Gregor Weiss

Trophoblast invasion and oxygenation of the placenta: measurements versus presumptions

Invasion of extravillous trophoblast into maternal tissues has a profound effect on the oxygenation of the placenta and hence the fetus. The main route of trophoblast invasion is interstitial invasion into the tissues of the decidua and myometrium. From this main route side branches reach the spiral arteries (endovascular trophoblast) as well as the uterine glands (endoglandular trophoblast) to open both structures toward the intervillous space. This enables histiotrophic nutrition in the first trimester and hemotrophic nutrition in the second and third trimesters of pregnancy. Failure of endovascular trophoblast invasion has profound effects on the oxygenation of the placenta. Interestingly, this does not lead to hypoxia as has long been presumed. Rather, all measurements available today point to increased oxygen levels within the placenta in patients with a failure of spiral artery transformation. This should lead to a rethink regarding pathological conditions such as intrauterine growth restriction and preeclampsia.

Amnion-Derived Mesenchymal Stromal Cells Show Angiogenic Properties but Resist Differentiation into Mature Endothelial Cells

Mesenchymal stromal cells derived from the human amnion (hAMSC) currently play an important role in stem cell research, as they are multipotent cells that can be isolated using noninvasive methods and are immunologically tolerated in vivo. The objective of this study was to evaluate their endothelial differentiation potential with regard to a possible therapeutic use in vascular diseases. hAMSC were isolated from human term placentas and cultured in Dulbeccos modified Eagles medium (DMEM) (non-induced hAMSC) or endothelial growth medium (EGM-2) (induced hAMSC). Induced hAMSC changed their fibroblast-like toward an endothelial-like morphology, and were able to take up acetylated low-density lipoprotein and form endothelial-like networks in the Matrigel assay. However, they did not express the mature endothelial cell markers von Willebrand factor and vascular endothelial-cadherin. Gene expression analysis revealed that induced hAMSC significantly downregulated pro-angiogenic genes such as tenascin C, Tie-2, vascular endothelial growth factor A (VEGF-A), CD146, and fibroblast growth factor 2 (FGF-2), whereas they significantly upregulated anti-angiogenic genes such as serpinF1, sprouty1, and angioarrestin. Analysis of protein expression confirmed the downregulation of FGF-2 and Tie-2 (27%±8% and 13%±1% of non-induced cells, respectively) and upregulation of the anti-angiogenic protein endostatin (226%±4%). Conditioned media collected from hAMSC enhanced viability of endothelial cells and had a stabilizing effect on endothelial network formation as shown by lactate dehydrogenase and Matrigel assay, respectively. In summary, endothelial induced hAMSC acquired some angiogenic properties but resisted undergoing a complete differentiation into mature endothelial cells by upregulation of anti-angiogenic factors. Nevertheless, they had a survival-enhancing effect on endothelial cells that might be useful in a variety of cell therapy or tissue-engineering approaches.

Evidence from the very beginning: endoglandular trophoblasts penetrate and replace uterine glands in situ and in vitro

STUDY QUESTIONnHow is histiotrophic nutrition of the embryo secured during the first trimester of pregnancy?nnnSUMMARY ANSWERnRather than specifically focusing on invasion into spiral arteries, extravillous trophoblasts also invade into uterine glands (endoglandular trophoblast) from the very beginning and open them toward the intervillous space.nnnWHAT IS KNOWN ALREADYnExtravillous trophoblasts can be found in close contact and within the lumen of uterine glands, sometimes replacing glandular epithelial cells.nnnSTUDY DESIGN, SIZE, DURATIONnAs well as extensive screening of specimens from first trimester placentation sites in situ we used a previously established three-dimensional co-culture in vitro model system of first trimester villous explants with non-invaded decidua parietalis.nnnPARTICIPANTS/MATERIALS, SETTING, METHODSnFirst trimester placentas were obtained from elective terminations of pregnancies (n = 48) at 5-11 weeks of gestational age. A subset was processed for confrontation co-culture (n = 31). Invaded decidua basalis was obtained from 20 placentas. All tissues were sectioned, subsequently immunostained and immunodoublestained with antibodies against keratin 7 (KRT7), major histocompatibility complex, class I, G (HLA-G), matrix metallopeptidase 9 (MMP9), von Willebrand factor (VWF) and the appropriate Immunoglobulin G (IgG) negative controls. Replacement of endothelial/epithelial cells by extravillous trophoblasts was quantified semi-quantitatively. Additionally, hematoxylin and eosin-stained archival specimens from early implantation sites were assessed.nnnMAIN RESULTS AND THE ROLE OF CHANCEnThe earliest available specimen was from around Day 10 after conception; already at this stage trophoblasts had penetrated into uterine glands and had started to replace the epithelium of the glands. Endoglandular trophoblasts replaced uterine glands in vitro and in situ and could be found in the lumen of invaded glands. Quantitative analysis revealed significantly more replacement of epithelial cells in glands (63.8 ± 22.1%) compared with endothelial cells in vessels (26.4 ± 8.8%). Accumulated detached glandular epithelial cells could be repeatedly observed in the lumen of invaded glands. Additionally, in areas of trophoblast invasion the glandular epithelium seemed to be completely disintegrated compared with glandular epithelium in the non-invaded parts of the decidua. Whole tissue specimens were used in vitro and in situ instead of cell lines; these systems mostly maintain the context of the in vivo situation.nnnLIMITATIONS, REASONS FOR CAUTIONnThis is a descriptive study supported by in vitro experiments. However, a histological section will always only be a snapshot and quantification from histological sections has its limitations.nnnWIDER IMPLICATIONS OF THE FINDINGSnThis study further strengthens the hypothesis of histiotrophic nutrition of the embryo prior to the establishment of the maternal blood flow toward the placenta. Invasion of uterine glands by endoglandular trophoblasts may have more impact on the outcome of early pregnancy than assumed up to now.

