservation of in vitro matured oocytes enhance their developmental potential after fertilization? SUMMARY ANSWER: LEA proteins improve the developmental potential of human in vitro matured oocytes following cryopreservation, mostly by downregulating FOS genes, reducing oxidative stress, and inhibiting the formation of ice crystals. WHAT IS KNOWN ALREADY: Various factors in the vitrification process, including cryoprotectant toxicity, osmotic stress, and ice crystal formation during rewarming, can cause fatal damage to oocytes, thereby affecting the oocytes developmental potential and subsequent clinical outcomes. Recent studies have shown that LEA proteins possess high hydrophilicity and inherent stress tolerance, and can reduce low-temperature damage, although the molecular mechanism it exerts protective effects is still unclear. STUDY DESIGN, SIZE, DURATION: Two LEA proteins extracted and purified by us were added to solutions for vitrification-warming of oocytes at concentrations of 10, 100, and 200 µg/mL, to determine the optimal protective concentration for each protein. Individual oocyte samples were collected for transcriptomic analysis, with each group consisting of three sample replicates. PARTICIPANTS/MATERIALS, SETTING, METHODS: Immature oocytes were collected from patients who were undergoing combined in vitro fertilization (IVF) treatment and who had met the designated inclusion and exclusion criteria. These oocytes underwent in vitro maturation (IVM) culture for experimental research. A fluorescence microscope was used to detect the levels of mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and calcium in the mitochondria of vitrified-warmed human oocytes treated with different concentrations of LEA proteins, and the protective effect of the protein on mitochondrial function was assessed. The levels of intracellular ice recrystallization inhibition (IRI) in human oocytes after vitrification-warming were characterized by the cryomicroscope, to determine the LEA proteins inhibitory effect on recrystallization. By analyzing transcriptome sequencing data to investigate the potential mechanism through which LEA proteins exert their cryoprotective effects. MAIN RESULTS AND THE ROLE OF CHANCE: The secondary structures of AfrLEA2 and AfrLEA3m proteins were shown to consist of a large number of α-helices and the proteins were shown to be highly hydrophilic, in agreement with previous reports. Confocal microscopy results showed that the immunofluorescence of AfrLEA2-FITC and AfrLEA3m-FITC-labeled proteins appeared to be extracellular and did not penetrate the cell membrane compared with the fluorescein isothiocyanate (FITC) control group, indicating that both AfrLEA2 and AfrLEA3m proteins were extracellular. The group treated with 100 µg/mL AfrLEA2 or AfrLEA3m protein had more uniform cytoplasmic particles and fewer vacuoles compared to the 10 and 200 µg/mL groups and were closest to the fresh group. In the 100 µg/mL groups, MMPs were significantly higher while ROS and calcium levels were significantly lower than those in the control group and were closer to the levels observed in fresh oocytes. Meanwhile, 100 µg/mL of AfrLEA2 or AfrLEA3m protein caused smaller ice crystal formation in the IRI assay compared to the control group treated with dimethylsulphoxide (DMSO) and ethylene glycol (EG); thus, the recrystallization inhibition was superior to that with the conventional cryoprotectants DMSO and EG. Further results revealed that the proteins improved the developmental potential of human oocytes following cryopreservation, likely by downregulating FOS genes and reducing oxidative stress. LIMITATIONS, REASONS FOR CAUTION: The in vitro-matured metaphase II (IVM-MII) oocytes used in the study, due to ethical constraints, may not accurately reflect the condition of MII oocytes in general. The AfrLEA2 and AfrLEA3m proteins are recombinant proteins and their synthetic stability needs to be further explored. WIDER IMPLICATIONS OF THE FINDINGS: LEA proteins, as a non-toxic and effective cryoprotectant, can reduce the cryoinjury of oocytes during cryopreservation. It provides a new promising method for cryopreservation of various cell types. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Key Research and Development Program of China (2022YFC2703000) and the National Natural Science Foundation of China (52206064). The authors declare no competing interest. TRIAL REGISTRATION NUMBER: N/A.

Preimplantation genetic testing as a means of preventing hereditary congenital myasthenic syndrome caused by RAPSN.

