Vanishing twins, spared cohorts, and the birthweight of periviable infants born to Black and white women in the United States.

Pregnancies ending before 26 weeks contribute 1% of births but 40% of infant deaths in the United States. The rate of these "periviable" births to non-Hispanic (NH) Black women exceeds four times that for NH whites. Small male periviable infants remain most likely to die. NH white periviable males weigh more than their NH Black counterparts. We argue that male infants born from twin gestations, in which one fetus died in utero (i.e., the vanishing twin syndrome), contribute to the disparity. We cannot directly test our argument because "vanishing" typically occurs before clinical recognition of pregnancy. We, however, describe and find associations that would emerge in vital statistics were our argument correct. Among male periviable singleton births from 288 monthly conception cohorts (January 1995 through December 2018), we found an average NH white advantage of 30 grams (759 grams versus 729 grams). Consistent with our argument, however, cohorts signaling relatively few survivors of the vanishing twin syndrome showed no disparity.

Prenatal exposure to ethion caused maternal and foetal toxicity in rats.

Ethion is a class II moderately toxic organothiophosphate pesticide. The main objective of this study was to evaluate the maternal and foetal toxicity of ethion in rats. Pregnant rats were divided into 5 groups. Group I served as control. Group II, III, IV, and V were orally administered with 0.86, 1.71, 3.43, and 6.9 mg/kg of ethion respectively, from gestational day (GD) 6-19. Dams were sacrificed on GD 20. Maternal toxicity was assessed by body weight gain, foetal resorptions, oxidative stress, liver and kidney function tests, and histopathology. Foetal toxicity was assessed by physical status, gross, teratological and histopathological examination. Ethion caused dose-dependent reduction in maternal body weight gain, increased resorptions, and reduced gravid uterine weights. Elevated MDA levels and altered levels of GSH, SOD and catalase were recorded in pregnant dam serum and tissues. SGOT, SGPT, total bilirubin, urea, uric acid, and creatinine were elevated in ethion groups indicating liver and kidney toxicity. Histology of uterus revealed myometrial degeneration and mucosal gland atrophy in uterus of pregnant dams and degenerative changes in placenta. It showed histological alterations in liver, kidney, and lungs. There was reduction in the foetal body weights and placental weights, and degenerative changes in the foetal liver and kidney. Gross evaluation of foetuses showed subcutaneous hematoma. Skeletal evaluation showed partial ossification of skull bones, costal separation, and agenesis of tail vertebrae, sternebrae, metacarpals and metatarsals. The findings reveal that prenatal exposure to ethion caused maternal and foetal toxicity in rats.

Toxic effects of 70% ethanol extract of Moringa stenopetala leaf (Baker f.) Cufod. (Moringaceae) on fetus and placenta of pregnant Wistar rats.

BACKGROUND: Moringa stenopetala leaves (Baker f.) Cufod. (Moringaceae) are used as a staple food and traditional medicine for treating various diseases like malaria, hypertension, stomach pain, diabetes, elevated cholesterol, and removing the retained placenta. Its prenatal toxicity study is minimal. Thus, this study aimed to assess the toxic effects of a 70% ethanol extract of Moringa stenopetala leaf on the fetuses and placentas of pregnant Wistar rats. METHOD: Fresh leaves of Moringa stenopetala were collected, dried at room temperature, ground to powder, and extracted using 70% ethanol. For this study, five groups of animals, each containing ten pregnant rats, were used. Groups I-III were experimental groups and treated with 250, 500, and 1000 mg/kg body weight of Moringa stenopetala leaf extract, respectively. Groups IV and V were pair-fed and ad libitum control groups. The extract was given during gestation days 6 to 12. The fetuses were recovered at day 20 of gestation and examined for the presence of developmental delays, gross external malformations, skeletal and visceral defects. Gross and histopathological changes in the placenta were also evaluated. RESULTS: Compared to the pair-fed control group, maternal daily food intake and weight gain were reduced in the 1000 mg/kg-treated group during the treatment and post-treatment periods. A significantly higher number of fetal resorptions was also seen in the 1000 mg/kg treatment group. The crown-rump length and fetal and placental weights were all significantly reduced in pregnant rats given 1000 mg/kg. However, there were no visible malformations in the visceral organs as well as external genitalia in all the treatment and control groups. About 40.7% of the fetuses in the 1000 mg/kg treated rats had no proximal hindlimb phalanges. In addition, light microscopic investigations of the placenta in the high-dose treated rats revealed structural changes in the decidual basalis, trophoblastic zone, and labyrinthine zones. CONCLUSION: In conclusion, consumption of M. stenopetala leaves at a higher dose may have toxic effects on the development of rat fetuses. At a higher dose, the plant extract increased the number of fetal resorptions, reduced the number of fetuses, decreased the fetal and placental weights, and alter the placental histopathology. Thus, it is recommended to limit the excess feeding of M. stenopetala leaves during gestation.

