Identification of tumor heterogeneity associated with KRAS/TP53 co-mutation status in lung adenocarcinoma based on single-cell RNA sequencing.

Lung cancer stands as the predominant cause of cancer-related mortality globally. Lung adenocarcinoma (LUAD), being the most prevalent subtype, garners extensive attention due to its notable heterogeneity, which significantly influences tumor development and treatment approaches. This research leverages single-cell RNA sequencing (scRNA-seq) datasets to delve into the impact of KRAS/TP53 co-mutation status on LUAD. Moreover, utilizing the TCGA-LUAD dataset, we formulated a novel predictive risk model, comprising seven prognostic genes, through LASSO regression, and subjected it to both internal and external validation sets. The study underscores the profound impact of KRAS/TP53 co-mutational status on the tumor microenvironment (TME) of LUAD. Crucially, KRAS/TP53 co-mutation markedly influences the extent of B cell infiltration and various immune-related pathways within the TME. The newly developed predictive risk model exhibited robust performance across both internal and external validation sets, establishing itself as a viable independent prognostic factor. Additionally, in vitro experiments indicate that MELTF and PLEK2 can modulate the invasion and proliferation of human non-small cell lung cancer cells. In conclusion, we elucidated that KRAS/TP53 co-mutations may modulate TME and patient prognosis by orchestrating B cells and affiliated pathways. Furthermore, we spotlight that MELTF and PLEK2 not only function as prognostic indicators for LUAD, but also lay the foundation for the exploration of innovative therapeutic approaches.

Overweight and obesity determined by body mass index criteria for Asian populations adversely affect assisted reproductive outcomes among Chinese women with polycystic ovary syndrome.

BACKGROUND: Obesity is a common feature in women with polycystic ovary syndrome (PCOS) and is associated with multiple adverse reproductive outcomes. However, the impact of overweight and obesity on reproductive outcomes of women with PCOS who underwent in vitro fertilization-embryo transfer (IVF-ET) is currently controversial and appropriate body mass index (BMI) levels differ across ethnic groups. METHODS: This was a retrospective study including 1066 women with PCOS receiving IVF treatment at our institution between January 2018 and June 2021, among whom 960 underwent their first fresh or frozen embryo transfer. Participants were categorized according to BMI cut-off values proposed by the World Health Organization for Asian populations: normal weight (BMI < 23 kg/m2), overweight (BMI: 23-24.9 kg/m2), and obesity (BMI ≥ 25 kg/m2). The effect of BMI on clinical and embryological outcomes was evaluated by descriptive statistics and logistic regression models with confounders adjusted. The dose-response relationship between BMI as a continuous variable and IVF outcomes is also explored. INTERVENTIONS: no RESULTS: Increasing BMI was associated with significantly lower numbers of total oocytes retrieved, metaphase II oocytes, two pronuclear (2PN) zygotes, and good-quality embryos among women with PCOS. Patients with PCOS with a BMI ≥ 23 kg/m2 had significantly lower live birth rates (41.9% vs. 49.1%; adjusted odds ratio [aOR], 0.75; 95% confidence interval [CI], 0.57-0.97) and implantation rates (35.8% vs. 43.9%; aOR, 0.76; 95% CI, 0.61-0.93) than those with normal BMI. Moreover, BMI showed a non-linear relationship (p for nonlinearity <0.001) with the number of 2PN zygotes with the curve becoming steeper as BMI surpassed 22.4 kg/m2. CONCLUSIONS: Patients with PCOS with a BMI ≥ 23 kg/m2 have lower live birth rates than those with a BMI < 23 kg/m2. Defining obesity and overweight with ethnicity-specific BMI cut-offs may help to improve IVF outcomes among PCOS patients.

Identification of Hb Lepore, Hb anti-Lepore, and α-globin gene triplications by long-read single-molecule real-time sequencing.

