Immune Cell Populations in Idiopathic Recurrent Pregnancy Loss and Unexplained Infertility in Venezuelan Admixed Women

BackgroundThe prognostic value of peripheral natural killer (pNK) cells, as a screening test in women with recur-rent pregnancy loss (RPL) and unexplained infertility, is still a matter for discussion. The purpose of this study was tocompare the percentage of circulating CD56+NK cells, CD69 and perforin markers between women with unexplainedinfertility and RPL with the healthy control group.Materials and MethodsIn this case-control study, the percentage of CD56+NK cells and activation markers (CD69and perforin levels) in the peripheral blood were measured in 25 women with unexplained infertility, 24 women withidiopathic RPL and 26 women from the healthy control group, using specific monoclonal antibodies by flow cytometry.ResultsThe percentage of CD56+NK cells was significantly higher in patients with infertility in comparison withthe healthy control group (P=0.007). There were not significant differences either in the total number of CD56+cellsbetween the RPL group and the control group (P=0.2) or between the RPL group and the infertile group (P=0.36).The percentage of CD69+lymphocytes in RPL group was significantly higher than in the infertility group (P=0.004).There was a statistically significant difference in Perforin levels between RLP and control (P=0.001) as well as RPLand infertile (P=0.002) groups.ConclusionAn increased percentage of CD56+NK cells in patients with unexplained infertility, an elevated expressionof CD69 on NK cells in patients with RPL and infertility and a high level of perforin on CD56+cells in the RPL groupmight be considered as immunological risk factors in these women.

Evaluation of FAS and caspase-3 in the endometrial tissue of patients with idiopathic infertility and recurrent pregnancy loss

Study question To what extent do genomic variants contribute to idiopathic recurrent pregnancy loss (RPL)? Summary answer Pathogenic uniparental disomy, copy number variations, and single nucleotide variants were identified in 18 of 83 couples and their embryos/fetuses with idiopathic RPL. What is known already RPL is defined as the loss of two or more pregnancies before 24 weeks gestation. The etiologies of RPL include antiphospholipid antibody syndrome, and anatomic, endocrinological and chromosomal abnormalities. About 50% of RPL is unexplained and is termed as idiopathic RPL. Chromosomal aberrations and genetic variants have been identified to be associated with idiopathic RPL, demonstrating that genomic factors underlie idiopathic RPL. Study design, size, duration We applied genome sequencing to study genomic variations that causing idiopathic RPL. Couples and their embryos/fetuses were collected and sequenced. Genomic variations from women and men experiencing RPL and their embryos/fetuses were analyzed to fully characterize the genomic contribution to idiopathic RPL. Participants/materials, setting, methods We recruited 83 Chinese couples with idiopathic RPL and collected 249 samples from their embryos/fetuses and peripheral blood. Genomic DNA of those samples were sequenced, and pathogenic chromosomal abnormalities and genetic variants were identified. Main results and the role of chance Generally, pathogenic variants were identified in RPL females, males and embryos/fetuses. One uniparental disomy, 9 copy number variations, and 1 homozygous and 1 de novo single nucleotide variants (SNVs) were identified pathogenic in 12 RPL embryos/fetuses. Five and three pathogenic SNVs were identified in 4 RPL females and 2 RPL males respectively. Many SNVs affect the genes involved in sexual development. In total, 18 of 83 (21.69%) couples with idiopathic RPL could be explained by genomic variation, providing information for clinical treatments. Limitations, reasons for caution The sample size is relatively small, and they are from one medical center. Wider implications of the findings Genomic variations in females, males and their embryos/fetuses can cause RPL, and identification of genetic causes will facilitate further precision intervention. Trial registration number not applicable

Should patients be screened for chronic endometritis before assisted reproductive technology?

