Bioavailability of the tumor necrosis factor alpha/regulated on activation, normal T cell expressed and secreted (RANTES) biosystem inside the gestational sac during the pre-immune stages of embryo development.

OBJECTIVES: In-vivo studies of the bioavailability of major components of the tumor necrosis factor alpha (TNFα) biosystem inside the gestational sac during embryogenesis have not been reported. We sought to determine the concentration of TNFα, soluble (s) TNFα receptors (sTNFR1, sTNFR2), and RANTES in the primate extraembryonic celomic fluid (ECF). METHODS: A validated timed-pregnant baboon animal model (N: 10) for experimental research in pregnancy was used to collect paired maternal blood and ECF samples in ongoing pregnancies. The concentrations (pg/dL) of TNFα, sTNFR1, sTNFR2, and RANTES were then determined by ELISA immunoassays. RESULTS: All animals delivered at term healthy newborns. The differential concentration of TNFα, sTNFR1, sTNFR2, and RANTES between the maternal plasma and the ECF could be determined with ratios for TNFα (5.4), sTNFR2 (1.85) and RANTES (3.59) that contrasted with that of sTNFR1 (0.07), which favored the gestational sac compartment. No significant correlations were noted between maternal plasma and ECF TNFR1, sTNFR2 and RANTES. There was a trend for a correlation between TNFα in maternal plasma and ECF (R=0.74; p=0.07). CONCLUSIONS: We report the physiological concentrations of TNFα, sTNFR1, sTNFR2, and RANTES in extraembryonic celomic fluid during embryogenesis in primates.

Preeclampsia at delivery is associated with lower serum vitamin D and higher antiangiogenic factors: a case control study.

BACKGROUND: Preeclampsia is characterized by decreased trophoblastic angiogenesis leading to abnormal invasion of spiral arteries, shallow implantation and resulting in compromised placentation with poor uteroplacental perfusion. Vitamin D plays an important role in pregnancy influencing implantation, angiogenesis and placental development. The objective of this study was to determine whether there is an association between serum vitamin D levels, and anti-angiogenic factors at the time of delivery and the occurrence of preeclampsia. METHODS: This nested case control study analyzed frozen serum samples at the time of delivery and related clinical data from women with singleton liveborn pregnancies who had participated in studies of the NICHD Stillbirth Collaborative Research Network. Women with a recorded finding of preeclampsia and who had received magnesium sulfate treatment prior to delivery were considered index cases (N = 56). Women without a finding of preeclampsia were controls (N = 341). RESULTS: Women with preeclampsia had 14.5% lower serum vitamin D levels than women in the control group (16.5 ng/ml vs. 19 ng/ml, p = 0.014) with 64.5% higher sFlt-1 levels (11,600 pg/ml vs. 7050 pg/ml, p < 0.001) and greater than 2 times higher endoglin levels (18.6 ng/ml vs. 8.7 ng/ml, < 0.001). After controlling for gestational age at delivery and maternal BMI, vitamin D levels were 0.88 times lower (P = 0.051), while endoglin levels were 2.5 times higher and sFlt-1 levels were 2.1 times higher than in control pregnancies (P < 0.001). CONCLUSIONS: Women with preeclampsia at time of delivery have higher maternal antiangiogenetic factors and may have lower maternal serum vitamin D levels. These findings may lead to a better understanding of the underlying etiology of preeclampsia as well as possible modifiable treatment options which could include assuring adequate levels of maternal serum vitamin D prior to pregnancy.

Maternal Morbidity in the Second Stage of Labor: Analysis to Simulate the Clinical Choice.

OBJECTIVE: The aim of the study is to analyze maternal morbidity in the second stage of labor in a manner that approximates clinical choice. STUDY DESIGN: The study design comprises secondary analysis of the Consortium for Safe Labor, which included 228,688 deliveries at 19 hospitals between 2002 and 2008. We included the 107,675 women who were undergoing a trial of labor without a prior uterine scar or history of substance abuse, who reached the second stage, with a liveborn, nonanomalous, vertex, singleton, at term of at least 2,500 g. Maternal complications included postpartum fever, hemorrhage, blood transfusion, thrombosis, intensive care unit (ICU) admission, hysterectomy, and death. For maternal complications, we simulated the clinical choice by comparing operative vaginal or cesarean deliveries to continued expectant management at every hour in the second stage. For neonatal complications, we modeled the risk of severe neonatal complication by second stage duration for spontaneous vaginal deliveries only, adjusting for maternal demographics, comorbidities, and delivery hospital. Severe neonatal complications included death, asphyxia, hypoxic-ischemic encephalopathy (HIE), seizure, sepsis with prolonged stay, need for mechanical ventilation, and 5-minute Apgar score <4. RESULTS: Maternal morbidity was higher with operative vaginal/cesarean delivery versus continued expectant management for every hour in the second stage, a difference that was statistically significant at hour 2 (18.4 vs. 14.7%; p <0.01). Overall, 951 (0.88%) deliveries were complicated by a severe neonatal complication. A second stage over 4 hours was associated with an adjusted odds of severe neonatal complication of 2.10 (95% confidence interval [CI]: 1.32-3.34) as compared with women who delivered in the first hour. CONCLUSION: There is a trade-off between maternal and neonatal morbidity in the second stage of labor. Serious neonatal complications rise throughout, however, there is no time at which maternal morbidity is improved with a cesarean or operative vaginal delivery. Strategies are needed to identify neonates at highest risk of complication for targeted intervention. KEY POINTS: · Severe neonatal complications increase with every hour in the second stage.. · Shortening the second stage is associated with higher maternal complications at every hour.. · There is a trade-off between maternal and neonatal morbidity in the second stage..