BACKGROUND: Congenital myasthenic syndrome is a heterogeneous group of inherited neuromuscular transmission disorders. Variants in RAPSN are a common cause of CMS, accounting for approximately 14%-27% of all CMS cases. Whether preimplantation genetic testing for monogenic disease (PGT-M) could be used to prevent the potential birth of CMS-affected children is unclear. METHODS: Application of WES (whole-exome sequencing) for carrier testing and guidance for the PGT-M in the absence of a genetically characterized index patient as well as assisted reproductive technology were employed to prevent the occurrence of birth defects in subsequent pregnancy. The clinical phenotypes of stillborn fetuses were also assessed. RESULTS: The family carried two likely pathogenic variants in RAPSN(NM_005055.5): c.133G>A (p.V45M) and c.280G>A (p.E94K). And the potential birth of CMS-affected child was successfully prevented, allowing the family to have offspring devoid of disease-associated variants and exhibiting a normal phenotype. CONCLUSION: This report constitutes the first documented case of achieving a CMS-free offspring through PGT-M in a CMS-affected family. By broadening the known variant spectrum of RAPSN in the Chinese population, our findings underscore the feasibility and effectiveness of PGT-M for preventing CMS, offering valuable insights for similarly affected families.

vizAPA: visualizing dynamics of alternative polyadenylation from bulk and single-cell data.

MOTIVATION: Alternative polyadenylation (APA) is a widespread post-transcriptional regulatory mechanism across all eukaryotes. With the accumulation of genome-wide APA sites, especially those with single-cell resolution, it is imperative to develop easy-to-use visualization tools to guide APA analysis. RESULTS: We developed an R package called vizAPA for visualizing APA dynamics from bulk and single-cell data. vizAPA implements unified data structures for APA data and genome annotations. vizAPA also enables identification of genes with differential APA usage across biological samples and/or cell types. vizAPA provides four unique modules for extensively visualizing APA dynamics across biological samples and at the single-cell level. vizAPA could serve as a plugin in many routine APA analysis pipelines to augment studies for APA dynamics. AVAILABILITY AND IMPLEMENTATION: https://github.com/BMILAB/vizAPA.

MiR-9-enriched mesenchymal stem cells derived exosomes prevent cystitis-induced bladder pain via suppressing TLR4/NLRP3 pathway in interstitial cystitis mice.

BACKGROUND: Inflammatory response of central nervous system is an important component mechanism in the bladder pain of interstitial cystitis/bladder pain syndrome (IC/BPS). Exosomes transfer with microRNAs (miRNA) from mesenchymal stem cell (MSCs) might inhibit inflammatory injury of the central nervous system. Herein, the purpose of our study was to explore the therapeutic effects by which extracellular vesicles （EVs） derived from miR-9-edreched MSCs in IC/BPS and further investigate the potential mechanism to attenuate neuroinflammation. METHODS: On the basis of IC/BPS model, we used various techniques including bioinformatics, cell and molecular biology, and experimental zoology, to elucidate the role and molecular mechanism of TLR4 in regulating the activation of NLRP3 inflammasome in bladder pain of IC/BPS, and investigate the mechanism and feasibility of MSC-EVs enriched with miR-9 in the treatment of bladder pain of IC/BPS. RESULTS: The inflammatory responses in systemic and central derived by TLR4 activation were closely related to the cystitis-induced pelvic/bladder nociception in IC/BPS model. Intrathecal injection of miR-9-enreched MSCs derived exosomes were effective in the treatment of cystitis-induced pelvic/bladder nociception by inhibiting TLR4/NF-κb/NLRP3 signal pathway in central nervous system of IC/BPS mice. CONCLUSIONS: This study demonstrated that miR-9-enreched MSCs derived exosomes alleviate neuroinflammaiton and cystitis-induced bladder pain by inhibiting TLR4/NF-κb/NLRP3 signal pathway in interstitial cystitis mice, which is a promising strategy against cystitis-induced bladder pain.

Thiolutin, a selective NLRP3 inflammasome inhibitor, attenuates cyclophosphamide-induced impairment of sperm and fertility in mice.

