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We directly sequenced cell-free DNA with high-throughput shotgun sequencing technology from plasma of pregnant women, obtaining, on average, 5 million sequence tags per patient sample. This enabled us to measure the over- and underrepresentation of chromosomes from an aneuploid fetus. The sequencing approach is polymorphism-independent and therefore universally applicable for the noninvasive detection of fetal aneuploidy. Using this method, we successfully identified all nine cases of trisomy 21 (Down syndrome), two cases of trisomy 18 (Edward syndrome), and one case of trisomy 13 (Patau syndrome) in a cohort of 18 normal and aneuploid pregnancies; trisomy was detected at gestational ages as early as the 14th week. Direct sequencing also allowed us to study the characteristics of cell-free plasma DNA, and we found evidence that this DNA is enriched for sequences from nucleosomes.

Circulating cell-free RNA in the blood provides a potential window into the health, phenotype, and developmental programs of a variety of human organs. We used high-throughput methods of RNA analysis such as microarrays and next-generation sequencing to characterize the global landscape circulating RNA in a cohort of human subjects. By focusing on genes whose expression is highly specific to certain tissues, we were able to identify the relative contributions of these tissues to circulating RNA and to monitor changes in tissue development and health. As one application of this approach, we performed a longitudinal study on pregnant women and analyzed their combined cell-free RNA transcriptomes across all three trimesters of pregnancy and after delivery. In addition to the analysis of mRNA, we observed and characterized noncoding species such as long noncoding RNA and circular RNA transcripts whose presence had not been previously observed in human plasma. We demonstrate that it is possible to track specific longitudinal phenotypic changes in both the mother and the fetus and that it is possible to directly measure transcripts from a variety of fetal tissues in the maternal blood sample. We also studied the role of neuron-specific transcripts in the blood of healthy adults and those suffering from the neurodegenerative disorder Alzheimer’s disease and showed that disease specific neural transcripts are present at increased levels in the blood of affected individuals. Characterization of the cell-free transcriptome in its entirety may thus provide broad insights into human health and development without the need for invasive tissue sampling.

Prenatal diagnosis in pregnancies at risk of single-gene disorders is currently performed using invasive methods such as chorionic villus sampling and amniocentesis. This is in contrast with screening for common aneuploidies, for which noninvasive methods with a single maternal blood sample have become standard clinical practice. We developed a protocol for noninvasive prenatal diagnosis of inherited single-gene disorders using droplet digital PCR from circulating cell-free DNA (cfDNA) in maternal plasma. First, the amount of cfDNA and fetal fraction is determined using a panel of TaqMan assays targeting high-variability single-nucleotide polymorphisms. Second, the ratio of healthy and diseased alleles in maternal plasma is quantified using TaqMan assays targeting the mutations carried by the parents. Two validation approaches of the mutation assay are presented. We collected blood samples from 9 pregnancies at risk for different single-gene disorders, including common conditions and rare metabolic disorders. We measured cases at risk of hemophilia, ornithine transcarbamylase deficiency, cystic fibrosis, β-thalassemia, mevalonate kinase deficiency, acetylcholine receptor deficiency, and DFNB1 nonsyndromic hearing loss. We correctly differentiated affected and unaffected pregnancies (2 affected, 7 unaffected), confirmed by neonatal testing. We successfully measured an affected pregnancy as early as week 11 and with a fetal fraction as low as 3.7% (0.3). Our method detects single-nucleotide mutations of autosomal recessive diseases as early as the first trimester of pregnancy. This is of importance for metabolic disorders in which early diagnosis can affect management of the disease and reduce complications and anxiety related to invasive testing.

Noninvasive prenatal diagnosis with cell-free DNA in maternal plasma is challenging because only a small portion of the DNA sample is derived from the fetus. A few previous studies provided size-range estimates of maternal and fetal DNA, but direct measurement of the size distributions is difficult because of the small quantity of cell-free DNA. We used high-throughput paired-end sequencing to directly measure the size distributions of maternal and fetal DNA in cell-free maternal plasma collected from 3 typical diploid and 4 aneuploid male pregnancies. As a control, restriction fragments of lambda DNA were also sequenced. Cell-free DNA had a dominant peak at approximately 162 bp and a minor peak at approximately 340 bp. Chromosome Y sequences were rarely longer than 250 bp but were present in sizes of <150 bp at a larger proportion compared with the rest of the sequences. Selective analysis of the shortest fragments generally increased the fetal DNA fraction but did not necessarily increase the sensitivity of aneuploidy detection, owing to the reduction in the number of DNA molecules being counted. Restriction fragments of lambda DNA with sizes between 60 bp and 120 bp were preferentially sequenced, indicating that the shotgun sequencing work flow introduced a bias toward shorter fragments. Our results confirm that fetal DNA is shorter than maternal DNA. The enrichment of fetal DNA by size selection, however, may not provide a dramatic increase in sensitivity for assays that rely on length measurement in situ because of a trade-off between the fetal DNA fraction and the number of molecules being counted.

