Gestational Time Points Research Articles

Contribution of Amniotic Fluid along Gestation to the Prediction of Perinatal Mortality in Women with Early Preterm Premature Rupture of Membranes

Introduction: To evaluate the largest vertical pocket (LVP) of amniotic fluid as a time-dependent factor to predict perinatal mortality in women with early preterm premature rupture of membranes (EPPROM). Material and Methods: Observational cohort study of singleton pregnancies with EPPROM <24 weeks. Termination of pregnancy (TOP) was considered if the LVP was <2 cm at 7 days. The maternal and neonatal characteristics of ongoing pregnancies were recorded. Prediction of perinatal mortality was estimated based on the influence of the LVP as a time-dependent factor after adjustment for maternal age, prior invasive procedure, and gestational age at EPPROM. Results: Of 104 women, 39 requested TOP. Neonatal survival to discharge was 40%, increasing to 74% if pregnancies achieved 24 weeks. LVP at admission <1 cm, latency to delivery, and gestational age at delivery were independent predictors of perinatal mortality. When evaluating the LVP at different time points of gestation, the highest perinatal mortality risk was established at 2 weeks (odds ratio 14.67, p < 0.001) after membrane rupture, being 5.75 (p = 0.05) the week after and 10.93 (p = 0.037) beyond 2 weeks of EPPROM. Discussion: When LVP measurement, gestational age at EPPROM, maternal age, and prior invasive procedure were considered, we found that the worst prognosis related to perinatal mortality was at 2 weeks after EPPROM.

ERα and GnRH co-localize in the hypothalamic neurons of the South American plains vizcacha, Lagostomus maximus (Rodentia, Caviomorpha).

Gonadotropin-releasing hormone (GnRH) is the key regulator of the hypothalamic-pituitary-gonadal axis. Estradiol (E2) affects GnRH synthesis and delivery. Hypothalamic estrogen receptors (ER) modulate GnRH expression acting as transcription factors. The South American plains vizcacha, Lagostomus maximus, is able to ovulate up to 800 oocytes per reproductive cycle, and shows continuous folliculogenesis with pre-ovulatory follicle formation and an ovulatory event at mid-gestation. The aim of this work was to analyze the hypothalamic expression of ER in the vizcacha at different gestational time-points, and its relationship with GnRH expression, serum luteinizing hormone (LH) and E2. The hormonal pattern of mid-gestating vizcachas was comparable to ovulating-females with significant increases in GnRH, LH and E2. Hypothalamic protein and mRNA expression of ERα varied during pregnancy with a significant increase at mid-gestation whereas ERβ mRNA expression did not show significant variations. Hypothalamic immunolocalization of ERα was observed in neurons of the diagonal band of Brocca, medial preoptic area (mPOA), periventricular, suprachiasmatic, supraoptic (SON), ventromedial, and arcuate nuclei, and medial eminence, with a similar distribution throughout gestation. In addition, all GnRH neurons of the mPOA and SON showed ERα expression with no differences across the reproductive status. The correlation between GnRH and ERα at mid-gestation, and their co-localization in the hypothalamic neurons of the vizcacha, provides novel information compared with other mammals suggesting a direct action of estrogen as part of a differential reproductive strategy to assure GnRH synthesis during pregnancy.

Placental hormone profiles as predictors of preterm birth in twin pregnancy: A prospective cohort study.

ObjectiveThe objective of the study was to analyse placental hormone profiles in twin pregnancies to determine if they could be used to predict preterm birth.Study designProgesterone, estradiol, estriol and corticotropin-releasing hormone were measured using competitive immunoassay and radioimmunoassay in serum and saliva samples of 98 women with twin pregnancies,at 3 or more gestational timepoints. Hormone profiles throughout gestation were compared between very preterm (<34 weeks; n = 8), preterm (<37 weeks; n = 40) and term (37+ weeks; n = 50) deliveries.ResultsNo significant differences were found between preterm and term deliveries in either absolute hormone concentrations or ratios. Estimated hormone concentrations and ratios at 26 weeks did not appear to predict preterm delivery. Salivary and serum hormone concentrations were generally poorly correlated.ConclusionOur results suggest that serial progesterone, estradiol, estriol and corticotropin-releasing hormone measurements in saliva and serum are not robust biomarkers for preterm birth in twin pregnancies.