OBJECTIVES: Hemoglobin (Hb) Lepore and Hb anti-Lepore are infrequent fusion gene variants that result from nonhomologous crossovers during meiosis. Conventional molecular testing methods may face challenges in identifying these variants. During Hb analysis using capillary electrophoresis, we encountered 6 cases with unusual Hb variants. Our aim was to identify the alterations in their globin genes. METHODS: Gap-polymerase chain reaction (PCR), reverse dot-blot assay (RDB), Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA), and long-read single-molecule real-time (SMRT) sequencing were used to confirm the presence of globin gene alterations. RESULTS: The routine thalassemia gene test kit using the gap-PCR and RDB techniques did not detect common gene variations. Direct sequencing failed to identify any known or unknown globin gene alterations. The MLPA analysis, however, revealed the possible presence of α-globin gene triplications as well as 2 types of fusion gene alterations. Further analysis using long-read SMRT sequencing accurately identified 3 rare gene variations: αααanti-3.7, Hb Lepore-Boston-Washington, and Hb anti-Lepore P-India. CONCLUSIONS: Conventional methods may overlook rare thalassemias or Hb variants. Long-read SMRT sequencing has the potential to identify breakpoints in fusion genes, demonstrating that it is a promising technique for detecting rare thalassemias.

A New Hemoglobin Variant: Hb Tangshan [HBA1: c.239C > T, CD79(GCG > GTG)(Ala > Val)] Detected by MALDI-TOF MS.

In this report we decribed a new α-chain variant found during the measurement of hemoglobin A1c (Hb A1c) using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS). MALDI-TOF MS analysis detected an α-chain variant with a mass of 15,155 Da. However, this Hb variant was not detected during Hb A1c measurement by cation-exchange high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) methods. Sanger sequencing validated the presence of a heterozygous missense mutation [HBA1: c.239C > T, CD79(GCG > GTG)(Ala > Val)]. The observed 28 Da mass difference exactly matches the theoretical mass difference (28 Da) resulting from the substitution of alanine (89.079) with valine (117.133). As this represents the initial documentation of the mutation, we named it Hb Tangshan after the proband's residence.

ALKBH5 controls the meiosis-coupled mRNA clearance in oocytes by removing the N 6-methyladenosine methylation.

N6-methyladenosine (m6A) maintains maternal RNA stability in oocytes. One regulator of m6A, ALKBH5, reverses m6A deposition and is essential in RNA metabolism. However, the specific role of ALKBH5 in oocyte maturation remains elusive. Here, we show that Alkbh5 depletion causes a wide range of defects in oocyte meiosis and results in female infertility. Temporal profiling of the maternal transcriptomes revealed striking RNA accumulation in Alkbh5-/- oocytes during meiotic maturation. Analysis of m6A dynamics demonstrated that ALKBH5-mediated m6A demethylation ensures the timely degradation of maternal RNAs, which is severely disrupted following Alkbh5-/- depletion. A distinct subset of transcripts with persistent m6A peaks are recognized by the m6A reader IGF2BP2 and thus remain stabilized, resulting in impaired RNA clearance. Additionally, reducing IGF2BP2 in Alkbh5-depleted oocytes partially rescued these defects. Overall, this work identifies ALKBH5 as a key determinant of oocyte quality and unveil the facilitating role of ALKBH5-mediated m6A removal in maternal RNA decay.

Leveraging senescence-oxidative stress co-relation to predict prognosis and drug sensitivity in breast invasive carcinoma.

Introduction: Female breast cancer has risen to be the most common malignancy worldwide, causing a huge disease burden for both patients and society. Both senescence and oxidative stress attach importance to cancer development and progression. However, the prognostic roles of senescence and oxidative stress remain obscure in breast cancer. In this present study, we attempted to establish a predictive model based on senescence-oxidative stress co-relation genes (SOSCRGs) and evaluate its clinical utility in multiple dimensions. Methods: SOSCRGs were identified via correlation analysis. Transcriptome data and clinical information of patients with breast invasive carcinoma (BRCA) were accessed from The Cancer Genome Atlas (TCGA) and GSE96058. SVM algorithm was employed to process subtype classification of patients with BRCA based on SOSCRGs. LASSO regression analysis was utilized to establish the predictive model based on SOSCRGs. Analyses of the predictive model with regards to efficacy evaluation, subgroup analysis, clinical association, immune infiltration, functional strength, mutation feature, and drug sensitivity were organized. Single-cell analysis was applied to decipher the expression pattern of key SOSCRGs in the tumor microenvironment. Additionally, qPCR was conducted to check the expression levels of key SOSCRGs in five different breast cancer cell lines. Results: A total of 246 SOSCRGs were identified. Two breast cancer subtypes were determined based on SOSCRGs and subtype 1 showed an active immune landscape. A SOSCRGs-based predictive model was subsequently developed and the risk score was clarified as independent prognostic predictors in breast cancer. A novel nomogram was constructed and exhibited favorable predictive capability. We further ascertained that the infiltration levels of immune cells and expressions of immune checkpoints were significantly influenced by the risk score. The two risk groups were characterized by distinct functional strengths. Sugar metabolism and glycolysis were significantly upregulated in the high risk group. The low risk group was deciphered to harbor PIK3CA mutation-driven tumorigenesis, while TP53 mutation was dominant in the high risk group. The analysis further revealed a significantly positive correlation between risk score and TMB. Patients in the low risk group may also sensitively respond to several drug agents. Single-cell analysis dissected that ERRFI1, ETS1, NDRG1, and ZMAT3 were expressed in the tumor microenvironment. Moreover, the expression levels of the seven SOSCRGs in five different breast cancer cell lines were quantified and compared by qPCR respectively. Conclusion: Multidimensional evaluations verified the clinical utility of the SOSCRGs-based predictive model to predict prognosis, aid clinical decision, and risk stratification for patients with breast cancer.

