Metabolic Profiles of Offspring Born From Biopsied Embryos from Toddlerhood to Preschool Age.

CONTEXT: Embryo biopsy, which is necessary for preimplantation genetic testing (PGT), has not been fully investigated regarding its potential influences and safety. Previous studies of children born from biopsied embryos (PGT children) have primarily centered around their growth and neuropsychological development, while there remains limited knowledge concerning their endocrine and metabolic parameters. OBJECTIVE: This study aims to examine the effect of trophectoderm (TE) biopsy on metabolic outcomes for PGT children. METHODS: A total of 1267 children from the Center for Reproductive Medicine, Shandong University, who were conceived through in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) with and without PGT, were analyzed in this study. Three sets of measurements pertaining to growth and metabolism were taken at each predetermined follow-up time point. The linear regression models within a generalized estimating equation were employed to examine the associations between the PGT and each outcome measure and the approach of false discovery rate was used to correct for multiple comparisons. RESULTS: After controlling for confounding factors and correcting for multiple comparisons, no statistically significant difference was identified in any of the measured variables between the PGT children and children conceived by IVF alone (IVF children) and children conceived through IVF using ICSI (ICSI children). The same is true also for age- or sex-based subgroup analyses. CONCLUSION: Between the ages of 1 and 5 years, there are no clinically adverse metabolic outcomes observed in PGT children, and their metabolic profiles are essentially identical to those of IVF children and ICSI children.

Excessive Exogenous Gonadotropins and Genetic and Pregnancy Outcomes After Euploidy Embryo Transfer: A Secondary Analysis of a Randomized Clinical Trial.

Importance: The safety of exogenous gonadotropin treatment, based on its effect on embryos and pregnancy outcomes, remains inconclusive. Objective: To evaluate the associations of different doses and durations of gonadotropins with embryonic genetic status and pregnancy outcomes after euploid embryo transfer in couples with infertility. Design, Setting, and Participants: This study was a post hoc analysis of a multicenter randomized clinical trial (RCT) conducted at 14 reproductive centers throughout China from July 2017 to June 2018 that evaluated the cumulative live birth rate with or without preimplantation genetic testing for aneuploidy (PGT-A) among couples with infertility and good prognosis. The PGT-A group from the original RCT was selected for secondary analysis. Patients were divided into 4 groups according to the total dosage of exogenous gonadotropins and treatment duration: group 1 (≤1500 IU and <10 days), group 2 (≤1500 IU and ≥10 days), group 3 (>1500 IU and <10 days), and group 4 (>1 500 IU and ≥10 days). Group 1 served as the control group. Data were analyzed from June through August 2023. Interventions: Blastocyst biopsy and PGT-A. Main outcomes and measures: The primary outcomes were embryonic aneuploidy, embryonic mosaicism, and cumulative live birth rates after euploid embryo transfer. Results: A total of 603 couples (mean [SD] age of prospective mothers, 29.13 [3.61] years) who underwent PGT-A were included, and 1809 embryos were screened using next-generation sequencing. The embryo mosaicism rate was significantly higher in groups 2 (44 of 339 embryos [13.0%]; adjusted odds ratio [aOR], 1.69 [95% CI, 1.09-2.64]), 3 (27 of 186 embryos [14.5%]; aOR, 1.98 [95% CI, 1.15-3.40]), and 4 (82 of 651 embryos [12.6%]; aOR, 1.60 [95% CI, 1.07-2.38]) than in group 1 (56 of 633 embryos [8.8%]). There were no associations between gonadotropin dosage or duration and the embryo aneuploidy rate. The cumulative live birth rate was significantly lower in groups 2 (83 of 113 couples [73.5%]; aOR, 0.49 [95% CI, 0.27-0.88]), 3 (42 of 62 couples [67.7%]; aOR, 0.41 [95% CI, 0.21-0.82]), and 4 (161 of 217 couples [74.2%]; aOR, 0.53 [95% CI, 0.31-0.89]) than in group 1 (180 of 211 couples [85.3%]). Conclusions and relevance: In this study, excessive exogenous gonadotropin administration was associated with increased embryonic mosaicism and decreased cumulative live birth rate after euploid embryo transfer in couples with a good prognosis. These findings suggest that consideration should be given to minimizing exogenous gonadotropin dosage and limiting treatment duration to improve embryo outcomes and increase the live birth rate. Trial Registration: ClinicalTrials.gov Identifier: NCT03118141.

Sono-anatomy of the middle cervical sympathetic ganglion verified with pathology.

