Methodological approach for an integrated female-specific study of anxiety and smoking comorbidity.

Two primary ovarian hormones that fluctuate across the female menstrual cycle-estradiol and progesterone-have been independently linked in separate literatures to nicotine reinforcement and anxiety psychopathology. We identify existing methodological limitations in these literatures, describe an example protocol that was developed to address such limitations, highlight case examples, and offer insights on the resulting advantages and challenges. This protocol was an observational, prospective, within-subjects study of female cigarette smokers who were followed over the course of a complete menstrual cycle. Non-treatment seeking, female cigarette smokers (N = 50), between the ages of 18-40 who have a normal menstrual cycle (25-35 days in length) were recruited from the community. Females with anxiety or mood psychopathology represented 38.0% of the sample. Salivary progesterone and estradiol were assessed each morning via at-home saliva collection methods. Self-reported within-day momentary ratings of anxiety and nicotine reinforcement were collected using ecological momentary assessment (EMA) via a mobile app. Protocol compliance was >85%. Within- and between-subjects heterogeneity was observed in the progesterone and estradiol, anxiety, and nicotine craving measures, especially in the context of anxiety psychopathology. We aimed to integrate the anxiety and nicotine dependence literatures and advance the empirical study of the role of ovarian hormones. This protocol reflects an intensive, yet feasible approach to collecting daily-level naturalistic data related to estradiol, progesterone, anxiety, and nicotine reinforcement.

Cell-free DNA-based prenatal screening via rolling circle amplification: Identifying and resolving analytic issues.

OBJECTIVE: A rolling circle amplification (RCA) based commercial methodology using cell-free (cf)DNA to screen for common trisomies became available in 2018. Relevant publications documented high detection but with a higher than expected 1% false positive rate. Preliminary evidence suggested assay variability was an issue. A multi-center collaboration was created to explore this further and examine whether subsequent manufacturer changes were effective. METHODS: Three academic (four devices) and two commercial (two devices) laboratories provided run date, chromosome 21, 18, and 13 run-specific standard deviations, number of samples run, and reagent lot identifications. Temporal trends and between-site/device consistency were explored. Proportions of run standard deviations exceeding pre-specified caps of 0.4%, 0.4% and 0.6% were computed. RESULTS: Overall, 661 RCA runs between April 2019 and July 30, 2022 tested 39,756 samples. In the first 24, subsequent 9, and final 7 months, proportions of capped chromosome 21 runs dropped from 39% to 22% to 6.0%; for chromosome 18, rates were 76%, 36%, and 4.0%. Few chromosome 13 runs were capped using the original 0.60%, but capping at 0.50%, rates were 28%, 16%, and 7.6%. Final rates occurred after reformulated reagents and imaging software modifications were fully implemented across all devices. Revised detection and false positive rates are estimated at 98.4% and 0.3%, respectively. After repeat testing, failure rates may be as low as 0.3%. CONCLUSION: Current RCA-based screening performance estimates are equivalent to those reported for other methods, but with a lower test failure rate after repeat testing.

Validation of a monoclonal unconjugated estriol antibody for use in prenatal maternal serum screening.

OBJECTIVES: Unconjugated estriol (uE3) is used as a marker for fetal aneuploidy in maternal serum screening tests. The goal of this study was to examine the validity of a new immunoassay for uE3 that uses a monoclonal antibody (m-uE3) rather than the more commonly used polyclonal antibody (p-uE3). SETTING: Assays were performed in the Special Chemistry laboratory at Women and Infants Hospital of Rhode Island. METHODS: Residual fresh (n = 100) and frozen (n = 533) second trimester serum samples from routine clinical care were tested using p-uE3 and/or m-uE3 assays. Assay results were compared between methods using Bland-Altman plots. A median equation was developed for m-uE3 results. Down syndrome risks were compared between the two assays in a case-control sample set (21 cases each matched with five controls for the completed week of gestation, duration of freezer storage and race). RESULTS: Log-transformed serum uE3 levels were highly correlated between the assays (r = 0.93, p < 0.001), with the m-uE3 assay levels yielding, on average, 23% higher (standard deviation of differences in log uE3 concentrations = 0.07) results. Assay and gestation-based median equations were calculated and used to convert m-uE3 concentrations to multiples of the median (MoM). The m-uE3 MoM values fit a log Gaussian distribution well with a log standard deviation of 0.11. Down syndrome risk results were not significantly different between assays. CONCLUSIONS: The m-uE3 assay, with results expressed in MoMs, is suitable for screening and as a monoclonal-based assay offers the advantage of a predictable and indefinite supply of antibody to perform the assay.