Mutational spectrum of HBD gene in the Chinese population: Description of 36 mutations including 11 novel variants.

INTRODUCTION: Mutations in the hemoglobin subunit delta (HBD) gene (MIM#142000) are associated with decreased levels of the Hemoglobin A2 (Hb A2 ) fraction. We aimed to examine the prevalence of HBD gene mutations and summarize their characteristics in the Chinese population. METHODS: Individuals who exhibited Hb A2 levels below 1.8%, with or without Hb A2 variant peaks, were chosen for further investigation. Hemoglobin analysis was conducted using capillary electrophoresis. Common α and ß-thalassemia in China were detected using gap-PCR and reverse dot blot hybridization. The presence of HBD gene mutations was confirmed by DNA sequencing. RESULTS: A total of 188 patients were identified as carriers of the HBD gene mutation, with a prevalence of approximately 0.46%. We discovered 36 types of mutations, 30 of which resulted in δ-globin variants, while the remaining 6 resulted in δ-thalassemia. The most common mutation was HBD:c.-127 T > C, accounting for 87.2% of δ-thalassemia cases. In addition, we identified 11 novel HBD gene mutations and found 10 cases compounded with other common thalassemias. CONCLUSION: We observed a high prevalence of HBD gene mutations in southern China. Our findings provide a genetic basis for screening for δ-thalassemia and enrich the spectrum of HBD gene mutations.

Prognosis stratification of patients with breast invasive carcinoma based on cysteine metabolism-disulfidptosis affinity.

PURPOSE: The rise of female breast cancer has created a significant global public health issue that requires effective solutions. Disulfidptosis, a recently identified form of cell death characterized by an excessive accumulation of disulfides, has unique initiatory and regulatory mechanisms. The formation of disulfide bonds is a metabolic event typically associated with cysteines. This study aims to explore the potential of the affinity between cysteine metabolism and disulfidptosis in risk stratification for breast invasive carcinoma (BRCA). METHODS: We used correlation analysis to decipher co-relation genes between cysteine metabolism and disulfidptosis (CMDCRGs). Both LASSO regression analysis and multivariate Cox regression analysis were employed to construct the prognostic signature. Additionally, we conducted investigations concerning subtype identification, functional enhancement, mutation landscape, immune infiltration, drug prioritization, and single-cell analysis. RESULTS: We developed and validated a six-gene prognostic signature as an independent prognostic predictor for BRCA. The prognostic nomogram, based on risk score, demonstrated a favorable capability in predicting survival outcomes. We identified distinct gene mutations, functional enhancements, and immune infiltration patterns between the two risk groups. Four clusters of drugs were predicted as potentially effective for patients in the low-risk group. We identified seven cell clusters within the tumor microenvironment of breast cancer, and RPL27A was found to be widely expressed in this environment. CONCLUSION: Multidimensional analyses confirmed the clinical utility of the cysteine metabolism-disulfidptosis affinity-based signature in risk stratification and guiding personalized treatment for patients with BRCA.

Modeling human ectopic pregnancies with trophoblast and vascular organoids.

