Recurrent Miscarriage Patients Research Articles

POSTER 17The cytokines IL-1b and IL-18 are upregulated in the placenta of recurrent miscarriage patients

POSTER 17The cytokines IL-1b and IL-18 are upregulated in the placenta of recurrent miscarriage patients

Determination of chlamydial load in recurrent miscarriage in relation to some female sex hormones: a case-control study.

Chlamydia trachomatis is a frequent cause of adverse pregnancy outcomes including recurrent spontaneous abortion (RSA). However, regulation of infectious load by host immune response is unknown. Female sex hormones are known to affect C. trachomatis infection. The aim of this study was to determine correlation of chlamydial infectious load and gestational age with concentration of progesterone/estrogen in RSA. Urine and non-heparinized blood were collected from patients with history of > 3 spontaneous abortions (n = 150, cases) and those with history of > 2 successful deliveries (n = 150, controls) from Department of Obstetrics and Gynecology, Safdarjung hospital, New Delhi, India. C. trachomatis positivity was determined by polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) and chlamydial load by real-time PCR. Estrogen and progesterone concentrations were estimated by ELISA and correlated with chlamydial load. 22/150 case patients were positive for C. trachomatis. 2,000-10,000 copies/mL of chlamydial load were detected in infected RSA patients. Progesterone concentration showed significant decrease while estrogen concentration was significantly increased in C. trachomatis-positive RSA patients versus controls. Chlamydial load and estrogen concentration were positively correlated while progesterone concentration was negatively correlated with chlamydial load. Gestational age was positively correlated with concentration of estrogen and negatively correlated with concentration of progesterone in infected-RSA women. Overall findings suggest that interplay between chlamydial copy load, hormonal changes such as increased expression of estrogen and decreased expression of progesterone, and advanced gestational age may be contributing as deciding factors for ensuing RSA during C. trachomatis-infection.

Microarray profiling of serum micro-RNAs in women with Chlamydia trachomatis-associated recurrent spontaneous abortion: A case control study

Chlamydia trachomatis infection is a major cause of sexually transmitted diseases and adverse pregnancy outcomes such as recurrent spontaneous abortion (RSA). Micro-RNAs (miRNAs) have been known to be upregulated/downregulated in various reproductive-associated diseases such as ectopic pregnancy, preterm birth and pre-eclampsia. However, there is paucity of literature on miRNA profile in C. trachomatis-infected RSA. The present study aimed to determine the profile of serum miRNAs followed by their validation in C. trachomatis-infected RSA and to find target genes involved in biological pathways. Non-heparinized blood and first void urine were collected from 30 non-pregnant women with RSA and 30 non-pregnant women with ≥2 successful deliveries (controls) attending Department of Obstetrics and Gynaecology, Safdarjung hospital, New Delhi, India. C. trachomatis detection was done in urine by PCR and chlamydial load was determined by quantitative real-time PCR (qRT-PCR). miRNA expression was studied by microarray analysis followed by in vitro validation by qRT-PCR. Analysis of target genes/pathways was characterized in silico. 06 RSA patients were infected with C. trachomatis, while chlamydial load was found to be 6000–12,000 copies/ml. 110 circulating miRNAs were expressed differentially in infected RSA patients compared with controls. Of these, 16 were overexpressed and 94 downregulated. 06 differentially expressed circulating miRNAs were selected to validate the microarray results. qRT-PCR data confirmed the reliability of the microarray results: miR-4443, -5100, −7975 showed statistically significant upregulation, while miR-6808–5p, −3148, -6727–5p were significantly downregulated in infected RSA patients versus controls. Chlamydial load was positively correlated with these upregulated miRNAs. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that target genes of miRNAs in RSA are involved in AMPK, Akt and mTOR signaling pathways. Overall results indicate that differentially expressed circulating miRNAs are involved in pathogenesis of C. trachomatis-associated RSA and have the potential to be used as biomarkers for predicting RSA.

Dysregulation of Histone Deacetylases Inhibits Trophoblast Growth during Early Placental Development Partially through TFEB-Dependent Autophagy-Lysosomal Pathway.

Dysregulated biological behaviors of trophoblast cells can result in recurrent spontaneous abortion (RSA)-whose underlying etiology still remains insufficient. Autophagy, a conserved intracellular physiological process, is precisely monitored throughout whole pregnancy. Although the exact mechanism or role remains elusive, epigenetic modification has emerged as an important process. Herein, we found that a proportion of RSA patients exhibited higher levels of autophagy in villus tissues compared to controls, accompanied with impaired histone deacetylase (HDAC) expression. The purpose of this study is to explore the connection between HDACs and autophagy in the pathological course of RSA. Mechanistically, using human trophoblast cell models, treatment with HDAC inhibitor (HDACI)-trichostatin A (TSA) can induce autophagy by promoting nuclear translocation and transcriptional activity of the central autophagic regulator transcription factor EB (TFEB). Specifically, overactivated autophagy is involved in the TSA-driven growth inhibition of trophoblast, which can be partially reversed by the autophagy inhibitor chloroquine (CQ) or RNA interference of TFEB. In summary, our results reveal that abnormal acetylation and autophagy levels during early gestation may be associated with RSA and suggest the potential novel molecular target TFEB for RSA treatment.

Recognition of the Subtypes Classification and Diagnostic Signature Based on RNA N6-Methyladenosine Regulators in Recurrent Spontaneous Abortion.

