Abortion Tissues Research Articles

Antioxidant Defenses, Oxidative Stress Responses, and Apoptosis Modulation in Spontaneous Abortion: An Immunohistochemistry Analysis of First-Trimester Chorionic Villi.

Oxidative stress (OS) and apoptosis are critical factors in placental development and function. Their interplay influences trophoblast proliferation, differentiation, and invasion, as well as vascular development. An imbalance between these processes can lead to pregnancy-related disorders such as preeclampsia, intrauterine growth restriction, and even spontaneous abortion. Our study seeks to elucidate the associations between preventive antioxidant/protective OS response factors-glutathione (GSH), MutT Homolog 1 (MTH1), and apoptotic regulation modulators-tumor protein p53 and B-cell lymphoma (Bcl-2) transcripts, in the context of spontaneous abortion (30 samples) versus elective termination of pregnancy (20 samples), using immunohistochemistry (IHC) to determine their proteomic expression in chorionic villi within abortive fetal placenta tissue samples. Herein, comparative statistical analyses revealed that both OS response factors, GSH and MTH1, were significantly under-expressed in spontaneous abortion cases as compared to elective. Conversely, for apoptotic regulators, p53 expression was significantly higher in spontaneous abortion cases, whereas Bcl-2 expression was significantly lower in spontaneous abortion cases. These findings suggest that a strong pro-apoptotic signal is prevalent within spontaneous abortion samples, alongside reduced anti-apoptotic protection, depleted antioxidant defenses and compromised oxidative DNA damage prevention/repair, as compared to elective abortion controls. Herein, our hypothesis that OS and apoptosis are closely linked processes contributing to placental dysfunction and spontaneous abortion was thus seemingly corroborated. Our results further highlight the importance of maintaining redox homeostasis and apoptotic regulation for a successful pregnancy. Understanding the mechanisms underlying this interplay is essential for developing potential therapies to manage OS, promote placentation, and avoid unwanted apoptosis, ultimately improving pregnancy outcomes. Antioxidant supplementation, modulation of p53 activity, and the enhancement of DNA repair mechanisms may represent potential approaches to mitigate OS and apoptosis in the placenta. Further research is needed to explore these strategies and their efficacy in preventing spontaneous abortion.

Enhancing Clinicopathological Diagnosis of Hydatidiform Mole Through the Combined Application of Histomorphologic Analysis, Immunohistochemical Analysis of p57 Expression, and Short Tandem Repeat Typing Method

This study aims to assess the early histomorphologic characteristics and investigate the role of the p57kip2 protein combined with STR genotyping for pathological diagnosis and typing of the hydatidiform mole (HM). A total of 73 induced abortion tissues were collected for pathological evaluation, including 14 cases with partial HM (PHM), 7 cases with complete HM (CHM), and 52 cases with non-molar pregnancies. Histopathological examination of moles was conducted using hematoxylin and eosin staining. DNA extraction from paraffin sections was performed using Fe3O4 nano-magnetic beads. Molecular diagnosis was performed using STR genotyping. Immunohistochemical analysis was used to determine the distribution and expression level of p57kip2 protein in HM. Significant differences were observed in the morphological indices of villous edema, cistern formation, trophoblastic inclusions, and trophoblastic hyperplasia between the PHM and CHM groups (P < 0.05). The central cistern formation and the trophoblast inclusion showed a significant difference between the HM and non-molar pregnancy (P < 0.05). Moreover, our findings revealed that p57kip2 expression contributed to distinguishing CHM from PHM. However, it could not distinguish PHM from non-mole pregnancy. Furthermore, the results of STR genotyping were consistent with pathological typing. In conclusion, the integration of pathomorphology, immunohistochemical staining, and molecular diagnostics holds great value for the diagnosis and classification of HM.