Study question What are the differences in menstrual blood lymphocytes between controls, patients with recurrent pregnancy loss (RPL) or with unexplained infertility (uINF)? Summary answer Compared with controls, RPL- and uINF patients had a different menstrual blood immune profile, including altered NK-subtypes indicating an altered cytotoxicity in these patients. What is known already T-cells, particularly regulatory T-cells, and uterine natural killer cells play a vital role as they are the main immunological regulators at the feto-maternal interface. However, an endometrial biopsy (EB) is required for the examination of these cells, necessitating an invasive and uncomfortable procedure. Moreover, the small tissue samples are mainly analyzed by immunohistochemistry, limiting the amount of analysable markers and ultimately results in controversial data. Menstrual blood (MB) has been used in proof-of-concept studies to analyse lymphocyte populations. So far, no study has investigated MB lymphocytes in a well-defined cohort of RPL- and uINF patients in comparison to healthy controls. Study design, size, duration In this prospective study, 46 healthy controls, 28 RPL patients and 11 uINF patients were included between 03/2021 and 02/2022. RPL was defined as ≥ 3 consecutive pregnancy losses. For being diagnosed with uINF, all standard-diagnostic procedures for infertility had to provide negative results in couples trying to conceive for >12 months. Participants/materials, setting, methods To establish the procedure, lymphocyte compositions in EB in 7 controls were compared with those in MB during 48h. The first 24h and the second 24h were collected separately. In all other RPL- and uINF-patients, as well as controls, peripheral blood (PB) and MB was collected. Density gradient centrifugation was performed to isolate PB and MB mononuclear cells. Cells were analyzed by flow cytometry focusing on the main lymphocyte populations and NK-cell subsets. Main results and the role of chance The first 24h of MB closely resembled the uterine lymphocyte composition in EB, with were CD3+ T-cells and CD56+ NK-cells being the predominant lymphocyte populations in EB and MB. We therefore based further analyses on the first 24h of menstrual shedding. In comparison to controls, RPL-patients showed significantly higher MB-CD56+-NK-cell numbers (mean±SD: 31.13±7.52 vs. 36.73±5.4; p = 0.002). Furthermore, MB-CD56dimCD16bright NK-cells within the CD56+-NK-cell population were decreased in individuals with RPL and uINF compared to controls (mean±SD: 20.4±11.53;16.34±14.65;15.7±5.91; Ctrl vs. RPL p = 0.011; Ctrl vs. uINF p = 0.02). Further, compared to controls and RPL patients, uINF patients had lower CD3+ T-cell counts (mean±SD Ctrl. vs. uINF: 47.93±11.11 vs. 38.81±5.04, p = 0.013), with decreased CD4+ (mean±SD Ctrl. vs. uINF: 25.78±7.91 vs. 15.66±4.88, p = 0.001) and CD8+ T-cells (mean±SD Ctrl. vs. uINF: 15.32±4.14 vs. 8.43±2.85, p < 0.001). Interestingly, uINF-patients showed higher CD25highCD127dim/neg regulatory T-cell counts than controls (mean±SD Ctrl. vs. uINF: 1.34±0.49 vs. 1.84±0.51, p = 0.009). The cytotoxicity receptors NKp46 and NKG2D were more present in uINF and RPL patients. Furthermore, RPL and uINF patients had significantly higher peripheral CD56+-NK-cell counts as compared to controls (mean±SD: 8.4±3.5;11.42±4.05;12.86±4.29; Ctrl vs. RPL p = 0.021; Ctrl vs. uINF p = 0.009). Limitations, reasons for caution The small sample size of our study limits our ability to extrapolate the findings to other populations, as well as perform further subgroup analyses in controls, RPL and uINF. Future research must concentrate on NK cell subpopulations in larger cohorts, potentially in a multi-center setting. Wider implications of the findings Strengths of this study include well phenotyped participants and that the immunological milieu of the endometrium is directly compared for EB and MB. In future studies, this non-invasive analysis might enable to identify and monitor patients who could profit from (immunomodulatory) medications, and thereby improve live birth rates. Trial registration number not applicable

