Elizabeth Eklund

Serum levels of the ovarian cancer biomarker HE4 are decreased in pregnancy and increase with age

OBJECTIVE The purpose of this study was to establish normal ranges for human epididymis protein 4 (HE4) serum levels in healthy women. STUDY DESIGN HE4 levels were measured in healthy women and analyzed by age, menopausal status, and pregnancy status. Upper 95th percentiles were determined for normal ranges. RESULTS Serum samples from 1101 healthy women and 67 pregnant women were analyzed. Above the age of 40 years significant elevations in HE4 concentrations emerged with advancing age. The upper 95th percentile for HE4 levels was 89 pmol/L for premenopausal women, 128 pmol/L for postmenopausal women, and 115 pmol/L for all women. There was a significant difference in the median serum HE4 levels in premenopausal women (46.6 pmol/L) compared with postmenopausal women (57.6 pmol/L; P < .001). In pregnant women, median HE4 concentrations were significantly lower than their premenopausal counterparts (P < .001). CONCLUSION HE4 serum concentrations vary significantly on the basis of age. These variations must be considered when the upper limit of normal for HE4 is determined.

Levels of antimullerian hormone in serum during the normal menstrual cycle.

OBJECTIVE To determine whether levels of antimüllerian hormone (AMH) in serum vary during the normal menstrual cycle, using the most recently developed immunoassay method. DESIGN Prospective cohort study. SETTING Local community. PATIENT(S) Women with normal menstrual cycles and between the ages of 18 and 45 years were recruited (n = 45). Blood samples were collected on 5 days within each cycle: two in the follicular phase and three after confirmed ovulation. Exclusion criteria were anovulatory cycles, incomplete sample collection, insufficient blood volume, or non-Caucasian ethnicity. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Serum samples were tested for levels of AMH using a new immunoassay method (Ansh Labs). The effects of body mass index (BMI) and smoking on serum AMH levels were considered. RESULT(S) Serum AMH levels varied significantly during the menstrual cycle, with the highest levels in the follicular phase. When the analysis was stratified by age, AMH variation during the menstrual cycle was significant only for women older than 30 years. Serum AMH levels were not significantly altered by BMI or smoking. CONCLUSION(S) The new AMH immunoassay revealed a follicular phase rise in serum levels, particularly in women over the age of 30 years. This is consistent with other reports finding an interaction of menstrual cycle variation in AMH and chronological age. Nonetheless, the extent of variation is small, and sampling on any day of the menstrual cycle is expected to adequately reflect ovarian reserve. CLINICAL TRIAL REGISTRATION NUMBER NCT01337999.

The clinical utility of DNA-based screening for fetal aneuploidy by primary obstetrical care providers in the general pregnancy population

Objective:To assess the clinical utility of cell-free DNA (cfDNA)-based screening for aneuploidies offered through primary obstetrical care providers to a general pregnancy population.Methods:Patient educational materials were developed and validated and providers were trained. Serum was collected for reflexive testing of cfDNA failures. Providers and patients were surveyed concerning knowledge, decision making, and satisfaction. Pregnancy outcome was determined by active or passive ascertainment.Results:Between September 2014 and July 2015, 72 providers screened 2,691 women. The five largest participating practices increased uptake by 8 to 40%. Among 2,681 reports, 16 women (0.6%) were screen-positive for trisomy 21, 18, or 13; all saw genetic professionals. Twelve were confirmed (positive predictive value (PPV), 75%; 95% CI, 48–93%) and four were false-positives (0.15%). Of 150 failures (5.6%), 79% had a negative serum or subsequent cfDNA test; no aneuploidies were identified. Of 100 women surveyed, 99 understood that testing was optional, 96 had their questions answered, and 95 received sufficient information. Pretest information was provided by the physician/certified nurse midwife (55) or office nurse/educator (40); none was provided by genetic professionals.Conclusion:This first clinical utility study of cfDNA screening found higher uptake rates, patient understanding of basic concepts, and easy incorporation into routine obstetrical practices. There were no reported cases of aneuploidy among cfDNA test failures.Genet Med advance online publication 12 January 2017

Evaluating first trimester maternal serum screening combinations for Down syndrome suitable for use with reflexive secondary screening via sequencing of cell free DNA: high detection with low rates of invasive procedures

Examine primary Down syndrome screening using combinations of first trimester serum markers, with and without sequencing of cell free DNA as a secondary reflexive test.

