Unexplained Spontaneous Abortion Research Articles

Strong association between angiotensin I-converting enzyme insertion/deletion polymorphism and unexplained recurrent spontaneous abortion of Sudanese women: a case-control study

This study investigated the link between angiotensin I-converting enzyme insertion/deletion (ACE I/D) polymorphism and unexplained spontaneous abortion. This retrospective analytical case-control was conducted at the Omdurman Maternity Hospital in Sudan. The current study contained 230 individuals, including 119 cases (women who had at least three abortions) of unknown cause and 119 controls (healthy women who had at least two full-term deliveries without spontaneous abortion). Patients and controls were provided five ml of ethylenediaminetetraacetic acid blood and answered questionnaires about their demographics, personal lives, and family histories. ACE I/D polymorphisms were assessed using a conventional polymerase chain reaction approach after total genomic DNA was isolated from blood leukocytes using the GF-1 blood DNA extraction kit. Data was analyzed using the Statistical Package for the Social Sciences version 24. ACE I/D polymorphism is strongly linked to unexplained spontaneous abortion, and women with the I/D and D/D genotypes are more likely to have it than those with the I/I genotype. The current study reveals that ACEI/D polymorphism increases pregnancy problems. Sudanese women may have spontaneous abortions due to the ACE I/D polymorphism.

Defective Homologous Recombination Repair By Up-Regulating Lnc-HZ10/Ahr Loop in Human Trophoblast Cells Induced Miscarriage.

Human trophoblast cells are crucial for healthy pregnancy. However, whether the defective homologous recombination (HR) repair of dsDNA break (DSB) in trophoblast cells may induce miscarriage is completely unknown. Moreover, the abundance of BRCA1 (a crucial protein for HR repair), its recruitment to DSB foci, and its epigenetic regulatory mechanisms, are also fully unexplored. In this work, it is identified that a novel lnc-HZ10, which is highly experssed in villous tissues of recurrent miscarriage (RM) vs their healthy control group, suppresses HR repair of DSB in trophoblast cell. Lnc-HZ10 and AhR (aryl hydrocarbon receptor) form a positive feedback loop. AhR acts as a transcription factor to promote lnc-HZ10 transcription. Meanwhile, lnc-HZ10 also increases AhR levels by suppressing its CUL4B-mediated ubiquitination degradation. Subsequently, AhR suppresses BRCA1 transcription; and lnc-HZ10 (mainly 1-447 nt) interacts with γ-H2AX; and thus, impairs its interactions with BRCA1. BPDE exposure may trigger this loop to suppress HR repair in trophoblast cells, possibly inducing miscarriage. Knockdown of murine Ahr efficiently recovers HR repair in placental tissues and alleviates miscarriage in a mouse miscarriage model. Therefore, it is suggested that AhR/lnc-HZ10/BRCA1 axis may be a promising target for alleviation of unexplained miscarriage.

Polystyrene Nanoplastics Activate Autophagy and Suppress Trophoblast Cell Migration/Invasion and Migrasome Formation to Induce Miscarriage.

Nanoplastics (NPs), as emerging pollutants, have attracted global attention. Nevertheless, the adverse effects of NPs on female reproductive health, especially unexplained miscarriage, are poorly understood. Defects of trophoblast cell migration and invasion are associated with miscarriage. Migrasomes were identified as cellular organelles with largely unidentified functions. Whether NPs might affect migration, invasion, and migrasome formation and induce miscarriage has been completely unexplored. In this study, we selected polystyrene nanoplastics (PS-NPs, 50 nm) as a model of plastic particles and treated human trophoblast cells and pregnant mice with PS-NPs at doses near the actual environmental exposure doses of plastic particles in humans. We found that exposure to PS-NPs induced a pregnant mouse miscarriage. PS-NPs suppressed ROCK1-mediated migration/invasion and migrasome formation. SOX2 was identified as the transcription factor of ROCK1. PS-NPs activated autophagy and promoted the autophagy degradation of SOX2, thus suppressing SOX2-mediated ROCK1 transcription. Supplementing with murine SOX2 or ROCK1 could efficiently rescue migration/invasion and migrasome formation and alleviate miscarriage. Analysis of the protein levels of SOX2, ROCK1, TSPAN4, NDST1, P62, and LC-3BII/I in PS-NP-exposed trophoblast cells, villous tissues of unexplained miscarriage patients, and placental tissues of PS-NP-exposed mice gave consistent results. Collectively, this study revealed the reproductive toxicity of nanoplastics and their potential regulatory mechanism, indicating that NP exposure is a risk factor for female reproductive health.

Analysis of copy number variations and possible candidate genes in spontaneous abortion by copy number variation sequencing.

