Extraembryonic Structures Research Articles

Особенности морфофункционального состояния маточно-плацентарной единицы и эмбриона мыши на ранних сроках экспериментального невынашивания беременности

Introduction. The majority of cases of habitual miscarriage are closely related to impaired maternal immune tolerance. Experimental mice models of abortion, where the critical time points are established to monitor pregnancy progression, are widely used to study the pathogenesis of early reproductive loss. However, the relationship between the embryonic development and extraembryonic structures with the immune microen-vironment is not well understood. The aim of the study is to compare the morphofunctional characteristics of the uteroplacental unit and embryo in early pregnancy in mice with experimental spontaneous, induced, and potentiated abortions. Materials and methods. Allogeneic physiological pregnancy was modeled using ♀CBA × ♂BALB/c mice mating. Spontaneous abortions were reproduced by mating ♀CBA × ♂DBA/2 mice. Induced abortions and potentiated abortions were triggered by intraperitoneal administration of the immunomodulator ß-heptylglycoside muramyl dipeptide (MDP) to pregnant females on gestation day (gd) 5 and gd 7 mated in following combinations ♀CBA × ♂BALB/c and ♀CBA × ♂DBA/2, respectively. Mice were withdrawn from the experiment on gd 8, then uterine horns and uteroplacental units were isolated. The material was fixed in 10% neutral formalin, and standard processing was performed for histological examination. In equatorial sections, the cross-sectional area of uteroplacental unit was measured, and maternal, embryonic, as well as extraembryonic structures were analyzed. An immunohistochemical assay with rabbit polyclonal anti-CD68 antibodies was conducted to detect macrophages, then the density of CD68+ cells per 1 mm2 of the decidual area was determined. Results. On day 8 of pregnancy, in the groups of induced and potentiated abortions cross-sectional area of the uteroplacental units was decreased compared with the groups of physiological pregnancy and sponta-neous abortions. Induced and potentiated abortions demonstrated vasodilation in the mesometric decidua zone and stagnation of blood in the blood vessels, compaction of trophoblast, and hemorrhage in decidua basalis whether spontaneous abortions showed occluded maternal vessels surrounded by edematous and vacuolated trophoblast cells.At the early stages of modeling of spontaneous and immuno-dependent abortions, disturbances in the devel-opment of the embryo and extraembryonic structures were revealed, ranging from initial signs of resorption to the destruction of the embryo and membranes.In viable embryos, density indices of CD68+ macrophages in the decidual membrane of the uteroplacental units did not differ significantly in all miscarriage cases, CD68+ cells were not detected in cases of embryo-nal resorption. Conclusion. The MDP treatment in a period after completion of implantation and before forming a mature placenta (gd 5–7) results in the inducing or the potentiating abortions in CBA females mated with by Balb/c or DBA/2 males, respectively. In both cases, we revealed disturbed chorion morphogenesis and embryonic development to the extent of its complete resorption and the absence of CD68+ macrophages in resorbed embryos’ microenvironment on day 8 of pregnancy. Keywords: miscarriage, models, muramyl dipeptide β-heptylglycoside, uteroplacental unit, macrophages, immunotolerance

From Mother or Father: Uniparental Embryos Uncover Parent-of-Origin Effects in Humans.

In mammals, both parents make unique contributions to the offspring and maternal and paternal genomes are required for development. Two recent papers in Cell Stem Cell (Leng etal., 2019; Sagi etal., 2019) study uniparental embryos and uniparental embryonic stem cells to interrogate parent-of-origin effects in human embryogenesis.

Carcinogenesis: the cancer cell-mast cell connection.

In mammals, inflammation is required for wound repair and tumorigenesis. However, the events that lead to inflammation, particularly in non-healing wounds and cancer, are only partly understood. Mast cells, due to their great plasticity, could orchestrate the inflammatory responses inducing the expression of extraembryonic programs of normal and pathological tissue formation. This heterogeneity of mast cells could allow a microenvironment to be recreated similar to the extraembryonic structures, i.e., amnion and yolk sac, which are needed for embryonic development. Mast cells could provide a framework for understanding the connection between inflammation and tumor growth, invasion and metastasis. In this way, the mast cells could express inflammatory phenotypes, which would enable the cancer stem cells to develop. Thus, the cancer cell uses mast cells to express the extraembryonic functions that are needed to allow the cancer stem cell to proliferate and invade. If so, then by using this appropriate inflammatory interstitial microenvironment, a cancer stem cell can reach maximum levels of growth and invasion inside the host. Therefore, the comparison of tumors with wounds that do not heal would be supported since both pathological processes use extraembryonic mechanisms by mast cells. The adoption of these mechanisms warrants tumor survival in an embryonic-like state.