Surveillance of SARS-CoV-2 Antibodies in Early 2020 among Rhode Island Pregnancies.

OBJECTIVE: Limited data are available on the performance of SARS-CoV-2 antibody assays and data collected during pregnancy vary widely. The objective of this study was to estimate the seroprevalence of antibodies against SARS-CoV-2 in pregnant individuals in Rhode Island and to evaluate whether the prevalence differed by month of collection, age, county of residence, or economic status as estimated by zip code. METHODS: Pre-pandemic (2019) and early pandemic (2020) serum samples, collected for prenatal screening between 15 and 22 weeks of gestation, were analyzed utilizing two SARS-CoV-2 immunoglobulin G (IgG) automated assays that targeted the viral nucleocapsid (anti-N) or spike (anti-S) receptor binding domain proteins. RESULTS: Among 756 pre-pandemic samples, one anti-S IgG and 13 anti-N IgG were identified. No samples were positive for both. Among 787 pandemic specimens, 16 (2.03%) were positive for both anti-N IgG and anti-S IgG. When stratified by month of collection, there was a significant increase in seropositivity rate (p=0.023). Seropositivity rates were associated with lower income levels (p=0.08) but this was not statistically significant. No trend by maternal age was found (p=0.70). CONCLUSIONS: When a positive result was defined as both anti-N IgG and anti-S IgG, false positives were unlikely. Based on this methodology, serology could be utilized to monitor infection trends during pregnancy.

Assessment of a Simplified Cell-Free DNA Method for Prenatal Down Syndrome Screening.

BACKGROUND: Prenatal screening for common trisomies via cell-free (cfDNA) is usually implemented by technologies utilizing massively parallel sequencing, stringent environmental controls, complex bioinformatics, and molecular expertise. An alternative and less complex methodology utilizes rolling circle amplification (RCA). Further evaluation of its performance and related requirements are warranted. METHODS: At 16 sites, women at 10 to 20 weeks gestation provided informed consent, relevant information, and 2 to 3 blood samples. Samples shipped for testing were processed and stored. Women were enrolled at primary cfDNA screening, or following such screening at referral for diagnostic testing. RCA testing occurred post-enrollment, over 11 months. Diagnostic results and delivery notes determined clinical truth. Detection rates were based on confirmed trisomic pregnancies; false-positive rates were based on unaffected pregnancies from the general population. RESULTS: Detection rate for the common trisomies was 95.9% (117/122, 95% CI, 90.5%-98.5%); overall false-positive rate was 1.00% (22/2,205, 0.65%-1.51%). Test failure rate after repeat testing was 0.04%. When assay standard deviations were below pre-specified levels, the overall false-positive rate was much lower at 0.30% (P < 0.001). Fetal sex calls were correct for 99.7%. One technician analyzed 560 samples over 2 weeks, a rate of 14 000/year. CONCLUSIONS: Our assessment of this simplified cfDNA-based system for prenatal screening for common trisomies performed in a prenatal screening laboratory is encouraging. Improved detection, low failure rates and rapid reporting can be achieved by collecting 2 samples. Future priorities should include achieving higher run precision using a single collection tube. CLINICALTRIALS.GOV REGISTRATION NUMBER: NCT03087357.

Serum Decorin, Biglycan, and Extracellular Matrix Component Expression in Preterm Birth.

Preterm birth is a leading cause of infant morbidity and mortality. Decorin and biglycan are proteoglycans that play key roles in maintaining the connective tissue matrix and tensile strength of human fetal membranes and have been previously linked to PPROM. Extracellular matrix proteins, such as matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9), TIMP metallopeptidase inhibitor 1 (TIMP-1), TIMP metallopeptidase inhibitor 2 (TIMP-2), and collagen VI (COL-6), have also been linked to PPROM and may have utility in a serum-based screening model for this condition. To define the natural course of serum decorin and biglycan expression throughout the duration of healthy pregnancy, to explore patterns of serum decorin and biglycan expression in serum of asymptomatic women who go on to develop spontaneous preterm labor, and to investigate the potential role for matrix metalloproteinases, their inhibitors, and collagen VI in a serum-based screening model to predict PPROM. Serum decorin level decreases less than 1% per week, and serum biglycan decreases by 2.9% per week over the duration of healthy pregnancy. Serum decorin and biglycan concentrations do not differ in spontaneous preterm labor cases compared with those in controls. Mean concentrations of MMP-2, MMP-9, TIMP-1, TIMP-2, and COL-6 do not differ in PPROM cases compared with those in controls. We have demonstrated that serum decorin and biglycan concentrations remain stable throughout the duration of normal pregnancy and are not early indicators of preterm labor, while common MMPs, TIMPs, and collagen VI are not early indicators of PPROM.

