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Plant and animal life forms evolved mechanisms for sensing and responding to gravity on Earth where homeostatic needs require responses. The lack of gravity, such as in the International Space Station (ISS), causes acute, intra-generational changes in the quality of life. These include maintaining calcium levels in bone, maintaining muscle tone, and disturbances in the vestibular apparatus in the ears. These problems decrease work efficiency and quality of life of humans not only during microgravity exposures but also after return to higher gravity on Earth or destinations such as Mars or the Moon. It has been hypothesized that lack of gravity during mammalian development may cause prenatal, postnatal and transgenerational effects that conflict with the environment, especially if the developing organism and its progeny are returned, or introduced de novo, into the varied gravity environments mentioned above. Although chicken and frog pregastrulation development, and plant root development, have profound effects due to orientation of cues by gravity-sensing mechanisms and responses, mammalian development is not typically characterized as gravity-sensing. Although no effects of microgravity simulation (MGS) on mouse fertilization were observed in two reports, negative effects of MGS on early mammalian development after fertilization and before gastrulation are presented in four reports that vary with the modality of MGS. This review will analyze the positive and negative mammalian early developmental outcomes, and enzymatic and epigenetic mechanisms known to mediate developmental responses to simulated microgravity on Earth and microgravity during spaceflight experiments. We will update experimental techniques that have already been developed or need to be developed for zero gravity molecular, cellular, and developmental biology experiments. [ABSTRACT FROM AUTHOR]

This review focuses on hypoxic stress and its effects on the placental lineage and the earliest differentiation events in mouse and human placental trophoblast stem cells (TSCs). Although the placenta is a decidual organ at the end of pregnancy, its earliest rapid growth and function at the start of pregnancy precedes and supports growth and function of the embryo. Earliest function requires that TSCs differentiate, however, "hypoxia" supports rapid growth, but not differentiation of TSCs. Most of the literature on earliest placental "hypoxia" studies used 2% oxygen which is normoxic for TSCs. Hypoxic stress happens when oxygen level drops below 2%. It decreases anabolism, proliferation, potency/stemness and increases differentiation, despite culture conditions that would sustain proliferation and potency. Thus, to study the pathogenesis due to TSC dysfunction, it is important to study hypoxic stress below 2%. Many studies have been performed using 0.5 to 1% oxygen in cultured mouse TSCs. From all these studies, a small number has examined human trophoblast lines and primary first trimester placental hypoxic stress responses in culture. Some other stress stimuli, aside from hypoxic stress, are used to elucidate common and unique aspects of hypoxic stress. The key outcomes produced by hypoxic stress are mitochondrial, anabolic, and proliferation arrest, and this is coupled with stemness loss and differentiation. Hypoxic stress can lead to depletion of stem cells and miscarriage, or can lead to later dysfunctions in placentation and fetal development. [ABSTRACT FROM AUTHOR]