Survivin from Flk1+ Endothelial Progenitor Cells in the Developing Embryo Ameliorates Maternal Diabetes–Induced Neural Tube Defects through Exosome

Neural tube defects (NTDs) are among the most common and severe structural birth defects and maternal diabetes induces NTDs. During mouse embryonic development, blood vessel formation initially occurs in the yolk sac, preceding neurulation which takes place from embryonic day 8 (E8) to E10.5. Our previous studies have demonstrated that maternal diabetes induces embryonic vasculopathy at early embryonic developmental stage. We hypothesize that early vasculopathy accounts for late failed neural tube closure in diabetic pregnancy. To test this hypothesis, we created the Flk1 promoter driven survivin (the cell cycle-regulated apoptosis inhibitor) transgenic mice. Maternal diabetes induced the loss of Flk1+ progenitors, impaired blood island formation and reduced blood vessel densities. Expression of survivin in the Flk1+ progenitors ameliorated diabetes-induced NTDs. Under nondiabetic conditions, survivin overexpression did not affect embryonic development. Overexpression of survivin significantly reduced diabetes-induced NTDs. Flk1+ progenitors produced survivin-containing exosomes. Immunocytochemistry staining indicated that survivin-containing exosomes were transported and incorporated into cells of the developing neuroepithelium, leading to blockage of neuroepithelial cell apoptosis induced by maternal diabetes. Furthermore, we isolated exosomes from Flk1+ progenitors derived from E7.5 embryos. In utero delivery of the survivin-containing exosomes by microinjection into the amniotic cavity of diabetic mice at E9 significantly inhibited maternal diabetes-induced neuroepithelial cell apoptosis and reduced NTDs. Taken together, these data revealed that the cross-talk between the extra-embryonic structures and the developing neuroepithelium through the survivin-containing exosome plays a critical role for the proper neural tube closure. Disclosure S. Cao: None. W. Shen: None. P. Yang: None.

ULTRASONIC PARAMETERS OF EXTRAEMBRYONIC STRUCTURES DURING THE PERIOD OF PLACENTAL COMPLEX FORMATION IN WOMEN WITH PELVIC INFLAMMATORY DISEASES

One of the most important triggers for PD development is the diffuse-perfusion failure of uteroplacental, intraplacental and fetus-placental circulation. In this case, there are violations of transport, trophic, endocrine, metabolic and antitoxic functions of the placenta, which leads to the pathology of the fetus and the newborn. In order to evaluate the functional status of the FPC at the stage of the primary placental dysfunction formation in women with pelvic inflammatory diseases, we used the determination of the echographic extraembryonic structures’ parameters (volume of the yolk sac, corpus luteum, choroidal and amniotic cavity, localization of the chorion) in the women of the main and control groups, 6-7 and 12-14 weeks of gestation. Determination of the yolk sac volume, corpus luteum, choroidal and amniotic cavity, chorion localization was carried out by the three-dimensional VOCAL echography program. According to the goal, an ultrasound examination was performed on 67 women in 6-7 and 12-14 weeks of gestation. The main group consisted of 37 women with signs of inflammatory diseases of female genital organs, a control group of 30 women was practically healthy Investigation of extraembryonic structures in women with genital tract infections is an actual method of diagnosing the development of primary placental dysfunction.