Maternal Marijuana Exposure, Feto-Placental Weight Ratio, and Placental Histology.

OBJECTIVE: Marijuana use is associated with placenta-mediated adverse pregnancy outcomes including fetal growth restriction, but the mechanism remains uncertain. The objective was to evaluate the association between maternal marijuana use and the feto-placental weight ratio (FPR). Secondarily, we aimed to compare placental histology of women who used marijuana to those who did not. STUDY DESIGN: This was a secondary analysis of singleton pregnancies enrolled in a multicenter and case-control stillbirth study. Prior marijuana use was detected by electronic medical record abstraction or cord homogenate positive for 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid. Prior tobacco use was detected by self-report or presence of maternal serum cotinine. Stillbirths and live births were considered separately. The primary outcome was FPR. Association of marijuana use with FPR was estimated with multivariable linear modeling adjusted for fetal sex, preterm birth, and tobacco use. Comparisons between groups for placental histology were made using Chi-square and stratified by live birth and stillbirth, term and preterm deliveries, and fetal sex. RESULTS: Of 1,027 participants, 224 were stillbirths and 803 were live births. Overall, 41 (4%) women used marijuana during the pregnancy. The FPR ratio was lower among exposed offspring but reached statistical significance only for term stillbirths (mean 6.84 with marijuana use vs. mean 7.8 without use, p < 0.001). In multivariable modeling, marijuana use was not significantly associated with FPR (p = 0.09). There were no differences in histologic placental features among those with and without marijuana use overall or in stratified analyses. CONCLUSION: Exposure to marijuana may not be associated with FPR. Similarly, there were no placental histologic features associated with marijuana exposure. Further study of the influence of maternal marijuana use on placental development and function is warranted to better understand the association between prenatal marijuana use and poor fetal growth. KEY POINTS: · Maternal marijuana exposure was not associated with the feto-placental weight ratio.. · Marijuana exposure was not associated with differences in placental histology.. · Concerning trend toward lower feto-placental weight ratios among marijuana-exposed stillbirths..

DLK1: A Novel Biomarker of Placental Insufficiency in Stillbirth and Live Birth.

OBJECTIVE: Delta-like homolog 1 (DLK1) is a growth factor that is reduced in maternal sera in pregnancies with small for gestational age neonates. We sought to determine if DLK1 is associated with stillbirth (SB), with and without placental insufficiency. STUDY DESIGN: A nested case-control study was performed using maternal sera from a multicenter case-control study of SB and live birth (LB). SB and LB were stratified as placental insufficiency cases (small for gestational age <5% or circulatory lesions on placental histopathology) or normal placenta controls (appropriate for gestational age and no circulatory lesions). Enzyme-linked immunosorbent assay (ELISA) was used to measure DLK1. The mean difference in DLK1 was compared on the log scale in an adjusted linear regression model with pairwise differences, stratified by term/preterm deliveries among DLK1 results in the quantifiable range. In exploratory analysis, geometric means were compared among all data and the proportion of "low DLK1" (less than the median value for gestational age) was compared between groups and modeled using linear and logistic regression, respectively. RESULTS: Overall, 234 SB and 234 LB were analyzed; 246 DLK1 values were quantifiable within the standard curve. Pairwise comparisons of case and control DLK1 geometric means showed no significant differences between groups. In exploratory analysis of all data, adjusted analysis revealed a significant difference for the LB comparison only (SB: 71.9 vs. 99.1 pg/mL, p = 0.097; LB: 37.6 vs. 98.1 pg/mL, p = 0.005). In exploratory analysis of "low DLK1," there was a significant difference between the odds ratio of having "low DLK1" between preterm cases and controls for both SB and LB. There were no significant differences in geometric means nor "low DLK1" between SB and LB. CONCLUSION: In exploratory analysis, more placental insufficiency cases in preterm SB and LB had "low DLK1." However, low DLK1 levels were not associated with SB. KEY POINTS: · Maternally circulating DLK1 is correlated with placental insufficiency.. · Maternally circulating DLK1 is not correlated with SB.. · DLK1 is a promising marker for placental insufficiency..

Computational medicine, present and the future: obstetrics and gynecology perspective.

