Maternal Immune Response Research Articles

Prostaglandin E2 Receptor 4 (EP4) Affects Trophoblast Functions via Activating the cAMP-PKA-pCREB Signaling Pathway at the Maternal-Fetal Interface in Unexplained Recurrent Miscarriage.

Implantation consists of a complex process based on coordinated crosstalk between the endometrium and trophoblast. Furthermore, it is known that the microenvironment of this fetal–maternal interface plays an important role in the development of extravillous trophoblast cells. This is mainly due to the fact that tissues mediate embryonic signaling biologicals, among other molecules, prostaglandins. Prostaglandins influence tissue through several cell processes including differentiation, proliferation, and promotion of maternal immune tolerance. The aim of this study is to investigate the potential pathological mechanism of the prostaglandin E2 receptor 4 (EP4) in modulating extravillous trophoblast cells (EVTs) in unexplained recurrent marriage (uRM). Our results indicated that the expression of EP4 in EVTs was decreased in women experiencing uRM. Furthermore, silencing of EP4 showed an inhibition of the proliferation and induced apoptosis in vitro. In addition, our results demonstrated reductions in β- human chorionic gonadotropin (hCG), progesterone, and interleukin (IL)-6, which is likely a result from the activation of the cyclic adenosine monophosphate (cAMP)- cAMP-dependent protein kinase A (PKA)-phosphorylating CREB (pCREB) pathway. Our data might provide insight into the mechanisms of EP4 linked to trophoblast function. These findings help build a more comprehensive understanding of the effects of EP4 on the trophoblast at the fetal–maternal interface in the first trimester of pregnancy.

ABO haemolytic disease of the newborn: Improved prediction by novel integration of causative and protective factors in newborn and mother.

Prediction of haemolytic disease of the foetus and newborn (HDFN) caused by maternal anti-A/-B enables timely therapy, thereby preventing the development of kernicterus spectrum disorder. However, previous efforts to establish accurate prediction methods have been only modestly successful. In a case-control study, we examined 76 samples from mothers and 76 samples from their newborns; 38 with and 38 without haemolysis. The IgG subclass profile of maternal anti-A and anti-B was determined by flow cytometry. Samples from newborns were genetically analysed for the A2 subgroup, secretor and FcγRIIa receptor alleles. Surprisingly, we found a correlation between the newborn secretor allele and haemolysis (p=0.034). No correlation was found for FcγRIIa alleles. The A2 subgroup was found only in newborns without haemolysis. Unexpectedly, different reaction patterns were found for maternal anti-A and anti-B; consequently, the results were treated separately. For the prediction of haemolysis in A-newborns, the maternal IgG1 subclass determination resulted in an accuracy of 83% at birth. For B-newborns, an accuracy of 91% was achieved by the maternal IgG2 subclass determination. We improved the prediction of ABO-HDFN by characterizing maternal anti-A and anti-B by flow cytometry and we presented genetic traits in newborns with correlation to haemolysis. We propose a new understanding of A- and B-substances as immunogens that enhance the maternal immune response and protect the newborn, and we suggest that the development of ABO-HDFN is different when caused by maternal anti-A compared to maternal anti-B.

Congenital Human Cytomegalovirus Infection: A Narrative Review of Maternal Immune Response and Diagnosis in View of the Development of a Vaccine and Prevention of Primary and Non-Primary Infections in Pregnancy.

Congenital cytomegalovirus infection (cCMV) may affect about 1% of all newborns all over the world as a result of either a primary or recurrent human cytomegalovirus (HCMV) infection. While about 90% of infants affected by cCMV are asymptomatic at birth, the remaining 10% are symptomatic often with neurodevelopmental impairment and sensorineural hearing loss. In view of identifying the best approach to vaccine prevention of cCMV, this review will examine the most important steps made in the study of the immune response to, and diagnosis of, HCMV infection. The maternal immune response and immune correlates of protection are being partially identified with a partial contribution given by our laboratory. The diagnosis of primary infection is often difficult to achieve in the first three months of pregnancy, which is the time primarily involved in virus transmission to the fetus in association with the most severe symptoms and sequelae. Prevention of cCMV is anticipated by prevention of primary infection in early pregnancy by means of different measures, such as (i) behavioral-educational measures, (ii) immunoglobulin administration, (iii) antiviral treatment with valaciclovir. However, the most promising approach to cCMV prevention appears to be the development of a non-living vaccine, including at least three viral antigens: gB, pentamer complex gHgLpUL128L, and pp65, which have been shown to be able to stimulate both the humoral and the cellular arms of the maternal immune response. Primary HCMV infection may be managed in pregnancy by counseling of the couples involved by a team of specialists that includes virologists, obstetricians, infectivologists and neonatologists.

