Hofbauer cells of M2a, M2b and M2c polarization may regulate feto-placental angiogenesis.

The human placenta comprises a special type of tissue macrophages, the Hofbauer cells (HBC), which exhibit M2 macrophage phenotype. Several subtypes of M2-polarized macrophages (M2a, M2b and M2c) exist in almost all tissues. Macrophage polarization depends on the way of macrophage activation and leads to the expression of specific cell surface markers and the acquisition of specific functions, including tissue remodeling and the promotion of angiogenesis. The placenta is a highly vascularized and rapidly growing organ, suggesting a role of HBC in feto-placental angiogenesis. We here aimed to characterize the specific polarization and phenotype of HBC and investigated the role of HBC in feto-placental angiogenesis. Therefore, HBC were isolated from third trimester placentas and their phenotype was determined by the presence of cell surface markers (FACS analysis) and secretion of cytokines (ELISA). HBC conditioned medium (CM) was analyzed for pro-angiogenic factors, and the effect of HBC CM on angiogenesis, proliferation and chemoattraction of isolated primary feto-placental endothelial cells (fpEC) was determined in vitro Our results revealed that isolated HBC possess an M2 polarization, with M2a, M2b and M2c characteristics. HBC secreted the pro-angiogenic molecules VEGF and FGF2. Furthermore, HBC CM stimulated the in vitro angiogenesis of fpEC. However, compared with control medium, chemoattraction of fpEC toward HBC CM was reduced. Proliferation of fpEC was not affected by HBC CM. These findings demonstrate a paracrine regulation of feto-placental angiogenesis by HBC in vitro Based on our collective results, we propose that the changes in HBC number or phenotype may affect feto-placental angiogenesis.

Expression of serum amyloid A4 in human trophoblast-like choriocarcinoma cell lines and human first trimester/term trophoblast cells.

Trophoblast invasion into uterine tissues represents a hallmark of first trimester placental development. As expression of serum amyloid A4 (SAA4) occurs in tumorigenic and invasive tissues we here investigated whether SAA4 is present in trophoblast-like human AC1-M59/Jeg-3 cells and trophoblast preparations of human first trimester and term placenta. SAA4 mRNA was expressed in non-stimulated and cytokine-treated AC1-M59/Jeg-3 cells. In purified trophoblast cells SAA4 mRNA expression was upregulated at weeks 10 and 12 of pregnancy. Western-blot and immunohistochemical staining of first trimester placental tissue revealed pronounced SAA4 expression in invasive trophoblast cells indicating a potential role of SAA4 during invasion.

Mesenchymal stromal cells from the human placenta promote neovascularization in a mouse model in vivo.

Cell transplantation is a promising strategy in regenerative medicine for revascularization of ischemic tissues. Based on our observation that placental mesenchymal stromal cells (PMSC) enhance endothelial cell viability in vitro via secretion of angiogenic factors, we asked whether PMSC support vascular growth in vivo. PMSC were isolated from amnion and placental endothelial cells (PLEC) from chorion and either separately or co-transplanted subcutaneously into immune-deficient mice. Co-transplantation resulted in a higher number of perfused human vessels (CD31+/vimentin+) containing mouse glycophorin A+ erythrocytes. Results indicate positive effects of PMSC on neovascularization in vivo, making them attractive candidates to create autologous PMSC/PLEC pairs for research and transplantation.

Distribution of decidual natural killer cells and macrophages in the neighbourhood of the trophoblast invasion front: a quantitative evaluation.