P-538 Liquid biopsy (niPOC) as a new approach to genetic studies in early pregnancy loss. First clinical experience in Spain

Abstract Study question How accurate is the use of liquid biopsy as a new tool for aneuploidy testing in cases of pregnancy losses? Summary answer This study demonstrates that genome-wide cfDNA testing in the maternal bloodstream (niPOC) constitutes a simple, tissue-independent, straightforward and reliable complement/alternative to traditional POC analysis. What is known already Around 50-70% of clinical miscarriages are caused by aneuploidies, mostly trisomies. To date, conventional cytogenetic and advanced molecular techniques are used for the analysis of POC to identify the genetic cause of miscarriage, providing valuable information for genetic counseling. However, both approaches are based in the direct analysis of the abortive tissue, which entails several limitations due to a risk of culture failure and/or maternal cell contamination. To solve these drawbacks, maternal cell-free DNA (cfDNA) testing emerges as a promising alternative due to the accumulated evidence. Study design, size, duration Retrospective study and first clinical experience, both conducted in our facilities from January 2021 to December 2023. Before carrying out the fetal tissues collection for the POC analysis, a blood sample was drawn to evaluate possible aneuploidies by cfDNA testing. Using NGS+STR POC results as the gold standard, results derived from both studies were compared to assess the percentage of concordance and the cases of non-informativeness (fetal fraction (FF) <2%), false positives, and false negatives. Participants/materials, setting, methods Herein we present a clinical validation (n = 152) and the first clinical experience (n = 103). Maternal peripheral blood samples were extracted for aneuploidy evaluation using Illumina’s sequencing platform. Genetic testing on POCs was performed using NGS (Thermo Fisher Scientific) to detect aneuploidies, and short tandem repeats to detect cases with MCC and/or polyploidy. In both groups (niPOC and POC), the percentage of concordance and cases of non-informativeness (by fetal fraction <2%, or CCM) were evaluated. Main results and the role of chance In the validation set, no significant differences in non-informativity were observed between POC and niPOC [9.2% (14/152) vs 13.8% (21/152)]. Excluding triploids, the sensitivity of niPOC compared with the pregnancy tissue sequencing result was 90.1%, specificity of 97.1% and an accuracy of 88%. In the first clinical experience, non-informativity was similar to that found in the validation (17/103, 16.5%). The percentage of abnormal cases was 43%, very close to the 50% expected according to the scientific literature. Regarding turnaround time, reports in niPOC were released in an average of 3.8 working days. Limitations, reasons for caution Current cfDNA testing technology fails in polyploidy, uniparental disomy identification, which is a potential cause of pregnancy loss. Aneuploidy detection is only feasible if the blood collection was performed before the fetal remains evacuation. Wider implications of the findings CfDNA testing before curettage or pharmacological treatment could be an alternative to POC analysis. If optimized, cfDNA testing could be used contingently with the molecular POC analysis in cases where maternal cell contamination is present. As a result, the overall success rate in the POC program could be substantially improved. Trial registration number Not applicable

Prenatal Diagnosis of Cystic Fibrosis by Celocentesis.

Celocentesis is a new sampling tool for prenatal diagnosis available from 7 weeks in case of couples at risk for genetic diseases. In this study, we reported the feasibility of earlier prenatal diagnosis by celocentesis in four cases of cystic fibrosis and one case of cystic fibrosis and β-thalassemia co-inherited in the same fetus. Celomic fluids were aspired from the celomic cavity between 8+2 and 9+3 weeks of gestation and fetal cells were picked up by micromanipulator. Maternal DNA contamination was tested and target regions of fetal DNA containing parental pathogenetic variants of CFTR and HBB genes were amplified and sequenced. Four of the five fetuses resulted as being affected by cystic fibrosis and, in all cases, the women decided to interrupt the pregnancy. In the other case, the fetus presented a healthy carrier of cystic fibrosis. The results were confirmed in three cases on placental tissue. In one case, no abortive tissue was obtained. In the last case, the woman refused the prenatal diagnosis to confirm the celocentesis data; the pregnancy is ongoing without complications. This procedure provides prenatal diagnosis of monogenic diseases at least four weeks earlier than traditional procedures, reducing the anxiety of patients and providing the option for medical termination of the affected fetus at 8-10 weeks of gestation, which is less traumatic and safer than surgical termination in the second trimester.

