Intrauterine hyperglycaemia during late gestation caused mitochondrial dysfunction in skeletal muscle of male offspring through CREB/PGC1A signaling.

BACKGROUND: Maternal diabetes mellitus can influence the development of offspring. Gestational diabetes mellitus (GDM) creates a short-term intrauterine hyperglycaemic environment in offspring, leading to glucose intolerance in later life, but the long-term effects and specific mechanism involved in skeletal muscle dysfunction in offspring remain to be clarified. METHODS: Pregnant mice were divided into two groups: The GDM group was intraperitoneally injected with 100 mg/kg streptozotocin on gestational days (GDs) 6.5 and 12.5, while the control (CTR) group was treated with vehicle buffer. Only pregnant mice whose random blood glucose level was higher than 16.8 mmol/L beginning on GD13.5 were regarded as the GDM group. The growth of the offspring was monitored, and the glucose tolerance test was performed at different time points. Body composition analysis and immunohistochemical methods were used to evaluate the development of lean mass at 8 weeks. The exercise capacity and grip strength of the male mouse offspring were assessed at the same period. Transmission electron microscopy was used to observe the morphology inside skeletal muscle at 8 weeks and as a foetus. The genes and proteins associated with mitochondrial biogenesis and oxidative metabolism were investigated. We also coanalyzed RNA sequencing and proteomics data to explore the underlying mechanism. Chromatin immunoprecipitation and bisulfite-converted DNA methylation detection were performed to evaluate this phenomenon. RESULTS: Short-term intrauterine hyperglycaemia inhibited the growth and reduced the lean mass of male offspring, leading to decreased endurance exercise capacity. The myofiber composition of the tibialis anterior muscle of GDM male offspring became more glycolytic and less oxidative. The morphology and function of mitochondria in the skeletal muscle of GDM male offspring were destroyed, and coanalysis of RNA sequencing and proteomics of foetal skeletal muscle showed that mitochondrial elements and lipid oxidation were consistently impaired. In vivo and in vitro myoblast experiments also demonstrated that high glucose concentrations impeded mitochondrial organisation and function. Importantly, the transcription of genes associated with mitochondrial biogenesis and oxidative metabolism decreased at 8 weeks and during the foetal period. We predicted Ppargc1α as a key upstream regulator with the help of IPA software. The proteins and mRNA levels of Ppargc1α in the skeletal muscle of GDM male offspring were decreased as a foetus (CTR vs. GDM, 1.004 vs. 0.665, p = 0.002), at 6 weeks (1.018 vs. 0.511, p = 0.023) and 8 weeks (1.006 vs. 0.596, p = 0.018). In addition, CREB phosphorylation was inhibited in GDM group, with fewer activated pCREB proteins binding to the CRE element of Ppargc1α (1.042 vs. 0.681, p = 0.037), Pck1 (1.091 vs. 0.432, p = 0.014) and G6pc (1.118 vs. 0.472, p = 0.027), resulting in their decreased transcription. Interestingly, we found that sarcopenia and mitochondrial dysfunction could even be inherited by the next generation. CONCLUSIONS: Short-term intrauterine hyperglycaemia significantly reduced lean mass in male offspring at 8 weeks, resulting in decreased exercise endurance and metabolic disorders. Disrupted organisation and function of the mitochondria in skeletal muscle were also observed among them. Foetal exposure to hyperglycaemia decreased the ratio of phosphorylated CREB and reduced the transcription of Ppargc1α, which inhibited the transcription of downstream genes involving in mitochondrial biogenesis and oxidative metabolism. Abnormal mitochondria, which might be transmitted through aberrant gametes, were also observed in the F2 generation.

Pediatric and adult obesity concerns in female health: a Mendelian randomization study.