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

During the first trimester of pregnancy, extravillous trophoblasts (EVTs) invade into the decidual interstitium to the first third of the myometrium, thereby anchoring the placenta to the uterus. They also follow the endovascular and endoglandular route of invasion; plug, line and remodel spiral arteries, thus being responsible for the establishment of hemotrophic nutrition with the beginning of the second trimester and invade and open uterine glands toward the intervillous space for a histiotrophic nutrition during the first trimester. The aim of this study was to provide proof that uterine veins are invaded by EVTs similar to uterine arteries and glands in first trimester of pregnancy. Therefore, serial sections from in situ first trimester placenta were immuno-single- and immuno-double-stained to distinguish in a first step between arteries and veins and secondly between invaded and non-invaded vessels. Subsequently, invasion of EVTs into uterine vessels was quantified. Our data show that uterine veins are significantly more invaded by EVTs than uterine arteries (29.2xa0±xa015.7xa0%) during early pregnancy. Counted vessel cross sections revealed significantly higher EVT invasion into veins (59.5xa0±xa07.9xa0%) compared to arteries (29.2xa0±xa015.7xa0%). In the lumen of veins, single EVTs were repeatedly found, beside detached glandular epithelial cells or syncytial fragments. This study allows the expansion of our hitherto postulated concept of EVT invasion during first trimester of pregnancy. We suggest that invasion of EVTs into uterine veins is responsible the draining of waste and blood plasma from the intervillous space during the first trimester of pregnancy.

Placental Mesenchymal Stromal Cells Derived from Blood Vessels or Avascular Tissues: What Is the Better Choice to Support Endothelial Cell Function?

Mesenchymal stromal cells (MSCs) are promising tools for therapeutic revascularization of ischemic tissues and for support of vessel formation in engineered tissue constructs. Recently, we could show that avascular-derived MSCs from placental amnion release soluble factors that exhibit survival-enhancing effects on endothelial cells (ECs). We hypothesize that MSCs derived from placental blood vessels might have even more potent angiogenic effects. Therefore, we isolated and characterized MSCs from placental chorionic blood vessels (bv-MSCs) and tested their angiogenic potential in comparison to amnion-derived avascular MSCs (av-MSCs). bv-MSCs express a very similar surface marker profile compared with av-MSCs and could be differentiated toward the adipogenic and osteogenic lineages. bv-MSCs exert immunosuppressive properties on peripheral blood mononuclear cells, suggesting that they are suitable for cell transplantation settings. Conditioned medium (Cdm) from av-MSCs and bv-MSCs significantly enhanced EC viability, whereas only Cdm from bv-MSCs significantly increased EC migration and network formation (Matrigel assay). Angiogenesis array analysis of av- and bv-MSC-Cdm revealed a similar secretion pattern of angiogenic factors, including angiogenin, interleukins-6 and -8, and tissue inhibitors of matrix metalloproteinase-1 and 2. Enzyme-linked immunosorbent assay analysis showed that, in contrast to av-MSCs, bv-MSCs secreted vascular endothelial growth factor. In direct coculture with bv-MSCs, ECs showed a significantly increased formation of vessel-like structures compared with av-MSCs. With regard to therapeutic treatment, bv-MSCs and particularly their Cdm might be valuable to stimulate angiogenesis especially in ischemic tissues. av-MSCs and their Cdm could be beneficial in conditions when it is required to promote the survival and stabilization of blood vessels without the risk of unmeant angiogenesis.