FEATURES OF DEVELOPMENT OF THE EMBRYO IN PREGNANT WOMEN WITH THE DYSFUNCTION OF CHILDBEARING

Summary. Introduction. Researching of ultrasound parameters of the embryo and extraembryonic structures under placental insufficiency against the background of non-pregnancy is not yet sufficiently studied, which determines its relevance.Goal. To develop normative parameters of formation of the embryo and extraembryonic structures, to evaluate the peculiarities of the development of the fetoplacental system during uncomplicated pregnancy and in the presence of manifestations of placental insufficiency in case of non-pregnancy by conducting complexultrasound, cardiotocographic examination.Matherials and metods. We examined 30 somatically healthy women with physiological course of pregnancy from 5 to 40 weeks of pregnancy and 32 pregnant women with symptoms of placental dysfunction and miscarriage in anamnesis.Results. In the group of pregnant women with recurrent pregnancy loss in the majority of cases (21) the dynamics of changes in heart rate of an embryo within the parameters of the physiological course of pregnancy.The functional state of the placenta in many cases due to the degree of its development in accordance with the gestational period and the preservation of compensatory - adaptive mechanisms. Ultrasonic placentometria of pregnant women in the control group during thegestational period showed the line thickness of the placenta gestating normal in 94% of pregnant women. At the same time, 2 (6.6%) pregnant women in the observed decrease in this indicator. ’Thick’ placenta was observed at 4 patients (13.3%)of pregnant control group.Conclusions. Wapost of maturity of the placenta, the gestational period is one of the most important conditions to ensure the adequate development of the fetus and its protection.

Expression of cancer-testis antigens of the Mage family in mouse oocytes and early embryos

Cancer-testis antigens of the Mage family (Melanoma antigens) are expressed predominantly in the spermatogenic and cancer cells, but some genes of this family are expressed ubiquitously. Expression patterns and functional role of Mage family antigens in the regulation of cellular processes in normal embryonic and definitive cells are virtually unknown. Comparative immunofluorescent analysis of Mage expression in mouse oocytes and early embryos identified the expression of Mage antigens at all stages studied. The greatest intensity of the fluorescent staining was detected in the epiblasts and the extraembryonic structures of the egg cylinder at E6.5 stage. At all studied developmental stages of the mouse oocyte and the early embryo, the localization of Mage antigens was found predominantly in the cytoplasm. Quantitative real-time PCR showed that expression levels of most Mage genes in cells of the epiblast and ectoplacental cone were similar, while the gene expression levels of Mage-a10, Mage-b16, and Mage-b18 were higher in cells of the ectoplacental cone than in epiblast cells. Thus, for the first time, our analysis has shown that the Mage family antigens are expressed at the early stages of mouse development and may be involved in the regulation of earliest events of embryogenesis.

Корекція кровотеч в ранні терміни гестації залежно від стану ембріона та екстраембріональних структур

The objective: the study of the status of the embryo and extraembryonic structures for bleeding that is not associated with the detachment of the chorion in the first trimester of gestation. Patients and methods. Were examined 34 patients in the gestational age 6-9 weeks (main group) with bleeding from the genital tract, in which, at the primary ultrasonic inspection revealed no detachment of the chorion. The control group included 30 women with uncomplicated course And trimester of gestation. The average age of the pregnant women of the main group was 25.3±3.1 years in the control group 26.4±4.3 years. All women underwent standard clinical and laboratory examination. Results. Studies have shown that prognostic markers of unfavorable development of the embryo in the early stages of pregnancy bleeding not related to chorion detachment was not detected. For the purpose of hemostasis in pregnant of the main group of prescribed drugs traneksamic acid 0.25 mg 3-4 times a day for 3 days. Traneksam – antifbrinolytic tool is able to block not only plasminogen, and plasmin that has already formed. It also stimulates the synthesis of collagen, through which increases the elasticity of the fibrin clot. Pregnant women in both groups received vitamin E 1 capsule per day magnesium products, Magnicum 1 tablet 3 times a day, folic acid 400 micrograms per day. Assessing clinical signs of disease in dynamics of treatment, it should be noted that the hemostasis in the main group was achieved in an average of 2.3±0.7 days. Conclusion. Bleeding in early gestation, not connected with the detachment of the chorion, the use of Traneksam provides fast absolute hemostasis, which in turn contributes to the normalization of tone of the uterus and the physiological course of placentation. Key words: bleeding in early pregnancy, the chorion detachment, hemostasis, hemostatic therapy, Traneksam.

Gene Expression Noise Enhances Robust Organization of the Early Mammalian Blastocyst.

A critical event in mammalian embryo development is construction of an inner cell mass surrounded by a trophoectoderm (a shell of cells that later form extraembryonic structures). We utilize multi-scale, stochastic modeling to investigate the design principles responsible for robust establishment of these structures. This investigation makes three predictions, each supported by our quantitative imaging. First, stochasticity in the expression of critical genes promotes cell plasticity and has a critical role in accurately organizing the developing mouse blastocyst. Second, asymmetry in the levels of noise variation (expression fluctuation) of Cdx2 and Oct4 provides a means to gain the benefits of noise-mediated plasticity while ameliorating the potentially detrimental effects of stochasticity. Finally, by controlling the timing and pace of cell fate specification, the embryo temporally modulates plasticity and creates a time window during which each cell can continually read its environment and adjusts its fate. These results suggest noise has a crucial role in maintaining cellular plasticity and organizing the blastocyst.

