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The physical connection of mother and offspring during pregnancy allows the bi-directional exchange of a small number of cells through the placenta. These cells, which can persist long-term in the recipient individual are genetically foreign to it and therefore fulfill the principle of microchimerism. Over the last years, pioneer research on microchimeric cells revealed their role in immune adaptation during pregnancy and priming of tolerogenic responses in the progeny. However, the mechanisms involved in cell transfer across the placenta barrier remain poorly investigated. In this review, we summarize the evidence of fetomaternal microchimerism, propose a mechanism for cell trafficking through the placenta and discuss the different models and techniques available for its analysis. Likewise, we aim to generate interest in the use of ex vivo placenta perfusion to investigate microchimerism in physiological and pathological settings.

Species differences are among the main reasons for the high failure rate of preclinical studies. A better awareness and understanding of these differences might help to improve the outcome of preclinical research. In reproduction, the placenta is the central organ regulating fetal exposure to a substance circulating in the maternal organism. Exact information about placental transfer can help to better estimate the toxic potential of a substance. From an evolutionary point of view, the chorioallantoic placenta is the organ with the highest anatomical diversity among species. Moreover, frequently used animal models in reproduction belong to rodents and lagomorphs, two groups that are characterized by the generation of an additional type of placenta, which is crucial for fetal development, but absent from humans: the inverted yolk sac placenta. Taken together, the translatability of placental transfer studies from laboratory animals to humans is challenging, which is supported by the fact that numerous species-dependent toxic effects are described in literature. Thus, reliable human-relevant data are frequently lacking and the toxic potential of chemicals and pharmaceuticals for humans can hardly be estimated, often resulting in recommendations that medical treatments or exposure to chemicals should be avoided for safety reasons. Although species differences of placental anatomy have been described frequently and the need for human-relevant research models has been emphasized, analyses of substances with species-dependent placental transfer have been performed only sporadically. Here, we present examples for species-specific placental transfer, including that of nanoparticles and pharmaceuticals, and discuss potential underlying mechanisms. With respect to the COVID 19-pandemic it might be of interest that some antiviral drugs are reported to feature species-specific placental transfer. Further, differences in placental structure and antibody transfer may affect placental transfer of ZIKA virus.

Developmental toxicity testing urgently requires the implementation of human relevant new approach methodologies (NAMs) that better recapitulate the peculiar nature of human physiology during pregnancy, especially the placenta and the maternal/fetal interface, which represent a key stage for the human lifelong health. Fit-for-purpose NAMs for the placental-fetal interface are desirable to improve the biological knowledge of environmental exposure at molecular level and to reduce the high cost, time and ethical impact of animal studies. This article reviews the state of the art on the available in vitro (placental, fetal and amniotic cell-based systems) and in silico NAMs of human relevance for developmental toxicity testing purposes, as well as of the available Adverse Outcome Pathways related to developmental toxicity. The OECD TG 414 for the identification and assessment of deleterious effects of prenatal exposure to chemicals on developing organisms will be discussed to delineate the regulatory context and to better debate what is missing and needed in the context of the developmental origins of health and disease hypothesis to significantly improve this sector. Starting from this analysis, the development of a novel human feto-placental organ-on-chip platform will be introduced as an innovative alternative tool for developmental toxicity testing, considering possible implementation and validation strategies to overcome the limitation of the current animal studies and NAMs available in regulatory toxicology and in the biomedical field.

Introduction Research on human placental development and function lacks a conclusive in vivo model. To investigate the intracellular molecular mechanisms in trophoblast cells, different cell lines have been established during the last decades. So far, none of these accomplishes all features of primary trophoblast, thus their suitability as well as the transferability of the results has been discussed. The aim of this study is to assess molecular markers and features matching different trophoblast subpopulations in trophoblastic cell lines to provide orientation on their suitability and relevance for distinct research questions. Methods The commonly used trophoblastic cell lines, BeWo, JEG-3, HTR-8/SVneo, AC1-M59, AC1-M32, ACH-3P and Swan71 were selected. qPCR and immunoblotting were used to determine expression of characteristic molecular markers. C14MC, C19MC and miR-371-3 miRNA expression were investigated by real time PCR. Proliferation, migration and network stabilization assays were performed. Hormone secretion was determined by chemiluminescent-immunoassays. DNA profiles were obtained by Short Tandem Repeat (STR)-genotyping. Results Immortalized cell lines differ from choriocarcinoma-derived ones in the expression of HLA-G, E-cadherin, N-cadherin, VE-cadherin, cadherin-11, cytokeratin 7, vimentin, ADAM12 and PRG2. Compared to choriocarcinoma-derived cell lines, expression of C19MC and hormone secretion were almost absent in immortalized cell lines. Conversely, they express C14MC and exhibit higher migration and network stabilization. Discussion The data presented will help justify the use of a cell line to evaluate distinct features of trophoblast biology and pathology. In general, characteristics and markers of choriocarcinoma derived cell lines seem to be more similar to in vivo trophoblast than immortalized cell lines and thus might be regarded as more suitable models.

