Overview of genetic causes of recurrent miscarriage and the diagnostic approach

Recurring miscarriage(RM)is a frustrating reproductive complication with variable etiology.Numerous genetic defects have been known to play a crucial role in the etiology of RM.Chromosomal abnormalities are frequently detected,while other genetic defects cannot be diagnosed through routine research,such as cryptic chromosomal anomalies,single nucleotide polymorphism,single-gene defect,and gene copy number variation.Diagnostic laboratories have recently used variable advanced techniques to detect potential genetic abnormalities in couples with RM and/or in products of conception.Here we aim to summarize the known genetic causes of RM,with a focus on the new diagnostic techniques.Knowledge of the genetic profile of miscarriages is important for prognosis and potential counseling planning,as well as the prenatal diagnostic strategy in subsequent pregnancies.

Expression of E2A in Mid-secretory Endometrium of Women Suffering from Recurrent Miscarriage

E2A is involved in promoting forkhead box P3（FOXP3） and retinoid-related orphan receptor gamma t（RORγt） gene transcription, which are pivotal transcription factors of T regulatory cells and Th17 cells, respectively. Little is known about the involvement of E2 A in pregnancy process. This study aimed to investigate the expression of E2 A, cytotoxic T-lymphocyte-associated protein 4（CTLA-4）, and Foxp3 in luteal phase endometrium of women suffering recurrent miscarriage（RM）（n=21） and control group（n=11） by immunohistochemistry, with the Vectra？ automated quantitative pathology imaging system for analysis. The percentage of E2 A＋ cells and CTLA-4＋ cells was significantly higher in the endometrium of women with RM than in the controls. There was positive correlation between E2 A and CTLA-4（r=0.523, P=0.002）, E2 A and FOXP3（r=0.380, P=0.032）, and FOXP3 and CTLA-4（r=0.625, P=0.000） in the mid-secretory phase of endometrium for all subjects. It was concluded that the abnormal expression of endometrial E2 A existed in mid-secretory endometrium of women with RM, and there was a positive correlation between E2 A and FOXP3, and E2 A and CTLA-4, suggesting the possible regulation role of E2 A involved in regulating endometrium receptivity.

Sperm DNA fragmentation, recurrent implantation failure and recurrent miscarriage

Evidence is increasing that the integrity of sperm DNA may also be related to implantation failure and recurrent miscarriage （RM）. To investigate this, the sperm DNA fragmentation in partners of 35 women with recurrent implantation failure （RIF） following in vitro fertilization, 16 women diagnosed with RM and seven recent fathers （control） were examined. Sperm were examined pre- and post-density centrifugation by the sperm chromatin dispersion （SCD） test and the terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL） assay. There were no significant differences in the age of either partner or sperm concentration, motility or morphology between three groups. Moreover, there were no obvious differences in sperm DNA fragmentation measured by either test. However, whilst on average sperm DNA fragmentation in all groups was statistically lower in prepared sperm when measured by the SCD test, this was not seen with the results from the TUNEL assay. These results do not support the hypothesis that sperm DNA fragmentation is an important cause of RIF or RM, or that sperm DNA integrity testing has value in such patients. It also highlights significant differences between test methodologies and sperm preparation methods in interpreting the data from sperm DNA fragmentation tests.

Single-cell Immune Landscape of Human Recurrent Miscarriage

t Successful pregnancy in placental mammals substantially depends on the establishment of maternal immune tolerance to the semi-allogenic fetus.Disorders in this process are tightly associated with adverse pregnancy outcomes including recurrent miscarriage(RM).However,an indepth understanding of the systematic and decidual immune environment in RM remains largely lacking.In this study,we utilized single-cell RNA-sequencing(scRNA-seq)to comparably analyze the cellular and molecular signatures of decidual and peripheral leukocytes in normal and unexplained RM pregnancies at the early stage of gestation.Integrative analysis identifies 22 distinct cell clusters in total,and a dramatic difference in leukocyte subsets and molecular properties in RM cases is revealed.Specifically,the cytotoxic properties of CD8^(+)effector T cells,nature killer(NK),and mucosal-associated invariant T(MAIT)cells in peripheral blood indicates apparently enhanced pro-inflammatory status,and the population proportions and ligand–receptor interactions of the decidual leukocyte subsets demonstrate preferential immune activation in RM patients.The molecular features,spatial distribution,and the developmental trajectories of five decidual NK(dNK)subsets have been elaborately illustrated.In RM patients,a dNK subset that supports embryonic growth is diminished in proportion,while the ratio of another dNK subset with cyto toxic and immune-active signature is significantly increased.Notably,a unique pro-inflammatory CD56^(+)CD16^(+)dNK subset substantially accumulates in RM decidua.These findings reveal a comprehensive cellular and molecular atlas of decidual and peripheral leukocytes in human early pregnancy and provide an in-depth insight into the immune pathogenesis for early pregnancy loss.