Prenatal diagnosis and molecular cytogenetic characterization of fetuses with central nervous system anomalies using chromosomal microarray analysis: a seven-year single-center retrospective study

Few existing reports have investigated the copy number variants (CNVs) in fetuses with central nervous system (CNS) anomalies. To gain further insights into the genotype–phenotype relationship, we conducted chromosomal microarray analysis (CMA) to reveal the pathogenic CNVs (pCNVs) that were associated with fetal CNS anomalies. We enrolled 5,460 pregnant women with different high-risk factors who had undergone CMA. Among them, 57 subjects with fetal CNS anomalies were recruited. Of the subjects with fetal CNS anomalies, 23 were given amniocentesis, which involved karyotype analysis and CMA to detect chromosomal abnormalities. The other 34 cases only underwent CMA detection using fetal abortive tissue. In this study, we identified five cases of chromosome aneuploid and nine cases of pCNVs in the fetuses, with a chromosomal aberration detection rate of 24.56% (14/57). In the 23 cases that were given both karyotype and CMA analysis, one case with trisomy 18 was detected by karyotyping. Moreover, CMA revealed a further three cases of pCNVs, including the 1p36.33p36.31, 7q11.23, and 1q21.1q21.2 microdeletions, with a 13.04% (3/23) increase in CMA yield over the karyotype analysis. Additionally, three cases of trisomy 13, one case of trisomy 21, and six cases of pCNVs were detected in the other 34 fetuses where only CMA was performed. Furthermore, a higher chromosomal aberration detection rate was observed in the extra CNS anomaly group than in the isolated CNS anomaly group (40.91% vs 14.29%). In conclude, several pathogenic CNVs were identified in the fetuses with CNS anomalies using CMA. Among the detected CNVs, ZIC2, GNB1, and NSUN5 may be the candidate genes that responsible for fetal CNS anomalies. Our findings provides an additional reference for genetic counseling regarding fetal CNS anomalies and offers further insight into the genotype–phenotype relationship.

Trophoblast-derived miR-410-5p induces M2 macrophage polarization and mediates immunotolerance at the fetal-maternal interface by targeting the STAT1 signaling pathway

BackgroundMacrophages phenotypic deviation and immune imbalance play vital roles in pregnancy-associated diseases such as spontaneous miscarriage. Trophoblasts regulate phenotypic changes in macrophages, however, their underlying mechanism during pregnancy remains unclear. Therefore, this study aimed to elucidate the potential function of trophoblast-derived miRNAs (miR-410-5p) in macrophage polarization during pregnancy.MethodsPatient decidual macrophage tissue samples in spontaneous abortion group and normal pregnancy group (those who had induced abortion for non-medical reasons) were collected at the Reproductive Medicine Center of Renmin Hospital of Wuhan University from April to December 2021. Furthermore, placental villi and decidua tissue samples were collected from patients who had experienced a spontaneous miscarriage and normal pregnant women for validation and subsequent experiments at the Shenzhen Zhongshan Obstetrics & Gynecology Hospital (formerly Shenzhen Zhongshan Urology Hospital), from March 2021 to September 2022. As an animal model, 36 female mice were randomly divided into six groups as follows: naive-control, lipopolysaccharide-model, agomir-negative control prevention, agomir-410-5p prevention, agomir-negative control treatment, and agomir-410-5p treatment groups. We analyzed the miR-410-5p expression in abortion tissue and plasma samples; and supplemented miR-410-5p to evaluate embryonic absorption in vivo. The main source of miR-410-5p at the maternal–fetal interface was analyzed, and the possible target gene, signal transducer and activator of transcription (STAT) 1, of miR-410-5p was predicted. The effect of miR-410-5p and STAT1 regulation on macrophage phenotype, oxidative metabolism, and mitochondrial membrane potential was analyzed in vitro.ResultsMiR-410-5p levels were lower in the spontaneous abortion group compared with the normal pregnancy group, and plasma miR-410-5p levels could predict pregnancy and spontaneous abortion. Prophylactic supplementation of miR-410-5p in pregnant mice reduced lipopolysaccharide-mediated embryonic absorption and downregulated the decidual macrophage pro-inflammatory phenotype. MiR-410-5p were mainly distributed in villi, and trophoblasts secreted exosomes-miR-410-5p at the maternal–fetal interface. After macrophages captured exosomes, the cells shifted to the tolerance phenotype. STAT1 was a potential target gene of miR-410-5p. MiR-410-5p bound to STAT1 mRNA, and inhibited the expression of STAT1 protein. STAT1 can drive macrophages to mature to a pro-inflammatory phenotype. MiR-410-5p competitive silencing of STAT1 can avoid macrophage immune disorders.ConclusionMiR-410-5p promotes M2 macrophage polarization by inhibiting STAT1, thus ensuring a healthy pregnancy. These findings are of great significance for diagnosing and preventing spontaneous miscarriage, providing a new perspective for further research in this field.