Medicine is, in its essence, decision making under uncertainty; the decisions are made about tests to be performed and treatments to be administered. Traditionally, the uncertainty in decision making was handled using expertise collected by individual providers and, more recently, systematic appraisal of research in the form of evidence-based medicine. The traditional approach has been used successfully in medicine for a very long time. However, it has substantial limitations because of the complexity of the system of the human body and healthcare. The complex systems are a network of highly coupled components intensely interacting with each other. These interactions give those systems redundancy and thus robustness to failure and, at the same time, equifinality, that is, many different causative pathways leading to the same outcome. The equifinality of the complex systems of the human body and healthcare system demand the individualization of medical care, medicine, and medical decision making. Computational models excel in modeling complex systems and, consequently, enabling individualization of medical decision making and medicine. Computational models are theory- or knowledge-based models, data-driven models, or models that combine both approaches. Data are essential, although to a different degree, for computational models to successfully represent complex systems. The individualized decision making, made possible by the computational modeling of complex systems, has the potential to revolutionize the entire spectrum of medicine from individual patient care to policymaking. This approach allows applying tests and treatments to individuals who receive a net benefit from them, for whom benefits outweigh the risk, rather than treating all individuals in a population because, on average, the population benefits. Thus, the computational modeling-enabled individualization of medical decision making has the potential to both improve health outcomes and decrease the costs of healthcare.

Cervicovaginal fluid proteomic analysis to identify potential biomarkers for preterm birth.

BACKGROUND: Spontaneous preterm birth is a leading cause of neonatal morbidity and mortality. Early identification of at-risk women by reliable screening tests could reduce the spontaneous preterm birth rate, but conventional methods such as obstetrical history and maternal cervical length screening identify only a minority of spontaneous preterm birth cases. Cervicovaginal fluid might prove to be a useful, readily available biological fluid for identifying spontaneous preterm birth biomarkers. OBJECTIVE: The objective of the study was to identify cervicovaginal fluid biomarkers of early spontaneous preterm birth in a high-risk cohort of pregnant women with a history of spontaneous preterm birth using targeted and shotgun proteomic analyses. STUDY DESIGN: A nested case control study (cases were spontaneous preterm birth <34 weeks in the current pregnancy; controls were spontaneous labor and delivery at 39-41 weeks) was performed using cervicovaginal fluid samples collected at 3 study visits (100/7 to 186/7 weeks, 190/7 to 236/7 weeks, and 280/7 to 316/7 weeks). All participants had a history of at least 1 prior spontaneous preterm birth. Targeted proteomic analysis was performed using a stable isotope-labeled proteome derived from endocervical and vaginal mucosal cells. This served as a standard to quantitate candidate protein levels in individual cervicovaginal fluid samples from the second and third study visits using liquid chromatography-multiple reaction monitoring mass spectrometry. The ratio of endogenous peptide area/stable isotope-labeled proteome-derived peptide area was used to measure levels of 42 peptides in 22 proteins. To maximize biomarker discovery in the cervicovaginal fluid samples, shotgun proteomic analysis also was performed utilizing liquid chromatography and ion trap mass spectrometry. A validation study was performed in second-trimester cervicovaginal fluid samples from an independent study group (12 spontaneous preterm birth cases, 19 term delivery controls) using enzyme-linked immunosorbent assay for 5 proteins expressed at higher levels in spontaneous preterm birth cases compared with controls in targeted or shotgun proteomic analyses. RESULTS: For targeted proteomics, cervicovaginal fluid samples from 33 cases and 32 controls at 190/7 to 236/7 weeks and 16 cases and 14 controls at 280/7 to 316/7 weeks from the same pregnancies were analyzed. When samples were compared between cases and controls, the relative abundance of 5 proteins was greater (P = .02-.05) in cases at both visits, while the relative abundance of 1 protein was lower (P = .03) in cases at both visits. For shotgun proteomics analyses, cervicovaginal fluid samples were pooled for 9 spontaneous preterm birth cases and 9 term delivery controls at each study visit. Shotgun proteomics yielded 28 proteins that were detected at levels >2 times higher and 1 protein that was detected at a level <0.5 times lower in spontaneous preterm birth cases compared with controls at all 3 study visits. Validation enzyme-linked immunosorbent assay for 5 proteins that were detected at higher levels in cervicovaginal fluid samples from spontaneous preterm birth cases compared with term delivery controls in proteomics analyses did not demonstrate statistically significant differences between spontaneous preterm birth cases and controls. CONCLUSIONS: Potential biomarkers of spontaneous preterm birth were identified by targeted and shotgun proteomics analyses in cervicovaginal fluid samples from high-risk, asymptomatic women. Many of the proteins detected at higher levels in cervicovaginal fluid samples from spontaneous preterm birth cases are extracellular matrix proteins and/or regulate cell membrane physiology. These proteins have substantial biological interest, but validation enzyme-linked immunosorbent assay for 5 of these proteins did not yield clinically useful biomarkers for spontaneous preterm birth.

Findings in Stillbirths Associated with Placental Disease.