Association between a history of depression and anti-müllerian hormone among late-reproductive aged women: the Harvard study of moods and cycles.

BACKGROUND: There is conflicting evidence regarding the association between a history of depression and risk of early menopause. In a cohort of premenopausal women, we investigated the association between depression history and ovarian reserve, as measured by anti-müllerian hormone (AMH). METHODS: The Harvard Study of Moods and Cycles (HSMC) was a prospective cohort study of women living in the Boston, MA metropolitan-area (1995-1999). Women aged 36-45 years at cohort entry (1995) were sampled from seven Boston metropolitan-area communities using census directories. We measured serum AMH in early-follicular phase venous blood specimens from 141 women with a Structured Clinical Interview for DSM-IV (SCID)-confirmed history of depression and 228 without such a history. We calculated prevalence ratios (PR) for the association between characteristics of depression history and low AMH (≤1.4 ng/mL), adjusting for several potential confounders. RESULTS: The prevalence of low AMH was similar among depressed (57.5%) and non-depressed (57.9%) women (Adjusted [Adj] PR = 0.90, 95% CI: 0.75, 1.08). Among depressed women, results were not appreciably different among those who had ever used antidepressants and those with comorbid anxiety. Modest inverse associations between depression and low AMH were seen among women aged 36-40 years (Adj PR = 0.75, 95% CI: 0.52, 1.09) and nulliparous women (Adj PR = 0.77, 95% CI: 0.59, 1.00). No dose-response association with greater duration or length of depressive symptoms was observed. CONCLUSIONS: Overall, the prevalence of low AMH was similar for depressed and non-depressed women 36-45 years of age. Surprisingly, among younger and nulliparous women, those with a history of depression had a slightly reduced prevalence of low AMH relative to those without such a history. These results do not indicate reduced ovarian reserve among women with a history of depression.

Adjusting antimüllerian hormone levels for age and body mass index improves detection of polycystic ovary syndrome.

OBJECTIVE: To examine whether accounting for a woman's age and body mass index (BMI) would improve the ability of antimüllerian hormone (AMH) to distinguish between women with (cases) and without (controls) polycystic ovarian syndrome (PCOS). DESIGN: An opportunistic case-control dataset of reproductive age women having evaluations for PCOS as defined by National Institutes of Health criteria. SETTING: Two medical centers in the United States enrolled women. Serum samples were analyzed for relevant analytes. PATIENTS: Women were between 18 and 39 years of age when samples and clinical information were collected. Residual samples had been stored for 2-17 years. AMH was measured via immunoassay. INTERVENTIONS: None; this was an observational study. MAIN OUTCOME MEASURES: Detection and false-positive rates for PCOS were computed for AMH results expressed as multiples of the median (MoM) both before and after adjustment for the woman's age and BMI. RESULTS: Using unadjusted AMH MoM results, 168 cases (78%) cases were at or beyond the 90th centile of controls (2.47 MoM). After accounting for each woman's age and BMI, 188 (87%) of those women were beyond the 90th centile of controls (2.20 MoM), a significant increase (P = .015). The adjusted AMH MoM levels fitted logarithmic normal distributions well (mean, standard deviation for controls and cases of 0.0000, 0.2765 and 0.6884, 0.2874, respectively) and this allowed for computation of patient-specific PCOS risks. CONCLUSIONS: Accounting for the woman's age and BMI resulted in significantly higher AMH-based detection rates for PCOS at a 10% false-positive rate, and patient-specific PCOS risks could be computed.

Measuring maternal serum screening markers for Down's syndrome in plasma collected for cell-free DNA testing.

Objectives To determine whether maternal plasma collected in cell-free DNA stabilizing tubes is suitable for measuring prenatal screening 'serum' markers. Methods Matched plasma and serum samples were collected from 41 second trimester and 42 first trimester non-Down's syndrome pregnancies. Second trimester samples were tested for alpha-fetoprotein, unconjugated estriol, human chorionic gonadotropin, and inhibin-A (Beckman Coulter DxI immunoassay). First trimester samples were tested for human chorionic gonadotropin and pregnancy-associated plasma protein A. Method comparisons performed for each marker compared plasma and serum results. Down's syndrome likelihood ratios in serum and plasma were compared. Results Plasma and serum results for all markers were highly correlated ( r > 0.983) but for all, plasma results differed, usually by proportional amounts. After conversion to multiples of the median using sample type-specific medians, the logarithmic standard deviations in serum and plasma did not differ (all p > 0.37). Likelihood ratios for the first and second trimester marker combinations were highly correlated and closely agreed (log likelihood ratios range 1.005 to 1.032; 1.000 indicates complete agreement). Conclusions These results using specialized plasma collection tubes are similar to those of our earlier study showing that plasma collected in EDTA tubes is suitable for 'serum' Down's syndrome screening. Laboratories must account for proportional changes by computing new plasma medians or modifying existing serum medians. Using a portion of the plasma from cell-free DNA collection tubes for 'serum screening' may have an advantage in programmes that are reflexively testing cell-free DNA, as only one sample type need be collected.

