Placental structural adaptation to maternal physical activity and sedentary behavior: findings of the DALI lifestyle study.

STUDY QUESTION: Are maternal levels of moderate-to-vigorous physical activity (MVPA) and sedentary time (ST) in obese pregnant women associated with placental structural adaptations for facilitating oxygen delivery to the fetus? SUMMARY ANSWER: Higher maternal MVPA and ST are associated with a higher density of villi, a proxy measure of placental surface area for oxygen delivery to the fetus, without further added placental vessels. WHAT IS KNOWN ALREADY: Physical activity during pregnancy intermittently reduces uterine blood flow, potentially limiting placental and fetal oxygen supply. The placenta can mount several adaptive responses, including enlargement of the surface area of villi and/or feto-placental vessels to accommodate fetal needs. Early research on the morphology and growth of the placenta with exercise interventions has shown inconsistencies and is lacking, particularly in non-lean pregnant women. STUDY DESIGN, SIZE, DURATION: This study is a secondary longitudinal analysis of the vitamin D and lifestyle intervention for gestational diabetes prevention (DALI) randomized controlled trial. The prospective study was conducted between 2012 and 2015 in nine European countries at 11 different sites. In this analysis, 92 pregnant women with a BMI ≥ 29 kg/m2 were combined into one cohort. PARTICIPANTS/MATERIALS, SETTING, METHODS: MVPA and percentage of time spent sedentary (% ST) were measured with accelerometers during gestation. Placental sections were immunostained for endothelial cell-specific CD34. Artificial intelligence (AI)-based stereology assessed villous density, number, and cross-sectional area of vessels on whole-slide images and in selected regions comprising peripheral villi only, where the majority of vascular adaptations occur. Expression of pro- and anti-angiogenic factors was quantified using molecular counting analysis. MAIN RESULTS AND THE ROLE OF CHANCE: In multivariable regression, higher levels of maternal MVPA (min/day) were associated with a higher density of villi in both whole-slide images (beta 0.12; 95% CI 0.05, 0.2) and selected regions (0.17; CI 0.07, 0.26). Unexpectedly, ST was also positively associated with density of villi (0.23; CI 0.04, 0.43). MVPA and ST were not associated with vessel count/mm2 villous area, vessel area, or pro- and anti-angiogenic factor mRNA expression. All estimates and statistical significance of the sensitivity analyses excluding smokers, women who developed gestational diabetes or pre-eclampsia and/or pregnancy-induced hypertension were similar in the main analysis. LIMITATIONS, REASONS FOR CAUTION: The placenta is a complex organ undergoing dynamic changes. While various adjustments were made to account for different maternal contributing factors, in addition to the outcome measures, various other factors could impact oxygen delivery to the fetus. WIDER IMPLICATIONS OF THE FINDINGS: For the first time, we evaluated the association between placental structures quantified using an AI-based approach with objectively measured physical activity and ST at multiple time points in pregnant women with obesity. The observed adaptations contribute to the advancement of our understanding of the hemodynamics and adaptations of the placental unit in response to MVPA and ST. However, our results might not be generalizable to lean pregnant women. STUDY FUNDING/COMPETING INTEREST(S): The DALI project has received funding from the European Community's 7th Framework Program (FP7/2007-2013) under grant agreement no. 242187. The funders had no role in study design, collection of data, analyses, writing of the article, or the decision to submit it for publication. The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: ISRCTN70595832.

Maternal COVID-19 causing intrauterine foetal demise with microthrombotic placental insufficiency: a case report.

