Unexplained Recurrent Pregnancy Loss Research Articles

Investigating into microbiota in the uterine cavity of the unexplained recurrent pregnancy loss patients in early pregnancy

IntroductionThe majority of unexplained recurrent pregnancy loss (URPL) cases have been attributed to immune abnormalities. Inappropriate changes in microbiota could lead to immune disorders. However, the specific role of uterine cavity microbiota in URPL remains unclear, and only a limited number of related studies are available for reference. MethodsWe utilized double-lumen embryo transfer tubes to collect uterine cavity fluid samples from pregnant women in their first trimester. Subsequently, we conducted 16S rRNA sequencing to analyze the composition and abundance of the microbiota in these samples. ResultsFor this study, we enlisted 10 cases of URPL and 28 cases of induced miscarriages during early pregnancy. Microbial communities were detected in all samples of the URPL group (100 %, n = 10), whereas none were found in the control group (0 %, n = 28). Among the identified microbes, Lactobacillus and Curvibacter were the two most dominant species. The abundance of Curvibacter is correlated with the number of NK cells in peripheral blood (r = −0.759, P = 0.018). DiscussionThis study revealed that during early pregnancy, Lactobacillus and Curvibacter were the predominant colonizers in the uterine cavity of URPL patients and were associated with URPL. Consequently, alterations in the dominant microbiota may lead to adverse pregnancy outcomes.

Maternal Serum Amyloid a Level as a Novel Marker of Primary Unexplained Recurrent Early Pregnancy Loss

Background: Recurrent abortion affects 1-2% of women. Serum Amyloid A belongs to a family of apolipoproteins produced in response to cytokines released by activated monocytes and macrophages, isolated in the last 50 years. Our current study emphasizes human SAA protein as a sensitive biochemical marker for primary unexplained recurrent miscarriage. The aim of the current study is to examine the hypothesis that primary unexplained REPL might be associated with high maternal serum levels of SAA, which in turn could lead to defective trophoblastic invasion into the decidua, and subsequent pregnancy failure and miscarriage. Patients and Method: A prospective study (case control study) in Al-Zahraa Maternity Hospital, Najaf, Iraq, from first of January to the first of December of 2019. The study was conducted among 91 who were divided into two groups Group 1: women with missed miscarriage in the first trimester with at least two consecutive primary unexplained REPLs and no previous live births were enrolled. Group 2: A control group was formed of women with miscarriage no history of REPL who had at least one previous uneventful pregnancy with no adverse outcomes. Serum samples were collected to measure SAA levels. Result: The main outcome was the association between SAA and primary unexplained REPL. A total number of 91 participants. Mean SAA level was significantly higher among women with REPL than among women in the control group (P<0.001). The SAA level was dependent indicator of primary unexplained REPL, P<0.001 Elevated SAA levels found among women with primary unexplained REPL could represent biomarker for this complication of pregnancy. Conclusions: The present findings suggest that SAA is potentially a novel marker for primary unexplained REPL that warrants further investigation. For example, studies could be conducted to compare SAA levels among women with a history of primary unexplained REPL during the pregnant and non-pregnant states, as well as before and after miscarriage. In future, such studies might guide the timing for initiation of new treatments, such as gene therapy or the use of immune-receptor antagonists.

Increased levels of villus-derived exosomal miR-29a-3p in normal pregnancy than uRPL patients suppresses decidual NK cell production of interferon-γ and exerts a therapeutic effect in abortion-prone mice