Antiphospholipid Antibody Titers and Clinical Outcomes in Patients with Recurrent Miscarriage and Antiphospholipid Antibody Syndrome： A Prospective Study

Background： The management of patients with recurrent miscarriage （RM） and antiphospholipid antibody syndrome （APS） includes prolonged treatment with heparin and aspirin, starting from the confirmation of pregnancy and continuing until 6 weeks after birth. This study was conducted to determine the relationship between changes in antiphospholipid antibody titers and clinical outcomes. The effect of a shortened treatment regimen was also evaluated. Methods： A prospective study of 123 patients with RM and APS between March 2012 and May 2014 was conducted. Patients were pretreated with a low dose of prednisone plus aspirin before pregnancy, and heparin was added after conception. The levels of antiphospholipid antibodies and pregnancy outcomes were evaluated. Results： All patients were positive for anti-β2-glycoprotein 1 （anti-β2-GP 1） IgM. Atier prepregnancy treatment with low-dose prednisone plus aspirin, 99 of 123 patients became pregnant, and 87 of those pregnancies resulted in successful live births, while 12 resulted in miscarriage, showing a success rate of 87.9%. In the live birth group, levels of anti-β2-GP1 were 56.8±49.0 RU/ml before the pretreatment regimen, 32. 1± 26.0 RU/ml after 2 months of pretreatment, and 24.1 ± 23. IRU/ml during early pregnancy （P 〈 0.05）. In the miscarriage group, antiphospholipid antibody titers were 52.8 ±30.7 RU/ml before pretreatment, 38.5 ±34.2 RU/ml after pretreatment, and 33.9 ±24.7 RU/ml during early pregnancy; the decrease in antiphospholipid antibodies was lower in the miscarriage group than in the live birth group （P 〈 0.05）. Of the 24 inferthe patients, the average antibody titer did not decline after pretreatment （P = 0.802）. Conclusions： Anti-[32-GP1 IgM was the predominant form of antibody in patients with RM and APS. The decreases in antiphospholipid antibody titers correlated with better pregnancy outcomes. The shorter treatment regimen was effective and economical.

CircRNA expression profiles in decidual tissue of patients with early recurrent miscarriage

Circular RNAs(circRNAs)are a novel class of endogenous noncoding RNAs that play important roles in gene expression regulation.This study aimed to evaluate the potential role of circRNAs in decidual tissue of patients with early recurrent miscarriage(RM).We constructed circRNA expression profiles in decidual tissue using microarray data.A total of 123 differentially expressed circRNAs,including 78 upregulated and 45 downregulated circRNAs were detected in the early RM group compared with the control group(P<0.05).Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis also revealed the enrichment of specific circRNAs.The verified circRNA-targeted miRNA-mRNA network was constructed,most of the circRNAs harbored miRNA binding sites.The network involved 3 circRNAs,27 microRNAs and 82 mRNAs.Hsa_circRNA_103092-miR-224-PRLR network was selected to verify by qRT-PCR.These results showed that circRNAs are aberrantly expressed in the decidual tissue in early RM patients and play potential roles in the development of early RM.It gives new insights into the mechanism of recurrent miscarriage.

Microchimerism in recurrent miscarriage

Maternal-fetal cell exchange during pregnancy results in acquisition of microchimerism, which can durably persist in both recipients. Naturally acquired microchimerism may impact maternal-fetal interaction in pregnancy. We conducted studies to ask whether microchimerism that a woman acquired from her own mother is detectable before or during pregnancy in women with recurrent miscarriage. Fetal microchimerism was also assayed. Women with primary idiopathic recurrent miscarriage （n=23） and controls （n=31） were studied. Genotyping was conducted for probands, their mothers and the fetus, a non-shared polymorphism identified and quantitative polymerase chain reaction performed to measure microchimerismin peripheral blood mononuclear cells. Preconception comparisons were made between recurrent miscarriage subjects and controls, using logistic regression and Wilcoxon rank sum. Longitudinal microchimerism in subsequent pregnancies of recurrent miscarriage subjects was described. There was a trend toward lower preconception detection of microchimerism in recurrent miscarriage versus controls, 6% vs. 19% （1/16 vs. 6/31, P=0.2）. During pregnancy, 3111 （27%） of recurrent miscarriage subjects who went on to have a birth had detection of microchimerism from their own mother, whereas neither of two subjects who went on to miscarry had detection （0/2）. This initial data suggest that microchimerism from a woman＇s own mother, while detectable in women with recurrent miscarriage, may differ from controls and according to subsequent pregnancy outcome. Further studies are needed to determine the cell types,quantities and any potential functional role of microchimerism in recurrent miscarriage.