Ruptured ectopic pregnancy (REP), a pregnancy complication caused by aberrant implantation, deep invasion, and overgrowth of embryos in fallopian tubes, could lead to rupture of fallopian tubes and accounts for 4%-10% of pregnancy-related deaths. The lack of ectopic pregnancy phenotypes in rodents hampers our understanding of its pathological mechanisms. Here, we employed cell culture and organoid models to investigate the crosstalk between human trophoblast development and intravillous vascularization in the REP condition. Compared with abortive ectopic pregnancy (AEP), the size of REP placental villi and the depth of trophoblast invasion are correlated with the extent of intravillous vascularization. We identified a key pro-angiogenic factor secreted by trophoblasts, WNT2B, that promotes villous vasculogenesis, angiogenesis, and vascular network expansion in the REP condition. Our results reveal the important role of WNT-mediated angiogenesis and an organoid co-culture model for investigating intricate communications between trophoblasts and endothelial/endothelial progenitor cells.

Sperm-borne microRNA-34c regulates maternal mRNA degradation and preimplantation embryonic development in mice.

BACKGROUND: Studies have shown that sperm-borne microRNAs (miRNAs) are involved in mammalian preimplantation embryonic development. In humans, spermatozoan miR-34c levels are correlated with in vitro fertilization outcomes, such as embryo quality and the clinical pregnancy and live birth rates. In rabbits and cows, miR-34c improves the developmental competence of embryos generated by somatic cell nuclear transfer. However, the mechanisms underlying the regulation of embryonic development by miR-34c remain unknown. METHODS: Female C57BL/6 mice (6-8 weeks old) were superovulated, and pronucleated zygotes were collected and microinjected with an miR-34c inhibitor or a negative-control RNA. The embryonic development of the microinjected zygotes was evaluated, and the messenger RNA (mRNA) expression profiles of the embryos at the two-cell, four-cell and blastocyst stages (five embryos per group) were determined by RNA sequencing analysis. Gene expression levels were verified by reverse transcription-quantitative polymerase chain reaction. Cluster analysis and heat map visualization were performed to detect differentially expressed mRNAs. Pathway and process enrichment analyses were performed using ontology resources. Differentially expressed mRNAs were systematically analyzed using the Search Tool for the Retrieval of Interacting Genes/Proteins database to determine their biological functions. RESULTS: Embryonic developmental potential was significantly reduced in zygotes microinjected with the miR-34c inhibitor compared with those microinjected with a negative-control RNA. Two-cell stage embryos microinjected with an miR-34c inhibitor presented altered transcriptomic profiles, with upregulated expression of maternal miR-34c target mRNAs and classical maternal mRNAs. Differentially expressed transcripts were mainly of genes associated with lipid metabolism and cellular membrane function at the two-cell stage, with cell-cycle phase transition and energy metabolism at the four-cell stage; and with vesicle organization, lipid biosynthetic process and endomembrane system organization at the blastocyst stage. We also showed that genes related to preimplantation embryonic development, including Alkbh4, Sp1, Mapk14, Sin3a, Sdc1 and Laptm4b, were significantly downregulated after microinjection of an miR-34c inhibitor. CONCLUSIONS: Sperm-borne miR-34c may regulate preimplantation embryonic development by affecting multiple biological processes, such as maternal mRNA degradation, cellular metabolism, cell proliferation and blastocyst implantation. Our data demonstrate the importance of sperm-derived miRNAs in the development of preimplantation embryos.

Inhibition of USP10 induces myeloma cell apoptosis by promoting cyclin D3 degradation.

The cell cycle regulator cyclin D3 (CCND3) is highly expressed in multiple myeloma (MM) and it promotes MM cell proliferation. After a certain phase of cell cycle, CCND3 is rapidly degraded, which is essential for the strict control of MM cell cycle progress and proliferation. In the present study, we investigated the molecular mechanisms regulating CCND3 degradation in MM cells. By utilizing affinity purification-coupled tandem mass spectrometry, we identified the deubiquitinase USP10 interacting with CCND3 in human MM OPM2 and KMS11 cell lines. Furthermore, USP10 specifically prevented CCND3 from K48-linked polyubiquitination and proteasomal degradation, therefore enhancing its activity. We demonstrated that the N-terminal domain (aa. 1-205) of USP10 was dispensable for binding to and deubiquitinating CCND3. Although Thr283 was important for CCND3 activity, it was dispensable for CCND3 ubiquitination and stability modulated by USP10. By stabilizing CCND3, USP10 activated the CCND3/CDK4/6 signaling pathway, phosphorylated Rb, and upregulated CDK4, CDK6 and E2F-1 in OPM2 and KMS11 cells. Consistent with these findings, inhibition of USP10 by Spautin-1 resulted in accumulation of CCND3 with K48-linked polyubiquitination and degradation that synergized with Palbociclib, a CDK4/6 inhibitor, to induce MM cell apoptosis. In nude mice bearing myeloma xenografts with OPM2 and KMS11 cells, combined administration of Spautin-l and Palbociclib almost suppressed tumor growth within 30 days. This study thus identifies USP10 as the first deubiquitinase of CCND3 and also finds that targeting the USP10/CCND3/CDK4/6 axis may be a novel modality for the treatment of myeloma.