PREDICTION OF ADVERSE PREGNANCY OUTCOME FOR THE FETUS ON THE BASIS OF ULTRASOUND INVESTIGATION OF THE EMBRYO AND EXTRAEMBRYONIC STRUCTURES

Aim. Identify the most informative ultrasound criteria for the pathology of the embryo and extraembryonic structures from the 8th to the 11th weeks of gestational age in order to predict the course and outcome of pregnancy for the fetus. Materials and methods. 215 pregnant women were examined during gestation from 8 to 11 weeks. Results. By calculation of sensitivity and specificity, the most informative parameters of unfavorable development of pregnancy were revealed. Parameters of adverse course and outcome of pregnancy for the fetus are the following highly specific signs: absence of yolk sac, inadequate development of yolk sac, inadequate vascularization of the chorion. Lowspecific signs are: tone of myometrium, inadequate vascularization of the yellow body. Conclusion. Ultrasound evaluation of the embryo and extraembryonic structures during gestation from 8 to 11 weeks makes possible predicting the features of the course and the unfavorable outcome of pregnancy for the fetus.

Characterization of egg white antibacterial properties during the first half of incubation: A comparative study between embryonated and unfertilized eggs

Egg white is an important contributor to the protection of eggs against bacterial contaminations during the first half of incubation (day zero to 12), prior to the egg white transfer into the amniotic fluid to be orally absorbed by the embryo. This protective system relies on an arsenal of antimicrobial proteins and on intrinsic physicochemical properties that are generally unfavorable for bacterial multiplication and dissemination. Some changes in these parameters can be observed in egg white during egg storage and incubation. The aim of this work was to characterize changes in the antibacterial potential of egg white in embryonated eggs (FE) during the first half of incubation using unfertilized eggs (UF) as controls. Egg white samples were collected at day zero, 4, 8, and 12 and analyzed for pH, protein concentration, and protein profile. Antibacterial properties of egg white proteins were evaluated against Listeria monocytogenes, Streptococcus uberis, Staphylococcus aureus, Escherichia coli, and Salmonella Enteritidis. During incubation, differential variations of egg white pH and protein concentrations were observed between UF and FE. At equal protein concentrations, similar activities against L. monocytogenes and S. uberis were observed for FE and UF egg white proteins. A progressive decline in these activities, however, was observed over incubation time, regardless of the egg group (UF or FE). SDS-PAGE analysis of egg white proteins during incubation revealed discrete changes in the profile of major proteins, whereas the stability of some less abundant antimicrobial proteins seemed more affected. To conclude, the antibacterial activity of egg white proteins progressively decreased during the first half of egg incubation, possibly resulting from the alteration of specific antimicrobial proteins. This apparent decline may be partly counterbalanced in embryonated eggs by the increase in egg white protein concentration. The antibacterial potential of egg white is very effective during early stages of embryonic development but its alteration during incubation suggests that extra-embryonic structures could then progressively ensure protective functions.

Distinct mechanisms regulate Cdx2 expression in the blastocyst and in trophoblast stem cells.

The first intercellular differences during mammalian embryogenesis arise in the blastocyst, producing the inner cell mass and the trophectoderm. The trophectoderm is the first extraembryonic tissue and does not contribute to the embryo proper, its differentiation instead forming tissues that sustain embryonic development. Crucial roles in extraembryonic differentiation have been identified for certain transcription factors, but a comprehensive picture of the regulation of this early specification is still lacking. Here, we investigated whether the regulatory mechanisms involved in Cdx2 expression in the blastocyst are also utilized in the postimplantation embryo. We analyzed an enhancer that is regulated through Hippo and Notch in the blastocyst trophectoderm, unexpectedly finding that it is inactive in the extraembryonic structures at postimplantation stages. Further analysis identified other Cdx2 regulatory elements including a stem-cell specific regulatory sequence and an element that drives reporter expression in the trophectoderm, a subset of cells in the extraembryonic region of the postimplantation embryo and in trophoblast stem cells. The cross-comparison in this study of cis-regulatory elements employed in the blastocyst, stem cell populations and the postimplantation embryo provides new insights into early mammalian development and suggests a two-step mechanism in Cdx2 regulation.