Embryonic chromosomal abnormalities represent a major causative factor in early pregnancy loss, highlighting the importance of understanding their role in spontaneous abortion. This study investigates the potential correlation between chromosomal abnormalities and spontaneous abortion using copy number variation sequencing (CNV-seq), a Next-Generation Sequencing (NGS) technology. We analyzed Copy Number Variations (CNVs) in 395 aborted fetal specimens from spontaneous abortion patients by CNV-seq. And collected correlated data, including maternal age, gestational week, and Body Mass Index (BMI), and analyzed their relationship with the CNVs. Out of the 395 cases, 67.09% of the fetuses had chromosomal abnormalities, including numerical abnormalities, structural abnormalities, and mosaicisms. Maternal age was found to be an important risk factor for fetal chromosomal abnormalities, with the proportion of autosomal trisomy in abnormal karyotypes increasing with maternal age, while polyploidy decreased. The proportion of abnormal karyotypes with mosaic decreased as gestational age increased, while the frequency of polyploidy and sex chromosome monosomy increased. Gene enrichment analysis identified potential miscarriage candidate genes and functions, as well as pathogenic genes and pathways associated with unexplained miscarriage among women aged below or over 35 years old. Based on our study, it can be inferred that there is an association between BMI values and the risk of recurrent miscarriage caused by chromosomal abnormalities. Overall, these findings provide important insights into the understanding of spontaneous abortion and have implications for the development of personalized interventions for patients with abnormal karyotypes.

Research progress of immune balance and genetic polymorphism in unexplained recurrent abortion

The etiology of recurrent spontaneous abortion (RSA) is extremely complex, as there are 40–50% of patients with unexplained miscarriages, known as unexplained RSA (URSA). URSA affects approximately 1–2% of females of childbearing age and has a massive impact on the physical and mental conditions of both patients and their families. The pathogenesis of the disease remains unclear, making its treatment complicated. In recent years, considerable progress has been made in the exploration of the URSA immune balance mechanism and it has been universally acknowledged that a balanced immune response (as abnormal immunity) may be the root cause of poor pregnancy outcomes. This review discussed and summarized the effects of immune cells and blocking antibodies (BAs) on URSA based on the current state of knowledge in this area. Additionally, molecular genetics also plays an essential role in the incidence rate of URSA since the role of genetic polymorphism in the pathogenesis of URSA has been thoroughly studied. Nonetheless, the outcomes of these studies are inconsistent, particularly across populations. This paper reviewed previous studies on URSA and maternal genetic polymorphism, focusing on and synthesizing the most important findings to date, and providing diagnostic recommendation for URSA patients with clinical symptoms.

Placental Pathology Findings in Unexplained Pregnancy Losses.

There are approximately 5 million pregnancies per year in the USA, with 1 million ending in miscarriage (a loss occurring prior to 20weeks of gestation) and over 20,000 ending in stillbirth at or beyond 20weeks of gestation. As many as 50% of these losses are unexplained. Our objective was to evaluate the effect of expanding the placental pathology diagnostic categories to include the explicit categories of (1) dysmorphic chorionic villi and (2) small placenta in examining previously unexplained losses. Using a clinical database of 1256 previously unexplained losses at 6-43weeks of gestation, the most prevalent abnormality associated with each loss was determined through examination of its placental pathology slides. Of 1256 cases analyzed from 922 patients, there were 878 (69.9%) miscarriages and 378 (30.1%) antepartum stillbirths. We determined the pathologic diagnoses for 1150/1256 (91.6%) of the entire series, 777/878 (88.5%) of the miscarriages (< 20weeks' gestation), and 373/378 (98.7%) of the stillbirths (≥ 20weeks' gestation). The most common pathologic feature observed in unexplained miscarriages was dysmorphic chorionic villi (757 cases; 86.2%), a marker associated with genetic abnormalities. The most common pathologic feature observed in unexplained stillbirths was a small placenta (128 cases; 33.9%). Our classification system reinforced the utility of placental examination for elucidating potential mechanisms behind pregnancy loss. The improved rate of diagnosis appeared to be the result of filling a gap in previous pregnancy loss classification systems via inclusion of the categories of dysmorphic chorionic villi and small placenta.

MiR-146b-5p downregulates IRAK1 and ADAM19 to suppress trophoblast proliferation, invasion, and migration in miscarriage†.