Killer-cell immunoglobulin-like receptors (KIRs) play a crucial role in the cytotoxic activity of natural killer (NK) cells, encompassing both inhibitory and activating types. A higher ratio of cytotoxic to inhibitory receptors may harm successful pregnancies by disrupting the uterine environment. Ongoing debates surround the impact of KIR gene variations on recurrent pregnancy loss (RPL) and infertility across populations. This study aimed to explore KIR gene polymorphisms in RPL and infertility among the Venezuelan admixed population. The Venezuelan population exhibits a genetic mix of Caucasian, African, and local Amerindian ancestry, distinguishing it from other Latin American admixed populations. This study included 100 controls and 86 patients: 73 women with idiopathic RPL (53 primary and 20 secondary) and 13 infertile patients (4 primary and 9 secondary). The frequency of activating receptors KIR2DS2 and KIR2DS3 was significantly lower (p < 0.05) in the whole patient group compared to controls. However, when analyzing the haplotypes and genotypes, the significance between patients and controls was lost. When comparing RPL and infertile patients, KIR2DS2, KIR2DL3, 2DL5, and 3DL1 were significantly less frequent in infertile women. In infertile women, KIR2DS3 frequency was increased compared to controls and RPL. The results suggest that the frequency of inhibitory receptors may differentiate patients with RPL and infertility. Further studies should ascertain the expression and function of KIRs in uterine NK cells in patients with RPL and infertility.

(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.

Sperm deoxyribonucleic acid (DNA) damage assessment in male partners of couples with recurrent pregnancy loss and unexplained infertility: a prospective cohort study

Chapter 3 - T helper cell pathology and recurrent pregnancy losses; Th1/Th2, Treg/Th17, and other T cell responses

The pathogenesis of recurrent pregnancy loss (RPL) is multifactorial and not completely elucidated. Dysregulated immunity was implicated with RPL, in which regulatory T cells (Tregs) are key. As Tregs development and function are regulated by forkhead box P3 (FOXP3) transcription factor, and as FOXP3 expression is genetically determined, a role for FOXP3 polymorphisms in RPL pathogenesis was suggested. To investigate the association of rs2294021, rs2232365, rs3761548, and rs141704699 FOXP3 variants with idiopathic RPL in Lebanese women. This retrospective case-control study included 386 RPL cases and 398 age-matched control women. Logistic odds ratios (OR) were estimated with 95% confidence interval after adjustment; a significance value of P<.05 was set. Significantly lower rs22944021 and rs2232365 minor allele frequency (MAF) was found in patients with idiopathic RPL in comparison with the control group. Furthermore, statistically significantly lower frequency of heterozygous and homozygous rs2294021 and rs2232365 genotypes was seen in controls, while significantly lower rs3761548 heterozygous genotype frequencies were found in the patient group. Obesity, antihypertension treatment, smoking, positive RPL family history, abortion state, and infertility treatment correlated negatively with rs2294021, while rs2232365 negatively correlated with obesity, and rs3761548 negatively correlated with infertility treatment. Marked linkage disequilibrium (LD) was noted among FOXP3 SNPs, with TGCC and CGAC haplotypes being positive, while CAAC, CACC, and TGAC haplotypes being negatively associated with RPL risk. Except for CGAC, the association of these haplotypes with RPL persisted after adjustment. FOXP3 gene variants and haplotypes are associated with altered incidence of RPL, proposing the role of Treg in RPL pathogenesis.

