The metabolic landscape of the maternal-fetal interface in missed miscarriage: a cross-sectional pilot multiomics study.

Several reports have revealed that human prolactin increases during pregnancy, not only in maternal and fetal serum but also in the amniotic fluid. The source and the role of prolactin in the amniotic fluid however, have not been clear up to now. In their incubation experiment, Riddick et al (29) proved that decidual tissue at term could secret immunoreactive prolactin (IR-PRL). In order to investigate the source of PRL in the amniotic fluid, we extracted IR-PRL from decidual or villous tissue in early normal pregnancies and measured it by a double antibody radioimmunoassay in this experiment. The results were the following: 1) We were able to measure IR-PRL from decidual or villous tissue in early pregnancy, the dilution curve of which paralleled pituitary standard prolactin. The cross reaction of a PRL standard preparation with HCG and HPL was not recognized within 10 micrograms/ml. 2) In human decidual tissue, the IR-PRL concentration began to increase at about the sixth week, arrived at the peak value, 81.06 +/- 2.13 ng/0.1 g dry weight (d.w.) in the eighth week, and did not change significantly after that. In human villous tissue, although the IR-PRL concentration was distinctly lower than it was in the decidual tissue, it increased gradually and reached the level of 37.44 +/- 7.16 ng/0.1g d.w. in the tenth week. 3) The extracted material (IR-PRL) from these two tissues in the 8th week, amniotic fluid at term, and pituitary standard PRL were passed through a Sephadex G-100 column (2 X 93 cm) with phosphate buffer and saline (0.01M, PH 7.4). In the chromatogram of these four test materials, one peak of IR-PRL was observed. The peak of IR-PRL of decidual and villous tissues and amniotic fluid revealed almost the same fraction number but elevated after the pituitary PRL preparation. 4) By gel filtration, the IR-PRL was able to be differentiated from HCG but not from HPL. The peak of HCG appeared earlier than the peak of IR-PRL and HPL. 5) The distribution of IR-PRL between human decidual and villous tissues was significantly different from that of HPL. IR-PRL concentration was higher in the decidual tissue than in the villous tissue, while HPL concentration was higher in the villous tissue than it was in the decidual tissue. From this observation, we could consider that the sources of these two hormones were different. The results of these experiments suggest that one of the sources of PRL in the amniotic fluid was the decidual tissue.

The transforming growth factor (TGF)-beta s are multifunctional cytokines, and they play a role in the controlled growth of trophoblasts. Moreover they are thought to be important in maternal-fetal interaction during early gestation. Human decidual and villous tissues in the first trimester were Northern blotted and amplified by reverse transcription-polymerase chain reaction to measure the expression of TGF-beta 1, -beta 2, and -beta 3 and their receptors, types I and II, at the first trimester of pregnancy. In addition, their cell-specific expression at the maternal-fetal interface was determined by in situ hybridization. Each isoform of TGF-beta was expressed in both decidual and villous tissues. Because most TGF-beta 1 gene expression was found in villous tissues, TGF-beta 2 mRNA was expressed preferentially in the decidual tissues. TGF-beta 3 transcripts were expressed in the nonpregnant endometrium. The results suggest that each isoform of TGF-beta plays some specific role in decidualization and placentation. Furthermore, it is predicted that they regulate the maternal-fetal interaction at early gestation.

Spontaneous abortion (SA) is a common adverse outcome of early pregnancy, yet its underlying pathophysiological mechanisms remain incompletely understood. Accumulating evidence suggests that dysregulated inflammatory responses at the maternal-fetal interface play a critical role in pregnancy loss. However, the potential associations between alterations in gut microbiota, metabolic disturbances, and localized decidual inflammation in patients with SA have not been systematically characterized. Women with SA (n = 30) and those with normal early pregnancy (NP, n = 28) were enrolled in this study. Proinflammatory cytokines were quantified in decidual tissue homogenates, and histopathological and molecular analyses were performed to evaluate inflammatory activation at the maternal-fetal interface. The gut microbiota composition was profiled using shotgun metagenomic sequencing, while metabolic alterations in the feces were assessed by untargeted metabolomics. Integrated multi-omics analyses were conducted to explore associations among gut microbial dysbiosis, metabolic perturbations, decidual inflammatory signaling, and molecular alterations. Compared with those from the NP group, the decidual tissues from the SA group exhibited significantly elevated levels of IL-1β and TNF-α (1.49-fold and1.51-fold, bothP < 0.0001), accompanied by pronounced histopathological abnormalities. Enhanced activation of the NF-κB signaling pathway was observed at the maternal-fetal interface in SA patients. Metagenomic analyses revealed distinct differences in the gut microbiota composition and community structure between the two groups, with differentially abundant bacterial taxa identified (LDA score > 2.0). Consistent with these findings, fecal metabolomic profiling clearly revealed differences between SA and NP patients, with differentially abundant metabolites (VIP > 1.0, adjusted P < 0.05) predominantly enriched in lipid metabolism, amino acid metabolism, and immune-related pathways. In addition, the expression of leucine-rich repeat-containing G protein-coupled receptor 6 was significantly upregulated (P < 0.0001) in the decidual tissue of SA patients. These findings indicate that SA is associated with localized inflammatory activation at the maternal-fetal interface, dysregulation of decidual molecular activity, gut microbiota dysbiosis, and metabolic perturbations. Integrated multi-omics analyses suggest potential interactions among these factors that may be linked to decidual dysfunction during early pregnancy, providing new insights into the complex pathophysiology of SA.