OBJECTIVE: The activation of the NLRP3 inflammasome has been implicated in male infertility. Our study aimed to investigate the therapeutic role of Thiolutin (THL), an inhibitor of the NLRP3 inflammasome, on oligoasthenospermia (OA) and to elucidate its mechanisms. MATERIALS AND METHODS: Semen from 50 OA and 20 healthy males were analyzed to assess the sperm quality and levels of inflammatory markers. Their correlation was determined using Pearson's correlation coefficient. The BALB/c mice were intraperitoneal injected by cyclophosphamide at 60 mg/kg/day for five days to induce OA, followed by a two-week treatment with THL or L-carnitine. Reproductive organ size and H&E staining were determined to observe the organ and seminiferous tubule morphology. ELISA and western blotting were utilized to measure sex hormone levels, inflammatory markers, and NLRP3 inflammasome levels. Furthermore, male and female mice were co-housed to observe pregnancy success rates. RESULTS: OA patients exhibited a decrease in sperm density and motility compared to healthy individuals, along with elevated levels of IL-1ß, IL-18 and NLRP3 inflammasome. In vivo, THL ameliorated OA-induced atrophy of reproductive organs, hormonal imbalance, and improved sperm density, motility, spermatogenesis and pregnancy success rates with negligible adverse effects on weight or liver-kidney function. THL also demonstrated to be able to inhibit the activation of NLRP3 inflammasome and associated proteins in OA mice. DISCUSSION: THL can improve sperm quality and hormonal balance in OA mice through the inhibition of NLRP3 inflammasome activation. Thus, THL holds promising potential as a therapeutic agent for OA.

The predictive value of uterine artery Doppler in the success rate of pregnancy from the first frozen embryo transfer during the implantation window.

BACKGROUND: Worldwide, frozen embryo transfer (FET) has become a new strategy for the treatment of infertility. The success of FET is closely related to endometrial receptivity. Does uterine artery Doppler during the implantation window predict pregnancy outcome from the first FET? METHODS: A total of 115 retrospectively collected cycles were included in the study, with 64 cycles of clinical pregnancy and 51 cycles of nonclinical pregnancy; There were 99 nonabsent end-diastolic flow (NAEDF) cycles and 16 absent end-diastolic flow (AEDF) cycles. The differences in uterine artery Doppler findings between different pregnancy outcomes were investigated. The clinical pregnancy rate and spontaneous abortion rate in the NAEDF and AEDF groups were compared. The predictive value of uterine artery Doppler during the implantation window in the success rate of pregnancy from the first FET was also investigated. RESULTS: Between the clinical pregnancy group and the nonclinical pregnancy group, there were no significant differences in the mean resistance index (mRI) (Z = -1.065, p = 0.287), mean pulsatility index (mPI) (Z = -0.340, p = 0.734), and mean peak systolic/end-diastolic velocity(mS/D) (Z = -0.953, p = 0.341); there were significant differences in the mean peak systolic velocity (mPSV) (Z = -1.982, p = 0.048) and mean end-diastolic velocity (mEDV) (Z = -2.767, p = 0.006). Between the NAEDF and AEDF groups, there was no significant difference in the clinical pregnancy rate (χ2 = 0.003, p = 0.959), and there was a significant difference in the spontaneous abortion rate (χ2 = 3.465, p = 0.019). Compared with uterine artery Doppler alone, its combination with artificial abortion history, waist-to-hip ratio, LH (Luteinizing hormone) of P (Progesterone) administration day, mPSV and mEDV had a higher predictive value regarding clinical pregnancy from the first FET [ROC-AUC 0.782, 95% CI (0.680-0.883) vs. 0.692, 95% CI (0.587-0.797)]. CONCLUSIONS: Uterine artery Doppler, particularly mPSV and mEDV during the implantation window, was useful for predicting clinical pregnancy, and AEDF was related to spontaneous abortion in the first trimester. Uterine artery Doppler combined with artificial abortion history, waist-to-hip ratio, LH of P administration day, mPSV and mEDV have a higher predictive value than uterine artery Doppler alone regarding the pregnancy from the first FET.

Preimplantation genetic testing and prenatal diagnosis in a family with pseudovaginal perineoscrotal hypospadias: A case report.