Capturing the ephemeral human pluripotent state.

During human development, pluripotency is present only in early stages of development. This ephemeral cell potential can be captured in vitro by obtaining pluripotent stem cells (PSC) with self-renewal properties, the human embryonic stem cells (hESC). However, diverse studies suggest the existence of a plethora of human PSC (hPSC) that can be derived from both embryonic and somatic sources, depending on defined culture conditions, their spatial origin, and the genetic engineering used for reprogramming. This review will focus on hPSC, covering the conventional primed hESC, naïve-like hPSC that resemble the ground-state of development, region-selective PSC, and human induced PSC (hiPSC). We will analyze differences and similarities in their differentiation potential as well as in the molecular circuitry of pluripotency. Finally, we describe the need for human feeder cells to derive and maintain hPSC, because they could emulate the interaction of in vivo pluripotent cells with extraembryonic structures that support development. Developmental Dynamics 245:762-773, 2016. © 2016 Wiley Periodicals, Inc.

Experimental Infection of Sheep at 45 and 60 Days of Gestation with Schmallenberg Virus Readily Led to Placental Colonization without Causing Congenital Malformations.

BackgroundMain impact of Schmallenberg virus (SBV) on livestock consists in reproductive disorders, with teratogenic effects, abortions and stillbirths. SBV pathogenesis and viral placental crossing remain currently poorly understood. Therefore, we implemented an experimental infection of ewes, inoculated with SBV at 45 or 60 days of gestation (dg).Methodology“Mourerous” breed ewes were randomly separated in three groups: eight and nine ewes were subcutaneously inoculated with 1 ml of SBV infectious serum at 45 and 60 dg, respectively (G45 and G60). Six other ewes were inoculated subcutaneously with sterile phosphate buffer saline as control group. All SBV inoculated ewes showed RNAemia consistent with previously published studies, they seroconverted and no clinical sign was reported. Lambs were born at term via caesarian-section, and right after birth they were blood sampled and clinically examined. Then both lambs and ewes were euthanatized and necropsied.Principal Findings/SignificanceNo lambs showed any malformation suggestive of SBV infection and none of them had RNAemia or anti-SBV antibodies prior to colostrum uptake. Positive SBV RNA detection in organs was rare in both G45 and G60 lambs (2/11 and 1/10, respectively). Nevertheless most of the lambs in G45 (9/11) and G60 (9/10) had at least one extraembryonic structure SBV positive by RTqPCR. The number of positive extraembryonic structures was significantly higher in G60 lambs. Time of inoculation (45 or 60 dg) had no impact on the placental colonization success rate but affected the frequency of detecting the virus in the offspring extraembryonic structures by the time of lambing. SBV readily colonized the placenta when ewes were infected at 45 or 60 dg but infection of the fetuses was limited and did not lead to congenital malformations.

Ultrasound Imaging of Early Extraembryonic Structures

ABSTRACT Transvaginal sonography is the most useful diagnostic method to visualize the early pregnancy, to determine whether it is intrauterine or extrauterine (ectopic), viable or not. Detailed examination of extraembryonic structures allows us to differentiate the types of early pregnancy failures and highlights the backgrounds of vaginal bleeding, as the most frequent symptom of the first trimester of gestation. The reliable ultrasonographic sign of an intrauterine pregnancy is visualization of double decidual ring, which represents the trophoblast's layer. The abnormality in the sonographic appearance of a gestational sac, a yolk sac, and a chorionic plate can predict subsequent embryonic damage and death. How to cite this article Nagy S, Papp Z. Ultrasound Imaging of Early Extraembryonic Structures. Donald School J Ultrasound Obstet Gynecol 2017;11(1):11-19.

Transmission and Scanning Electron Microscopy of the Accessory Cells and Chorion During Development of Ciona intestinalis Type B Embryos and the Impact of Their Removal on Cell Morphology.