Objectives: Cervical discomfort and other symptoms may be attributable to the middle cervical sympathetic ganglion. The aim of this study was to explore the sonographic features of this ganglion in anatomical specimens and cadavers and evaluate the feasibility of its visualization using high-resolution ultrasonography. Methods: We examined three cervical sympathetic-ganglion specimens and two fresh cadavers using high-resolution ultrasound to explore the sonographic features of this ganglion. Basic imaging characteristics examined included the shape, echo intensity, and location of the ganglion. Core-needle biopsy was performed to examine the suspected middle cervical sympathetic ganglion in the two fresh cadavers and verify the accuracy of the sonographic identification via pathological examination. Results: The middle cervical sympathetic ganglion appeared on high-resolution ultrasonography as an oval-shaped hypoechoic structure, with at least one continuous hypoechoic line connected to each ending in the anatomical specimens and fresh cadavers, and it was distinctly different from the adjacent lymph nodes. Discussion: Based on an adequate understanding of both its location and sonographic features, the direct visualization of the middle cervical sympathetic ganglion using high-resolution ultrasonography is feasible.

Transient receptor potential M2 channel in the hypothalamic preoptic area and its impact on thermoregulation during menopause.

BACKGROUND: Decreased estrogen levels can cause abnormal thermosensitivity of the preoptic area (POA) in the hypothalamus during menopause, which may cause hot flashes. Thermosensitive transient receptors (ThermoTRPs) affect the thermosensitivity of neurons. It is worth exploring whether ThermoTRPs change under low estrogen state and participate in the abnormal thermoregulation of POA. METHODS: Adult female Sprague-Dawley rats were randomly divided into sham operation (SHAM), ovariectomy (OVX) and estrogen treatment after ovariectomy (OVX+E) groups. Under 10 â„ƒ, 18 â„ƒ, 25 â„ƒ, 37 â„ƒ and 45 â„ƒ incubations, their skin temperature was monitored and the expression of TRPA1, TRPM8, TRPM2, and TRPV1 in POA were investigated. RESULTS: The skin temperature of ovariectomized rats changed faster and more dramatically under different incubation temperatures. The results at mRNA level show that only the expression of TRPM2 decreased in POA of OVX group compared with the other two groups at 25 â„ƒ, TRPA1 expression in POA of the three groups increased at 10 â„ƒ, TRPM8 increased at 10 â„ƒ and 18 â„ƒ, TRPV1 increased at 10 â„ƒ and 45 â„ƒ, while the expression of TRPM2 decreased at 10 â„ƒ and 18 â„ƒ and increased at 37 â„ƒ and 45 â„ƒ. In all these cases, the magnitudes of the changes were less in the OVX group relative to the other two groups. The further immunohistochemical and Western blot results of TRPM2 and the activated TRPM2 positive cells labeled by c-Fos were consistent with the results of mRNA level. CONCLUSIONS: The expression and thermosensitivity of TRPM2 in POA changed greatly under different incubation temperatures, but the changes in ovariectomized rats were less. This may be the key factor triggering thermoregulation dysfunction under low estrogen and may cause hot flashes.

H3K27me3-modulated Hofbauer cell BMP2 signalling enhancement compensates for shallow trophoblast invasion in preeclampsia.

BACKGROUND: Preeclampsia (PE) is a common hypertensive pregnancy disorder associated with shallow trophoblast invasion. Although bone morphogenetic protein 2 (BMP2) has been shown to promote trophoblast invasion in vitro, its cellular origin and molecular regulation in placenta, as well as its potential role in PE, has yet to be established. Additionally, whether BMP2 and/or its downstream molecules could serve as potential diagnostic or therapeutic targets for PE has not been explored. METHODS: Placentas and sera from PE and healthy pregnant women were subjected to multi-omics analyses, immunoblots, qPCR, and ELISA assays. Immortalized trophoblast cells, primary cultures of human trophoblasts, and first-trimester villous explants were used for in vitro experiments. Adenovirus expressing sFlt-1 (Ad Flt1)-induced PE rat model was used for in vivo studies. FINDINGS: We find globally decreased H3K27me3 modifications and increased BMP2 signalling in preeclamptic placentas, which is negatively correlated with clinical manifestations. BMP2 is derived from Hofbauer cells and epigenetically regulated by H3K27me3 modification. BMP2 promotes trophoblast invasion and vascular mimicry by upregulating BMP6 via BMPR1A-SMAD2/3-SMAD4 signalling. BMP2 supplementation alleviates high blood pressure and fetal growth restriction phenotypes in Ad Flt1-induced rat PE model. INTERPRETATION: Our findings demonstrate that epigenetically regulated Hofbauer cell-derived BMP2 signalling enhancement in late gestation could serve as a compensatory response for shallow trophoblast invasion in PE, suggesting opportunities for diagnostic marker and therapeutic target applications in PE clinical management. FUNDING: National Key Research and Development Program of China (2022YFC2702400), National Natural Science Foundation of China (82101784, 82171648, 31988101), and Natural Science Foundation of Shandong Province (ZR2020QH051, ZR2020MH039).