PURPOSE: Adulthood and childhood obesity are both associated with reproductive diseases and gynecological cancers in females. However, the causal factors associated with these observations have yet to be identified. Mendelian randomization is a process that is independent of inverse bias and confounding and can act as a random control trial in which genetic groups are settled during meiosis, thus representing an effective tool with which to investigate causality. METHODS: We carried out several Mendelian randomization trials based on the combined genetic scores of 75 adult-associated and 15 childhood-associated body mass index (BMI) single nucleotide polymorphisms (SNPs), databases for several gynecological cancers and reproductive diseases from the UK Biobank (with 194,153 participants), using the traditional inverse-variance weighted (IVW) method as the main method. RESULTS: Elevated adult-associated BMI scores (odds ratio [OR] = 1.003; 95% confidence interval [CI]: 1.001-1.004) and childhood-associated BMI scores (OR = 1.003; 95% CI: 1.001-1.004) were related to a higher risk of the polycystic ovarian syndrome (PCOS), as determined by the traditional IVW method. The random IVW method further revealed a nominal negative causal association between childhood-associated BMI and subsequent endometriosis (OR = 0.995; 95% CI: 0.991-0.999). CONCLUSIONS: Consistent with observational consequences, our findings indicated that adulthood obesity may play role in the development of PCOS and that childhood obesity can increase the risk of PCOS but may reduce the incidence of endometriosis in later life. Further research is now needed to validate our findings and identify the precise mechanisms involved.

BNC1 Promotes Spermatogenesis by Regulating Transcription of Ybx2 and Papolb via Direct Binding to Their Promotor Elements.

BNC1 is a transcription factor that is crucial for spermatogenesis and male fertility, although the underlying mechanism remains unclear. To study BNC1's specific role in spermatogenesis, we characterized a previously developed mouse model carrying a truncating mutation in Bnc1 (termed Bnc1+/tr for heterozygotes and Bnc1tr/tr for homozygotes) and found that the mutation decreased BNC1 protein levels and resulted in germ cell loss by apoptosis. Given that loss of functional Bnc1 is known to result in decreased expression of the spermatogenesis genes Ybx2 and Papolb, we aimed to explore whether and how BNC1 promotes transcription of Ybx2 and Papolb to mediate its role in spermatogenesis. We confirmed significant reduction in YBX2 and PAPOLB protein levels in testis tissue from Bnc1+/tr and Bnc1tr/tr males compared with wild-type mice (Bnc1+/+). Consistently, knockdown of Bnc1 led to downregulation of Ybx2 and Papolb in CRL-2196 cells in vitro. To investigate if BNC1 directly induces Ybx2 and Papolb gene expression, chromatin immunoprecipitation using mouse testicular tissue and luciferase reporter assays in HEK293 cells were used to identify functional binding of BNC1 to the Ybx2 and Papolb promoters at defined BNC1 binding sites. Taken together, this study reveals a mechanism for BNC1's role in spermatogenesis by directly binding to BNC1 binding elements in the promoter regions of both Ybx2 and Papolb and inducing transcription of these important spermatogenesis genes.

Sperm parameters and anti-Müllerian hormone remain stable with Helicobacter pylori infection: a cross-sectional study.

BACKGROUND AND AIMS: It has been reported that Helicobacter pylori (HP) infection was more prevalent in infertile populations. HP infection could lead to decreased sperm parameters, and treating the HP infection could improve the quality of sperm. However, studies investigating the relationship between infertility and HP infection are still limited, and more evidence is required. Therefore, we performed the present study to investigate the impact of HP infection on sperm quality in males and on ovarian reserve in females. METHODS: A total of 16,522 patients who visited the Second Hospital of Zhejiang University from January 2016 to June 2019 due to abdominal discomfort and underwent a 13/14C-urea breath HP test were included in this retrospective cross-sectional study. Among them, 565 had performed sperm analysis or ovarian reserve tests in the past three months and were involved for further analyses. Sperm parameters were examined with a computer-assisted sperm analysis system, and serum anti-Müllerian hormone (AMH) and sex hormones were tested with an electrochemiluminescence method. RESULTS: Among 363 patients who underwent the sperm test, 136 (37.47%) had HP infection. Among 202 patients who underwent the AMH test, 55 (27.23%) had HP infection. There was no difference in sperm concentration and motility between the HP+ and HP- groups (P > 0.05). Further subgroup analyses stratified into 5-year age groups confirmed that there was no significant difference in sperm parameters (P > 0.05). When pooled with previously published data, no significant difference in sperm concentration or motility was found (P > 0.05). Meanwhile, this study found that the serum AMH level was similar between the HP+ and HP- groups (P > 0.05). Further subgroup analyses confirmed that there was no significant difference in serum AMH level (P > 0.05). CONCLUSIONS: There were no differences in sperm parameters and AMH levels based on history of HP infection among Chinese patients.

