UNASSIGNED: Recurrent miscarriage (RM) remains unsolved in > 50% of patients and causes physical and psychological problems in women without specific risk factors for miscarriage. For a successful pregnancy, acceptance of the endometrium and invasion of trophoblast cells into the endometrium is necessary.
UNASSIGNED: This study aimed to use computational analysis to identify key genes and related pathways in endometrial and trophoblast cells derived from RM samples.
UNASSIGNED: In this bioinformatics study, we explored the differential expression of genes in endometrial and trophoblast cells by analyzing the GSE165004 and GSE76862 datasets, respectively with the limma package in R software. Subsequently, overlapped genes between 2 datasets were selected, gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed. The overlapped genes were integrated to construct a protein-protein interaction network and hub genes selection.
UNASSIGNED: We observed 41 overlapped genes between endometrial and trophoblast cells, and future analysis was accomplished in overlapped and nonoverlapped genes. Kyoto Encyclopedia of Genes and Genomes analysis indicated that overlapped genes were significantly enriched in the complement and coagulation cascades, pluripotency of stem cells, and synthesis and degradation of ketone bodies. Gene ontology analysis suggested that the genes were enriched in the cell cycle, apoptosis, and cell division. The top 10 genes included: IRS1, FGF2, MAPK6, MAPK1, MAPK3, MAPK8, MAPK9, PLK1, PRKACA, and PRKCA were identified from the PPI network.
UNASSIGNED: This study identified the key genes and potential molecular pathways underlying the development of RM. This could provide novel insights to determine the possible mechanisms and interventional strategies associated with miscarriage.

Benz[a]anthracene (BaA), a hazardous polycyclic aromatic hydrocarbon classified by the EPA, is a probable reproductive toxicant. Epidemiological studies suggest that BaA exposure may be a risk factor for recurrent miscarriage (RM). However, the underlying mechanisms are not well understood. This study identified DEC1 as a key gene through RNA-seq and single-cell RNA sequencing analysis. DEC1 expression was found to be downregulated in villous tissues from women with RM and in primary extravillous trophoblasts (EVTs) exposed to BaA. BaA suppressed DEC1 expression by promoting abnormal methylation patterns. Further analysis revealed that ARHGAP5 is a direct target of DEC1 in EVTs, where DEC1 inhibits trophoblast invasion by directly regulating ARHGAP5 transcription. Additionally, BaA destabilized matrix metalloproteinase 2 (MMP2) by activating the aryl hydrocarbon receptor (AhR) and promoting E3 ubiquitin ligase MID1-mediated degradation. In a mouse model, BaA induced miscarriage by modulating the DEC1/ARHGAP5 and MID1/MMP2 axes. Notably, BaA-induced miscarriage in mice was prevented by DEC1 overexpression or MID1 knockdown. These findings indicate that BaA exposure leads to miscarriage by suppressing the DEC1/ARHGAP5 pathway and enhancing the MID1/MMP2 pathway in human EVTs.

OBJECTIVE: The purpose of this study was to determine the etiologies of recurrent miscarriage in our hospital and whether its diagnostic approach followed the recommendations of the American Society of Reproductive Medicine (ASRM) guidelines published in 2012 and the National Institute for Health and Care Excellence (NICE) guidelines published in 2011.
METHODS: This was a retrospective study. The medical records of 158 patients diagnosed with recurrent miscarriage between 2013 and 2018 at Santander University Hospital were reviewed. The Institutional Review Board of HUS approved the study in May 2020.
RESULTS: The most common etiologies identified were protein S deficiency, thrombophilia, and cervical insufficiency, with incidence rates of 25.9%, 10.7%, and 3.8%, respectively. Moreover, the most frequently requested diagnostic tests were for protein S, protein C, and anti-phospholipid IgG. Abnormal results for protein S were obtained in 49% of the patients, whereas lupus anticoagulant was abnormal in 12.8%, and Factor V Leiden gene mutations in 8.5% of the patients. Three substantial deviations from the recommended diagnostic approach for recurrent miscarriage by international guidelines were identified in our population: the lack of request for cytogenetic analysis of pregnancy tissue, request for cytogenetic analysis for the parents in only 0.6% of the study sample, and the request for imaging tests to assess uterine anatomy in only 6.3% of the studied population. Both the ASRM and NICE guidelines were only partially followed with a combined adherence rate of 66.5%.
CONCLUSIONS: The diagnostic approach for recurrent miscarriage poses important clinical challenges when compared to the recommendations of international guidelines. Therefore, the development of a local recurrent miscarriage assessment protocol is proposed in our institution.