Purpose Several roles are attributed to the myometrium including sperm and embryo transport, menstrual discharge, control of uterine blood flow, and labor. Although being a target of diabetes complications, the influence of high glucose on this compartment has been poorly investigated. Both miRNAs and IGF1R are associated with diabetic complications in different tissues. Herein, we examined the effects of high glucose on the expression of miRNAs and IGF1R signaling pathway in the human myometrium. Methods Human myometrial explants were cultivated for 48 h under either high or low glucose conditions. Thereafter, the conditioned medium was collected for biochemical analyses and the myometrial samples were processed for histological examination as well as miRNA and mRNA expression profiling by qPCR. Results Myometrial structure and morphology were well preserved after 48 h of cultivation in both high and low glucose conditions. Levels of lactate, creatinine, LDH and estrogen in the supernatant were similar between groups. An explorative screening by qPCR arrays revealed that 6 out of 754 investigated miRNAs were differentially expressed in the high glucose group. Data validation by single qPCR assays confirmed diminished expression of miR-215-5p and miR-296-5p, and also revealed reduced miR-497-3p levels. Accordingly, mRNA levels of IGF1R and its downstream mediators FOXO3 and PDCD4, which are potentially targeted by miR-497-3p, were elevated under high glucose conditions. In contrast, mRNA expression of IGF1, PTEN, and GLUT1 was unchanged. Conclusions The human myometrium responds to short-term exposure (48 h) to high glucose concentrations by regulating the expression of miRNAs, IGF1R and its downstream targets.

Recent trends in 3D cell culturing has placed organotypic tissue models at another level. Now, not only is the microenvironment at the cynosure of this research, but rather, microscopic geometrical parameters are also decisive for mimicking a tissue model. Over the years, technologies such as micromachining, 3D printing, and hydrogels are making the foundation of this field. However, mimicking the topography of a particular tissue-relevant substrate can be achieved relatively simply with so-called template or morphology transfer techniques. Over the last 15 years, in one such research venture, we have been investigating a micro thermoforming technique as a facile tool for generating bioinspired topographies. We call them MatriGrid

A complex network composed of at least 1900 microRNA (miRNA) species orchestrates the development and function of the human placenta. These molecules regulate genes and pathways operating major functional processes in trophoblast cells such as proliferation, invasion, differentiation, and metabolism. Nevertheless, the cellular localization and role of most placental miRNAs remain to be determined. The existence of eutherian- (C14MC) and primate-specific miRNA clusters (C19MC), together with human placenta-specific miRNAs, indicate the relevance of these molecules in evolution and diversification of the placenta, including the acquisition of its unique features in humans. They may be related also to diseases that are exclusively present in primates, such as preeclampsia. Changes in the miRNA expression profile have been reported in several placental pathologies. Which miRNAs are involved in the pathomechanism of these diseases or act to maintain placental homeostasis is uncertain. Placenta-derived miRNAs are packed into extracellular vesicles (EVs) and distributed through the maternal circulation to distant organs, where they contribute to adaptations required during pregnancy. Similarly, the placenta also receives molecular information from other tissues to adapt fetoplacental metabolic demands to the maternal energetic supply. These processes can be impaired in pathologic conditions. Therefore, the collection of circulating placental miRNAs constitutes potentially a minimally-invasive approach to assess the fetoplacental status and to diagnose pregnancy diseases. Future therapies may include manipulation of miRNA levels for prevention and treatment of placental complications to protect maternal health and fetal development.