BACKGROUND: Pregnant women have an increased risk of getting infected with SARS-CoV-2 and are more prone to severe illness. Data on foetal demise in affected pregnancies and its underlying aetiology is scarce and pathomechanisms remain largely unclear. CASE: Herein we present the case of a pregnant woman with COVID-19 and intrauterine foetal demise. She had no previous obstetric or gynaecological history, and presented with mild symptoms at 34 + 3 weeks and no signs of foetal distress. At 35 + 6 weeks intrauterine foetal death was diagnosed. In the placental histopathology evaluation, we found inter- and perivillous fibrin depositions including viral particles in areas of degraded placental anatomy without presence of viral entry receptors and SARS-CoV-2 infection of the placenta. CONCLUSION: This case demonstrates that maternal SARS-CoV-2 infection in the third trimester may lead to an unfavourable outcome for the foetus due to placental fibrin deposition in maternal COVID-19 disease possibly via a thrombogenic microenvironment, even when the foetus itself is not infected.

Early human trophoblast development: from morphology to function.

Human pregnancy depends on the proper development of the embryo prior to implantation and the implantation of the embryo into the uterine wall. During the pre-implantation phase, formation of the morula is followed by internalization of blastomeres that differentiate into the pluripotent inner cell mass lineage, while the cells on the surface undergo polarization and differentiate into the trophectoderm of the blastocyst. The trophectoderm mediates apposition and adhesion of the blastocyst to the uterine epithelium. These processes lead to a stable contact between embryonic and maternal tissues, resulting in the formation of a new organ, the placenta. During implantation, the trophectoderm cells start to differentiate and form the basis for multiple specialized trophoblast subpopulations, all of which fulfilling specific key functions in placentation. They either differentiate into polar cells serving typical epithelial functions, or into apolar invasive cells that adapt the uterine wall to progressing pregnancy. The composition of these trophoblast subpopulations is crucial for human placenta development and alterations are suggested to result in placenta-associated pregnancy pathologies. This review article focuses on what is known about very early processes in human reproduction and emphasizes on morphological and functional aspects of early trophoblast differentiation and subpopulations.

The challenges of histology education.

Generations of students have struggled to learn histology. They have found the subject extremely dry and the nomenclature very challenging. Insidiously, histology is fading from the minds of stakeholders. Unless this is rectified, it is not inconceivable that the subject will be further diluted in the medical curriculum. What contributing factors could there be? Via a survey and focus group discussions, international faculty (n = 111) were asked what the challenges in teaching histology were. Both qualitative and quantitative data were collected and stratified by the number of teaching years. A collaborative teaching session with a pathologist was also delivered to the YLLSoMM1 students (n = 280), who gave opinions about it. Of the international faculty, 85 (±1)% responded. Among those, 60 (±1)% felt that the pedagogy should be reformed (e.g., by gamification). Interestingly, 30 (±1)% opined that organ system histology should be moved into the clinical years. Notably, 70 (±1)% of teachers preferred to teach face to face (FTF) using either microscopes or virtual microscopy. Among the students, 71 (±1)% reported self-teaching from online resources. Significantly, 88 (±1)% of M1 students agreed that having the pathologist co-teach histology was beneficial. Some teachers, and most students, struggle with histology. There is a generational divide in opinions concerning how histology should be taught. The traditionalists wish to maintain the status quo while the reformists want changes. Learners want to be engaged with hybrid approaches aided by the pathologist. They also welcome more clinical context during histology lessons.

mRNA and Protein Expression in Human Fetal Membrane Cells: Potential Biomarkers for Preterm Prelabor Rupture of the Fetal Membranes?

Clinically, unique markers in fetal membrane cells may contribute to the search for biomarkers for preterm prelabor rupture of the fetal membranes (pPROM) in maternal blood. pPROM is associated with overwhelming inflammation and premature cellular senescence causing "biological microfractures" of the fetal membranes. We hypothesize that these pathological processes are associated with the shedding of fetal membrane cells into the maternal circulation. The aim of this study was to identify markers expressed exclusively in fetal membrane cells to facilitate their isolation, characterization, and determination of biomarker potential in maternal blood. We have (1), by their transcriptomic profile, identified markers that are upregulated in amnion and chorion tissue compared to maternal white blood cells, and (2), by immunohistochemistry, confirmed the localization of the differentially expressed proteins in fetal membranes, placenta, and the placental bed of the uterus. RNA sequencing revealed 31 transcripts in the amnion and 42 transcripts in the chorion that were upregulated. Among these, 22 proteins were evaluated by immunohistochemistry. All but two transcripts were expressed both on mRNA and protein level in at least one fetal membrane cell type. Among these remaining 20 proteins, 9 proteins were not significantly expressed in the villous and extravillous trophoblasts of the placenta.