Use of high-frequency ultrasound to study the prenatal development of cranial neural tube defects and hydrocephalus in Gldc-deficient mice.

ObjectiveWe used non‐invasive high‐frequency ultrasound (HFUS) imaging to investigate embryonic brain development in a mouse model for neural tube defects (NTDs) and non‐ketotic hyperglycinemia (NKH).MethodUsing HFUS, we imaged embryos carrying loss of function alleles of Gldc encoding glycine decarboxylase, a component of the glycine cleavage system in mitochondrial folate metabolism, which is known to be associated with cranial NTDs and NKH in humans. We serially examined the same litter during the second half of embryonic development and quantified cerebral structures. Genotype was confirmed using PCR. Histology was used to confirm ultrasound findings.ResultsHigh‐frequency ultrasound allowed in utero detection of two major brain abnormalities in Gldc‐deficient mouse embryos, cranial NTDs (exencephaly) and ventriculomegaly (corresponding with the previous finding of post‐natal hydrocephalus). Serial ultrasound allowed individual embryos to be analysed at successive gestational time points. From embryonic day 16.5 to 18.5, the lateral ventricle volume reduced in wild‐type and heterozygous embryos but increased in homozygous Gldc‐deficient embryos.ConclusionExencephaly and ventriculomegaly were detectable by HFUS in homozygous Gldc‐deficient mouse embryos indicating this to be an effective tool to study CNS development. Longitudinal analysis of the same embryo allowed the prenatal onset and progression of ventricle enlargement in Gldc‐deficient mice to be determined. © 2017 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.

Early Intra-Amniotic Gene Transfer using Lentiviral Vectors in Murine Model

Gene transfer after intra-amniotic injection has, in general, been of low efficiency and limited to epithelial cells in the skin, pulmonary and gastrointestinal system. We have shown that early gestational administration results in a more efficient gene transfer to developmentally accessible stem cell populations in the skin and eye. In this presentation, we present a comprehensive analysis of patterns of tissue expression seen after early intra-amniotic gene transfer (IAGT) using lentiviral vectors. To assess the influence of developmental stage on tissue expression, injections were administered from the late head fold/early somite stage (E8) to E18. In early gestation (E8-10), green fluorescent protein (GFP) expression was observed in multiple organs, derived from all three germ layers. Remarkably, GFP expression was observed in tissues derived from mesoderm and neural ectoderm at E8, whereas expression was limited to only epithelial cells of ectoderm- and endoderm-derived organs after E11. The amount and duration of gene expression was much higher after IAGT at early gestational time points. The observed temporal patterns of gene expression correspond to the predicted developmental accessibility of organ-specific cell populations. This model may be useful for the analyses of mechanisms of genetic and/or developmental disease and for the development of prenatal gene therapy for specific disorders.

Assessing extensive cardiac echography examination for detecting foetal congenital heart defects during early and late gestation: a systematic review and meta-analysis

Background Extensive cardiac echography examination (ECEE) can identify cardiac heart defects (CHDs) in utero. However, there is not yet a clear appraisal of ECEE’s performance in detecting foetal CHDs as a function of gestational time. In this study, we assessed the diagnostic efficacy of ECEE in detecting CHDs during early gestation (< 18 weeks) and late gestation (≥ 18 weeks).Methods MEDLINE, EMBASE, and Cochrane CENTRAL databases were searched for ECEE studies that had a prenatal diagnosis later validated by echocardiography, catheterization, surgery, or autopsy and allowed construction of 2 × 2 tables. Pooled sensitivity, specificity, and diagnostic odds ratio of ECEE during early and late gestation were calculated. Summary receiver operating characteristic curves were plotted to obtain area under the curve (AUC) values.Results ECEE in late gestation had a slightly superior sensitivity relative to that in early gestation (0.90 vs 0.86, respectively), while ECEE during both early and late gestation displayed near-perfect specificity (1.00 vs 1.00, respectively). ECEE in late gestation displayed a superior diagnostic odds ratio relative to that in late gestation (3,502.81 vs 1,076.79, respectively). ECEE during both early and late gestation displayed similar overall diagnostic accuracy in terms of AUC (0.9861 vs 0.9783, respectively).Conclusions ECEE performed before the 18-week gestational time point provides nearly equivalent diagnostic accuracy to ECEE performed on or after the 18-week gestational time point. However, ECEE conducted prior to week 11 may not be advisable due to the immature development of foetal heart structures.