Exploring the clinical and cellular mechanisms of LncRNA-KCNQ1OT1/miR-29a-3p/SOCS3 molecular axis in cases of unexplained recurrent spontaneous abortion

Objective The objective of this study is to explore the functions and mechanisms of the LncRNA-KCNQ1OT1/miR-29a-3p/SOCS3 molecular pathway in the context of unexplained recurrent spontaneous abortion (URSA). Methods We conducted qRT-PCR to assess the levels of LncRNA-KCNQ1OT1, miR-29a-3p, and SOCS3 in both abortion tissues from women who experienced URSA and healthy early pregnant women. A dual-luciferase assay was employed to investigate whether miR-29a-3p targets SOCS3. Furthermore, RNA IP and RNA Pull-Down assays were employed to confirm the interaction between KCNQ1OT1 and SOCS3 with miR-29a-3p. RNA FISH was used to determine the cellular localization of KCNQ1OT1. Additionally, trophoblast cells (HTR8/SVneo) were cultured and the CCK-8 assay was utilized to assess cell proliferation, while flow cytometry was employed to analyze cell apoptosis. Results Compared to abortion tissues obtained from healthy early pregnant individuals, those from women who experienced URSA displayed a notable downregulation of KCNQ1OT1 and SOCS3, accompanied by an upregulation of miR-29a-3p. Suppression of KCNQ1OT1 resulted in the inhibition of cell proliferation and the facilitation of apoptosis in HTR8/SVneo cells. Our findings suggest that KCNQ1OT1 may exert a regulatory influence on SOCS3 through a competitive binding mechanism with miR-29a-3p. Notably, KCNQ1OT1 exhibited expression in both the cytoplasm and nucleus, with a predominant localization in the cytoplasm. Furthermore, we observed a negative regulatory relationship between miR-29a-3p and SOCS3, as the miR-29a-3p mimic group demonstrated significantly reduced cell proliferation and an increased rate of apoptosis when compared to the negative control (NC mimic) group. Additionally, the SOCS3 Vector group exhibited a substantial improvement in proliferation capability and a marked reduction in the apoptosis rate in comparison to the NC Vector group. The miR-29a-3p mimic + SOCS3 Vector group demonstrated a remarkable enhancement in proliferation and a reduction in apoptosis when compared to the miR-29a-3p mimic group. Conclusion The competitive binding of miR-29a-3p to LncRNA-KCNQ1OT1 appears to result in the elevation of SOCS3 expression, consequently fostering the proliferation of trophoblast cells while concomitantly suppressing apoptosis.

Genetic analysis of chorionic villus tissues in early missed abortions

Chromosomal abnormalities are the most common etiology of early spontaneous miscarriage. However, traditional karyotyping of chorionic villus samples (CVSs) is limited by cell culture and its low resolution. The objective of our study was to investigate the efficiency of molecular karyotyping technology for genetic diagnosis of early missed abortion tissues. Chromosome analysis of 1191 abortion CVSs in early pregnancy was conducted from August 2016 to June 2021; 463 cases were conducted via copy-number variations sequencing (CNV-seq)/quantitative fluorescent-polymerase chain reaction (QF-PCR) and 728 cases were conducted using SNP array. Clinically significant CNVs of CVSs were identified to clarify the cause of miscarriage and to guide the couples’ subsequent pregnancies. Among these, 31 cases with significant maternal cell contamination were removed from the study. Among the remaining 1160 samples, 751 cases (64.7%) with genetic abnormalities were identified, of which, 531 (45.8%) were single aneuploidies, 31 (2.7%) were multiple aneuploidies, 50 (4.3%) were polyploidies, 54 (4.7%) were partial aneuploidies, 77 (6.6%) had submicroscopic CNVs (including 25 with clinically significant CNVs and 52 had variants of uncertain significance), and 8 cases (0.7%) were uniparental disomies. Our study suggests that both SNP array and CNV-seq/QF-PCR are reliable, robust, and high-resolution technologies for genetic diagnosis of miscarriage.

The role of PCD in sexual dimorphism of dioecious Spinacia oleracea L.