The trophoblast plug during early pregnancy: a deeper insight

During the first trimester of pregnancy, foetal endovascular trophoblasts invade into maternal spiral arteries, accumulate and form plugs in the lumen of the vessels. These plugs only allow blood plasma to seep through. Hence, during the first trimester of pregnancy, a first flow of fluids through the placental intervillous space is established, resulting in a physiological oxygen gradient between mother and foetus. The trophoblast plugs block spiral arteries until the beginning of the second trimester (11–14xa0weeks). In parallel, uterine glands are invaded and opened by endoglandular trophoblasts towards the intervillous space of the placenta, without showing the formation of plugs (Moser et al. in Hum Reprod 25:1127–1136, 2010, Hum Reprod Oxf Engl 30:2747–2757, 2015). This enables histiotrophic nutrition of the embryo prior to onset of maternal blood flow into the placenta. Failure of these endovascular and endoglandular invasion processes may lead to miscarriage or pregnancy disorders such as intrauterine growth restriction (IUGR). After dissolution of the plugs, the onset of maternal blood flow allows maternal blood cells to enter the intervillous space and oxygen concentrations rise up. In this study, we demonstrate for the first time serial cross sections through a trophoblast plug in a first trimester placental bed specimen. Invaded and plugged arteries as well as invaded uterine glands in week 11 of gestation are visualized with specific immunohistochemical double staining techniques. We show that spiral artery plugs appear throughout the placental invasion zone and illustrate erythrocytes stowed due to trophoblast plugs. In addition, we give evidence of the presence of MMP-1 in plugs of invaded spiral arteries. The results reveal a better understanding and a closer insight into the morphological appearance of trophoblast plugs and the consequences for placental and uterine blood flow.

Placental Fractalkine Is Up-Regulated in Severe Early-Onset Preeclampsia

The pathogenesis of preeclampsia (PE) includes the release of placental factors into the maternal circulation, inducing an inflammatory environment in the mother. One of the factors may be the proinflammatory chemokine fractalkine, which is expressed in the syncytiotrophoblast of human placenta, from where it is released into the maternal circulation by constitutive shedding. We examined whether placental fractalkine is up-regulated in severe early-onset PE and whether the proinflammatory cytokines tumor necrosis factor (TNF)-α and IL-6 are able to increase the expression of fractalkine. Gene expression analysis, enzyme-linked immunosorbent assay, and immunohistochemistry consistently showed increased fractalkine expression in placentas from severe early-onset PE, compared to gestational age-matched controls. Expression of a disintegrin and metalloproteinases (ADAMs) 10 and 17, which convert transmembrane fractalkine into the soluble form, was significantly increased in these cases. Incubation of first-trimester placental explants with TNF-α provoked a significant increase in fractalkine expression and release of the soluble form, whereas IL-6 had no effect. TNF-α-mediated up-regulation of placental fractalkine was reversed in the presence of the aspirin-derivative salicylate, which impaired activation of NF-κB p65 in TNF-α-treated explants. On the basis of data from placental explants, we suggest that increased maternal TNF-α may up-regulate the expression and release of placental fractalkine, which, in turn, may contribute to an exaggerated systemic inflammatory response in PE.

Establishing principles of macromolecular crowding for in vitro fibrosis research of the vocal fold lamina propria.

Vocal fold fibrosis represents a major disease burden. Screening of antifibrotic compounds could be facilitated by an in vitro fibrogenesis system. Limitations of existing models might be overcome by implication of the excluded volume effect.

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

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

Anti-Mullerian hormone concentrations in individual follicular fluids within one stimulated IVF cycle resemble blood serum values

PurposeAnti-Mullerian hormone (AMH) is commonly known as the most potent marker for ovarian reserve due to its decline as female age increases. While serum AMH (sAMH) levels have been intensively investigated, there is less data regarding AMH concentrations in follicular fluid (FF), since FF has usually been designated as waste product during oocyte collection in assisted reproductive technologies. This pilot study investigated follicle AMH concentrations (fAMH) of several follicles per ovary, individually collected with the Steiner-Tan needle, and compared them to sAMH concentrations in women undergoing IVF treatment. We hypothesized that there is no difference of fAMH concentrations in individual follicles and that these concentrations resemble the sAMH value of the patient.MethodsPatients were stimulated with a gonadotropin-releasing hormone antagonist ovarian hyperstimulation protocol. On the day of oocyte retrieval, serum samples and FF from all individual follicles from one stimulated IVF cycle were collected and individually analyzed for AMH concentrations.ResultsIntracyclic mean fAMH values (nfolliclexa0=xa02–14) were significantly correlated to sAHM values (ρxa0=xa00.85, pxa0<xa00.001) and showed a trend to be negatively associated with age (ρxa0=xa0−0.43, pxa0=xa00.06). Mean intrapatient fAMH concentrations differed significantly (pxa0<xa00.001). Furthermore, significant correlations of sAMH with individual fAMH values of the first five follicles of each patient were observed.ConclusionsIn conclusion, our results clearly showed that individual fAMH concentrations reflected sAMH values and that fAMH concentrations did not significantly differ within one patient. In future studies, it will be interesting to correlate individual fAMH values to the respective embryo development and overall pregnancy outcome in order to improve IVF treatments and to refrain from embryo overproduction.