Second-Generation Human Immunodeficiency Virus Integrase Inhibitors Induce Differentiation Dysregulation and Exert Toxic Effects in Human Embryonic Stem Cell and Mouse Models.

BACKGROUND: Each year, approximately 1.1 million children are exposed in utero to human immunodeficiency virus antiretrovirals, yet their safety is often not well characterized during pregnancy. The Tsepamo study reported a neural tube defect signal in infants exposed to the integrase strand transfer inhibitor (InSTI) dolutegravir from conception, suggesting that exposure during early fetal development may be detrimental. METHODS: The effects of InSTIs on 2 human embryonic stem cell (hESC) lines were characterized with respect to markers of pluripotency, early differentiation, and cellular health. In addition, fetal resorptions after exposure to InSTIs from conception were analyzed in pregnant mice. RESULTS: At subtherapeutic concentrations, second-generation InSTIs bictegravir, cabotegravir, and dolutegravir decreased hESC counts and pluripotency and induced dysregulation of genes involved in early differentiation. At therapeutic concentrations, bictegravir induced substantial hESC death and fetal resorptions. It is notable that first-generation InSTI raltegravir did not induce any hESC toxicity or differentiation, at any concentration tested. CONCLUSIONS: Exposure to some InSTIs, even at subtherapeutic concentrations, can induce adverse effects in hESCs and pregnant mice. Given the increasingly prevalent use of second-generation InSTIs, including in women of reproductive age, it is imperative to further elucidate the effect of InSTIs on embryonic development, as well as their long-term safety after in utero exposure.

A microRNA or messenger RNA point of departure estimates an apical endpoint point of departure in a rat developmental toxicity model.

Traditional developmental toxicity testing practice examines fetal apical endpoints to identify a point of departure (POD) for risk assessment. A potential new testing paradigm involves deriving a POD from a comprehensive analysis of molecular-level change. Here, the rat ketoconazole endocrine-mediated developmental toxicity model was used to test the hypothesis that maternal epigenomic (miRNA) and transcriptomic (mRNA) PODs are similar to fetal apical endpoint PODs. Sprague-Dawley rats were exposed from gestation day (GD) 6-21 to 0, 0.063, 0.2, 0.63, 2, 6.3, 20, or 40 mg/kg/day ketoconazole. Dam systemic, liver, and placenta PODs, along with GD 21 fetal resorption, body weight, and skeletal apical PODs were derived using BMDS software. GD 21 dam liver and placenta miRNA and mRNA PODs were obtained using three methods: a novel individual molecule POD accumulation method, a first mode method, and a gene set method. Dam apical POD values ranged from 2.0 to 38.6 mg/kg/day; the lowest value was for placenta histopathology. Fetal apical POD values were 10.9-20.3 mg/kg/day; the lowest value was for fetal resorption. Dam liver miRNA and mRNA POD values were 0.34-0.69 mg/kg/day, and placenta miRNA and mRNA POD values were 2.53-6.83 mg/kg/day. Epigenomic and transcriptomic POD values were similar across liver and placenta. Deriving a molecular POD from dam liver or placenta was protective of a fetal apical POD. These data support the conclusion that a molecular POD can be used to estimate, or be protective of, a developmental toxicity apical POD.

Fetal resorption coincides with dysregulated LH secretion in AMH-overexpressing mice.