OBJECTIVE: Placental disease is a leading cause of stillbirth. Our purpose was to characterize stillbirths associated with placental disease. STUDY DESIGN: The Stillbirth Collaborative Research Network conducted a prospective, case-control study of stillbirths and live births from 2006 to 2008. This analysis includes 512 stillbirths with cause of death assignment and a comparison group of live births. We compared exposures between women with stillbirth due to placental disease and those due to other causes as well as between women with term (≥ 37 weeks) stillbirth due to placental disease and term live births. RESULTS: A total of 121 (23.6%) out of 512 stillbirths had a probable or possible cause of death due to placental disease by Initial Causes of Fetal Death. Characteristics were similar between stillbirths due to placental disease and other stillbirths. When comparing term live births to stillbirths due to placental disease, women with non-Hispanic black race, Hispanic ethnicity, lack of insurance, or who were born outside of the United States had higher odds of stillbirth due to placental disease. Nulliparity and antenatal bleeding also increased risk of stillbirth due to placental disease. CONCLUSION: Multiple discrete exposures were associated with stillbirth caused by placental disease. The relationship between these factors and utility of surveillance warrants further study.

Associations Between Features of Placental Morphology and Birth Weight in Dichorionic Twins.

Low birth weight is associated with perinatal and long-term morbidity and mortality, and may be a result of abnormal placental development and function. In studies of singletons, associations have been reported between features of placental morphology and birth weight. Evaluating similar associations within twin pairs offers a unique opportunity to control for key confounders shared within a twin pair, including gestational age, parental characteristics, and intrauterine environment. Data from 3 studies in the United States that were completed from 2012 to 2013, 2006 to 2008, and 1959 to 1966 were used in our analysis of 208 sets of dichorionic twins with unfused placentas. We used linear regression to model difference in birth weight within a twin pair as a function of differences in placental characteristics (i.e., thickness, 2-dimensional surface area, intraplacental difference in diameter). After controlling for sex discordance, a 75.3- cm2 difference in placental surface area, which reflects the interquartile range, was associated with a difference in birth weight of 142.1 g (95% confidence interval (CI): 62.9, 221.3). The magnitude of the association also may be larger for same-sex male pairs than same-sex female pairs (males: 265.8 g, 95% CI: 60.8, 470.8; females: 133.0 g, 95% CI: 15.7, 250.3). Strong associations between surface area and birth weight are consistent with reported results for singleton pregnancies.

Associations Between the Features of Gross Placental Morphology and Birthweight.

The placenta plays a critical role in regulating fetal growth. Recent studies suggest that there may be sex-specific differences in placental development. The purpose of our study was to evaluate the associations between birthweight and placental morphology in models adjusted for covariates and to assess sex-specific differences in these associations. We analyzed data from the Stillbirth Collaborative Research Network's population-based case-control study conducted between 2006 and 2008, which recruited cases of stillbirth and population-based controls in 5 states. Our analysis was restricted to singleton live births with a placental examination (n = 1229). Characteristics of placental morphology evaluated include thickness, surface area, difference in diameters, shape, and umbilical cord insertion site. We used linear regression to model birthweight as a function of placental morphology and covariates. Surface area had the greatest association with birthweight; a reduction in surface area of 83 cm2, which reflects the interquartile range, is associated with a 260.2-g reduction in birthweight (95% confidence interval, -299.9 to -220.6), after adjustment for other features of placental morphology and covariates. Reduced placental thickness was also associated with lower birthweight. These associations did not differ between males and females. Our results suggest that reduced placental thickness and surface area are independently associated with lower birthweight and that these relationships are not related to sex.

Smith-Lemli-Opitz Mutations in Unexplained Stillbirths.

OBJECTIVE: Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive syndrome caused by a defect in cholesterol biosynthesis with mutations in 7-dehydrocholesterol reductase (DHCR7). A total of 3% of Caucasians carry DHCR7 mutations, theoretically resulting in a homozygote frequency of 1/4000. However, SLOS occurs in only 1/20,000 to 60,000 live births. Our objective was to assess DHCR7 mutations in unexplained stillbirths. STUDY DESIGN: Prospective, multicenter, population-based case-control study of all stillbirths and a representative sample of live births enrolled in five geographic areas. Cases with stillbirth due to obstetric complications, infection, or aneuploidy, and those with poor quality deoxyribonucleic acid (DNA) were excluded. DNA was extracted from placental tissue stored at -80°C, and exons 3 to 9 of the DCHR7 gene were amplified, purified, and subjected to bidirectional sequencing to identify mutations. RESULTS: One-hundred forty four stillbirths were unexplained and had adequate DNA for analysis. Nine stillbirths of 139 (6.5%) had a single mutation in one allele in coding exons 3 to 9 of DHCR7 (Table 1). One case (0.7%) was a compound heterozygote for mutations in exons 3 to 9 of DHCR7; this fetus had no clinical or histologic features of SLOS. CONCLUSION: We detected SLOS mutations in only 0.7% of stillbirths. This does not support a strong association between unrecognized DHCR7 mutations and stillbirth.