RATIONALE: Pseudovaginal perineoscrotal hypospadias (PPSH) is a rare autosomal recessive disorder of sex development caused by biallelic mutations in SRD5A2. PPSH is characterized by a vaginal-like blind ending perineal opening, penoscrotal hypospadias, and impaired masculinization. PATIENT CONCERNS: We reported preimplantation genetic testing and prenatal diagnosis in a family with PPSH. DIAGNOSIS: Whole-exome sequencing of the family identified 2 SRD5A2 pathogenic variants (c.578A>G and c.607G>A). Haplotype analysis showed that the variants were inherited from the previous generation of this family. INTERVENTIONS: During subsequent in vitro fertilization, preimplantation genetic testing was performed on 9 embryos. One unaffected embryo was transferred, resulting in a singleton pregnancy. OUTCOMES: The prenatal diagnosis at 20 weeks' gestation confirmed the fetus was unaffected. A healthy female infant weighing 3100 g and measuring 50 cm was delivered vaginally at 39+5 weeks of gestation. LESSONS SUBSECTIONS: This case highlights the use of preimplantation genetic testing and prenatal diagnosis to prevent the transmission of PPSH in families at risk. Our approach provides an effective strategy for identification and management of families with autosomal recessive disorders like PPSH.

ELF1/PRR11/ARP2/3 promoted trophoblast cells proliferation and motility in early pregnancy.

BACKGROUND/OBJECTIVE: Early pregnancy loss (EPL) is a common adverse pregnancy outcome with an incidence of approximately 10-30%. There are many factors that cause EPL, among which the lack of proliferation and invasive properties of trophoblast cells can lead to embryonic development. Therefore, in this study, the molecular biology of trophoblast cells was investigated. METHODS: Placental villous tissues from EPL patients were collected to explore ELF1 and PRR11 gene expression. The proliferation and migration of trophoblast cells were assessed by MTT, crystalline violet staining, and traswell assays, respectively. Western blotting and RT-qPCR were performed to investigate the relationship between ELF1, PRR11, and ARP2/3. F-actin polymerization and FAK activation were evaluated by immunofluorescence and western blotting. Ultimately, ELF1/PRR11/ARP2/3 expression was verified in the EPL mice model RESULTS: ELF1 and PRR11 were lowly expressed in placental villous tissues from EPL. The overexpression of ELF1 and PRR11 promoted proliferation and migration of trophoblast cells. Moreover, while ELF1 bound to the PRR11 promoter and promoted transcriptional activation. Finally, ELF1/PRR11/ARP2/3 showed low expression in the placental tissue of EPL mice. CONCLUSION: Our study suggested that PRR11 promoted the motility of trophoblast cells by binding to the ARP2/3 complex to promote F-actin polymerization and FAK activation. In addition, ELF1 bound to the initiation site of PRR11 to promote its transcription. ELF1/PRR11/ARP2/3 may play an important role in EPL.

Interaction of air pollution and meteorological factors on IVF outcomes: A multicenter study in China.

BACKGROUND: Previous studies revealed associations between air-pollutant exposure and in vitro fertilization (IVF) outcomes. However, modification effects of air pollution on IVF outcomes by meteorological conditions remain elusive. METHODS: This multicenter retrospective cohort study included 15,217 women from five northern Chinese cities during 2015-2020. Daily average concentrations of air pollutants (PM2.5, PM10, O3, NO2, SO2, and CO) and meteorological factors (temperature, relative humidity, wind speed, and sunshine duration) during different exposure windows were calculated as individual approximate exposure. Generalized estimating equations models and stratified analyses were conducted to assess the associations of air pollution and meteorological conditions with IVF outcomes and estimate potential interactions. RESULTS: Positive associations of wind speed and sunshine duration with pregnancy outcomes were detected. In addition, we observed that embryo transfer in spring and summer had a higher likelihood to achieve a live birth compared with winter. Exposure to PM2.5, SO2, and O3 was adversely correlated with pregnancy outcomes in fresh IVF cycles, and the associations were modified by air temperature, relative humidity, and wind speed. The inverse associations of PM2.5 and SO2 exposure with biochemical pregnancy were stronger at lower temperatures and humidity. Negative associations of PM2.5 with clinical pregnancy were only significant at lower temperatures and wind speeds. Moreover, the effects of O3 on live birth were enhanced by higher wind speed. CONCLUSIONS: Our results suggested that the associations between air-pollutant exposure and IVF outcomes were modified by meteorological conditions, especially temperature and wind speed. Women undergoing IVF treatment should be advised to reduce outdoor time when the air quality was poor, particularly at lower temperatures.