Role of anti-Müllerian hormone and testosterone in follicular growth: a cross-sectional study.

BACKGROUND: Anti-Müllerian hormone (AMH) is now considered the best serum biomarker of ovarian reserve, while basal sex hormones are classic markers used for assessing ovarian reserve. The interaction between AMH and sex hormones are complicated and not sufficiently addressed. In this study, we took diminished ovarian reserve (DOR) and polycystic ovarian syndrome (PCOS) as two extremes of ovarian reserve (deficient and excessive respectively) to investigate the role of AMH and sex hormones in follicular growth. METHODS: A retrospective cross-sectional survey was performed. The patients assessed AMH and basal sex hormones in the Second Hospital of Zhejiang University from April 2016 to March 2019 were involved in this study. Serum AMH and sex hormone concentrations were tested with electrochemiluminescence method. Stepwise linear regression and binary logistic regression was used to determine the predictors of AMH level and to explore the involved factors determining DOR and PCOS. RESULTS: In the present study, we found that age and follicle-stimulating hormone (FSH) were main negative correlation factors, and luteinizing hormone (LH) and testosterone (T) were main positive factors of AMH. In DOR group, age, FSH and estradiol (E2) increased and T decreased, while in PCOS group, LH and T increased. Binary logistic regression found that age, weight, FSH, E2, and T were the significant factors which independently predicted the likelihood of DOR, and that age, body mass index (BMI), AMH, LH, and T predicted the likelihood of PCOS. CONCLUSIONS: Our study demonstrated that age, FSH, and T were factors that most closely correlated with AMH level, and T was involved in both DOR and PCOS. Since DOR and PCOS are manifested with insufficient AMH and excessive AMH respectively, it is suggested that total testosterone correlated with AMH closely and plays an important role in follicular growth. More attention should be given to testosterone level during controlled ovarian hyperstimulation (COH) process.

Basonuclin 1 deficiency causes testicular premature aging: BNC1 cooperates with TAF7L to regulate spermatogenesis.

Basonuclin (BNC1) is expressed primarily in proliferative keratinocytes and gametogenic cells. However, its roles in spermatogenesis and testicular aging were not clear. Previously we discovered a heterozygous BNC1 truncation mutation in a premature ovarian insufficiency pedigree. In this study, we found that male mice carrying the truncation mutation exhibited progressively fertility loss and testicular premature aging. Genome-wide expression profiling and direct binding studies (by chromatin immunoprecipitation sequencing) with BNC1 in mouse testis identified several spermatogenesis-specific gene promoters targeted by BNC1 including kelch-like family member 10 (Klhl10), testis expressed 14 (Tex14), and spermatogenesis and centriole associated 1 (Spatc1). Moreover, biochemical analysis showed that BNC1 was associated with TATA-box binding protein-associated factor 7 like (TAF7L), a germ cell-specific paralogue of the transcription factor IID subunit TAF7, both in vitro and in testis, suggesting that BNC1 might directly cooperate with TAF7L to regulate spermatogenesis. The truncation mutation disabled nuclear translocation of the BNC1/TAF7L complex, thus, disturbing expression of related genes and leading to testicular premature aging. Similarly, expressions of BNC1, TAF7L, Y-box-binding protein 2 (YBX2), outer dense fiber of sperm tails 1 (ODF1), and glyceraldehyde-3-phosphate dehydrogenase, spermatogenic (GAPDHS) were significantly decreased in the testis of men with non-obstructive azoospermia. The present study adds to the understanding of the physiology of male reproductive aging and the mechanism of spermatogenic failure in infertile men.

Intrauterine exposure to hyperglycemia retards the development of brown adipose tissue.