Toll-like receptor-4 null mutation causes fetal loss and fetal growth restriction associated with impaired maternal immune tolerance in mice

Maternal immune adaptation to accommodate pregnancy depends on sufficient availability of regulatory T (Treg) cells to enable embryo implantation. Toll-like receptor 4 is implicated as a key upstream driver of a controlled inflammatory response, elicited by signals in male partner seminal fluid, to initiate expansion of the maternal Treg cell pool after mating. Here, we report that mice with null mutation in Tlr4 (Tlr4−/−) exhibit impaired reproductive outcomes after allogeneic mating, with reduced pregnancy rate, elevated mid-gestation fetal loss, and fetal growth restriction, compared to Tlr4+/+ wild-type controls. To investigate the effects of TLR4 deficiency on early events of maternal immune adaptation, TLR4-regulated cytokines and immune regulatory microRNAs were measured in the uterus at 8 h post-mating by qPCR, and Treg cells in uterus-draining lymph nodes were evaluated by flow cytometry on day 3.5 post-coitum. Ptgs2 encoding prostaglandin-endoperoxide synthase 2, cytokines Csf2, Il6, Lif, and Tnf, chemokines Ccl2, Cxcl1, Cxcl2, and Cxcl10, and microRNAs miR-155, miR-146a, and miR-223 were induced by mating in wild-type mice, but not, or to a lesser extent, in Tlr4−/− mice. CD4+ T cells were expanded after mating in Tlr4+/+ but not Tlr4−/− mice, with failure to expand peripheral CD25+FOXP3+ NRP1− or thymic CD25+FOXP3+ NRP1+ Treg cell populations, and fewer Treg cells expressed Ki67 proliferation marker and suppressive function marker CTLA4. We conclude that TLR4 is an essential mediator of the inflammation-like response in the pre-implantation uterus that induces generation of Treg cells to support robust pregnancy tolerance and ensure optimal fetal growth and survival.

Association of reduced maternal sHLA-G5 isoform levels and elevated TNF-α/IL-4 cytokine ratio with Recurrent Pregnancy Loss: A study on South Indian women.

Inflammation is of critical importance in successful implantation during pregnancy. However, the establishment of maternal immune tolerance towards semi-allograft foetus is more exigent and is achieved predominantly by human leukocyte antigen-G (HLA-G) isoforms with a special emphasis on soluble HLA-G5 (sHLA-G5). Constant inflammation and lack of resolution by anti-inflammatory milieu, due to aberrant expression of critical immunoregulatory molecules such as sHLA-G5 and dysfunctional T helper cells 1 and 2 (Th1-Th2) cytokine shift, can lead to adverse pregnancy outcomes including recurrent pregnancy loss (RPL). Serum samples of 270 pregnant women (135 healthy parous and 135 with a history of RPL) were evaluated for the concentrations of sHLA-G5, interleukin-4 (IL-4) and tumour necrosis factor-alpha (TNF-α) using sandwich enzyme-linked immunosorbent assay (ELISA) and found elevated levels of sHLA-G5 and IL-4 in controls and higher TNF-α levels and TNF-α:IL-4 ratio in patients (P<.05). Stratified data analysis based on the time of sample collection, that is the first and second trimesters exhibited higher sHLA-G5 and IL-4 in both first and second trimesters in controls than patients, while they displayed lower levels concerning TNF-α and TNF-α:IL-4 ratio (P<.05). However, within patients and controls in the first or second trimesters, there was a significant variation concerning sHLA-G5 alone. Further, the outcome of pregnancies studied in the present investigation revealed a significant elevation in sHLA-G5 levels among women with successful pregnancies compared with women who experienced pregnancy loss, therefore, concluding the potential application of sHLA-G5 isoform as a marker in assisting improved pregnancy outcomes.

Influence of Vitamin D3 Levels and T Cell-Related Cytokines in Human Milk on Coronavirus Disease 2019 Infection in Lactating Women.

Background: Vitamin D deficiency was associated with an increased risk of coronavirus disease 2019 (COVID-19) infection. Vitamin D deficient mothers are more likely to have infants with vitamin D deficiency, affecting their immunity and protection against infection. This study aimed at comparing the concentrations of vitamin D3 and T cell-related cytokines in milk between mothers with confirmed COVID-19 polymerase chain reaction (PCR) test, mothers with viral infections suggestive of COVID-19, and mothers without infection. Materials and Methods: Concentrations of vitamin D3 and T cell-related cytokines in milk samples were determined by ELISA from 10 mothers who had a positive COVID-19 PCR test, 10 mothers with viral symptoms suggestive of COVID-19, and 20 mothers without infection. Results: Vitamin D3 concentration in human milk was higher in women without infection than in women with viral symptoms or COVID-19 PCR. Interleukin-2 level in milk was higher in the no-infection group than the COVID-19 PCR group but it did not differ with the viral symptoms group. Vitamin D3 did not correlate with any cytokines in human milk. Prenatal vitamin intake did not affect the vitamin D3 in human milk. The percentage of milk from mothers with <20 ng/mL of vitamin D3 was 50% in the COVID-19 PCR group, 60% in the viral symptoms group, and 5% in the no-infection group. Conclusions: Vitamin D3 level in breast milk may influence maternal immunity against COVID-19 infection. A larger study is needed to evaluate the relationship between vitamin D3 concentration in breast milk, maternal immune response, and the incidence of COVID-19 infection in lactating mothers.