Correlation between methylenetetrahydrofolate reductase gene-specific methylation and recurrent spontaneous abortion.

OBJECTIVE: To investigate the correlation between methylenetetrahydrofolate reductase (MTHFR) gene-specific methylation and recurrent spontaneous abortion (RSA). METHODS: A total of 50 RSA patients who visited our hospital were recruited in the study group; 50 multiparous women who underwent physical examinations during the same period were enrolled in the control group. The levels of homocysteine, folic acid, and vitamin B12 and their MTHFR gene polymorphism and specific methylation were measured in both groups. The Logistic regression equation was used to analyze the correlation between MTHFR gene-specific methylation and RSA. RESULTS: The methylated allele MM was not found in the control group, and the frequency in the study group was 1.19%. The frequency of the MU genotype in the study group 32.93% was higher than that in the control group 12.45%. The frequency of methylated alleles of CC and CT genotypes carrying MTHFR C677T polymorphism in the study group was higher than that in the control group (P < 0.05). There was no significant difference in the TT genotype between the two groups (P > 0.05). Multivariate Logistic regression analysis exhibited that patients with methylated alleles of CC genotype had a risk of RSA increased by 1.167 times, and the risk increased by 2.500 times in patients with methylated alleles of CT genotype (P < 0.05). 83.33% of RSA patients carrying methylated alleles affected hyperhomocysteinemia. In patients with elevated homocysteine levels, the risk of RSA caused by methylated allele was significantly increased by 7.321 times. CONCLUSION: MTHFR gene-specific methylation can significantly increase the risk of RSA.

Exploration of the interchromosomal effects in preimplantation genetic testing for structural rearrangements based on next-generation sequencing.

BACKGROUND: To investigate the interchromosomal effect (ICE) in chromosome translocation carriers. METHODS: Data on preimplantation genetic testing aneuploidy and structural rearrangements (translocation) were retrospectively collected and classified into a reciprocal translocation group, a Robertsonian translocation group and a control group. According to the carrier's gender and age, all cases underwent further subgroup difference analysis of de novo abnormal embryo rates and the number of chromosomes involved in de novo abnormal embryos. RESULTS: Among the 283 couples who participated in this study, 1076 blastocysts from 352 cycles were collected, and 246 de novo abnormal embryos were included. There was a significant difference in the rate of de novo abnormal embryos among the three groups (p < .05) but no significant difference in the number of de novo abnormal chromosomes in the abnormal embryos (p > .05). Gender and age (classified by 35 years old) had no effect on the de novo abnormal embryo ratios among the translocation carriers (p > .05). However, the de novo abnormal ratio increased with age. The embryo constitution reflected no significant difference between the translocation groups (p > .05). CONCLUSION: The ICE was detected for the translocation carriers. The de novo abnormal embryo ratio increased with age. Gender had no effect on the de novo abnormal embryo ratio. Translocation status played a more important role than age and gender.

Logistic regression analyses of factors affecting the euploidy of blastocysts undergoing in vitro fertilization and preimplantation genetic testing.

Embryo chromosomal abnormalities are considered as the main cause of low pregnancy rate for in vitro fertilization (IVF). Recently, a new metric of success in assisted reproductive technology, that is, the ability to achieve at least 1 euploid blastocyst for transfer, has been brought into focus among clinicians. Our study aimed to investigate the effects of different factors on the euploidy of blastocysts undergoing IVF and preimplantation genetic testing (PGT). This retrospective observational study included 493 cycles underwent IVF/intracytroplasmatic sperm injection intended to obtain trophectoderm biopsy for PGT from June 2016 to December 2019 at a single academic fertility center. Logistic regression was adopted to analyze the clinical characteristics and embryonic data related to the ability to achieve at least 1 euploid blastocyst for transfer. The study took 1471 blastocysts from 493 cycles as samples for PGT. Among them, 149 cycles (30.22%) had no euploid blastocyst and 344 cycles (69.78%) had at least 1 euploid blastocyst. A multivariate logistic analysis suggested that maternal age >36, abnormal parental karyotype, nonfirst cycles and blastocysts number per cycle <3 were the risk factors for no euploid blastocyst. The parental karyotype, maternal age, number of cycles, and number of blastocysts per cycle were the dominant factors affecting the ability to achieve at least 1 euploid blastocyst for transfer and therefore could be regarded as potential predictors for genetic counseling.