ObjectiveRecurrent pregnancy loss (RPL) patients have higher absolute numbers of decidual natural killer (dNK) cells with elevated intracellular IFN-γ levels leading to a pro-inflammatory cytokine milieu, which contributes to RPL pathogenesis. The main objective of this study was twofold: first to explore the regulatory effects and mechanisms of villus-derived exosomes (vEXOs) from induced abortion patients or RPL patients at the level of intracellular IFN-γ in dNK cells; second to determine the validity of application of vEXOs in the treatment of unexplained RPL (uRPL) through in vitro experiments and mouse models.MethodsExosomes were isolated from villus explants by ultracentrifugation, co-cultured with dNK cells, and purified by enzymatic digestion and magnetically activated cell sorting. Flow cytometry, enzyme-linked immunosorbent assays, and RT-qPCR were used to determine IFN-γ levels. Comparative miRNA analysis of vEXOs from induced abortion (IA) and uRPL patients was used to screen potential candidates involved in dNK regulation, which was further confirmed by luciferase reporter assays. IA-vEXOs were electroporated with therapeutic miRNAs and encapsulated in a China Food and Drug Administration (CFDA)-approved hyaluronate gel (HA-Gel), which has been used as a clinical biomaterial in cell therapy for > 30 years. In vivo tracking was performed using 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyaine iodide (DiR) labelling. Tail-vein and uterine horn injections were used to evaluate therapeutic effects of the engineered exosomes in an abortion-prone mouse model (CBA/J × DBA/2 J). Placental growth was evaluated based on placental weight. IFN-γ mRNA levels in mouse placentas were measured by RT-qPCR.ResultsIFN-γ levels were significantly higher in dNK cells of uRPL patients than in IA patients. Both uRPL-vEXOs and IA-vEXOs could be efficiently internalized by dNK cells, whereas uRPL-vEXOs could not reduce the expression of IFN-γ by dNK cells as much as IA-vEXOs. Mechanistically, miR-29a-3p was delivered by vEXOs to inhibit IFN-γ production by binding to the 3′ UTR of IFN-γ mRNA in dNK cells. For in vivo treatment, application of the HA-Gel effectively prolonged the residence time of vEXOs in the uterine cavity via sustained release. Engineered vEXOs loaded with miR-29a-3p reduced the embryo resorption rate in RPL mice with no signs of systemic toxicity.ConclusionOur study provides the first evidence that villi can regulate dNK cell production of IFN-γ via exosome-mediated transfer of miR-29a-3p, which deepens our understanding of maternal–fetal immune tolerance for pregnancy maintenance. Based on this, we developed a new strategy to mix engineered vEXOs with HA-Gel, which exhibited good therapeutic effects in mice with uRPL and could be used for potential clinical applications in uRPL treatment.Graphical

Immunological parameters of maternal peripheral blood as predictors of future pregnancy outcomes in patients with unexplained recurrent pregnancy loss.

Unexplained recurrent pregnancy loss (URPL), affecting approximately 1%-5% of women, exhibits a strong association with various maternal factors, particularly immune disorders. However, accurately predicting pregnancy outcomes based on the complex interactions and synergistic effects of various immune parameters without an automated algorithm remains challenging. In this historical cohort study, we analyzed the medical records of URPL patients treated at Xiangya Hospital, Changsha, China, between January 2020 and October 2022. The primary outcomes included clinical pregnancy and miscarriage. Predictors included complement, autoantibodies, peripheral lymphocytes, immunoglobulins, thromboelastography findings, and serum lipids. Least absolute shrinkage and selection operator (LASSO) analysis and logistic regression analysis was performed for model development. The model's performance, discriminatory, and clinical applicability were assessed using area under the curve (AUC), calibration curve, and decision curve analysis, respectively. Additionally, models were visualized by constructing dynamic and static nomograms. In total, 502 patients with URPL were enrolled, of whom 291 (58%) achieved clinical pregnancy and 211 (42%) experienced miscarriage. Notable differences in complement, peripheral lymphocytes, and serum lipids were observed between the two outcome groups. Moreover, URPL patients with elevated peripheral NK cells (absolute counts and proportion), decreased complement levels, and dyslipidemia demonstrated a significantly increased risk of miscarriage. Four models were developed in this study, of which Model 2 demonstrated superior performance with only seven predictors, achieving an AUC of 0.96 (95% CI: 0.93-0.99) and an accuracy of 0.92. A web-based platform was established to visually present model 2 and to facilitate its utilization by clinicians in outpatient settings (available from: https://yingrongli.shinyapps.io/liyingrong/). Our findings suggest that the implementation of such prediction models could serve as valuable tools for providing comprehensive information and facilitating clinicians in their decision-making processes.