The formation of non-hermaphroditic, i.e. male or female, flowers is a rare event in the plant kingdom. S. oleracea provides an ideal unisexual floral developmental system for studying the structural development of floral organs. These species forms non-hermaphroditic flowers; the pistil is fertile in the female flower, but the development of the stamens stops at an early phase and this organ atrophies and becomes functionless, while the male flowers form four fertile stamens, however there is not any trace of the pistil, it aborts at a much early stage. We searched for the presence of programmed cell death (PCD) in the abortive tissues during the ontogenetic development of these flowers. These results show curicial information on how the fertile sex organ in spinach differentiates and develops while arresting the development of the other aborted sex organ ; the presence of PCD occur in unisexual flower development in rhe very early stage and continue short time. We also found that stamen development in the female flower and pistil development in the male flower were subject to changes that did not result in large-scale structural changes. The PCD data obtained are the first study of spinach in the literature. This type of studies are shedding additional light on the sexual specialization hypothesis. Moreover, the ability to manipulate or control the flowering of the dioecious plant by simple means holds great potential, both from an economic aspect and to increase food production for an ever-growing human population

Analysis of genetic etiology and related factors in 1 065 women with spontaneous abortions

To explore the genetic etiology and related factors in 1 065 women with spontaneous abortions. All patients have presented at the Center of Prenatal Diagnosis of Nanjing Drum Tower Hospital from January 2018 to December 2021. Chorionic villi and fetal skin samples were collected, and the genomic DNA was assayed by chromosomal microarray analysis (CMA). For 10 couples with recurrent spontaneous abortions but normal CMA results for abortive tissues, non-in vitro fertilization-embryo transfer (IVF-ET) pregnancies and no previous history of live births and no structural abnormalities of the uterus, peripheral venous blood samples were collected. Genomic DNA was subjected to trio-whole exome sequencing (trio-WES). Candidate variants were verified by Sanger sequencing and bioinformatics analysis. Multifactorial unconditional logistic regression analysis was carried out to analyze the factors that may affect chromosomal abnormality in spontaneous abortions, such as the age of the couple, number of previous spontaneous abortions, IVF-ET pregnancy and history of live birth. The incidence of chromosomal aneuploidies in spontaneous abortions during the first trimester was compared in young or advanced-aged patients by chi-square test for liner trend. Among the 1 065 spontaneous abortion patients, 570 cases (53.5%) of chromosomal abnormalities were detected in spontaneous abortion tissues, which included 489 cases (45.9%) of chromosomal aneuploidies and 36 cases (3.4%) of pathogenic/likely pathogenic copy number variations (CNVs). Trio-WES results have revealed one homozygote variant and one compound heterozygote variants in two pedigrees, both of which were inherited from the parents. One likely pathogenic variant was detected in the patient from two pedigrees. Multifactorial unconditional Logistic regression analysis suggested that age of patient was an independent risk factor of chromosome abnormalities (OR = 1.122, 95%CI: 1.069-1.177, P < 0.001), the number of previous abortions and IVF-ET pregnancy were independent protective factors for chromosomal abnormalities (OR = 0.791, 0.648; 95%CI: 0.682-0.916, 0.500-0.840; P = 0.002, 0.001), whilst the age of husband and history of live birth were not (P > 0.05). The incidence of aneuploidies in the abortive tissues has decreased with the number of previous spontaneous abortions in young patients (χ² = 18.051, P < 0.001), but was not significantly correlated with the number of previous spontaneous abortions in advanced-aged patients with spontaneous abortions (P > 0.05). Chromosomal aneuploidy is the main genetic factor for spontaneous abortion, though CNVs and genetic variants may also underlie its genetic etiology. The age of patients, number of previous abortions and IVF-ET pregnancy are closely associated with chromosome abnormalities in abortive tissues.

Trophoblast retrieval from the cervical canal to predict abnormal pregnancy early in gestation: a pilot study

BackgroundThe current detection of fetal chromosomal abnormalities by non-invasive prenatal testing (NIPT) mainly relies on the cell free DNA(cfDNA) in the maternal blood. However, a gestational age of less than 12 weeks or a high maternal BMI affects cfDNA fetal fraction and further the detection by NIPT negatively. In this study, we aim to retrieve the trophoblast cells from the maternal cervix to develop a new sampling method for NIPT enabling an earlier use of NIPT.MethodsWe enrolled three patients who wanted to undergo induced abortion at Beijing Hospital between January 2022 and March 2022. Peripheral blood, cervix specimen, and the abortion tissue were collected and processed for each patient. Allele frequencies of the mutated gene loci of the maternal blood and the cervix sample were compared and the Sex Determining Region Y (SRY) gene was tested.ResultsThe allele frequencies of the mutated gene loci showed no significant difference between the maternal blood and the cervix sample. But we successfully detected signal of the SRY gene in the cervix sample of the only patient carrying a male fetus.ConclusionsThe detection of the SRY gene in a cervix sample indicated a successful retrieval of trophoblast cells from the cervix canal. Further study needs to be conducted to verify our finding before its application to the clinical settings.