STUDY QUESTION: Do decidual natural killer (dNK) cells and decidual macrophages (dMph) become enriched in the vicinity of the trophoblast invasion front? SUMMARY ANSWER: Morphometric image analysis and areal cell density calculations, which excluded observer bias, showed an enrichment of decidual leukocytes in the neighbourhood of the trophoblast invasion front. WHAT IS KNOWN ALREADY: In previous studies, the number of decidual leukocytes was visually counted in medium- or high power fields. These methods, however, cannot reveal the exact spatial relationship between leukocytes and invasive trophoblast cells, and are therefore prone to subjective errors. Thus, a more objective approach is required. STUDY DESIGN, SIZE, DURATION: Applying a new method of morphometric image analysis, leukocyte populations were studied in human tissue fragments derived from first trimester placentation sites (n = 7) as well as in co-cultures of first trimester decidual tissue with placental villi of the same pregnancy representing an appropriate in vitro model of trophoblast invasion (n = 15). PARTICIPANTS/MATERIALS, SETTINGS, METHODS: First trimester decidual tissue was obtained from women undergoing elective terminations of pregnancy at 7-10 weeks of gestational age. Tissue sections were double-stained immunohistochemically for markers of dNK cells or dMph on one hand, and for invasive extravillous trophoblast cells on the other. To analyse the distribution of leukocytes, distinct cell compartments as well as cell neighbourhood areas were defined. Finally, relative areal cell densities were calculated and these data were compared with those of an in vitro model of trophoblast invasion as well as with tissue fragments derived from decidua parietalis without trophoblast cells. MAIN RESULTS AND THE ROLE OF CHANCE: At first trimester placentation sites, a higher density of dNK cells as well as of dMph was found in close proximity to the invasive trophoblast (P ≤ 0.01), compared with the average areal cell density of decidual leukocytes in the tissue with exclusion of the trophoblast. The highest areal cell density of leukocytes was determined up to a distance of 20 µm from the trophoblast cells, whereas in more distant regions it was even lower than average, indicating a migration of these leukocytes towards the trophoblast invasion front. In the three-dimensional co-culture model, however, we found an enrichment of dMph (P ≤ 0.01) but not of dNK cells (P > 0,05) in the neighbourhood of the invasive trophoblast. LIMITATIONS, REASONS FOR CAUTION: The morphometric image analysis depends on intense immunohistochemical staining that is free of background and cross-reactivity. WIDER IMPLICATIONS OF THE FINDINGS: The presented method will be useful not only for the investigation of recurrent miscarriage but also in the fields of tumour immunology and inflammation. STUDY FUNDING/COMPETING INTEREST(S): The study was supported by the European Commission (Network of Excellence 'The Control of Embryo Implantation (EMBIC)', FP6-512040, lead researcher: P.S.), and by the Franz Lanyar Foundation of the Medical University of Graz, Austria (Grant #347). None of the authors declared a conflict of interests.

OS066. Intrauterine CYP2J2 expression and circulatingepoxyeicosatrienoic acid levels in preeclampsia.

INTRODUCTION: The cytochrome P450 (CYP)-system regulates vascular functions, inflammation, and angiogenesis that are mechanistically important in preeclampsia. OBJECTIVES: The aim of this study was to analyze the dysregulation of the Cytochrome P450 in the pathogenesis of preeclampsia. METHODS: We performed microarray screening of placenta and decidua from 25 preeclamptic women and 23 controls. Results were confirmed by realtime RT-PCR, immunohistochemistry and Serum of patients were analyzed by HPLC tandem mass spectrometry. For functional testing we did cardiomyocyte contraction bioassay and myograph studies. The reduced uterine perfusion pressure (RUPP) rat model was proceed for interventional study. RESULTS: In microarray studies the CYP subfamily 2J polypeptide 2 (CYP2J2) was upregulated in preeclamptic decidual tissue (3.9 fold, p<0.0001) and in preeclamptic placenta (1.55 fold, p<0.001). RT-PCR confirmed the upregulation and immunohistochemistry, localized CYP2J2 in trophoblasts of villi and deciduas at week 12 and term. The CYP2J2 metabolites were analyzed by HPLC tandem mass spectrometry. 5,6- epoxyeicosatrienoic acids (EET), 14,15-EET, and the corresponding dihydroxyeicosatrienoic acids (DHET), were elevated in preeclamptic women compared to controls in the latter two-thirds of pregnancy and after delivery. Stimulation of the trophoblast-derived cell line SGHPL-4 with the preeclampsia-associated cytokine tumor necrosis factor-a enhanced CYP2J2 gene and protein expression. For functional testing, 5,6-EET increased the beating rate of neonatal cardiomyocytes in a bioassay and downregulated large-conductance calcium-activated potassium channel KCa 1.1 activity. In the RUPP rat model of preeclampsia, we observed elevated EET, DHET, and preeclamptic features that were ameliorated by the CYP inhibitor MsPPOH. Uterine arterial rings of rats also dilated in response to MsPPOH. CONCLUSION: Our data implicate CYP2J2 in the pathogenesis of preeclampsia and as a potential candidate for the disturbed uteroplacental remodeling, leading to hypertension and endothelial dysfunction.

