Feto-maternal Interface Research Articles

Molecular mechanisms of environmental toxin cadmium at the feto-maternal interface investigated using an organ-on-chip (FMi-OOC) model

Human labor is associated with feto-maternal-derived signals that coordinate to initiate delivery. Exposure to environmental chemicals can prematurely trigger labor-initiating signals at the feto-maternal interface (FMi: decidua, amniochorion), leading to spontaneous preterm birth (PTB). Testing the association between environmental chemical exposure and PTB is difficult due to many limitations in vivo or in vitro. Physiological organ-on-chips (OOCs) are potential alternatives for studying mechanisms leading to PTB. The presented study tested the effect of maternal exposure to cadmium (Cd), an environmental toxin, using the FMi-OOC that incorporates maternal decidua cells and three different fetal cells (chorion, amnion mesenchymal, and amnion epithelial cells). Cd transport through the FMi and its impact on cell cycle, cell death, and inflammation were analyzed. Cd treatment resulted in significant cell death and a pro-inflammatory environment in the maternal decidua, but had minimal effect on the fetal chorion cells, and no effect in the fetal amnion cells compared to controls. The maternal response, but lack of fetal response, indicates that Cd-mediated adverse effects originate from maternal pathophysiology rather than fetal-derived triggers of preterm labor. This study demonstrates that the FMi-OOC can indeed predict the response of FMi upon exposure to chemicals, opening the possibility for using OOC models for environmental toxin screens.

Cytokines, Hormones and Cellular Regulatory Mechanisms Favoring Successful Reproduction

Its semi-allogeneic nature renders the conceptus vulnerable to attack by the maternal immune system. Several protective mechanisms operate during gestation to correct the harmful effects of anti-fetal immunity and to support a healthy pregnancy outcome. Pregnancy is characterized by gross alterations in endocrine functions. Progesterone is indispensable for pregnancy and humans, and it affects immune functions both directly and via mediators. The progesterone-induced mediator - PIBF - acts in favor of Th2-type immunity, by increasing Th2 type cytokines production. Except for implantation and parturition, pregnancy is characterized by a Th2-dominant cytokine pattern. Progesterone and the orally-administered progestogen dydrogesterone upregulate the production of Th2-type cytokines and suppress the production of Th1 and Th17 cytokine production in vitro. This is particularly relevant to the fact that the Th1-type cytokines TNF-α and IFN-γ and the Th17 cytokine IL-17 have embryotoxic and anti-trophoblast activities. These cytokine-modulating effects and the PIBF-inducing capabilities of dydrogesterone may contribute to the demonstrated beneficial effects of dydrogesterone in recurrent spontaneous miscarriage and threatened miscarriage. IL-17 and IL-22 produced by T helper cells are involved in allograft rejection, and therefore could account for the rejection of paternal HLA-C-expressing trophoblast. Th17 cells (producing IL-17 and IL-22) and Th22 cells (producing IL-22) exhibit plasticity and could produce IL-22 and IL-17 in association with Th2-type cytokines or with Th1-type cytokines. IL-17 and IL-22 producing Th cells are not harmful for the conceptus, if they also produce IL-4. Another important protective mechanism is connected with the expansion and action of regulatory T cells, which play a major role in the induction of tolerance both in pregnant women and in tumour-bearing patients. Clonally-expanded Treg cells increase at the feto-maternal interface and in tumour-infiltrating regions. While in cancer patients, clonally-expanded Treg cells are present in peripheral blood, they are scarce in pregnancy blood, suggesting that fetal antigen-specific tolerance is restricted to the foeto-maternal interface. The significance of Treg cells in maintaining a normal materno-foetal interaction is underlined by the fact that miscarriage is characterized by a decreased number of total effector Treg cells, and the number of clonally-expanded effector Treg cells is markedly reduced in preeclampsia. In this review we present an overview of the above mechanisms, attempt to show how they are connected, how they operate during normal gestation and how their failure might lead to pregnancy pathologies.

Study of relationships between maternal HLA-G gene polymorphism and intrauterine infection with risk of congenital malformations