Brown adipose tissue (BAT) is an exclusive tissue of nonshivering thermogenesis. It is fueled by lipids and glucose and involved in energy and metabolic homeostasis. Intrauterine exposure to hyperglycemia during gestational diabetes mellitus may result in abnormal fetal development and metabolic phenotypes in adulthood. However, whether intrauterine hyperglycemia influences the development of BAT is unknown. In this study, mouse embryos were exposed to the intrauterine hyperglycemia environment by injecting streptozocin into pregnant mice at 1 d post coitum (dpc). The structure of BAT was examined by hematoxylin and eosin staining and immunohistochemical analysis. The glucose uptake in BAT was measured in vivo by [18F]-fluoro-2-deoxyglucose-micro-positron emission tomography. The gene expression in BAT was determined by real-time PCR, and the 5'-C-phosphate-G-3' site-specific methylation was quantitatively analyzed. Intrauterine hyperglycemia exposure resulted in the impaired structure of BAT and decreased glucose uptake function in BAT in adulthood. The expressions of the genes involved in thermogenesis and mitochondrial respiratory chain in BAT, such as Ucp1, Cox5b, and Elovl3, were down-regulated by intrauterine hyperglycemia exposure at 18.5 dpc and at 16 wk of age. Furthermore, higher methylation levels of Ucp1, Cox5b, and Elovl3 were found in offspring of mothers with streptozotocin-induced diabetes. Our results provide the evidence for enduring inhibitory effects of intrauterine hyperglycemia on BAT development in offspring. Intrauterine hyperglycemia is associated with increased DNA methylation of the BAT specific genes in offspring, which support an epigenetic involvement.-Yu, D.-Q., Lv, P.-P., Yan, Y.-S., Xu, G.-X., Sadhukhan, A., Dong, S., Shen, Y., Ren, J., Zhang, X.-Y., Feng, C., Huang, Y.-T., Tian, S., Zhou, Y., Cai, Y.-T., Ming, Z.-H., Ding, G.-L., Zhu, H., Sheng, J.-Z., Jin, M., Huang, H.-F. Intrauterine exposure to hyperglycemia retards the development of brown adipose tissue.

Construction of implantation failure related lncRNA-mRNA network and identification of lncRNA biomarkers for predicting endometrial receptivity.

Insufficient endometrial receptivity is a major factor leading to implantation failure (IF), and the traditional way of morphological observation of endometrium cannot determine the condition of receptivity sufficiently. Considering that long-noncoding RNAs (lncRNAs) regulate endometrial receptivity and competing endogenous RNA (ceRNA) mechanism works in plenty of biological processes, ceRNA is likely to function in the pathology of IF. In the present study, we aim to construct an implantation failure related lncRNA-mRNA network (IFLMN), and to identify the key lncRNAs as the candidates for predicting endometrial receptivity. The global background network was constructed based on the presumed lncRNA-miRNA and miRNA-mRNA pairs obtained from lncRNASNP and miRTarBase. Differentially expressed genes (DEGs) of IF were calculated using the data of GSE26787, and then re-annotated as differentially expressed mRNAs (DEMs) and lncRNAs (DELs). IFLMN was constructed by hypergeometric test, including 255 lncRNA-mRNA pairs, 10 lncRNAs, and 212 mRNAs. Topological analysis determined the key lncRNAs with the highest centroid. Functional enrichment analyses were performed by unsupervised clustering, GO classification, KEGG pathway, and co-expression module analyses, achieving six key lncRNAs and their ceRNA sub-networks, which were involved in immunological activity, growth factor binding, vascular proliferation, apoptosis, and steroid biosynthesis in uterus and prepared endometrium for embryo implantation. Sixteen endometrial samples were collected during mid-luteal phase, including 8 recurrent implantation failure (RIF) or recurrent miscarriage (RM) women and 8 controls who conceived successfully. Quantitative real-time PCR was performed to compare the expression of the above six lncRNAs, which validated that the expression of all these lncRNAs was significantly elevated in endometrium of RIF/RM patients. Further studies are needed to investigate the underlying mechanism, and the lncRNAs may be developed into predictive biomarkers for endometrial receptivity.

Maternal high estradiol exposure alters CDKN1C and IGF2 expression in human placenta.