Recurrent spontaneous abortion (RSA) is a common reproductive disease in female patients that seriously affects the quality of life of patients. N6-methyladenosine (m6A), as the most common modification, plays an important role in various biological behaviors; however, the relationship between m6A and RSA is still unknown. In the present study, we utilized RNA sequencing data and clinical information of RSA patients and normal women in the GEO database to identify the expression profiles of m6A regulators in RSA. Based on the m6A regulators' expression profiles, we constructed a random forest model consisting of 4 genes to predict the prevalence of RSA patients, including FMR1, METTL14, LRPPRC, and RBMX. The predictive performance of the nomogram was constructed and validated. Not only that, consensus clustering was performed to divide RSA patients into 3 clusters based on the expression of m6A regulators and calculated the m6A scores and immune infiltration of patients in different clusters. It was found that the TH1-type immune response was dominant in the A cluster, the B-type immune activity was poor, and the C cluster was the strongest. In addition, on the basis of m6A typing, we further used the differentially expressed genes between clusters to perform consensus clustering verification, and the results were consistent with the previous findings. In conclusion, the m6A regulators played an indispensable role in the occurrence of RSA, and the m6A-based typing could effectively identify the immune characteristics of different RSA patients to a certain extent, providing a new direction and strategy for the diagnosis and treatment of RSA patients.

Jiawei Shoutai Pill promotes decidualization by regulating the SGK1/ENaC pathway in recurrent spontaneous abortion

Ethnopharmacological relevanceJiawei Shoutai Pill (JWSTW) is a traditional herbal formula for recurrent spontaneous abortion (RSA). Although JWSTW significantly improves the clinical symptoms of RSA patients, its molecular mechanism remains unclear. Aim of studyThis study evaluated the expression and function of the serum/glucocorticoid regulated kinase 1/epithelial sodium channel (SGK1/ENaC) pathway and decidualization level in RSA patients and mice. It also investigated the therapeutic effects and potential mechanisms of JWSTW. Materials and methods30 early RSA patients and 30 normal pregnant women undergoing induced abortion during the same period were included in the study. Decidual tissues were collected, and HE staining, immunohistochemistry, Western blot, and RT-PCR were used to detect protein and mRNA expression levels of SGK1, ENaC-a, estrogen Rreceptor β (ERβ), and progesterone receptor (PR) in patients’ decidual tissues. Protein expression levels of prolactin receptor (PRLR) and insulin-like growth factor binding protein 1 (IGFBP-1) were also detected. A classical RSA mouse model was constructed, and the mice were randomly divided into four groups: normal, model, dydrogesterone (DQYT) (0.33 g/kg/d), and JWSTW (1.66 g/kg/d). The normal and model groups received the same volume of distilled water by gavage for 8 and 14 days after pregnancy. On the 14th day of pregnancy, the embryonic loss rate of each group, the number of offspring born to naturally delivered mice, and the protein or mRNA expression levels of key factors of the SGK1/ENaC pathway (SGK1, ENaC-a, ERβ, and PR), decidual proliferation marker (Ki67), mesenchymal-epithelial transition (E-cadherin and Vimentin), and decidualization markers (PRLR and IGFBP-1) in mouse decidual tissue on the eighth day of pregnancy were observed. ResultsThe decidual tissue structure of RSA patients was abnormal. Immunohistochemical analysis revealed significantly reduced positive expression of SGK1, ENaC-a, ERβ, and PR proteins in the decidual tissue of RSA patients (P < 0.001). Western blot and RT-PCR analyses demonstrated significantly decreased protein and mRNA expression of SGK1, ENaC-a, ERβ, and PR in the decidual tissue of RSA patients (all P < 0.05). Additionally, protein expression of PRLR and IGFBP-1 was significantly reduced (both P < 0.001). The RSA mouse model exhibited a significant increase in embryo loss rate and decreased litter size (both P < 0.001). Treatment with DQYT and JWSTW rescued the embryo loss rate and litter size to varying extents (all P < 0.05). The protein or mRNA expression levels of SGK1, ENaC-a, ERβ, PR, Ki67, E-cadherin, vimentin, PRLR, and IGFBP-1 in RSA mice were improved to different degrees after treatment with DQYT and JWSTW (all P < 0.05). ConclusionsAbnormal SGK1/ENaC signaling pathway regulation is closely associated with early endometrial damage in RSA patients. JWSTW promotes endometrial proliferation and mesenchymal-epithelial transition through the SGK1/ENaC signaling pathway, improving endometrial shedding. Consequently, JWSTW is a potential treatment for RSA.

P-494 Relevance of intravenous immunoglobulins in patients with unexplained recurrent miscarriages that occurred at the same gestational weeks every time