Correlation between follicular fluid levels of sRAGE and vitamin D in women with PCOS.

PURPOSE: The pro-inflammatory advanced glycation end products (AGEs) and their anti-inflammatory soluble receptors, sRAGE, play a role in the pathogenesis of PCOS. There is a correlation between vitamin D (vit D) and sRAGE in the serum, whereby vit D replacement increases serum sRAGE levels in women with PCOS, thus incurring a protective anti-inflammatory role. OBJECTIVE: This study aims to compare levels of sRAGE, N-carboxymethyl-lysine (CML; one of the AGEs), and 25-hydroxy-vit D in the follicular fluid (FF) of women with or without PCOS, and to evaluate the correlation between sRAGE and 25-hydroxy-vit D in the FF. MATERIAL AND METHODS: Women with (n = 12) or without (n = 13) PCOS who underwent IVF were prospectively enrolled. RESULTS: Women with PCOS had significantly higher anti-Mullerian hormone levels, higher number of total retrieved and mature oocytes, and higher number of day 3 and day 5 embryos formed. Compared to women without PCOS, women with PCOS had significantly lower FF sRAGE levels. In women with PCOS, in women without PCOS, and in all participants together, there was a significant positive correlation between sRAGE and 25-hydroxy-vit D. sRAGE positively correlated with CML in women without PCOS but not in women with PCOS. CONCLUSIONS: In women with PCOS, the low ovarian levels of the anti-inflammatory sRAGE suggest that sRAGE could represent a biomarker and a potential therapeutic target for ovarian dysfunction in PCOS. Whether there is a direct causal relationship between sRAGE and vit D in the ovaries remains to be determined.

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.

Antimüllerian Hormone Levels Are Not Altered by Glucose Challenge or a Meal.

BACKGROUND: Measurement of antimüllerian hormone (AMH) is used to assess ovarian reserve. Circulating levels of AMH correlate with antral follicle count, with relatively high levels indicating an ample reserve of primary and preantral follicles in the ovary. AMH levels are stable with dilution and freezer storage, and are not altered by hemolysis or menstrual cycle day in young women of reproductive age. We sought to examine whether glucose challenge or food intake modifies AMH levels compared with fasting. METHODS: Residual plasma samples were available from 54 pregnant women under fasting conditions and then 1, 2, and 3 h after ingestion of a 100-g glucose challenge. These samples were collected as part of routine clinical care to identify gestational diabetes (GDM) at 24-28 weeks of gestation. Twelve of these women met criteria for GDM based on an increased glucose level at a minimum of 2 time points. A second set consisted of serum samples collected from 8 nonpregnant women at fasting and 1 h after a meal. Levels of AMH were measured using an ultrasensitive assay (Ansh Labs, Webster, TX). A 2-way ANOVA (sample timing and GDM status) or matched t-test was performed. AMH measurements were subject to a logarithmic transformation before analysis. RESULTS: Median AMH levels in pregnant women at 1, 2, or 3 h after glucose challenge did not differ compared with AMH levels at fasting or by diagnosis of GDM. Similarly, there was no difference in median AMH levels in nonpregnant women of reproductive age at fasting and after a meal. CONCLUSION: AMH levels are not altered by glucose or food intake.

Where have all the trisomies gone?