A large proportion of miscarriages are classified as unexplained miscarriages since no cause is identified. No reliable biomarkers or treatments are available for these pregnancy losses. While our transcriptomic sequencing has revealed substantial upregulation of miR-146b-5p in unexplained miscarriage villous tissues, its role and associated molecular processes have yet to be fully characterized. Our work revealed that relative to samples from normal pregnancy, miR-146b-5p was significantly elevated in villous tissues from unexplained miscarriage patients and displayed promising diagnostic potential. Moreover, miR-146b-5p agomir contributed to higher rates of embryonic resorption in ICR mice. When overexpressed in HTR-8/SVneo cells, miR-146b-5p attenuated the proliferative, invasive, and migratory activity of these cells while suppressing the expression of MMP9 and immune inflammation-associated cytokines, including IL1B, IL11, CXCL1, CXCL8, and CXCL12. Conversely, inhibition of its expression enhanced proliferation, migration, and invasion abilities. Mechanistically, IL-1 receptor-associated kinase-1 and a disintegrin and metalloproteinase 19 were identified as miR-146b-5p targets regulating trophoblast function, and silencing IL-1 receptor-associated kinase-1 had similar effects as miR-146b-5p overexpression, while IL-1 receptor-associated kinase-1 overexpression could partially reverse the inhibitory impact of this microRNA on trophoblasts. miR-146b-5p may inhibit trophoblast proliferation, migration, invasion, and implantation-associated inflammation by downregulating IL-1 receptor-associated kinase-1 and a disintegrin and metalloproteinase 19, participating in the pathogenesis of miscarriage and providing a critical biomarker and a promising therapeutic target for unexplained miscarriage.

Gene Expression Levels of CSF-1 and CSF-1R Endometrial under The Influence of Prolactin Level in Unexplained Miscarriage: A Case-Control Study.

Hormones such as prolactin, by influencing expression of the endometrial genes, play a pivotal role in embryo implantation and development. The present study aimed to evaluate serum level of prolactin and its effect on altering expression level of colony-stimulating factor-1 (CSF-1) and colony-stimulating factor-1 receptor (CSF-1R) genes in endometrial tissue during in vitro fertilization (IVF) pregnancy in the infertile women and recurrent pregnancy loss (RPL), compared to fertile women, who lost their pregnancies at gestational age <20 weeks. In this case-control study, 40 infertile women, 40 IVF pregnant women with RPL and 40 fertile women who lost their pregnancies at <20 weeks of gestation for unknown reasons were selected. Prolactin serum level was assessed using ELISA technique and expression of CSF-1 and CSF-1R genes was determined in endometrial tissue, using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Mean prolactin level of the infertile group was 24.38 ± 1.43 ng/mL and it had statistically significant relationship with the fertile group (P<0.001). Expression level of the CSF-1 and CSF-1R genes were higher in the fertile than infertile groups by 2.88 times (P<0.0001) and 2.64 times (P<0.0001), while it was respectively 2.28 (P<0.0001) and 1.69 (P<0.0001) times higher compared to the RPL group. Risk factors for pregnancy loss, such as aging, increased body mass index (BMI), smoking and diabetes caused decreasing changes in gene expression (CSF-1 and CSF-1R ) and the differences were statistically significant, except in the infertile group. The present study showed a significant relationship of CSF-1 and CSF-1R expression levels with pregnancy loss. Risk factors such as aging, obesity, smoking and diabetes decreased both genes expression levels.

Implantation Failure/ Reccurent miscarriages- TreatmentPOSTER 5Adalimumab for recurrent miscarriages and implantation failure

IntroductionRecurrent miscarriages and implantation failures are a frequent and still unresolved clinical situation, without any recommended therapies. In euploid implantation, the impact of dys-immune reaction such as allograft rejection could raise the use of immunomodulatory therapies. TNFa antagonists were recently reported in small case series and here we present a multicenter retrospective cohort study. Patients and MethodsPatients referred to our center for unexplained miscarriages and or implantation failure and which were refractory to previous various immunomodulatory therapies (HBPM, steroids, hydroxychloroquine) received adalimumab at 40 mg every 2 weeks a month before implantation and until 9 weeks of gestation. The primary objective was the number of pregnancies with alive baby and the safety was the second objective. ResultsA total of 42 patients with implantation failure and /or recurrent miscarriage (previous number of failures 9 [4-19], received adalimumab alone or associated with other immunomodulatory therapies. Among them, 27 were pregnant and were analyzed for the efficacy of adalimumab. Among these 27 patients, 16 women (59%) experienced a pregnancy with alive baby and 11 (41%) experienced recurrent miscarriages. The number of previous failures was not significantly different between the women with recurrent miscarriages and others (11 [4-19] vs 9 [4-15]. The number of associated immunosuppressive therapies was not significantly different between women with and without recurrent failures: low molecular weight heparin in 1 and 5 cases, steroids in 5 and 12 cases, hydroxychloroquine in 2 and 6 cases, respectively. There was no case with malformation or teratogenicity in both groups and no cases of maternal serious side effect. ConclusionIn a recurrent miscarriages and implantation failures women with particular high rates of failures, adalimumab seems to allow a good rate of alive babies with a good maternal and fetal tolerance.