Oxidative and carbonyl stress in pregnant women with obstructive sleep apnea

PurposePregnant women are particularly susceptible to sleep-disordered breathing. Obstructive sleep apnea (OSA) in pregnancy is associated with poor pregnancy and fetal outcomes. Oxidative stress caused by intermittent hypoxemia and reoxygenation may impact pregnancy health. We hypothesize that pregnant women with OSA have a pronounced oxidative stress profile.MethodsA case-control study was performed to study oxidative stress markers in the serum of pregnant women with or without OSA. Patients with OSA were identified between 2003 and 2009. Contemporaneous controls were pregnant subjects without apnea, gasping, or snoring around the time of delivery. Serum markers of oxidative and carbonyl stress were measured by spectrophotometric/fluorometric methods. Multiple linear regression analysis was used with a model including age, body mass index at delivery, history of diabetes, and gestational age.ResultsSerum samples from 23 OSA cases and 41 controls were identified. Advanced oxidation protein products, a marker for oxidative stress, and advanced glycation end products (AGEs), a marker for carbonyl stress, were significantly lower in women with OSA than in controls (p value <0.0001). Total antioxidant capacity was higher in women with OSA in comparison to controls (p value <0.0001). The difference in AGEs remained significant even after adjusting for confounders.ConclusionContrary to our hypothesis, the results of this study suggest that pregnant women with OSA have higher antioxidant capacity and lower oxidative and carbonyl stress markers compared to controls, suggesting a possible protective effect of intermittent hypoxia. Whether OSA in pregnancy impacts oxidative stress differently than OSA in the general population remains to be confirmed.

A First-Trimester Biomarker Panel for Predicting the Development of Gestational Diabetes:

OBJECTIVE Serum markers measured early in pregnancy have been associated with the later diagnosis of gestational diabetes mellitus (GDM). This study aims to explore the performance of a panel of first-trimester biochemical markers for the prediction of GDM. METHODS A case-control study was performed that included 12 women who developed GDM and 60 controls matched for maternal and gestational age at blood collection. Levels of pregnancy-associated plasma protein A (PAPP-A), soluble endoglin, pregnancy protein 13, and adiponectin (Adipo) were measured on residual sera used in first-trimester screening for Down syndrome. Data were analyzed by nonparametric methods. A receiver operating characteristic curve was used to calculate the detection rate (DR) obtained with a panel of significant predictors for GDM. RESULTS Multiples of the median values for Adipo and PAPP-A were significantly reduced in GDM cases versus matched controls. Combination of Adipo and PAPP-A yielded a DR of 63.6% at a false-positive rate of 10%. Addition of body mass index (BMI) to this panel increased DR to 72.7%. CONCLUSION This study suggests that first-trimester screening with Adipo, PAPP-A, and BMI may effectively identify women at high risk for the development of GDM.Objective: Serum markers measured early in pregnancy have been associated with the later diagnosis of gestational diabetes mellitus (GDM). This study aims to explore the performance of a panel of first-trimester biochemical markers for the prediction of GDM. Methods: A case–control study was performed that included 12 women who developed GDM and 60 controls matched for maternal and gestational age at blood collection. Levels of pregnancy-associated plasma protein A (PAPP-A), soluble endoglin, pregnancy protein 13, and adiponectin (Adipo) were measured on residual sera used in first-trimester screening for Down syndrome. Data were analyzed by nonparametric methods. A receiver operating characteristic curve was used to calculate the detection rate (DR) obtained with a panel of significant predictors for GDM. Results: Multiples of the median values for Adipo and PAPP-A were significantly reduced in GDM cases versus matched controls. Combination of Adipo and PAPP-A yielded a DR of 63.6% at a false-positive rate of 10%. Addition of body mass index (BMI) to this panel increased DR to 72.7%. Conclusion: This study suggests that first-trimester screening with Adipo, PAPP-A, and BMI may effectively identify women at high risk for the development of GDM.

Activin subunit and receptor expression in normal and cleft human fetal palate tissues.

Craniofacial malformations, such as cleft palate, present serious complications in the newborn and are often of unknown etiology. Activin BA subunit deletion leads to cleft palate in mice, but the expression of this protein in the human palate has not been explored. Our goal was to determine the spatial and temporal expression of inhibin/activin subunits; the binding protein, follistatin; and activin receptors in the human fetal palate. Residual human fetal palate tissues, with or without cleft, were collected during routine autopsy at Women and Infants Hospital. Inhibin/activin alpha and beta subunits, follistatin, and activin receptor protein and mRNA expression were studied by immunocytochemistry and reverse-transcriptase polymerase chain reaction (RT-PCR) experiments, respectively. Dimeric activin A levels were compared in cleft and normal palate tissue homogenates by immunoassay. Activin BA, follistatin, and activin receptor type IIA proteins were observed in normal and cleft palate tissues throughout pregnancy (gestational weeks 11 to 40). Proteins were predominantly found in developing bone cells, with no significant group differences. Inhibin/activin BA subunit, follistatin, and activin receptor mRNAs were also detected in normal and cleft fetal palate tissues, but inhibin alpha and BB subunit were absent. Inhibin/activin BA subunit expression was consistent with the presence of dimeric activin A, but levels did not differ significantly between cleft and control tissues. Inhibin/activin BA subunit, follistatin, and activin receptor proteins and mRNAs are present in the human fetal palate. These data suggest that activin signalling has the potential to be associated with human palate development.

Comparison of Inhibin Alpha Subunit and Antimüllerian Hormone Immunoreactivity in Granulosa Cell and Mucinous Ovarian Tumors.