IFPA Meeting 2011 workshop report III: Placental immunology; epigenetic and microRNA-dependent gene regulation; comparative placentation; trophoblast differentiation; stem cells.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialised topics. At IFPA meeting 2011 there were twelve themed workshops, five of which are summarized in this report. These workshops related to various aspects of placental biology: 1) immunology; 2) epigenetics; 3) comparative placentation; 4) trophoblast differentiation; 5) stem cells.

Placental markers of folate-related metabolism in preeclampsia.

The etiology and degree of clinical symptoms of preeclampsia depend on genotypic and phenotypic maternal and trophoblast factors, and elevated levels of plasma homocysteine (Hcy) are one of the pathogenetic factors of preeclampsia. To assess the impact of the folate-related metabolism, we characterized the indices of this metabolism in 40 samples from uncomplicated term placentas and 28 samples from preeclamptic pregnancies by quantifying the total content of folate, methionine (Met), Hcy and related cysteine, and glutathione (GSH) in compliance with the 677 C/T genotype of methylene tetrahydrofolate reductase (MTHFR). The prevalence of MTHFR genotypes was not significantly different between the two groups. The polymorphism of MTHFR was not unambiguously connected with the content of total placental Met, Hcy and related cysteine, and GSH either in uncomplicated or in complicated pregnancies. By contrast, the combination of the heterozygous MTHFR genotype with folate deficiency in the samples from preeclamptic pregnancies was characterized by a statistically significant decrease in the Met content, a trend toward increased Hcy levels and a tight association between metabolically directly and indirectly related compounds, e.g. positive relation between Hcy versus cysteine and folate versus GSH and negative relation between folate versus Hcy and both Hcy and cysteine versus GSH. We demonstrated the expression of cystathionine-ß-synthase (CBS) in human placenta at term by RT-PCR and western blot analysis, for the first time, and confirmed its catalytic activity and the accumulation of cysteine and CBS in placental explants cultivated in the presence of elevated Hcy concentrations. We suggest that disturbance in placental folate-related metabolism may be one of the pathogenetic factors in preeclampsia.

Placental and trophoblastic in vitro models to study preventive and therapeutic agents for preeclampsia.

In the field of preeclampsia, enormous efforts are ongoing to identify biomarkers predicting the syndrome already in the first trimester of pregnancy. At the same time, there is the need for in vitro models to test such biomarkers prior to their use in clinical trials. In addition, in vitro models may accelerate the development and evaluation of the benefit of any putative therapeutics. Therefore, in vitro systems have been established to evaluate the release of biomarkers and measure the effect of putative therapeutics using placental villous explants as well as the choriocarcinoma cell line BeWo. For explants, a cryogenic method to freeze, transport and thaw villous explants was developed to use such tissues for a multi-site tissue culture evaluation. Here we focus on three out of many in vitro models that have been established for human placental trophoblast. (1) Choriocarcinoma cell lines such as BeWo, Jeg-3 and Jar cells (2) isolated primary trophoblast cells, and (2) villous explants from normal placentas delivered at term. Cell lines were used to assess the effect of differentiation and fusion on the expression and release of a preeclampsia marker (placental protein 13; PP13) and beta-hCG. Moreover, cell lines were used to study the effect of putative preeclampsia therapeutics such as vitamins C and E, heparin and aspirin on marker release and viability. Cryopreservation of villous explants enabled shipment to a remote laboratory and testing of parameters in different countries using explants from one and the same placenta. Recently published data make it tempting to speculate that the choriocarcinoma cell line BeWo as well as fresh and cryogenically stored placental villous explants may well serve as in vitro models to study preventive and therapeutic agents in the field of preeclampsia.

Effects of vitamins C and E, acetylsalicylic acid and heparin on fusion, beta-hCG and PP13 expression in BeWo cells.