Paradoxical expression of NRP1 in decidual stromal and immune cells reveals a novel inflammation balancing mechanism during early pregnancy.

To investigate the balancing mechanisms between decidualization-associated inflammation and pregnancy-related immunotolerance. Decidual samples from women with normal pregnancy (n = 58) or unexplained spontaneous miscarriage (n = 13), peripheral blood from normal pregnancy and endometria from non-pregnancy (n = 10) were collected. Primary endometrial stromal cells (ESCs), decidual stromal cells (DSCs), decidual immune cells (DICs) and peripheral blood mononuclear cells (PBMCs) were isolated. The plasmid carrying neuropilin-1 (NRP1) gene was transfected into ESC for overexpression. To induce decidualization in vitro, ESCs were treated with a combination of 10nM estradiol, 100nM progesterone and 0.5mM cAMP. Anti-Sema3a and anti-NRP1 neutralizing antibodies were applied to block the ligand-receptor interactions. RNA-seq analysis was performed to identify differentially expressed genes in DSCs and DICs, and NRP1 expression was verified by Western blotting and flow cytometry. The secretion of inflammatory mediators was measured using a multifactor cytometric bead array. The effects of Sema3a-NRP1 pathway on DICs were determined by flow cytometry. Statistical differences between groups were compared using the T test and one way or two-way ANOVA. Combined with five RNA-seq datasets, NRP1 was the only immune checkpoint changing oppositely between DSCs and DICs. The decreased expression of NRP1 in DSCs allowed intrinsic inflammatory responses required for decidualization, while its increased expression in DICs enhanced tolerant phenotypes beneficial to pregnancy maintenance. DSC-secreted Sema3a promoted immunosuppression in DICs via NRP1 binding. In women with miscarriage, NRP1 was abnormally elevated in DSCs but diminished in decidual macrophages and NK cells. NRP1 is a multifunctional controller that balances the inflammatory states of DSCs and DICs in gravid uterus. Abnormal expression of NRP1 is implicated in miscarriage.

Immunohistochemical study of MMP-2, MMP-9, EGFR and IL-8 in decidual and trophoblastic specimens of recurrent pregnancy loss cases

Background and Aim: Unexplained recurrent pregnancy loss has been a a challenging research task to experts since there is no explicit pathophysiological mechanism and therefore, the treatment remains elusive. Immunological imbalance and morphological abnormalities are under investigation. This study aims to evaluate the implication of MMP-2, MMP-9, EGFR, and IL-8 in recurrent pregnancy loss cases. Materials & Methods: The study was carried out through comparison among two groups; the unexplained miscarriage group which consisted of 22 women, and the control group consisted of 18 women, who had electively terminated their pregnancies. Both groups were in the first trimester of gestation. The specimens included the trophoblast, decidua basalis, and decidua parietalis. The study was conducted via immunohistochemical methods. Antibodies were used against MMP-2, MMP-9, EGFR, and IL-8. The results were presented at a contingency table and were statistically analyzed with the Chi-Square Test (X2). Results: There were remarkable disparities in some cases in the comparison of the two groups. MMP-9 was detected significantly high in recurrent pregnancy loss (RPL) cases, both on trophoblastic and decidual specimens (p-value < .00001), MMP-2 displayed no difference among the two groups (mild to moderate detection on trophoblast and almost negative on decidual tissues). EGFR was highly detected in trophoblastic tissue (p-value = .014). IL-8 detection was particularly different in both trophoblast and decidua parietalis of the two groups (p-value < .01). Conclusion: The study revealed both morphological and immunological dysregulations that might participate in the RPL pathogenesis.

ASSOCIATION BETWEEN RECURRENT PREGNANCY LOSS AND MATERNAL THROMBOPHILIA MUTATIONS

Pregnant women with hereditary thrombophilia have an increased risk of RPL. The role of genetic thrombophilia in women with recurrent pregnancy loss have been evaluated and assumed it to be a causing factor for recurrent pregnancy loss and other reproductive complications, which includes Factor V Leiden mutation, Prothrombin, MTHFR and PAİ 4G/5G mutation. In this study we aimed to investigate the prevalence of these molecular defects in subjects with a history of early RPL. 35 women with one or more consecutive unexplained first trimester miscarriages were detected. The presence of these mutations was assessed by polymerase chain reaction analysis. We detected a combined mutation of one or more genes in 33 women, 20% of patients with at least one homozygous mutation (7 women), oonly two women (5.7%) did not have any thrombophilic mutation. These results suggest that thrombophilic mutations have an important role in etiology of RPL.

Whole exome sequencing in unexplained recurrent miscarriage families identified novel pathogenic genetic causes of euploid miscarriage.