BackgroundEffects of vitamin D deficiency in pregnancy have been associated with some adverse pregnancy outcomes. The 25-hydroxyvitamin D3-1α-hydroxylase (CYP27B1) is integral to the vitamin D metabolic pathway. The enzyme catalyzes localized conversion of pro-hormone 25-hydroxyvitamin D3 to active 1,25-dihydroxyvitamin D3. Our aim was to investigate the expression of CYP27B1 at the fetal-maternal interface in the first trimester pregnancy and to determine whether CYP27B1 was associated with recurrent miscarriage (RM).MethodsExpressions of CYP27B1 mRNA and protein in villi and decidua from 20 women undergoing primary miscarriage, 20 women with RM and 20 women with normal pregnancy were evaluated by western blot, and quantitative real-time PCR. The co-localization of CYP27B1 and certain cytokines including IL-10, IFN-γ, TNF-α, and IL-2 expression were examined using immunohistochemistry and confocal microscopy.ResultsWomen with RM had a significantly lower expression of CYP27B1 mRNA and protein in villous and decidual tissues compared with the normal pregnant women (P = 0.000 in villus, P = 0.002 in decidua for mRNA; P = 0.036 in villus, P = 0.007 in decidua for protein.). Compared with the normal pregnancy, immunostaining for CYP27B1 was significantly decreased in villous trophoblasts and decidual glandular epithelial cells in RM women. No significant differences in the localization of CYP27B1, IL-10, IFN-γ, TNF-α, and IL-2 expression were identified between the normal pregnant and RM women.ConclusionsWomen with RM have a lower level of CYP27B1 expression in chorionic villi and decidua compared with normal pregnant women, suggesting that reduced CYP27B1 expression may be associated with RM. The consistent localization of CYP27B1 and IL-10, IFN-γ, TNF-α, and IL-2 expression in villous and decidual tissues suggests the importance of the local production of 1,25(OH)2D3 at the fetal-maternal interface to regulate cytokine responses.

The role of maternal–foetal interface inflammation mediated by NLRP3 inflammasome in the pathogenesis of recurrent spontaneous abortion

The disruption of the inflammatory microenvironment in the uterus affects pregnancy outcome. However, the exact quantification and distribution of leukocyte subpopulations in the uterus in preeclampsia (PE) have not been clearly characterized. Inflammasomes promote the release of proinflammatory cytokines interleukin (IL)-β and IL-18. A higher expression of NLRP3 inflammasome in placentas contributes to excessive inflammation in PE. However, related studies on the uterus are scarce. We aimed to investigate changes in the infiltration of leukocyte subpopulations in decidual and uterine tissues, and explore the role of activation of uterine NLRP3 inflammasomes in PE. Decidual tissues were collected from normotensive pregnant women and preeclamptic women. A PE-like model was established via administration of lipopolysaccharide to normal pregnant rats. Uterine and decidual tissues were collected from all experimental groups. It was found that the number of leukocytes was significantly elevated in decidual and uterine tissues in PE patients compared to normal controls. The leukocytes (predominantly macrophages and NK cells) particularly infiltrated into the decidua and uterine decidua in PE-like rats, and these were sparse in the myometrium. The NLRP3 immunoreactivity in the uterus was extremely little in control rats, its immunoreactivity and caspase-1 immunoreactivity were significantly elevated in the PE-like rats; the mRNA expression results also indicated an upward trend in the activation of NLRP3 inflammasomes. These results support that leucocyte infiltration in the decidua and uterine deciduas, and the activation of NLRP3 inflammasome in the uterus, which participate in the pathogenesis, are responsible for the excessive inflammation at the maternal-fetal interface during PE.

Intrauterine environment and fetal allergic sensitization.