Epidural Pulsation Accelerates the Drainage of Brain Interstitial Fluid.

Unhindered transportation of substances in the brain extracellular space (ECS) is essential for maintaining brain function. Regulation of transportation is a novel strategy for treating ECS blockage-related brain diseases, but few techniques have been developed to date. In this study, we established a novel approach for accelerating the drainage of brain interstitial fluid (ISF) in the ECS using minimally invasive surgery, in which a branch of the external carotid artery is separated and implanted epidurally (i.e., epidural arterial implantation [EAI]) to promote a pulsation effect on cerebrospinal fluid (CSF) in the frontoparietal region. Tracer-based magnetic resonance imaging was used to evaluate the changes in ISF drainage in rats 7 and 15 days post-EAI. The drainage of the traced ISF from the caudate nucleus to ipsilateral cortex was significantly accelerated by EAI. Significant increases in the volume fraction of the ECS and molecular diffusion rate were demonstrated using the DECS-mapping technique, which may account for the mechanisms underlying the changes in brain ISF. This study provides a novel perspective for encephalopathy treatment via the brain ECS.

Does the Risk of Embryo Abnormality Increase in PCOS Women? A Secondary Analysis of a Multicenter, Randomized Controlled Trial.

CONTEXT: Some studies have reported the early miscarriage rate is higher in polycystic ovary syndrome (PCOS) women. However, there is a lack of evidence as to whether the risk of embryo abnormalities increases in PCOS women. OBJECTIVE: This work aimed to evaluate the association between PCOS and embryo ploidy. METHODS: A secondary analysis of a multicenter, randomized controlled trial was conducted from July 2017 to June 2018. The original intent was to identify whether preimplantation genetic test for aneuploidy (PGT-A) improves the live birth rate as compared with in vitro fertilization (IVF). From 14 reproductive centers, 190 patients diagnosed with PCOS and 1:1 age-matched non-PCOS patients were chosen from a PGT-A group. A total of 380 patients with 1118 embryos were included in our study. Intervention included women diagnosed with PCOS, and the main outcome measures were embryonic aneuploidy and embryonic mosaic. RESULTS: After adjusting for potential confounders, the rate of embryonic aneuploidy and embryonic mosaic in the PCOS group were comparable with the control group (embryonic aneuploid rate PCOS group: 14.0% vs control group: 18.3%, adjusted OR [95% CI]: 0.78 [0.54, 1.12]; P = .19; embryonic mosaic rate 10.9% vs 10.1%, adjusted OR [95% CI]: 0.91 [0.59, 1.40]; P = .66). We further stratified PCOS women into 4 groups according to phenotype. The rate of aneuploid and mosaic embryos was comparable between each PCOS phenotype and control group. There was still no significant difference of embryonic aneuploid and embryo mosaic rates among the 4 phenotypes. CONCLUSION: The risk of aneuploid and mosaic embryos did not increase in PCOS women. Thus, we suggest that the miscarriage rate arising from abnormal embryonic chromosomes could be similar between PCOS and non-PCOS women.

Glutamatergic and GABAergic neurons in the preoptic area of the hypothalamus play key roles in menopausal hot flashes.

During menopause, when estrogen levels are low, abnormalities in the hypothalamic preoptic area (POA) of the thermoregulatory center can cause hot flashes. However, the involved neural population has not been identified. Proteomics showed that under low estrogen, differentially expressed proteins in the hypothalamus were associated with glutamatergic and GABAergic synapses. RNAscope, Western blotting and qRT-PCR indicated that the number of glutamatergic neurons in the POA was decreased, while the number of GABAergic neurons was increased. Chemogenetics showed that the rat body temperature decreased slowly after glutamatergic neurons were activated and increased quickly after glutamatergic neurons were inhibited, while it increased quickly after GABAergic neurons were activated and decreased slowly after GABAergic neurons were inhibited. RNAscope, immunofluorescence, Western blotting and qRT-PCR further showed that glutamate decarboxylase (GAD) 1 expression in the POA was increased, while GAD2 expression in the POA was decreased; that thermosensitive transient receptor potential protein (ThermoTRP) M (TRPM) 2 expression in glutamatergic neurons was decreased, while TRPM8 expression in GABAergic neurons was increased; and that estrogen receptor (ER) α and ß expression in the POA was decreased, and ERα and ERß expressed in both glutamatergic and GABAergic neurons. Estrogen therapy corrected these abnormalities. In addition, CUT&Tag and Western blot after injection of agonists and inhibitors of ERs showed that ERα and ERß were both transcription factors in glutamatergic and GABAergic synapses. Mechanistically, during menopause, estrogen may regulate the transcription and expression of GADs and ThermoTRPs through ERs, impacting the number and function of glutamatergic and GABAergic neurons, resulting in unbalanced heat dissipation and production in the POA and ultimately triggering hot flashes.