Doxorubicin (DOX) is one of the most commonly used drugs for the treatment of childhood cancers, including leukemia and lymphomas. Despite the high survival rate, female leukemia survivors are at higher risk of ovarian failure and infertility later in life. Treatment with chemotherapeutic drugs like DOX is associated with damage in ovarian follicles, but the affectation grade of granulosa cells remains unclear. To assess and avoid the possible side-effects of DOX, early biomarkers of ovarian injury and chemotherapy-induced ovarian toxicity should be identified. MicroRNAs (miRNAs) have emerged in recent years as a promising new class of biomarkers for drug-induced tissue toxicity. In this study, the effects of DOX on cell viability, steroidogenesis, and miRNA expression were studied in primary granulosa cells (GCs) and in two cellular models (COV434 and KGN cells). We report that compared to other chemotherapeutic drugs, DOX treatment is more detrimental to granulosa cells as observed by decrease of cell viability. Treatment with DOX changes the expression of the aromatase gene (CYP19A1) and the secretion of 17β-estradiol (E2) in a cell-specific manner. miR-132-3p is dose-dependently increased by DOX in all cellular models. In absence of DOX, miR-132-3p overexpression in COV434 cells has no effect on E2 secretion or CYP19A1 expression. Altogether, these findings contribute to understanding the hormonal disbalance caused by DOX in human ovarian cells and suggest miR-132 as a putative sensor to predict DOX-induced ovarian toxicity.

Non-coding RNAs (ncRNAs) play a central and regulatory role in almost all cells, organs, and species, which has been broadly recognized since the human ENCODE project and several other genome projects. Nevertheless, a small fraction of ncRNAs have been identified, and in the placenta they have been investigated very marginally. To date, most examples of ncRNAs which have been identified to be specific for fetal tissues, including placenta, are members of the group of microRNAs (miRNAs). Due to their quantity, it can be expected that the fairly larger group of other ncRNAs exerts far stronger effects than miRNAs. The syncytiotrophoblast of fetal origin forms the interface between fetus and mother, and releases permanently extracellular vesicles (EVs) into the maternal circulation which contain fetal proteins and RNA, including ncRNA, for communication with neighboring and distant maternal cells. Disorders of ncRNA in placental tissue, especially in trophoblast cells, and in EVs seem to be involved in pregnancy disorders, potentially as a cause or consequence. This review summarizes the current knowledge on placental ncRNA, their transport in EVs, and their involvement and pregnancy pathologies, as well as their potential for novel diagnostic tools.

Since the full development of the ex vivo dual perfusion model of the human placenta cotyledon, the technique has provided essential insight into how nutrients, lipids, gases, immunoglobulins, endocrine agents, pharma-ceuticals, chemicals, nanoparticles, micro-organisms and parasites might traverse the maternofetal barrier. Additionally, the model has been instrumental in gaining a better understanding of the regulation of vascular tone, endocrinology and metabolism within this organ. The human placenta is unique amongst species in its anatomy and transfer modalities. This orthologous diversity therefore requires an appropriate consideration of placental transfer rates of compounds, particles and micro-organisms specific to humans. Different research centres have adapted this model with a wide variation in perfusion parameters, including in the establishment of perfusion, perfusate composition, gassing regime, cannulation method, flow rates, perfused tissue mass, and also in the application of quality control measures. The requirement to harmonise and stan-dardise perfusion practice between centres is largely driven by the need to obtain consistency in our under -standing of placental function, but also in the qualification of the model for acceptance by regulatory agencies in drug and toxicology testing. A pilot study is proposed, aiming to describe how existing inter-centre variation in perfusion methodology affects placental metabolism, protein synthesis, oxygen consumption, the materno-fetal transfer of key molecular markers, and placental structure.