Missed abortions are common complications that occur in early pregnancy, and impaired trophoblast functions have been indicated to be associated with their pathogenesis. The long noncoding RNAs (lncRNAs) Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1), HOX Transcript Antisense RNA (HOTAIR) and Maternally expressed gene 3 (MEG3) have been demonstrated to serve a crucial regulatory role in the mobility of trophoblast cells and embryo implantation. However, the expression profile and role of each of these three lncRNAs in patients with a missed abortion remain unclear. The expression of MALAT1, HOTAIR, and MEG3 in decidual and villous tissues from 26 patient exhibiting a missed abortion and 26 healthy controls was detected using reverse-transcription quantitative PCR. Serum TNF-α, IL-1β, IL-6 and IL-10 levels were measured using ELISA, and serum estradiol and progesterone levels were measured with electrochemiluminescence immunoassays. Additionally, the correlations between lncRNA expression and the levels of cytokines and hormones were further analyzed. MALAT1, HOTAIR and MEG3 expression was significantly higher in villous tissues of patients exhibiting a missed abortion compared with healthy controls. MALAT1 expression was higher in decidual tissues of patients exhibiting a missed abortion compared with healthy controls. Serum IL-10 levels were significantly lower in patients exhibiting a missed abortion compared with healthy controls. Serum estradiol and progesterone levels were significantly lower in the group of patients exhibiting a missed abortion compared with the control group. Furthermore, MALAT1 expression in villous tissue was inversely related to serum progesterone levels. The results of the current study suggest that MALAT1 may be associated with the pathogenesis of missed abortions.

Decidual endothelium, Notch1 and TGF β , gatekeepers for Treg accumulation at the maternal–fetal interface

The endemic nature of SARS-CoV-2 underscores the imperative to elucidate its effects on pregnancy, particularly the potential for vertical transmission and its influence on the early maternal–fetal interface. Here, we performed an extensive cohort study involving 761 pregnant women who voluntarily terminated their pregnancies during the first trimester. Analyses conducted using RT‒qPCR, fluorescence in situ hybridisation, and indirect immunofluorescence showed low detection levels of SARS-CoV-2 infection within villous and decidual tissues. Single-cell RNA sequencing analysis revealed an absence of cell populations demonstrating significant coexpression of ACE2 and TMPRSS2, potentially providing a mechanistic explanation for the rare incidence of viral infection within placental tissues. The maternal systemic inflammatory response was activated, and the levels of IL-31, IL-5, and GRO-α were significantly elevated during acute infection. Furthermore, increased IgG titres were negatively correlated with TNF-β concentrations, suggesting a protective immunomodulatory function of IgG antibodies. Conversely, both bulk and single-cell transcriptomic analyses revealed pronounced, cell type-specific antiviral and immune responses within the placental microenvironment. A pervasive interferon-stimulated gene signature was identified, accompanied by the emergence of M2-like macrophages with diminished antigen presentation capacity during convalescence. Importantly, maternal SARS-CoV-2 infection disrupted intercellular communication networks, notably impairing the activity of the WNT and TGF-β signalling pathways in trophoblasts, thereby altering their differentiation trajectories. In summary, within this large cohort of first-trimester samples, we identified infrequent instances of SARS-CoV-2 infection in both villous and decidual tissues. Nonetheless, infection substantially perturbed the placental immune milieu and trophoblast dynamics, which may adversely affect pregnancy outcomes.

Differential expression and the anti-apoptotic effect of human placental neurotrophins and their receptors

191. Lower memory T cell proportions in decidual tissue from pre-eclamptic pregnancies compared to healthy controls

Accumulating evidence suggests the association between abnormal angiogenesis at the maternal-fetal interface and recurrent pregnancy loss (RPL); nonetheless, the mechanism remains largely unknown. Previous studies have reported the clinical effect of the traditional Chinese medicine Danshen in the treatment of RPL. This study aimed to investigate whether salvianolic acid B (SalB), the primary water-soluble component of Danshen, could reduce the embryonic absorption rate (EAR) by increasing placental and decidual angiogenesis in RPL mice and to explore the possible mechanism. The decidual and chorionic tissues were collected from normal pregnancies and unknown recurrent pregnancy loss (URPL) patients. Western blotting was used to determine the expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) in the tissues. Different doses of SalB and/or the VEGF inhibitor PTC299 were intragastrically administered to normal and RPL pregnant mice daily at 0.5 day of pregnancy for 10 days. The EAR, mean placental weight (MPW), and micro vessel density (MVD) were determined in placental and decidual tissues, and the number of live pups per litter was counted. The expression of VEGF and HIF-1α in the tissues was evaluated using western blotting and immunohistochemistry. The VEGF protein levels in decidual and chorionic tissues were significantly lower, and HIF-1α levels were significantly higher than that of normal pregnancies. The EAR was significantly higher, MVD, MPW, and protein levels of VEGF in the placental and decidual tissues of RPL mice were significantly lower than those in normal mice. In contrast, the protein levels of HIF-1α were significantly higher in RPL mice than in normal mice. SalB restored the morphological changes in the uterus of RPL mice, as well as the number of blood vessels in the placenta and decidua, and ameliorated adverse embryonic development in mice (such as neural tube defects and reduced crown-rump length), thereby increasing the pups per. Additionally, SalB increased the VEGF/VEGFR2/p-VEGFR2 levels, placental and decidual MVD, and MPW and decreased the HIF-1α levels and EAR in a dose-dependent manner. A positive association of daily SalB dose (0-100mg/kg) with the VEGF levels, placental and decidual MVD and MPW, and a negative association of daily SalB dose with the HIF-1α levels and EAR were observed.PTC299 reversed the aforementioned increases and decreases in the daily SalB intake of RPL mice. Among these, the daily dose of 100mg/kg of SalB was deemed optimal, and a daily dose of SalB exceeding 100mg/kg did not entirely induce these changes. No correlation between HIF-1α and VEGF was observed in decidual and placental/ chorionic tissues from normal pregnancies and URPL patients, and from normal and RPL mice, but a negative correlation between the two factors was observed in the tissues from RPL mice in the presence of SalB with or without PTC299. SalB ameliorates RPL by restoring physiological HIF-1α/VEGF balance and placental angiogenesis. Optimal daily dose was 100mg/kg, which demonstrated the absence of embryotoxicity or mitigated the embryotoxicity of RPL mice, while higher doses (400mg/kg) showed lesser improvement and increased hepatotoxicity. The promotion of placental and decidual angiogenesis may be an effective strategy for treating unexplained RPL.