Stillbirth, Inflammatory Markers, and Obesity: Results from the Stillbirth Collaborative Research Network.

BACKGROUND: Obesity is associated with increased risk of stillbirth, although the mechanisms are unknown. Obesity is also associated with inflammation. Serum ferritin, C-reactive protein, white blood cell count, and histologic chorioamnionitis are all markers of inflammation. OBJECTIVE: This article determines if inflammatory markers are associated with stillbirth and body mass index (BMI). Additionally, we determined whether inflammatory markers help to explain the known relationship between obesity and stillbirth. STUDY DESIGN: White blood cell count was assessed at admission to labor and delivery, maternal serum for assessment of various biomarkers was collected after study enrollment, and histologic chorioamnionitis was based on placental histology. These markers were compared for stillbirths and live births overall and within categories of BMI using analysis of variance on logarithmic-transformed markers and logistic regression for dichotomous variables. The impact of inflammatory markers on the association of BMI categories with stillbirth status was assessed using crude and adjusted odds ratios (COR and AOR, respectively) from logistic regression models. The interaction of inflammatory markers and BMI categories on stillbirth status was also assessed through logistic regression. Additional logistic regression models were used to determine if the association of maternal serum ferritin with stillbirth is different for preterm versus term births. Analyses were weighted for the overall population from which this sample was derived. RESULTS: A total of 497 women with singleton stillbirths and 1,414 women with live births were studied with prepregnancy BMI (kg/m2) categorized as normal (18.5-24.9), overweight (25.0-29.9), or obese (30.0 + ). Overweight (COR, 1.48; 95% confidence interval [CI]: 1.14-1.94) and obese women (COR, 1.60; 95% CI: 1.23-2.08) were more likely than normal weight women to experience stillbirth. Serum ferritin levels were higher (geometric mean: 37.4 ng/mL vs. 23.3, p < 0.0001) and C-reactive protein levels lower (geometric mean: 2.9 mg/dL vs. 3.3, p = 0.0279), among women with stillbirth compared with live birth. Elevated white blood cell count (15.0 uL × 103 or greater) was associated with stillbirth (21.2% SB vs. 10.0% live birth, p < 0.0001). Histologic chorioamnionitis was more common (33.2% vs. 15.7%, p < 0.0001) among women with stillbirth compared with those with live birth. Serum ferritin, C-reactive protein, and chorioamnionitis had little impact on the ORs associating stillbirth with overweight or obesity. Adjustment for elevated white blood cell count did not meaningfully change the OR for stillbirth in overweight versus normal weight women. However, the stillbirth OR for obese versus normal BMI changed by more than 10% when adjusting for histologic chorioamnionitis (AOR, 1.38; 95% CI: 1.02-1.88), indicating confounding. BMI by inflammatory marker interaction terms were not significant. The association of serum ferritin levels with stillbirth was stronger among preterm births (p = 0.0066). CONCLUSION: Maternal serum ferritin levels, elevated white blood cell count, and histologic chorioamnionitis were positively and C-reactive protein levels negatively associated with stillbirth. Elevated BMIs, both overweight and obese, were associated with stillbirth when compared with women with normal BMI. None of the inflammatory markers fully accounted for the relationship between obesity and stillbirth. The association of maternal serum ferritin with stillbirth was stronger in preterm than term stillbirths.

A genome-wide association study of early spontaneous preterm delivery.

Preterm birth is the leading cause of infant morbidity and mortality. Despite extensive research, the genetic contributions to spontaneous preterm birth (SPTB) are not well understood. Term controls were matched with cases by race/ethnicity, maternal age, and parity prior to recruitment. Genotyping was performed using Affymetrix SNP Array 6.0 assays. Statistical analyses utilized PLINK to compare allele occurrence rates between case and control groups, and incorporated quality control and multiple-testing adjustments. We analyzed DNA samples from mother-infant pairs from early SPTB cases (20(0/7)-33(6/7) weeks, 959 women and 979 neonates) and term delivery controls (39(0/7)-41(6/7) weeks, 960 women and 985 neonates). For validation purposes, we included an independent validation cohort consisting of early SPTB cases (293 mothers and 243 infants) and term controls (200 mothers and 149 infants). Clustering analysis revealed no population stratification. Multiple maternal SNPs were identified with association P-values between 10×10(-5) and 10×10(-6). The most significant maternal SNP was rs17053026 on chromosome 3 with an odds ratio (OR) 0.44 with a P-value of 1.0×10(-6). Two neonatal SNPs reached the genome-wide significance threshold, including rs17527054 on chromosome 6p22 with a P-value of 2.7×10(-12) and rs3777722 on chromosome 6q27 with a P-value of 1.4×10(-10). However, we could not replicate these findings after adjusting for multiple comparisons in a validation cohort. This is the first report of a genome-wide case-control study to identify single nucleotide polymorphisms (SNPs) that correlate with SPTB.