P–401 Frozen-thawed embryo-transfer adjuvant therapy: one size DOES NOT fit all

Abstract Study question Does adjuvant therapy after frozen-thawed embryo-transfer (FRET) with CardioAspirin and Prednisone enhance clinical pregnancy rate (CPR) and live birth rate (LBR)? Summary answer Adjuvant therapy enhanced CPR and LBR in study-group. A significant correlation was found confronting blastocyst FRET in study-group versus controls. What is known already Embryo implantation is a rate-limiting step of FRET cycles. It’s a complex process resulting from a balance between inflammation pathways and maternal immune tolerance. Low-dose aspirin unlocks Prostaglandin-F2 synthesis by Cyclooxygenase–1, thus increasing uterine vascular permeability and attachment reaction while reducing vasoconstriction. Pregnancy results from a balance between helper and regulatory T-cells (Treg), the latter protect the embryo from maternal immune attack. Treg cells’ immunosuppressive function is pivotal in pregnancy establishment. Prednisone increases the proportion of Treg cells thus inhibiting inflammation. Many therapy schedules for implantation enhancement are currently used worldwide, although there is no consistent shared evidence. Study design, size, duration Retrospective cohort-control study including 237 subjects who underwent FRET after artificial endometrial-preparation from January 2018 to March 2020. Estrogenic stimulation was either oral or transdermic. The study-group received luteal support (vaginal Progesterone 600 mg/die) and adjuvant therapy (CardioAspirin and Prednisone 25–5 mg); the control-group received luteal support only. Pregnancy test (PT) was scheduled 10–14 days post-transfer (blastocysts or cleavage stage embryos). Second PT and ultrasound were performed 7 days later if the first was positive. Participants/materials, setting, methods Patients referred to Padua University Hospital’s Human Reproduction Pathophysiology Unit. Exclusion criteria: &amp;gt;50/&amp;lt;18 years, fresh embryo-transfer cycles, oocyte-thawing cycles, natural/natural-modified cycles. Male factor was the prevalent fertility issue. Single embryo-transfer was performed in both groups. Mean endometrial thickness was 9 mm trilaminar in both groups. Statistical analysis were carried out using JMP Pro 14 software. Categorical variables were analyzed using Chi-square test or Fisher’s exact test where appropriate. Main results and the role of chance In the study-group, 87 subjects were given luteal support and adjuvant therapy, while in the control-group, 150 subjects received luteal support only. Groups were homogeneous for age, number of embryos transferred, endometrial thickness, endometrial features (trilaminarity) and fertilization tecniques (108 IVF/ 127 ICSI). CPR and LBR were significantly higher in the study-group. CPR was 31.4% in study-group versus 14.8% in controls (p = 0.002), LBR was 27.4% in study-group versus 11.6% in controls (p = 0.002). Since heterogeneity between groups was found regarding the type of embryo transferred (55.3% cleavage-stage versus 44.7% blastocyst, p &amp;lt; 0.01), the groups were split analyzed basing upon the type if embryo transferred. In the cleavage-stage FRET condition no relevant correlation was found between groups. However in blastocyst-FRET group CPR (34.5% study-group versus 18% controls, p = 0.04) and LBR (30.9% study-group versus 12% controls, p = 0.017) were significantly higher in the study-group, thus showing that adjuvant therapy could improve CPR and LBR. Limitations, reasons for caution Limited sample size negatively impacts the study’s power. It would be appropriate to expand the sample to obtain more reliable results. Wider implications of the findings: Although no unanimous consent exists for tout-court adjuvant therapy administration, scientific literature shows that such therapy can help patients with repeated implantation failures or anti-nuclear-antibodies positivity. Assuming that a single-therapy-regimen could perfectly fit all patients is not realistic. We have to move towards patient-tailored adjuvant therapy thinking. Trial registration number Not applicable

P–408 Inadequate increase in Tim–3 on peripheral NK cells after blastocyst transfer is associated with early miscarriage