Cytokines and Pentraxin 3 Levels in Unexplained Recurrent Pregnancy Loss: Role of Oral Micronized Progesterone Therapy as Immunomodulator on Their Levels and Pregnancy Outcome, a Prospective Comparative Study

Cytokines and Pentraxin 3 Levels in Unexplained Recurrent Pregnancy Loss: Role of Oral Micronized Progesterone Therapy as Immunomodulator on Their Levels and Pregnancy Outcome, a Prospective Comparative Study

Centromeric AA motif in KIR as an optimal surrogate marker for precision definition of alloimmune reproductive failure

Throughout pregnancy, the decidua is predominantly populated by NK lymphocytes expressing Killer immunoglobulin-like receptors (KIR) that recognize human leukocyte antigen-C (HLA-C) ligands from trophoblast cells. This study aims to investigate the association of KIR-HLA-C phenotypes in couples facing infertility, particularly recurrent pregnancy loss (RPL) and recurrent implantation failure (RIF), in comparison to a reference population and fertile controls. This observational, non-interventional retrospective case–control study included patients consecutively referred to our Reproductive Immunology Unit from 2015 to 2019. We analyzed the frequencies of KIR and HLA-C genes. As control groups, we analyzed a reference Spanish population for KIR analysis and 29 fertile controls and their male partners for KIR and HLA-C combinations. We studied 397 consecutively referred women with infertility and their male partners. Among women with unexplained RPL (133 women) and RIF (176 women), the centromeric (cen)AA KIR genotype was significantly more prevalent compared to the reference Spanish population (p = 0.001 and 0.02, respectively). Furthermore, cenAA was associated with a 1.51-fold risk of RPL and a 1.2-fold risk of RIF. Conversely, the presence of BB KIR showed a lower risk of reproductive failure compared to non-BB KIR (OR: 0.12, p < 0.001). Women and their partners with HLA-C1C1/C1C1 were significantly less common in the RPL-Group (p < 0.001) and RIF-Group (p = 0.002) compared to the control group. Moreover, the combination of cenAA/C1C1 in women with C1C1 partners was significantly higher in the control group than in the RPL (p = 0.009) and RIF (p = 0.04) groups, associated with a 5-fold increase in successful pregnancy outcomes. In our cohort, the cenAA KIR haplotype proved to be a more accurate biomarker than the classic AA KIR haplotype for assessing the risk of RPL and RIF, and might be particularly useful to identify women at increased risk among the heterogeneous KIR AB or Bx population. The classification of centromeric KIR haplotypes outperforms classical KIR haplotypes, making it a better indicator of potential maternal–fetal KIR-HLA-C mismatch in patients.

Expanding the Role of Chromosomal Microarray Analysis in the Evaluation of Recurrent Pregnancy Loss

Multiple factors contribute to recurrent pregnancy loss (RPL). This review highlights the latest international guidelines for RPL workup, including immunological testing, by the American Society for Reproductive Medicine (ASRM), the European Society of Human Reproduction and Embryology (ESHRE), and the Royal College of Obstetricians and Gynaecologists (RCOG). These three societies recommend testing for antiphospholipid syndrome. ESHRE and RCOG also recommend thyroid peroxidase antibody testing, whereas ASRM does not. All guidelines advise against testing of natural killer cells, cytokines, antinuclear antibodies, human leukocyte antigen (HLA) compatibility, anti-HLA antibodies, and anti-sperm antibodies. However, when following ASRM, ESHRE or RCOG diagnostic guidelines, over 50% of cases have no identifiable cause. Genetic testing of products of conception (POC) can improve our understanding of unexplained RPL as aneuploidy is a common cause of RPL. Based on studies reporting results from chromosomal microarray analysis (CMA) of POC, we propose a novel algorithm for RPL evaluation. The algorithm involves following evidence-based societal guidelines (published by ASRM, ESHRE, or RCOG), excluding parental karyotyping, in combination with CMA testing of miscarriage tissue. When utilizing this new evaluation algorithm, the number of unexplained cases of RPL decreases from over 50% to less than 10%. As a result, most patients are provided an explanation for their loss and healthcare costs are potentially reduced. Patients with an otherwise negative workup with euploid POC, are classified as “truly unexplained RPL”. These patients are excellent candidates for enrollment in randomized, controlled trials examining novel immunological testing and treatment protocols.