Predictors of response to 17-alpha hydroxyprogesterone caproate for prevention of recurrent spontaneous preterm birth.

BACKGROUND: Prematurity is the leading cause of neonatal morbidity and death among nonanomalous neonates in the United States. Intramuscular 17-alpha hydroxyprogesterone caproate injections reduce the risk of recurrent prematurity by approximately one third. Unfortunately, prophylactic 17-alpha hydroxyprogesterone caproate is not always effective, and one-third of high-risk women will have a recurrent preterm birth, despite 17-alpha hydroxyprogesterone caproate therapy. The reasons for this variability in response are unknown. Previous investigators have examined the influence of a variety of factors on 17-alpha hydroxyprogesterone caproate response but have analyzed data that used a fixed outcome of term delivery to define progesterone response. OBJECTIVE: We hypothesized that the demographics, history, and pregnancy course among women who deliver at a similar gestational age with 17-alpha hydroxyprogesterone caproate for recurrent spontaneous preterm birth prevention differs when compared with those women who deliver later with 17-alpha hydroxyprogesterone caproate and that these associations could be refined by the use of a contemporary definition of 17-alpha hydroxyprogesterone caproate "responder." STUDY DESIGN: This was a planned secondary analysis of a prospective, multicenter, longitudinal study of women with ≥1 previous documented singleton spontaneous preterm birth at <37 weeks gestation. Data were collected at 3 prespecified gestational age epochs during pregnancy. All women who were included in this analysis received 17-alpha hydroxyprogesterone caproate during the studied pregnancy. We classified women as a 17-alpha hydroxyprogesterone caproate responder or nonresponder by calculating the difference in delivery gestational age between the 17-alpha hydroxyprogesterone caproate-treated pregnancy and her earliest spontaneous preterm birth. Responders were defined as those with pregnancy that extended ≥3 weeks later with 17-alpha hydroxyprogesterone caproate, compared with the delivery gestational age of their earliest previous spontaneous preterm birth. Data were analyzed with the use of chi-square test, t-test, and logistic regression. RESULTS: One hundred fifty-five women met the inclusion criteria. The 118 responders delivered later on average (37.7 weeks gestation) than the 37 nonresponders (33.5 weeks gestation; P < .001). Among responders, 32% (38/118 women) had a recurrent spontaneous preterm birth. Demographics (age, race/ethnicity, education, and parity) were similar between groups. In the regression model, the gestational age of the previous spontaneous preterm birth (odds ratio, 0.68; 95% confidence interval, 0.56-0.82; P < .001), vaginal bleeding/abruption in the current pregnancy (odds ratio, 0.24; 95% confidence interval, 0.06-0.88; P = .031), and first-degree family history of spontaneous preterm birth (odds ratio, 0.37; 95% confidence interval, 0.15-0.88; P = .024) were associated with response to 17-alpha hydroxyprogesterone caproate. Because women with a penultimate preterm pregnancy were more likely to be 17-alpha hydroxyprogesterone caproate nonresponders, we performed an additional limited analysis examining only the 130 women whose penultimate pregnancy was preterm. In regression models, the results were similar to those in the main cohort. CONCLUSION: Several historic and current pregnancy characteristics define women who are at risk for recurrent preterm birth at a similar gestational age, despite 17-alpha hydroxyprogesterone caproate therapy. These data should be studied prospectively in larger cohorts and combined with genetic and environmental data to identify women who are most likely to benefit from this intervention.

Factor V Leiden, prothrombin G20210A, and methylene tetrahydrofolate reductase mutations and stillbirth: the Stillbirth Collaborative Research Network.

BACKGROUND: An evaluation for heritable thrombophilias is recommended in the evaluation of stillbirth. However, the association between thrombophilias and stillbirth remains uncertain. OBJECTIVE: We sought to assess the association between maternal and fetal/placental heritable thrombophilias and stillbirth in a population-based, case-control study in a geographically, racially, and ethnically diverse population. STUDY DESIGN: We conducted secondary analysis of data from the Stillbirth Collaborative Research Network, a population-based case-control study of stillbirth. Testing for factor V Leiden, prothrombin G20210A, methylene tetrahydrofolate reductase C677T and A1298C, and plasminogen activating inhibitor (PAI)-1 4G/5G mutations was done on maternal and fetal (or placental) DNA from singleton pregnancies. Data analyses were weighted for oversampling and other aspects of the design. Odds ratios (OR) were generated from univariate models regressing stillbirth/live birth status on each thrombophilia marker. RESULTS: Results were available for ≥1 marker in 488 stillbirths and 1342 live birth mothers and 405 stillbirths and 990 live birth fetuses. There was an increased odds of stillbirth for maternal homozygous factor V Leiden mutation (2/488; 0.4% vs 1/1380; 0.0046%; OR, 87.44; 95% confidence interval, 7.88-970.92). However, there were no significant differences in the odds of stillbirth for any other maternal thrombophilia, even after stratified analyses. Fetal 4G/4G PAI-1 (OR, 0.63; 95% confidence interval, 0.43-0.91) was associated with decreased odds of stillbirth. Other fetal thrombophilias were similar among groups. CONCLUSION: Most maternal and fetal thrombophilias were not associated with stillbirth. Maternal factor V Leiden was weakly associated with stillbirth, and the fetal PAI-1 4G/4G polymorphism was associated with live birth. Our data do not support routine testing for heritable thrombophilias as part of an evaluation for possible causes of stillbirth.