INTRODUCTION: The increased maternal estradiol (E2) concentrations induced by assisted reproductive technology (ART) result in lower birth weight of offspring, which is associated with increased risk of adult diseases. However, the exact mechanism remains unknown. The present study investigated the effect of high E2 exposure on the expression of imprinted genes CDKN1C and IGF2 in human placentas and the DNA methylation status of their differential methylation regions (DMRs). METHODS: The mRNA expression of CDKN1C and IGF2 in human placentas and the human trophoblast cells (HTR8) treated with E2 were investigated by reverse transcription-real time polymerase chain reaction (PCR). The DNA methylation of their DMRs were investigated by sodium bisulfite sequencing. RESULTS: CDKN1C and IGF2 were significantly up-regulated in ART conceived placentas. The mean birth weight of ART singletons was significantly lower than that of naturally conceived (NC) ones, with the increased percentage of small-for-gestational-age (SGA) birth. The DNA methylation was significantly down-regulated in the DMR of CDKN1C (KvDMR1) and up-regulated in the DMR of IGF2 (H19 DMR) in ART placentas. The treatment of E2 altered the expression of the two genes and the DNA methylation of their DMRs in HTR8 to a similar tendency as in vivo. DISCUSSION: The maternal high E2 levels after ART up-regulate the expression of imprinted genes in human placentas through epigenetic modifications, which influences the growth potential of the offspring. Further studies are needed to follow up the growth and development of the ART offspring.

Ovarian stimulation perturbs methylation status of placental imprinting genes and reduces blood pressure in the second generation offspring.

OBJECTIVE(S): Assisted reproductive technology (ART) is associated with DNA methylation dysfunction of offspring. However, it is unclear whether ovarian stimulation (OS) is responsible for DNA methylation dysfunction of offspring STUDY DESIGN: We built the first-generation (F1) and second-generation (F2) offspring mice model of ovarian stimulation. Bodyweight of F1 and F2 were measured. Expression levels of several imprinted genes (Impact, H19, Igf2, Plagl1, Mest, and Snrpn) in F1 placenta were tested. Methylation status of Plagl1 and H19 promoters was examined with bisulfite sequencing. Glucose tolerance, blood pressure, and heart rate were evaluated in F2 mice. RESULTS: The OS F1 showed elevated bodyweights in the 2nd, 3rd and 4th weeks, but the difference disappeared in the 5th week. Plagl1 was down-regulated in OS F1. Promoters of Plagl1 and H19 were also hypermethylated in OS F1. F2 of OS mice had the similar bodyweight and glucose tolerance compared with the control F2. However, F2 of OS ♂F1+OS♀ F1 showed the decreased systolic pressure, diastolic pressure, and heart rate. CONCLUSIONS: Ovarian stimulation perturbs expression levels and methylation status of imprinted genes in offspring. The effect of ovarian stimulation may be passed to F2.

Maternal High Estradiol Exposure is Associated with Elevated Thyroxine and Pax8 in Mouse Offspring.

Our previous studies have shown that maternal high estradiol (E2) environment increased the risk of thyroid dysfunction in offspring. However, the mechanism involved remains unexplored. To evaluate the thyroid function of offspring after high E2 exposure and to explore the underlying mechanism, we established a high E2 mouse model of early pregnancy, and detected thyroid hormones of their offspring. In thyroids of offspring, the expressions of Tg, Nis, Tpo, Pax8, and Titf1 and CpG island methylation status of Pax8 and genes involved in methylation were analyzed. We found that thyroxine (T4) and FT4 levels of offspring were obviously increased in the high-E2 group, especially in females. In both 3- and 8-week-old offspring of the high-E2 group, Pax8 was significantly up-regulated in thyroid glands, accompanied by the abnormal CpG island methylation status in the promoter region. Furthermore, Dnmt3a and Mbd1 were obviously down-regulated in thyroids of the high E2 group. Besides, the disturbance of thyroid function in females was more severe than that in males, implying that the effects were related to gender. In summary, our study indicated that maternal high E2 exposure disturbed the thyroid function of offspring through the dysregulation and abnormal DNA methylation of Pax8.

IGFBP1 Involved in the Decreased Birth Weight Due to Fetal High Estrogen Exposure in Mice.