Seminal Oxidative Stress and Sperm DNA Fragmentation in Men from Couples with Infertility or Unexplained Recurrent Pregnancy Loss

(1) Background: This case–control study examined whether men from couples with unexplained recurrent pregnancy loss (RPL) or infertility exhibited higher seminal oxidative stress (OS) and sperm DNA fragmentation (SDF) compared to fertile controls. (2) Methods: The study included 30 participants from each group: unexplained RPL, unexplained infertility, and proven fertility. Data were collected at Aalborg University Hospital tertiary RPL and fertility treatment clinics (Aalborg, Denmark), excluding couples with mixed conditions for homogeneity. Semen samples were analyzed using computer-aided sperm analysis (CASA) for concentration, motility, and morphology. SDF was assessed via a CASA-based sperm chromatin dispersion test. OS was measured as static oxidation-reduction potential (sORP). (3) Results: The results showed no significant OS differences between groups. The RPL group had significantly lower SDF levels than the control group. A significant positive correlation between SDF and OS was observed in the infertility group. Overall, this study did not find significant differences in OS levels between men from couples with unexplained RPL or infertility and fertile controls, while SDF levels were lower in the RPL group compared to controls. (4) Conclusion: In conclusion, despite the existing literature suggesting that OS and SDF are negative prognostic factors, our findings suggest they may not be reliable diagnostic markers for RPL and infertility.

Overexpression of regulatory T cells in patients with unexplained recurrent pregnancy loss: friend or foe?

This study aimed to investigate the role of regulatory T cells in patients with unexplained recurrent pregnancy loss (URPL). We retrospectively analyzed 136 women who had experienced two or more miscarriages before 24 weeks of gestation for no obvious reason from May 2018 to October 2021. The basic clinical data of the patients and expression of lymphocyte subsets such as regulatory T cells (Tregs) and natural killer cells (NKs) by flow cytometry were collected to explore the risk factors of pregnancy outcome in URPL patients. A total of 136 URPL patients were enrolled in this study. Eventually, 50 patients attained clinical pregnancy. The median age was 31.8 ± 4.6 years in patients with clinical pregnancy. The univariate and multivariate logistic regression analyses indicated that Tregs was associated with the pregnancy outcomes of patients with URPL (odds ratio 0.63, 95% confidence interval 0.50-0.80). More importantly, a U-shaped association was found between Tregs and pregnancy outcome (p < 0.001), with either higher or lower Tregs levels adversely affecting pregnancy outcome. Tregs levels that are either too high or too low can harm pregnancy outcomes. It was expected to be a very promising quantitative biomarker for predicting pregnancy outcomes in URPL patients.

Identification of cryptic balanced translocations in couples with unexplained recurrent pregnancy loss based upon embryonic PGT-A results.

The goal of this study is to determine whether any balanced translocation (BT) had been missed by previous karyotyping in patients with unexplained recurrent pregnancy loss (uRPL). This case series included 48 uRPL-affected couples with normal karyotypes. The embryos from these couples have all undergone preimplantationtesting for aneuploidies (PGT-A). Based on the PGT-A's results, 48 couples could be categorized into two groups: 17 couples whose multiple embryos were detected with similar structural variations (SVs, segmental/complete) and 31 couples without such findings but who did not develop any euploid embryo despite at least three high-quality blastocysts being tested. The peripheral blood sample of each partner was then collected for mate-pair sequencing (MPseq) to determine whether any of them were BT carriers. MPseq analyses identified 13 BTs in the 17 couples whose multiple embryos had similar SVs detected (13/17, 76.47%) and three BTs in the 31 couples without euploid embryo obtained (3/31, 9.7%). Among the 16 MPseq-identified BTs, six were missed due to the limited resolution of G-bandingkaryotyping analysis, and the rest were mostly owing to thesimilar banding patterns and/or comparable sizes shared by the two segments exchanged. A normal karyotype does not eliminate the possibility of carrying BT for couples with uRPL. The use of PGT-A allows us to perceive the "carrier couples" missed by karyotyping analysis, providing an increased risk of finding cryptic BTs if similar SVs are always detected on two chromosomes among multiple embryos. Nonetheless, certain carriers with translocated segments of sub-resolution may still go unnoticed.