The imbalance in the Th17/Regulatory T (Treg) cell ratio is associated with recurrent pregnancy loss (RPL). This study aimed to determine a cut-off for the Th17/Treg cell ratio to predict pregnancy outcomes in RPL and evaluate the effectiveness of intravenous immunoglobulin (IVIG) based on this cut-off value. This retrospective cohort study included 49 idiopathic RPL and 75 controls. The subgroups of IL-17+ T cell to Foxp3+ T cell ratios in peripheral blood were measured using flow cytometry. The cut-off values of Th17/Treg cell ratios were determined by the ROC curve to distinguish between RPL and controls. The IVIG treatment effectiveness in pregnancy outcome was compared between high- and low-ratio groups. Pearson correlation assessed the Th17/Treg cell ratio's relationship with NK cell cytotoxicity (NKC), NK cell percentage, and Th1/Th2 cell ratio. Using the ROC curve, we identified six Th17/Treg cell ratio markers with diagnostic value, and the following two, CD3+IL-17+ T cell/CD3+Foxp3high T cell ratio (sensitivity at 97%) and CD4+IL-17+ T cell/CD3+Foxp3high T cell ratio (specificity at 93.61%), showed the highest statistical significance in diagnosing idiopathic RPL. Among the six diagnostic markers, in terms of predicting pregnancy outcomes with IVIG treatment, CD3+IL-17+ T cell/CD4+Foxp3+ T cell ratio was the most valuable prognostic marker. In RPL women with high CD3+IL-17+ T cell/CD4+Foxp3+ T cell ratio (≥ 1.096), the live birth rate (LBR) was improved with IVIG treatment. (IVIG treatment, 78.57%vs. no IVIG, 28.57%, p = 0.026). On the other hand, RPL women with low CD3+IL-17+ T cell/CD4+Foxp3+ T cell ratio did not demonstrate the effectiveness of IVIG (LBRs with IVIG treatment, 50.00%vs. no IVIG, 84.62%, p = 0.219). In a correlation study, the CD3+IL-17+ T cell/CD4+Foxp3+ T cell ratio was an independent prognostic marker, showing no correlation with NKC, NK cell percentage, and Th1/Th2 cell ratio. The CD3+IL-17+ T/CD4+Foxp3+ T cell ratio may serve as a valuable marker for understanding the pathogenesis of RPL, predicting pregnancy outcomes, and selecting candidates for immunotherapy. Our study demonstrates that IVIG treatment can significantly improve LBR in women with a high CD3+IL-17+ T/CD4+Foxp3+ T ratio, offering a promising therapeutic approach for this challenging condition.

To evaluate the relationship between idiopathic recurrent pregnancy loss (RPL) and oxidative stress (OS) by means of thiol/disulfide homeostasis via a novel technique. Thirty-nine pregnant women diagnosed with idiopathic RPL were compared with 50 healthy pregnant women without a history of abortion. Idiopathic RPL was defined as experiencing two or more consecutive miscarriages prior to 20 weeks of gestation with the presence of normal karyotypes of couple and/or abortus materials, negative maternal screening for anticardiolipin, anti β 2 glycoprotein antibodies and lupus anticoagulant, normal thyroid stimulating hormone, prolactin and hemoglobin A1C levels and normal pelvic sonography and/or hysterosalpingography. A new and fully automated method was used to measure plasma native thiol, total thiol and disulfide levels, based on the reduction of dynamic disulfide bonds to functional thiol groups by sodium borohydrate. Women with idiopathic RPL had significantly lower plasma levels of native thiol (341.89 ± 50.0 μmol/L vs. 390.84 ± 38.5 μmol/L, P < 0.001) and total thiol (386.18 ± 51.7 μmol/L vs. 435.78 ± 42.3 μmol/L, P < 0.001). Disulfide/thiol and disulfide/total thiol ratios were significantly higher in the study group. The native thiol/total thiol ratio was significantly lower in patients with idiopathic RPL. No difference was measured in disulfide, albumin and total protein plasma levels. The main outcome of our study indicates a relation between idiopathic RPL and OS. More importantly, the new method used in our study proposes a promising, practical and daily applicable test for evaluating patients with idiopathic RPL.