Case report: A reciprocal translocation-free and pathogenic DUOX2 mutation-free embryo selected by complicated preimplantation genetic testing resulted in a healthy live birth.

Preimplantation genetic testing (PGT) is an effective approach to improve clinical outcomes and prevent transmission of genetic imbalances by selecting embryos free of disease-causing genes and chromosome abnormalities. In this study, PGT was performed for a challenging case in which a couple simultaneously carried a maternal subchromosomal reciprocal translocation (RecT) revealed by fluorescence in situ hybridization involving the chromosome X (ChrX) and heterozygous mutations in dual oxidase 2 (DUOX2). Carriers of RecT are at increased risk for infertility, recurrent miscarriages, or having affected children due to the unbalanced gametes produced. DUOX2 mutation results in congenital hypothyroidism. Pedigree haplotypes for DUOX2 was constructed after the mutations were verified by Sanger sequencing. Since male carriers of X-autosome translocations may exhibit infertility or other abnormalities, pedigree haplotype for chromosomal translocation was also constructed to identify embryo with RecT. Three blastocysts were obtained by in vitro fertilization and underwent trophectoderm biopsy, whole genomic amplification, and next-generation sequencing (NGS). A blastocyst lacking copy number variants and RecT but carrying the paternal gene mutation in DUOX2, c.2654G>T (p.R885L) was used for embryo transfer, resulting in a healthy female infant whose genetic properties were confirmed by amniocentesis. Cases containing RecT and single gene disorder are rare. And the situation is more complicated when the subchromosomal RecT involving ChrX cannot be identified with routine karyotype analysis. This case report contributes significantly to the literature and the results have shown that the NGS-based PGT strategy may be broadly useful for complex pedigrees.

A degradome-based prognostic signature that correlates with immune infiltration and tumor mutation burden in breast cancer.

Introduction: Female breast cancer is the most common malignancy worldwide, with a high disease burden. The degradome is the most abundant class of cellular enzymes that play an essential role in regulating cellular activity. Dysregulation of the degradome may disrupt cellular homeostasis and trigger carcinogenesis. Thus we attempted to understand the prognostic role of degradome in breast cancer by means of establishing a prognostic signature based on degradome-related genes (DRGs) and assessed its clinical utility in multiple dimensions. Methods: A total of 625 DRGs were obtained for analysis. Transcriptome data and clinical information of patients with breast cancer from TCGA-BRCA, METABRIC and GSE96058 were collected. NetworkAnalyst and cBioPortal were also utilized for analysis. LASSO regression analysis was employed to construct the degradome signature. Investigations of the degradome signature concerning clinical association, functional characterization, mutation landscape, immune infiltration, immune checkpoint expression and drug priority were orchestrated. Cell phenotype assays including colony formation, CCK8, transwell and wound healing were conducted in MCF-7 and MDA-MB-435S breast cancer cell lines, respectively. Results: A 10-gene signature was developed and verified as an independent prognostic predictor combined with other clinicopathological parameters in breast cancer. The prognostic nomogram based on risk score (calculated based on the degradome signature) showed favourable capability in survival prediction and advantage in clinical benefit. High risk scores were associated with a higher degree of clinicopathological events (T4 stage and HER2-positive) and mutation frequency. Regulation of toll-like receptors and several cell cycle promoting activities were upregulated in the high-risk group. PIK3CA and TP53 mutations were dominant in the low- and high-risk groups, respectively. A significantly positive correlation was observed between the risk score and tumor mutation burden. The infiltration levels of immune cells and the expressions of immune checkpoints were significantly influenced by the risk score. Additionally, the degradome signature adequately predicted the survival of patients undergoing endocrinotherapy or radiotherapy. Patients in the low-risk group may achieve complete response after the first round of chemotherapy with cyclophosphamide and docetaxel, whereas patients in the high-risk group may benefit from 5-flfluorouracil. Several regulators of the PI3K/AKT/mTOR signaling pathway and the CDK family/PARP family were identified as potential molecular targets in the low- and high-risk groups, respectively. In vitro experiments further revealed that the knockdown of ABHD12 and USP41 significantly inhibit the proliferation, invasion and migration of breast cancer cells. Conclusion: Multidimensional evaluation verified the clinical utility of the degradome signature in predicting prognosis, risk stratification and guiding treatment for patients with breast cancer.