RAPID COMMUNICATION: Expression of an endogenous retroviral element, during early gestation in beef heifers.

Endogenous retroviral gene elements have been implicated in development and formation of the feto-maternal interface. A variant of the syncytin endogenous retroviral envelope gene family, , was recently found in ruminants. We hypothesized that mRNA would be differentially expressed in utero-placental tissues and would fluctuate during key time points of early gestation in beef heifers. Commercial Angus crossbred heifers ( = 46; ∼15 mo of age; BW = 362.3 ± 34.7kg) housed in 6-animal pens were fed daily with native grass hay and supplemented with cracked corn to gain 0.3 kg/d. The heifers were estrus synchronized, artificially inseminated, (d of breeding= d 0) and ovariohysterectomized on d 16, 22, 28, 34, 40, and 50 ( = 9, 6, 6, 7, 6, and 5, respectively) of gestation and at d 16 of the estrous cycle for non-bred, non-pregnant controls (NP; = 7). Harvested tissues were separated into maternal caruncle (CAR), intercarunclar endometrium (ICAR), and fetal membranes, (FM; chorioallantois, d 22 and later). All tissues were obtained from the ipsilateral uterine horn to the CL. Statistical analyses were conducted via the GLM procedure of SAS. Maternal CAR expression of was greater ( = 0.003) on d 50 by 81.5-fold compared to NP controls. At d 50 expression of in CAR was 190.3-fold greater than ( < 0.0001) ICAR. Fetal membranes had greater ( < 0.002) expression of from d 22 until d 50 of gestation compared to maternal ICAR (d 16 not analyzed). Expression of in FM was greater ( < 0.004) than in CAR until d 40 of gestation. Therefore, we conclude that is differentially expressed in utero-placental tissues and may be involved in the establishment of pregnancy. The expression of in maternal tissues is completely novel and indicates unique functions of syncytin in ruminant pregnancy.

Changes in Circulating ProAMH and Total AMH during Healthy Pregnancy and Post-Partum: A Longitudinal Study.

Circulating Anti-Müllerian hormone (AMH) is derived from the gonads, and is a mixture of the prohormone (proAMH), which does not bind to AMH receptors, and receptor-competent AMH. The functions of a hormone are partially defined by the factors that control its levels. Ovarian reserve accounts for 55~75% of the woman-to-woman variation in AMH level, leaving over 25% of the biological variation to be explained. Pregnancy has been reported to decrease circulating AMH levels, but the observations are inconsistent, with the effect of pregnancy on the bioactivity of AMH being unknown. We have therefore undertaken a longitudinal study of circulating proAMH and total AMH during pregnancy. Serum samples were drawn at 6–8 gestational time-points (first trimester to post-partum) from 25 healthy women with prior uneventful pregnancies. The total AMH and proAMH levels were measured at each time-point using ELISA. The level of circulating total AMH progressively decreased during pregnancy, in all women (p<0.001). On average, the percentage decline between the first trimester and 36–39 weeks’ gestation was 61.5%, with a standard deviation of 13.0% (range 30.4–81.2%). The percentage decline in total AMH levels associated with maternal age (R = -0.53, p = 0.024), but not with the women’s first trimester AMH level. The postpartum total AMH levels showed no consistent relationship to the woman’s first trimester values (range 31–273%). This raises the possibility that a fundamental determinant of circulating AMH levels is reset during pregnancy. The ratio of proAMH to total AMH levels exhibited little or no variation during pregnancy, indicating that the control of the cleavage/activation of AMH is distinct from the mechanisms that control the total level of AMH.