Abstract Study question Are intravenous immunoglobulins (IVIG) effective for the selected patients who experienced unexplained recurrent miscarriages (RM) that occurred at the same gestational weeks every time? Summary answer IVIG before and after the gestational week-limit (GWL), where miscarriage occurred at the same gestational weeks every time,may improve pregnancy outcomes in RM patients. What is known already Treatment for patients with unexplained RM are challenging. Since the efficacy of immunotherapy with paternal mononuclear cells has been denied in 1999, some treatment options (e.g., intralipid, G-CSF) have been published with conflicting results. In terms of IVIG, the same story has been described. Because previous studies for IVIG treatment included very heterogeneous group of patients, different dose, different intervals of IVIG, and different starting time of infusions. Recently, a double-blind, randomized, placebo-controlled study for unexplained RM women has been published in 2022, which revealed that IVIG (0.4g/kg) for five consecutive days at 4-5 weeks of gestation improved pregnancy outcomes. Study design, size, duration We performed a retrospective study between September 2013 and May 2021 in two fertility centers. We included 106 patients in the study who experienced 2 or more miscarriages between 5 and 21 weeks of gestation that occurred at the same gestational weeks every time, who had all negative results for our routine RM work up and still unsuccessful result after treating these conditions, and whose products of conceptus (POC) revealed at least one normal karyotype. Participants/materials, setting, methods IVIG (Venoglobulin IH 5%, Japan Blood Products Organization, 0.4g/kg) was injected twice before and after 1 week of GWL. If the GWL was 8 weeks of gestation, IVIG should be injected 7 and 9 weeks. We defined successful IVIG treatment as live birth. When the pregnancy ended in miscarriage, POC was performed as much as possible. All participants provided written informed consent, and Institutional Review Board approval was obtained. Main results and the role of chance Of 106 patients, average age was 37.5 years (28-47 years) and the mean number of previous miscarriages was 3.1. Total 128 cycles of IVIG attempt resulted in 90 (70.3%) successful pregnancies, 36 miscarriages and 2 biochemical pregnancies. After excluding abnormal karyotype of POC (N = 7), 68.6% (83/121) was successful. Per patients, 81.1% (86/106) had at least one live birth. Of 32 patients who experienced miscarriages at the first IVIG attempt, 16 patients got pregnant. Following second IVIG attempt resulted in 12 live births (75.0%). The average weight of live birth babies was 3038g and the birth week was 38.8 weeks of gestation (34-41 wk) for singleton pregnancy (N = 83), whereas 2529g with 35.9 weeks of gestation (33-37 wk) for twin pregnancy (N = 7). Four (4/83, 4.8%) and five (5/7, 71.4%) preterm deliveries were observed for singleton and twin pregnancy, respectively. One baby showed pulmonary hemorrhage resulting in hospitalization in the Neonatal Intensive Care Unit, other live birth babies were without any abnormal findings. Hypertensive disorders of pregnancy were noted in two women resulting in cesarian section delivery. Limitations, reasons for caution This study was retrospective study without any control group and was conducted at only two fertility centers. In future clinical research, it is necessary to conduct randomized controlled trials using placebo group to demonstrate the effectiveness of IVIG before and after the GWL using euploid blastocysts. Wider implications of the findings IVIG before and after the GWL adjusted for each patient may improve pregnancy outcomes in RM patients. Although IVIG is expensive, the current method requires only two IVIG injections, that may be acceptable for much more patients who want to avoid miscarriages and who didn’t use IVIG for economic reasons. Trial registration number not applicable

O-221 Regenerative endometrial PRGF (plasma rich in growth factors) treatment in patients with thin endometrium, recurrent implantation failure, and recurrent miscarriages: a retrospective, self-controlled, cohort study

Abstract Study question What reproductive outcomes are observed in patients submitted to endometrial PRGF therapy due to thin endometrium (ThE), recurrent implantation failure (RIF) and recurrent miscarriages (RM)? Summary answer Following endometrial PRGF treatment, success rates were significantly increased in ThE and RIF patients, whereas in the RM group pregnancy loss rates were not affected. What is known already Adequate development of a functional endometrium is a prerequisite for successful embryo implantation in ART cycles. In contrast, the management of patients with thin endometrial lining or recurrent implantation failure has been an ongoing challenge with none of the proposed surgical, hormonal, and pharmacological interventions achieving satisfactory results. In recent years, the use of plasma rich in growth factors (PRGF) - successfully applied in other medical fields - has become a novel treatment option in reproductive medicine. It was used experimentally both for ovarian follicle activation and to enhance endometrial receptivity although published studies are small with often contradictory results. Study design, size, duration All consecutive patients (n = 107) who underwent endometrial PRGF treatment (n = 150) in a single, private centre between 2016-2022 were included in this retrospective analysis. Patients were recruited into ThE (n = 64), RIF (n = 36) and RM (n = 7) groups, respectively. Live birth / ongoing pregnancy rates per embryo transfer were compared to success rates obtained in previous embryo transfers preceding the endometrial PRGF intervention. External Ethics Committee approval was obtained for a comparative pilot study including above patient groups. Participants/materials, setting, methods PRGF was obtained by processing the patients’ autologous blood sample in an in-house validated open system. Endometrial PRGF interventions were performed using a thawed PRGF sample with a series of intrauterine instillations (43%) or with a combined approach performing hysteroscopic subendometrial infiltration during early follicular phase (57%). Endometrial preparation was conducted using an artificial hormone replacement protocol with high-dose oral estrogens and vaginal progesterone. Most embryo transfers involved the replacement of a single, vitrified-thawed blastocyst. Main results and the role of chance A total of 107 patients underwent 150 endometrial PRGF treatments and 131 subsequent embryo transfers. Altogether 19 (13%) embryo transfers were cancelled, higher in ThE than in the RIF group (16 vs 7.1%). In the ThE group (64 patients, 98 PRGF cycles and 107 controls), positive pregnancy (41 vs 32%, NS), clinical pregnancy (35 vs 22%, p = 0.049) and ongoing pregnancy/live birth rates (24 vs 4.7%, p &amp;lt; 0.0001) per embryo transfer were significantly higher in the endometrial PRGF treatment group compared to previous embryo transfers. In the RIF group (36 patients, 42 PRGF cycles and 101 controls), positive pregnancy (59 vs 20%, p &amp;lt; 0.0001), clinical pregnancy (44 vs 11%, p &amp;lt; 0.0001) and ongoing pregnancy/live birth rates (33 vs 7.9%, p &amp;lt; 0.0001) per embryo transfer were significantly higher in the endometrial PRGF treatment group compared to previous embryo transfers. In the RM group (7 patients, 10 PRGF cycles and 15 controls), positive pregnancy (50 vs 60%), clinical pregnancy (20 vs 27%) and ongoing pregnancy/live birth rates (0 vs 0%) per embryo transfer were not significantly different and no ongoing pregnancies were achieved. So far, 20 singletons and 1 set of twins have been confirmed to be born from the above PRGF cycles (12 pregnancies still ongoing). Limitations, reasons for caution Heterogeneity of clinical severity between included ThE patients could affect observed reproductive outcomes. The self-controlled design of the study might have influenced the comparison between pre-, and post-intervention pregnancy rates, although this also highlights the poor-prognostic nature of included participants. RM patients were too few to evaluate pregnancy loss rates. Wider implications of the findings This preliminary, retrospective study has shown that regenerative therapy using autologous PRGF is a safe, affordable, and efficient treatment option for ThE and RIF patients. Further randomized studies are warranted, although they are hampered by patient selection issues and the lack of applicable efficient treatment options in the non-intervention group. Trial registration number not applicable