Study Question: Does low molecular weight heparin (LMWH) require heparin-binding epidermal growth factor (EGF)-like growth factor (HBEGF) signaling to induce extravillous trophoblast differentiation and decrease apoptosis during oxidative stress?Summary Answer: LMWH increased HBEGF expression and secretion, and HBEGF signaling was required to stimulate trophoblast extravillous differentiation, increase invasion in vitro and reduce trophoblast apoptosis during oxidative stress.What Is Known Already: Abnormal trophoblast differentiation and survival contribute to placental insufficiency syndromes, including preeclampsia and intrauterine growth restriction. Preeclampsia often manifests as a pro-thrombotic state, with unsuccessful transformation of the spiral arteries that reduces oxygen supply and can produce placental infarction. LMWH improves placental function by increasing blood flow. Recent data suggest that the actions of LMWH transcend its anti-coagulative properties, but the molecular mechanism is unknown. There is evidence that LMWH alters the expression of human HBEGF in trophoblast cells, which regulates human trophoblast pathophysiology. HBEGF, itself, is capable of increasing trophoblast survival and invasiveness.Study Design, Size, Duration: First-trimester placental explants and the HTR-8/SVneo cell line, established using extravillous trophoblast outgrowths from first-trimester villous explants, were treated in vitro with LMWH to examine the effects on HBEGF signaling and trophoblast function under normal physiological and pathological conditions. A highly specific antagonist of HBEGF and other inhibitors of HBEGF downstream signaling were used to determine the relationship between LMWH treatment and HBEGF.Participants/materials, Setting, Methods: Placental tissues (n = 5) were obtained with IRB approval and patient consent from first-trimester terminations. Placental explants and HTR-8/SVneo cells were cultured on plastic or Matrigel™ and treated with a therapeutic dose of LMWH (Enoxaparin; 10 IU/ml), with or without CRM197, pan Erb-B2 Receptor Tyrosine Kinase (ERBB) inhibitor, anti-ERBB1 or ERBB4 blocking antibodies, or pretreatment of cells with heparitinase I. Extravillous differentiation was assessed by immunocytochemistry to determine the relative levels of integrins α6β4 and α1β1. Trophoblast invasiveness was assessed in villous explants by measuring outgrowth from villous tips cultured on Matrigel, and by invasion assays with HTR-8/SVneo cells cultured on Matrigel-coated transwell insert. Placental explants and HTR-8/SVneo cells were exposed to oxidative stress in a hypoxia-reoxygenation (H-R) model, measuring cell death by TUNEL assay, caspase 3 cleavage, and BCL-2α expression.Main Results and the Role Of Chance: LMWH induced extravillous differentiation, according to trophoblast invasion assays and integrin (α6β4-α1β1) switching. Treatment with LMWH rescued cytotrophoblasts and HTR-8/SVneo cells from apoptosis during exposure to reoxygenation injury, based on TUNEL, caspase 3 cleavage and BCL-2α expression. Experiments using CRM197, ERBB1 and ERBB4 blocking antibodies, pan-ERBB inhibitor and removal of cell surface heparin demonstrated that the effects of LMWH on trophoblast invasion and survival were dependent upon HBEGF signaling.Large Scale Data: N/A.Limitations, Reasons For Caution: The primary limitation of this study was the use of only in vitro experiments. Patient demographics from elective terminations were not available.Wider Implications Of the Findings: These data provide new insights into the non-coagulation-related aspects of perinatal LMWH treatment in the management of placental insufficiency disorders.Study Funding/competing Interest(s): This research was supported by grants from the National Institutes of Health (HD071408 and HL128628), the March of Dimes, and the W. K. Kellogg Foundation. There were no conflicts or competing interests. [ABSTRACT FROM AUTHOR]

The article discusses a study on the diagnosis of birth defects of genetic origin in utero after invasive extraction of fetal tissues. The study performed noninvasive testing with circulating cell-free fetal DNA. It examined intact trophoblast cells from Papanicolaou smears collected from pregnant women and employed multiplex targeted DNA sequencing to profile matched maternal, placental and fetal samples.