Our previous study indicated that maternal high-estrogen environment in the first trimester is correlated with increased risks of low birth weight (LBW) and adult diseases. The present study aimed to establish an animal model to confirm such an effect in mice, and to further explore the mechanism involved. A mouse model with high estradiol (E2) exposure during early pregnancy was established, and the birth weight, growth after birth, and expression levels of insulin-like growth factor-binding protein 1 (IGFBP1) of pups were examined. Meanwhile, IGFBP1 expression after treatment of E2 was examined in a HepG2 hepatoma cell line. We found that after exposure to a high-E2 environment the weight of the pups decreased significantly, not only before but also after birth. Meanwhile, both mRNA and protein expressions of IGFBP1 were elevated in placenta and liver tissues. Furthermore, the level of IGFBP1 in the HepG2 cell line was elevated by the treatment of E2, whereas this effect was blocked by estrogen receptor antagonist ICI 182780. In summary, maternal high estrogen up-regulates expression of IGFBP1 in placenta and fetal livers, which contributes to LBW and decreases body weight in offspring.

High Maternal Serum Estradiol Levels Induce Dyslipidemia in Human Newborns via a Hepatic HMGCR Estrogen Response Element.

UNLABELLED: While the intrauterine environment is essential for the health of offspring, the impact of high maternal serum estradiol (E2) on lipid metabolism in offspring and the mechanisms are unknown. We found that ovarian stimulation (OS) could result in high E2 levels in women throughout pregnancy. Strikingly, their newborns showed elevated total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) levels that were positively related with E2 in newborns. In vitro, E2 dose-dependently stimulated TC and LDL-C secretion, and increased expression of the cholesterol synthesis rate-limiting enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) in HepG2 cells and mouse fetal hepatocytes. In vivo, high maternal E2 was detected and fetal livers also showed significantly higher HMGCR expression in an OS mouse model. Notably, an estrogen response element (ERE) was identified in the HMGCR promoter, indicating that high maternal serum E2 could up-regulate HMGCR expression in fetal hepatocytes via an ERE that in turn induces elevated levels of TC and LDL-C in offspring. CONCLUSION: OS can induce a high maternal E2 environment, which up-regulates HMGCR expression in fetal hepatocytes via an ERE in the promoter, and induces elevated levels of TC and LDL-C in newborns that may be related to increased risk of metabolic disease in adulthood.

The association between polymorphism of INSR and polycystic ovary syndrome: a meta-analysis.

Polycystic ovary syndrome (PCOS) is the most common gynecological endocrine disorder. The genetic background is believed to play a crucial role in the pathogenesis of PCOS. In recent years, the role of insulin receptor (INSR) polymorphisms in PCOS predisposition has attracted much attention. We performed a meta-analysis to investigate the association between the single nucleotide polymorphisms (SNPs) of INSR and PCOS. Published literature from Pubmed, Embase, and Cochrane CENTRAL was retrieved up until 7 August 2014. A total of 20 case-control studies including 23,845 controls and 17,460 PCOS cases with an average Newcastle-Ottawa quality assessment scale (NOS) score of 6.75 were analyzed. Ninety-eight SNPs distributed in 23 exons and the flanking regions of INSR were investigated, among which 17 SNPs were found to be associated with PCOS. Three SNPs detected in more than three studies were selected for further analyses. Twelve studies including 1158 controls and 1264 PCOS cases entered the analysis of rs1799817, but no significant association was found for every genotype (p > 0.05). Further subgroup stratification by ethnicity and weight did not lead to discovery of significant correlation (p > 0.05). For rs2059806, four studies including 442 controls and 524 PCOS cases were qualified for meta-analysis, and no significant association with PCOS was found for any genotype (p > 0.05). Four studies including 12,830 controls and 11,683 PCOS cases investigated the correlation between rs2059807 and PCOS, and five of the six cohorts indicated a significant impact. Our current meta-analysis suggests no significant correlation between rs1799817/rs2059806 SNPs and susceptibility of PCOS, while rs2059807 could be a promising candidate SNP that might be involved in the susceptibility of PCOS.

Altered thyroid hormone profile in offspring after exposure to high estradiol environment during the first trimester of pregnancy: a cross-sectional study.