Female anti-Müllerian hormone (AMH) overexpressing (Thy1.2-AMHTg/0) mice experience fetal resorption (miscarriage) by mid-gestation. This study examined whether the ovary, uterine implantation sites and hypothalamus are potential sites of AMH action, as AMH type-2 receptor (AMHR2) expression is reported in each tissue. Pregnancy in Thy1.2-AMHTg/0 mice was compared to wild-type (WT) mice via histological examination of implantation sites, hormone assays, embryo culture and embryo transfer. Uterine AMH and AMHR2 expression was examined by RT-qPCR and immunohistochemistry. The first signs of fetal resorption in the Thy1.2-AMHTg/0 dams occurred at embryonic day 9.5 (E9.5) with 100% of fetuses resorbing by E13.5. Cultured embryos from Thy1.2-AMHTg/0 dams had largely normal developmental rates but a small proportion experienced a minor developmental delay relative to embryos from WT dams. However, embryos transferred from WT donor females always failed to survive to term when transferred into Thy1.2-AMHTg/0 dams. Amh and Amhr2 mRNA was detected in the gravid uterus but at very low levels relative to expression in the ovaries. Progesterone and estradiol levels were not significantly different between WT and Thy1.2-AMHTg/0 dams during pregnancy but luteinizing hormone (LH) levels were significantly elevated in Thy1.2-AMHTg/0 dams at E9.5 and E13.5 relative to WT dams. Collectively, these experiments suggest that AMH overexpression does not cause fetal resorption through an effect on oocytes or preimplantation embryo development. The Thy1.2-AMHTg/0 fetal resorption phenotype is nearly identical to that of transgenic LH overexpression models, suggesting that neuroendocrine mechanisms may be involved in the cause of the miscarriage.

BCG-trained innate immunity leads to fetal growth restriction by altering immune cell profile in the mouse developing placenta.

Trained immunity is a new concept illustrating that innate immune cells are able to undergo a long-term metabolic and epigenetic reprogramming after infection or vaccination, thus displaying either a pro- or an anti-inflammatory phenotype during a sequential unrelated challenge. Innate immune cells such as natural killer (NK) cells and macrophages constitute a large part of the decidual leukocyte population at the maternal-fetal interface, playing an important role in placental development and as such in fetal growth and development. In this study, we hypothesized that training the innate immune cells before pregnancy could have an impact on pregnancy. To test this hypothesis, we used CBA/J x DBA/2 mouse model to investigate pregnancy outcomes and leukocyte population at the maternal-fetal interface. Although we were not able to show a beneficial effect of LPS-tolerogenic training on fetal resorption, Bacillus Calmette-Guérin (BCG) training, known to prime innate immune cells to be proinflammatory, led to fetal growth restriction, without aggravating the fetal resorption rate. We also found that BCG training led to less NK cells and macrophages at the maternal-fetal interface at the early stage of placentation (E9.5), associated with a down-regulation of Ccr3 and Lif mRNA expression. This induced altered leucocyte population profile can be an explanation for the subsequent fetal growth restriction. These data suggest that preconceptional infections-induced trained immunity could influence pregnancy outcomes.

Toll-like receptor-4 null mutation causes fetal loss and fetal growth restriction associated with impaired maternal immune tolerance in mice.

Maternal immune adaptation to accommodate pregnancy depends on sufficient availability of regulatory T (Treg) cells to enable embryo implantation. Toll-like receptor 4 is implicated as a key upstream driver of a controlled inflammatory response, elicited by signals in male partner seminal fluid, to initiate expansion of the maternal Treg cell pool after mating. Here, we report that mice with null mutation in Tlr4 (Tlr4-/-) exhibit impaired reproductive outcomes after allogeneic mating, with reduced pregnancy rate, elevated mid-gestation fetal loss, and fetal growth restriction, compared to Tlr4+/+ wild-type controls. To investigate the effects of TLR4 deficiency on early events of maternal immune adaptation, TLR4-regulated cytokines and immune regulatory microRNAs were measured in the uterus at 8 h post-mating by qPCR, and Treg cells in uterus-draining lymph nodes were evaluated by flow cytometry on day 3.5 post-coitum. Ptgs2 encoding prostaglandin-endoperoxide synthase 2, cytokines Csf2, Il6, Lif, and Tnf, chemokines Ccl2, Cxcl1, Cxcl2, and Cxcl10, and microRNAs miR-155, miR-146a, and miR-223 were induced by mating in wild-type mice, but not, or to a lesser extent, in Tlr4-/- mice. CD4+ T cells were expanded after mating in Tlr4+/+ but not Tlr4-/- mice, with failure to expand peripheral CD25+FOXP3+ NRP1- or thymic CD25+FOXP3+ NRP1+ Treg cell populations, and fewer Treg cells expressed Ki67 proliferation marker and suppressive function marker CTLA4. We conclude that TLR4 is an essential mediator of the inflammation-like response in the pre-implantation uterus that induces generation of Treg cells to support robust pregnancy tolerance and ensure optimal fetal growth and survival.