Abstract Study question Whether the changing peripheral levels of Tim–3/Galectin–9 (Gal–9) and PD–1/PL–1 over 4 weeks after ET in ongoing pregnancies is different from pregnancies destined to miscarry. Summary answer A significant and sustained increase of Tim–3 in pNK cells was observed in pregnancies which were ongoing but not in pregnancies which later miscarried. What is known already The importance of maternal immune adaptation and tolerance to the implanting embryo, an allograft, has been extensively investigated for decades. Immune checkpoint molecules, like T-cell immunoglobulin mucin–3 (Tim–3) and programed cell death–1 (PD–1), are co-stimulatory receptors negatively regulating immune responses. During pregnancy, Tim–3 and PD–1 are expressed by several immune cells in the decidua and participate in the maternal-fetal immune interactions to mediate maternal immune tolerance through binding to their ligands Gal–9 and progressed death-ligand 1 (PD-L1) produced by trophoblast and immune cells. In addition to the implantation site, Tim–3 and PD–1 expressions in peripheral lymphocytes are modified during pregnancy. Study design, size, duration A prospective observational study includes 81 women who achieved ongoing pregnancy and 17 women who suffered from miscarriage after single day–5 blastocyst transfer. All the subjects were recruited from November 2018 to January 2020 in a university teaching hospital. Participants/materials, setting, methods Women undergoing single blastocyst transfer after in-vitro-fertilization (IVF) or intracytoplasmic sperm injection (ICSI) treatment were recruited on the day of ET following informed, written consent. They had serial blood samples taken on the day of ET, and 4, 5, 6 and 7 weeks of gestation for measurement of (1) membranous Tim–3 and PD–1 expression on various peripheral lymphocytes by flow cytometry; and (2) serum concentrations of ligands Gal–9 and PD-L1 by ELISA. Main results and the role of chance The comparisons between two groups showed there was no significant difference between the 2 groups in baseline levels among all the parameters measured. In women who achieved ongoing pregnancy, a significant and sustained increase of Tim–3 in either peripheral NK (pNK) subsets was observed at 4-week, 5-week, 6-week and 7-week gestations compared to the baseline (Tim–3+CD56dimNK 39.14±1.51%, 41.14±1.62%, 41.34±1.94%, and 41.69±2.12% vs. 30.27±1.49%; Tim–3+CD56brihgtNK cells, 24.54±1.71%, 25.43±1.54%, 27.26±1.88% and 24.70±1.64% vs. 19.08±1.13%), and the concentration of serum PD-L1 was significantly increased at 6-week and 7-week gestations (48.33±17.78 pg/ml, 52.53±20.60 pg/ml) when compared to the day of blastocyst transfer (41.40±16.01 pg/ml). The expressions of Tim–3 in T, NKT cells and PD–1 in NK, T, NKT cells were not significantly changed across the 5 time points. In women who conceived but later miscarried, all the parameters examined from 4–7 weeks of gestation were not significantly different when compared with the baseline measurement. The only measurement which showed a significant difference between the 2 groups and across all time points after ET was the proportion of Tim–3+CD56dimNK cells which was significantly higher in women who achieved ongoing pregnancies compared with women who destined to miscarry from 4 to 7 weeks of gestation. Limitations, reasons for caution It is uncertain if the observation would be different between miscarriage associated with aneuploid embryo or euploid embryo as we had not been able to obtain karyotyping result in most of the miscarriage cases. Wider implications of the findings: Our preliminary observation suggests that the proportion of Tim–3+pNK cells as early as 4-week gestation could be a potential immuno-bio-marker to predict if a pregnancy is likely to progress normally or result in a miscarriage. Clearly, the finding in this study needs to be confirmed in a larger cohort study Trial registration number not applicable.

O-129 Lactobacillus deplete vaginal microbial composition is associated with chromosomally normal miscarriage and local inflammation

Abstract Study question To investigate the vaginal microbial composition and the local immune response in chromosomally normal and abnormal miscarriages and compare this to uncomplicated pregnancies delivering at term. Summary answer We show that euploid miscarriage is associated with a significantly higher prevalence of Lactobacillus spp. deplete vaginal microbial communities compared to aneuploid miscarriage. What is known already Emerging evidence supports the role of the vaginal microbiota in adverse pregnancy outcome, but the underlying mechanisms are poorly understood. A dominance of Lactobacillus spp. in pregnancy provides protection against pathogenic bacteria by producing lactic acid and antimicrobial compounds. A depletion in Lactobacillus spp. is often linked to adverse pregnancy outcomes.Current work also implicates the reproductive tract microbiota as a key modulator of local inflammatory and immune pathways. We have previously shown that miscarriage is associated with vaginal dysbiosis but without knowledge of the cytogenetic status of those miscarriages or the local immune profile. Study design, size, duration This study was a prospective observational cohort study based at Queen Charlotte’s &amp; Chelsea Hospital, Early Pregnancy Unit, London between March 2014-February 2019. Vaginal swabs were collected from the posterior vaginal fornix of 167 patients. Participants/materials, setting, methods We used 16S rRNA gene based metataxonomics to interrogate the vaginal microbiota in a cohort of 167 women, 93 miscarriage patients (54 euploid and 39 aneuploid using molecular cytogenetics) and 74 women who delivered at term and correlate this with the aneuploidy status of the miscarriages. We also measured the concentrations of IL-2, IL-4, IL-6, IL-8, TNF-α, IFN-γ, IL-1β, IL-18 and IL-10 in cervical vaginal fluid using Human Magnetic Luminex Screening Assay (8-plex). Main results and the role of chance We show that euploid miscarriage is associated with a significantly higher prevalence of Lactobacillus spp. deplete vaginal microbial communities compared to aneuploid miscarriage (P=0.008). In women having Lactobacillus spp. deplete vaginal microbial communities, euploid miscarriage associates with higher concentrations of pro-inflammatory cytokines IL-1β, IL-8, IL-6 (P&amp;lt;0.001, P=0.01 and P&amp;lt;0.001 respectively) and lower concentrations of anti-inflammatory cytokines IL10 (P&amp;lt;0.001) when compared to viable term pregnancy. We identified Prevotella bivia and Streptococcus as particularly common in euploid miscarriage and as drivers of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α). Co-occurrence network analyses revealed low levels of co-occurrence between Lactobacillus crispatus and other organisms and strong co-occurrence between Streptococcal species. Our data show a combination of both an adverse vaginal microbiota and a cytokine response to it influences early pregnancy outcome. Although this may be a reflection of intrinsic maternal immune response, it appears that the cytokine response is largely driven by the bacterial taxa present in the vagina, which presents an opportunity for specific, directed intervention. The negative co-occurrence between L.crispatus and all other organisms suggests a possible therapeutic role for probiotics containing this organism. The influence of Streptococci also suggests a potential benefit of targeted antibiotics with probiotics for some patients. Limitations, reasons for caution There were no longitudinal samples in this cohort and our results are based on the assumption that the vaginal microbial composition is stable throughout the first trimester.Future longitudinal studies with larger sample sizes are needed to corroborate these findings and provide insights to the mechanisms that trigger the inflammatory response. Wider implications of the findings These findings support the hypothesis that the vaginal microbiota plays an important aetiological role in euploid miscarriage and may represent a target to modify the risk of pregnancy loss. Trial registration number n/a