BACKGROUND: Recurrent miscarriage (RM) is defined as the occurrence of at least two or three subsequent miscarriages within the 20th -24th weeks of pregnancy. The primary objective of this study was to investigate whether histidine-rich glycoprotein C633T single nucleotide polymorphism (HRG C633T SNP) statistically correlates with the occurrence of RM among Iranian women.
RESULTS: Blood samples from 200 women were taken at the outset of the study. Then, the blood samples of 100 women who had a record of RM (case group) were compared with the other 100 women\'s blood samples who had no record of RM (control group). Following DNA extraction, the polymorphism of histidine-rich glycoprotein C633T (HRG C633T) for every case was specified and all women were genotyped by the amplification-refractory mutation system (ARMS) method. The results of the study revealed that there was a statistically significant difference between T/T genotype (OR = 3.5, CI (1.39-8.77), p = 0.007), and C/T genotype (OR = 1.83, CI (0.99-3.37), p = 0.05) in the case and control groups. Also, a statistically significant association was observed in T allelic frequency in the RM participants compared to the control group (OR = 2.01, CI (1.31-3.09), p = 0.01).
CONCLUSIONS: The present study determined that there was a statistically significant relationship between HRG C633T SNP and increased RM regarding allelic and genotypical aspects. Moreover, it became apparent that women with homozygous T/T genotype were more susceptible to the risk of RM.

Part II of the Australasian guideline for the investigation and management of recurrent pregnancy loss (RPL) provides evidence-based guidance on the management of RPL provided. The implications of inherited and acquired thrombophilia with respect to RPL and suggestions for clinical management are provided. Autoimmune factors, including human leukocyte antigen, cytokines, antinuclear antibodies and coeliac antibodies, and guidance for management are discussed. Infective, inflammatory and endometrial causes of RPL are discussed in detail. Environmental and lifestyle factors, male factor and unexplained causes are outlined. Levels of evidence and grades of consensus are provided for all evidence-based statements.

Guidelines for the investigation and management of recurrent pregnancy loss (RPL) have been developed in Europe, USA and UK, but there is currently no Australasian guideline. The Australasian Certificate of Reproductive Endocrinology and Infertility Consensus Expert Panel on Trial Evidence group has prepared a two-part guideline to provide guidance on the management of RPL. In Part I chromosomal, anatomical, and endocrine factors are outlined along with relevant recommendations for clinical management, levels of evidence and grades of consensus. In Part II thrombophilia, autoimmune factors, infective, inflammatory, and endometrial causes, environmental and lifestyle factors, male factor and unexplained causes will be outlined.

Recurrent miscarriage (RM) is a distressing pregnancy complication with an unknown etiology. Increasing evidence indicates the relevance of dysregulation of human trophoblast stem cells (hTSCs), which may play a role in the development of RM. However, the potential molecular regulatory mechanism underlying the initiation and maintenance of hTSCs is yet to be fully elucidated. In this study, we performed data analysis and identified Forkhead box M1 (FOXM1) as a potential factor associated with RM. FOXM1 is a typical transcription factor known for its involvement in various pathophysiological processes, while the precise function of FOXM1 functions in hTSCs and RM remains incompletely understood. Utilizing RNA-seq, CUT&Tag, ChIP-qPCR, and sodium bisulfite conversion methods for methylation analysis, we elucidate the underlying regulatory mechanisms of FOXM1 in hTSCs and its implications in RM. Our findings demonstrate the relative high expression of FOXM1 in proliferating cytotrophoblasts (CTBs) compared to differentiated extravillous cytotrophoblasts (EVTs) and syncytiotrophoblasts (STBs). Besides, we provide evidence supporting a significant correlation between FOXM1 downregulation and the incidence of RM. Furthermore, we demonstrate the significant role of FOXM1 in regulating hTSCs proliferation and cell cycle through the transcriptional regulation of CDKN3, CCNB2, CCNA2, MAD2L1 and CDC25C. Notably, we observed a correlation between the downregulation of FOXM1 in RM and hypermethylation in its promoter region. Collectively, these results provide insights into the impact of FOXM1 on trophoblast regulation and offer a novel perspective on RM.