Poor ovarian response is associated with air pollutants: A multicentre study in China.

BACKGROUND: Human evidence on the association between air pollution and ovarian response is scarce. Poor ovarian response (POR) with an incidence of 5-35% is a tricky problem in IVF treatment. METHODS: In this large-scale multicentre study, we included 2186 women with POR (< 4 oocytes retrieved) and 7033 women with a normal ovarian response (10-15 oocytes retrieved), who underwent their first in vitro fertilization treatment in five cities in northern China during 2015-2020. Average concentrations of six air pollutants (PM2.5, PM10, O3, NO2, CO, and SO2) during different exposure windows (5 days, 1, 3, 6, and 12 months) before oocyte pick up (OPU) were calculated using data from the air monitoring station nearest to the residential site as approximate individual exposure. Logistic regression models were employed to assess the association between exposure to air pollutants and the risk of POR. Stratification analyses were conducted based on female age. Sensitivity analyses were performed in poor responders identified by Bologna criteria and women with unexpected POR. FINDINGS: We detected that increased SO2 exposure during all exposure windows before OPU was associated with a higher risk of POR, especially for women ≤ 30 years old. In the stratified analysis, the effect sizes were larger for the unexpected poor ovarian response. INTERPRETATION: The findings provide human evidence for adverse effects of exposure to ambient air pollutants on ovarian response and underscore the need to reduce ambient air pollution exposure in women of reproductive age to protect human fertility. FUNDING: This study was granted from the National Key Research and Development Program (2018YFC1004203), the Major Special Construction Plan for Discipline Construction Project of China Medical University (3110118033), the Shengjing Freelance Researcher Plan of Shengjing Hospital of China Medical University, and the National Natural Science Foundation of China (82071601), the Central Government Special Fund for Local Science and Technology Development (2020JH6/10500006).

Prolonged Cryopreservation Negatively Affects Embryo Transfer Outcomes Following the Elective Freeze-All Strategy: A Multicenter Retrospective Study.

Background: With the development of embryo freezing and warming technology, frozen-thawed embryo transfer (FET) has been widely utilized. However, studies investigating the association between cryopreservation duration and FET outcomes are limited and controversial, and previous studies did not conduct stratification analyses based on demographic or clinical characteristics. Methods: This multicenter retrospective study included 17,826 women who underwent their first FET following the freeze-all strategy during the period from January 2014 to December 2018. Duration of cryopreservation was categorized into five groups: 3-8 weeks, 8-12 weeks, 12-26 weeks, 26-52 weeks, and >52 weeks. Modified Poisson regression and multivariate logistic regression were used to assess the association between cryostorage time of vitrified embryos and transfer outcomes. Moreover, further stratification analyses were performed according to variables with p <0.05 in multivariate models. Results: In this large multicenter study, we observed that storage duration was inversely associated with the possibility of pregnancy and live birth (p <0.001), but not with the risk of ectopic pregnancy and miscarriage. Stratification analyses based on maternal age, the number of oocytes retrieved, and condition of embryo transferred indicated that the inverse correlation was significant in the subpopulation with characteristics: (1) less than 40 years old, (2) more than 3 oocytes retrieved, and (3) only high-quality blastocysts transferred. Conclusion: The results of this large, multicenter, retrospective study suggested that prolonged cryopreservation was inversely associated with the probability of pregnancy and live birth. Therefore, for patients who adopt a freeze-all strategy, early FET might achieve a better outcome.

Association between exposure to ambient air pollutants and the outcomes of in vitro fertilization treatment: A multicenter retrospective study.