Placental Villous Explant Culture 2.0: Flow Culture Allows Studies Closer to the In Vivo Situation.

During pregnancy, freely floating placental villi are adapted to fluid shear stress due to placental perfusion with maternal plasma and blood. In vitro culture of placental villous explants is widely performed under static conditions, hoping the conditions may represent the in utero environment. However, static placental villous explant culture dramatically differs from the in vivo situation. Thus, we established a flow culture system for placental villous explants and compared commonly used static cultured tissue to flow cultured tissue using transmission and scanning electron microscopy, immunohistochemistry, and lactate dehydrogenase (LDH) and human chorionic gonadotropin (hCG) measurements. The data revealed a better structural and biochemical integrity of flow cultured tissue compared to static cultured tissue. Thus, this new flow system can be used to simulate the blood flow from the mother to the placenta and back in the most native-like in vitro system so far and thus can enable novel study designs.

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.

IJMS Special Issue "Molecular and Cellular Mechanisms of Preeclampsia"-Editorial.

Over the last few decades, massive research efforts have been put into deciphering the etiology of the pregnancy pathology preeclampsia[...].

Traditional and New Routes of Trophoblast Invasion and Their Implications for Pregnancy Diseases.

Historically, invasion of placental trophoblasts was thought to be extremely specific, only invading into the connective tissues of the maternal uterus and finally reaching and transforming the uterine spiral arteries. Only recently, identification of new routes of trophoblast invasion into different structures of the maternal uterus has been achieved. Thorough morphological analysis has resulted in the identification of trophoblasts invading into glands, veins, and lymph vessels of the uterine wall. These new routes pave the way for a re-evaluation of trophoblast invasion during normal placental development. Of course, such new routes of trophoblast invasion may well be altered, especially in pregnancy pathologies such as intra-uterine growth restriction, preeclampsia, early and recurrent pregnancy loss, stillbirth, and spontaneous abortion. Maybe one or more of these pregnancy pathologies show alterations in different pathways of trophoblast invasion, and, thus, etiologies may need to be redefined, and new therapies may be developed.

Galectin 13 (PP13) Facilitates Remodeling and Structural Stabilization of Maternal Vessels during Pregnancy.

Galectins regulate cell growth, proliferation, differentiation, apoptosis, signal transduction, mRNA splicing, and interactions with the extracellular matrix. Here we focus on the galectins in the reproductive system, particularly on a group of six galectins that first appears in anthropoid primates in conjunction with the evolution of highly invasive placentation and long gestation. Of these six, placental protein 13 (PP13, galectin 13) interacts with glycoproteins and glycolipids to enable successful pregnancy. PP13 is related to the development of a major obstetric syndrome, preeclampsia, a life-threatening complication of pregnancy which affects ten million pregnant women globally. Preeclampsia is characterized by hypertension, proteinuria, and organ failure, and is often accompanied by fetal loss and major newborn disabilities. PP13 facilitates the expansion of uterine arteries and veins during pregnancy in an endothelial cell-dependent manner, via the eNOS and prostaglandin signaling pathways. PP13 acts through its carbohydrate recognition domain that binds to sugar residues of extracellular and connective tissue molecules, thus inducing structural stabilization of vessel expansion. Further, decidual PP13 aggregates may serve as a decoy that induces white blood cell apoptosis, contributing to the mother's immune tolerance to pregnancy. Lower first trimester PP13 level is one of the biomarkers to predict the subsequent risk to develop preeclampsia, while its molecular mutations/polymorphisms that are associated with reduced PP13 expression are accompanied by higher rates of preeclampsia We propose a targeted PP13 replenishing therapy to fight preeclampsia in carriers of these mutations.