Preeclampsia is one of the leading causes for maternal and fetal morbidity. Placental protein 13 (PP13) is a placenta specific protein and with its decreased maternal serum levels in the first trimester it is one of the most promising markers to predict the syndrome in early pregnancy. In clinical trials attempts to prevent preeclampsia have already been made using low-dose aspirin, low-molecular-weight heparin, and antioxidants such as vitamins C and E. Here we investigated the effect of these agents on PP13 and beta-hCG levels using choriocarcinoma cell lines as surrogates for primary villous trophoblast. Five different cell lines were triggered with forskolin and cultured for 48 h. Amongst the five tested cell lines BeWo cells showed the strongest increase in PP13 mRNA after forskolin treatment compared to controls. Hence these cells were used to investigate the effect of varying concentrations of vitamin C, acetylsalicylic acid (ASA), Trolox) and heparin on cell fusion and PP13 and beta-hCG levels. The response to vitamin C was a dose-dependent increase in protein expression, while the other drugs showed only modest effects. Since first trimester PP13 has been shown to be significantly decreased in women subsequently developing preeclampsia, this data might point to a beneficial effect of very early vitamin C treatment of such women already in the early first trimester of pregnancy.

Caspases rather than calpains mediate remodelling of the fodrin skeleton during human placental trophoblast fusion.

Fusion of cytotrophoblasts with the overlying syncytiotrophoblast is an integral step in differentiation of the human placental villous trophoblast. Multiple factors, such as growth factors, hormones, cytokines, protein kinases, transcription factors and structural membrane proteins, were described to modulate trophoblast fusion. However, the knowledge on remodelling of the membrane-associated cytoskeleton during trophoblast fusion is very limited. This study describes the link between remodelling of spectrin-like alpha-fodrin and intercellular trophoblast fusion. Experiments with primary trophoblasts isolated from term placentas and the choriocarcinoma cell line BeWo revealed a biphasic strategy of the cells to achieve reorganization of alpha-fodrin. Syncytialization of trophoblasts was accompanied by down-regulation of alpha-fodrin mRNA, whereas the full-length alpha-fodrin protein was cleaved into 120 and 150 kDa fragments. Application of calpeptin and calpain inhibitor III did not affect alpha-fodrin fragmentation in primary term trophoblasts and forskolin-treated BeWo cells, but decreased secretion of beta human chorionic gonadotropin. In contrast, inhibitors of caspases 3, 8 and 9 attenuated generation of the 120 kDa fragment and a general caspase inhibitor completely blocked fragmentation, suggesting an exclusive function of caspases in alpha-fodrin remodelling. Immunofluorescence double staining of human placenta revealed co-localization of active caspase 8 with alpha-fodrin positive vesicles in fusing villous cytotrophoblasts. These results suggest that caspase-dependent fragmentation of alpha-fodrin may be important for reorganization of the sub-membranous cytoskeleton during trophoblast fusion.

Fusion of villous trophoblast can be visualized by localizing active caspase 8.

Villous cytotrophoblast differentiation and subsequent fusion with the overlying syncytiotrophoblast depend on multiple factors such as growth factors, cytokines, hormones, protein kinases, transcription factors, structural membrane proteins and proteases. Caspase 8, an aspartate-specific cysteine protease, is mainly known for its role in programmed cell death, but was also demonstrated to be crucial for villous trophoblast differentiation. This study aimed to localize active caspase 8 in the villous trophoblast layer of human first trimester placenta. To this end, immunofluorescence double staining was performed, using a monoclonal rabbit antibody against cleaved caspase 8 in combination with antibodies against cytokeratin 7, chorionic gonadotropin beta subunit (beta hCG), beta-actin, placental protein 13 (PP13), alpha-fodrin and Ki-67. Immunofluorescence revealed cleaved caspase 8 in one out of 422 villous cytotrophoblasts resting on the basement membrane, in one out of 759 perinuclear regions within the syncytiotrophoblast and in few trophoblasts located between these two layers. Double staining of cleaved caspase 8 and Ki-67 antigen revealed that caspase 8 is activated only in cytotrophoblasts which have left the cell cycle. The staining suggests that caspase 8 is activated in villous cytotrophoblasts just prior to fusion of these cells and escorts the nuclei from the mononucleated to the syncytial state.

[Effect of homocysteine on the structure and functions of human placenta trophoblasts].

Elevated level of homocysteine in blood serum of pregnant women is the risk factor for placental malfunction and fetal abnormalities. Our study has shown the activation of apoptosis, inhibition of proliferation, destruction of placental trophoblast and activation of the transsulfuration pathway under elevated homocysteine level in the incubation medium in the range of 20-80 microM. The activation of the transsulfuration pathway indicates that placenta may to some extent withstand elevated homocysteine level.

IFPA meeting 2008 workshops report.