Can whole exome sequencing (WES) followed by trio bioinformatics analysis identify novel pathogenic genetic causes of first trimester euploid miscarriage? We identified genetic variants in six candidate genes that indicated plausible underlying causes of first-trimester euploid miscarriage. Previous studies have identified several monogenic causes of Mendelian inheritance in euploid miscarriages. However, most of these studies are without trio analyses and lack cellular and animal models to validate the functional effect of putative pathogenic variants. Eight unexplained recurrent miscarriage (URM) couples and corresponding euploid miscarriages were included in our study for whole genome sequencing (WGS) and WES followed by trio bioinformatics analysis. Knock-in mice with Rry2 and Plxnb2 variants and immortalized human trophoblasts were utilized for functional study. Additional 113 unexplained miscarriages were included to identify the mutation prevalence of specific genes by multiplex PCR. Whole blood from URM couples and their <13 weeks gestation miscarriage products were both collected for WES, and all variants in selected genes were verified by Sanger sequencing. Different stage C57BL/6J wild-type mouse embryos were collected for immunofluorescence. Ryr2N1552S/+, Ryr2R137W/+, Plxnb2D1577E/+, and Plxnb2R465Q/+ point mutation mice were generated and backcrossed. Matrigel-coated transwell invasion assays and wound-healing assays were performed using HTR-8/SVneo cells transfected with PLXNB2 small-interfering RNA and negative control. Multiplex PCR was performed focusing on RYR2 and PLXNB2. Six novel candidate genes, including ATP2A2, NAP1L1, RYR2, NRK, PLXNB2, and SSPO, were identified. Immunofluorescence staining showed that ATP2A2, NAP1L1, RyR2, and PLXNB2 were widely expressed from the zygote to the blastocyst stage in mouse embryos. Although compound heterozygous mice with Rry2 and Plxnb2 variants did not show embryonic lethality, the number of pups per litter was significantly reduced when backcrossing Ryr2N1552S/+ ♂ with Ryr2R137W/+ ♀ or Plxnb2D1577E/+ ♂ with Plxnb2R465Q/+ ♀ (P < 0.05), which were in accordance with the sequencing results of Family 2 and Family 3, and the proportion of Ryr2N1552S/+ offspring was significantly lower when Ryr2N1552S/+ female mice were backcrossed with Ryr2R137W/+ male mice (P < 0.05). Moreover, siRNA-mediated PLXNB2 knockdown inhibited the migratory and invasive abilities of immortalized human trophoblasts. Besides, additional 10 variants of RYR2 and PLXNB2 were detected in 113 unexplained euploid miscarriages by multiplex PCR. The relatively small number of samples is a limitation of our study which may result in the identification of variants in unique candidate genes with no definitive although plausible causal effect. Larger cohorts are needed to replicate these findings and additional functional research is needed to confirm the pathogenic effects of these variants. Moreover, the sequencing coverage restricted the detection of low-level parental mosaic variants. For first-trimester euploid miscarriage, variants in unique genes may be underlying genetic etiologies and WES on trio could be an ideal model to identify potential genetic causes, which could facilitate individualized precise diagnostic and therapeutic regimens in the future. This study was supported by grants from the National Key Research and Development Program of China (2021YFC2700604), National Natural Science Foundation of China (31900492, 82101784, 82171648), Basic Science Center Program of the National Natural Science Foundation of China (31988101), Key Research and Development Program of Shandong Province (2021LCZX02), Natural Science Foundation of Shandong Province (ZR2020QH051), Natural Science Foundation of Jiangsu Province (BK20200223), Taishan Scholars Program for Young Experts of Shandong Province (tsqn201812154) and Young Scholars Program of Shandong University. The authors declare no conflicts of interest. N/A.

Uterine artery Doppler and Sub-Endometrial blood flow and Serum Progesterone level in patients with Unexplained Recurrent Miscarriage

Background: Unexplained Miscarriage that occurs repeatedly is still a difficult issue for couples and professionals. It affects 2–5% of females attempting to conceive and represents a considerable challenge for physicians. Aim of the Work: This study compares women who have unexplained recurrent miscarriages to healthy, fertile women in order to assess uterine artery blood flow, sub-endometrial blood flow, and serum progesterone levels in these women. Patients and Methods: At Al-Galaa Maternity Teaching Hospital, our research was carried out, and patients were divided into two groups. The case group: 50 individuals disclosed a history of recurrent, unexplained miscarriages, and the control group: 50 individuals who had at least one term birth and no history of miscarriage. Results: pulstility index of the uterine artery especially the mean value, and flow index, vascular index and vascular flow index of the endometrial and sub-endometrial area, in addition to the serum progesterone level are crucial factors in case of unexplained recurrent miscarriage. They affect unexplained recurrent miscarriage incidence significantly. The pulstility index of the uterine artery has its influence on unexplained recurrent miscarriage with 74% accuracy. The flow index has 95% accuracy, and the vascular flow index has 86% accuracy in case unexplained recurrent miscarriage. The serum progesterone level has 70% accuracy effect on unexplained recurrent miscarriage. Conclusion: the study documented that the uterine artery Doppler, endometrial and sub-endometrial blood flow level and serum progesterone level affect the incidence of unexplained recurrent miscarriage with accuracy ranging between 70% and 95%.