This study aims for assessing relationships between maternal HLA-G gene polymorphisms (rs41551813, rs12722477, rs41557518) and intrauterine infection with the risk of congenital malformations (CM) in infants. We studied 331 women who had offspring with CMs, and 408 women with one or more healthy children. Influence of the intrauterine infection was analyzed on the basis of laboratory tests. Diagnostics of bacterial vaginosis and vulvovaginal candidiasis by microscopic examination were conducted. Viral infections (herpes simplex virus type 2, cytomegalovirus, human papilloma virus type 16/18) as well as Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma urealyticum, Gardnerella vaginalis; Trichomonas vaginalis and Toxoplasma gondii were detected by enzyme-linked immunoassay or polymerase chain reaction (PCR) techniques. The data were obtained from the medical cards of the surveyed women. The gene polymorphisms were typed for Thr31Ser (rs41551813, HLA-G*01:03) in exon 2, Leu110Ile (rs12722477, HLA-G*01:04) and 1597 delС (rs41557518, HLA-G*01:05N) in exon 3 HLA-G using real-time PCR followed by melting analysis. The study showed that maternal age was not a significant risk factor for CMs in the fetus/newborns. Meanwhile, the maternal intrauterine infections were shown to be a significant risk factor for CMs in their infants (OR = 1.57 (1.08-2.29); p = 0.002). It was found that the 110 Ile allele (HLA-G *01:04) was a risk factor for CMs incidence in the fetus/newborns (OR = 1.57 (1.08-2.29), p = 0.01). No association was found between the maternal rs41551813 and rs41557518 HLA-G genetic polymorphisms and CMs in the infants. Hence, intrauterine infections and maternal 110 Ile allele (HLA-G *01:04) may be suggested as risk factors for birth defects in the children. Our results will be useful in understanding the molecular mechanisms of immune disorders in feto-maternal interface.

Advances in understanding the crosstalk between mother and fetus on iron utilization

A full-term pregnancy comes with significant demand for iron. Not meeting this demand has adverse effects on maternal health and on the intrauterine and postnatal development of the infant. In the infant, some of these adverse effects cannot be reversed by postnatal iron supplementation, highlighting the need to tackle iron deficiency in utero. Achieving this requires sound understanding of the pathways that govern iron transfer at the fetomaternal interface. Two pathways are emerging as key players in this context; the hepcidin/ferroportin axis pathway and the iron regulatory protein (IRPs) pathway. In late gestation, suppression of maternal hepcidin, by as yet unknown factors, is required for increasing iron availability to the growing fetus. In the placenta, the rate of iron uptake by transferrin receptor TfR1 at the apical/maternal side and of iron release by ferroportin FPN at the basal/fetal side is controlled by IRP1. In fetal hepatocytes, build up of fetal iron stores requires post-translational inhibition of FPN by the cell-autonomous action of hepcidin. In the fetal liver, FPN is also subject to additional control at the transcriptional level, possibly by the action of hypoxia-inducible factor HIF2α. The rates of apical iron uptake and basal iron release in the placenta are modulated according to iron availability in the maternal blood and the placenta's own needs. This placental modulation ensures that the amount of iron delivered to the fetal circulation is maintained within a normal range, even in the face of mild maternal iron deficiency or overload. However, when maternal iron deficiency or overload are extreme, placental modulation is not sufficient to maintain normal iron supply to the fetus, resulting in fetal iron deficiency and overload respectively. Thus, the rate of iron transfer at the fetomaternal interface is subject to several regulatory signals operating simultaneously in the maternal liver, the placenta and the fetal liver. These regulatory signals act in concert to maintain normal iron supply to the fetus within a wide range of maternal iron states, but fail to do so when maternal iron deficiency or overload are extreme. The limitations of existing experimental models must be overcome if we are to gain better understanding of the role of these regulatory signals in normal and complicated pregnancy. Ultimately, that understanding could help identify better markers of fetal iron demand and underpin novel iron replacement strategies to treat maternal and fetal iron deficiency.

Clinical, pathologic, and epidemiologic features of nocardioform placentitis in the mare

Placentitis is the leading cause of infectious abortion in the horse and contributes to roughly 19% of all abortions in the United States. A type of placental infection, nocardioform placentitis (NP) is associated with gram-positive branching actinomycetes localized within the ventral body of the feto-maternal interface to create a lymphoplasmacytic mucoid lesion. While the etiology of this disease is poorly described, this placental infection continues to cause episodic abortions in addition to weak and/or growth retarded neonates. The goal of the present study was to perform a comprehensive analysis of pregnancies associated with a nocardioform-affected placenta and make inferences into the epidemiology of this elusive disease. To do so, 264 mares were enrolled in the study, with 145 as having suspected disease (n = 145; NP) either based on pregnancy-related complications or postpartum placental evaluation, while an additional 119 were enrolled as healthy pregnancies (n = 119; CON). Following diagnosis as either NP or CON based on gross and histopathology at the University of Kentucky Veterinary Diagnostic Laboratory, information was gathered on the mares and neonates for comparisons between diseased and healthy pregnancies. Clinically, a significant portion of diseased mares had clinical indications of NP, including premature mammary gland development, thickening of the placenta noted on transrectal ultrasonography, and separation between the chorioallantois and endometrium noted on abdominal ultrasonography, while vulvar discharge was not commonly noted. Additionally, NP was correlated with increased mare age, decreased gestational length, and decreased neonatal weight, although neonatal IgG and WBC were comparable to CON. Incidence of NP was not correlated with last breeding date, pre- and post-breeding therapeutics, parity, prophylactic medications, or housing. Additionally, NP did not affect postpartum fertility. While NP was associated with a poor neonatal outcome (abortion and/or growth retarded neonate), this did not appear to be influenced by the bacteria isolated (Amycolatopsis spp. vs. Crossiella equi), and mares diagnosed with NP do not appear to be infectious to other pregnant mares nor have repetitive years of the disease. Interestingly, lesion size was positively correlated with last breeding date, as mares bred later in the breeding season correlating with a larger placental lesion. In conclusion, while the etiology of NP continues to elude researchers, the epidemiology of this disease has gained clarity, providing inferences into the management of suspect mares.