Performance and Diagnostic Value of Genome-Wide Noninvasive Prenatal Testing in Multiple Gestations.

OBJECTIVE: To evaluate the accuracy and diagnostic value of genome-wide noninvasive prenatal testing (NIPT) for the detection of fetal aneuploidies in multiple gestations, with a focus on dichorionic-diamniotic twin pregnancies. METHODS: We performed a retrospective cohort study including data from pregnant women with a twin or higher-order gestation who underwent genome-wide NIPT at one of the eight Belgian genetic centers between November 1, 2013, and March 1, 2020. Chorionicity and amnionicity were determined by ultrasonography. Follow-up invasive testing was carried out in the event of positive NIPT results. Sensitivity and specificity were calculated for the detection of trisomy 21, 18, and 13 in the dichorionic-diamniotic twin cohort. RESULTS: Unique NIPT analyses were performed for 4,150 pregnant women with a multiple gestation and an additional 767 with vanishing gestations. The failure rate in multiple gestations excluding vanishing gestations ranged from 0% to 11.7% among the different genetic centers. Overall, the failure rate was 4.8%, which could be reduced to 1.2% after single resampling. There were no common fetal trisomies detected among the 86 monochorionic-monoamniotic and 25 triplet cases. Two monochorionic-diamniotic twins had an NIPT result indicative of a trisomy 21, which was confirmed in both fetuses. Among 2,716 dichorionic-diamniotic twin gestations, a sensitivity of 100% (95% CI 74.12-100%) and a specificity of 100% (95% CI 99.86-100%) was reached for trisomy 21 (n=12). For trisomy 18 (n=3), the respective values were 75% (95% CI 30.06-95.44%) sensitivity and 100% (95% CI 99.86-100%) specificity, and for trisomy 13 (n=2), 100% (95% CI 20.65-100%) sensitivity and 99.96% (95% CI 99.79-99.99%) specificity. In the vanishing gestation group, 28 NIPT results were positive for trisomy 21, 18, or 13, with only five confirmed trisomies. CONCLUSION: Genome-wide NIPT performed accurately for detection of aneuploidy in dichorionic-diamniotic twin gestations.

Applications of noninvasive prenatal testing in vanishing twin syndrome pregnancies after treatment of assisted reproductive technology in a single center.

OBJECTIVE: The objective of the study is to assess the clinical application of noninvasive prenatal testing (NIPT) for VTS pregnancies after the treatment of assisted reproductive technology (ART). METHOD: This was a retrospective study on VTS pregnancies through ART treatment. Participants underwent NIPT at 11 to 13 weeks gestation by sequencing. Resampling was recommended for both positive and testing failure cases. For NIPT positive results, participants were advised to have invasive testing. Clinical outcomes were obtained by telephone interview. RESULTS: In total of 579 cases, testing failure rates after first sampling and resampling were 7.6% and 1.4%, respectively. Twelve positive results were reported by NIPT. But only one true positive was confirmed, giving a PPV of 8%. A total of 576 cases completed the follow-up (including 533 NIPT negative, 12 positive, and 31 testing failure) and three cases lost follow-up. Among the 536 cases with NIPT negative results, 504 (94.0%) resulted in live-birth and 29 (5.4%) resulted in miscarriage or stillbirths. No false-negative result was reported. CONCLUSION: NIPT has the potential to be used in prenatal screening for VTS pregnancies. For the pregnant women who obtained positive and testing failure results, resampling after 15 weeks of gestation is recommended.

Adoptive cell therapy with induced regulatory T cells normalises the abortion rate in abortion-prone mice.

Ovarian hormones drive invivo generation of regulatory T cells (Tregs) during pregnancy. Little is known about the therapeutic potential of invitro hormone-derived Tregs in pregnancy loss. We investigated the effects of hormone-induced Tregs in a murine model of abortion. CD4+CD25- T cells were isolated from the spleens of CBA/J mice and stimulated with either 17ß-oestradiol (E2), progesterone (P4) or transforming growth factor-ß1 (TGFB1) plus retinoic acid (RA) for 4 days to generate induced Tregs (iTregs). On Days 1-4 of gestation, DBA/2-mated pregnant CBA/J female mice (abortion prone) were injected intravenously with iTregs or Tregs isolated from normal BALB/c-mated pregnant CBA/J mice (np-Tregs). On Day 14, the number of resorbed fetuses was assessed. Serum interferon (IFN)-γ and uterine forkhead box p3 (Foxp3) expression was analysed by ELISA and immunohistochemistry respectively. Using a 3H-thymidine incorporation assay, isolated CD4+CD25+ Tregs induced by the different treatments suppressed the proliferation of CD4+CD25- T cells. Adoptive transfer of iTregs (from all induction groups) significantly decreased fetal resorption in abortion-prone mice. There were no significant changes in serum IFN-γ concentrations after the adoptive transfer of iTregs or np-Tregs. Immunohistochemistry revealed significantly higher Foxp3 expression in gravid uteri from mice injected with np-Tregs and P4-induced iTregs than in the phosphate-buffered saline-treated group. The findings of this study indicate a potential therapeutic benefit of invitro-induced Tregs in patients with recurrent abortion.