Gestational hormone profiles predict human maternal behavior at 1-year postpartum

In many non-human species, including primates, gestational reproductive hormones play an essential role in the onset of maternal motivation and behaviors. We investigated the associations between prepartum estradiol and progesterone and maternal behavior at 1-year postpartum in 177 women. Blood was obtained at five gestational time points and an index of quality of maternal care was determined using a well-validated mother-child interaction protocol. Women who exhibited higher quality maternal care at 1-year postpartum were characterized by unique gestational profiles of estradiol, progesterone and the estrogen to progesterone ratio; specifically by slower accelerations and levels of these hormone trajectories beginning in midgestation. Further, it appeared that both fetal sex and parity moderated these findings, with first time mothers and mothers of females showing stronger associations. In sum, these data document persisting associations between prepartum hormone profiles and human maternal behavior. More broadly, these findings add to the growing literature highlighting the perinatal period as one of critical neurodevelopment in the lifespan of the human female.

13 The renin–angiotensin–aldosterone system and superimposed pre-eclampsia in women with chronic kidney disease and chronic hypertension

Introduction Systemic renin–angiotensin–aldosterone system (RAAS) activity is augmented in non-pregnant women with chronic kidney disease (CKD) and chronic hypertension (CHT), but its activity in pregnancy and superimposed pre-eclampsia is poorly understood. Objectives (i) To compare longitudinal changes in plasma renin and angiotensinogen concentrations in pregnant women with CKD and CHT with healthy controls. (ii) To compare differences in plasma renin and angiotensinogen concentrations in pregnant women with CKD and CHT with and without superimposed pre-eclampsia and women with pre-eclampsia without pre-existing disease. Methods Plasma renin activity and angiotensinogen were quantified in samples from women with CKD or CHT, women with pre-eclampsia without pre-existing disease and healthy controls recruited to a longitudinal prospective cohort study at two London Academic Health Centers. Demographics and pregnancy outcomes were extracted from patient records. RAAS component concentrations were compared across gestational ages using repeated measures analysis. Results One hundred and ninety-five women were recruited (CKD=80; Primary CHT=31; Pre-eclampsia without pre-existing disease=19; Healthy controls=65). Eighteen women with CKD (22.5%) and 10 women (32.3%) with primary CHT developed superimposed pre-eclampsia. Plasma renin activity was lower in women with CKD or CHT with compared to healthy controls at all gestational time points ( p p 0.003) or healthy controls ( p Conclusion Women with CKD or CHT have suppressed plasma renin activity during pregnancy compared with healthy controls. After diagnosis of superimposed pre-eclampsia women with CKD or CHT have lower plasma renin than women without superimposed pre-eclampsia, but there are no differences between women with pre-eclampsia and superimposed pre-eclampsia, suggestive of a common pathophysiological pathway.

Fetal and Neonatal Diastolic Myocardial Strain Rate: Normal Reference Ranges and Reproducibility in a Prospective, Longitudinal Cohort of Pregnancies

Normative fetal diastolic strain rate values have not been comprehensively reported. The aims ofthis study were to report normative data for diastolic strain rate parameters across gestation and upon delivery, determine the effect of advancing gestational age on these measures, and compare interobserver variability at multiple gestational age time points. Sixty gravid women were enrolled before 20weeks' gestation. The following measures were obtained by two blinded observers at five time points across gestation and at 4 to 8weeks' postnatal age: global left ventricular circumferential strain rate peak E and A waves, global left and right ventricular longitudinal strain rate peak E and A waves, and mitral and tricuspid valve E/A ratios. Reproducibility was assessed using intraclass correlation and Bland-Altman analysis. Least square means analysis was used to evaluate for changes across gestational age. Left ventricular longitudinal and circumferential diastolic strain rate values decreased across gestation, while right ventricular longitudinal values remained stable. With delivery, left ventricular values remained fairly stable, while right ventricular values increased. Intraclass correlations for diastolic strain rate values were 0.68 to 0.94 at ≥24weeks' gestation and 0.25 to 0.82 for values at 20 to 21weeks' gestation. Intraclass correlations ranged from 0.49 to 0.90 for mitral valve and tricuspid valveE/A ratios across gestation. Fetal measures of diastolic strain rate have acceptable reproducibility after 24weeks' gestation. The described changes across gestation likely reflect intrinsic myocardial adaptation to loading conditions. These reference ranges can be used to assess effects of various disease states on fetal myocardial deformation.