Workshops are an important part of the IFPA annual meeting. At the IFPA meeting 2008 diverse topics were discussed in 12 themed workshops. Topics covered included: immunology of placentation; galectins and trophoblast invasion; signaling in implantation and invasion; markers to identify trophoblast subpopulations; placental pathology; placental toxicology; stereology; placental transport of fatty acids; placental mesenchymal stem cells; comparative placentation; trophoblast and neoplasia; trophoblast differentiation. This report is a summary of the various topics covered.

Trophoblastic invasion in vitro and in vivo: similarities and differences.

BACKGROUND: The basic mechanisms of trophoblast invasion are not completely understood. This may be due to the lack of suitable in vitro models which enable experimental modulation of this complex process. In the present study, a three-dimensional co-culture model is used for comparing two factors considered to be implicated in the regulation of trophoblast invasion, the expression of HLA-G and apoptosis, in vitro and in vivo. METHODS: Tissue fragments from human first trimester decidua parietalis were put in close contact with spheroids of AC-1M59 trophoblast/choriocarcinoma hybrid cells as a model of the invasive trophoblast. Cryostat sections from these co-cultures were immunohistochemically stained and compared with first trimester placentation sites in vivo. RESULTS: Only the invasive trophoblast-derived cells showed an intensive staining for HLA-G, whereas the cells on the periphery of the confrontation culture exhibited only a weak staining. A similar staining pattern was found in vivo. Both in vitro and in vivo CD45(+) apoptotic leukocytes were frequently detected in close proximity to the invasive trophoblastic cells. CONCLUSIONS: In this co-culture system, key factors considered to be implicated in trophoblast invasion in vivo can also be demonstrated in vitro. Therefore, it may help in finding strategies for the management of diseases associated with deficient trophoblast invasion.

Effects of oxygen on cell turnover and expression of regulators of apoptosis in human placental trophoblast.

Pre-eclampsia (PE) and intrauterine growth restriction (IUGR) are associated with aberrant cell turnover, including increased apoptosis, in placental villous trophoblast. The increased apoptosis is associated with exaggerated expression of p53, which promotes cell cycle arrest or apoptosis via downstream proteins such as p21 or Bax. These changes in apoptosis and p53 expression are purported to result from exposure to altered oxygen tension. Using a model of villous trophoblast turnover, we examined the effect of 20%, 6% and 1% ambient oxygen (O(2)) on apoptosis, necrosis, proliferation and expression of p53 and related regulators of cell turnover, compared to both fresh tissue. Altered O(2) tension exerted an effect on cell turnover in cultured term villous tissue: cytotrophoblast proliferation was increased by culture in 20% O(2) and reduced in 1% O(2) (median proliferative index: fresh tissue=0.32%, 20% O(2)=0.9%, 6% O(2)=0.28%, 1% O(2)=0.07%). Apoptosis was increased in all culture environments, but was significantly enhanced by culture in 1% O(2) (median apoptotic index: fresh tissue=0.64%, 20% O(2)=2.96%, 6% O(2)=3.81%, 1% O(2)=9.2%). Necrotic cell death was also increased by culture in 1% O(2) compared to 6% and 20% O(2). The expression of p53, p21 and Mdm2 in both cytotrophoblast and stromal cells was increased following culture in 1% O(2). There was no alteration in the expression of Bax or Bcl-2. This study provides evidence that p53 is elevated in trophoblast following exposure to hypoxia. The potential role of the p53-pathway in the control of cell turnover in villous trophoblast and the regulation of p53 by altered O(2) tension merits further investigation.

Spatial and temporal distribution of Tie-1 and Tie-2 during very early development of the human placenta.

Vasculogenesis in the human placenta comprises differentiation and growth of newly forming blood vessels derived from hemangiogenic stem cells within the mesenchymal core of villi. In a second stage, angiogenesis leads to the expansion and remodeling of the already existing vessels. At present, relatively little is known about the regulatory mechanisms of vasculogenesis and angiogenesis during very early placentation. Using placental villous tissues from days 22 to 48 of pregnancy, we analyzed the spatial and temporal expression of Tie-1 and Tie-2 in parallel to vascular maturation in the human placenta. In immunohistochemistry both receptors, Tie-1 and Tie-2 show a cell and villous type specific expression during this early phase of placental development. Especially, cytotrophoblast and hemangiogenic cell cords in mesenchymal villi and Hofbauer cells in immature intermediate villi have the strongest immunoreactivities. Western blot analysis showed that no significant changes were detected for Tie-1 and Tie-2 as pregnancy advanced. Moreover, phospho-Tie-2 levels did not change significantly in parallel to pregnancy ages. We conclude that both receptors are involved in angiogenesis as well as vascular modulation of early vessels. Due to their spatial distribution we speculate on an additional role in regulation of villous and extravillous trophoblastic behavior.