P-512 Cell-mediated immunity as a biomarker of predisposition in recurrent miscarriage.

Abstract Study question Are alterations in the peripheral cellular immune system a potential risk factor for recurrent miscarriage? Summary answer Percentage alteration of some circulating immune cell subpopulations could be used as biomarkers or potential therapeutic targets in recurrent miscarriage. What is known already About 50% of patients with recurrent miscarriage (RM) have no known cause of the disease. The maternal immune system plays an important role in maternal-fetal tolerance, so an imbalance in the maternal immune system could trigger reproductive failure in women. Antiphospholipid syndrome is the only immunological alteration with sufficient evidence of association with RM, however, there are many other immunological parameters under investigation that appear to mediate the maternal-fetal relationship such as KIR receptors and HLA-C. Other authors are focusing their studies on reproductive immunology at the cellular level, looking at the role of different lymphocyte subpopulations in RM. Study design, size, duration Prospective observational analytical study (cases and controls) conducted in the Immunology Department of the Hospital General Universitario Gregorio Marañón (Madrid, Spain) from 2019 to 2022. It included a group of 49 non-pregnant women with a clinical history of recurrent miscarriages of unknown cause, and two control groups, one with 40 women of childbearing age who had at least one live newborn and another with 60 women of childbearing age who had no history of pregnancy. Participants/materials, setting, methods The inclusion criteria for the study were: two or more gestational losses (&amp;lt; 22 weeks of pregnancy). Patients with severe uterine pathologies, genetic disorders, thrombophilias, obesity, infections, coeliac disease without gluten restriction, uncontrolled endocrine disorders, immunomodulatory treatment, autoimmune disorders and documented RM were excluded from the study. An immunophenotypic study of T, B, NK and NKT lymphocyte subpopulations and monocytic series was performed. Cell characterization was carried out by 6-colour flow cytometry in peripheral blood. Main results and the role of chance When comparing the cellular profile of RM patients with the profile of both control groups, we observed alterations in the percentage levels of CD3+ T cells, CD8+ T cells, NKT cells and non-switch and class switch memory B cells in the patient group, suggesting the presence of a predominantly pro-inflammatory peripheral cellular immune profile in RM patients. In contrast to previous studies by our research group, we did not observe significant differences between the percentage levels of NK cells and activated CD8+ T cells (HLA DR+) in the patient and control groups, which may be explained by the high and exhaustive patient selection in this study. After association study of these alterations with RM, we identified levels of NKT cells &amp;gt; 5%, CD8+ T cells &amp;gt; 26.50%, non-switch memory B cells &amp;gt; 22% and class switch memory B cells &amp;gt; 32% as new biomarkers of RM risk. In addition, the combination of at least two of these new biomarkers was found to significantly increase the likelihood of developing the disease, suggesting that RM is a complex pathology in which multiple factors may influence the pathogenesis of the disease. Limitations, reasons for caution Among the limitations of our study, we can highlight the collection of control group variables from databases developed in previous studies, in addition to a small sample size. Wider implications of the findings The determination of these immunological biomarkers in RM allows us to evaluate the role of immunomodulatory therapies on these biomarkers in order to evaluate possible new treatments to help prevent immune-mediated reproductive failure, thus contributing to a more personalized medicine. Trial registration number Not applicable

O-304 Whole-exome sequencing in women with reproductive failure is a potentially useful diagnostic test in clinical practice