Novel deleterious splicing variant in HFM1 causes gametogenesis defect and recurrent implantation failure: concerning the risk of chromosomal abnormalities in embryos.

Poor ovarian response (POR) affects approximately 9% to 24% of women undergoing in vitro fertilization (IVF) cycles, resulting in fewer eggs obtained and increasing clinical cycle cancellation rates. The pathogenesis of POR is related to gene variations. Our study included a Chinese family comprising two siblings with infertility born to consanguineous parents. Poor ovarian response (POR) was identified in the female patient who had multiple embryo implantation failures occurring in subsequent assisted reproductive technology cycles. Meanwhile, the male patient was diagnosed with non-obstructive azoospermia (NOA). Whole-exome sequencing and rigorous bioinformatics analyses were conducted to identify the underlying genetic causes. Moreover, the pathogenicity of the identified splicing variant was assessed using a minigene assay in vitro. The remaining poor-quality blastocyst and abortion tissues from the female patient were detected for copy number variations. We identified a novel homozygous splicing variant in HFM1 (NM_001017975.6: c.1730-1G > T) in two siblings. Apart from NOA and POI, biallelic variants in HFM1 were also associated with recurrent implantation failure (RIF). Additionally, we demonstrated that splicing variants caused abnormal alternative splicing of HFM1. Using copy number variation sequencing, we found that the embryos of the female patients had either euploidy or aneuploidy; however, both harbored chromosomal microduplications of maternal origin. Our results reveal the different effects of HFM1 on reproductive injury in males and females, extend the phenotypic and mutational spectrum of HFM1, and show the potential risk of chromosomal abnormalities under the RIF phenotype. Moreover, our study provides new diagnostic markers for the genetic counseling of POR patients.

Expert consensus on the detection of genome-wide copy number variations in abortive tissues and family reproductive consultation

Chromosomal aberrations including numerical abnormalities and segment duplications/deletions, as genome-wide copy number variations (CNVs), are a leading cause for spontaneous abortion. Analysis of abortive tissues for such CNVs can detect potential genomic variations in the couple and provide guidance for the choice of appropriate method to avoid further miscarriage or birth of child with chromosomal disorders. With evidence-based clinical data, an expert group jointly formed by the Genetic Disease Prevention and Control Group, Committee for Birth Defects Prevention and Control, Chinese Association of Preventive Medicine; the Clinical Genetics Group, the Society of Medical Genetics, Chinese Medical Association; the Professional Committee for Prenatal Diagnosis of Genetic Diseases, the Society of Medical Geneticists, Chinese Medical Doctor Association has discussed and formulated this consensus, with an aim to provide guidance for the application of genomic CNVs detection for the abortive tissue and genetic counseling for family reproduction.

F8 gene inversion and duplication cause no obvious hemophilia A phenotype.

Hemophilia A (HA, OMIM#306700) is an X-linked recessive bleeding disorder caused by the defects in the F8 gene, which encodes coagulation factor VIII (FVIII). Intron 22 inversion (Inv22) is found in about 45% of patients with severe hemophilia A. Here, we reported a male without obvious hemophilia A phenotype but bearing an inherited segmental variant duplication encompassing F8 as well as Inv22. The duplication was approximately 0.16Mb and involved from exon 1 to intron 22 of F8. This partial duplication and Inv22 in F8 was first found in the abortion tissue of his older sister with recurrent miscarriage. The genetic testing of his family revealed that his phenotypically normal older sister and mother also had this heterozygous Inv22 and a 0.16Mb partial duplication of F8, while his father was genotypically normal. The integrity of the F8 gene transcript was verified by sequencing of the adjacent exons at the inversion breakpoint, which explained why this male had no phenotype for hemophilia A. Interestingly, although he had no significant hemophilia A phenotype, the expression of C1QA in his mother, sister, and the male subject was only about half of that in his father and normal population. Our report broadens the mutation spectrum of F8 inversion and duplication and its pathogenicity in hemophilia A.