Spawned ascidian oocytes are surrounded by a membrane called the chorion (or vitelline coat) and associated with two populations of maternally-supplied cells. Outside the chorion are follicle cells, which may affect the buoyancy of eggs. Inside the chorion are test cells, which during oogenesis provision the egg and which after fertilisation contribute to the larval tunic. The structure of maternal cells may vary between species. The model ascidian Ciona intestinalis has been recently split into two species, currently named type A and type B. The ultrastructure of extraembryonic cells and structures from type A embryos has been reported. Here we describe the ultrastructure of follicle and test cells from C. intestinalis type B embryos. Test cells are about 5 µm in diameter and line the inside of the chorion of developing embryos in a dense sheet. Follicle cells are large (> 100 µm long) and spike-shaped, with many large vesicles. Terminal electron dense granules are found towards the tips of spikes, adjacent to cytoplasm containing numerous small electron dense bodies connected by filaments. These are probably vesicles containing material for the terminal granules. Removal of maternal structures and cells just after fertilisation, as commonly used in many experiments manipulating C. intestinalis development, has been reported to affect embryonic patterning. We examined the impact of this on embryonic ectoderm cells by scanning electron microscopy. Cells of embryos that developed without maternal structures still developed cilia, but had indistinct cell boundaries and a more flattened appearance than those that developed within the chorion.

Retinoic Acid and the Development of the Endoderm

Retinoic acid (RA) is an important signaling molecule in the development of the endoderm and an important molecule in protocols used to generate endodermal cell types from stem cells. In this review, we describe the RA signaling pathway and its role in the patterning and specification of the extra embryonic endoderm and different endodermal organs. The formation of endoderm is an ancient evolutionary feature and RA signaling appears to have coevolved with the vertebrate lineage. Towards that end, we describe how RA participates in many regulatory networks required for the formation of extraembryonic structures as well as the organs of the embryo proper.

Acyl-CoA:lysocardiolipin acyltransferase (Lycat): a potent pathogenic factor in antiphospholipid syndrome (APS)-related recurrent pregnancy failure

To validate a novel model system to study the pathogenesis of recurrent miscarriages in APS patients. Experimental animal study. Elevated titers of antiphospholipid antibodies (aPL) have a close association with many obstetric disorders. Cadiolipin (CL), a major phospholipid of mitochondria, regulates membrane permeability and potential. Remodeling enzyme Lycat is required for both CL maturation and repair of damaged acyl chains. At present, APS animal models were generally created by immunization of exogenous antigens. This study presents the first genetic model in which elevated Lycat leads to a drastic rise of aPL, concomitant with a high rate of in utero fetal loss. Mice over-expressing Lycat were generated by BAC transgenesis (Tg). Fertility were compared between Tg and WT mice. Mutant implantation sites were sectioned for histology, anti-phospho-H3 IF, TUNEL and in situ hybridization. Altered global gene expression of the mutant conceptus was first tested by NextGENe RNA-seq technology, followed by Q-PCR. Serum aPL were tested by ELISA. At least 3 samples were included in each assay for each genotype. Unpaired student's t-test was performed between WT and Tg groups. All Tg female mice showed elevated aPL, whose levels are positively correlated to the severity of fetal loss. Pregnant Tg females produced fewer pups than the controls (Tg, 2.3±0.2, v.s. WT, 8.5±1.4; p<0.01). Degenerating conceptus revealed faulty extraembryonic structures involving trophoblast giant cells, chorion and amnion. As a result, the embryos developmentally arrested by E7.5. Absence of EOMES, Hand1, PL1 and Tpbp indicates defective trophoblast differentiation; while altered expression of Gas1 and Foxh1 suggests mis-instructed embryogenesis. Decreased synthesis of hemoglobins by decidual cells indicates severe oxygen starvation in the mutants. Pathogenic effects of elevated Lycat are multifold, in which oxygen homeostasis, trophoblast differentiation and embryo growth are all affected.

A Big Catch for Germ Cell Tumour Research

A Big Catch for Germ Cell Tumour Research

Morphological evaluation of the placenta and fetal membranes during canine pregnancy from early implantation to term

To describe the histological changes of fetal adnexa throughout the physiological pregnancy, canine samples were obtained during natural delivery and caesarean section, as well as during ovariohysterectomy performed at any stage of undesired pregnancies (N=12). The first period of pregnancy (multiple samples collected at 10, 12, 14days) was consistent with pre- and peri-implantation events, i.e. apposition and initial invasion steps into the endometrium. The second period (multiple samples collected at 18, 38, 40, 45days) was related to the development of extra-embryonic structures, placenta establishment and labyrinth formation. At the end of this period the maximum morphological complexity of the endotheliochorial placenta was achieved, characterized by complete erosion of the endometrial epithelium and underlying interstitium with exposure of maternal capillaries to the chorial cells. The third period of gestation (multiple samples collected at 50, 53, 57, 60, 63days) was characterized by enhancement either of placental and extra-embryonic tissues.