Geralyn Lambert-Messerlian

DNA sequencing of maternal plasma to detect Down syndrome: An international clinical validation study

Purpose: Prenatal screening for Down syndrome has improved, but the number of resulting invasive diagnostic procedures remains problematic. Measurement of circulating cell-free DNA in maternal plasma might offer improvement.Methods: A blinded, nested case-control study was designed within a cohort of 4664 pregnancies at high risk for Down syndrome. Fetal karyotyping was compared with an internally validated, laboratory-developed test based on next-generation sequencing in 212 Down syndrome and 1484 matched euploid pregnancies. None had been previously tested. Primary testing occurred at a CLIA-certified commercial laboratory, with cross validation by a CLIA-certified university laboratory.Results: Down syndrome detection rate was 98.6% (209/212), the false-positive rate was 0.20% (3/1471), and the testing failed in 13 pregnancies (0.8%); all were euploid. Before unblinding, the primary testing laboratory also reported multiple alternative interpretations. Adjusting chromosome 21 counts for guanine cytosine base content had the largest impact on improving performance.Conclusion: When applied to high-risk pregnancies, measuring maternal plasma DNA detects nearly all cases of Down syndrome at a very low false-positive rate. This method can substantially reduce the need for invasive diagnostic procedures and attendant procedure-related fetal losses. Although implementation issues need to be addressed, the evidence supports introducing this testing on a clinical basis.

DNA sequencing of maternal plasma reliably identifies trisomy 18 and trisomy 13 as well as Down syndrome: an international collaborative study.

Purpose:To determine whether maternal plasma cell–free DNA sequencing can effectively identify trisomy 18 and 13.Methods:Sixty-two pregnancies with trisomy 18 and 12 with trisomy 13 were selected from a cohort of 4,664 pregnancies along with matched euploid controls (including 212 additional Down syndrome and matched controls already reported), and their samples tested using a laboratory-developed, next-generation sequencing test. Interpretation of the results for chromosome 18 and 13 included adjustment for CG content bias.Results:Among the 99.1% of samples interpreted (1,971/1,988), observed trisomy 18 and 13 detection rates were 100% (59/59) and 91.7% (11/12) at false-positive rates of 0.28% and 0.97%, respectively. Among the 17 samples without an interpretation, three were trisomy 18. If z-score cutoffs for trisomy 18 and 13 were raised slightly, the overall false-positive rates for the three aneuploidies could be as low as 0.1% (2/1,688) at an overall detection rate of 98.9% (280/283) for common aneuploidies. An independent academic laboratory confirmed performance in a subset.Conclusion:Among high-risk pregnancies, sequencing circulating cell–free DNA detects nearly all cases of Down syndrome, trisomy 18, and trisomy 13, at a low false-positive rate. This can potentially reduce invasive diagnostic procedures and related fetal losses by 95%. Evidence supports clinical testing for these aneuploidies.Genet Med 2012:14(3):296–305

Day 3 serum inhibin-B is predictive of assisted reproductive technologies outcome

OBJECTIVE To determine if women with day 3 serum inhibin-B concentrations < 45 pg/mL (conversion factor to SI unit, 1.00) demonstrate a poorer response to ovulation induction and assisted reproductive technologies outcome relative to women with inhibin-B values > or = 45 pg/mL. DESIGN Analysis of inhibin-B, FSH, and E2 concentrations in day 3 serum samples. SETTING Academic clinical practice. PATIENT(S) One hundred fifty-six women who underwent 178 assisted reproductive technology (ART) cycles with luteal phase GnRH agonist suppression plus hMG and urofollitropin stimulation. MAIN OUTCOME MEASURE(S) Serum E2 on day of hCG, number of oocytes retrieved per patient, fertilization rate, cleavage rate, clinical pregnancy rate (PR) per initiated cycle, cancellation rate per initiated cycle, and spontaneous abortion rate. RESULT(S) Women with day 3 serum inhibin-B < 45 pg/mL demonstrated 70% of the E2 response, had 66.6% of the number of oocytes retrieved per patient, with 28% of the clinical PR per initiated cycle, and three times the cancellation rate per initiated cycle than women with day 3 inhibin-B > or = 45 pg/mL. After controlling for age, day 3 serum FSH, day 3 serum E2, patient cycle number, and method of ART, day 3 serum inhibin-B > or = 45 pg/mL was noted to be prognostic of the number of oocytes retrieved and clinical PR. The adjusted odds ratio of clinical pregnancy for those with day 3 serum inhibin-B > or = 45 pg/mL versus those with inhibin-B < 45 pg/mL was 6.8 (95% confidence interval 1.8 to 25.6). CONCLUSION(S) Women with low day 3 serum inhibin-B concentrations demonstrate a poorer response to ovulation induction and are less likely to conceive a clinical pregnancy through ART relative to women with high day 3 inhibin-B.