The concept of biological identity has been traditionally a central issue in immunology. The assumption that entities foreign to a specific organism should be rejected by its immune system, while self-entities do not trigger an immune response is challenged by the expanded immunotolerance observed in pregnancy. To explain this "immunological paradox", as it was first called by Sir Peter Medawar, several mechanisms have been described in the last decades. Among them, the intentional transfer and retention of small amounts of cells between a mother and her child have gained back attention. These microchimeric cells contribute to expanding allotolerance in both organisms and enhancing genetic fitness, but they could also provoke aberrant alloimmune activation. Understanding the mechanisms used by microchimeric cells to exert their function in pregnancy has proven to be challenging as per definition they are extremely rare. Profiting from studies in the field of transplantation and cancer research, a synergistic effect of microchimerism and cellular communication based on the secretion of extracellular vesicles (EVs) has begun to be unveiled. EVs are already known to play a pivotal role in feto-maternal tolerance by transferring cargo from fetal to maternal immune cells to reshape their function. A further aspect of EVs is their function in antigen presentation either directly or on the surface of recipient cells. Here, we review the current understanding of microchimerism in the feto-maternal tolerance during human pregnancy and the potential role of EVs in mediating the allorecognition and tropism of microchimeric cells.

The transport of drugs across the placenta is a point of great importance in pharmacotherapy during pregnancy. However, the knowledge of drug transport in pregnancy is mostly based on experimental clinical data, and the underlying biological mechanisms are not fully understood. In this review, we summarize the current knowledge of drug transporters in the human placenta. We only refer to human data since the placenta demonstrates great diversity among species. In addition, we describe the experimental models that have been used in human placental transport studies and discuss their availability. A better understanding of placental drug transporters will be beneficial for the health of pregnant women who need drug treatment and their fetuses.

Members of the innate immune system, innate lymphoid cells (ILCs), encompass five major populations (Natural Killer (NK) cells, ILC1s, ILC2s, ILC3s, and lymphoid tissue inducer cells) whose functions include defense against pathogens, surveillance of tumorigenesis, and regulation of tissue homeostasis and remodeling. ILCs are present in the uterine environment of humans and mice and are dynamically regulated during the reproductive cycle and pregnancy. These cells have been repurposed to support pregnancy promoting maternal immune tolerance and placental development. To accomplish their tasks, immune cells employ several cellular and molecular mechanisms. They have the capacity to remember a previously encountered antigen and mount a more effective response to succeeding events. Memory responses are not an exclusive feature of the adaptive immune system, but also occur in innate immune cells. Innate immune memory has already been demonstrated in monocytes/macrophages, neutrophils, dendritic cells, and ILCs. A population of decidual NK cells characterized by elevated expression of NKG2C and LILRB1 as well as a distinctive transcriptional and epigenetic profile was found to expand during subsequent pregnancies in humans. These cells secrete high amounts of interferon-γ and vascular endothelial growth factor likely favoring placentation. Similarly, uterine ILC1s in mice upregulate CXCR6 and expand in second pregnancies. These data provide evidence on the development of immunological memory of pregnancy. In this article, the characteristics, functions, and localization of ILCs are reviewed, emphasizing available data on the uterine environment. Following, the concept of innate immune memory and its mechanisms, which include epigenetic changes and metabolic rewiring, are presented. Finally, the emerging role of innate immune memory on reproduction is discussed. Advances in the comprehension of ILC functions and innate immune memory may contribute to uncovering the immunological mechanisms underlying female fertility/infertility, placental development, and distinct outcomes in second pregnancies related to higher birth weight and lower incidence of complications.

Developmental toxicity testing urgently requires the implementation of human-relevant new approach methodologies (NAMs) that better recapitulate the peculiar nature of human physiology during pregnancy, especially the placenta and the maternal/fetal interface, which represent a key stage for human lifelong health. Fit-for-purpose NAMs for the placental–fetal interface are desirable to improve the biological knowledge of environmental exposure at the molecular level and to reduce the high cost, time and ethical impact of animal studies. This article reviews the state of the art on the available in vitro (placental, fetal and amniotic cell-based systems) and in silico NAMs of human relevance for developmental toxicity testing purposes; in addition, we considered available Adverse Outcome Pathways related to developmental toxicity. The OECD TG 414 for the identification and assessment of deleterious effects of prenatal exposure to chemicals on developing organisms will be discussed to delineate the regulatory context and to better debate what is missing and needed in the context of the Developmental Origins of Health and Disease hypothesis to significantly improve this sector. Starting from this analysis, the development of a novel human feto–placental organ-on-chip platform will be introduced as an innovative future alternative tool for developmental toxicity testing, considering possible implementation and validation strategies to overcome the limitation of the current animal studies and NAMs available in regulatory toxicology and in the biomedical field.