Intravenous Immunoglobulin Treatment of Recurrent Miscarriage:an Update of Placebo-controlled Trials

Background Immunological disturbances which may be treated with intravenous immunoglobulin (IvIg) play a significant role in the majority of patients with recurrent miscarriage (RM). The present study aimed to review the current knowledge about IvIg treatment in RM primarily based on results from published placebo controlled trials. Seven placebo controlled trials were identified comprising a total of 343 patients. The background variables, the treatment protocols and the results were extremely different between the trials. Among the patients with secondary RM, a meta analysis showed that the pooled odds ratio for live birth among IvIg treated women compared with women infused with placebo was 1.69 (95 % CI = 0.72～3.96, not significant). IvIg also seemed to be efficacious in patients with repeated second trimester intrauterine fetal deaths. A new big placebo controlled trial should be conducted which focus on RM patients with secondary RM or recurrent second trimester fetal deaths. Sufficient IvIg doses should be given with optimal time intervals.

Effects of Heat Clearing and Stasis Resolving Method on PTS,Inflammatory Factors and T-lymphocyte Subsets in Peripheral Blood of URM Patients with Suppressed Internal Heat

[Objectives]To explore the effects of heat clearing and stasis resolving method on prethrombotic state,inflammatory factors and T-lymphocyte subsets in peripheral blood of unexplained recurrent miscarriage(URM)patients with suppressed internal heat.[Methods]Thirty cases of URM patients with suppressed internal heat and 30 normal women were collected,and characteristics of changes in peripheral serum D-dimer(D-D),fibrin degradation product(FDP),fibrinogen(FIB),IL-6,IL-10 and TNF-α,CD,CD,CD,CD,CDlevels were detected.URM patients were treated with traditional Chinese medicine for clearing heat and resolving blood stasis for 3 menstrual cycles,and the changes of indicators before and after treatment were observed.[Results]Compared with normal women,the peripheral serum levels of D-D,IL-6,TNF-αand CDin URM patients with suppressed internal heat were increased(P<0.05),while the IL-10 lymphocyte level was significantly decreased(P<0.05);compared with that before treatment,the contents of D-D,IL-6,TNF-αand CDdecreased after 3 menstrual cycles(P<0.05),while the contents of IL-10 and CDT lymphocytes increased significantly(P<0.05).[Conclusions]The heat clearing heat and stasis resolving method can effectively improve the prethrombotic state of URM,and the action mechanism may be related to the regulation of immune and peripheral blood inflammatory factors.

Epidemiological Survey and Risk Factor Analysis of Recurrent Spontaneous Miscarriages in Infertile Women at Large Infertility Centers

Background：A higher frequency of spontaneous miscarriage has been observed in infertile couples,and there is a higher prevalence of infertility among patients with a history of recurrent spontaneous miscarriages （RSMs;〉2 miscarriages）.This study aimed to determine the proportion of infertile patients with RSM and examine risk factors associated in patients with RSM being treated with assisted reproductive technologies.Methods：This cross-sectional observational study was conducted at six reproductive medicine centers in three cities of China.Data of 751 patients with at least one spontaneous miscarriage were analyzed.Demographic data and etiological factors associated with infertility were compiled and compared between patients with a single spontaneous miscarriage （SSM） and those with RSM.Results：Two hundred （26.6%,95% confidence interval [CI]：23.50-29.95%） patients experienced RSMs and 551 （73.4%） had a single miscarriage.The odds of RSM increased with increasing age （odds ratio [OR] =1.06）,uterine disorders （OR =2.09）,endocrine disorders （OR =2.48）,and immune disorders （OR =2.98）.Higher education level,masters or above,and a pelvic cavity disorder were associated with lower risk of RSM （OR =0.27 and 0.46,respectively）.Late spontaneous miscarriages were more frequent in patients with RSM than in those with a SSM （31.5% vs.14.2%,respectively,P 〈 0.001） and were associated with a history of uterine cavity procedures （OR =2.095） and cervical factors related to infertility （OR =4.136,95% CI：1.012-16.90）.Conclusions：Compared to patients with only a SSM,the conditions of patients with RSM are more complicated.To increase the success rate of assisted reproductive technology,factors including uterus cavity adhesion,cervical relaxation,endocrine disorders,and immune disorders should be treated before assisted reproduction is initiated.These data may provide treatment guidance for infertile patients with a history of RSM.