The ubiquitination and acetylation of histones are associated with male reproductive disorders induced by chronic exposure to arsenite.

Exposure to arsenic, which occurs via various routes, can cause reproductive toxicity. However, the mechanism for arsenic-induced reproductive disorders in male mice has not been extensively investigated. Here, 6-week-old male mice were dosed to 0, 5, 10, or 20 ppm sodium arsenite (NaAsO2), an active form of arsenic, in drinking water for six months. For male mice exposed to arsenite, fertility was lower compared to control mice. Moreover, for exposed mice, there were lower sperm counts, lower sperm motility, and higher sperm malformation ratios. Further, the mRNA and protein levels of the gonadotropin-regulated testicular RNA helicase (DDX25) and chromosome region maintenance-1 protein (CRM1), along with proteins associated with high mobility group box 2 (HMGB2), phosphoglycerate kinase 2 (PGK2), and testicular angiotensin-converting enzyme (tACE) were lower. Furthermore, chronic exposure to arsenite led to lower H2A ubiquitination (ubH2A); histone H3 acetylation K18 (H3AcK18); and histone H4 acetylations K5, K8, K12, and K16 (H4tetraAck) in haploid spermatids from testicular tissues. These alterations disrupted deposition of protamine 1 (Prm1) in testes. Overall, the present results indicate that the ubiquitination and acetylation of histones is involved in the spermiogenesis disorders caused by chronic exposure to arsenite, which points to a previously unknown connection between the modification of histones and arsenite-induced male reproductive toxicity.

Comparison of the rates of preterm birth and low birth weight of vanishing twin and primary pregnancies conceived with assisted reproductive technology.

OBJECTIVES: The purpose of this study was to compare the rate of preterm birth, low birth weight, and foetal growth restriction in assisted reproductive technology (ART) singleton pregnancies diagnosed with vanishing twin (VT) syndrome to those of ART pregnancies that were originally singleton pregnancies. METHODS: In this retrospective study, 177 pregnancies diagnosed with VT syndrome were matched and compared with 218 primary singleton pregnancies. The preterm birth and low birth weight rates of these two groups were evaluated. All pregnancies were conceived through ART and delivered at Peking University First Hospital and Hebei Xingtai Infertility Hospital from 2014 to 2016. RESULTS: The preterm delivery rate (20.90 vs. 8.72%, p<0.05) was significantly higher in the ART singletons with VT syndrome than in the control singleton group. The proportion of low-birth-weight (<2500 g) infants was also higher in the VT group than in the primary singleton group (10.73 vs. 3.67%, p<0.05). In addition, the preterm birth rate of the naturally conceived singletons was significantly lower than that of the ART singletons (6.00 vs. 14.18%, p<0.05). CONCLUSIONS: ART singleton pregnancies with VT syndrome have higher rates of preterm birth and low-birth-weight new-borns than ART pregnancies that were originally singleton pregnancies.

Heme Oxygenase Protects against Placental Vascular Inflammation and Abortion by the Alarmin Heme in Mice.