BPDE, the Migration and Invasion of Human Trophoblast Cells, and Occurrence of Miscarriage in Humans: Roles of a Novel lncRNA-HZ09.

Recurrent miscarriage (RM) affects 1%-3% of pregnancies. However, in almost 50% of cases, the cause is unknown. Increasing evidence have shown that benzo(a)pyrene [B(a)P], a representative of polycyclic aromatic hydrocarbons (PAHs), is correlated with miscarriage. However, the underlying mechanisms of B(a)P/benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE)-induced trophoblast cell dysfunctions and miscarriage remain largely unknown. The objective was to discover the role(s) of a novel lncRNA, lnc-HZ09, in the regulation of BPDE-inhibited migration and invasion of trophoblast cells and the occurrence of miscarriage. Human trophoblast cells were treated with 0, 0.25, 0.5, BPDE with or without corresponding lnc-HZ09 silencing or overexpression. Using these cells, we evaluated cell migration and invasion, the mRNA and protein levels of members of the PLD1/RAC1/CDC42 pathway, the regulatory roles of lnc-HZ09 in PLD1 transcription and mRNA stability, and lnc-HZ09 transcription and stability. Human villous tissues were collected from RM () group and their matched healthy control (HC, ) group. We evaluated the levels of BPDE-DNA adducts, lnc-HZ09, and the mRNA and protein expression of members of the PLD1/RAC1/CDC42 pathway, and correlated their relative expression levels. We further constructed 0, 0.05 or B(a)P-induced mouse miscarriage model (each ), in which the mRNA and protein expression of members of the Pld1/Rac1/Cdc42 pathway were measured. We identified a novel lnc-HZ09. Human trophoblast cells treated with lnc-HZ09 exhibited less cell migration and invasion. In addition, the levels of this lncRNA were higher in villous tissues from women with recurrent miscarriage than those from healthy individuals. SP1-mediated PLD1 mRNA levels were lower, and HuR-mediated PLD1 mRNA stability was less in trophoblast cells overexpressing lnc-HZ09. However, trophoblast cells treated with MSX1 had higher levels of lnc-HZ09, and METTL3-mediated m6A methylation on lnc-HZ09 resulted in greater lnc-HZ09 RNA stability. In BPDE-treated human trophoblast cells and in RM villous tissues, MSX1-mediated lnc-HZ09 transcription and METTL3-mediated lnc-HZ09 stability were both greater. In our mouse miscarriage model, B(a)P-treated mice had lower mRNA and protein levels of members of the Pld1/Rac1/Cdc42 pathway. These results suggest that in human trophoblast cells, BPDE exposure up-regulated lnc-HZ09 level, suppressed PLD1/RAC1/CDC42 pathway, and inhibited migration and invasion, providing new insights in understanding the causes and mechanisms of unexplained miscarriage. https://doi.org/10.1289/EHP10477.

Does hysteroscopy surgery enhance subsequent reproductive outcomes in women with unexplained infertility?

Introduction and Aim: Embryo implantation and a healthy placenta depend on the uterine cavity and endometrium, thus any reproductive surgery for unexplained infertility must evaluate these. Hysteroscopy cures intrauterine adhesions, submucous fibroids, endometrial polyps, and endometriosis and directly visualizes the uterus cavity. This study investigated whether hysteroscopy could diagnose uterine disorders in women with recurrent, unexplained miscarriages and improve reproductive outcomes.&#x0D;  &#x0D; Methods: This observational retrospective study included 58 patients who had recurrent pregnancy loss and were hospitalized in the Kamal AL-Samarrai Hospital's reproduction unit / Physiopathology section in Baghdad, Iraq between January 2021 and February 2022. The participants were divided into two groups based on whether they had received hysteroscopic surgery. IVF was performed on patients who underwent hysteroscopy surgery. A comparative analysis was made for clinical factors and uterine pathology among the two groups. Data were subjected to statistical analysis.&#x0D;  &#x0D; Results: The patients in this study ranged in age from 20 to 42 years, with an average of 3265 years, and a BMI ranging from (20-39) with an average of 29.270.48 kg/m2. Out of the 58 women analyzed, 31 (53.4%), 13 (22.4%), and 48 (82.7%) had a previous cesarean, laparoscopy, and HSG procedures, respectively. 28 patients (48.2%) who underwent hysteroscopic surgery were detected with uterine disorders; IUA (28%), fibroids (25%), submucosal polyps (21.4%), endometrial hyperplasia (17.8%), and endometriosis (7.1%). No significant difference was seen for the duration of infertility, type of infertility, or previous IVF attempt failure between the hysteroscopic and control groups (p&gt;0.05). In women who underwent IVF procedures after hysteroscopic surgery, the clinical pregnancy rate was lower as compared to women without surgery.&#x0D; Conclusion: Hysteroscopy can diagnose some intrauterine diseases early enough for surgery. Undiagnosed infertility in women with RPL suggests a hysteroscopic examination before in vitro fertilization. 