Karyotype versus microarray testing for genetic abnormalities after stillbirth.

BACKGROUND: Genetic abnormalities have been associated with 6 to 13% of stillbirths, but the true prevalence may be higher. Unlike karyotype analysis, microarray analysis does not require live cells, and it detects small deletions and duplications called copy-number variants. METHODS: The Stillbirth Collaborative Research Network conducted a population-based study of stillbirth in five geographic catchment areas. Standardized postmortem examinations and karyotype analyses were performed. A single-nucleotide polymorphism array was used to detect copy-number variants of at least 500 kb in placental or fetal tissue. Variants that were not identified in any of three databases of apparently unaffected persons were then classified into three groups: probably benign, clinical significance unknown, or pathogenic. We compared the results of karyotype and microarray analyses of samples obtained after delivery. RESULTS: In our analysis of samples from 532 stillbirths, microarray analysis yielded results more often than did karyotype analysis (87.4% vs. 70.5%, P<0.001) and provided better detection of genetic abnormalities (aneuploidy or pathogenic copy-number variants, 8.3% vs. 5.8%; P=0.007). Microarray analysis also identified more genetic abnormalities among 443 antepartum stillbirths (8.8% vs. 6.5%, P=0.02) and 67 stillbirths with congenital anomalies (29.9% vs. 19.4%, P=0.008). As compared with karyotype analysis, microarray analysis provided a relative increase in the diagnosis of genetic abnormalities of 41.9% in all stillbirths, 34.5% in antepartum stillbirths, and 53.8% in stillbirths with anomalies. CONCLUSIONS: Microarray analysis is more likely than karyotype analysis to provide a genetic diagnosis, primarily because of its success with nonviable tissue, and is especially valuable in analyses of stillbirths with congenital anomalies or in cases in which karyotype results cannot be obtained. (Funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development.).

Cluster analysis of spontaneous preterm birth phenotypes identifies potential associations among preterm birth mechanisms.

OBJECTIVE: We sought to use an innovative tool that is based on common biologic pathways to identify specific phenotypes among women with spontaneous preterm birth (SPTB) to enhance investigators' ability to identify and to highlight common mechanisms and underlying genetic factors that are responsible for SPTB. STUDY DESIGN: We performed a secondary analysis of a prospective case-control multicenter study of SPTB. All cases delivered a preterm singleton at SPTB ≤34.0 weeks' gestation. Each woman was assessed for the presence of underlying SPTB causes. A hierarchic cluster analysis was used to identify groups of women with homogeneous phenotypic profiles. One of the phenotypic clusters was selected for candidate gene association analysis with the use of VEGAS software. RESULTS: One thousand twenty-eight women with SPTB were assigned phenotypes. Hierarchic clustering of the phenotypes revealed 5 major clusters. Cluster 1 (n = 445) was characterized by maternal stress; cluster 2 (n = 294) was characterized by premature membrane rupture; cluster 3 (n = 120) was characterized by familial factors, and cluster 4 (n = 63) was characterized by maternal comorbidities. Cluster 5 (n = 106) was multifactorial and characterized by infection (INF), decidual hemorrhage (DH), and placental dysfunction (PD). These 3 phenotypes were correlated highly by χ(2) analysis (PD and DH, P < 2.2e-6; PD and INF, P = 6.2e-10; INF and DH, (P = .0036). Gene-based testing identified the INS (insulin) gene as significantly associated with cluster 3 of SPTB. CONCLUSION: We identified 5 major clusters of SPTB based on a phenotype tool and hierarch clustering. There was significant correlation between several of the phenotypes. The INS gene was associated with familial factors that were underlying SPTB.

The phenotype of spontaneous preterm birth: application of a clinical phenotyping tool.