Both infectious as non-infectious inflammation can cause placental dysfunction and pregnancy complications. During the first trimester of human gestation, when palatogenesis takes place, intrauterine hematoma and hemorrhage are common phenomena, causing the release of large amounts of heme, a well-known alarmin. We postulated that exposure of pregnant mice to heme during palatogenesis would initiate oxidative and inflammatory stress, leading to pathological pregnancy, increasing the incidence of palatal clefting and abortion. Both heme oxygenase isoforms (HO-1 and HO-2) break down heme, thereby generating anti-oxidative and -inflammatory products. HO may thus counteract these heme-induced injurious stresses. To test this hypothesis, we administered heme to pregnant CD1 outbred mice at Day E12 by intraperitoneal injection in increasing doses: 30, 75 or 150 µmol/kg body weight (30H, 75H or 150H) in the presence or absence of HO-activity inhibitor SnMP from Day E11. Exposure to heme resulted in a dose-dependent increase in abortion. At 75H half of the fetuses where resorbed, while at 150H all fetuses were aborted. HO-activity protected against heme-induced abortion since inhibition of HO-activity aggravated heme-induced detrimental effects. The fetuses surviving heme administration demonstrated normal palatal fusion. Immunostainings at Day E16 demonstrated higher numbers of ICAM-1 positive blood vessels, macrophages and HO-1 positive cells in placenta after administration of 75H or SnMP + 30H. Summarizing, heme acts as an endogenous "alarmin" during pregnancy in a dose-dependent fashion, while HO-activity protects against heme-induced placental vascular inflammation and abortion.

Vanishing Twins Conceived Through Fresh In Vitro Fertilization: Obstetric Outcomes and Placental Pathology.

OBJECTIVE: To characterize the obstetric outcomes and placental pathology in live births arising from vanishing twin pregnancies compared with nonreduced in vitro fertilization (IVF) pregnancies. METHODS: This is a retrospective cohort study of live births resulting from fresh embryo transfers after IVF cycles with autologous oocytes from 2004 through 2017 at a large academic fertility center. Clinical information and pathology reports were reviewed. Placental diagnoses were coded using established nosology by expert placental pathologists. Analysis of variance, Kruskal-Wallis, Pearson's χ, and Fisher exact tests were used, as appropriate, to compare pathology categories between pregnancy outcomes. Mixed effects logistic regression models were generated to reveal the association between pregnancy outcome and placenta pathology, controlling for pregnancies arising in the same woman and various suspected confounders. RESULTS: Of 905 fresh autologous IVF cycles with placental pathology available for review, we identified 73 vanishing twin pregnancies (8.1%), 556 singleton pregnancies (61.4%), and 276 twin pregnancies (30.5%). Vanishing twin syndrome was not associated with preterm delivery, route of delivery, growth restriction or other obstetric outcomes as compared with IVF singleton pregnancies. However, vanishing twin syndrome pregnancies showed distinctive placental pathologies including an increased rate of small placentas (less than the 10th percentile by weight), with more anatomical abnormalities than IVF singleton pregnancies (odds ratio 1.73, 95% CI 0.94-3.19; adjusted odds ratio 2.15, 95% CI 1.08-4.28). The frequency of placental vascular and inflammatory pathologies associated with IVF vanishing twin syndrome pregnancies were similar to that of IVF singleton pregnancies. Loss of a twin after 8 weeks of gestation was not associated with greater risks of placental pathologies. CONCLUSION: In vitro fertilization pregnancies affected by vanishing twin syndrome did not have significant differences in obstetric or perinatal outcomes as compared with twin or singleton gestations. However, early twin loss was potentially associated with differences in placental development associated with a higher rate of small placentas and other anatomic pathologies.

PDL1 blockage increases fetal resorption and Tfr cells but does not affect Tfh/Tfr ratio and B-cell maturation during allogeneic pregnancy.

A successful pregnancy requires sophisticated regulation of uterine microenvironment to guarantee the existence of semi-allogeneic conceptus without immune rejection. T follicular regulatory (Tfr) cells exert a suppressive effect on Tfh-cell expansion, B-cell response, and antibody production. Although accumulating evidence has demonstrated that dysregulations of Tfr cells can bring on various immunological diseases, their immunomodulatory roles during pregnancy still remain unheeded. Herein, we introduced an allogeneic normal-pregnant mouse model and found that CD4+CXCR5hiPD-1hiFoxp3+ Tfr cells were preferentially accumulated in the uterus at mid-gestation and displayed a distinct phenotype. In addition, the absence of PDL1 resulted in increased fetal resorption by favoring Tfr cells accumulation and upregulating PD-1 expression on these cells. However, PDL1 blockade affected neither the ratio of Tfh/Tfr cells nor the maturation and differentiation of B cells. Overall, our results are the first to present a correlation of Tfr cells accumulation with healthy allogeneic pregnancy and PDL1 blockade-induced miscarriage, and to indicate that appropriate assembly of Tfr cells is important for pregnancy maintenance. Since blockade of PD-1-PDL1 pathway leads to more Tfr cells and fetal losses, the reproductive safety must be taken into consideration when PD-1/PD-L1 checkpoint blockade immunotherapy is used in pregnancy.