BACKGROUND: The increasing number of babies conceived by in vitro fertilization and embryo transfer (IVF-ET) shifts concern from pregnancy outcomes to long-time health of offspring. Maternal high estradiol (E2) is a major characteristic of IVF-ET and lasts throughout the first trimester of pregnancy. The fetal thyroid develops during this period and may thus be affected by exposure to the supra-physiological E2. The aim of this study is to investigate whether the high E2 maternal environment in the first trimester increases the risk of thyroid dysfunction in children born following IVF-ET. METHODS: A cross-sectional survey design was used to carry out face-to-face interviews with consecutive children attending the hospital. A total of 949 singletons born after fresh embryo transfer (ET) (n=357), frozen ET (n=212), and natural conception (NC) (n=380), aged 3 to 10 years old, were included. All children were thoroughly examined. Meanwhile, another 183 newborns, including 55 fresh ET, 48 frozen ET, and 80 NC were studied. Levels of serum T3, FT3, T4, FT4, and TSH and levels of maternal E2 at different stages of the first trimester were examined. RESULTS: The mean serum E2 levels of women undergoing fresh ET during the first trimester of pregnancy were significantly higher than those of the women undergoing frozen ET or following NC. The thyroid hormone profile, especially the levels of T4, FT4, and TSH, were significantly increased in 3- to 10-year-old children conceived by fresh ET compared to NC. The same tendency was confirmed in newborns. However, levels of T4 and TSH in the frozen ET group were nearer to that of the NC group. Furthermore, levels of T4 and FT4 in fresh ET were positively correlated with maternal serum levels of E2 during early pregnancy. CONCLUSIONS: The maternal high E2 environment in the first trimester is correlated with increased risk of thyroid dysfunction. Frozen ET could reduce risks of thyroid damage in children conceived by IVF. Further studies are needed to confirm these findings and to better determine the underlying molecular mechanisms and clinical significance. TRIAL REGISTRATION: ChicCTR-OCC-14004682 (22-05-2014).

High maternal serum estradiol environment in the first trimester is associated with the increased risk of small-for-gestational-age birth.

CONTEXT: There are increasing concerns that a disrupted endocrine environment may disturb the growth of the fetus. Assisted reproductive technology (ART) situates gamete/embryo in a supraphysiological estradiol (E2) environment and, thus, provides an ideal model to investigate this problem. OBJECTIVE: Our objective was to investigate whether the maternal high-E2 environment in the first trimester increases the risks of low birth weight (LBW) and small-for-gestational-age (SGA) birth. METHODS: In total, 8869 singletons born after fresh embryo transfer (ET) (n = 2610), frozen ET (n = 1039), and natural conception (NC) (n = 5220) and their mothers were included. Birth weight, LBW, SGA, and maternal serum E2 levels were investigated. RESULTS: The mean serum E2 levels of women undergoing fresh ET at 4 and 8 weeks of gestation were significantly higher than those of the women undergoing frozen ET and the women with NC (P < .01). Serum E2 levels of women undergoing fresh ET at 4 and 8 weeks of gestation were positively correlated to those on the day of human chorionic gonadotropin (hCG) administration (r = 0.5 and r = 0.4, respectively; P < 0.01). The birth weight after fresh ET was significantly lower than that after frozen ET and NC (P < 0.01), with increased incidence of LBW and SGA (P < .05). Furthermore, in the fresh ET group, singletons of mothers with high E2 levels (≥10460 pmol/L on the day of hCG administration) had higher risks of LBW (P < .01) and SGA (P < .01) than those with low E2 levels, and maternal serum E2 level on the day of hCG administration negatively correlated with the birth weight (P < .01). CONCLUSIONS: The maternal high-E2 environment in the first trimester is correlated with increased risks of LBW and SGA. Evaluation of serum E2 before ET should be adopted to reduce the possibility of high E2 exposure to gamete/embryo.

Insufficient maintenance DNA methylation is associated with abnormal embryonic development.

BACKGROUND: Early pregnancy loss (EPL) is a frustrating clinical problem, whose mechanisms are not completely understood. DNA methylation, which includes maintenance methylation and de novo methylation directed by DNA methyltransferases (DNMTs), is important for embryo development. Abnormal function of these DNMTs may have serious consequences for embryonic development. METHODS: To evaluate the possible involvement of DNA methylation in human EPL, the expression of DNMT proteins and global methylation of DNA were assessed in villous or decidua from EPL patients. The association of maintenance methylation with embryo implantation and development was also examined. RESULTS: We found that DNMT1 and DNMT3A were both expressed in normal human villous and decidua. DNMT1 expression and DNA global methylation levels were significantly down-regulated in villous of EPL. DNMT3A expression was not significantly changed in the EPL group compared to controls in either villous or decidua. We also found that disturbance of maintenance methylation with a DNMT1 inhibitor may result in a decreased global DNA methylation level and impaired embryonic development in the mouse model, and inhibit in vitro embryo attachment to endometrial cells. CONCLUSIONS: Our results demonstrate that defects in DNA maintenance methylation in the embryo, not in the mother, are associated with abnormal embryonic implantation and development. The findings of the current study provide new insights into the etiology of EPL.