Abstract Study question Could whole-exome sequencing (WES) be useful in clinical practice for primary infertile female involving oocyte or embryo defects and unexplained recurrent miscarriage (RM) patients? Summary answer Identifying key genes involved in oocyte and embryo development helps explain approximately 17% cases of reproductive failure. WES results will help optimize clinical therapeutic treatment. What is known already Dozens of genes have been reported to be the genetic causes of human infertility. Variants in these genes have specific effects on certain processes of human early reproduction and result in a spectrum of phenotypes, such as oocyte maturation arrest, fertilization defects, cleavage failure, early embryonic lethality, and recurrent miscarriage. Study design, size, duration This retrospective cohort study was conducted from July 2020 to August 2022. A total of 129 affected females were enrolled. Most of them were primary infertile women who suffered repeated failure of in vitro fertilization (≥2 cycles) due to abnormal development of oocytes or embryos (PI group, n = 125). Our cohort also comprised a small number of unexplained RM subjects without offspring (RM group, n = 4). All patients and their spouses had normal karyotype. Participants/materials, setting, methods The peripheral blood samples from all the participants and all of their available family members were obtained for DNA extraction. Genomic DNA samples from the probands were subjected to WES to identify candidate variants. Subsequently, the variants were validated by Sanger sequencing or qPCR. Familial co-segregation analyses were then carried out within the family members. The relationship between phenotype and genotype by clinical tests and the clinical records of the patients was studied. Main results and the role of chance Firstly, variants that are potentially relevant to the phenotypes of female infertility were identified in 17.05% of cases (n = 22), including TUBB8 in 14 cases, PATL2 in 2 cases, ZP2, ZP3, PANX1, TLE6, NLRP2 and NLRP7 in 1 case for each. A breakdown by phenotype revealed that we identified variants in 16.00% (n = 20) of PI group and 50% (n = 2) of RM group. Secondly, 16 TUBB8 variants were identified in 14 cases, of which 8 were novel, 3 were previously reported, and 5 were novel amino acid change occurring at the same position as another previously reported change. We found 3 TUBB8 variants (p.C211R, p.T232I, p.A342T) in 3 embryonic arrest families and 1 TUBB8 mutation (p.A102V) in a RM family, which were maternally inherited. Thirdly, 75 patients in PI group without phenotypic variant underwent ≥1 cycles of ovulation and/or frozen embryo transfer after WES, and 49.33% had achieved clinical pregnancy (n = 37). Limitations, reasons for caution The genomic DNA of parents were not available in all these 22 families, so we cannot determine whether the variant is de novo or inherited in some cases. Additionally, further functional studies should be performed to prove that these novel mutations affect protein function. Wider implications of the findings The present study expands the kinds of variants and phenotypic spectrum of gene mutations with regard to female reproductive failure. Genetic tests for causative genes involved in oocyte and embryo defects were recommended for primary infertile patients who suffered from these conditions and for unexplained RM patients. Trial registration number not applicable

P-529 High Live Birth Rates After Low Dose Immunization Therapy (LIT) Pre and Post Pregnancy With Partner Lymphocytes In Patients With Recurrent Miscarriage (RM)

Abstract Study question Could Lymphocyte Immunization Therapy effectively improve pregnancy and live birth rate in Unexplained Recurrent Miscarriage patients? Summary answer The use of paternal lymphocytes pre and post pregnancy significantly increases the pregnancy success and Live Birth Rate In Patients With Recurrent Miscarriage (RM) What is known already RM affects 1–2% of couples who attempt to conceive and has been defined as three consecutive pregnancy losses within 20 weeks of pregnancy confirmation. Although many potential causes have been established for RM, about 50% of these remain idiopathic and unexplained owing to immunology factors. Meta-analyses have observed better effectiveness and safety of LIT in treating couples with RM shown improvement in pregnancy outcomes. Still, results are conflicting due to different screening criteria and therapeutic protocols. Objective of present study is to evaluate effectiveness of low dose LIT in patients with uRM and Th1/Th2/Treg paradigm disorders Study design, size, duration In self-controlled, prospective study, after IEC clearance and obtaining Informed consent, 19 RM patients underwent immunization with partner lymphocytes from August 2021 to October 2022. Inclusion criteria were at least three clinical pregnancies that culminated in abortion before 20 weeks of gestation or at least three cycles of IVF/ICSI with at least two PGT+ embryos and &amp;gt;8mm endometrium during ET and no pregnancy, normal coagulation tests, autoimmune antibodies negative, normal couple karyotyping. Participants/materials, setting, methods Maternal BMI of 30 and age of 45 was the upper limit with peripheral blood Th1/Th2/Treg cells proportions and concentrations of TNF-a, IFN-g, TGF-β1, IL-2, IL-4, IL-6, IL-10, and blocking antibodies were detected using flowcytometry/ELISA as per Seragen’s Immuno profiling platform. 30-50ml paternal blood was used to purify lymphocytes which were intradermally administered to the wife before Embryo Transfer and post-pregnancy confirmation. Main results and the role of chance Proportion of Th1 cells was significantly decreased while the proportions of Th2 cells and Treg cells were significantly increased in immunotherapy patients after treatment. In addition, the concentration of TGF-β1 in serum was significantly higher after immunotherapy than before. The concentration of TNF-a, IFN-g was significantly high before therapy were significantly decreased after therapy. LIT effectively induced the production of blocking antibodies in all the patients. 16/19 (84%) uRM patients underwent ET post LIT became pregnant and 4 of the pregnant patient had delivered healthy babies (21%). 42% (8/19) of patients have reported uneventful ongoing pregnancies crossed &amp;gt;14weeks. 4 patients who were pregnant for more than 12-14 weeks had miscarriages (21%). 1 patient (5%) did not conceive. 1 patient (5%) conceived spontaneously and crossed 20 weeks of uneventful gestation. 1 patient (5%) opted to try to conceive naturally hence ET is not planned. The cumulative positive uneventful pregnancy outcome was 68%, approximately the same as the figures reported earlier. LIT is associated with high live birth rates, especially in women with RM. It not only ameliorates patient’s cellular immune function but also further increases patient’s pregnancy success rate with high safety, which is worthy of clinical application and promotion. Limitations, reasons for caution This prospective self-control study with small sample size lacks randomized control group. Beneficial effects observed in this study are in sync with published data. Larger study with patients recruited based on inclusion criteria of unexplained recurrent miscarriage due to immunological factors is proposed for recommending LIT as routine therapy. Wider implications of the findings Our study findings supports LIT as a beneficial treatment for RM/RIF in IVF patients. LIT may be considered safe and effective therapy in individual cases and based on the immunoprofiling (Th1/Th2/Treg paradigm disorders), after all other potential causes of RM or RIF have been ruled out. Trial registration number Not applicable