BACKGROUND: Exposure to ambient air pollution has been reported to be inversely correlated with human reproductive health. However, the results of previous studies exploring the association between air pollution and in vitro fertilization (IVF) outcomes are conflicting, and further research is needed to clarify this association. OBJECTIVES: This study aimed to investigate the associations between exposure to air pollutants and IVF outcomes. METHODS: We conducted a multicenter retrospective cohort study involving 20,835 patients from four cities in Northern China, contributing to 11,787 fresh embryo transfer cycles, 9050 freeze-all cycles, and 17,676 frozen-thawed embryo transfer (FET) cycles during 2014-2018. We calculated the daily average concentrations of six criteria air pollutants (PM2.5, PM10, O3, NO2, CO, and SO2) during different exposure windows in IVF treatment timeline using data from the air monitoring station nearest to the residential site as approximate individual exposure. Generalized estimation equation models were used to assess the association between air pollution exposure and IVF outcomes. RESULTS: Exposure to O3, NO2, and CO during most exposure windows in fresh embryo transfer cycles were correlated with lower possibilities of biochemical pregnancy, clinical pregnancy, and live birth. An inverse association of exposure to O3 and SO2 with pregnancy outcomes was observed in FET cycles. In addition, we found a significant association of exposure to air pollutants with a higher risk of ectopic pregnancy and lower oocyte yield. CONCLUSIONS: Our study provided large-scale human evidence of the association between air pollution and adverse human reproductive outcomes in the population opting for IVF. Thus, exposure to air pollutants in the population opting for IVF should be limited to improve treatment outcomes.

Testicular STAC3 regulates Leydig cell steroidogenesis through potentiating mitochondrial membrane potential and StAR processing.

SH3 and cysteine-rich protein 3 (STAC3), a small adapter protein originally identified as a core component of excitation-contraction coupling machinery, regulates the voltage-induced Ca2+ release in skeletal muscle. However, the possibility of additional, as yet unknown, non-muscle effects of STAC3 cannot be ruled out. Herein, we provide the evidence for the expression and functional involvement of STAC3 in spermatogenesis. STAC3 expression was localized in the testicular interstitium of rodent and human testes. By using the cytotoxic drug ethylene dimethane sulfonate (EDS), STAC3 expression was observed to be decreased sharply in rat testis after selective withdrawal of Leydig cells (LCs), and reappeared immediately after LCs repopulation, indicating that testicular expression of STAC3 mainly stems from LCs. From a functional standpoint, in vivo lentiviral vector-mediated suppression of STAC3 resulted in a significant decrease in testosterone production, and thereafter caused impairment of male fertility by inducing oligozoospermia and asthenospermia. The indispensible involvement of STAC3 in testicular steroidogenesis was validated using the in vivo knockdown model with isolated primary LCs as well as in vitro experiments with primary LCs. By generating the TM3Stac3-/- cells, we further revealed that STAC3 depletion attenuated mitochondrial membrane potential and StAR processing in db-cAMP-stimulated LCs. Thus, the inhibitory effect of STAC3 deficiency on testicular steroidogenesis may be ascribed to a disturbed mitochondrial homeostasis. Collectively, the present results strongly suggest that STAC3 may function as a novel regulator linking mitochondrial homeostasis and testicular steroidogenesis in LCs. Our data underscore an unexpected reproductive facet of this muscle-derived factor.

miR­132 is upregulated in polycystic ovarian syndrome and inhibits granulosa cells viability by targeting Foxa1.

Polycystic ovary syndrome (PCOS) is one of the most common endocrine metabolic disorders characterized by hyperandrogenism, polycystic ovaries and ovulatory dysfunction. Several studies have suggested that the aberrant expression of microRNAs (miRNAs/miRs) plays an important role in the pathogenesis of PCOS; however, the role and underlying mechanisms of miR­132 in the development of PCOS remain unclear. In the present study, the expression of miR­132 in granulosa cells (GCs) derived from 26 patients with PCOS and 30 healthy controls was detected by reverse transcription­quantitative PCR (RT­qPCR). The apoptosis of GCs was examined using a TUNEL assay. The human ovarian granulosa­like tumor cell line, KGN, was cultured for Cell Counting Kit­8 assays following the overexpression or knockdown of miR­132. TargetScan was applied to identify the potential targets of miR­132, which was further verified by a luciferase assay, RT­qPCR and western blotting. The expression of miR­132 was decreased in GCs from patients with PCOS. Moreover, the GCs of patients with PCOS exhibited significantly increased apoptotic nuclei. Furthermore, the overexpression of miR­132 inhibited the viability of KGN cells. In addition, the results verified that miR­132 directly targeted forkhead box protein A1 (Foxa1), the knockdown of which suppressed KGN cell viability. On the whole, the findings of the present study demonstrated that miR­132 inhibited cell viability and induced apoptosis by directly interacting with Foxa1. Thus, miR­132 may be a potential target for the treatment of patients with PCOS.