Disturbed Placental Imprinting in Preeclampsia Leads to Altered Expression of DLX5, a Human-Specific Early Trophoblast Marker.

BACKGROUND: Preeclampsia is a complex and common human-specific pregnancy syndrome associated with placental pathology. The human specificity provides both intellectual and methodological challenges, lacking a robust model system. Given the role of imprinted genes in human placentation and the vulnerability of imprinted genes to loss of imprinting changes, there has been extensive speculation, but no robust evidence, that imprinted genes are involved in preeclampsia. Our study aims to investigate whether disturbed imprinting contributes to preeclampsia. METHODS: We first aimed to confirm that preeclampsia is a disease of the placenta by generating and analyzing genome-wide molecular data on well-characterized patient material. We performed high-throughput transcriptome analyses of multiple placenta samples from healthy controls and patients with preeclampsia. Next, we identified differentially expressed genes in preeclamptic placentas and intersected them with the list of human imprinted genes. We used bioinformatics/statistical analyses to confirm association between imprinting and preeclampsia and to predict biological processes affected in preeclampsia. Validation included epigenetic and cellular assays. In terms of human specificity, we established an in vitro invasion-differentiation trophoblast model. Our comparative phylogenetic analysis involved single-cell transcriptome data of human, macaque, and mouse preimplantation embryogenesis. RESULTS: We found disturbed placental imprinting in preeclampsia and revealed potential candidates, including GATA3 and DLX5, with poorly explored imprinted status and no prior association with preeclampsia. As a result of loss of imprinting, DLX5 was upregulated in 69% of preeclamptic placentas. Levels of DLX5 correlated with classic preeclampsia markers. DLX5 is expressed in human but not in murine trophoblast. The DLX5high phenotype resulted in reduced proliferation, increased metabolism, and endoplasmic reticulum stress-response activation in trophoblasts in vitro. The transcriptional profile of such cells mimics the transcriptome of preeclamptic placentas. Pan-mammalian comparative analysis identified DLX5 as part of the human-specific regulatory network of trophoblast differentiation. CONCLUSIONS: Our analysis provides evidence of a true association among disturbed imprinting, gene expression, and preeclampsia. As a result of disturbed imprinting, the upregulated DLX5 affects trophoblast proliferation. Our in vitro model might fill a vital niche in preeclampsia research. Human-specific regulatory circuitry of DLX5 might help explain certain aspects of preeclampsia.

Human trophoblast invasion: new and unexpected routes and functions.

Until recently, trophoblast invasion during human placentation was characterized by and restricted to invasion into uterine connective tissues and the uterine spiral arteries. The latter was explained to connect the arteries to the intervillous space of the placenta and to guarantee the blood supply of the mother to the placenta. Today, this picture has dramatically changed. Invasion of endoglandular trophoblast into uterine glands, already starting at the time of implantation, enables histiotrophic nutrition of the embryo prior to perfusion of the placenta with maternal blood. This is followed by invasion of endovenous trophoblasts into uterine veins to guarantee the drainage of fluids from the placenta back into the maternal circulation throughout pregnancy. In addition, invasion of endolymphatic trophoblasts into the lymph vessels of the uterus has been described. Only then, invasion of endoarterial trophoblasts into spiral arteries takes place, enabling hemotrophic nutrition of the fetus starting with the second trimester of pregnancy. This new knowledge paves the way to identify changes that may occur in pathological pregnancies, from tubal pregnancies to recurrent spontaneous abortions.

Expression of matrix metalloproteinase 12 is highly specific for non-proliferating invasive trophoblasts in the first trimester and temporally regulated by oxygen-dependent mechanisms including HIF-1A.