Maternal vitamin C deficiency does not reduce hippocampal volume and β-tubulin III intensity in prenatal Guinea pigs

Marginal vitamin C (vitC) deficiency affects 5% to 10% of adults including subpopulations such as pregnant women and newborns. Animal studies link vitC deficiency to deleterious effects on the developing brain, but exactly how the brain adapts to vitC deficiency and the mechanisms behind the observed deficits remain largely unknown. We hypothesized that vitC deficiency in utero may lead to a decreased neuronal maturation and increased cellular death giving rise to alterations of the hippocampal morphology in a guinea pig model. Brains from prenatal guinea pig pups (n=9-10 in each group) subjected to either a sufficient (918mg vitC/kg feed) or deficient (100mg vitC/kg feed) maternal dietary regimen were assessed with regards to hippocampal volume and β-tubulin isotype III staining intensity at 2 gestational time points (45 and 56). We found a distinct differential regional growth pattern of the hippocampus with a clear effect of gestational age, whereas vitC status did not affect either investigated parameters. Within hippocampal subdivisions, the overall expansion of the hippocampus from gestational day 45 to 56 was found to reside in the dentate gyrus. In conclusion, the present study found that hippocampal volume and β-tubulin isotype III intensity in the prenatal guinea pig were influenced by gestational day but not by maternal vitC intake.

25-Hydroxyvitamin D in pregnancy and genome wide cord blood DNA methylation in two pregnancy cohorts (MoBa and ALSPAC)

The aim of the study was to investigate whether maternal mid-pregnancy 25-hydroxyvitamin D concentrations are associated with cord blood DNA methylation.DNA methylation was assessed using the Illumina HumanMethylation450 BeadChip, and maternal plasma 25-hydroxyvitamin D was measured in 819 mothers/newborn pairs participating in the Norwegian Mother and Child Cohort (MoBa) and 597 mothers/newborn pairs participating in the Avon Longitudinal Study of Parents and Children (ALSPAC).Across 473,731CpG DNA methylation sites in cord blood DNA, none were strongly associated with maternal 25-hydroxyvitamin D after adjusting for multiple tests (false discovery rate (FDR)>0.5; 473,731 tests). A meta-analysis of the results from both cohorts, using the Fisher method for combining p-values, also did not strengthen findings (FDR>0.2). Further exploration of a set of CpG sites in the proximity of four a priori defined candidate genes (CYP24A1, CYP27B1, CYP27A1 and CYP2R1) did not result in any associations with FDR<0.05 (56 tests). In this large genome wide assessment of the potential influence of maternal vitamin D status on DNA methylation, we did not find any convincing associations in 1416 newborns. If true associations do exist, their identification might require much larger consortium studies, expanded genomic coverage, investigation of alternative cell types or measurements of 25-hydroxyvitamin D at different gestational time points.