Rare heterotopic pregnancy after frozen embryo transfer: a case report and literature review.

BACKGROUND: Heterotopic pregnancy occurred after frozen embryo transfer with two D3 embryos, and the case had a history of bilateral salpingectomy due to salpingocyesis. An ectopic heterotopic pregnancy was implanted in the left psoas major muscle, which has not been previously reported. CASE PRESENTATION: A 33-year-old woman presented with left back pain after curettage due to foetal arrest in the uterus without vaginal bleeding and spotting, and painkillers relieved the pain initially. When the painkillers ceased to work, the patient returned to the hospital. The ß-human chorionic gonadotropin (ß-hCG) level remained increased compared with the time of curettage, and a diagnosis of retroperitoneal abdominal pregnancy was suggested by ultrasonography and computerized tomography (CT) with the gestational sac implanted in the left psoas major muscle at the left hilum level. Laparotomy was performed to remove the ectopic pregnancy. During the operation, we carefully separated the adipose tissue between the space of the left kidney door and left psoas major muscle, peeled away the gestational sac that was approximately 50 mm × 40 mm with a 25-mm-long foetal bud, and gave a local injection of 10 mg of methotrexate in the psoas major muscle. Fifty days later, ß-hCG decreased to normal levels. CONCLUSION: It is necessary to pay more attention to the main complaints to exclude rare types of ectopic pregnancies of the pelvis and abdomen after embryo transfer.

Genetic and preimplantation diagnosis of cystic kidney disease with ventriculomegaly.

Ventriculomegaly with cystic kidney disease (VMCKD) is a rare and severe disorder characterized by cerebral ventriculomegaly, greatly elevated maternal serum alpha-fetoprotein (MSAFP) or amniotic fluid alpha-fetoprotein (AFAFP) levels and kidney disease similar to Finnish congenital nephrosis. Recessive mutations in the CRB2 (NM_173689) gene have been shown to cause the syndrome. Here, we described a nonconsanguineous Chinese family with two fetuses affected with VMCKD. A novel compound heterozygous mutation was identified in the CRB2 gene with co-segregation. One mutation [c.1960G>C (p.A654P)] was inherited from the father, while another mutation [c.3078_c.3093delGGCGCGGCCCCGGCCC (p.L1026Lfs*110)] was inherited from the mother. Preimplantation genetic testing for monogenic disease (PGT-M) was performed for the carrier couple with full informed consent and successfully blocked the inheritance of the disease. Our study has important implications on molecular diagnosis and genetic counseling for VMCKD and extends the mutation spectrum in CRB2 gene.

Identification of the key pathways and genes related to polycystic ovary syndrome using bioinformatics analysis.

Polycystic ovary syndrome (PCOS) is the most common hormonal and metabolic disorder among women of reproductive age, but the mechanisms underlying this unique pathogenesis remain unknown. This study was therefore designed to identify candidate genes involved in the pathogenesis of PCOS, using bioinformatics analysis. The gene expression profiles of GSE34526 from 7 PCOS patients and 3 controls were downloaded from Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were identified using GCBI online tool. Expression levels of candidate genes were verified using quantitative RT-PCR (qRT-PCR) and Western blot. 426 DEGs were identified by GCBI, including 418 up-regulated and 8 down-regulated genes. Function and pathway enrichment analyses showed that these DEGs were significantly enriched in inflammation and immune-related pathways. Additionally, protein-protein interaction (PPI) network and module analyses showed that two modules involved the Toll-like receptor signaling pathway were ranked among the most upregulated modules, and the candidate genes involved in this signaling pathway consisted of TLR1, TLR2, TLR8, and CD14. Finally, expression levels of TLR2, TLR8 and CD14 were significantly increased in samples from PCOS patients. Collectively, the results suggested that the Toll-like receptor signaling pathway might play an important role in the pathogenesis of PCOS.