Deficiency of RARα Suppresses Decidualization via Downregulating CEBPB Transcription in Women With Recurrent Implantation Failure.

Background: Recurrent implantation failure (RIF) is a disease associated with endometrial receptivity dysfunction. Retinoic acid receptor alpha (RARα) is an important protein in many biological processes, such as differentiation and development. However, the exact underlying mechanism whereby RARα affects RIF remains unknown. This study investigated RARα expression and its contribution in the mid-luteal phase endometria of patients with RIF. Methods: The expression levels of RARα and CCAAT/enhancer-binding protein (C/EBP) ß in the endometria of the RIF and normal group were investigated using western blotting and immunohistochemistry. In in vitro experiments, immortal telomerase-transformed human endometrial stromal cells (T-HESCs) were incubated with medroxyprogesterone-17-acetate (MPA) and cyclic adenosine monophosphate (cAMP) for 4 days to induce decidualization. The expression levels of the decidualization markers prolactin (PRL) and insulin-like growth factor-binding protein-1 (IGFBP-1) were determined using quantitative polymerase chain reaction. RARα was knocked down using a small interfering RNA, and C/EBPß was overexpressed from an adenoviral vector. The transcriptional regulation of CEBPB by RARα was determined by chromatin immunoprecipitation (ChIP) assay and luciferase assays. Results: We found that the expression levels of RARα decreased in the mid-luteal endometria of RIF patients. After 4 days of decidualization induction in vitro, RARα knockdown impaired the decidualization of T-HESCs and downregulated the expression of C/EBPß. The restoration of C/EBPß expression rescued the RARα knockdown-induced suppression of T-HESC decidualization. In ChIP analysis of lysates from decidualized T-HESCs, the CEBPB promoter region was enriched in chromatin fragments pulled down using an anti-RARα antibody. However, the relationship between CEBPB transcription and RARα expression levels was only observed when the decidualization of T-HESCs was induced by the addition of cAMP and MPA. To identify the binding site of RARα/retinoid X receptor α, we performed luciferase assays. Mutation of the predicted binding site in CEBPB (-2,009/-1,781) decreased the transcriptional activity of the reporter. To confirm this mechanism, the expression levels of C/EBPß in the mid-luteal endometria of RIF patients were determined and found to decrease with decreased RARα expression levels. Conclusion: A deficiency of RARα expression in the mid-luteal endometrium inhibits decidualization due to the downregulation of CEBPB transcription. This is a potential mechanism contributing to RIF.

Non-Assisted Hatching Trophectoderm Biopsy Does Not Increase The Risks of Most Adverse Maternal and Neonatal Outcome and May Be More Practical for Busy Clinics: Evidence From China.

OBJECTIVE: This study was conducted in order to investigate whether non-assisted hatching trophectoderm (TE) biopsy increases the risks of adverse perinatal outcomes in livebirths following elective single cryopreserved-thawed blastocyst transfer. PATIENTS AND METHODS: A total of 5,412 cycles from 4,908 women who achieved singleton livebirths between 2013 and 2019 were included in this retrospective cohort study. All embryos in this study were fertilized by intracytoplasmic sperm injection (ICSI) and cryopreserved through vitrification. The main intervention is to open the zona pellucida (ZP) of day 5/6 blastocyst immediately for biopsy without pre-assisted hatching. The main outcome measures are the common maternal and neonatal outcomes, including hypertensive disorders of pregnancy (HDPs), gestational diabetes mellitus (GDM), abnormal placentation, abnormalities in umbilical cord and amniotic fluid, preterm birth, cesarean section, low birth weight, postpartum hemorrhage, and prolonged hospital stay (both mothers and infants). The generalized estimation equation (GEE) was used to control the effects of repeated measurements. The non-conditional logistic regression model was used to examine the associations between embryo biopsy status and each adverse perinatal event. Given that the selection bias and changes in learning curve might affect the results, we selected 1,086 similar (matching tolerance = 0.01) cycles from the ICSI group via propensity score matching (PSM) for second comparisons and adjustment (conditional logistic regression). RESULTS: After adjusting for confounders, we confirmed that the non-assisted hatching protocol did not increase the risks of most adverse maternal and neonatal outcomes. Despite this, there were increased risks of GDM (aOR: 1.522, 95% CI: 1.141-2.031) and umbilical cord abnormalities (aOR: 11.539, 95% CI: 1.199-111.067) in the biopsy group. In the second comparisons after PSM, GDM incidence in the biopsy group was still higher (7.26% vs. 5.16%, P = 0.042), yet all measurement outcomes were equally likely to occur in both groups after the second adjustment. CONCLUSIONS: The non-assisted hatching TE biopsy does not increase the risks of most adverse perinatal outcomes. However, there is a higher GDM incidence in the biopsy group, and this association warrants further study. Considering its safety and simplicity, the non-assisted hatching protocol has the potential to become the preferred option for TE biopsy, especially in busy clinics and IVF laboratories.