Microvesicles and exosomes released by amnion epithelial cells under oxidative stress cause inflammatory changes in uterine cells†.

Extracellular vesicles play a crucial role in feto-maternal communication and provide an important paracrine signaling mechanism in pregnancy. We hypothesized that fetal cells-derived exosomes and microvesicles (MVs) under oxidative stress (OS) carry unique cargo and traffic through feto-maternal interface, which cause inflammation in uterine cells associated with parturition. Exosomes and MVs, from primary amnion epithelial cell (AEC) culture media under normal or OS-induced conditions, were isolated by optimized differential centrifugation method followed by characterization for size (nanoparticle tracking analyzer), shape (transmission electron microscopy), and protein markers (western blot and immunofluorescence). Cargo and canonical pathways were identified by mass spectroscopy and ingenuity pathway analysis. Myometrial, decidual, and cervical cells were treated with 1 × 107 control/OS-derived exosomes/MVs. Pro-inflammatory cytokines were measured using a Luminex assay. Statistical significance was determined by paired T-test (P< 0.05). AEC produced cup-shaped exosomes of 90-150nm and circular MVs of 160-400nm. CD9, heat shock protein 70, and Nanog were detected in exosomes, whereas OCT-4, human leukocyte antigen G, and calnexin were found in MVs. MVs, but not exosomes, were stained for phosphatidylserine. The protein profiles for control versus OS-derived exosomes and MVs were significantly different. Several inflammatory pathways related to OS were upregulated that were distinct between exosomes and MVs. Both OS-derived exosomes and MVs significantly increased pro-inflammatory cytokines (granulocyte-macrophage colony-stimulating factor, interleukin 6 (IL-6), and IL-8) in maternal cells compared with control (P< 0.05). Our findings suggest that fetal-derived exosomes and MVs under OS exhibited distinct characteristics and a synergistic inflammatory role in uterine cells associated with the initiation of parturition.

Study of relationships between HLA-G gene polymorphism, intrauterine infection and recurrent miscarriage in women

The relationship between the HLA-G gene polymorphism (rs41551813, rs12722477, rs41557518), intrauterine infection and recurrent miscarriage (RM) in women were studied. The case group consisted of 180 patients with RM, defined as two or more consecutive miscarriages (min = 2; max = 8) at up to 20 weeks of gestation, and with clinically confirmed pregnancies and non-viable fetuses. At the time of examination. the women were enrolled from the Genetic Counseling Center at the Kemerovo Regional Clinical Hospital, Kemerovo, Russia, and were not pregnant. Each patient underwent a gynecological examination. We excluded women with a history of medical abortion, birth, and ectopic pregnancies. In addition, we excluded women with endocrine (e.g. diabetes) disorders. To exclude other known causes of spontaneous abortion, the following tests were performed: ultrasound examination of pelvic organs, and karyotyping in women and men. The women’s mean age in the RM group, was 29.6±4.8 (SD) years. The control group comprised 408 fertile women. These women didn’t have a history of spontaneous abortion, or a family history of congenital malformations. They have born, at least, 1-2 healthy children. Women’s mean age at birth of last child was 26.8±5.2 (SD) years. Influence of the intrauterine infection was analyzed on the basis of laboratory tests. Diagnostics of bacterial vaginosis and vulvo-vaginal candidiasis by microscopic examination was conducted. Viral agent infections (herpes simplex virus type 2, cytomegalovirus, human papilloma virus type 16/18), Chlamydia trachomatis, Mycoplasma hominis, Ureaplasma urealyticum, Gardnerella vaginalis and Trichomonas vaginalis were detected by enzyme-linked immunoassay and polymerase chain reaction (PCR). The data were obtained from the medical cards of the surveyed women. All the women gave a written informed consent before participating in the study. Typing of polymorphisms of Thr31Ser (rs41551813, HLA-G*01:03) in exon 2, Leu110Ile (rs12722477, HLA-G*01:04) and 1597 delС (rs41557518, HLA-G*01:05N) in exon 3 HLA-G genes were performed by realtime PCR followed by melting analysis. The study showed that the intrauterine infection was not a risk factor for RM (p = 0.30) in the examined women. It was found that the 110 Ile allele (HLA-G *01:04) was a risk factor for RM both in women with intrauterine infection [ORa = 4.50 (2.41-8.38), p = 2.09e-06], and in women without infection [ORa = 2.46 (1.44-4.21), p = 0.0009]. The cooperative influence of genetic and infections factors with the risk of RM in women was revealed [ORa+f = 3.50 (2.01-6.09), p = 8.78e-06]. Our results will be useful in understanding the molecular mechanisms of immune disorders in fetomaternal interface, and for choosing the strategy of management and treatment in women with RM.