Study Question: Is protein expression of the muscle segment homeobox gene family member MSX1 altered in the human secretory endometrium by cell type, developmental stage or fertility?Summary Answer: MSX1 protein levels, normally elevated in the secretory phase endometrium, were significantly reduced in endometrial biopsies obtained from women of infertile couples.What Is Known Already: Molecular changes in the endometrium are important for fertility in both animals and humans. Msx1 is expressed in the preimplantation mouse uterus and regulates uterine receptivity for implantation. The MSX protein persists a short time, after its message has been down-regulated. Microarray analysis of the human endometrium reveals a similar pattern of MSX1 mRNA expression that peaks before the receptive period, with depressed expression at implantation. Targeted deletion of uterine Msx1 and Msx2 in mice prevents the loss of epithelial cell polarity during implantation and causes infertility.Study Design, Size Duration: MSX1 mRNA and cell type-specific levels of MSX1 protein were quantified from two retrospective cohorts during the human endometrial cycle. MSX1 protein expression patterns were compared between fertile and infertile couples. Selected samples were dual-labeled by immunofluorescence microscopy to localize E-cadherin and β-catenin in epithelial cells.Participants/materials, Setting Methods: MSX1 mRNA was quantified by PCR in endometrium from hysterectomies (n = 14) determined by endometrial dating to be in the late-proliferative (cycle days 10-13), early-secretory (cycle days 14-19) or mid-secretory (cycle days 20-24) phase. MSX1 protein was localized using high-throughput, semi-quantitative immunohistochemistry with sectioned endometrial biopsy tissues from fertile (n = 89) and infertile (n = 89) couples. Image analysis measured stain intensity specifically within the luminal epithelium, glands and stroma during the early-, mid- and late- (cycle days 25-28) secretory phases.Main Results and the Role Of Chance: MSX1 transcript increased 5-fold (P < 0.05) between the late-proliferative and early secretory phase and was then down-regulated (P < 0.05) prior to receptivity for implantation. In fertile patients, MSX1 protein displayed strong nuclear localization in the luminal epithelium and glands, while it was weakly expressed in nuclei of the stroma. MSX1 protein levels accumulated throughout the secretory phase in all endometrial cellular compartments. MSX1 protein decreased (P < 0.05) in the glands between mid- and late-secretory phases. However, infertile patients demonstrated a broad reduction (P < 0.001) of MSX1 accumulation in all cell types throughout the secretory phase that was most pronounced (∼3-fold) in stroma and glands. Infertility was associated with persistent co-localization of E-cadherin and β-catenin in epithelial cell junctions in the mid- and late-secretory phases.Limitations, Reasons For Caution: Details of the infertility diagnoses and other patient demographic data were not available. Therefore, patients with uterine abnormalities (Mullerian) could not be distinguished from other sources of infertility. Antibody against human MSX2 is not available, limiting the study to MSX1. However, both RNAs in the human endometrium are similarly regulated. In mice, Msx1 and Msx2 are imperative for murine embryo implantation, with Msx2 compensating for genetic ablation of Msx1 through its up-regulation in a knockout model.Wider Implications Of the Findings: This investigation establishes that the MSX1 homeobox protein accumulation is associated with the secretory phase in endometrium of fertile couples, and is widely disrupted in infertile patients. It is the first study to examine MSX1 protein localization in the human endometrium, and supported by genetic findings in mice, suggests that genes regulated by MSX1 are linked to the loss of epithelial cell polarity required for uterine receptivity during implantation.Study Funding/competing Interests: This research was supported by the NICHD National Cooperative Reproductive Medicine Network grant HD039005 (M.P.D.), NIH grants HD068524 (S.K.D.), HD071408 (D.R.A., M.P.D.), and HL128628 (S.D.), the Intramural Research Program of the NICHD, March of Dimes (S.K.D., S.D.) and JSPS KAKENHI grant 26112506 (Y.H.). There were no conflicts or competing interests. [ABSTRACT FROM AUTHOR]

Objective: To determine the frequency of echogenic intracardiac focus (EIF) by race/ethnicity. Methods: We performed a retrospective analysis from January 1996 through June 2003. We reviewed all initial sonograms from 14 to 23 weeks gestation in singleton pregnancies. Mothers on admission for delivery provided race/ethnicity. Results: There were 8207 ultrasounds and deliveries that met study criteria. There were 4636 (56.5%) Caucasian, 2087 (25.4%) African-American, 1261 (15.4%) Hispanic and 223 (2.7 %) Asian subjects. There were 347 (4.2%) EIF detected. The frequency by race/ethnicity varied significantly (p < 0.0001). Conclusions: This large, population-based study showed that fetuses born to Asian mothers were significantly more likely to have an EIF. This racial difference should be taken into account when counseling patients about the potential for Down syndrome. [ABSTRACT FROM AUTHOR]

Objective: To determine the effect of sildenafil, a phosphodiesterase type 5 inhibitor, on trophoblast invasiveness.Design: Laboratory investigation.Setting: Academic medical center.Patient(s): Placental tissues discarded after first-trimester terminations were obtained from patients with informed consent.Intervention(s): A cell line, HTR-8/SVneo, established from first-trimester cytotrophoblast, and villous explants, was treated with or without sildenafil, guanosine 3',5'-cyclic monophosphate (cGMP) analog, cGMP inhibitor, or L-NAME (N(G)-nitro-L-arginine methyl ester hydrochloride) and cultured on fibronectin or Matrigel. Integrins α6β4 and α1β1 were detected by immunocytochemistry.Main Outcome Measure(s): Trophoblast outgrowth from villous tips, cytotrophoblast cell invasion, and integrin immunostaining were assessed in cytotrophoblast and explant cultures.Result(s): Integrin expression in trophoblast cells ex vivo switched from α6 to α1, and invasiveness increased, when exposed to sildenafil or cGMP agonist. Either cGMP antagonist or L-NAME blocked integrin switching and invasion induced by sildenafil. Elevation of nitric oxide pharmacologically induced invasion, but not when cGMP antagonist was present.Conclusion(s): Sildenafil altered trophoblast phenotype through a process dependent on nitric oxide availability and cGMP accumulation. In addition to its vasoactivity, sildenafil directly stimulates trophoblast extravillous differentiation, which would be favorable for implantation and reduce risk for adverse pregnancy outcomes. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the effect of infertility and intracytoplasmic sperm injection (ICSI) on DNA methylation of offspring.Design: Microarray analysis of DNA methylation in archived neonatal bloodspots of in vitro fertilization (IVF)/ICSI-conceived children compared with controls born to fertile and infertile parents.Setting: Academic research laboratory.Patient(s): Neonatal blood spots of 137 newborns conceived spontaneously, through intrauterine insemination (IUI), or through ICSI using fresh or cryopreserved (frozen) embryo transfer.Intervention(s): None.Main Outcome Measure(s): The Illumina Infinium HumanMethylation450k BeadChip assay determined genome-wide DNA methylation. Methylation differences between conception groups were detected using a Bioconductor package, ChAMP, in conjunction with Adjacent Site Clustering (A-clustering).Result(s): The methylation profiles of assisted reproductive technology and IUI newborns were dramatically different from those of naturally (in vivo) conceived newborns. Interestingly, the profiles of ICSI-frozen (FET) and IUI infants were strikingly similar, suggesting that cryopreservation may temper some of the epigenetic aberrations induced by IVF or ICSI. The DNA methylation changes associated with IVF/ICSI culture conditions and/or parental infertility were detected at metastable epialleles, suggesting a lasting impact on a child's epigenome.Conclusion(s): Both infertility and ICSI alter DNA methylation at specific genomic loci, an effect that is mitigated to some extent by FET. The impact of assisted reproductive technology and/or fertility status on metastable epialleles in humans was uncovered. This study provides an expanded set of loci for future investigations on IVF populations. [ABSTRACT FROM AUTHOR]