Tobacco smoking is an important public health issue recognized by the world health organization as one of the most serious, preventable risk factors for developing a series of pregnancy pathologies. Maternal smoking is positively associated with intrauterine growth restriction (IUGR) and gestational diabetes (GDM), but negatively associated with preeclampsia (PE). In this review, we examine epidemiological, clinical and laboratory studies of smoking effects on immunoregulation during pregnancy, trophoblast function, and placental vasculature development and metabolism. We aim to identify effects of tobacco smoke components on specific placental compartments or cells, which may contribute to the understanding of the influences of maternal smoking on placenta function in normal and pathological pregnancies. Data corroborates that in any trimester, smoking is unsafe for pregnancy and that its detrimental effects outweigh questionable benefits. The effects of maternal smoking on the maternal immune regulation throughout pregnancy and the impact of different tobacco products on fetal growth have not yet been fully understood. Smoking cessation rather than treatment with replacement therapies is recommended for future mothers because also single components of tobacco and its smoke may have detrimental effects on placental function.

This opinion paper briefly presents arguments that support the unlikelihood of an impact on female fertility from current covid-19 vaccines.

ZusammenfassungDas Mammakarzinom gehört zu den am häufigsten in der Schwangerschaft auftretenden Malignomen. Da Frauen zunehmend erst in fortgeschrittenem Alter gebären, ist anzunehmen, dass die Inzidenz schwangerschaftsassoziierter Mammakarzinome zukünftig weiter steigen wird. Aufgrund schwangerschaftsbedingter Veränderungen und zurückhaltender Diagnostik kommt es häufig dazu, dass diese Karzinome erst in fortgeschrittenen Stadien detektiert werden und somit meist einer systemischen adjuvanten Therapie bedürfen. Verfügbare Daten bezüglich des optimalen chemotherapeutischen Managements sind limitiert. Insbesondere zur Anwendung des Target-Agents Trastuzumab, das in der Therapie des HER2-überexprimierenden Mammakarzinoms der nicht schwangeren Frau einen entscheidenden Beitrag zur Verbesserung der Prognose leisten konnte, fehlen dezidierte Informationen bezüglich des Wirk- und Sicherheitsprofils in der Schwangerschaft sowie auch hinsichtlich etwaiger Langzeiteffekte auf das Kind. Innerhalb der derzeit verfügbaren Literatur konnten 38 Schwangerschaften unter Trastuzumab zur Therapie eines Mammakarzinoms analysiert werden. Es lassen sich daraus Informationen gewinnen und Rückschlüsse ziehen, die zukünftig die Therapieoptionen der schwangeren Mammakarzinompatientin individualisieren und entscheidend verbessern können.

The cynomolgus monkey is increasingly considered in toxicological research as the most appropriate model for humans due to the species' close physiological contiguity, including reproductive physiology. Here, literature on the cynomolgus monkey placenta is reviewed in regards to its similarity to the human placenta and particularly for its immunological role, which is not entirely mirrored in humans. Pertinent original data are included in this article. The cynomolgus monkey placenta is evaluated based on three aspects: first, morphological development; second, the spatial and temporal appearance of maternal and fetal immune cells and certain immune cell products of the innate and adaptive immune systems; and third, the expression of relevant immune tolerance-related molecules including the homologs of anti-human leucocyte antigen, indoleamine 2,3-dioxygenase, FAS/FAS-L, annexin II, and progesterone. Parameters relevant to the immunological role of the placenta are evaluated from the immunologically immature stage of gestational day (GD) 50 until more mature stages close to birth. Selected comparisons are drawn with human and other laboratory animal placentas. In conclusion, the cynomolgus monkey placenta has a high degree of morphological and physiological similarity to the human placenta. However, there are differences in the topographical distribution of cell types and immune tolerance-related molecules. Three basic features are recognized: (1) the immunological capacity of the placenta changes throughout the lifetime of the organ; (2) these immunological changes include multiple parameters such as morphological adaptations, cell type involvement, and changes in immune-relevant molecule expression; and (3) the immune systems of two genetically disparate individuals (mother and child) are functionally intertwined at the maternal-fetal interface.