Expression Patterns of Analgesic Metabolising Machinery in 1st and 2nd Trimester Human Fetal Liver and Gonads

Use of over-the-counter analgesics during pregnancy is widespread globally. Most analgesic compounds can freely diffuse through the placental feto-maternal interface and reach the developing fetus. Current literature suggests an endocrine disruptor (ED) potential of in utero exposure to these compounds. The liver is the primary site of contact with EDs in the fetus. Exposure of the fetal gonads can also alter reproductive function with potential intergenerational effects. We aimed to characterise the metabolic capability of these fetal organs. RNA sequencing was performed in 80 second trimester human fetal livers and 48 fetal gonads (balanced for fetal age and fetal sex). Samples were collected from elective terminations of normal pregnancies (liver 11-19 weeks, FeGo study: REC 04/S0802/21, and gonads 6-17 weeks, as previously described1. RNA was extracted and Illumina NextSeq was used to produce 76 bp single end (liver) or paired end 2x50 bp (gonads) sequencing reads. Reads were quality controlled, aligned to the human reference genome and quantified at gene regions. Statistical analyses involved an ANOVA model of two-way interactions between fetal sex and fetal age. All organs expressed phase I and II metabolising enzymes and drug transporters involved in the pharmacokinetic and pharmacodynamic pathways of over-the-counter analgesics. The human fetal liver expressed ABCC2, ABCC3, ABCC4 and ABCG2 receptors at similar levels between males and females. Expression of cytochrome p450 enzymes CYP2A6, CYP2C8, CYP2C9, CYP2E1, CYP3A4 involved in metabolism of the analgesics paracetamol and ibuprofen, all increased with gestational age in the liver. Expression of GSTM1, GSTP1, GSTT1, SULT1A1, SULT1A3, SULT1A4, SULT1E1, SULT2A1, UGT2B4, UGT2B7 and UGT2B15 metabolising enzymes also increased during gestation, while fetal hepatic GSTP1 expression showed a significant 2-way interaction between both sex and age. Fetal gonads expressed ABCC4 and ABCG2 transporters, with transcript levels demonstrating significant sex-specific and gestational age differences. Fewer analgesic metabolising enzymes were expressed in the gonads than the fetal liver, including CYP2E1, GSTP1 and SULT1A1, all significantly altered by gestation and fetal sex. Our results reveal expression of major analgesics metabolic and transport components within the human fetal liver, ovaries and testes between gestation weeks 7-19. Significant sex alterations in transcript levels also suggest sexually dimorphic metabolic activity of these organs during fetal life. In conclusion, analgesics can be transported into fetal liver and gonad cells and metabolised into bioactive forms, posing toxicity risks for the developing fetus.1. Lecluze E, Rolland AD, Filis P, et al. Dynamics of the transcriptional landscape during human fetal testis and ovary development. Hum Reprod. 2020;35(5):1099-1119.

Molecular Orchestration of Epithelial Mesenchymal Transition During Trophoblast and Placental Development

In Eutherian mammals, placenta is a highly specialized extra-embryonic organ that provides the feto-maternal interface and modulates the uterine environment conducive to successful pregnancy. Placenta originates from trophectoderm cells of the blastocyst and is comprised of various lineages of trophoblast cells, endothelial and smooth muscle cells. Since trophectoderm cells are epithelial in nature, it is obvious that epithelial mesenchymal transition (EMT) is a hallmark of placentation. In this report, we aimed to identify the molecular signature that regulates EMT during trophoblast and placental development in mice. On E7.5 onwards the trophoblast stem cells located in ectoplacental cone rapidly divides, differentiate leading to formation of placenta proper. Functional EMT transcriptome in mouse implantation sites on E7.5 and E9.5 were analyzed using a RT2 profiler real time PCR array scanning 84 EMT related genes. Validation of the array was performed using real time q-PCR and western blot analyses. Array result showed that there was an overall reduction of EMT gene expression in the implantation site as gestation progressed from E7.5 to E9.5 albeit the levels of EMT gene expression were substantial on both days. The differentially expressed EMT genes were further assessed during a) placental development using mouse placental samples from various days of gestation, b) induction of either EMT or differentiation in trophoblast stem (TS) cells using western blot analysis. The genes thus identified include transcription factors (Esr1, Snai2, Stat3, Foxc2), mesenchymal markers (Vim, Fn1), extracellular matrix and cell adhesion genes (Col1a2, Col3a1, Itga5, Vcan), migration and motility genes (Msn, Fn1, Pdgfrb). Extent of EMT gene expression was highest in placenta from E12.5 of mouse gestation. In addition, ectopic administration of EMT inducer led to the induction of EMT in TS cells as evidenced by EMT gene expression profile. Furthermore, differentiation of TS cells to other trophoblast lineages led to decline in EMT gene expression. Our data thus provides new insights into the importance of EMT during trophoblast stem cell differentiation and placental morphogenesis.