A Comparative Analysis of Anticardiolipin, Anti-Β2-Glycoprotein-1, and Lupus Anticoagulants in Saudi Women with Recurrent Spontaneous Abortions.

Association and pathogenesis of antiphospholipid antibodies (APAs) in patients with Antiphospholipid syndrome (APS) as well as systemic lupus erythematosus (SLE) suffering from unexplained spontaneous abortions is controversial. Moreover, the majority of the published evidence is based on the patient histories rather than the study groups. The main objective of the present study is to do a comparative analysis of the high levels of antiphospholipid antibodies (APAs) including anticardiolipin (ACA), anti-β2-glycoprotein-1 (β2 GP1), and Lupus anticoagulants (LA) and their association with the gestational age of abortion and duration past abortion in Saudi women. In this study, 100 women living in Makkah city, located in the Western region of Saudi Arabia, with a mean age of 30.5 ± 12.60 years (mean ± standard deviation), having a previous history of recurrent spontaneous abortions were enrolled as case group and 100 healthy pregnant women previously having one or more successful pregnancies were selected as control group. Based on the gestational age of the patient’s miscarriages, our results revealed consistently and significantly high levels of ACA, β2 GP1, and LA, being greatest at more than 15 weeks when compared with 10 weeks and 11−15 weeks, respectively. Based on duration of past miscarriages, a slightly increased level was observed at ≤4 years after their first miscarriage. In addition, less or more equal levels of these antibodies were observed at 5−12 and more than 12 years in all the cases of miscarriages. We concluded an elevated pattern of APAs in these patients with an increase in the age of abortion. A comparison between the levels of ACA, β2 GP1, and LA further indicated a highly significant level of LA in all the cases of abortions (p < 0.0005).

RhoB Promotes Endometrial Stromal Cells Decidualization Via Semaphorin3A/PlexinA4 Signaling in Early Pregnancy.

Endometrial decidualization refers to a series of morphological changes and functional remodeling of the uterine endometrium to accept the embryo under the effect of estrogen and progesterone secreted by ovaries after ovulation. During decidualization, endometrial stromal cells (ESCs) proliferate and differentiate into decidual stromal cells, undergoing cytoskeletal rearrangement-mediated morphological changes and expressing decidualization markers, such as insulin-like growth factor-binding protein-1 and prolactin. Ras homology (Rho) proteins, a family of small G proteins, are well known as regulators of cellular morphology and involved in multiple other cellular processes. In this study, we found ras homolog family member B (RHOB) was the most significantly upregulated gene in the Rho protein family after the in vitro decidualization of human primary ESCs. RhoB expression was induced mainly by 3',5'-cyclic adenosine 5'-monophosphate (cAMP) / protein kinase A (PKA) / cyclic adenosine monophosphate-response element binding protein signaling and partly by progesterone signaling. Knockdown of RhoB in ESCs greatly inhibited actin cytoskeletal rearrangement, cell morphological transformation, and upregulation of insulin-like growth factor-binding protein-1, suggesting an indispensable role of RhoB in decidualization. Mechanistically, the downstream target of RhoB was semaphorin3A (Sema3A), which mediated its signaling via interacting with the receptor, plexinA4. More importantly, decreased expression of RhoB, Sema3A, and plexinA4 were detected in deciduas from patients with unexplained spontaneous miscarriage. Collectively, our results indicate that RhoB/Sema3A/plexinA4 signaling plays a positive role in endometrial decidualization and relates to unexplained spontaneous miscarriage, which is worthy of further exploration so as to provide new insights into therapeutic strategies for pregnancy diseases associated with poor decidualization.