P-504 Pregnancy outcome of intravenous immunoglobulin in women with recurrent pregnancy loss

Abstract Study question Does intravenous immunoglobulin(IVIG) increase live births in recurrent miscarriage(RM) women with cellular immune abnormality such as high NK cell level and cytotoxicity and TNF-α/IL-10 ratio? Summary answer IVIG treatment increased the live birth rate of RM women with abnormal cellular immunity. What is known already Various causes of RM have been identified but even with a detailed evaluation, almost half of the cases have unidentified etiologies. Immune imbalance is one of the proposed potential etiologies of these idiopathic RM. To regulate abnormal cellular immunity, treatment such as IVIG is proposed to improve pregnancy outcomes. However, the use of IVIG in RM is still controversial. In addition, one of the widely used indicators of immune imbalance in pregnancy is the NK cell level, but there is a disagreement in setting the cut-off value. The percentage varies from 12 to 18%. Study design, size, duration RM was defined as women with two or more spontaneous abortions and in total, 987 RM women visited Department of Obstetrics and Gynaecology, Konyang University Hospital(KYUH) from January 2007 to December 2020. Only those with a full evaluation and known treatment outcome were included. A total 204 idiopathic RM and 209 RM women with known aetiology were enrolled. Participants/materials, setting, methods We investigated the pregnancy outcome by sorting the patients into seven subgroups depending on abnormal cellular immunity including NK cell level, NK cell cytotoxicity and TNF-α/IL-10 ratio. Then, to verify the cut-off value(16.1%) of NK cell level which we set in our previous study, patients were classified into three groups according to their NK cell level: 1) &amp;lt;12%(low), 2) 12-16%(moderate), 3) &amp;gt;16%(high). Main results and the role of chance Among all RM women with at least one abnormal cellular immunity were treated with IVIG and the overall live birth rate (LBR) was 80.2%. The group which did not have IVIG treatment showed an overall LBR of 78.0%. Within the 7 groups with abnormal cellular immunity, the group with both high NK cell toxicity and TNF-α/IL-10 ratio showed the highest LBR, 100% of LBR and the group with both high NK cell level and TNF-α/IL-10 ratio showed the lowest treatment outcome, 71.4%. To verify the adequacy of our cut-off value, after excluding those with high NK cell cytotoxicity and TNF-α/IL-10 ratio, 152 patients were classified into low, moderate, and high level of NK cells. LBR were 81.0%, 72.5%, and 76.9%. Limitations, reasons for caution The study was designed retrospectively, resulting in numerous follow-up losses. It is a single-centered study that may not be enough to generalize the diagnosis method and treatments from these findings. Moreover, IVIG is an expensive drug that may lead to certain patient preferences. Wider implications of the findings The study may provide evidence in selecting RM patients with immune abnormalities to be treated with IVIG and raise their pregnancy outcomes. We can also apply these results to the reproductive failure group. A detailed evaluation and an evidence-based treatment are important. Trial registration number not applicable

Alpha-enolase 1 knockdown facilitates the proliferation and invasion of villous trophoblasts by upregulating COX-2.

Enolase 1 (ENO1) is a metabolic enzyme which participates in pyruvate synthesis and ATP production in cells. Previously, differential expression of ENO1 was discovered in villous tissues between recurrent miscarriage and induced abortion. This study was designed to explore whether ENO1 influences the proliferation and invasion of villous trophoblasts and the related molecular mechanisms. First, ENO1 expression in placental villus tissues collected from recurrent miscarriage (RM) patients and women for induced abortion as well as in trophoblast-derived cell lines was detected by RT-qPCR and western blotting. ENO1 localization and expression in villus tissues were further confirmed through immunohistochemistry staining. Then, the effects of ENO1 downregulation on trophoblast Bewo cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) process were evaluated by CCK-8 assay, transwell assay, and western blotting. As for the regulatory mechanism of ENO1, the expression of COX-2, c-Myc and cyclin D1 in Bewo cells after ENO1 knockdown was finally evaluated by RT-qPCR and western blotting. ENO1 was mainly localized in the cytoplasm, with very small amounts in the nucleus of trophoblast cells. ENO1 expression in the villi tissues of RM patients was significantly increased, when compared with the villous tissues of healthy controls. Furthermore, Bewo cells, a trophoblast cell line with relatively higher expression of ENO1, was used to downregulate the ENO1 expression by ENO1-siRNA transfection. ENO1 knockdown significantly facilitated Bewo cell growth, EMT process, migration, and invasion. ENO1 silencing markedly elevated COX-2, c-Myc, and cyclin D1 expression. ENO1 may participate in the development of RM via suppressing the growth and invasion of villous trophoblasts via reducing the expression of COX-2, c-Myc, and cyclin D1.

MITA Promotes Macrophage Proinflammatory Polarization and Its circRNA-Related Regulatory Mechanism in Recurrent Miscarriage.