P–390 The intrauterus administration of peripheral blood mononuclear cells increase the pregnancy rates for the patients with advanced maternal age after single euploid embryo transfer

Abstract Study question Does the intrauterine administration of peripheral blood mononuclear cells (PBMCs) effect the outcome of IVF for patients with advanced maternal age when the euploid embryos after PGT-A are transferred? Summary answer The implantation rates were significantly higher after the intrauterine application of PBMCs in patients with advanced maternal age (AMA) before transfer of the euploid embryo. What is known already The aneuploidy rates of blastocysts in IVF is in range 45–70% depending on different factors. Besides that, the endometrium plays an important role in achieving optimal outcomes of assisted reproductive technologies. It has been proposed that intrauterine administration of peripheral blood mononuclear cells modulates maternal immune response to favor implantation. Study design, size, duration The effect of the intrauterine application of PBMCs to improve the implantation rates in the group of patients with advanced maternal age was studied. Two group of patients (PBMCs-group and non-PBMCs-group_ were formed. Single euploid embryo was transferred for each patient. Participants/materials, setting, methods: The ploidy status of 373 blastocysts from 82 AMA-patients was analyzed by the method of next generation sequencing (NGS). PBMCs were applied for 39 women with the mean age 39.2±3.2 y.o. before embryo transfer (Group 1). For 43 patients with the mean age 38.2±2.1 y.o. single euploid embryo transfers were performed without PBMCs administration (Group 2). Chi-squared test was used for data analysis. The study’s protocol was approved by the Center’s IRB. Main results and the role of chance Totally the rate of euploid embryos was 27.1% (101 blastocysts). In the mentioned study 55.0% of examined blastocysts were aneuploid (205 embryos) and 17.9% of blastocysts were detected as mosaic (67 embryos). Single euploid embryo was transferred in each case in the patients of both experimental groups. The implantation rate was significantly higher in Group 1 with PBMCs application comparing with non-PBMCs experimental Group 2 (38.5% (15 pregnancies) vs. 23.3% (10 pregnancies), df = 1, χ2 = 5.487, χ2critical = 3.841, P = 0.020). Limitations, reasons for caution The embryo biopsy was performed only for blastocyst with top-quality morphology. Wider implications of the findings: The implantation rates were significantly higher when the intrauterine application of PBMCs in patients with advanced maternal age before the transfer of the euploid embryo (P = 0.020). The randomized studies to improve our knowledge in immunogenic therapy in reproductive medicine should be performed. Trial registration number -

P–414 HLA-G expression as a new prognostic marker of successful implantation

Abstract Study question Is the low HLA-G expression a determinant of early reproductive loss? Summary answer Low expression of HLA-G is associated with pregnancy complications and can be one of the reasons of spontaneous abortion (such as RPL). What is known already The dysregulated maternal immune responses to invading embryos may play role in RIF, RPL, and second- and third-trimester obstetrical conditions. HLA-G is a molecule that was first known to confer protection to the fetus from destruction by the immune system of its mother, thus critically contributing to fetal–maternal tolerance due to inducing displacement of pro-inflammatory to Th1 cell-mediated response of Th2, has a positive influence on the process of implantation. HLA-G is mainly restricted to the fetal–maternal interface on the extravillous cytotrophoblast, to placenta, amnion. Study design, size, duration It was a prospective complex cohort study from 2016–2020 years with pathomorphological investigations. The purpose of this study is to investigate the HLA-G and KIR2DL4 expression in chorionic villous among 3 groups (study included 118 cases of abortion material): group 1 – 36 cases after missed abortion with normal karyotype, group 2 – 36 cases after missed abortion with polyploidy and group 3 – 46 cases after induced abortion group (normal pregnancy). Participants/materials, setting, methods Criteria of inclusion: abortive material from 3 groups of women with missed or after induced abortion; 6–12 weeks, singleton pregnancy, cytogenetic of chorionic villous was obligatorily - normal fetal karyotype and polyploidy of fetus. Pathomorophological investigation included H&amp;E stain, IHC and confocal laser scanning microscopy. Immunohistochemical examination included quantitative and qualitative assessment of the expression of Anti-HLA-G (mouse monoclonal) in an extra villous trophoblast and Anti-KIR2DL1+KIR2DL3 + KIR2DL4 + KIR2DS4 (rabbit polyclonal) in chorionic villi. Main results and the role of chance The immunohistochemical study showed homogenous distribution HLA-G expression in extravillous trophoblast cells (EVT) and KIR2DL4 expression in chorion villous both in missed abortion groups and in induced abortion group. HLA-G expression average relative area in 1 and 2 groups was not statistically different (in 1 group with normal karyotype 33,9±3,5 and in 2 group 38,6±2,8). But the expression of HLA-G in 3 group was strictly higher (55,6 ±2,4). The average relative area of KIR2DL4 receptor wasn’t statistically different among 3 groups. However, the histological picture both missed abortion groups (for the genetic\immunological reasons for rejection) is the only one - this is a missed abortion of an early terms of gestation. In a histological study of missed abortion, as our study shows, the histological picture is similar in 1 and 2 groups. Thus, in 1 group with a normal karyotype of the fetus (before conducting the chorion cytogenetic study in the genetics laboratory) in 59.2% the histological examination determined a picture of an impaired early pregnancy with signs of trophoblast chromosomal pathology. Thus, without a cytogenetic study of the chorion, it is impossible to clearly determine whether the chromosomal pathology of the fetus is the cause of missed abortion. Limitations, reasons for caution There is no limitations, reasons for caution. Wider implications of the findings: Thus, HLA-G molecule has a leading role in the onset and successful prolongation of pregnancy, implantation, placentation and fetal development. Trial registration number 98–2019