New insights into COL26A1 in thyroid carcinoma: prognostic prediction, functional characterization, immunological drug target and ceRNA network.

Background: Thyroid carcinoma (THCA) is one of the most commonly diagnosed malignancies. Collagen is the main component in extracellular matrix. Rising studies have determined the oncogenic effect of collagen in cancer progression, which is intriguing to be further explored. Collagen type XXVI alpha 1 chain (COL26A1) is a newly discovered collagen subtype, functions of which still remain poorly demonstrated in THCA. Methods: Based on the transcriptome data from The Cancer Genome Atlas (TCGA) and other public databases, we conducted investigations of COL26A1 in THCA with respects to diagnostic/prognostic prediction, functional characterization, immune infiltration, chemical drug target and non-coding RNA regulatory network. Furthermore, quantitative real-time polymerase chain reaction (qRT-PCR) and western blot were used to verify the expression of COL26A1 in THCA. Results: COL26A1 was significantly upregulated in THCA, and the high COL26A1 expression inferred poor prognosis [hazard ratio (HR) =4.76; 95% confidence interval (CI): 1.36-16.73; P=0.015]. The diagnostic area under the curve (AUC) of COL26A1 achieved 0.736 (95% CI: 0.669-0.802). COL26A1 was also identified as an independent prognostic predictor for THCA (HR =3.928; 95% CI: 3.716-4.151; P<0.001). Besides, logistic regression analysis indicated that age >45 years [odds ratio (OR) =1.532; 95% CI: 1.081-2.176; P=0.017], pathological stage III (OR =2.055; 95% CI: 1.314-3.184; P=0.001), tall cell subtype (OR =5.533; 95% CI: 2.420-14.957; P<0.001), residual tumor R1 (OR =1.844; 95% CI: 1.035-3.365; P=0.041) and extrathyroidal extension (OR =1.800; 95% CI: 1.225-1.660; P=0.003) were risk factors associated with high COL26A1 expression in THCA. Functional characterizations implied that COL26A1 was associated with immunological processes and oncogenic signaling pathways. High COL26A1 expression was accompanied by more abundant infiltration of immune cells and higher stromal/immune score. In addition, most immune checkpoints were significantly positively co-expressed with COL26A1, including PD-1, PD-L1 and CTLA4. Drugs including trichloroethylene, acetamide and thioacetamide etc. that can decrease the expression of COL26A1 were also identified. The predicted long noncoding RNA (lncRNA)-microRNA (miRNA)-COL26A1 regulatory axes were successfully deciphered. qRT-PCR and western blot verified the upregulation of COL26A1 in THCA. Conclusions: Our work has primarily appraised COL26A1 as a promising biomarker for diagnosis/prognosis and immuno/therapeutic target in THCA.

Trends in the prevalence, prenatal diagnosis, and outcomes of births with chromosomal abnormalities: a hospital-based study in Zhejiang Province, China during 2014-2020.

BACKGROUND: To investigate the prevalence and prenatal diagnosis rate of chromosomal abnormalities (CA) in Zhejiang Province, China. METHODS: We estimated the annual changes in the detected prevalence of CA and prenatal diagnosis rate among 681,590 births in Zhejiang Province, China, between 2014 and 2020. Data were derived from the provincial birth defects surveillance system, which represents 30% of annual births in Zhejiang Province. The effect of maternal age was also evaluated. RESULTS: The detected prevalence of sex chromosomal abnormalities (1.70-7.30 per 10,000 births, Ptrend < 0.001) and microdeletion and microduplication (0.30-6.81 per 10,000 births, Ptrend < 0.001) gradually increased, contributing to an upward trend in overall CA (12.09-39.22 per 10,000 births). The diagnosis rate before 22 gestational weeks constantly increased from 20.8 to 70.1% for trisomy 21 (Ptrend = 0.003). The prevalence rate ratio for maternal age of ≥ 35 years was higher than that for maternal age of 25-29 years for trisomy 21 (5.40, 95% confidence interval [CI] 4.59-6.35) and sex chromosomal abnormalities (3.28, 95% CI 2.48-4.33). CONCLUSIONS: The rising prevalence of CA in China may be attributable to the elevated maternal age and the innovation of prenatal diagnosis tools, Thus, studies should pay attention to the rare CA that were previously ignored, and select rational screening tools.