MITA (also called STING), a master regulator of DNA-mediated innate immune activation, is a potential therapeutic target for viral infection and virus-related diseases. The circRNA-mediated ceRNA network plays important roles in gene regulation and may contribute to many human diseases. However, the relationship between MITA and recurrent miscarriage (RM) and its circRNA-related regulatory mechanisms remain unclear. In this study, we validated that the decidual M1/M2 ratio was upregulated in RM patients, suggesting the vital roles of decidual macrophages in the pathogenesis of RM. We found that MITA was highly expressed in decidual macrophages of RM patients and validated that MITA could promote apoptosis and macrophage proinflammatory polarization in THP-1-derived macrophage (TDM) cells. Using circRNA sequencing and bioinformatic analysis, we screened out a novel circRNA (circKIAA0391) that is overexpressed in decidual macrophages of RM patients. Mechanistically, we found that circKIAA0391 could promote the apoptosis and proinflammatory polarization of TDM cells by sponging the miR-512-5p/MITA axis. This study provides a theoretical basis for further understanding the impact of MITA on macrophages and its circRNA-related regulatory mechanisms, which may have a crucial immunomodulatory function in the pathophysiology of RM.

USP7 prevents recurrent spontaneous abortion by targeting EZH2 to regulate trophoblast proliferation, apoptosis, migration and invasion through the Wnt/β-catenin pathway†.

Trophoblasts are significant components of the placenta and play crucial roles in maternal-fetal crosstalk. Adequate trophoblast migration and invasion are essential for embryo implantation and a healthy pregnancy. Ubiquitin-specific protease 7 (USP7), a member of the deubiquitinating enzyme family, regulates the processes of migration and invasion in multiple tumor cells. However, the effects of USP7 on trophoblasts and its possible mechanism in the development of recurrent spontaneous abortion (RSA) are still unclear. In this study, we analyzed the expression of USP7 in villous tissues obtained from RSA patients and healthy controls, and then GNE-6776 (a USP7-specific inhibitor) and USP7 siRNA were used in a trophoblast cell line, HTR-8/SVneo, to further assess the effect of USP7 on the biological function of trophoblasts. Our results provide convincing evidence that USP7 is downregulated in the placental villous tissues of RSA patients. USP7 was found to have a crucial role in the proliferation, apoptosis, migration, invasion and epithelial-mesenchymal transition (EMT) process of trophoblast cells. Further experiments revealed that USP7 directly interacted with enhancer of zeste homolog 2 (EZH2) and regulated the Wnt/β-catenin signaling pathway in trophoblasts. Taken together, these findings indicate the vital role of USP7 in regulating trophoblast proliferation, migration and invasion, thus affecting the pathogenesis of RSA, providing new insights into the important role of USP7 in the maternal-fetal interface.

SHP2 participates in decidualization by activating ERK to maintain normal nuclear localization of progesterone receptor.

The establishment and maintenance of embryo implantation and pregnancy require decidualization of endometrial stromal cells. This paper reveals that SHP2 ensures the correct subcellular localization of progesterone receptor, thereby safeguarding the process of decidualization. Decidualization is the process of conversion of endometrial stromal cells into decidual stromal cells, which is caused by progesterone production that begins during the luteal phase of the menstrual cycle and then increases throughout pregnancy dedicated to support embryonic development. Decidualization deficiency is closely associated with various pregnancy complications, such as recurrent miscarriage (RM). Here, we reported that Src-homology-2-containing phospho-tyrosine phosphatase (SHP2), a key regulator in the signal transduction process downstream of various receptors, plays an indispensable role in decidualization. SHP2 expression was upregulated during decidualization. SHP2 inhibitor RMC-4550 and shRNA-mediated SHP2 reduction resulted in a decreased level of phosphorylation of ERK and aberrant cytoplasmic localization of progesterone receptor (PR), coinciding with reduced expression of IGFBP1 and various other target genes of decidualization. Solely inhibiting ERK activity recapitulated these observations. Administration of RMC-4550 led to decidualization deficiency and embryo absorption in mice. Moreover, reduced expression of SHP2 was detected in the decidua of RM patients. Our results revealed that SHP2 is key to PR's nuclear localization, thereby indispensable for decidualization and that reduced expression of SHP2 might be engaged in the pathogenesis of RM.

Inhibition of TMUB1 blocks apoptosis and NF-κB pathway-mediated inflammation in recurrent spontaneous abortion.

Approximately 50% of cases with recurrent spontaneous abortion (RSA) have unexplained etiology. Aberrant expression of transmembrane and ubiquitin-like domain containing 1 (TMUB1) is closely related to a series of diseases, including RSA. However, the function and underlying mechanism of TMUB1 in the occurrence of RSA has not been described. TMUB1 expression was detected in the placental villous tissues of 30 women with normal miscarriages and 12 women with RSA. The pregnant mice were injected intraperitoneally with lipopolysaccharide (LPS) to induce abortion. Human chorionic trophoblast cells were treated with LPS. Pathological analysis of placental tissues was performed by hematoxylin and eosin staining. TMUB1 was highly expressed in the placental villous tissues of RSA patients compared to the patients who underwent induced abortions. After LPS administration, the mice exhibited high embryo absorption and pathological alterations, as well as presented an increase in inflammation and apoptosis (the etiology of RSA induction) in placental tissues. Moreover, the upregulated expression of TMUB1 was also found in placental tissues of LPS-induced mice, and further investigation showed that TMUB1 deficiency blocked embryo loss as well as inhibited apoptotic rate and inflammation after LPS activation. Furthermore, we found that the loss of TMUB1 suppressed the phosphorylation of IkappaB kinase (IKK) α/β and attenuated cytoplasmic-nuclear translocation of nuclear factor-κB (NF-κB) p65 in LPS-induced cells. Our results indicate that TMUB1 may involve in the modulation of apoptosis and NF-κB pathway-mediated inflammation in RSA. Therefore, TMUB1 may develop as a potential biomarker for RSA treatment.