Immune cell composition at the feto-maternal interface in health and disease

Abstract Objectives: We hypothesize that innate and adaptive leukocyte infiltration, function and localization within the placenta is altered in health and disease and that immune cell interactions play a crucial role in maintaining immune tolerance at the fetal-maternal interface. Methods: To evaluate the complexity and differences of the immune landscape in decidua basalis we examine tissue samples from healthy women and women who developed gestational diabetes (GDM). We use flow cytometry (18-plex), fluorescent multiplex immunohistochemistry and gene expression analysis. Results: Preliminary data suggest abundant innate immune cell infiltration (macrophages and neutrophils) as well as accumulation of T-cell and NK cell subsets at the fetal-maternal interface. In gestational diabetes mellitus pregnancies, we were able to observe lower T cell infiltration when compared to healthy, which was not seen in obese pregnant women. However, in both groups (obese and GDM) there was a trend for lower T regulatory cell infiltration. For monocyte and macrophage populations a trend towards lower immune cell infiltration was observed in both obese and GDM pregnancies when compared to healthy. 70–80% of macrophages show M2 polarization, which suggest that they have an important role in anti-inflammatory processes during pregnancies. We could observe also abundant neutrophil infiltration (up to 20% of all leukocytes), although there was no difference between heathy and diseased pregnancies. Conclusion: Our study comprehends a detailed picture of the immune landscape at the fetal-maternal interface in normal and GMD pregnancies, which will aid our understanding of possible dysfunctional immune mechanisms.

Parental bias in expression and interaction of genes in the equine placenta

Most autosomal genes in the placenta show a biallelic expression pattern. However, some genes exhibit allele-specific transcription depending on the parental origin of the chromosomes on which the copy of the gene resides. Parentally expressed genes are involved in the reciprocal interaction between maternal and paternal genes, coordinating the allocation of resources between fetus and mother. One of the main challenges of studying parental-specific allelic expression (allele-specific expression [ASE]) in the placenta is the maternal cellular remnant at the fetomaternal interface. Horses (Equus caballus) have an epitheliochorial placenta in which both the endometrial epithelium and the epithelium of the chorionic villi are juxtaposed with minimal extension into the uterine mucosa, yet there is no information available on the allelic gene expression of equine chorioallantois (CA). In the current study, we present a dataset of 1,336 genes showing ASE in the equine CA (https://pouya-dini.github.io/equine-gene-db/) along with a workflow for analyzing ASE genes. We further identified 254 potentially imprinted genes among the parentally expressed genes in the equine CA and evaluated the expression pattern of these genes throughout gestation. Our gene ontology analysis implies that maternally expressed genes tend to decrease the length of gestation, while paternally expressed genes extend the length of gestation. This study provides fundamental information regarding parental gene expression during equine pregnancy, a species with a negligible amount of maternal cellular remnant in its placenta. This information will provide the basis for a better understanding of the role of parental gene expression in the placenta during gestation.

Mesenchymal stem cells induce expansion of regulatory T cells in abortion-prone mice.

Recurrent pregnancy loss (RPL) is one of the most common complications of early pregnancy associated in most cases with local or systemic immune abnormalities such as the diminished proportion of regulatory T cells (Tregs). Mesenchymal stem cells (MSCs) have been shown to modulate the immune responses by de novo induction and expansion of Tregs. In this study, we analyzed the molecular and cellular mechanisms involved in Treg-associated pregnancy protection following MSCs administration in an abortion-prone mouse mating. In a case-control study, syngeneic abdominal fat-derived MSCs were administered intraperitoneally (i.p) to the DBA/2-mated CBA/J female mice on day 4.5 of pregnancy. Abortion rate, Tregs proportion in spleen and inguinal lymph nodes, Ho1, Foxp3, Pd1 and Ctla4 genes expression at the feto-maternal interface were then measured on day 13.5 of pregnancy using flow cytometry and quantitative RT-PCR, respectively. The abortion rate in MSCs-treated mice reduced significantly and normalized to the level observed in normal pregnant animals. We demonstrated a significant induction of Tregs in inguinal lymph nodes but not in the spleen following MSCs administration. Administration of MSCs remarkably upregulated the expression of Ho1, Foxp3, Pd1 and Ctla4 genes in both placenta and decidua. Here, we show that MSCs therapy could protect the fetus in the abortion-prone mice through Tregs expansion and upregulation of Treg-related genes. These events could establish an immune-privileged microenvironment, which participates in the regulation of detrimental maternal immune responses against the semi-allogeneic fetus.