P-366 Tumor necrosis factor-alpha and milk fat globule-epidermal growth factor 8: novel biomarkers to predict implantation failure and pregnancy loss

Abstract Study question Can implantation failure (IF) and pregnancy loss be predicted in serum prior to in vitro fertilization (IVF)? Summary answer Tumor necrosis factor (TNFα) and milk fat globule-epidermal growth factor 8 (MFG-E8) levels may serve as serum markers to predict IF and pregnancy loss. What is known already In a normal pregnancy, mediators such as TNFα are released creating a physiological inflammatory response. However, an exaggerated release of TNFα has been associated with IF and recurrent pregnancy loss (RPL). Recent studies demonstrated that TNFα up-regulates the expression of inflammatory factors such as MFG-E8. MFG-E8 is known to modulate implantation by acting at various levels of the trophoblast and endometrial compartments. Hence an overexpression of this protein may result in apoptosis, endometrial damage, and impaired implantation. Study design, size, duration This multicentric prospective controlled pilot clinical study was conducted from January 2016 to January 2020 and included 30 women in their natural cycle in which serum MFG-E8, TNFα, estradiol (E2), and progesterone (P4) levels were quantified in the early proliferative (cycle day 2) and secretory phases (urinary LH + 7 days). Additionally, an endometrial biopsy was obtained on urinary LH + 7 days for MFG-E8 and TNF α protein and gene expression analysis. Participants/materials, setting, methods Women ages 21-35y were recruited from 3 groups: fertile controls (C), unexplained IF (following 3 failed good quality embryo transfers), and RPL (at least 2 unexplained first trimester miscarriages). Patients with history of uterine surgery, abnormal uterine cavity (fibroids, endometrial polyps, adhesions, adenomyosis, and congenital uterine abnormalities), hydrosalpinx, diminished ovarian reserve, harboring chromosomal rearrangements, thrombophilia, or autoimmune diseases were excluded. Main results and the role of chance Ten women were included in each group. No statistical differences were found in age, BMI, AMH, baseline FSH, and baseline antral follicle count among cohorts. Mean serum E2 and P4 levels were similar among all groups in both the proliferative and secretory phases: E2 proliferative (C 69.19±26.64  pg/ml, IF 64.19±32.56  pg/ml, RPL proliferative 57.44±38.51; p = 0.55), E2 secretory (C 164.10±52.57  pg/ml, IF 172.57±121, RPL 173.81±.97.35; p = 0.25), P4 proliferative (C 0.45±0.15 ng/ml, IF 0.45±0.19 ng/ml, RPL 0.53±0.18 ng/ml; p = 0.85), P4 secretory (C 7.42±4.06 ng/ml, IF 7.8±4.56 ng/ml, RPL 8.05±4.38 ng/ml; p = 0.74). Mean serum TNFα levels were significantly higher in both, the proliferative and secretory phases for the RPL group (proliferative RPL 9.98±4.47  pg/ml, IF 4.73±2.56  pg/ml, C 3.42±1.01  pg/ml; p = 0.001 vs secretory RPL 8.67±4.45  pg/ml, C 3.35±0.94  pg/ml, IF 3.85±1.01  pg/ml; p = 0.03). Mean serum MFG-E8 levels were significantly higher in the IF group during the proliferative phase (IF 373±201  pg/ml, RPL 201±115  pg/ml, C 225.58±109.73pg/ml; p = 0.03), but not in the secretory phase (IF 237±101  pg/ml, RPL 189±116  pg/ml, C 199.41±112.43  pg/ml; p = 0.15). Endometrial MFG-E8 mRNA levels were significantly lower in the IF and RPL group compared to C (p = 0.03). TNFα mRNA levels were not statistically significant among groups (p = 0.12). Limitations, reasons for caution This is a pilot study to assess feasibility. Due to the small sample size, the effects of more subtle covariates would not have been detected. Future larger studies are warranted. Wider implications of the findings These novels differentially expressed serum and endometrial markers may provide information on the physiology of implantation and could generate the basis for non-invasive diagnostic tools and therapeutic use of MFG-E8/TNFα antagonists in women with IF and RPL. Trial registration number IIT-2014-100366

Low dose aspirin versus low dose aspirin plus omega 3 versus low molecular weight heparin in treatment of unexplained recurrent miscarriage

Introduction: recurrent miscarriages are the loss of 3 or more consecutive pregnancies before the 20th week of gestation. It is classified into primary and secondary. Objective: to evaluate the effectiveness of low dose aspirin vs. low dose aspirin and omega 3 vs. LMWH in treatment of unexplained recurrent miscarriage. Materials and methods: This comparative prospective research was conducted on 60 1st trimester pregnant female with a history of two or more unexplained spontaneous consecutive abortion aged from 18 to 36 years old with gestational age (5-7 weeks) and single intrauterine pregnancy. Patients were subdivided equally into 3 groups: Group A: received a daily dose of 75mg of aspirin, group B: received a daily dose of 75 mg of aspirin in addition to 300mg of omega -3 fatty acids and group C: received a daily dose of 4000 IU of enoxaparin. Results: There was an insignificant difference in pregnancy outcome among the three groups. 12 (60%) cases in group A, 15 (75%) cases in group B and 14 cases in group C completed 20 weeks of pregnancy.