Association of functional superoxide gene polymorphism with chlamydia trachomatis-associated recurrent spontaneous abortion

Oxidative stress generated by Chlamydia trachomatis infection is associated with reproductive complications such as recurrent spontaneous abortion. Aim of prospective study was to evaluate whether single nucleotide polymorphisms (SNPs) of SOD1 and SOD2 gene are associated with C. trachomatis-infected recurrent spontaneous abortion (RSA). 150 patients with history of RSA and 150 patients with history of successful deliveries were recruited from Department of Obstetrics and Gynecology, Safdarjung hospital, New Delhi, India. Urine and non-heparinized blood samples were collected and C. trachomatis was detected by polymerase chain reaction (PCR). Using qualitative real time PCR, SNPs rs4998557 (SOD1) and rs4880 (SOD2) were screened in enrolled patients. Level of 8-hydroxyguanosine (8-OHdG), 8-isoprostane (8-IP), progesterone and estrogen was determined by enzyme-linked immunosorbent assays and correlated with SNPs. Significant differences were found in frequency of AA genotype of SOD1 gene among RSA patients versus controls, (82% and 54.66%, respectively; p = 0.02; OR 0.40; CI 95%). Frequency of AA genotype of SOD1 gene among RSA patients with C. trachomatis infection was 87.33%, while in uninfected RSA patients was 71.33% (p < 0.0001; OR 8; CI 95%). No significant relation was found between SOD2 (rs4880) genotype and RSA. Furthermore, significant increase in 8-OHdG, 8-IP and estrogen and significant decrease in progesterone was observed among patients carrying AA genotype. Findings suggest the clinical importance of AA genotype along with 8-OHdG, 8-IP and estrogen and progesterone in screening C. trachomatis-infected RSA women.

Metastasis-associated lung adenocarcinoma transcript 1 induces methyl-CpG-binding domain protein 4 in mice with recurrent spontaneous abortion caused by anti-phospholipid antibody positivity

IntroductionAntiphospholipid syndrome is an autoimmune disease characterized by pregnancy-related morbidity, related to persistent positivity of antiphospholipid antibodies (APL). One of the characteristics of pregnancy-related morbidity in patients with antiphospholipid syndrome is recurrent spontaneous abortion (RSA). This study aimed to examine the mechanism through which metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) regulates methyl-CpG-binding domain protein 4 (MBD4) expression in APL-positive RSA. MethodsClinical samples were subjected to microarray analysis to filter differentially expressed genes. RSA mice with APL positivity were generated, followed by adenoviral vector injection to artificially upregulate MALAT1. The effects of MALAT1 on the biological behavior of trophoblast cells were assessed. The downstream mechanism of MALAT1 was analyzed using subcellular fractionation and bioinformatics prediction, and the relationship between MALAT1 and CREB binding protein (CREBBP) or MBD4 was investigated in trophoblast cells. ResultsMALAT1 was downregulated in APL-positive RSA patients. MALAT1 was predominantly localized in the nucleus and recruited CREBBP to mediate the MBD4 transcription. In the APL-positive RSA mice overexpressing MALAT1, the expression of soluble Fms-related tyrosine kinase 1 and anticardiolipin antibody and the embryonic resorption rate were decreased, indicating that MALAT1 reduced the occurrence of RSA in mice. Moreover, MALAT1 enhanced proliferation, migration, and invasion of trophoblast cells through recruiting CREBBP to promote MBD4 expression. Silencing of CREBBP or MBD4 increased embryonic resorption rate in RSA mice overexpressing MALAT1. DiscussionMALAT1 suppresses APL-positive RSA by promoting MBD4 transcription through recruitment of CREBBP to the MBD4 promoter region.

Impact of body mass index on peripheral and uterine immune status in the window of implantation in patients with recurrent reproductive failure

This study aimed to investigate whether maternal obesity affects the immune status of peripheral blood and endometrium in patients with recurrent reproductive failure classified according to their body mass index (BMI). A total of 228 repeated implantation failure (RIF) and 266 recurrent miscarriage (RM) patients were enrolled in the study and further subdivided into three groups according to their BMI: (i) normal weight (18.5≤ BMI <23); (ii) overweight (23≤ BMI <25); and (iii) obese (BMI ≥25). Peripheral blood and endometrium samples were collected in the mid-luteal phase before IVF treatment or natural pregnancy. Peripheral immunocytes were analyzed by flow cytometry, while uterine immune cells were subjected to immunohistochemistry. In RM patients, significantly increased peripheral helper T cells and decreased cytotoxic T cells, NK cells were observed in the obese group compared with the normal-weight group. Meanwhile, in the endometrium, the percentage of NK cell, macrophage cell, M2 macrophage cell, and Treg cell significantly reduced with increased BMI in RIF patients, and the percentage of NK cell and M2 macrophage cell significantly decreased with increased BMI in RM patients. In conclusion, obesity may cause endometrial immune disorder in recurrent reproductive failure women, but was only associated with the peripheral immune change in RM patients.