A Novel Exon Polymorphism of TP53 Associated with Recurrent Pregnancy Loss in a Chinese Han Population.

BACKGROUND: To identify recurrent pregnancy loss (RPL)-related genetic variants in exons of TP53 gene in a population of Chinese Han women. METHODS: This study is a case control study. The cases comprised 90 Chinese Han women with RPL. Another 90 women with at least one child and not more than one miscarriage were recruited as the controls. All exons of TP53 were amplified from genomic DNA and sequenced. RESULTS: A total of five single-nucleotide polymorphisms (SNPs) were identified in both RPL and control women, namely rs1642785 G>C, rs1042522 G>C, rs4968187 G>A, rs17884306 G>A, and rs55817367 A>G. A significant difference was only observed for rs17884306 between cases and controls. The wild type G allele was associated with an increased risk of RPL. AA+GA genetic variants significantly decreased the risk of RPL compared with GG variant (odds ratio [OR] = 0.315, 95% confidence interval [CI]: 0.125 - 0.793, p = 0.014). Linkage disequilibrium exits between rs17884306 and rs1642785 and A-C double mutant haplotype showed significantly lower risk of RPL compared with G-G wild type haplotype (OR = 0.303, 95% CI: 0.117 - 0.786, p = 0.014). Model based-multifactor dimensionality reduction indicated that the influence of rs17884306 had interaction with the genotypes of four other loci (all p < 0.05). However, rs17884306 G>A did not cause amino acid substitution. CONCLUSIONS: Our study showed that rs17884306 c.826G>A was a novel polymorphism associated with RPL in Chinese Han women. Moreover, the influence of this SNP on RPL is not associated with p53 amino acid sequence.

Association between endometrial thickness and birth weight in fresh IVF/ICSI embryo transfers: a retrospective cohort study of 9273 singleton births.

RESEARCH QUESTION: What is the association between endometrial thickness (EMT) on HCG trigger day and outcomes related to birth weight in fresh IVF and intracytoplasmic sperm injection (ICSI) embryo transfer cycles? DESIGN: A retrospective cohort study of 9273 singleton live births born to women undergoing fresh IVF/ICSI cycles in a single centre between January 2014 and December 2018. Multivariable logistic regression was used to investigate the associations between EMT, low birth weight (LBW) and small for gestational age (SGA). Multivariable-adjusted linear regression models incorporating restricted cubic splines were used to investigate the dose-response relationship between EMT, birth weight and birth weight z-score, respectively. An EMT of 8 mm was set as a reference value. RESULTS: Compared with women with an EMT of 8.0 to < 14.0 mm the risk of delivering a SGA infant was increased with EMT < 8.0 mm (adjusted odds ratio [aOR] 1.78, 95% confidence interval [CI] 1.09 to 2.90) and decreased with EMT ≥ 14.0 mm (aOR 0.57, 95% CI 0.35 to 0.93, respectively). Birth weights of infants born to women with an EMT of 8.0 mm compared with women with EMT of 5.0, 6.0, and 7.0 mm were lower by 120 g (95% CI -175 g to -66 g), 80 g (95% CI -116 g to -44 g), and 40 g (95% CI -58 g to -22 g) respectively; infant birth weight z-scores were also decreased by 0.19 (95% CI -0.27 to -0.10), 0.12 (95% CI -0.18 to -0.07) and 0.06 (95% CI -0.09 to -0.03), respectively. CONCLUSIONS: A thinner endometrium was associated with lower birth weight and birth weight z-score, and higher risk of SGA. Women with a thin endometrium warrant special attention during pregnancy.

circFAM120A participates in repeated implantation failure by regulating decidualization via the miR-29/ABHD5 axis.