Sequential steps during vasculogenesis and angiogenesis in the very early human placenta.

Development of blood vessels takes place via two subsequent processes, vasculogenesis and angiogenesis. During vasculogenesis, formation of first blood vessels is achieved by differentiation of hemangiogenic stem cells from pluripotent mesenchymal cells, while during angiogenesis new blood vessels form from already existing vessels. The combination of our data with those from the literature leads us to depict the chronological steps of cell differentiation in the mesenchymal core of placental villi during vasculogenesis and angiogenesis. This current opinion will focus on the temporal and spatial expression of VEGF and its receptors VEGFR-1 and VEGFR-2, and the angiopoietin receptors Tie-1 and Tie-2 in parallel to vascular maturation in human placental villi during very early stages of placental development. There is evidence that the interplay of a variety of growth factors secreted from different cell types during development is needed to trigger as well as maintain placental vasculogenesis and angiogenesis.

Bi-potential behaviour of cytotrophoblasts in first trimester chorionic villi.

Murine trophoblast stem (TS) cells express fibroblast growth factor receptor 2 (FGFR2) and are maintained in their proliferative state by fibroblast growth factor 4 (FGF4). We show in this report that in the first trimester human placenta FGFR2 expression is similarly found in a subset of villous cytotrophoblast and in proximal anchoring columns. Western analysis demonstrated declining FGFR2 protein expression as gestation advanced, suggesting a similar role for FGF in early human trophoblast proliferation. Mouse TS cell differentiation is known to occur along two distinct transcriptionally-regulated pathways; extravillous trophoblast (EVT) cells invade the uterine wall to promote maternal blood flow whilst syncytiotrophoblast lines chorionic villi in the labyrinth. Similar differentiation steps occur in the human placenta though the fate of human trophoblast stem cells is presently unknown. To investigate the mechanisms underlying human cytotrophoblast differentiation we have developed a novel cultured floating first trimester villous explant model in which denuded first trimester villi spontaneously regenerate syncytiotrophoblast following 48 h of culture. Addition of FGF4 and heparin inhibited syncytiotrophoblast regeneration in favor of forming clumps of cytotrophoblast. Proximal cells in these clumps were FGFR2 immuno-reactive and proliferative, intermediate parts expressed alpha5beta1-integrin, while the distal portion expressed HLA-G and the invasive integrin alpha1beta1 indicating differentiation to the EVT phenotype. In contrast, non-denuded villi exposed to FGF4 exhibited similar proliferation of the cytotrophoblast; however, these cells did not express any of the invasive EVT markers. We conclude that FGFR2-positive chorionic cytotrophoblasts exhibit bi-potential behaviour, being capable of forming either syncytiotrophoblast or EVT. We suggest bipotential trophoblast progenitor cells persist during first trimester human placental development.

Extravillous trophoblast apoptosis--a workshop report.

During normal pregnancy, extravillous trophoblast cells invade maternal uterine tissues. The interstitial trophoblast penetrates decidual tissues reaching the inner third of the myometrium. A subset of the interstitial trophoblast, the intramural/endovascular trophoblast transforms uterine spiral arteries into large-bore conduits to enable the adequate supply of nutrients and oxygen to the placenta and thus the fetus. Control of invasion is still a mystery and therefore, in this workshop report already existing concepts as well as new models are discussed. Maternal cells such as macrophages and endothelial cells have a clear impact on trophoblast invasion and apoptosis. However, the trophoblast cells need to be susceptible to undergo apoptosis. Thus, an intrinsic program within the trophoblast needs to be activated before induction from the outside can be successful. Quantification of apoptosis further clarified that apoptosis of interstitial trophoblast is not the ultimate means to lead to pathologically shallow invasion. On the other hand, apoptosis of intramural/endovascular trophoblast seems to be highly relevant for a correct transformation of spiral arteries.