Macrophage migration inhibitory factor in human early pregnancy events and association with placental pathologies.

Macrophage migration inhibitory factor (MIF) is a versatile cytokine acting as an important regulator of innate and adaptive immunity and implicated in many physiological and pathological processes. It is abundantly expressed at the feto-maternal interface and proposed to have a role in establishing and maintaining a healthy pregnancy. This review presents the current literature data regarding the MIF role in early pregnancy events and its association with some of the placental pathological conditions, including infection, preeclampsia, gestational diabetes mellitus and choriocarcinoma. General information regarding MIF structure and function is followed by an overview of its expression in reproductive tissues and in pregnancy. Futher, we discuss MIF's involvement in the survival of decidual stromal cells, placenta of the first trimester of pregnancy, and in trophoblast cell functions studied in vitro. Current findings associating this cytokine to placental infection, preeclampsia, gestational diabetes mellitus and choriocarcinoma are presented in the final part.

Interleukin-1 beta is significantly upregulated in the decidua of spontaneous and recurrent miscarriage placentas

BackgroundPregnancy is an extraordinarily complex immunological process. For successful pregnancy maintenance the maternal immune system must adapt to and tolerate the semi-allogenic fetus at the fetomaternal interface of the placenta. This balance is regulated by cytokines with a predominant T helper 2 (Th-2) system and a suppressed inflammatory T helper 1 (Th-1) response. This study investigates the role of the Th-1 pro-inflammatory cytokine Interleukin-1 beta (IL-1β) and its role in early pregnancy loss. Patients and methodsIn order to identify differences in IL- β levels a TaqMan® Human Cytokine Network Array, with placental tissue obtained from patients with healthy pregnancies (n = 15) and recurrent miscarriage (n = 15), was carried out. Protein expression of IL-1β in the decidua of healthy pregnancies (n = 15), spontaneous (n = 18) and recurrent miscarriages (n = 15), was investigated by immunohistochemistry. The identification of IL-1β expressing cells in the decidua was done with double-immunofluorescence. ResultsGene expression analysis identified a nearly 54-times higher expression of IL-1β in placental tissue of patients suffering from recurrent abortion. Immunohistochemistry confirmed a significant upregulation of IL-1β in the decidua of recurrent miscarriage specimens (p = 0.01) as well as in the decidua of women with spontaneous abortion (p = 0.001). Double-immunofluorescence identified decidual stoma cells as IL-1β expressing cells. ConclusionSignificant upregulation of IL-1β may be associated with an imbalanced immune system and a procoagulant state that could be responsible for early pregnancy loss. These results provide new evidence of the complex interplay of IL-1β at the fetomaternal interface and its crucial role in miscarriage processes.

Extracellular vesicle mediated feto-maternal HMGB1 signaling induces preterm birth.

Preterm birth (PTB; <37 weeks of gestation) impacts ∼11% of all pregnancies and contributes to 1 million neonatal deaths worldwide annually. An understanding of the feto-maternal (F-M) signals that initiate birthing (parturition) at term is critical to design strategies to prevent their premature activation, resulting in PTB. Although endocrine and immune cell signaling are well-reported, fetal-derived paracrine signals capable of transitioning quiescent uterus to an active state of labor are poorly studied. Recent reports have suggested that senescence of the fetal amnion membrane coinciding with fetal growth and maturation generates inflammatory signals capable of triggering parturition. This is by increasing the inflammatory load at the feto-maternal interface (FMi) tissues (i.e., amniochorion-decidua). High mobility group box 1 protein (HMGB1), an alarmin, is one of the inflammatory signals released by senescent amnion cells via extracellular vesicles (exosomes; 40-160 nm). Increased levels of HMGB1 in the amniotic fluid, cord and maternal blood are associated with term and PTB. This study tested the hypothesis that senescent amnion cells release HMGB1, which is fetal signaling capable of increasing FMi inflammation, predisposing them to parturition. To test this hypothesis, exosomes from amnion epithelial cells (AECs) grown under normal conditions were engineered to contain HMGB1 by electroporation (eHMGB1). eHMGB1 was characterized (quantity, size, shape, markers and loading efficiency), and its propagation through FMi was tested using a four-chamber microfluidic organ-on-a-chip device (FMi-OOC) that contained four distinct cell types (amnion and chorion mesenchymal, chorion trophoblast and decidual cells) connected through microchannels. eHMGB1 propagated through the fetal cells and matrix to the maternal decidua and increased inflammation (receptor expression [RAGE and TLR4] and cytokines). Furthermore, intra-amniotic injection of eHMGB1 (containing 10 ng) into pregnant CD-1 mice on embryonic day 17 led to PTB. Injecting carboxyfluorescein succinimidyl ester (CFSE)-labeled eHMGB1, we determined in vivo kinetics and report that eHMGB1 trafficking resulting in PTB was associated with increased FMi inflammation. This study determined that fetal exosome mediated paracrine signaling can generate inflammation and induce parturition. Besides, in vivo functional validation of FMi-OOC experiments strengthens the reliability of such devices to test physiologic and pathologic systems.