Repeated implantation failure (RIF) is a major problem that limits the pregnancy rate associated with assisted reproductive technology. However, the pathogenesis of RIF is still unknown. Recently, the expression levels of circular RNAs (circRNAs) were profiled in the endometrial tissues of patients with RIF. However, the exact role of circRNAs in RIF remains unclear. In our study, we found that circFAM120A levels were significantly down-regulated in the endometrium at the window of implantation in RIF patients compared with non-RIF controls. The suppression of circFAM120A expression inhibited decidualization in human endometrial stromal cells (hESCs). Furthermore, RNA-seq analysis after circFAM120A knockdown revealed ABHD5 as a potential downstream target gene of circFAM120A. As expected, down-regulating ABHD5 in hESCs also inhibited decidualization. Using the starBase and TargetScan databases, we predicted that miR-29 may interact with ABHD5, based on nucleotide sequence matching. Luciferase reporter assay showed that miR-29 bound to the 3' UTR of ABHD5 at the predicted complementary sites. Moreover, miR-29 mimics efficiently reduced ABHD5 expression levels and suppressed the decidualization process, whereas a miR-29 inhibitor partly rescued ABHD5 mRNA expression level and decidualization reduced by the knockdown of circFAM120A. Therefore, circFAM120A modulated decidualization in RIF through the miR-29/ABHD5 axis.

Thin endometrium is associated with the risk of hypertensive disorders of pregnancy in fresh IVF/ICSI embryo transfer cycles: a retrospective cohort study of 9,266 singleton births.

BACKGROUND: Thin endometrial thickness (EMT) has been suggested to be associated with reduced incidence of pregnancy rate after in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) treatment, but the effect of thin endometrium on obstetric outcome is less investigated. This study aims to determine whether EMT affects the incidence of obstetric complications in fresh IVF/ICSI-embryo transfer (ET) cycles. METHODS: We conducted a retrospective cohort study collecting a total of 9266 women who had singleton livebirths after fresh IVF/ICSI-ET treatment cycles at the Center for Reproductive Medicine Affiliated to Shandong University between January 2014 and December 2018. The women were divided into three groups according to the EMT: 544 women with an EMT ≤8 mm, 6234 with an EMT > 8-12 mm, and 2488 with an EMT > 12 mm. The primary outcomes were the incidence of obstetric complications including hypertensive disorders of pregnancy (HDP), gestational diabetes mellitus (GDM), placental abruption, placenta previa, postpartum hemorrhage (PPH) and cesarean section. Multivariable logistic regression analysis was performed to calculate the odds ratios (ORs) and 95% confidence intervals (CIs) for associations between the EMT measured on the day of human chorionic gonadotropin (HCG) trigger and the risk of the outcomes of interest. RESULTS: The HDP incidence rate of pregnant women was highest in EMT ≤ 8 mm group and significantly higher than those in EMT from > 8-12 mm and EMT > 12 mm group, respectively (6.8% versus 3.6 and 3.5%, respectively; P = 0.001). After adjustment for confounding variables by multivariate logistic regression analysis, a thin EMT was still statistically significant associated with an increased risk of HDP. Compared with women with an EMT > 8-12 mm, women with an EMT ≤8 mm had an increased risk of HDP (aOR = 1.853, 95% CI 1.281-2.679, P = 0.001). CONCLUSION: A thin endometrium (≤8 mm) was found to be associated with an increased risk of HDP after adjustment for confounding variables, indicating that the thin endometrium itself is a risk factor for HDP. Obstetricians should remain aware of the possibility of HDP when women with a thin EMT achieve pregnancy through fresh IVF/ICSI-ET treatment cycles.

Roles of TGF-ß Superfamily Proteins in Extravillous Trophoblast Invasion.

Following embryo implantation, extravillous trophoblasts (EVTs) invade the maternal decidua to a certain extent during early pregnancy, which is critical for normal placentation and successful pregnancy in humans. Although sharing a similar protein structure, the transforming growth factor-ß (TGF-ß) superfamily members exert divergent functions in regulating EVT invasion, which contributes to a relative balance of TGF-ß superfamily proteins in precisely modulating this process at the maternal-fetal interface during the first trimester of pregnancy. This review details recent advances in our understanding of the functions of TGF-ß superfamily members and their corresponding receptors, signaling pathways, and downstream molecular targets in regulating human EVT invasion from studies using various in vitro or ex vivo experimental models. Also, the relevance of these discoveries about TGF-ß superfamily members to adverse pregnancy outcomes is summarized. The application of 3D culture trophoblast organoids, single-cell sequencing, and microfluidic assays in EVT invasion studies will help better reveal the molecular mechanisms through which TGF-ß superfamily members regulate human EVT invasion, shedding light on the development of innovative strategies for predicting, diagnosing, treating, and preventing adverse human pregnancy outcomes related to EVT invasion dysfunction.