Oral administration of Bifidobacterium longum ES1 reduces endometrial inflammation in women with recurrent pregnancy loss.

Over-activation of endometrial inflammasome NALP-3 (Nod-like receptor family pyrin domain containing 3) can be found in recurrent pregnancy loss (RPL) women probably due to leaky gut and passage into circulation of lipopolysaccharides (LPS). Leaky gut can be caused by exposure to gluten in RPL women genetically predisposed to celiac disease, positive for Human Leukocyte Antigen (HLA)-DQ2/DQ8 haplotype. Oral administration of Bifidobacterium longum ES1 (GliadinES®) can inactivate gluten peptides toxicity to epithelial gut cells and improve gut barrier. We investigated by enzyme-linked immunoassay: (a) serum levels of LPS and zonuline (a marker of leaky gut); (b) LPS, NALP-3, caspase-1, interleukine (IL)-1β and IL-18 concentration in endometrial fluids, in untreated women with uncomplicated pregnancies (negative HLA-DQ2/DQ8 haplotype) (n=22) and in women with unexplained RPL, HLA-DQ2/DQ8 positive (n=22), before and after daily oral administration for 3 months of GliadinES®. RLP women showed higher serum levels of LPS (p<0.0001) and higher concentration of LPS (p<0.0001), NALP-3 (p<0.01); Caspase-1 (p<0.0001), IL-1β (p<0.0001), and IL-18 (p<0.0001) in endometrial fluids compared to controls. GliadinES® treatment significantly reduced serum levels of both LPS (p<0.0001) and zonuline (p<0.01), as well as LPS (p<0.5), NALP-3 (p<0.01), Caspase-1 (p<0.001), IL-1β (p<0.001), and IL-18 (p<0.01) concentrations in endometrial fluids of RPL women. RPL women positive for HLA-DQ2/DQ8 haplotype show increased circulating and endometrial levels of LPS and endometrial inflammasome NALP-3 over-activation. Oral administration of GliadinES® can reduce gut permeability, decrease serum levels of LPS and, contextually, improve endometrial inflammation in this specific subset of RPL women.

Additional Cover

The cover image is based on the Original Article Insufficient GDF15 expression predisposes women to unexplained recurrent pregnancy loss by impairing extravillous trophoblast invasion by Chunzi Lyu et al., https://doi.org/10.1111/cpr.13514. image

#286 : Circus Recurrent Pregnancy Loss by Modulating SETD1B/H3K4me2/me3 LoopK40 Regulates Cell Survival and Participates in