Altered endometrial expression of endothelial nitric oxide synthase in women with unexplained recurrent miscarriage and infertility

Angiogenesis and an adequate blood supply are critical for several steps in human early pregnancy. Some studies have reported angiogenesis- and vasoconstriction-related genes are associated with recurrent pregnancy loss (RPL), but their sample size was limited. This study was conducted to investigate the genetic association between these angiogenesis- and vasoconstriction-related genes and idiopathic RPL, using meta-analyses. A systematic review of the published literature from MEDLINE and EMBASE databases was conducted and investigations of an angiogenesis- and vasoconstriction-related gene polymorphism in RPL reported more than three times were selected. Aggregating data from eligible studies were integrated into meta-analyses by means of random effects models. Of 185 potentially relevant studies, 18 case-control studies comprising a total of 2397 RPL patients and 1760 controls were included into the meta-analyses. Among these genetic association studies were 4 reports of vascular endothelial growth factor (VEGF) (-1154G>A) polymorphisms, 4 reports of p53 (codon72) and 10 reports of endothelial nitric oxide synthase (eNOS) (B/A, Glu298Asp) with RPL. The integrated results showed that VEGF (-1154G>A), p53 (codon 72) and eNOS (Glu298Asp) polymorphisms were significantly associated with RPL, and their summary odd ratios [95% confidence interval (CI)] were 1.51 (1.13-2.03), 1.84(1.07-3.16) and 1.37 (1.11-1.69), respectively. The summary odd ratio of the eNOS (B/A) polymorphism in RPL was 1.15 (0.94-1.41), and failed to show significance at meta-analysis. Meta-analyses of available data showed significant associations between the VEGF (-1154G>A), p53 (codon72) and eNOS (Glu298Asp) polymorphisms and idiopathic RPL. These angiogenesis- and vasoconstriction-related genes jointly confer higher susceptibility to idiopathic RPL.

Sildenafil citrate (SC), a PDE5 inhibitor, a drug for erectile dysfunction (ED) and pulmonary hypertension (PAH), was found to exert a positive effect on pregnancy outcomes when administered intravaginally before conception. In our previous studies, sildenafil increased endometrial thickness and significantly decreased peripheral blood NK cell activity after the intravaginal administration in women with recurrent pregnancy loss (RPL). No data are available to confirm the effect of sildenafil on maternal T cell populations involved in shaping fetal-maternal tolerance and NK cell activity. Thus, the present study aimed to establish if SC influences NKT cells or the axis of Th17/Treg cells and Th1/Th2 cytokine production. Materials and methods: Twenty-one healthy fertile women and twenty-two nonpregnant women with idiopathic RPL were studied. The ELISA method was used to evaluate the production of cytokines, including IL-2, IL-12p40, IL-4, IL-10, IL-6, IL-17, IL-21, TGF-β, TNF-α, and IFN-γ in PBMC culture supernatants before and after supplementation with the physiological concentration of SC. The percentages of NKT (CD56+CD3+CD44+CD161+), Treg (CD4+CD25+FOXP3+) and Th17 (CD4+CD25+IL-17A+) cells were determined with flow cytometry method. Results: Unexpectedly, we found that the PBMCs of patients with RPL produced a significantly lower level of inflammatory cytokines (TNF-α and IL-6) and a higher level of anti-inflammatory cytokines (TGF-β and IL-10). SC significantly decreased IL-6, IL-12 and increased TGF-β cytokine concentration in fertile women. In the case of RPL patients’ PBMCs, SC improved the production of TNF-α and IL-10. Conclusions: Lower concentration of proinflammatory cytokines in idiopathic RPL women compared to fertile women might suggest the exhaustion of the immune system. The emphasized production of IL-10 by SC partially explains the previously observed downregulation of NK cell activity in RPL patients. The immunomodulatory effect of the drug might be utilized in anti-inflammatory therapies and help achieve positive pregnancy outcomes in women with reproductive failure due to a Th1/Th2 imbalance.