Maternal thyroid hypofunction and pregnancy outcome.

OBJECTIVE: To estimate whether maternal thyroid hypofunction is associated with complications. METHODS: A total of 10,990 patients had first- and second-trimester serum assayed for thyroid-stimulating hormone (TSH), free thyroxine (freeT4), and antithyroglobulin and antithyroid peroxidase antibodies. Thyroid hypofunction was defined as 1) subclinical hypothyroidism: TSH levels above the 97.5th percentile and free T4 between the 2.5th and 97.5th percentiles or 2) hypothyroxinemia: TSH between the 2.5th and 97.5th percentiles and free T4 below the 2.5th percentile. Adverse outcomes were evaluated. Patients with thyroid hypofunction were compared with euthyroid patients (TSH and free T4 between the 2.5th and 97.5th percentiles). Patients with and without antibodies were compared. Multivariable logistic regression analysis adjusted for confounders was used. RESULTS: Subclinical hypothyroidism was documented in 2.2% (240 of 10,990) in the first and 2.2% (243 of 10,990) in the second trimester. Hypothyroxinemia was documented in 2.1% (232 of 10,990) in the first and 2.3% (247 of 10,990) in the second trimester. Subclinical hypothyroidism was not associated with adverse outcomes. In the first trimester, hypothyroxinemia was associated with preterm labor (adjusted odds ratio [aOR] 1.62; 95% confidence interval [CI] 1.00–2.62) and macrosomia (aOR 1.97; 95% CI 1.37–2.83). In the second trimester, it was associated with gestational diabetes (aOR 1.7; 95% CI 1.02–2.84). Fifteen percent (1,585 of 10,990) in the first and 14% (1,491 of 10,990) in the second trimester had antithyroid antibodies. When both antibodies were positive in either trimester, there was an increased risk for preterm premature rupture of membranes (P=.002 and P<.001, respectively). CONCLUSION: Maternal thyroid hypofunction is not associated with a consistent pattern of adverse outcomes. LEVEL OF EVIDENCE: II

Quad screen as a predictor of adverse pregnancy outcome

Objective: To estimate the effect of second-trimester levels of maternal serum alpha-fetoprotein (AFP), human chorionic gonadotrophin (hCG), unconjugated estriol (uE3), and inhibin A (the quad screen) on obstetric complications by using a large, prospectively collected database (the FASTER database). Methods: The FASTER trial was a multicenter study that evaluated first- and second-trimester screening programs for aneuploidy in women with singleton pregnancies. As part of this trial, patients had a quad screen drawn at 15–18 6/7 weeks. We analyzed the data to identify associations between the quad screen markers and preterm birth, intrauterine growth restriction, preeclampsia, and fetal loss. Our analysis was performed by evaluating the performance characteristics of quad screen markers individually and in combination. Crude and adjusted effects were estimated by multivariable logistic regression analysis. Patients with fetal anomalies were excluded from the analysis. Results: We analyzed data from 33,145 pregnancies. We identified numerous associations between the markers and the adverse outcomes. There was a relatively low, but often significant, risk of having an adverse pregnancy complication if a patient had a single abnormal marker. However, the risk of having an adverse outcome increased significantly if a patient had 2 or more abnormal markers. The sensitivity and positive predictive values using combinations of markers is relatively low, although superior to using individual markers. Conclusion: These data suggest that components of the quad screen may prove useful in predicting adverse obstetric outcomes. We also showed that the total number and specific combinations of abnormal markers are most useful in predicting the risk of adverse perinatal outcome. Level of Evidence: II-2