Background and Aims: The epigenetic mechanisms involved in the etiology of unexplained recurrent pregnancy loss (uRPL) is largely unknown. This study aims to investigate the molecular mechanisms of circRNAs modulating decidua function by regulating methylation modification of histones and whereby participating in uRPL. Method: RNA sequencings on decidua of uRPL couples and endometrial stromal cells (ESCs) after overexpressed with circSTK40 were performed to confirm the regulatory relationship between circSTK40 and SETD1B. Functional experiments including TUNEL, autophagy double-label system, the tests of glucose intake, lactic acid production were performed to determine the effects of circSTK40 and SETD1B on ESCs. Mechanism studies were conducted using RNA pulldown, RNA binding protein immunoprecipitation, chromatin immunoprecipitation et al. Results: The expression levels of circSTK40 and SETD1B were significantly downregulated in the decidua of uRPL patients, and a positive regulatory relationship was observed between them. Both circSTK40 and SETD1B promoted cell survival and participated in the maintenance of pregnancy by regulating apoptosis, autophagy and glycolysis of decidualized ESCs. After the knockdown of SETD1B, the protective effect of circSTK40 could be eliminated, resulting in elevated apoptosis, reduced autophagy level and increased cellular glycolysis, leading to pregnancy loss. Regarding mechanism studies, circSTK40 is directly bound to SETD1B and histone H3 to promote their interaction and increase the methylation level of H3K4. H3K4me2 and H3K4me3 reversely bound to the promoter region of SETD1B and enhanced its transcription, resulting in upregulation of SETD1B, and consequently formed a SETD1B/H3K4me2/3 regulatory loop. Conclusion: CircSTK40 influences cell survival by modulating the SETD1B/H3K4me2/me3 regulatory loop. Downregulation of circSTK40 and SETD1B in decidua may contribute to uRPL via promoting cell apoptosis. Our findings indicate a novel epigenetic mechanism for uRPL pathogenesis involving circSTK40 activity and histone methylation modification.

Role of the CD40–CD40L expression level pathway in the diagnosis of unexplained recurrent pregnancy loss

Background Unexplained recurrent spontaneous pregnancy loss (URPL) lacks effective treatment and reliable early diagnosis and prediction. Immunologic dysfunction can be an underlying cause of recurrent pregnancy loss (RPL). Considering the regulatory role of CD40–CD40L in immune responses, we explored its clinical significance in URPL. Methods The 108 women with URPL who were treated in Hebei Yanda Hospital from January 2020 to December 2022 were selected as study subjects, and another 108 healthy women who were not pregnant and matched with the age and body mass index of the study group were selected as the control group. CD40 and CD4 + CD25 + Treg cells in peripheral blood mononuclear cells (PBMCs) and CD40L in peripheral blood platelets were measured by flow cytometry. The predictive value of CD40–CD40L in URPL for the risk of RPL was determined by receiver operating characteristic (ROC) curves. The correlations of CD40–CD40L with CD4 + CD25 + Treg cells and serum pro-inflammatory factors were assessed by Pearson’s analysis. Results CD40 on the surface of PBMCs and CD40L on the surface of platelets were up-regulated in URPL patients. CD40 in combination with CD40L had high predictive value for the risk of RPL in URPL patients. Peripheral blood CD40–CD40L was positively linked to IL-17 and IL-23, and negatively to CD4 + CD25 + Treg cells and IL-10 in URPL patients. Conclusions The CD40–CD40L pathway expression in peripheral blood can help predict the risk of RPL in URPL patients.

Statement of Retraction [A novel association between cytotoxin-associated gene A (CagA) positive strain of Helicobacter pylori and unexplained recurrent early pregnancy loss]

Statement of Retraction [A novel association between cytotoxin-associated gene A (CagA) positive strain of Helicobacter pylori and unexplained recurrent early pregnancy loss]

Unexplained recurrent pregnancy loss and markers of arterial vascular health: An experimental case-control study.

To investigate whether women with unexplained recurrent pregnancy loss have impaired arterial vascular health compared with controls, and to evaluate whether this is modifiable by exercise. Experimental case-control pilot study. University medical centre in the Netherlands. Twelve women with unexplained recurrent pregnancy loss, 11 nulliparous women and 19 primiparous women with a history of uncomplicated pregnancies. In all three groups we measured baseline characteristics, metabolic components and arterial vascular health, and repeated this in women with unexplained recurrent pregnancy loss after 1 month of protocolled and supervised cycle training. Differences in arterial vascular health between women with unexplained recurrent pregnancy loss and controls, and the effect of exercise on arterial vascular health in women with unexplained recurrent pregnancy loss. Women with unexplained recurrent pregnancy loss have a significantly increased carotid intima media thickness in comparison with both controls (both P < 0.01), a significantly decreased brachial endothelial dependent flow-mediated vasodilation in comparison with both controls (nulliparous: P < 0.01; primiparous: P = 0.05) and a significantly decreased femoral endothelial dependent flow-mediated vasodilation in comparison with primiparous women (P = 0.01). The endothelium independent glyceryl trinitrate response was similar in all groups. With 1 month of exercise, the carotid intima media thickness decreased significantly by 7% (P = 0.05) and the femoral FMD increased significantly by 10% (P = 0.01) in women with unexplained recurrent pregnancy loss. Women with unexplained recurrent pregnancy loss have an impaired vascular health in comparison with controls. This impaired arterial vascular health can be improved by exercise.