Key Gene and Functional Pathways Identified in Unexplained Recurrent Spontaneous Abortion Using Targeted RNA Sequencing and Clinical Analysis.

Identifying the mechanisms underlying unexplained recurrent spontaneous abortion (URSA) can help develop effective treatments. This study provides novel insights into the biological characteristics and related pathways of differentially expressed genes (DEGs) in URSA. Nineteen patients with URSA and three healthy fertile women with regular menstruation (control group) were recruited. RNA was extracted from the two groups to determine the differential expression of immunoregulatory gene sequences. Gene ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment analyses were used to identify the biological functions and pathways of the identified DEGs. A protein-protein interaction (PPI) network was constructed using the STRING database. Furthermore, qRT-PCR and ELISA were performed to validate the differential expression of the hub genes. We also explored the regulatory mechanism of Th1/Th2 imbalance. A total of 99 DEGs were identified, comprising 94 upregulated and five downregulated genes. Through GO analysis, nine immune cell function-related clusters were selected, and genes with significant differential expression were primarily enriched in eight immune regulatory functions related to the KEGG signalling pathway. Subsequently, five hub genes (TLR2, CXCL8, IFNG, IL2RA, and ITGAX) were identified using Cytoscape software; qRT-PCR confirmed the differential expression among the hub genes, whereas ELISA revealed a significant difference in extracellular IFN-γ and IL-8 levels. The levels of Th1 (IFN-γ) and the Th1/Th2 ratio were higher in the peripheral blood of URSA patients than in control group patients. These findings suggest that the occurrence of URSA may be associated with the abnormal expression of some specific immunoregulatory genes involved in T-cell activation and differentiation. Among the identified DEGs, IFNG may play a key role in regulating maternal immune response. Although further validation is required, our data provide an important theoretical basis for elucidating the pathogenesis of recurrent spontaneous abortion.

Genes and Pathways Affecting Sheep Productivity Traits: Genetic Parameters, Genome-Wide Association Mapping, and Pathway Enrichment Analysis

Ewe productivity is a composite and maternal trait that is considered the most important economic trait in sheep meat production. The objective of this study was the application of alternative genome-wide association study (GWAS) approaches followed by gene set enrichment analysis (GSEA) on the ewes’ genome to identify genes affecting pregnancy outcomes and lamb growth after parturition in Iranian Baluchi sheep. Three maternal composite traits at birth and weaning were considered. The traits were progeny birth weight, litter mean weight at birth, total litter weight at birth, progeny weaning weight, litter mean weight at weaning, and total litter weight at weaning. GWASs were performed on original phenotypes as well as on estimated breeding values. The significant SNPs associated with composite traits at birth were located within or near genes RDX, FDX1, ARHGAP20, ZC3H12C, THBS1, and EPG5. Identified genes and pathways have functions related to pregnancy, such as autophagy in the placenta, progesterone production by the placenta, placental formation, calcium ion transport, and maternal immune response. For composite traits at weaning, genes (NR2C1, VEZT, HSD17B4, RSU1, CUBN, VIM, PRLR, and FTH1) and pathways affecting feed intake and food conservation, development of mammary glands cytoskeleton structure, and production of milk components like fatty acids, proteins, and vitamin B-12, were identified. The results show that calcium ion transport during pregnancy and feeding lambs by milk after parturition can have the greatest impact on weight gain as compared to other effects of maternal origin.

Reduced Placental CD24 in Preterm Preeclampsia Is an Indicator for a Failure of Immune Tolerance.