Pregnant women have been carefully observed during the COVID-19 pandemic, as the pregnancy-specific immune adaptation is known to increase the risk for infections. Recent evidence indicates that even though most pregnant have a mild or asymptomatic course, a severe course of COVID-19 and a higher risk of progression to diseases have also been described, along with a heightened risk for pregnancy complications. Yet, vertical transmission of the virus is rare and the possibility of placental SARS-CoV-2 infection as a prerequisite for vertical transmission requires further studies. We here assessed the severity of COVID-19 and onset of neonatal infections in an observational study of women infected with SARS-CoV-2 during pregnancy. Our placental analyses showed a paucity of SARS-CoV-2 viral expression ex vivo in term placentae under acute infection. No viral placental expression was detectable in convalescent pregnant women. Inoculation of placental explants generated from placentas of non-infected women at birth with SARS-CoV-2 in vitro revealed inefficient SARS-CoV-2 replication in different types of placental tissues, which provides a rationale for the low ex vivo viral expression. We further detected specific SARS-CoV-2 T cell responses in pregnant women within a few days upon infection, which was undetectable in cord blood. Our present findings confirm that vertical transmission of SARS-CoV-2 is rare, likely due to the inefficient virus replication in placental tissues. Despite the predominantly benign course of infection in most mothers and negligible risk of vertical transmission, continuous vigilance on the consequences of COVID-19 during pregnancy is required, since the maternal immune activation in response to the SARS-CoV2 infection may have long-term consequences for children’s health.

Regeneration of the endometrial stromal compartment in premenopausal women is likely maintained by the perivascular endometrial mesenchymal stem/stromal cells (eMSC) expressing sushi domain containing 2 (SUSD2). The fate of SUSD2+ eMSC during pregnancy and their role in decidualization is not fully known. The aim of our study was to determine the effect of progesterone on the stemness of the SUSD2+ eMSC isolated from non-pregnant uterine samples. Secondary objectives were to characterize the functional capacity including differentiation and clonogenicity assays of SUSD2+ eMSC isolated from decidua at full term and compare it to the capacity of those isolated from non-pregnant uterine samples. Progesterone treatment induced changes in the decidual gene expression profile in non-pregnant SUSD2+ eMSC. Data analysis of a publicly available single cell RNA-seq data set revealed differential expression of several mesenchymal and epithelial signature genes between the SUSD2+ eMSC and the decidual stromal cells, suggesting mesenchymal-to-epithelial transition occurs during decidualization. Histological analysis revealed a significantly lower abundance of SUSD2+ eMSC in 1st trimester and full term samples compared to non-pregnant samples, p = 0.0296 and 0.005, respectively. The differentiation and the colony forming capacity did not differ significantly between the cells isolated from non-pregnant and pregnant uterine samples. Our results suggest that SUSD2+ eMSC undergo decidualization in vitro, while maintaining MSC plasma membrane phenotype. Human eMSC seem to play an important role in the course of endometrial decidualization and embryo implantation. Pregnancy reduced the abundance of SUSD2+ eMSC, however eMSC function remains intact.

Extracellular vesicle (EV)-mediated communication has been implicated in the cooperative alliance between trophoblast and immune cells toward maternal tolerance and placentation. Syncytiotrophoblast cells secrete EVs directly into the maternal circulation, which are taken up by immune cells, endothelial cells, and other cell types. Initial evidence also shows that EVs produced by immune cells are, in turn, incorporated by trophoblast cells and modulate placental responses. Non-coding RNAs (ncRNAs), proteins, and lipid mediators transported in EVs are able to influence proliferation, differentiation, cytokine production, and immunological responses of recipient cells. The molecular alphabet and cellular targets involved in this dialogue are being revealed. Nevertheless, several questions regarding the whole content, surface markers, and biological functions of EVs still remain to be investigated in both physiological and pathological conditions. Analysis of circulating EVs in maternal blood has the potential to serve as a minimally invasive approach to monitoring placental functions and immunological features of pregnancy, aiding in the diagnostics of complications. This review addresses the immunomodulatory properties of EVs and their tasks in the communication between placental and immune cells.