Gut dysbiosis induces the development of pre-eclampsia through bacterial translocation.

OBJECTIVE: Pre-eclampsia (PE) is one of the malignant metabolic diseases that complicate pregnancy. Gut dysbiosis has been identified for causing metabolic diseases, but the role of gut microbiome in the pathogenesis of PE remains unknown. DESIGN: We performed a case-control study to compare the faecal microbiome of PE and normotensive pregnant women by 16S ribosomal RNA (rRNA) sequencing. To address the causative relationship between gut dysbiosis and PE, we used faecal microbiota transplantation (FMT) in an antibiotic-treated mouse model. Finally, we determined the microbiome translocation and immune responses in human and mouse placental samples by 16S rRNA sequencing, quantitative PCR and in situ hybridisation. RESULTS: Patients with PE showed reduced bacterial diversity with obvious dysbiosis. Opportunistic pathogens, particularly Fusobacterium and Veillonella, were enriched, whereas beneficial bacteria, including Faecalibacterium and Akkermansia, were markedly depleted in the PE group. The abundances of these discriminative bacteria were correlated with blood pressure (BP), proteinuria, aminotransferase and creatinine levels. On successful colonisation, the gut microbiome from patients with PE triggered a dramatic, increased pregestational BP of recipient mice, which further increased after gestation. In addition, the PE-transplanted group showed increased proteinuria, embryonic resorption and lower fetal and placental weights. Their T regulatory/helper-17 balance in the small intestine and spleen was disturbed with more severe intestinal leakage. In the placenta of both patients with PE and PE-FMT mice, the total bacteria, Fusobacterium, and inflammatory cytokine levels were significantly increased. CONCLUSIONS: This study suggests that the gut microbiome of patients with PE is dysbiotic and contributes to disease pathogenesis.

First-trimester screening for trisomies in pregnancies with vanishing twin.

OBJECTIVES: To examine multiples of the median (MoM) values of serum free beta-human chorionic gonadotropin (ß-hCG) and pregnancy-associated plasma protein-A (PAPP-A) in a large series of pregnancies with a vanishing twin, determine the association of these values with the interval between embryonic death and blood sampling, and develop a model that would allow incorporation of these metabolites in first-trimester combined screening for trisomy. METHODS: This was a retrospective study comparing maternal serum free ß-hCG and PAPP-A levels at 11-13 weeks' gestation in 528 dichorionic pregnancies with a vanishing twin, including 194 (36.7%) with an empty gestational sac and 334 (63.3%) with a dead embryo, with those in 5280 normal singleton pregnancies matched for method of conception and date of examination. In vanishing-twin pregnancies with a dead embryo, marker levels were examined in relation to the estimated time between embryonic death and maternal blood sampling. RESULTS: First, in pregnancies with a vanishing twin, median free ß-hCG MoM was not significantly different from that in normal singleton pregnancies (1.000; 95% CI, 0.985-1.016 vs 0.995; 95% CI, 0.948-1.044; P = 0.849). Second, PAPP-A MoM was higher in vanishing-twin pregnancies than in normal singleton pregnancies (1.000; 95% CI, 0.985-1.015), both in the group with an empty gestational sac (1.165; 95% CI, 1.080-1.256; P = 0.0001) and in that with a dead embryo (1.175; 95% CI, 1.105-1.249; P < 0.0001). Third, in vanishing-twin pregnancies with a dead embryo, PAPP-A MoM was related inversely to the interval between estimated gestational age at embryonic demise and blood sampling (P < 0.0001). Fourth, in first-trimester screening for trisomy 21 in singleton pregnancies, the estimated detection rate, at a 5% false-positive rate, was 82% in screening by a combination of maternal age and fetal nuchal translucency thickness, and this increased to 86% with the addition of serum free ß-hCG and to 91% with the addition of serum PAPP-A. Fifth, similar performance of screening can be achieved in pregnancies with a vanishing twin, provided the appropriate adjustments are made to the level of PAPP-A for the interval between estimated gestational age at embryonic demise and blood sampling. CONCLUSIONS: First-trimester screening for trisomy in pregnancies with a vanishing twin should rely on a combination of maternal age, fetal nuchal translucency thickness and serum free ß-hCG, as in singleton pregnancy, without the use of serum PAPP-A. Alternatively, PAPP-A can be included but only after appropriate adjustment for the interval between estimated gestational age at fetal demise and blood sampling. Copyright © 2019 ISUOG. Published by John Wiley & Sons Ltd.