Introduction: CD24 is a mucin-like glycoprotein expressed at the surface of hematopoietic and tumor cells and was recently shown to be expressed in the first trimester placenta. As it was postulated as an immune suppressor, CD24 may contribute to maternal immune tolerance to the growing fetus. Preeclampsia (PE), a major pregnancy complication, is linked to reduced immune tolerance. Here, we explored the expression of CD24 in PE placenta in preterm and term cases. Methods: Placentas were derived from first and early second trimester social terminations (N = 43), and third trimester normal term delivery (N = 67), preterm PE (N = 18), and preterm delivery (PTD) (N = 6). CD24 expression was determined by quantitative polymerase chain reaction (qPCR) and Western blotting. A smaller cohort included 3–5 subjects each of term and early PE, and term and preterm delivery controls analyzed by immunohistochemistry. Results: A higher expression (2.27-fold) of CD24 mRNA was determined in the normal term delivery compared to first and early second trimester cases. The mRNA of preterm PE cases was only higher by 1.31-fold compared to first and early second trimester, while in the age-matched PTD group had a fold increase of 5.72, four times higher compared to preterm PE. The delta cycle threshold (ΔCt) of CD24 mRNA expression in the preterm PE group was inversely correlated with gestational age (r = 0.737) and fetal size (r = 0.623), while correlation of any other group with these parameters was negligible. Western blot analysis revealed that the presence of CD24 protein in placental lysate of preterm PE was significantly reduced compared to term delivery controls (p = 0.026). In immunohistochemistry, there was a reduction of CD24 staining in villous trophoblast in preterm PE cases compared to gestational age-matched PTD cases (p = 0.042). Staining of PE cases at term was approximately twice higher compared to preterm PE cases (p = 0.025) but not different from normal term delivery controls. Conclusion: While higher CD24 mRNA expression levels were determined for normal term delivery compared to earlier pregnancy stages, this expression level was found to be lower in preterm PE cases, and could be said to be linked to reduced immune tolerance in preeclampsia.

Interleukin-6-634 G/C gene polymorphisms in recurrent pregnancy loss among Egyptian women: Does it make a difference?

The balance between maternal immune responses and tolerance is considered as especially critical issue in the dilemma of recurrent pregnancy loss (RPL). Interleukin 6 (IL6) plays a fundamental role in fetal implantation and maintenance of pregnancy. This study aimed to explore the association between IL6–634 G/C gene polymorphisms and their serum levels in females with RPL. An observational case-control study involved 102 controls and 102 cases with RPL. Genotyping of IL6 polymorphism −634 G/C was done with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and their serum levels were measured. There was no statistically significant difference between controls and RPL cases as regard serum IL6 level. The percentage of distribution for allele G was 88.7%, and 78% in controls and cases respectively (p = 0.005). The risk of RPL was decreased by 60% in carriers of allele G (OR = 0.4, 95%CI: 0.2–0.8, p = 0.003). Besides, the homozygote genotype GG (OR = 0.5, 95%CI: 0.28–0.95, p = 0.031), was linked with decreased risk for RPL. The homozygous GG genotype and G allele were associated with decreased risk of RPL in Egyptian females.

Spermatozoa Induce Maternal Mononuclear Cells for Production of Antibody with Cytotoxic Activity on Paternal Blood Mononuclear Cells.

ObjectiveThe maternal immune response to paternal antigens is induced at insemination. We believe that pregnancy protective alloantibodies, such as anti-paternal cytotoxic antibody (APCA), may be produced against the paternal antigens in the context of stimulated immunity at insemination and that they increase during pregnancy. APCA is necessary for pregnancy. It is directed towards paternal human leucocyte antigens (HLAs) and has cytotoxic activity against paternal leucocytes. The present study aims to determine whether APCA is produced by the maternal peripheral blood mononuclear cells (PBMCs) in contact with the husband’s spermatozoa and to evaluate the relation of APCA production with HLA class I and II expressions by spermatozoa in fertile couples.Materials and Methods This cross-sectional study included 30 fertile couples with at least one child. The maternal PBMCs were co-cultured with the husband’s spermatozoa and the supernatant was assessed for the presence of IgG by ELISA. Cytotoxic activity of the supernatant on the husband’s PBMCs was assessed by the complement-dependent cytotoxicity (CDC) assay. Results IgG was produced in all co-cultures, and the mean level of supernatant IgG was 669 ng/ml. The cytotoxic activity of the supernatant was observed in all the supernatant obtained from the co-cultures. The mean percentage of APCA in supernatant was 73.93%. ConclusionBased on the results of this study it can be concluded that APCA may be a natural anti-sperm antibody (ASA), which can be produced during exposure to spermatozoa and may have some influence before pregnancy. Further research is required to determine the role of APCA before pregnancy.