Problem Extracellular vesicles (EVs) released by the placenta are packed with biological information and play a major role in fetomaternal communication. Here, we describe a comprehensive set-up for the enrichment and characterization of EVs from human placenta perfusion and their application in further assays. Method of study Human term placentas were used for 3 h ex vivo one-sided perfusions to simulate the intervillous circulation. Thereafter, populations of small (sEVs) and large EV (lEVs) were enriched from placental perfusate via serial ultracentrifugation. Following, EV populations were characterized regarding their size, protein concentration, RNA levels, expression of surface markers as well as their uptake and miRNA transfer to recipient cells. Results The sEV and lEV fractions from an entire perfusate yielded, respectively, 294±32 µg and 525±96 µg of protein equivalents and 2.6±0.5 µg and 3.6±0.9 µg of RNA. The sEV fraction had a mean diameter of 117±47 nm, and the lEV fraction presented 236±54 nm. CD63 was strongly detected by dot blot in sEVs, whereas only traces of this marker were found in lEVs. Both EV fractions were positive for the trophoblast marker PLAP (placental alkaline phosphatase) and annexin A1. EV internalization in immune cells was visualized by confocal microscopy, and the transfer of placental miRNAs was detected by quantitative real-time PCR (qPCR). Conclusions Enriched EV populations showed characteristic features of sEVs and lEVs. EV uptake and transfer of miRNAs to recipient cells demonstrated their functional integrity. Therefore, we advocate the ex vivo one-sided placenta perfusion as a robust approach for the collection of placental EVs.

Members of the placenta-specific miRNA cluster C19MC, including miR-519d, are secreted by fetal trophoblast cells within extracellular vesicles (EVs). Trophoblast-derived EVs can be internalized by the autologous trophoblast and surrounding maternal immune cells, resulting in coordination of cellular responses. The study of functions and targets of placental miRNAs in the donor and recipient cells may contribute to the understanding of the immune tolerance essential in pregnancy. Here, we report that miR-519d-3p levels correlate positively with cell proliferation and negatively with migration in trophoblastic cell lines. Inhibition of miR-519d-3p in JEG-3 cells increases caspase-3 activation and apoptosis. PDCD4 and PTEN are targeted by miR-519d-3p in a cell type-specific manner. Transfection of trophoblastic cell lines with miR-519d mimic results in secretion of EVs containing elevated levels of this miRNA (EVmiR-519d). Autologous cells enhance their proliferation and decrease their migration ability when treated with EVmiR-519d. NK92 cells incorporate EV-delivered miR-519d-3p at higher levels than Jurkat T cells. EVmiR-519d increases the proliferation of Jurkat T cells but decreases that of NK92 cells. Altogether, miR-519d-3p regulates pivotal trophoblast cell functions, can be transferred horizontally via EVs to maternal immune cells and exerts functions therein. Vesicular miRNA transfer from fetal trophoblasts to maternal immune cells may contribute to the immune tolerance in pregnancy.

PurposeInfertility is a debilitating situation that millions of women around the world suffer from, but the causal relationship between infertility and endometriosis is still unclear. We hypothesize that the immune cell populations of uterine natural killer cells (uNK) and plasma cells (PC) which define chronic endometritis could differ in patients with or without endometriosis and therefore be the link to endometriosis-associated infertility.MethodsOur retrospective study includes 173 patients that underwent an endometrial scratching in the secretory phase of the menstrual cycle and subsequently immunohistochemical examination for uNK cells and PC. Sixty-seven patients were diagnosed with endometriosis, 106 served as the control cohort.ResultsThe risk for an elevated number of uNK cells in women with endometriosis is not increased as compared to the control group. Our findings suggest that patients with endometriosis are 1.3 times more likely to have chronic endometritis (CE) as compared to those without and that the treatment with doxycycline might increase pregnancy rates. Endometriosis and an increased number of uNK cells seem to be unrelated.ConclusionsIn contrast to the lately published connection between endometriosis, infertility and increased uNK cells, we could not find any evidence that patients with endometriosis are more prone to elevated uterine uNK cells. Counting of PC in endometrial biopsies might be a new approach in the search of biomarkers for the nonsurgical diagnosis of endometriosis since our findings suggest a connection.