Anhydramnios secondary to anuria before 22 weeks of gestational age and congenital bilateral renal agenesis before 26 weeks of gestational age are collectively referred to as early-pregnancy renal anhydramnios. Early-pregnancy renal anhydramnios occurs in at least 1 in 2000 pregnancies and is considered universally fatal when left untreated because of severe pulmonary hypoplasia precluding ex utero survival The Renal Anhydramnios Fetal Therapy (RAFT) trial is a nonrandomized, nonblinded, multicenter clinical trial designed to assess the efficacy, safety, and feasibility of amnioinfusions for patients with pregnancies complicated by early-pregnancy renal anhydramnios. The primary objective of this study is to determine the proportion of neonates surviving to successful dialysis, defined as use of a dialysis catheter for ≥14 days. A consortium of 9 North American Fetal Therapy Network (NAFTNet) centers was formed, and the RAFT protocol was refined in collaboration with the NAFTNet Scientific Committee. Enrollment in the trial began in April 2020. Participants may elect to receive amnioinfusions or to join the nonintervention observational expectant management group. Eligible pregnant women must be at least 18 years of age with a fetal diagnosis of isolated early-pregnancy renal anhydramnios. In addition to the primary study objective stated above, secondary objectives include (1) to assess maternal safety and feasibility of the serial amnioinfusion intervention (2) to perform an exploratory study of the natural history of untreated early pregnancy renal anhydramnios (3) to examine correlations between prenatal imaging and lung specific factors in amniotic fluid as predictive of the efficacy of serial percutaneous amnioinfusions and (4) to determine short- and long-term outcomes and quality of life in surviving neonates and families enrolled in RAFT The RAFT trial is the first clinical trial to investigate the efficacy, safety, and feasibility of amnioinfusions to treat the survival-limiting pulmonary hypoplasia associated with anhydramnios. Although the intervention offers an opportunity to treat a condition known to be almost universally fatal in affected neonates, the potential burdens associated with end-stage kidney disease from birth must be acknowledged. ClinicalTrials.gov identifier: NCT03101891.

Preeclampsia is one of the most severe pregnancy complications and a leading cause of maternal death. However, early diagnosis of preeclampsia remains a clinical challenge. Alterations in the normal immune adaptations necessary for the maintenance of a healthy pregnancy are central features of preeclampsia. However, prior analyses primarily focused on the static assessment of select immune cell subsets have provided limited information for the prediction of preeclampsia. Here, we used a high-dimensional mass cytometry immunoassay to characterize the dynamic changes of over 370 immune cell features (including cell distribution and functional responses) in maternal blood during healthy and preeclamptic pregnancies. We found a set of eight cell-specific immune features that accurately identified patients well before the clinical diagnosis of preeclampsia (median area under the curve (AUC) 0.91, interquartile range [0.82-0.92]). Several features recapitulated previously known immune dysfunctions in preeclampsia, such as elevated pro-inflammatory innate immune responses early in pregnancy and impaired regulatory T (Treg) cell signaling. The analysis revealed additional novel immune responses that were strongly associated with, and preceded the onset of preeclampsia, notably abnormal STAT5ab signaling dynamics in CD4 T cell subsets (AUC 0.92, = 8.0E-5). These results provide a global readout of the dynamics of the maternal immune system early in pregnancy and lay the groundwork for identifying clinically-relevant immune dysfunctions for the prediction and prevention of preeclampsia.

ObjectiveTo investigate factors associated with positive postpartum depression (PPD) screen in pregnancies complicated by fetal congenital cardiac anomaly.Study designWe reviewed all records of pregnancies complicated by fetal congenital cardiac anomaly receiving prenatal, intrapartum and postpartum care at our single center, October 2016-October 2019. Maternal, obstetric, and neonatal data were compared between women with and without a positive PPD screen at the 6-week postpartum visit.ResultsOut of 415 women referred for fetal congenital cardiac anomaly, 86 women had complete inclusion criteria. Twenty-four women (28%) had a positive PPD screen. The frequencies of planned future infant surgery (73.9 vs. 26.2%, p = 0.01) and neonatal death prior to postpartum visit (12.5 vs. 0%, p = 0.02) were significantly higher among women with a positive PPD screen.ConclusionIn pregnancies complicated by fetal congenital cardiac anomaly, mothers of infants with planned future surgery or neonatal death are at significant risk for postpartum depression.