Objective To examine the expression pattern of biomarker proteins in extravillous trophoblast (EVT) cells obtained noninvasively by trophoblast retrieval and isolation from the cervix (TRIC) in patients with early pregnancy loss compared with control patients with uncomplicated term delivery. Design Case-control study. Setting Academic medical center. Patient(s) Women with either early pregnancy loss (EPL, n = 10) or an uncomplicated term delivery (N = 10). Intervention(s) Endocervical specimens obtained from ongoing pregnancies at gestational ages of 5–10 weeks to generate an archive of EVT cells isolated by TRIC, with medical records examined to select specimens matched for gestational age at the time of endocervical sampling. Main Outcome Measure(s) Known serum biomarkers for adverse pregnancy outcome that are expressed by EVT cells were evaluated by semiquantitative immunocytochemistry, using antibodies against endoglin (ENG), FMS-like tyrosine kinase-1 (FLT-1), α-fetoprotein (AFP), pregnancy-associated plasma protein-A (PAPP-A), galectin-13 (LGALS13), galectin-14 (LGALS14), and placental growth factor (PGF). Result(s) The EVT purity was over 95% in all specimens, based on chorionic gonadotropin expression; however, the number of EVT cells obtained was significantly lower in women with EPL than the control group. There was a statistically significant elevation of AFP, ENG, and FLT-1, and statistically significant reduction of PAPP-A, LGALS14, and PGF in the EPL group compared with controls. Conclusion(s) In this pilot study, EVT cells isolated by TRIC early in gestation exhibited altered protein expression patterns before an EPL compared with uncomplicated term pregnancies. [ABSTRACT FROM AUTHOR]