Functional expression of large-conductance calcium-activated potassium channels in human endometrium: a novel mechanism involved in endometrial receptivity and embryo implantation.

BACKGROUND: Large-conductance calcium-activated potassium channels (BK(Ca) channels) mediate physiological processes in nonexcitable cells. OBJECTIVE: The aim of the study was to determine BK(Ca) channel expression in human endometrium and its role in endometrial receptivity and embryo implantation. METHODS: BK(Ca) channel expression in human endometrium is described at different phases of the menstrual cycle using quantitative real time-PCR and Western blot techniques. Their effects on embryo implantation were examined using JAr spheroid attachment assays and in vivo mouse model. We examined their effects on endometrial receptivity factors, nuclear factor-κB (NF-κB) activity using quantitative real time-PCR, Western blot, and EMSA analyses. Changes in electrophysiological properties and cytosolic free Ca(2+) were measured in endometrial cells with or without specific BK(Ca) blocker or transfected with BK(Ca) small interfering RNA using patch-clamp and fluorescence analyses, respectively. RESULTS: BK(Ca) channels are expressed in human endometrial cells in a phase-related fashion during the menstrual cycle (proliferative, 0.20 ± 0.02, vs. mid-secretory, 0.72 ± 0.07; P < 0.01). Blocking BK(Ca) channel function or knockdown of endogenous BK(Ca) channel expression not only decreased JAr spheroid attachment rate and embryo implantation rate in mice but also significantly reduced the expression levels of endometrial receptive factors, including leukemia inhibitory factor, integrin ß3, claudin-4, and DKK-1, in human endometrial cells. Blocking BK(Ca) channels also reduced BK(Ca)-regulated NF-κB activity, cytosolic Ca(2+) concentrations, and membrane potentials in human endometrial cells. CONCLUSIONS: These observations demonstrate that BK(Ca) channels: 1) are expressed in endometrial cells; 2) affect embryo implantation by mediating endometrial receptive factors; and 3) alter the activity of NF-κB and homeostasis of Ca(2+) in the human endometrial cells.

Sustained endoplasmic reticulum stress as a cofactor of oxidative stress in decidual cells from patients with early pregnancy loss.

BACKGROUND: Oxidative stress is a common pathological background for different etiologies of early pregnancy loss (EPL). It has been suggested that elevated reactive oxygen species trigger endoplasmic reticulum (ER) stress by influencing ER function. However, it is unclear whether ER stress is associated with EPL. OBJECTIVES: The aim of the study was to determine whether and how ER stress occurs during the development of EPL. APPROACHES: Proteomic analysis was performed on decidua from women with EPL, and then ER stress markers, redox status, apoptotic features, and cell viability were analyzed in EPL decidual cells (DCs). RESULTS: EPL decidua were characterized by decreased levels of glucose-regulated protein 78 (GPR78) and valosin-containing protein and burdened with ubiquitinated proteins. Evidence of ER stress-induced apoptosis in EPL DCs was demonstrated by extensive dilation of ER, morphological features of apoptosis, and activation of caspase-4 and caspase-12. Furthermore, H(2)O(2) reduced the viabilities in both EPL and control DCs, whereas EPL DCs were more vulnerable to additional OS challenge than the controls as a result of failed induction of GRP78 expression. The cell survival percentages of DCs were dose-dependently reduced by H(2)O(2) and could be reversed in the presence of vitamin E. This effect was partly mediated by reducing the amount of misfolded proteins rather than regulating GRP78 expression. CONCLUSIONS: The sum of these observations demonstrate for the first time that sustained ER stress occurs in EPL DCs and the potentially vicious relationship between ER stress and oxidative stress is likely to play an important role in the development of EPL.