Do high endothelial venules (HEVs) appear in the uterus of healthy and pathological pregnancies? Our study reveals that HEVs are present in the non-pregnant endometrium and decidua parietalis (decP) but decline upon placentation in decidua basalis (decB) and are less abundant in decidual tissues from idiopathic, recurrent pregnancy losses (RPLs). RPL is associated with a compromised decidual vascular phenotype. Endometrial (n = 29) and first trimester decidual (n = 86, 6-12th week of gestation) tissue samples obtained from endometrial biopsies or elective pregnancy terminations were used to determine the number of HEVs and T cells. In addition, quantification of HEVs and immune cells was performed in a cohort of decidual tissues from RPL (n = 25). Position and frequency of HEVs were determined in non-pregnant endometrial as well as decidual tissue sections using immunofluorescence (IF) staining with antibodies against E-selectin, intercellular adhesion molecule, von Willebrand factor, ephrin receptor B4, CD34 and a carbohydrate epitope specific to HEVs (MECA-79). Immune cell distribution and characterization was determined by antibodies recognizing CD45 and CD3 by IF staining- and flow cytometry-based analyses. Antibodies against c-c motif chemokine ligand 21 (CCL21) and lymphotoxin-beta were used in IF staining and Western blot analyses of decidual tissues. Functional HEVs are found in high numbers in the secretory endometrium and decP but decline in numbers upon placentation in decB (P ≤ 0.001). Decidua parietalis tissues contain higher levels of the HEV-maintaining factor lymphotoxin beta and decP-associated HEVs also express CCL21 (P ≤ 0.05), a potent T-cell chemoattractant. Moreover, there is a positive correlation between the numbers of decidual HEVs and the abundance of CD3+ cells in decidual tissue sections (P ≤ 0.001). In-depth analysis of a RPL tissue collection revealed a decreased decB (P ≤ 0.01) and decP (P ≤ 0.01) HEV density as well as reduced numbers of T cells in decB (P ≤ 0.05) and decP (P ≤ .001) sections when compared with age-matched healthy control samples. Using receiver-operating characteristics analyses, we found significant predictive values for the ratios of CD3/CD45 (P < 0.001) and HEVs/total vessels (P < 0.001) for the occurrence of RPL. Analyses were performed in first trimester decidual tissues from elective terminations of pregnancy or non-pregnant endometrium samples from patients diagnosed with non-endometrial pathologies including cervical polyps, ovarian cysts and myomas. First trimester decidual tissues may include pregnancies which potentially would have developed placental disorders later in gestation. In addition, our cohort of non-pregnant endometrium may not reflect the endometrial vascular phenotype of healthy women. Finally, determination of immune cell distributions in the patient cohorts studied may be influenced by the different modes of tissue derivation. Pregnancy terminations were performed by surgical aspiration, endometrial tissues were obtained by biopsies and RPL tissues were collected after spontaneous loss of pregnancy. In this study, we propose an inherent mechanism by which the endometrium and in particular the decidua control T-cell recruitment. By demonstrating reduced HEV densities and numbers of T cells in decB and decP tissues of RPL samples we further support previous findings reporting an altered vascular phenotype in early pregnancy loss. Altogether, the findings provide important information to further decipher the etiologies of unexplained RPL. This study was supported by the Austrian Science Fund (P31470 B30 to M.K.) and by the Austrian National Bank (17613ONB to J.P.). There are no competing interests to declare. N/A.

Hystero-embryoscopy as a tool for RPL immunological research at the maternal-fetal interface.