Increased oxidative stress from early pregnancy in women who develop preeclampsia

Preeclampsia (PE) is a pregnancy-specific disorder, defined as new onset of maternal hypertension and proteinuria after 20 weeks of gestation. Our earlier study has shown increased maternal oxidative stress at delivery to be associated with poor birth outcome in PE. However, these results were observed when the pathology had progressed and may have been secondary to the effects of the disorder. To understand the role of antioxidant defense mechanisms in PE right from early pregnancy, in this prospective study, we measured malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione (GSH) concentrations in maternal blood at 3 time-points of gestation [16–20 weeks (T1), 26–30 weeks (T2), at delivery (T3)] and in cord blood. Gene expression of SOD and GPx and protein levels of endothelial nitric oxide synthase (eNOS) enzyme were also analyzed in the placenta. MDA levels were higher at T1 (p < 0.01) and T2 (p < 0.01) in women with PE as compared with control. GPx levels were higher at T3 (p < 0.05) while SOD levels were lower at T2 (p < 0.05), T3 (p < 0.01) and in cord (p < 0.01) in PE. GSH levels at T1 (p < 0.05) and expression of GPx in the placenta were lower in PE as compared with control. In conclusion, this study demonstrates that women who develop PE exhibit increased oxidative stress right from 16 to 20 weeks of gestation. This may alter placental development and lead to fetal programming of adult non-communicable disease in the offspring.

Nicotine ameliorates schizophrenia-like cognitive deficits induced by maternal LPS exposure: a study in rats.

ABSTRACTMaternal exposure to infectious agents is a predisposing factor for schizophrenia with associated cognitive deficits in offspring. A high incidence of smoking in these individuals in adulthood might be, at least in part, due to the cognitive-enhancing effects of nicotine. Here, we have used prenatal exposure to maternal lipopolysaccharide (LPS, bacterial endotoxin) at different time points as a model for cognitive deficits in schizophrenia to determine whether nicotine reverses any associated impairments. Pregnant rats were treated subcutaneously with LPS (0.5 mg/kg) at one of three neurodevelopmental time periods [gestation days (GD) 10-11, 15-16, 18-19]. Cognitive assessment in male offspring commenced in early adulthood [postnatal day (PND) 60] and included: prepulse inhibition (PPI), latent inhibition (LI) and delayed non-matching to sample (DNMTS). Following PND 100, daily nicotine injections (0.6 mg/kg, subcutaneously) were administered, and animals were re-tested in the same tasks (PND 110). Only maternal LPS exposure early during fetal neurodevelopment (GD 10-11) resulted in deficits in all tests compared to animals that had been prenatally exposed to saline at the same gestational time point. Repeated nicotine treatment led to global (PPI) and selective (LI) improvements in performance. Early but not later prenatal LPS exposure induced consistent deficits in cognitive tests with relevance for schizophrenia. Nicotine reversed the LPS-induced deficits in selective attention (LI) and induced a global enhancement of sensorimotor gating (PPI).

A Human Bone Marrow-Derived Stromal Cell Population with Hemogenic Potential

During ontogeny, definitive hematopoietic stem/progenitor cells (HSC) are thought to arise from vascular endothelial cells, through an endothelial-to-hematopoietic transition, a natural process that occurs in unique, specialized embryonic hemogenic endothelial cells. Developmental studies, and experiments using pluripotent stem cells in an effort to recapitulate this process and thereby gain a better understanding of the emergence of definitive hematopoiesis, have collectively led to the prevailing view that the hemogenic endothelium constitutes a transient population of cells within the embryo that rapidly disappears during development and is absent in the adult. Herein, we provide the first evidence that at early time points of gestation, prior to the establishment of hematopoiesis, a unique subpopulation of Stro-1+ cells present within the inner part of the developing human bone marrow co-expresses APLNR, a marker of angiogenic mesoderm. Moreover, these Stro-1+APLNR+ cells express multiple other markers described for hemogenic endothelium, and subsequently contribute to the vasculature, cartilage, and bone. Importantly, we also show that cells expressing these same markers of primitive mesoderm/hemogenic endothelium persist at low frequency within the adult marrow. These adult-derived cells can be extensively expanded in vitro without loss of potential, but lack hematopoietic colony-forming potential in vitro. However, upon transplantation into a fetal microenvironment, clonally-derived populations of these adult Stro1+ isolated stromal progenitors (SIPs) not only contribute to the vasculature and nascent BM niches, but also efficiently generate, at a clonal level, hematopoietic stem cells (HSC) that are capable of robust, multilineage hematopoietic reconstitution, with generation of both myeloid and lymphoid cells upon serial transplantation. In conclusion, our studies have thus uncovered the latent potential of a highly expandable population of seemingly vestigial adult human somatic cells, whose ontogenic history includes a phenotype identical to that described for hemogenic endothelium. We have also shown that, if provided with the appropriate/necessary inductive factors, these unique adult cells are capable of giving rise to hematopoietic cells that fulfill the gold standard criteria for bona fide HSC. Therefore, these cells could potentially be more amenable to reprogramming technologies, to produce HSC that could be used to treat/cure a broad variety of blood diseases. DisclosuresNo relevant conflicts of interest to declare.