This review focuses on hypoxic stress and its effects on the placental lineage and the earliest differentiation events in mouse and human placental trophoblast stem cells (TSCs). Although the placenta is a decidual organ at the end of pregnancy, its earliest rapid growth and function at the start of pregnancy precedes and supports growth and function of the embryo. Earliest function requires that TSCs differentiate, however, "hypoxia" supports rapid growth, but not differentiation of TSCs. Most of the literature on earliest placental "hypoxia" studies used 2% oxygen which is normoxic for TSCs. Hypoxic stress happens when oxygen level drops below 2%. It decreases anabolism, proliferation, potency/stemness and increases differentiation, despite culture conditions that would sustain proliferation and potency. Thus, to study the pathogenesis due to TSC dysfunction, it is important to study hypoxic stress below 2%. Many studies have been performed using 0.5 to 1% oxygen in cultured mouse TSCs. From all these studies, a small number has examined human trophoblast lines and primary first trimester placental hypoxic stress responses in culture. Some other stress stimuli, aside from hypoxic stress, are used to elucidate common and unique aspects of hypoxic stress. The key outcomes produced by hypoxic stress are mitochondrial, anabolic, and proliferation arrest, and this is coupled with stemness loss and differentiation. Hypoxic stress can lead to depletion of stem cells and miscarriage, or can lead to later dysfunctions in placentation and fetal development. Birth Defects Research 109:1330-1344, 2017. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Survival of trophoblast cells in the low oxygen environment of human placentation requires metalloproteinase-mediated shedding of HBEGF and downstream signaling. A matrix metalloproteinase (MMP) antibody array and quantitative RT-PCR revealed upregulation of MMP2 post-transcriptionally in human first trimester HTR-8/SVneo trophoblast cells and placental villous explants exposed to 2% O 2 . Specific MMP inhibitors established the requirement for MMP2 in HBEGF shedding and upregulation. Because α-amanitin inhibited the upregulation of HBEGF, differentially expressed genes were identified by next-generation sequencing of RNA from trophoblast cells cultured at 2% O 2 for 0, 1, 2 and 4 h. Nine genes, all containing HIF-response elements, were upregulated at 1 h, but only HSPA6 (HSP70B') remained elevated at 2-4 h. The HSP70 chaperone inhibitor VER 155008 blocked upregulation of both MMP2 and HBEGF at 2% O 2 , and increased apoptosis. However, both HBEGF upregulation and apoptosis were rescued by exogenous MMP2. Proximity ligation assays demonstrated interactions between HSP70 and MMP2, and between MMP2 and HBEGF, supporting the concept that MMP2-mediated shedding of HBEGF, initiated by HSP70, contributes to trophoblast survival at the low O 2 concentrations encountered during the first trimester, and is essential for successful pregnancy outcomes. Trophoblast survival during human placentation, when oxygenation is minimal, required HSP70 activity, which mediated MMP2 accumulation and the transactivation of anti-apoptotic ERBB signaling by HBEGF shedding.

Previous studies showed that cultured mouse trophoblast stem cells (mTSCs) have the most rapid proliferation, normal maintenance of stemness/potency, the least spontaneous differentiation, and the lowest level of stress-activated protein kinase (SAPK) when incubated at 2% O 2 rather than at the traditional 20% O 2 or hypoxic (0.5% and 0% O 2 ) conditions. Switching from 2% O 2 induced fast SAPK responses. Here we tested the dose response of AMP-activated protein kinase (AMPK) in its active form (pAMPK Thr172P) at O 2 levels from 20-0%, and also tested whether pAMPK levels show similar rapid changes when mTSC cultures were switched from the optimal 2% O 2 to other O 2 conditions. There was a delayed increase in pAMPK levels ~6-8 h after switching conditions from 20% to 2%, 0.5%, or 0% O 2 . Altering O 2 conditions from 2% to either 20%, 0.5%, or 0% led to rapid increase in pAMPK levels within 1 h, similar to the previously reported SAPK response in mTSC cells removed from 2% O 2 . Twelve hours of 0.5% O 2 exposure led to cell program changes in terms of potency loss and suppressed biosynthesis, as indicated by levels of phosphorylated inactive acetyl CoA carboxylase (pACC). Phosphorylation of ACC was inhibited by the AMPK inhibitor Compound C. However, unlike other stressors, AMPK does not mediate hypoxia-induced potency loss in mTSCs. These results suggest an important aspect of stem cell biology, which demands rapid stress enzyme activation to cope with sudden changes in external environment, e.g., from least stressful (2% O 2 ) to more stressful conditions.

A contributing factor to poor placental perfusion, leading to intrauterine growth restriction and preeclampsia, is the failure of invading extravillous trophoblast (EVT) cells to remodel the maternal uterine arteries during the first and second trimesters of pregnancy. Noninvasive assessment of EVT cells in ongoing pregnancies is possible beginning three weeks after conception, using trophoblast retrieval and isolation from the cervix (TRIC). Seven proteins were semi-quantified by immunofluorescence microscopy in EVT cells obtained between gestational weeks 6 and 20 from pregnancies with normal outcomes (N = 29) and those with intrauterine growth restriction or preeclampsia (N = 12). Significant differences were measured in expression of PAPPA, FLT1, ENG, AFP, PGF, and LGALS14, but not LGALS13 or the lineage marker KRT7. These findings provide for the first time direct evidence of pathology-associated protein dysregulation in EVT cells during early placentation. The TRIC platform provides a novel approach to acquire molecular signatures of EVT cells that can be correlated with pregnancy outcome., Competing Interests: D.R.A., S.D. and M.P.D. have pending patents and receive payment for intellectual property that has been licensed on their behalf by Wayne State University to PerkinElmer, Inc.