There is growing evidence that changes in the eutopic endometrial immune profile are a cause of endometriosis-associated infertility. Women affected by endometriosis experience a 2-fold increased risk of infertility compared to healthy controls. In our study we aimed to map out endometrial expressions of uterine natural killer cells, plasma cells, macrophages and the chemokine CXC-motif ligand 1 (CXCL1) as well as its main receptors CXC-motif receptor 2 (CXCR2) and Syndecan-1 in infertility-patients with endometriosis. 36 infertility patients were included of which 19 suffered from endometriosis and 17 served as a control cohort. All patients underwent endometrial scratching in the secretory phase and immunohistochemical staining which was evaluated by three independent observers. In endometriosis-patients, a higher concentration of macrophages coincided with an elevated number of uterine natural killer cells or plasma cells. Patients with endometriosis also showed a higher endothelial expression of VEGF-A. Furthermore, absence of stromal expression of SDC-1 was associated with an elevated level of uNK in general. Therefore, our study links endometriosis to an altered immune cell population in the eutopic endometrium, which might be a new approach to diagnosing endometriosis in infertility patients.

Die Einnahme der eigenen Plazenta durch die Mutter im Wochenbett geht auf traditionelle Anwendungen in der abendländischen und asiatischen Komplementärmedizin zurück. Hebammen in der Wochenbettbetreuung sehen sich einer gestiegenen Nachfrage gegenüber, jedoch mangelt es an evidenzbasierten Empfehlungen zur sogenannten Plazentophagie. Die Autoren der vorliegenden Übersichtsarbeit recherchierten Risiken und potenziellen Nutzen, um die Einnahme der verarbeiteten Plazenta als Heilmittel im Wochenbett zu bewerten.

Wenn Frauen einen unerfullten Kinderwunsch haben, kann es auch hilfreich sein, das Endometrium zu untersuchen. Findet sich hier eine immunologische Fehlregulation, etwa Plasmazellen, kann diese behandelt werden und die Chance auf eine Schwangerschaft erhohen.

Dienogest (DNG) administration is a well-established treatment for endometriosis but bleeding irregularities remain its main disadvantage. Changes in diet, mainly to vegetable consumption, are beneficial in the treatment of estrogen-related pathologies but their use for endometriosis has been poorly studied. In this study, addition of the phytochemical 3,3′-diindolylmethane (DIM) to DNG therapy has been investigated in in vitro and ex vivo models for endometriosis and in a small cohort of women with endometriosis. Endometrial Ishikawa cells were treated with DNG or DIM at dosages from 10−10 M to 10−5 M for up to 72 h. Cell proliferation was measured by assessing BrdU incorporation. Endometrial tissue from women with endometriosis and controls was incubated with DNG or a combination of DNG and DIM. Tissue viability was determined using a modified colorimetric MTS assay. 17β-estradiol secretion was quantified by an electro-chemiluminescence immunoassay. Finally, DNG as monotherapy or in combination with DIM was randomly administered to women with endometriosis (n = 8) over 3 months. Bleeding patterns and associated pelvic pain were assessed by Visual Analogue Scale (VAS). DNG and DIM significantly reduced cell proliferation in Ishikawa cells. Ex vivo, DIM reduced viability and estradiol secretion specifically in endometriotic but not in normal endometrial tissue. This effect was enhanced by combination with DNG. Endometriosis associated pelvic pain was significantly reduced in patients taking the DNG-DIM combination therapy compared to those taking DNG alone. Bleeding pattern (number and duration of episodes) was significantly improved by addition of DIM to the DNG treatment. In conclusion, addition of DIM enhances effects of DNG ex vivo and may ameliorate bleeding patterns in endometriosis patients.

The use of placenta preparations as an individual puerperal remedy can be traced back to historical, traditional practices in Western and Asian medicine. To evaluate the ingestion of processed placenta as a puerperal remedy, the potential risks (trace elements, microorganisms) and possible benefit (hormones in the placental tissue) of such a practice are discussed in this article based on a literature review.Die Einnahme der eigenen Plazenta durch die Mutter als individuelles Heilmittel im Wochenbett ist zurückzuführen auf historische, traditionelle Anwendungen in abendländischer und asiatischer komplementärer Medizin. In dieser Übersichtsarbeit wurden mittels Literaturrecherche sowohl die möglichen Risiken (Spurenelemente, Mikroorganismen) als auch möglicher Nutzen (Hormone im Plazentagewebe) erfasst, um die Einnahme der verarbeiteten Plazenta als Heilmittel im Wochenbett zu bewerten.