Osseous metaplasia of the endometrium is a rare entity in which there is presence of mature or immature bone tissue in the endometrium. It is a rare disorder that usually leads to secondary infertility and is frequently associated with recurrent miscarriages and can be rarely asymptomatic. We present the case of a patient with endometrial ossification associated with secondary infertility. The patient presented with excessive vaginal discharge and was undergoing evaluation for secondary infertility. Transvaginal sonography showed a hyperechoic elongated lesion in the endometrium suggestive of endometrial calcification with posterior acoustic shadowing. Diagnostic hysteroscopy was done and the endometrial cavity showed multiple white-colored, solid, fan-shaped structures and bony spicules all over the endometrium, which were removed with a hysteroscopic grasper and sent for histopathological examination (HPE). The HPE confirmed the presence of bony trabeculae along with secretory endometrial glands. There have only been a few cases of endometrial osseous metaplasia reported in India and it is usually an overlooked cause of infertility. Although rare, we should consider this as a probable cause of secondary infertility when the characteristic ultrasound features are visualized and that hysteroscopic resection is the gold standard treatment.

OBJECTIVE: We aimed to perform a systematic review and meta-analysis addressing the efficacy of levothyroxine therapy in pregnant women with subclinical hypothyroidism considering most recent evidence and subgroups of interest for clinical practice.
METHODS: PubMed, Embase, and Cochrane Central were searched from inception for randomized controlled trials (RCTs) comparing levothyroxine with placebo or no intervention in pregnant women with subclinical hypothyroidism. We used a random-effects model and conducted subgroup analyses based on thyroid peroxidase antibody status, thyroid stimulating hormone levels, fertility treatment, and recurrent miscarriage.
RESULTS: We included 11 RCTs comprising 2,749 pregnant women with subclinical hypothyroidism. Patients treated with levothyroxine (1,439; 52.3%) had significantly lower risk of pregnancy loss (risk ratio 0.69; 95% confidence interval 0.52-0.91; p < 0.01; 6 studies). However, there was no significant association between levothyroxine and live birth (risk ratio 1.01; 95% confidence interval 0.99-1.03; p = 0.29; 8 studies). No statistically significant interaction was observed across subgroups (p > 0.05).
CONCLUSIONS: Levothyroxine replacement therapy for subclinical hypothyroidism during pregnancy may decrease pregnancy loss when early prescribed. Nevertheless, further investigation is needed in patients with thyroid stimulating hormone above four milliunits per liter, especially when associated with recurrent miscarriage or infertility.

Recurrent miscarriage (RM) is related to the dysfunction of extravillous trophoblast cells (EVTs), but the comprehensive mechanisms remain largely unexplored. We analyzed single-cell RNA sequencing (scRNA-seq), bulk RNA sequencing and microarray datasets obtained from Gene Expression Omnibus (GEO) database to explore the hub genes in the mechanisms of RM. We identified 1724 differentially expressed genes (DEGs) in EVTs from the RM, and they were all expressed along the trajectory of EVTs. These DEGs were associated with hypoxia and glucose metabolism. Single-cell Regulatory Network Inference and Clustering (SCENIC) analysis revealed that E2F transcription factor (E2F) 8 (E2F8) was a key transcription factor for these DEGs. And the expression of ENO1 can be positively regulated by E2F8 via RNA sequencing analysis. Subsequently, we performed immunofluorescence assay (IF), plasmid transfection, western blotting, chromatin immunoprecipitation (ChIP), real-time quantitative polymerase chain reaction (qRT-PCR), and transwell assays for validation experiments. We found that the expression of alpha-Enolase 1 (ENO1) was lower in the placentas of RM. Importantly, E2F8 can transcriptionally regulate the expression of ENO1 to promote the invasion of trophoblast cells by inhibiting secreted frizzled-related protein 1/4 (SFRP1/4) to activate Wnt signaling pathway. Our results suggest that ENO1 can promote trophoblast invasion via an E2F8-dependent manner, highlighting a potential novel target for the physiological mechanisms of RM.