During first trimester pregnancy, trophoblast cells invade from the placenta into the maternal decidua where they anchor the placenta and remodel luminal structures like spiral arteries. This process depends on proteases secreted by invading trophoblasts, which degrade extracellular matrix (ECM). We here aimed to identify proteases particularly important for trophoblast invasion. We generated a list of proteases capable of degrading decidual ECM and trophoblast integrins using MEROPS database and compared expression of these proteases between primary trophoblasts isolated from first trimester placenta (FT, n = 3), representing an invasive phenotype, vs trophoblasts isolated from term pregnancy (TT, n = 3), representing a non-invasive trophoblast phenotype. Matrix metalloproteinase 12 (MMP12) revealed highest expression levels in FT, with absent expression in TT. In situ hybridisation and immunofluorescence localised MMP12 specifically to extravillous trophoblasts (evCT) whilst Ki67 co-staining revealed that proliferating trophoblasts of the cell columns were almost negative for MMP12. Quantification revealed a decline in MMP12 positive evCT at the end of first trimester, when oxygen levels start rising. MMP12 promoter analysis identified potential binding sites for hypoxia-inducible factor (HIF-1) and other oxygen-sensitive transcription factors. Moreover, MMP12 protein was increased by low oxygen in FT in vitro and by addition of a HIF-1α activator. Collectively, MMP12 is a highly expressed protease specific for invasive evCT during the first trimester. MMP12 down regulation by increasing oxygen concentration enables temporal expression control of MMP12 and involves several mechanisms including HIF-1α. These findings suggest MMP12 involved in trophoblast invasion during the first trimester.

Correction to: Expression of matrix metalloproteinase 12 is highly specific for non-proliferating invasive trophoblasts in the first trimester and temporally regulated by oxygen-dependent mechanisms including HIF-1A.

In the original publication, the contribution of Dr. Christian Eyth as equal first author was not indicated. This has been corrected confirming that U. Hidden and C. Eyth contributed equally to this work.

CD74-Downregulation of Placental Macrophage-Trophoblastic Interactions in Preeclampsia.

RATIONALE: We hypothesized that cluster of differentiation 74 (CD74) downregulation on placental macrophages, leading to altered macrophage-trophoblast interaction, is involved in preeclampsia. OBJECTIVE: Preeclamptic pregnancies feature hypertension, proteinuria, and placental anomalies. Feto-placental macrophages regulate villous trophoblast differentiation during placental development. Disturbance of this well-balanced regulation can lead to pathological pregnancies. METHODS AND RESULTS: We performed whole-genome expression analysis of placental tissue. CD74 was one of the most downregulated genes in placentas from preeclamptic women. By reverse transcriptase-polymerase chain reaction, we confirmed this finding in early-onset (<34 gestational week, n=26) and late-onset (≥34 gestational week, n=24) samples from preeclamptic women, compared with healthy pregnant controls (n=28). CD74 protein levels were analyzed by Western blot and flow cytometry. We identified placental macrophages to express CD74 by immunofluorescence, flow cytometry, and RT-PCR. CD74-positive macrophages were significantly reduced in preeclamptic placentas compared with controls. CD74-silenced macrophages showed that the adhesion molecules ALCAM, ICAM4, and Syndecan-2, as well as macrophage adhesion to trophoblasts were diminished. Naive and activated macrophages lacking CD74 showed a shift toward a proinflammatory signature with an increased secretion of tumor necrosis factor-α, chemokine (C-C motif) ligand 5, and monocyte chemotactic protein-1, when cocultured with trophoblasts compared with control macrophages. Trophoblasts stimulated by these factors express more CYP2J2, sFlt1, TNFα, and IL-8. CD74-knockout mice showed disturbed placental morphology, reduced junctional zone, smaller placentas, and impaired spiral artery remodeling with fetal growth restriction. CONCLUSIONS: CD74 downregulation in placental macrophages is present in preeclampsia. CD74 downregulation leads to altered macrophage activation toward a proinflammatory signature and a disturbed crosstalk with trophoblasts.

Predicting the Risk to Develop Preeclampsia in the First Trimester Combining Promoter Variant -98A/C of LGALS13 (Placental Protein 13), Black Ethnicity, Previous Preeclampsia, Obesity, and Maternal Age.