IFITM proteins inhibit placental syncytiotrophoblast formation and promote fetal demise.

Elevated levels of type I interferon (IFN) during pregnancy are associated with intrauterine growth retardation, preterm birth, and fetal demise through mechanisms that are not well understood. A critical step of placental development is the fusion of trophoblast cells into a multinucleated syncytiotrophoblast (ST) layer. Fusion is mediated by syncytins, proteins deriving from ancestral endogenous retroviral envelopes. Using cultures of human trophoblasts or mouse cells, we show that IFN-induced transmembrane proteins (IFITMs), a family of restriction factors blocking the entry step of many viruses, impair ST formation and inhibit syncytin-mediated fusion. Moreover, the IFN inducer polyinosinic:polycytidylic acid promotes fetal resorption and placental abnormalities in wild-type but not in Ifitm-deleted mice. Thus, excessive levels of IFITMs may mediate the pregnancy complications observed during congenital infections and other IFN-induced pathologies.

The evaluation of embryotoxicity of Ligusticum chuanxiong on mice and embryonic stem cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Chuanxiong (Chuanxiong Rhizoma, CR), the dried rhizome of Ligusticum chuanxiong Hort, has been used during pregnancy for more than 2000 years. However, the embryotoxicity of CR was not evaluated so far. The purpose of this study was to examine the safety and rational use of CR during pregnancy on mice and mouse embryonic stem cell (ES), and to explore the mechanism of embryotoxicity. AIM OF THE STUDY: This study was carried out to evaluate embryotoxicity of CR decoction in vivo and in vitro, and to explore the mechanism of embryotoxicity from the perspective of bone metabolism. MATERIALS AND METHODS: In animal experiments, pregnant mice were randomly assigned into 5 groups, i.e. mice were orally treated with CR decoction at dosages of 0 (distilled water, as negative controls), 2, 8, 32 g/kg/d (low, medium and high-dose group), and vitamin A (as positive controls), respectively. Maternal and embryo-fetal parameters were registered after cesarean section. The fetal skeletal development was further assessed with the alizarin red S and Hematoxylin-Eosin staining (H&E staining) and fluorescent imaging. Meanwhile, the mouse embryonic stem cell test model (EST model) was established to objectively evaluate the toxicity of CR on the embryo development. The median inhibitory proliferation values (IC50) for both the mouse embryonic stem cell D3 (ES) and mouse embryonic fibroblast 3T3 (3T3) were detected with MTT assays. After removal of inhibiting factor (LIF), mouse embryonic stem cells spontaneously differentiated into cardiomyocytes, the expression of specific myosin heavy chain gene (ß-MHC) contained in cardiomyocytes were detected by q-PCR quantitative analysis, and median inhibitory differentiation concentration (ID50) of ES was obtained. The development toxicity calculation formula was used to determine the embryotoxicity grade of CR decoction. finally, based on the successful induction of osteoblasts, the molecular mechanism of CR embryotoxicity was preliminarily studied based on BMP-Smads signal pathway. RESULTS: Compared with the negative control group, high, medium, and low doses of CR decoction had no significant effect on the maternal body weight and uterine weight (P > 0.05), as well as on the maternal liver, heart, and kidneys. The observation results showed that high dose of CR decoction significantly increase the number of absorbed fetuses (P < 0.05). The EST model was successfully established, the IC50 3T3, IC50 ES and ID50 ES of CR were 9.39 mg/mL, 18.78 mg/mL, and 10.20 mg/mL, respectively. CR was classified as weak embryonic development toxicity by the EST linear discriminant formula. Meanwhile, osteoblasts were successfully induced in vitro, the relative expression levels of BMP2, BMPR2, Smad1, and Smad5 were down-regulated in varying degrees after 3, 6, and 9 days of treatment with different concentration gradients of CR decoction. CONCLUSIONS: Combining in vivo and in vitro experiments, CR showed a potential embryotoxicity. The mechanism of embryotoxicity may be related to inhibiting the expression of key genes in the BMP-SMADs signaling pathway. In the clinical application, the normal dosage of CR is safe to a certain extent. However, when the dosage is too high (160 g/60 kg/d), there may be a risk of embryotoxicity.