Role of Serum and Placental Tissue IL6 in Unexplained Recurrent Pregnancy Loss

Role of Serum and Placental Tissue IL6 in Unexplained Recurrent Pregnancy Loss

Decrease in peripheral natural killer cell level during early pregnancy predicts live birth among women with unexplained recurrent pregnancy loss: a prospective cohort study

BACKGROUNDPrevious studies have suggested that the trophoblast cells inhibit the proliferation of peripheral natural killer (pNK) cells and the level of pNK cells decrease in middle and late pregnancy stage among healthy women. The change of pNK cells level during early pregnancy and the relationship between the change of pNK level and pregnancy outcomes in women with unexplained recurrent pregnancy loss (URPL) has not been sufficiently explored. OBJECTIVEThis study aims to characterize the level of pre-pregnancy pNK to early pregnancy among women with URPL and whether the change in the level of pNK cells in early pregnancy from pre-pregnancy can predict pregnancy outcomes. STUDY DESIGNIn this prospective cohort study, 1758 women with recurrent pregnancy loss were recruited between January 2017 and December 2021, of which 252 URPL women had pre-pregnancy and early pregnancy (4-6 weeks gestation) pNK measurements. These 252 women were divided into two groups: those with a lower gestational pNK levels (Group 1) compared to pre pregnancy, and those without (Group 2). Their respective outcomes on live birth and pregnancy loss were comparatively analyzed using Chi-square and Student’s t test. Candidate influence factors for live birth were selected using the Akaike information criterion (AIC). Then the participates were randomly divided into training and testing groups. Multivariable logistic regression model was performed, and nomogram was calculated to assess the possibility of live birth. Predictive accuracy was discriminated by area under the receiver operating characteristic curve (ROC), validated by plotting the predicted probabilities and the observed probabilities. Hosmer-Lemeshow test was used to assess the goodsess of fit. RESULTSWhen early gestational pNK cells levels were compared with pre pregnancy pNK cells levels, 61.5% (154) women had a comparatively lower early gestational pNK cells levels versus 38.9% (98) women with increase or no change of their pNK cells levels. The live birth rate in Group 1 was 89.0% (137/154), which was significantly higher than 49.0% (48/98) of Group 2 (p<0.001). Decrease of pNK cells level (odds ratio [OR]=1.36, 95% CI 1.22-1.55, p<0.001) and anti-Muellerian hormone (AMH) (OR=1.41, 95% CI 1.14-1.81, p=0.003) were important predicting factors for higher live birth. Female BMI (OR=0.97, 95% CI 0.82-1.15, p=0.763) and parity (OR=1.61, 95% CI 0.71-4.12, p=0.287) were also predicting factors. Furthermore, the area under the ROC curve of the model to diagnose of live birth was 0.853 with a sensitivity of 81.6% and specificity of 78.0% using the training dataset. And the Hosmer−Lemeshow test showed that the model was a good fit (p = 6.068). CONCLUSIONWe report a comparative decrease in pNK cells levels in over 60% URPL women at 4-6 weeks of gestation, when compared to their pre-pregnancy pNK cells level. Compared to pre pregnancy pNK cells levels, a decrease pNK cells level during early pregnancy might be a useful predictor for LBR in women with URPL.

[The impact of the number of previous miscarriages on embryo euploid rate and pregnancy outcomes in patients with unexplained recurrent pregnancy loss].