Deciphering biomarkers of endometriosis by proteomic analysis of eutopic endometrium in infertile patients.

AIM: Endometriosis is associated with infertility. The aim of this study was to examine the overall proteomic changes of eutopic endometrium in infertile women with endometriosis. METHODS: Tandem mass tags combined with multidimensional liquid chromatography and mass spectrometry analyses were used to screen the proteomic profiles of eutopic endometrium from infertile patients with endometriosis (N = 4), compared with that from patients without endometriosis (N = 4). Quantitative proteomic analysis, functional categories and significant pathway analysis were investigated subsequently. RESULTS: In total, 6.698 proteins were identified, among which 5,812 proteins were quantified. Compared with controls, proteomic analysis showed some differentially expressed proteins: 16 up-regulated proteins and 23 down-regulated proteins. Bioinformatics analysis indicated that differentially expressed proteins were involved in humoral immune response pathways, antimicrobial humoral response and regulation of nitric oxide biosynthetic process. Besides, our results showed that alpha-1-acid glycoprotein 2, complement factor B and zinc transporter Zip14 were important resources for investigating potential mechanism of infertility in infertile women with endometriosis. CONCLUSIONS: This study provided a reference proteome map of eutopic endometrium from infertile women with endometriosis. The long-term benefits of using those markers to stratify clinical treatment warrant further investigation.

MiR-148a-3p may contribute to flawed decidualization in recurrent implantation failure by modulating HOXC8.

PURPOSE: To evaluate whether miR-148a-3p overexpression is associated with disrupted decidualization of recurrent implantation failure (RIF). METHODS: Endometrial miRNA and mRNA expression profiles during the implantation window derived from women with and without RIF were identified using microarray and RT-qPCR. Immortalized human endometrial stromal cells (HESCs) were cultured for proliferation and in vitro decidualization assays after enhancing miR-148a-3p expression or inhibiting putative target gene homeobox C8 (HOXC8) expression. RT-qPCR, western blot, and luciferase reporter assays were used to confirm the relationship between miR-148a-3p and HOXC8 gene. RESULTS: MiR-148a-3p was significantly upregulated in RIF endometrial tissues. Forced expression of miR-148a-3p notably attenuated HESC in vitro decidualization. Mechanistic studies revealed that miR-148a-3p directly bounds to the HOXC8 3' untranslated region (3'UTR) and suppressed HOXC8 expressions in both mRNA and protein levels. Further investigations demonstrated that inhibition of HOXC8 in HESCs induced similar effects on decidual process as those induced by miR-148a-3p overexpression. CONCLUSION: Taken together, our findings suggested that elevated miR-148a-3p might account for flawed decidualization in RIF by negatively regulating HOXC8, raising the possibility that miR-148a-3p might be a novel therapeutic target in RIF.

Trophoblast H2S Maintains Early Pregnancy via Regulating Maternal-Fetal Interface Immune Hemostasis.

CONTEXT: Dysregulated immune hemostasis occurs in unexplained recurrent spontaneous abortion (URSA). Synthesized by cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE), hydrogen sulfide (H2S) promotes regulatory T-cell differentiation and regulates immune hemostasis; yet, its role in URSA is elusive. OBJECTIVE: To determine if H2S plays a role in early pregnancy and if dysregulated H2S signaling results in recurrent spontaneous abortion. DESIGN: First trimester placenta villi and decidua were collected from normal and URSA pregnancies. Protein expression was examined by immunohistochemistry and immunoblotting. Human trophoblast HTR8/SVneo and JEG3 cells were treated with H2S donors; HTR8/SVneo cells were transfected with CBS ribonucleic acid interference (RNAi) or complementary deoxyribonucleic acid. Cell migration and invasion were determined by transwell assays; trophoblast transcriptomes were determined by RNA sequencing (RNA-seq). Wild-type, CBS-deficient, and CBA/J × DBA/2 mice were treated with CBS and CSE inhibitors or H2S donors to determine the role of H2S in early pregnancy in vivo. RESULTS: CBS and CSE proteins showed cell-specific expressions, but only CBS decreased in the villous cytotrophoblast in URSA versus normal participants. H2S donors promoted migration and invasion and MMP-2 and VEGF expression in human placenta trophoblast cells that contain SV40 viral deoxyribonucleic acid sequences (HTR8/SVneo) and human placenta trophoblast cells (JEG3 cells), similar to forced CBS expression in HTR8/SVneo cells. The CBS-responsive transcriptomes in HTR8/SVneo cells contained differentially regulated genes (ie, interleukin-1 receptor and prostaglandin-endoperoxide synthase 2) that are associated with nuclear factor-κB-mediated inflammatory response. In vivo, dysregulated CBS/H2S signaling significantly increased embryonic resorption and decidual T-helper 1/T-helper 2 imbalance in mice, which was partially rescued by H2S donors. CONCLUSION: CBS/H2S signaling maintains early pregnancy, possibly via regulating maternal-fetal interface immune hemostasis, offering opportunities for H2S-based immunotherapies for URSA.