The inhibin alpha subunit protein is used in the histopathologic diagnosis of granulosa cell tumors (GCTs), and as a serum marker for disease progression. Yet, the availability of antibodies for inhibin has been limited. Serum antimüllerian hormone (AMH) levels have also been described as a GCT marker. The goal of this study was to compare inhibin and AMH immunoreactivity in tissues and serum from GCT (n=6) using existing and new antibodies. Expression was also explored in cases of mucinous tumors (n=15), where inhibin is also a serum marker in some cases. Immunocytochemistry was performed using a commercial and newly developed inhibin alpha subunit and AMH antibodies. Serum levels were examined with total inhibin and AMH immunoassays. Inhibin alpha subunit and AMH were equivalent markers of GCT in both tissue and serum. In mucinous samples, inhibin alpha subunit was detected in tumor and stromal cells, and levels in serum were also frequently elevated. In contrast, AMH protein was detected in mucinous tissues, but there was no evidence of secretion in serum. The new inhibin alpha subunit and AMH antibodies provide needed resources for examination of granulosa cell and mucinous tumors.

Expression of transcription factors controlling alpha inhibin gene expression in placental tissues from pregnancies affected by fetal Down syndrome

Maternal serum inhibin A is a clinically accepted marker in assessing risk of fetal Down syndrome during pregnancy. Second trimester levels of inhibin A are, on average, about two times higher in affected than in control pregnancies, although the pathological basis of this alteration is not understood. The genes for inhibin A subunits are not located on chromosome 21, eliminating a simple dosage effect. Nevertheless, previous studies have shown up-regulated inhibin A subunit mRNA levels in placental tissue from pregnancies affected with Down syndrome. Inhibin A is a dimeric protein consisting of an alpha and betaA subunit. It has been previously established, using in vitro studies, that the alpha subunit synthesis plays a key role in production of dimeric inhibin. The alpha inhibin promoter has been characterized in placenta and shown to lack a TATA element, but to contain a repeat TG element, multiple initiation sites and several binding sites for cis-acting elements. We sought to determine the factors regulating alpha inhibin expression in Down syndrome placenta, among candidates known to regulate alpha inhibin gene expression in reproductive tissues (placenta and/or ovary). Among the candidates studied were the alpha inhibin transcription repressors; Wilms’ tumor suppressor (WT1), inducible cAMP early repressor (ICER), and CCAAT/enhancer-binding protein beta (CEBP/beta) as well as the alpha inhibin transcription activators; nuclear receptor 5A2 (NR5A2, also known as LRH-1), GATA factors, cAMP response element binding protein (CREB), and transcription factor activating proteins 2 alpha and 2 gamma (TFAP2A and TFAP2C). The mRNA expression of these regulatory factors and inhibin subunits was compared in Down syndrome and unaffected placental tissues. Residual placental tissue was collected after routine examination with approval of the Institutional Review Board for Human Studies at Women and Infants Hospital. Samples were obtained in the second trimester after spontaneous loss or termination and in the third trimester after delivery. Chorionic villous tissue was sampled by Perinatal Pathology staff and stored in RNA later at 80 C before processing. Total RNA was extracted using Trizol, digested with deoxyribonuclease on Qiagen RNeasy columns, and analyzed for quality using a bioanalyzer. The total RNA was then reverse transcribed with avian myeloblastosis virus-reverse transcriptase. Following reverse transcription, the mRNA levels of select genes were determined by real-time PCR on a 7300 real-time PCR system using SYBR Green PCR master mix, as previously described. Primers were designed according to the complete human cDNA sequences of the above genes. The primer sequences are provided in Table 1. The relative mRNA level for each gene of interest was obtained by normalizing to the mRNA level of a control gene, ribosomal protein L19 (RPL19). Specificity of all the real-time PCR reactions was confirmed by a single peak in the melt curves and a single band of the predicted size after agarose gel electrophoresis of the PCR products (data not shown). Ten samples from Down syndrome pregnancy and eight control samples were collected in the second and third trimesters of pregnancy, at gestational weeks 14 to 39. The mean gestational age at sample collection was 26weeks ( 10) in each group. There was no difference in the distribution of fetal gender with 60% female in each group (three control samples were not assigned a fetal gender). Maternal age was slightly, but not significantly, higher in women having Down

Feasibility of using plasma rather than serum in first and second trimester multiple marker Down's syndrome screening

Objective To compare maternal plasma with serum for measuring markers currently used in first and second trimester screening for Downs syndrome. Setting A laboratory-based investigation of two sample types in assays used in prenatal screening for Downs syndrome. Methods A paired data-set included both plasma and serum from 101 pregnant women. A nested case/control data-set included only plasma samples from 34 first and 23 second trimester Downs syndrome pregnancies, each matched with six euploid controls. Analyte levels were measured and converted to multiples of the median (MoM). Results In the paired data-set, each of the five analytes (alphafetoprotein, unconjugated estriol, human chorionic gonadotropin, inhibin-A and pregnancy-associated plasma protein A) in serum and plasma was highly correlated (r > 0.970) and after conversion to MoM, the resulting distributions were equivalent (P > 0.7). In the matched data-set, plasma-based median MoM levels in cases were consistent with the published serum counterparts for all markers. Conclusions This study provides strong evidence that current serum-based prenatal screening can be performed equally well using plasma samples. This may prove useful, especially if secondary screening using a DNA-based test requires maternal plasma.