BACKGROUND: LGALS13 (placental protein 13 [PP13]) promoter DNA polymorphisms was evaluated in predicting preeclampsia (PE), given PP13's effects on hypotension, angiogenesis, and immune tolerance. METHODS: First-trimester plasma samples (49 term and 18 intermediate) of PE cases matched with 196 controls were collected from King's College Hospital, London, repository. Cell-free DNA was extracted and the LGALS13 exons were sequenced after PCR amplification. Expression of LGALS13 promoter reporter constructs was determined in BeWo trophoblast-like cells with luciferase assays. Adjusted odds ratio (OR) was calculated for the A/A genotype combined with maternal risk factors. RESULTS: The A/A, A/C, and C/C genotypes in the -98 promoter position were in Hardy-Weinberg equilibrium in the control but not in the PE group (p < 0.036). The dominant A/A genotype had higher frequency in the PE group (p < 0.001). The A/C and C/C genotypes protected from PE (p < 0.032). The ORs to develop term and all PE, calculated for the A/A genotype, previous PE, body mass index (BMI) >35, black ethnicity, and maternal age >40 were 15.6 and 11.0, respectively (p < 0.001). In luciferase assays, the "-98A" promoter variant had lower expression than the "-98C" variant in non-differentiated (-13%, p = 0.04) and differentiated (-26%, p < 0.001) BeWo cells. Forskolin-induced differentiation led to a larger expression increase in the "-98C" variant than in the "-98A" variant (4.55-fold vs. 3.85-fold, p < 0.001). CONCLUSION: Lower LGALS13 (PP13) expression with the "A" nucleotide in the -98 promoter region position (compared to "C") and high OR calculated for the A/A genotype in the -98A/C promoter region position, history of previous PE, BMI >35, advanced maternal age >40, and black ethnicity could serve to aid in PE prediction in the first trimester.

GDM alters paracrine regulation of feto-placental angiogenesis via the trophoblast.

Feto-placental angiogenesis and vascular development are tightly regulated by pro- and anti-angiogenic factors. Villous trophoblast may be a major source of these factors. It forms the classical placental barrier between mother and fetus, and is thus exposed to maternal influences as well. Metabolic and hormonal derangements in gestational diabetes mellitus (GDM) affect feto-placental angiogenesis and vascular growth. Here we hypothesized that GDM alters the trophoblast secretome, which will modulate the paracrine regulation of feto-placental angiogenesis. Primary term trophoblasts were isolated from normal (n=6) and GDM (n=6) pregnancies. Trophoblast conditioned medium (CM) was used to investigate paracrine effects of normal and GDM-exposed trophoblasts on feto-placental endothelial cells (fpECs; n=7), using functional assays for 2D network formation, wound healing, chemotaxis, and proliferation. Gene expression of 23 pro- and anti-angiogenic factors was analyzed. Four trophoblast-derived paracrine regulators of angiogenesis were specifically measured in CM. CM from GDM trophoblasts increased 2D network formation of fpEC by 2.4-fold (P<0.001), whereas wound healing was attenuated by 1.8-fold (P=0.02) and chemo-attraction to the CM was reduced by 33±9% (P=0.02). The effect of CM on proliferation was unchanged between normal and GDM trophoblasts. Expression analysis of pro- and anti-angiogenic molecules in normal and GDM trophoblasts revealed significant differences in ANGPT2, HGF, KISS1 and PLGF expression. Analysis of secreted proteins demonstrated reduced pigment epithelium derived factor and tumor necrosis factor-α secretion by GDM trophoblasts. GDM alters the balance of trophoblast derived, angiogenesis modulating paracrine factors. This may contribute to GDM-associated changes in placental angiogenesis and vascular structure.

Expression of CD24 and Siglec-10 in first trimester placenta: implications for immune tolerance at the fetal-maternal interface.