The impact of maternal plasma DNA fetal fraction on next generation sequencing tests for common fetal aneuploidies

Maternal plasma contains circulating cell‐free DNA fragments originating from both the mother and the placenta. The proportion derived from the placenta is known as the fetal fraction. When measured between 10 and 20 gestational weeks, the average fetal fraction in the maternal plasma is 10% to 15% but can range from under 3% to over 30%. Screening performance using next‐generation sequencing of circulating cell‐free DNA is better with increasing fetal fraction and, generally, samples whose values are less than 3% or 4% are unsuitable. Three examples of the clinical impact of fetal fraction are discussed. First, the distribution of test results for Down syndrome pregnancies improves as fetal fraction increases, and this can be exploited in reporting patient results. Second, the strongest factor associated with fetal fraction is maternal weight; the false negative rate and rate of low fetal fractions are highest for women with high maternal weights. Third, in a mosaic, the degree of mosaicism will impact the performance of the test because it will reduce the effective fetal fraction. By understanding these aspects of the role of fetal fraction in maternal plasma DNA testing for aneuploidy, we can better appreciate the power and the limitations of this impressive new methodology.

DNA sequencing of maternal plasma to identify Down syndrome and other trisomies in multiple gestations

Studies on prenatal testing for Down syndrome (trisomy 21), trisomy 18, and trisomy 13 by massively parallel shotgun sequencing (MPSS) of circulating cell free DNA have been, for the most part, limited to singleton pregnancies. If MPSS testing is offered clinically, it is important to know if these trisomies will also be identified in multiple pregnancies.

First- and second-trimester thyroid hormone reference data in pregnant women: a FaSTER (First- and Second-Trimester Evaluation of Risk for aneuploidy) Research Consortium study

OBJECTIVE The purpose of this study was to calculate first and second trimester reference ranges and within-woman correlations for TSH, free T4, and thyroid antibodies. STUDY DESIGN TSH, free T4, and thyroid antibodies were measured in paired sera from 9562 women in the FaSTER trial of Down syndrome screening. RESULTS The median first trimester TSH (1.05 mIU/L) is lower than the second (1.23 mIU/L); and 98th centile is higher (4.15 vs 3.77 mIU/L). Within-woman paired TSH correlations are moderately strong (r(2) = 0.64). Among women with first trimester TSH values above the 98th centile, second trimester values are over the 95th centile in 68%. Median first trimester free T4 values (1.10 ng/dL) are higher than second (1.01 ng/dL). Paired free T4 measurements correlate weakly (r(2) = 0.23). Among women with first trimester free T4 values below the 2nd centile, second trimester values are below the 5th centile in 32%. Antibody measurements correlate strongly between trimesters (thyroperoxidase r(2) = 0.79, thyroglobulin r(2) = 0.83). CONCLUSION TSH and free T4 measurements require gestation-specific reference ranges.

Variations in Serum Mullerian Inhibiting Substance Between White, Black and Hispanic Women

OBJECTIVE To compare serum müllerian inhibiting substance (MIS) levels between white, black, and Hispanic women to determine whether ovarian aging occurs at a different time course for women of different racial groups. DESIGN Longitudinal study of serum MIS levels in women of different race and ethnicity over two different time points. SETTING Womens Interagency HIV Study, a multicenter prospective cohort study. PATIENT(S) Serum samples obtained from 809 participants (122 white, 462 black, and 225 Hispanic women). INTERVENTION(S) Comparison of serum MIS between women of different race and ethnicity at two time points (median age 37.5 years and 43.3 years). MAIN OUTCOME MEASURE(S) Variation in MIS by race and ethnicity over time, controlling for age, body mass index, HIV status, and smoking. RESULT(S) Compared with white women, average MIS values were lower among black (25.2% lower) and Hispanic (24.6% lower) women, adjusting for age, body mass index, smoking, and HIV status. CONCLUSION(S) There is an independent effect of race and ethnicity on the age-related decline in MIS over time.

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.