Identification of distinct seminal plasma cytokine profiles associated with male age and lifestyle characteristics in unexplained recurrent pregnancy loss

BackgroundSeminal plasma contains a wide range of cytokines, chemokines and growth factors. Part of these signalling molecules assist in inducing a state of active maternal immune tolerance towards the fetus. Disbalances in seminal plasma content may contribute to pregnancy loss. This study investigated cytokine expression profiles in seminal plasma of male partners of couples with unexplained recurrent pregnancy loss (RPL) and the association with clinical and lifestyle characteristics, including smoking, alcohol consumption and body mass index (BMI). MethodsIn the seminal plasma of 52 men who visited a specialised RPL clinic the levels of 25 pre-selected cytokines, chemokines and growth factors were measured by Bio-Plex assay or ELISA. Two-way hierarchical cluster analysis was performed. Identified patient clusters were compared on clinical and lifestyle characteristics. ResultsTwo distinct cytokine expression profiles in the seminal plasma were revealed by cluster analysis. Patient cluster I showed relatively higher levels of pro-inflammatory cytokines, including IL-1α, IL-1β, IL-6, IL-8, IL-12, IL-18 and TNF-α, compared to Patient cluster II. Men belonging to Patient cluster I were significantly older and had significantly more lifestyle risk factors compared to men in Patient cluster II. ConclusionCluster analysis suggested the existence of a less favourable pro-inflammatory cytokine expression profile, being present in part of men affected by RPL and associated with advanced male age and lifestyle risk factors. These findings may serve as a starting point for further research into underlying mechanisms and ultimately lead to novel diagnostic and therapeutic approaches for couples with RPL.

Glycoproteins Presenting Galactose and N-Acetylgalactosamine in Human Seminal Plasma as Potential Players Involved in Immune Modulation in the Fertilization Process.

In light of recent research, there is increasing evidence showing that extracellular semen components have a significant impact on the immune reaction of the female partner, leading to the tolerogenic response enabling the embryo development and implantation as well as further progress of healthy pregnancy. Seminal plasma glycoproteins are rich in the unique immunomodulatory glycoepitopes that may serve as ligands for endogenous lectins that decorate the surface of immune cells. Such interaction may be involved in modulation of the maternal immune response. Among immunomodulatory glycans, Lewis type antigens have been of interest for at least two decades, while the importance of T/Tn antigens and related structures is still far from understanding. In the current work, we applied two plant lectins capable of distinguishing glycoepitopes with terminal GalNAc and Gal to identify glycoproteins that are their efficient carriers. By means of lectin blotting and lectin affinity chromatography followed by LC-MS, we identified lactotransferrin, prolactin inducible protein as well as fibronectin and semenogelins 1 and 2 as lectin-reactive. Net-O-glycosylation analysis results indicated that the latter three may actually carry T and/or Tn antigens, while in the case of prolactin inducible protein and lactotransferrin LacdiNAc and lactosamine glycoepitopes were more probable. STRING bioinformatics analysis linked the identified glycoproteins in the close network, indicating their involvement in immune (partially innate) processes. Overall, our research revealed potential seminal plasma ligands for endogenous Gal/GalNAc specific lectins with a possible role in modulation of maternal immune response during fertilization.

Maternal Antibody Response, Neutralizing Potency, and Placental Antibody Transfer After Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection.

To characterize maternal immune response after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection during pregnancy and quantify the efficiency of transplacental antibody transfer. We conducted a prospective cohort study of pregnant patients who tested positive for SARS CoV-2 infection at any point in pregnancy and collected paired maternal and cord blood samples at the time of delivery. An enzyme-linked immunosorbent assay (ELISA) and neutralization assays were performed to measure maternal plasma and cord blood concentrations and neutralizing potency of immunoglobulin (Ig)G, IgA, and IgM antibodies directed against the SARS-CoV-2 spike protein. Differences in concentrations according to symptomatic compared with asymptomatic infection and time from positive polymerase chain reaction (PCR) test result to delivery were analyzed using nonparametric tests of significance. The ratio of cord to maternal anti-receptor-binding domain IgG titers was analyzed to assess transplacental transfer efficiency. Thirty-two paired samples were analyzed. Detectable anti-receptor-binding domain IgG was detected in 100% (n=32) of maternal and 91% (n=29) of cord blood samples. Functional neutralizing antibody was present in 94% (n=30) of the maternal and 25% (n=8) of cord blood samples. Symptomatic infection was associated with a significant difference in median (interquartile range) maternal anti-receptor-binding domain IgG titers compared with asymptomatic infection (log 3.2 [3.5-2.4] vs log 2.7 [2.9-1.4], P=.03). Median (interquartile range) maternal anti-receptor-binding domain IgG titers were not significantly higher in patients who delivered more than 14 days after a positive PCR test result compared with those who delivered within 14 days (log 3.3 [3.5-2.4] vs log 2.67 [2.8-1.6], P=.05). Median (range) cord/maternal antibody ratio was 0.81 (0.67-0.88). These results demonstrate robust maternal neutralizing and anti-receptor-binding domain IgG response after SARS-CoV-2 infection, yet a lower-than-expected efficiency of transplacental antibody transfer and a significant reduction in neutralization between maternal blood and cord blood. Maternal infection does confer some degree of neonatal antibody protection, but the robustness and durability of protection require further study.

Antibody Response to Coronavirus Disease 2019 (COVID-19) Messenger RNA Vaccination in Pregnant Women and Transplacental Passage Into Cord Blood.

In Brief Coronavirus disease 2019 (COVID-19) vaccination in pregnancy induces a robust maternal immune response, with transplacental antibody transfer detectable in cord blood as early as 16 days after the first dose.