Objective: To investigate the impact of the number of previous miscarriages on embryo euploid rate and pregnancy outcomes after preimplantation genetic testing for aneuploidies (PGT-A) in patients with unexplained recurrent pregnancy loss (uRPL). Methods: A retrospective cohort study was conducted. 799 women with uRPL who underwent PGT-A for the first time between January 2015 and December 2021 at the Reproductive center of Shandong University were enrolled. These patients were divided into three groups according to the number of previous miscarriages (2, 3, and≥4). Stratified analysis was conducted according to female age (≤37 years and>37 years). The embryo euploidy rate, good-quality blastocyst formation rate, cumulative live birth rate, and cumulative clinical pregnancy loss rate of three groups were compared in younger and older patients, respectively. Meanwhile, the cumulative live birth rate, clinical pregnancy loss rate, and embryo euploidy rate were analyzed by multivariate logistic regression analysis. Results: Patients' age was (34.7±5.1) years old. In the three groups with 2, 3 and ≥4 previous miscarriages, there was no significant difference in the embryo euploidy rate between groups in the younger [48.9% (539/1 103), 50.6% (354/700) and 52.1% (152/292), P=0.567] and older [26.2% (103/393), 28.8% (55/191) and 20.5% (16/78), P=0.377] age population. Compared with 2 and 3 previous miscarriages, the cumulative live birth rate was significantly decreased [52.6% (153/291), 52.8% (93/176) and 34.3% (25/73), P=0.014] and the cumulative clinical pregnancy loss rate was significantly increased [15.8% (31/196), 15.3% (18/118) and 46.9% (23/49), P<0.001] in younger women with ≥4 miscarriages. After adjusting for maternal age, BMI, AMH, endometrial thickness on hCG trigger day and antral follicle count, the number of previous miscarriages ≥4 was a relevant factor for cumulative live birth rate (OR=0.461, 95%CI: 0.263-0.807, P=0.007) and the cumulative clinical pregnancy loss rate (OR=4.382, 95%CI: 2.165-8.873, P<0.001) in younger patients, but it was not significantly correlated with the cumulative live birth rate, cumulative clinical pregnancy loss rate and embryo euploidy rate in patients with advanced age. Conclusion: In uRPL patients,≥4 previous miscarriages decreased cumulative live birth rate and increased cumulative clinical pregnancy loss rate in women aged≤37 years old.

Impaired decidualization and relative increase of PROK1 expression in the decidua of patients with unexplained recurrent pregnancy loss showing insulin resistance

A recent meta-analysis revealed that patients with unexplained recurrent pregnancy loss (RPL) show higher insulin resistance compared to healthy controls. However, the etiology of RPL remains unknown. Prokineticin (PROK1), a pleiotropic uterine endometrial protein, is important for implantation and decidualization and is regulated by hypoxia and insulin. In this study, we investigated the decidualization status and the role of PROK1 in the decidua of patients with unexplained RPL showing insulin resistance. Thirty-two patients with unexplained RPL were included in this study. Following the diagnosis of a miscarriage, the decidua and villi of the patient were surgically collected. Fasting blood glucose and insulin levels were measured, and HOMA-β was calculated. Using IHC and ELISA, the expression of IGFBP-1, PRL and PROK1 in the decidua and IGF-2 in the villi were analyzed in patients with euploid miscarriage with a high HOMA-β index (n = 8) and compared to controls (euploid miscarriage with normal HOMA-β: n = 12, aneuploid miscarriage with normal HOMA-β: n = 12). The co-localization of PROK1 and IGFBP-1 was observed in the decidua by IHC. In the decidua of RPL patients with high HOMA-β, the expression levels of IGFBP-1 and PRL were significantly lower, whereas the PROK1/IGFBP-1 ratio was significantly higher compared to that of the controls. IGF-2 expression in villi was significantly lower in RPL patients with high HOMA-β. Impaired decidualization and excessive PROK1 production may have pathological implications in patients with unexplained RPL with insulin resistance, especially under the state of hyper insulin production.