Evaluation and Analysis of Absence of Homozygosity (AOH) Using Chromosome Analysis by Medium Coverage Whole Genome Sequencing (CMA-seq) in Prenatal Diagnosis.

Absence of homozygosity (AOH) is a genetic characteristic known to cause human diseases mainly through autosomal recessive or imprinting mechanisms. The importance and necessity of accurate AOH detection has become more clinically significant in recent years. However, it remains a challenging task for sequencing-based methods thus far. In this study, we developed and optimized a new bioinformatic algorithm based on the assessment of minimum sequencing coverage, optimal bin size, the Z-score threshold of four types of allele count and the frequency for accurate genotyping using 28 AOH negative samples, and redefined the AOH detection cutoff value. We showed the performance of chromosome analysis by five-fold coverage whole genome sequencing (CMA-seq) for AOH identification in 27 typical prenatal/postnatal AOH positive samples, which were previously confirmed by chromosomal microarray analysis with single nucleotide polymorphism array (CMA/SNP array). The blinded study indicated that for all three forms of AOH, including whole genomic AOH, single chromosomal AOH and segmental AOH, and all kinds of sample types, including chorionic villus sampling, amniotic fluid, cord blood, peripheral blood and abortive tissue, CMA-seq showed equivalent detection power to that of routine CMA/SNP arrays (750K). The subtle difference between the two methods is that CMA-seq is prone to detect small inconsecutive AOHs, while CMA/SNP array reports it as a whole. Based on our newly developed bioinformatic algorithm, it is feasible to detect clinically significant AOH using CMA-seq in prenatal diagnosis.

Elevated PGT promotes proliferation and inhibits cell apoptosis in preeclampsia by Erk signaling pathway

Prostaglandins participate in maternal recognition of pregnancy, implantation and maintenance of gestation. Prostaglandin transporter (PGT), as a candidate molecule of prostaglandin carriers, might be involved in the pathogenesis of preeclampsia. In preeclampsia (PE) patients’ placental tissue, we identified PGT by RNA sequencing, measured its expression pattern by quantitative real-time PCR and Western blot. PGT was found to be upregulated in preeclamptic placental tissue. The expression pattern of PGT in PE was double confirmed by eight Gene Expression Omnibus (GEO) databases. In abortion tissues at 6–8 weeks, we then observed the cellular location of PGT by Immunofluorescence technique (IF) and found PGT located in trophoblast cell of the placenta of early pregnancy. In vitro studies revealed that forced expression of PGT in HTR8/Sveno cell inhibited its apoptosis, but promoted its proliferation by activating Erk signaling. In vivo study, we used reduced uterine perfusion pressure (RUPP) rat model and L-NAME-induced preeclampsia-like rats to study the possible role of PGT in preeclampsia. And PGT was found to be upregulated in both preeclampsia rat models by Immunohistochemical (IHC) staining. Newly identified PGT plays an important role in trophoblast proliferation via Erk signaling, providing new insights for understanding the pathogenesis of PE.

Expression of LINE-1 retrotransposon in early human spontaneous abortion tissues

The aim of this study is to investigate a new mechanism that may affect spontaneous abortions (SA): Can long interspersed nuclear element-1 (LINE-1) insertions in embryo cells lead to early SA? The method involves prospective study on new mechanism of human early SA. Twenty SA tissues and 10 induced abortion (IA) tissues were utilized for this experiment. Western Blot, Immunohistochemistry (IHC), and reverse transcription-polymerase chain reaction were used to analyze different LINE-1 proteins and mRNA expression between early SA tissues and early IA tissues. SPSS software version 21.0 was used for statistical analysis. Western Blot demonstrated that the LINE-1 protein expression in SA tissues (Mean: 60.2%) is higher than in IA tissues (Mean: 30.3%) in 91% of the compared samples. reverse transcription-polymerase chain reaction showed that LINE-1 mRNA expression in SA tissues (Mean: 64.2%) is higher than in IA tissues (Mean: 29.2%) in 6 primer pairs in 89% of the compared samples. IHC showed that the LINE-1 protein expression in SA tissues (Mean: 59.2%) is higher than in IA tissues (Mean: 28.8%) in 83% of the compared samples. Expression of LINE-1 in early SA tissues is higher than in IA tissues, LINE-1 may lead to early SA and LINE-1 plays a role in early SA, this shows that a new mechanism may be involved in SA.