Complex preimplantation genetic tests for Robertsonian translocation, HLA, and X-linked hyper IgM syndrome caused by a novel mutation of CD40LG gene.

PURPOSE: To perform complex preimplantation genetic tests (PGT) for aneuploidy screening, Robertsonian translocation, HLA-matching, and X-linked hyper IgM syndrome (XHIGM) caused by a novel mutation c.156 G>T of CD40LG gene. METHODS: Reverse transcription PCR (RT-PCR) and Sanger sequencing were carried out to confirm the causative variant of CD40LG gene in the proband and parents. Day 5 and D6 blastocysts, obtained by in vitro fertilization (IVF) with intracytoplasmic sperm injection, underwent trophectoderm (TE) biopsy and whole genomic amplification (WGA) and next generation sequencing (NGS)-based PGT to detect the presence of a maternal CD40LG mutation, aneuploidy, Robertsonian translocation carrier, and human leukocyte antigen (HLA) haplotype. RESULTS: Sanger sequencing data of the genomic DNA showed that the proband has a hemizygous variant of c. 156 G>T in the CD40LG gene, while his mother has a heterozygous variant at the same position. Complementary DNA (cDNA) of CD40LG amplification and sequencing displayed that no cDNA of CD40LG was found in proband, while only wild-type cDNA of CD40LG was amplified in the mother. PGT results showed that only one of the six tested embryos is free of the variant c.156 G>T and aneuploidy and having the consistent HLA type as the proband. Meanwhile, the embryo is a Robertsonian translocation carrier. The embryo was transplanted into the mother's uterus. Amniotic fluid testing results are consistent with that of PGT. A healthy baby girl was delivered, and the peripheral blood testing data was also consistent with the testing results of transplanted embryo. CONCLUSIONS: The novel mutation of c. 156 G>T in CD40LG gene probably leads to XHIGM by nonsense-meditated mRNA decay (NMD), and complex PGT of preimplantation genetic testing for monogenic disease (PGT-M), aneuploidy (PGT-A), structural rearrangement (PGT-SR), and HLA-matching (PGT-HLA) can be performed in pedigree with both X-linked hyper IgM syndrome and Robertsonian translocation.

Overexpression of PTEN regulated by miR-19b and miR-494 in the villous of recurrent spontaneous abortion patients.

Recurrent spontaneous abortion (RSA) can be attributed to multiple factors, and abnormal invasion and apoptosis of trophoblast cells have attracted extensive attention. Our study aimed to investigate the expression of PTEN and miRNAs with potential regulatory relationships in the placental villi of RSA patients. Nineteen RSA patients and sixteen healthy women at reproductive age undergoing induced abortion (IA) were enrolled in the present study. The expression of PTEN and miRNAs were investigated using real-time polymerase chain reaction (PCR) and Western blotting, further verification between PTEN and potential miRNAs used cell culture and transfection, and luciferase activity assays were used to determine whether PTEN is directly regulated by potential miRNA. The results indicated that both PTEN mRNA and protein expression levels were upregulated in RSA patients, but a significant difference was only observed in protein expression level (p < 0.001). Through real-time PCR pre-scanning, the results of nine potential miRNAs revealed three significantly upregulated miRNAs (miR-494, miR-146a, and miR-21) and one significantly downregulated miRNA (miR-19b). The results of further verification regarding miR-19b and miR-494 suggested that upregulated miR-19b, cooperating with downregulated miRNA-494, could inhibit PTEN expression. In conclusion, the findings suggest that the overexpression of PTEN plays an important role in the pathogenesis of RSA, with miR-19b directly regulating PTEN and working with miR-494, all of which participating in abnormal effects of villous' trophoblastic cell may be a critical event.