Long-term cryopreservation and frozen embryo transfer do not impact clinical and neonatal outcomes: a retrospective cohort study of slow-frozen early-cleavage human embryos.

This study aimed to evaluate the effect of the cryopreservation duration (up to 160 months) on the clinical and neonatal outcomes of slow-frozen early-cleavage human embryos. Clinical data collected between February 2013 and August 2017 were included in this retrospective study. Cases were classified into five groups by the duration of cryopreservation: Group 1, 6-12 months; Group 2, 13-36 months; Group 3, 37-60 months; Group 4, 61-84 months; and Group 5, >84 months. The embryo survival rate, implantation rate, clinical pregnancy rate, live-birth rate, newborn sex ratio, singleton gestational age, singleton birth weight and malformation rate were compared between the groups. The cryopreservation duration did not significantly affect the rates of clinical pregnancy (P = 0.119) and live birth (P = 0.354), the newborn sex ratio (P = 0.614) or the singleton gestational age (P = 0.212) and birthweight (P = 0.212). Although decreases in the embryo survival and implantation rates were observed in groups 4 and 5 compared with those in groups 1-3, these differences were not statistically significant (P = 0.329, P = 0.279, respectively). Long-term cryopreservation does not appear to adversely affect the clinical and neonatal outcomes of slow-frozen early-cleavage human embryos.

Maternal inheritance of glucose intolerance via oocyte TET3 insufficiency.

Diabetes mellitus is prevalent among women of reproductive age, and many women are left undiagnosed or untreated1. Gestational diabetes has profound and enduring effects on the long-term health of the offspring2,3. However, the link between pregestational diabetes and disease risk into adulthood in the next generation has not been sufficiently investigated. Here we show that pregestational hyperglycaemia renders the offspring more vulnerable to glucose intolerance. The expression of TET3 dioxygenase, responsible for 5-methylcytosine oxidation and DNA demethylation in the zygote4, is reduced in oocytes from a mouse model of hyperglycaemia (HG mice) and humans with diabetes. Insufficient demethylation by oocyte TET3 contributes to hypermethylation at the paternal alleles of several insulin secretion genes, including the glucokinase gene (Gck), that persists from zygote to adult, promoting impaired glucose homeostasis largely owing to the defect in glucose-stimulated insulin secretion. Consistent with these findings, mouse progenies derived from the oocytes of maternal heterozygous and homozygous Tet3 deletion display glucose intolerance and epigenetic abnormalities similar to those from the oocytes of HG mice. Moreover, the expression of exogenous Tet3 mRNA in oocytes from HG mice ameliorates the maternal effect in offspring. Thus, our observations suggest an environment-sensitive window in oocyte development that confers predisposition to glucose intolerance in the next generation through TET3 insufficiency rather than through a direct perturbation of the oocyte epigenome. This finding suggests a potential benefit of pre-conception interventions in mothers to protect the health of offspring.

Age-Dependent Metabolomic Profile of the Follicular Fluids From Women Undergoing Assisted Reproductive Technology Treatment.