Approximately 1 in 4 pregnant women in the United States undergo labor induction. The onset and establishment of labor, particularly induced labor, is a complex and dynamic process influenced by multiple endocrine, inflammatory, and mechanical factors as well as obstetric and pharmacological interventions. The duration from labor induction to the onset of active labor remains unpredictable. Moreover, prolonged labor is associated with severe complications for the mother and her offspring, most importantly chorioamnionitis, uterine atony, and postpartum hemorrhage. While maternal immune system adaptations that are critical for the maintenance of a healthy pregnancy have been previously characterized, the role of the immune system during the establishment of labor is poorly understood. Understanding maternal immune adaptations during labor initiation can have important ramifications for predicting successful labor induction and labor complications in both induced and spontaneous types of labor. The aim of this study was to characterize labor-associated maternal immune system dynamics from labor induction to the start of active labor. Serial blood samples from fifteen participants were collected immediately prior to labor induction (baseline) and during the latent phase until the start of active labor. Using high-dimensional mass cytometry, a total of 1,059 single-cell immune features were extracted from each sample. A multivariate machine-learning method was employed to characterize the dynamic changes of the maternal immune system after labor induction until the establishment of active labor. A cross-validated linear sparse regression model (least absolute shrinkage and selection operator, LASSO) predicted the minutes since induction of labor with high accuracy (R = 0.86, p = 6.7e-15, RMSE = 277 min). Immune features most informative for the model included STAT5 signaling in central memory CD8 + T cells and pro-inflammatory STAT3 signaling responses across multiple adaptive and innate immune cell subsets. Our study reports a peripheral immune signature of labor induction, and provides important insights into biological mechanisms that may ultimately predict labor induction success as well as complications, thereby facilitating clinical decision-making to improve maternal and fetal well-being.

Background Preterm birth (PTB) is a serious health problem. PTB complications is the main cause of death in infants under five years of age worldwide. The ability to accurately predict risk for PTB during early pregnancy would allow early monitoring and interventions to provide personalized care, and hence improve outcomes for the mother and infant. Objective This study aims to predict the risks of early preterm (< 35 weeks of gestation) or very early preterm (≤ 26 weeks of gestation) deliveries by using high-resolution maternal urinary metabolomic profiling in early pregnancy. Design A retrospective cohort study was conducted by two independent preterm and term cohorts using high-density weekly urine sampling. Maternal urine was collected serially at gestational weeks 8 to 24. Global metabolomics approaches were used to profile urine samples with high-resolution mass spectrometry. The significant features associated with preterm outcomes were selected by Gini Importance. Metabolite biomarker identification was performed by liquid chromatography tandem mass spectrometry (LCMS-MS). XGBoost models were developed to predict early or very early preterm delivery risk. Setting and participants The urine samples included 329 samples from 30 subjects at Stanford University, CA for model development, and 156 samples from 24 subjects at the University of Alabama, Birmingham, AL for validation. Results 12 metabolites associated with PTB were selected and identified for modelling among 7,913 metabolic features in serial-collected urine samples of pregnant women. The model to predict early PTB was developed using a set of 12 metabolites that resulted in the area under the receiver operating characteristic (AUROCs) of 0.995 (95% CI: [0.992, 0.995]) and 0.964 (95% CI: [0.937, 0.964]), and sensitivities of 100% and 97.4% during development and validation testing, respectively. Using the same metabolites, the very early PTB prediction model achieved AUROCs of 0.950 (95% CI: [0.878, 0.950]) and 0.830 (95% CI: [0.687, 0.826]), and sensitivities of 95.0% and 60.0% during development and validation, respectively. Conclusion Models for predicting risk of early or very early preterm deliveries were developed and tested using metabolic profiling during the 1st and 2nd trimesters of pregnancy. With patient validation studies, risk prediction models may be used to identify at-risk pregnancies prompting alterations in clinical care, and to gain biological insights of preterm birth.

We examined the association of maternal obesity with risk of stillbirth, focusing on whether the pattern of results varied by gestational age or maternal race-ethnicity or parity. Analyses included 4,012 stillbirths and 1,121,234 liveborn infants delivered in California from 2007-2010. We excluded stillbirths due to congenital anomalies, women with hypertensive disorders or diabetes, and plural births, to focus on fetuses and women without these known contributing conditions. We used Poisson regression to estimate relative risks (RR) and 95% confidence intervals (CI). Separate models were run for stillbirths delivered at 20-23, 24-27, 28-31, 32-36, 37-41 weeks, relative to liveborn deliveries at 37-41 weeks. For stillbirth at 20-23 weeks, RRs were elevated for all race-ethnicity and parity groups. The RR for a 20-unit change in BMI (which reflects the approximate BMI difference between a normal weight and an Obese III woman) was 3.5 (95% CI 2.2, 5.6) for nulliparous white women and ranged from 1.8 to 5.0 for other sub-groups. At 24-27 weeks, the association was significant (p<0.05) only for multiparous non-Hispanic whites; at 28-31 weeks, for multiparous whites and nulliparous whites and blacks; at 32-36 weeks, for multiparous whites and nulliparous blacks; and at 37-41 weeks, for all groups except nulliparous blacks. The pattern of results was similar when restricted to stillbirths due to unknown causes and somewhat stronger when restricted to stillbirths attributable to obstetric causes. Increased risks were observed across all gestational ages, and some evidence of heterogeneity of the associations was observed by race-ethnicity and parity.