OBJECTIVE: Spontaneous preterm birth (SPTB) is a complex condition that is likely a final common pathway with multiple possible causes. We hypothesized that a comprehensive classification system appropriately could group women with similar STPB causes and could provide an explanation, at least in part, for the disparities in SPTB that are associated with race and gestational age at delivery. STUDY DESIGN: This was a planned analysis of a multicenter, prospective study of singleton SPTBs. Women with SPTB at <34 weeks' gestation were included. We defined 9 potential SPTB phenotypes based on clinical data: infection/inflammation, maternal stress, decidual hemorrhage, uterine distention, cervical insufficiency, placental dysfunction, premature rupture of the membranes, maternal comorbidities, and familial factors. Each woman's condition was evaluated for each phenotype. Delivery gestational age was compared between those with and without each phenotype. Phenotype profiles were also compared between women with very early (20.0-27.9 weeks' gestation) SPTB vs those with early SPTB (28.0-34.0 weeks' gestation) and between African American and white women. Statistical analysis was by t test and χ(2) test, as appropriate. RESULTS: The phenotyping tool was applied to 1025 women with SPTBs who delivered at a mean 30.0 ± 3.2 (SD) weeks' gestation. Of these, 800 women (78%) had ≥2 phenotypes. Only 43 women (4.2%) had no phenotypes. The 281 women with early SPTBs were more likely to have infection/inflammation, decidual hemorrhage, and cervical insufficiency phenotypes (all P ≤ .001). African American women had more maternal stress and cervical insufficiency but less decidual hemorrhage and placental dysfunction compared with white women (all P < .05). Gestational age at delivery decreased as the number of phenotypes that were present increased. CONCLUSION: Precise SPTB phenotyping classifies women with SPTBs and identifies specific differences between very early and early SPTB and between African American and white women.

Fetal growth and risk of stillbirth: a population-based case-control study.

BACKGROUND: Stillbirth is strongly related to impaired fetal growth. However, the relationship between fetal growth and stillbirth is difficult to determine because of uncertainty in the timing of death and confounding characteristics affecting normal fetal growth. METHODS AND FINDINGS: We conducted a population-based case-control study of all stillbirths and a representative sample of live births in 59 hospitals in five geographic areas in the US. Fetal growth abnormalities were categorized as small for gestational age (SGA) (<10th percentile) or large for gestational age (LGA) (>90th percentile) at death (stillbirth) or delivery (live birth) using population, ultrasound, and individualized norms. Gestational age at death was determined using an algorithm that considered the time-of-death interval, postmortem examination, and reliability of the gestational age estimate. Data were weighted to account for the sampling design and differential participation rates in various subgroups. Among 527 singleton stillbirths and 1,821 singleton live births studied, stillbirth was associated with SGA based on population, ultrasound, and individualized norms (odds ratio [OR] [95% CI]: 3.0 [2.2 to 4.0]; 4.7 [3.7 to 5.9]; 4.6 [3.6 to 5.9], respectively). LGA was also associated with increased risk of stillbirth using ultrasound and individualized norms (OR [95% CI]: 3.5 [2.4 to 5.0]; 2.3 [1.7 to 3.1], respectively), but not population norms (OR [95% CI]: 0.6 [0.4 to 1.0]). The associations were stronger with more severe SGA and LGA (<5th and >95th percentile). Analyses adjusted for stillbirth risk factors, subset analyses excluding potential confounders, and analyses in preterm and term pregnancies showed similar patterns of association. In this study 70% of cases and 63% of controls agreed to participate. Analysis weights accounted for differences between consenting and non-consenting women. Some of the characteristics used for individualized fetal growth estimates were missing and were replaced with reference values. However, a sensitivity analysis using individualized norms based on the subset of stillbirths and live births with non-missing variables showed similar findings. CONCLUSIONS: Stillbirth is associated with both growth restriction and excessive fetal growth. These findings suggest that, contrary to current practices and recommendations, stillbirth prevention strategies should focus on both severe SGA and severe LGA pregnancies. Please see later in the article for the Editors' Summary.

Maternal serum serpin B7 is associated with early spontaneous preterm birth.

OBJECTIVE: We sought to identify serum biomarkers of early spontaneous preterm birth (SPTB) using semiquantitative proteomic analyses. STUDY DESIGN: This was a nested case-control study of pregnant women with previous SPTB. Maternal serum was collected at 19-24 and 28-32 weeks' gestation, and analyzed by liquid chromatography-multiple reaction monitoring/mass spectrometry. Targeted and shotgun proteomics identified 31 candidate proteins that were differentially expressed in pooled serum samples from spontaneous preterm (cases [<34 weeks]) and term (controls) deliveries. Candidate protein expression was compared in individual serum samples between cases and controls matched by age and race groups, and clinical site. Protein expression was verified by Western blot in the placenta and fetal membranes from cases and controls. RESULTS: Serum samples were available for 35 cases and 35 controls at 19-24 weeks, and 16 cases and 16 controls at 28-32 weeks. One protein, serpin B7, yielded serum concentrations that differed between cases and controls. The mean concentration of serpin B7 at 28-32 weeks was 1.5-fold higher in women with subsequent preterm deliveries compared to controls; there was no difference at 19-24 weeks. Higher levels of serpin B7 at both gestational age windows were associated with a shorter interval to delivery, and higher levels of serpin B7 in samples from 28-32 weeks were associated with a lower gestational age at delivery. Western blotting identified serpin B7 protein in placenta, amnion, and chorion from cases and controls. CONCLUSION: Targeted and shotgun serum proteomics analyses associated 1 protein, serpin B7, with early SPTB. Our results require validation in other cohorts and analysis of the possible mechanistic role of serpin B7 in parturition.