Celocentesis for Early Prenatal Diagnosis in Couples at-Risk for β-Thalassemia and Sicilian (δβ)0-Thalassemia

The procedures commonly used for prenatal diagnosis (PND) of thalassemia are villocentesis or amniocentesis, respectively, at the 11th and 16th weeks of gestation. Their main limitation is essentially due to the late gestation week in which diagnosis is performed. The celomic cavity is accessible between the 7th and 9th weeks of gestation and it has been demonstrated that it contains embryonic erythroid precursor cells as a source of fetal DNA for earlier invasive PND of thalassemia and other monogenic diseases. In this study, we report the use of celomatic fluids obtained from nine women with high-risk pregnancies for Sicilian (δβ)0-thalassemia [(δβ)0-thal] deletion (NG_000007.3: g.64336_77738del13403) and β-thalassemia (β-thal). Fetal cells were isolated by a micromanipulator, and nested polymerase chain reaction (PCR) and short tandem repeats (STRs) analysis were performed. Prenatal diagnosis was successfully performed in all examined cases. One fetus was a compound heterozygote for (δβ)0- and β-thal, three fetuses were found to be carriers of β-thal, four fetuses carriers of a Sicilian δβ deletion, and one fetus without parental mutations. Accidentally, a rare case of paternal triploidy was observed. The genotypic analysis, carried out both by amniocentesis and on abortive tissue or after birth, showed concordance with results obtained on fetal celomic DNA. Our results unequivocally show that fetal DNA can be obtained by nucleated fetal cells present in the celomatic fluid and demonstrate, for the first time, that PND of Sicilian (δβ)0-thal and β-thal is feasible at an earlier time in pregnancy than other procedures.

Screening of Candidate Pathogenic Genes for Spontaneous Abortion Using Whole Exome Sequencing

Spontaneous abortion is a common disease in obstetrics and reproduction. This study aimed to screen candidate pathogenic genes for spontaneous abortion using whole-exome sequencing. Genomic DNA was extracted from abortion tissues of spontaneous abortion patients and sequenced using the Illumina HiSeq2500 high-throughput sequencing platform. Whole exome sequencing was performed to select harmful mutations, including SNP and insertion and deletion sites, associated with spontaneous abortion. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses and gene fusion analyses were performed. MUC3A and PDE4DIP were two novel mutation genes that were screened and verified by PCR in abortion tissues of patients. A total of 83,633 SNPs and 13,635 Indel mutations were detected, of which 29172 SNPs and 3093 Indels were screened as harmful mutations. The 7 GO-BP, 4 GO-CC, 9 GO-MF progress, and 3 KEGG pathways were enriched in GO and KEGG pathway analyses. A total of 746 gene fusion mutations were obtained, involving 492 genes. MUC3A and PDE4DIP were used for PCR verification because of their high number of mutation sites in all samples. There are extensive SNPs and Indel mutations in the genome of spontaneous abortion tissues, and the effect of these gene mutations on spontaneous abortion needs further experimental verification.

Molecular cytogenetic analysis of partial monosomy 10p and trisomy 10q resulting from familial pericentric inversion (10): a first case report in Chinese population

BackgroundChromosome aberrations of 10p monosomy and 10q trisomy resulting from parental pericentric inversion 10 are extremely rare, and to date, very few reports have been published on the matter.Case PresentationA 30-year-old pregnant woman with recurrent pregnancy loss is enrolled in this research. In this pregnancy, spontaneous abortion occurred in the first trimester of her pregnancy. Chromosomal microarray analysis of the abortion tissue showed a partial 10p monosomy (arr[GRCh37] 10p15.3p11.21(100,047_34,848,853) × 1) and a duplication of 10q (arr[GRCh37] 10q26.13q26.3(126,093,990_135,426,386) × 3). Further parental karyotype analysis indicated that the chromosomal abnormalities in the fetus was resulted from paternal pericenric inversion inv(10)(p11.21q26.13). This study presents the first case of a large deletion of 10p combined with 10q trisomy, resulting in pregnancy loss. Of these two manifestations, the large deletion of chromosome 10p may be the primary reason for spontaneous abortion in this subject.ConclusionsThis study presents the first case of partial 10p monosomy associated with 10q trisomy in Chinese population. It provides more information on the chromosome aberration of 10p monosomy and 10q trisomy and further strengthens the application value of microarray in the molecular etiological diagnosis of recurrent spontaneous abortion.