Cell Specific Expression of Vascular Endothelial Growth Factor Receptor-2 (Flk-1/KDR) in Developing Mice Embryo and Supporting Maternal Uterine Tissue during Early Gestation (D4-D7).

BackgroundVascular endothelial growth factor (VEGF) and the corresponding receptors play key role in vascu- logenesis and angiogenesis processes. VEGF is one of the prime candidates in regulating embryo implantation by increasing vascular permeability. VEGF receptor-2, also called Flk-1/KDR, is one of the prime receptor which is actively involved in the execution of various functions of VEGF. However, precise role of this receptor during early gestation period is yet to be addressed. In the present study, expression of Flk-1/KDR during peri-implantation mice uterus as well as fetal-maternal tissues from day 4-day 7 (D4-D7) of gestation was investigated.Materials and MethodsIn this experimental study, localization of Flk-1/KDR was investigated by immunohisto- chemistry and immunofluorescence techniques, in paraffin embedded tissue sections. Flk-1/KDR protein and mRNA expressions were investigated by western blotting and quantitative reverse transcription polymerase chain reaction (qRT-PCR), respectively. Effects of ovarian steroids on expression of Flk-1/KDR were also assessed by estrogen and progesterone antagonist treatment.ResultsUterine tissue on D4 showed strong expression of Flk-1/KDR in luminal and uterine glandular epithelium. On D5 and D6, differential expression of Flk-1/KDR was evidenced in certain cell types of the embryo, maternal tissues and fetal-maternal interface with varied intensity. Flk-1/KDR was specifically expressed in the ectoplacental cone (EPC) and various cells of the embryo on D7. Flk-1/KDR expression was not evidenced in the estradiol-17β (E2) and progesterone (P4) antagonist treated uterus. Western blotting result revealed presence of Flk-1/KDR protein in the all gestation days, except antagonist treated uterus. qRT-PCR analysis showed significant increase of Flk-1/KDR mRNA transcript on D6 and D7.ConclusionSpatial-temporal expression of Flk-1/KDR during peri-implntation period in mice uterus especially in the feto-maternal interface was observed. This spatio-temporal specificity as well as increased expression of Flk-1/KDR could be one of the determinants for establishment of fetal-maternal cross talk during the critical period of develop- ment.

In the porcine placenta, aromatase (CYP19) is specifically expressed in trough-like structures of the trophoblast at the basis and flanks of chorionic folds

Aromatase was localized in the porcine placenta by immunohistochemistry during the first peak of maternal estrogen concentrations (D25; n=3), their nadir around midgestation (D50; n=3) and their second increase in late gestation (D110; n=3). On D25, aromatase was highly expressed throughout the trophoblast. On D50, its expression was restricted to the trophoblast lining the trough-like fossal regions between the bases of chorionic folds. However, immunostaining was detected only in a minor proportion of these locations, with staining intensity being generally only weak to moderate. On D110, distinct to intense immunostaining was found in virtually all trophoblast fossal regions and in similar structures at the flanks of chorionic folds adjacent to the free margins of secondary and tertiary endometrial folds. A weak staining of questionable specificity was occasionally observed in endometrial glands. In other cell types of the utero-placental compartment, aromatase was undetectable. The results suggest that in pigs placental estrogens could be involved in the formation of a more complex geometry of the feto-maternal interface as pregnancy progresses.

Mitochondrial dysfunction in placental trophoblast cells experiencing gestational diabetes mellitus.