Female fertility declines with age, and this natural variation culminates in reproductive senescence. Human follicular fluids are rich in low-molecular weight metabolites which are responsible for the maturation of oocytes. The metabolomic approaches are powerful tools to study biochemical markers of oocyte quality in the follicular fluids. It is necessary to identify and quantify the reliable metabolites in follicular fluids reflecting oocyte developmental potential. The goal of this study is to conduct a metabolomic analysis of the follicular fluids in women of different ages and study the metabolomic profile of the follicular fluids in relationship with oocyte quality in assisted reproductive technology (ART) treatment. A total of 30 women seeking for ART treatment at the Women's Hospital, Zhejiang University School of Medicine from October 2014 to April 2015 were recruited for the present study. Fifteen women aged from 39 to 47 were grouped as advanced maternal age, and the other 15 women aged from 27 to 34, as young controls. Ovarian stimulation and oocyte retrieval were conducted using a regular protocol involving mid-luteal pituitary down-regulation and controlled ovarian stimulation. Follicular fluids from mature follicles were collected and centrifuged for analyses. Liquid Chromatography-Mass Spectrometry (LC-MS) and Gas Chromatography-Mass Spectroscopy (GC-MS) were used to perform the quantitative metabolomic analysis. The follicular fluid levels of 311 metabolites and the metabolic significance were assessed. 70 metabolites showed significant differences between women with young and advanced ages. Follicular fluids from women with advanced age showed significantly higher levels of creatine, histidine, methionine, trans-4-hydroxyproline, choline, mevalonate, N2,N2-dimethylguanosine and gamma-glutamylvaline, as compared to those from the young age group. 8 metabolites were found significantly correlated with maternal age positively. Moreover, 3 metabolites were correlated with the number of oocytes retrieved, and 5 metabolites were correlated with cleaved embryo numbers, both negatively. The follicular fluids from women undergoing ART treatment exhibited age-dependent metabolomic profile. Metabolites associated with oocyte quality were identified, suggesting them as potential biomarkers for oocyte maturation and ART outcomes.

Preimplantation Genetic Testing Prevented Intergenerational Transmission of X-Linked Alport Syndrome.

BACKGROUND: Alport syndrome (AS) is a hereditary renal basement membrane disease that can lead to end-stage renal disease in young adults. It can be diagnosed by genetic analysis, being mostly caused by mutations in COL4A3, COL-4A4, and COL4A5. To date, there is no radical cure for this disease. OBJECTIVES: The aim of this study was to avoid the transmission of AS within an affected family by selecting healthy embryos for uterine transfer. The embryos were identified by preimplantation genetic testing for monogenic disorders (PGT-M). METHODS: We used next-generation sequencing (NGS) to identify mutations in the proband and his parents. The results of NGS were confirmed by Sanger sequencing. Targeted NGS combined with targeted single-nucleotide polymorphism haplotyping was used for the in vitro identification of COL4A5 mutations in human embryos to prevent their intergenerational transmission. RESULTS: The c.349_359delGGACCTCAAGG and c.360_361insTGC mutations in COL4A5 were identified in a family affected by X-linked AS. Whole-genome sequencing by NGS with targeted haplotyping was performed on biopsied trophectoderm cells. A healthy baby was born after transfer of a single freeze-thawed blastocyst. CONCLUSIONS: The use of targeted NGS for identifying diagnostic markers combined with targeted haplotyping is an easy and efficient PGT-M method for preventing intergenerational transmission of AS.

A Rapid NGS-Based Preimplantation Genetic Testing for Chromosomal Abnormalities in Day-3 Blastomere Biopsy Allows Embryo Transfer Within the Same Treatment Cycle.

Nowadays, most of the preimplantation genetic testing (PGT) is performed with a strategy of comprehensive chromosome screening and trophectoderm biopsy. Nevertheless, patients with ovarian insufficiency may not have competent blastocysts. In the present study, we aimed to establish the value of multiple annealing and looping-based amplification cycle (MALBAC)-based next-generation sequencing (NGS) for PGT in day-3 embryos. A total of 94.3% (1168/1239) of embryos yielded informative results, and the overall embryo euploid rate was 21.9% (256/1168). Overall, 225 embryos were transferred in 169 cycles with a clinical pregnancy rate of 49.1% (83/169). The live birth and implantation rates were 47.3% (80/169) and 44.4% (100/225), respectively. Double embryos transfer showed higher clinical pregnancy and live birth rates compared with single embryo transfer, but the implantation rates were similar (44.2% vs. 44.6%, P > 0.05). The euploid rate for reciprocal translocations (16.1%) was significantly lower than that for Robertsonian translocations (28.0%, P < 0.01) and inversions (28.0%, P < 0.01). However, higher percentages of embryos with de novo abnormalities were observed with Robertsonian translocations (23.3%, P < 0.01) and inversions (30.5%, P < 0.01) than with reciprocal translocations (11.6%). We demonstrated that NGS for PGT on day-3 embryos is an effective clinical application, particularly for patients with a diminished ovarian reserve and limited embryos.