During pregnancy, the fetal-maternal interface establishes immune tolerance between the fetus and the mother. CD24, a mucin-like glycoprotein expressed at the surface of hematopoietic cells and diverse tumor cells, is known to interact with the sialic acid-binding immunoglobulin-type lectins (Siglecs). This interaction was assessed as a candidate complex for the immune suppression response in the placenta. CD24 was affinity purified from term placenta and characterized by SDS-PAGE, Western blot and ELISA. Binding of recombinant Siglecs to placental CD24 was evaluated by ELISA. The expression of CD24 and Siglec-10 in first trimester placental tissues was investigated by immunohistochemistry and immunofluorescence. Placental CD24 had an apparent molecular weight of 30-70 kDa consistent with its high degree of N- and O-linked glycosylation. EDTA-sensitive CD24-Siglec-10 interaction via the terminal sialic acid glycan residues of CD24 was observed. CD24 did not interact with Siglec-3 or Siglec-5. During the first trimester, and already in gestational week (GA) 8, CD24 showed high expression in villous and extravillous cytotrophoblasts. There was also a mild expression in stromal cells, while syncytiotrophoblasts were negative. Co-localization of CD24 with Siglec-10 was observed in endometrial glands and in first trimester decidual cells in close vicinity to extracellular trophoblasts. This study is the first to demonstrate the early presence of CD24 in the placenta cytotrophoblast layers, placental bed and maternal uterine glands. The presence of the CD24-Siglec-10 in these regions of fetal-maternal interactions suggests a possible role in mediating immune tolerance at the fetal-maternal interface.

Extravillous trophoblasts invade more than uterine arteries: evidence for the invasion of uterine veins.

During the first trimester of pregnancy, extravillous trophoblasts (EVTs) invade into the decidual interstitium to the first third of the myometrium, thereby anchoring the placenta to the uterus. They also follow the endovascular and endoglandular route of invasion; plug, line and remodel spiral arteries, thus being responsible for the establishment of hemotrophic nutrition with the beginning of the second trimester and invade and open uterine glands toward the intervillous space for a histiotrophic nutrition during the first trimester. The aim of this study was to provide proof that uterine veins are invaded by EVTs similar to uterine arteries and glands in first trimester of pregnancy. Therefore, serial sections from in situ first trimester placenta were immuno-single- and immuno-double-stained to distinguish in a first step between arteries and veins and secondly between invaded and non-invaded vessels. Subsequently, invasion of EVTs into uterine vessels was quantified. Our data show that uterine veins are significantly more invaded by EVTs than uterine arteries (29.2 ± 15.7 %) during early pregnancy. Counted vessel cross sections revealed significantly higher EVT invasion into veins (59.5 ± 7.9 %) compared to arteries (29.2 ± 15.7 %). In the lumen of veins, single EVTs were repeatedly found, beside detached glandular epithelial cells or syncytial fragments. This study allows the expansion of our hitherto postulated concept of EVT invasion during first trimester of pregnancy. We suggest that invasion of EVTs into uterine veins is responsible the draining of waste and blood plasma from the intervillous space during the first trimester of pregnancy.

Biobanking of different body fluids within the frame of IVF-a standard operating procedure to improve reproductive biology research.

PURPOSE: The aim of the present study was to develop a standard operating procedure (SOP) for the collection, transport, and storage of human cumulus cells, follicular fluid, blood serum, seminal plasma, embryo culture supernatant, and embryo culture supernatant control obtained within the IVF process under approved protocols and written informed consent from participating patients. The SOP was developed at the Kinderwunsch Institut Schenk, Dobl, Austria, together with Biobank Graz of the Medical University of Graz, Austria. METHODS: The SOP provides comprehensive details of laboratory procedures and sampling of the different fluids within the IVF process. Furthermore, information on sample coding, references of involved laboratory techniques (e.g., oocyte retrieval with a Steiner-TAN needle), ethical approvals, and biobanking procedures are presented. RESULTS: The result of the present study is a standard operating procedure. CONCLUSIONS: The SOP ensures a professional way for collection and scientific use of IVF samples by the Kinderwunsch Institut Schenk, Dobl, Austria, and Biobank Graz of the Medical University of Graz, Austria. It can be used as a template for other institutions to unify specimen collection procedures in the field of reproductive health research.