Amniotic fluid levels of phospholipase A2 in fetal rats with retinoic acid induced myelomeningocele: the potential “second hit” in neurologic damage

Objectives: There is growing evidence of ongoing, in utero neurological damage in fetuses with myelomeningocele (MMC). Phospholipase A2 (PLA2) has known neurotoxic properties and is predominantly present in its secretory isoform (sPLA2) in meconium, the passage of which is increased in MMC fetuses. The objective of this study was to determine if amniotic fluid (AF) levels of PLA2 are elevated in a rat model of MMC.Methods: Timed pregnant Sprague–Dawley rats were gavage fed 60 mg/kg/bodyweight retinoic acid (RA) in olive oil at embryonic day 10 (E10). Amniocentesis was performed at multiple gestational time points on MMC fetuses, RA-exposed fetuses without MMC and control fetuses. AF PLA2 levels were analyzed by a fluorescent enzyme activity assay. PLA2 isoforms were determined by measuring activity in the presence of specific inhibitors.Results: There was no difference in AF PLA2 activity between groups on E15. PLA2 activity was significantly increased in MMC fetuses on E17, E19 and E21 (p < 0.001). Secretory PLA2 primarily accounted for the overall greater activity.Conclusions: PLA2 levels are elevated in the AF of fetal rats with MMC and may contribute to ongoing neural injury. This pathway may be a useful drug target to limit ongoing damage and better preserve neurologic function.

Polygenic inheritance of cryptorchidism susceptibility in the LE/orl rat.