Mitochondrial dysfunction is known to occur in diabetic phenotypes including type 1 and 2 diabetes mellitus. The incidence of gestational diabetes mellitus (GDM) is increasing and defined as the onset of a diabetic phenotype during pregnancy. The role of placental mitochondria in the aetiology of GDM remains unclear and is an emerging area of research. Differing mitochondrial morphologies within the placenta may influence the pathogenesis of the disorder. This study observed mitochondrial dysfunction in GDM placenta when assessing whole tissue. Upon further investigation into mitochondrial isolates from the cytotrophoblast and syncytiotrophoblast, mitochondrial dysfunction appears exaggerated in syncytiotrophoblast. Assessing mitochondrial populations individually enabled the determination of differences between cell lineages of the placenta and established varying levels of mitochondrial dysfunction in GDM, in some instances establishing significance in pathways previously inconclusive or confounded when assessing whole tissue. This research lays the foundation for future work into mitochondrial dysfunction in the placenta and the role it may play in the aetiology of GDM. Mitochondrial dysfunction has been associated with diabetic phenotypes, yet the involvement of placental mitochondria in gestational diabetes mellitus (GDM) remains inconclusive. This is in part complicated by the different mitochondrial subpopulations present in the two major trophoblast cell lineages of the placenta. To better elucidate the role of mitochondria in this pathology, this study examined key aspects of mitochondrial function in placentas from healthy pregnancies and those complicated by GDM in both whole tissue and isolated mitochondria. Mitochondrial content, citrate synthase activity, reactive oxygen species production and gene expression regulating metabolic, hormonal and antioxidant control was examined in placental tissue, before examining functional differences between mitochondrial isolates from cytotrophoblast (Cyto-Mito) and syncytiotrophoblast (Syncytio-Mito). Our study observed evidence of mitochondrial dysfunction across multiple pathways when assessing whole placental tissue from GDM pregnancies compared with healthy controls. Furthermore, by examining isolated mitochondria from the cytotrophoblast and syncytiotrophoblast cell lineages of the placenta we established that although both mitochondrial populations were dysfunctional, they were differentially impacted. These data highlight the need to consider changes in mitochondrial subpopulations at the feto-maternal interface when studying pregnancy pathologies.

The Emerging Role of the Prokineticins and Homeobox Genes in the Vascularization of the Placenta: Physiological and Pathological Aspects.

Vasculogenesis and angiogenesis are key processes of placental development, which occur throughout pregnancy. Placental vasculogenesis occurs during the first trimester of pregnancy culminating in the formation of hemangioblasts from intra-villous stem cells. Placental angiogenesis occurs subsequently, forming new blood vessels from existing ones. Angiogenesis also takes place at the fetomaternal interface, allowing essential spiral arteriole remodeling to establish the fetomaternal circulation. Vasculogenesis and angiogenesis in animal models and in humans have been studied in a wide variety of in vitro, physiological and pathological conditions, with a focus on the pro- and anti-angiogenic factors that control these processes. Recent studies revealed roles for new families of proteins, including direct participants such as the prokineticin family, and regulators of these processes such as the homeobox genes. This review summarizes recent advances in understanding the molecular mechanisms of actions of these families of proteins. Over the past decade, evidence suggests increased production of placental anti-angiogenic factors, as well as angiogenic factors are associated with fetal growth restriction (FGR) and preeclampsia (PE): the most threatening pathologies of human pregnancy with systemic vascular dysfunction. This review also reports novel clinical strategies targeting members of these family of proteins to treat PE and its consequent effects on the maternal vascular system.

Uterine Scarring Leads to Adverse Pregnant Consequences by Impairing the Endometrium Response to Steroids.

Uterine surgical scarring is an increasing risk factor for adverse pregnant consequences that threaten fetal-maternal health. The detailed molecular features of scar implantation remain largely unknown. We aim to study the pathologic features of uterine surgical scarring and the mechanisms of compromised pregnancy outcomes of scar implantation. We generated a mouse model of uterine surgical scarring with a uterine incision penetrating the myometrium to endometrium to examine the pathologic changes and transcriptome profiles of uterine scarring at various postsurgery (PS) time points, as well as features of the feto-maternal interface during scar implantation. We found that uterine surgical scar recovery was consistently poor at PS3 until PS90, as shown by a reduced number of endometrial glands, inhibition of myometrial smooth muscle cell growth but excessive collagen fiber deposition, and massive leukocyte infiltration. Transcriptome annotation indicated significant chronic inflammation at the scarring site. At the peri-implantation and postimplantation stages, abnormal expression of various steroid-responsive genes at the scarring site was in parallel with lumen epithelial cell hyperplasia, inappropriate luminal closure, and disorientation of the implanted embryo, restricted stromal cell proliferation, and defective decidualization. High embryonic lethality (around 70%) before E10.5 was observed, and the small amount of survival embryos at E10.5 exhibited restricted growth and aberrant placenta defects including overinvasion of trophoblast cells into the decidua and insufficient fetal blood vessel branching in the labyrinth. The findings indicate that chronic inflammation and compromised responses to steroids in uterine scar tissues are the pivotal molecular basis for adverse pregnancy consequences of scar implantation.