Susceptibility to inherited cryptorchidism in the LE/orl rat may be associated with genetic loci that influence developmental patterning of the gubernaculum by the fetal testis. Cryptorchidism in the LE/orl rat is associated with a unique combination of homozygous minor alleles at multiple loci, and the encoded proteins are co-localized with androgen receptor (AR) and Leydig cells in fetal gubernaculum and testis, respectively. Prior studies have shown aberrant perinatal gubernacular migration, muscle patterning defects and reduced fetal testicular testosterone in the LE/orl strain. In addition, altered expression of androgen-responsive, cytoskeletal and muscle-related transcripts in the LE/orl fetal gubernaculum suggest a role for defective AR signaling in cryptorchidism susceptibility. The long-term LE/orl colony and short-term colonies of outbred Crl:LE and Crl:SD, and inbred WKY/Ncrl rats were maintained for studies. Animals were intercrossed (LE/orl X WKY/Ncrl), and obligate heterozygotes were reciprocally backcrossed to LE/orl rats to generate 54 F2 males used for genotyping and/or linkage analysis. At least five fetuses per gestational time point from two or more litters were used for quantitative real-time RT-PCR (qRT-PCR) and freshly harvested embryonic (E) day 17 gubernaculum was used to generate conditionally immortalized cell lines. We completed genotyping and gene expression analyses using genome-wide microsatellite markers and single nucleotide polymorphism (SNP) arrays, PCR amplification, direct sequencing, restriction enzyme digest with fragment analysis, whole genome sequencing (WGS), and qRT-PCR. Linkage analysis was performed in Haploview with multiple testing correction, and qRT-PCR data were analyzed using ANOVA after log transformation. Imaging was performed using custom and commercial antibodies directed at candidate proteins in gubernaculum and testis tissues, and gubernaculum cell lines. LE/orl rats showed reduced fertility and fecundity, and higher risk of perinatal death as compared with Crl:LE rats, but there were no differences in breeding outcomes between normal and unilaterally cryptorchid males. Linkage analysis identified multiple peaks, and with selective breeding of outbred Crl:LE and Crl:SD strains for alleles within two of the most significant (P < 0.003) peaks on chromosomes 6 and 16, we were able to generate a non-LE/orl cryptorchid rat. Associated loci contain potentially functional minor alleles (0.25-0.36 in tested rat strains) including an exonic deletion in Syne2, a large intronic insertion in Ncoa4 (an AR coactivator) and potentially deleterious variants in Solh/Capn15, Ankrd28, and Hsd17b2. Existing WGS data indicate that homozygosity for these combined alleles does not occur in any other sequenced rat strain. We observed a modifying effect of the Syne2(del) allele on expression of other candidate genes, particularly Ncoa4, and for muscle and hormone-responsive transcripts. The selected candidate genes/proteins are highly expressed, androgen-responsive and/or co-localized with developing muscle and AR in fetal gubernaculum, and co-localized with Leydig cells in fetal testis. The present study identified multiple cryptorchidism-associated linkage peaks in the LE/orl rat, containing potentially causal alleles. These are strong candidate susceptibility loci, but further studies are needed to demonstrate functional relevance to the phenotype. Association data from both human and rat models of spontaneous, nonsyndromic cryptorchidism support a polygenic etiology of the disease. Both the present study and a human genome-wide association study suggest that common variants with weak effects contribute to susceptibility, and may exist in genes encoding proteins that participate in AR signaling in the developing gubernaculum. These findings have potential implications for the gene-environment interaction in the etiology of cryptorchidism. Sequences were deposited in the Rat Genome Database (RGD, http://rgd.mcw.edu/). This work was supported by: R01HD060769 from the Eunice Kennedy Shriver National Institute for Child Health and Human Development (NICHD), 2P20GM103446 and P20GM103464 from the National Institute of General Medical Sciences (NIGMS), and Nemours Biomedical Research. The authors have no competing interests to declare.

OP21.07: Fetal and neonatal diastolic cardiac strain rate: normal reference ranges and reproducibility in a prospective, longitudinal cohort of pregnancies

Background: Normative fetal diastolic strain rate values have not been comprehensively reported. The aims of this study were to report normative data for diastolic strain rate parameters across gestation and upon delivery, determine the effect of advancing gestational age on these measures, and compare interobserver variability at multiple gestational age time points. Methods: Sixty gravid women were enrolled before 20 weeks’ gestation. The following measures were obtained by two blinded observers at five time points across gestation and at 4 to 8 weeks’ postnatal age: global left ventricular circumferential strain rate peak E and A waves, global left and right ventricular longitudinal strain rate peak E and A waves, and mitral and tricuspid valve E/A ratios. Reproducibility was assessed using intraclass correlation and Bland-Altman analysis. Least square means analysis was used to evaluate for changes across gestational age. Results: Left ventricular longitudinal and circumferential diastolic strain rate values decreased across gestation, while right ventricular longitudinal values remained stable. With delivery, left ventricular values remained fairly stable, while right ventricular values increased. Intraclass correlations for diastolic strain rate values were 0.68 to 0.94 at $24 weeks’ gestation and 0.25 to 0.82 for values at 20 to 21 weeks’ gestation. Intraclass correlations ranged from 0.49 to 0.90 for mitral valve and tricuspid valve E/A ratios across gestation. Conclusions: Fetal measures of diastolic strain rate have acceptable reproducibility after 24 weeks’ gestation. The described changes across gestation likely reflect intrinsic myocardial adaptation to loading conditions. These reference ranges can be used to assess effects of various disease states on fetal myocardial deformation. (J Am Soc Echocardiogr 2016;29:663-9.)

The role of microRNA miR223 in immune adaptation for pregnancy and fetal-placental development

The role of microRNA miR223 in immune adaptation for pregnancy and fetal-placental development