Providing reliable prenatal screening performance estimates is critical for patient counseling and policy-making. Women who choose prenatal screening for aneuploidy are likely to be concerned not only with the common aneuploidies but with all causes of intellectual disability and serious birth defects. Sequential prenatal screening (combined serum and ultrasound testing) for aneuploidy detection commonly is offered as a primary screening test. Among women identified as screen positive, cell-free (cf)DNA has been added recently as a secondary, noninvasive screening option, before the consideration of invasive diagnostic testing (eg, amniocentesis and karyotype). With the anticipation of lower costs in the future, cfDNA might be an alternative to sequential screening in the general population. Sequential and cfDNA tests are both noninvasive, and both identify common aneuploidies. Screening via cfDNA detects more common chromosome abnormalities (eg, trisomy 21, sex trisomies). Sequential screening can identify other aneuploidies (eg, triploidy), as well as chromosome abnormalities associated with fetal structural abnormalities. When the advantages and disadvantages of routine sequential screening with routine cfDNA screening are compared, one important measure is the proportion and severity of chromosome abnormalities identified. When reporting these detection rates, authors need to carefully consider the impact of multiple well-described biases. For women to make informed choices in situations of this type, determining reliable comparative performance estimates is crucial.

Cut-off levels for hyperandrogenemia among Samoan women: An improved methodology for deriving normative data in an obese population.

OBJECTIVE: To define biochemical hyperandrogenemia (HA) among a population-based sample of reproductive-aged Samoan women, taking into consideration their high BMI levels. DESIGN AND METHODS: A secondary analysis was performed among a cross-sectional sample of Samoan women aged 25-39years (n=494) who were part of a larger genome-wide association study (GWAS) of adiposity. Women indicating pregnancy/lactation, hysterectomy, oophorectomy, cancer treatment, or use of contraceptive injections were excluded from the study. We analyzed the distribution of free androgen index (FAI) values to establish normative androgen data among Samoan women of reproductive age. Using the lowest tertile of body mass index (BMI), we defined HA as free androgen index (FAI) values >95(th) FAI percentile in that subsample. We compared the anthropometric and metabolic characteristics of women with HA to women with normal androgen levels. RESULTS: HA was defined as FAI>8.5. Using this definition, 14% of women were classified as hyperandrogenemic. Women with HA had significantly higher average BMI values, abdominal circumferences, fasting triglycerides, and insulin levels as well as significantly lower adiponectin levels. CONCLUSION: This study is the first to define normative androgen values among Samoan women with a quantitative assessment of the relationship between adiposity and androgen levels. The uniquely high BMI levels in the population not only provide important clinical insight into normative androgen values among Samoan women, but they also serve as references for the clinical assessment of HA, taking into consideration BMI, in other populations.

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.

OBJECTIVES: 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. METHODS: Samples from 40 Down syndrome cases were matched with five control samples and tested for PAPP-A, free ß, AFP, inhibin-A and PlGF. Results were converted to weight-adjusted multiples of the median (MoM) and population parameters computed. Monte Carlo simulation modeled Down syndrome detection and false positive rates for various marker combinations. After reflexive DNA testing, the revised detection and false positive rates were also computed. RESULTS: At a primary false positive rate of 20%, the baseline combination (maternal age, PAPP-A and free ß) detected 86.9%. Adding AFP or PlGF increased detection to 89.8% and 89.5%, respectively. Adding AFP and PlGF, AFP and inhibin-A, or all three markers, detected 93.7%, 94.1% and 95.5%, respectively. Modeling reflexive cf DNA testing results in little loss in detection (1%), but false positive rates fall to 0.2%. CONCLUSION: First trimester reflexive testing does not require nuchal translucency measurements, and has high detection and very low rates of invasive procedures. However, timing of DNA sample collection and the costs of sample collection and DNA testing need to be considered before implementation.

Evaluation of patient education materials: the example of circulating cell free DNA testing for aneuploidy.

Informed consent is the process by which the treating health care provider discloses appropriate information to a competent patient so that the patient may make a voluntary choice to accept or refuse treatment. When the analysis of circulating cell free DNA (ccfDNA) became commercially available in 2011 through the Prenatal Diagnostic Laboratory at Women & Infants Hospital of Providence, Rhode Island to "high-risk" women, it provided an opportunity to examine how commercial laboratories informed potential consumers. We identified, via an internet search, four laboratories offering such testing in the United States and one in Europe. We evaluated patient educational materials (PEMs) from each using the Flesch Reading Ease method and a modified version of the Suitability Assessment of Materials (SAM) criteria. Pamphlets were also reviewed for their inclusion of content recommendations from the International Society for Prenatal Diagnosis, the National Society of Genetic Counselors, the American College of Obstetricians and Gynecologists jointly with the Society of Maternal Fetal Medicine, and the American College of Genetics and Genomics. Reading levels were typically high (10th-12th grade). None of the pamphlets met all SAM criteria evaluated nor did any pamphlet include all recommended content items. To comply with readability and content recommendations more closely, Women & Infants Hospital created a new pamphlet to which it applied the same criteria, and also subjected it to focus group assessment. These types of analyses can serve as a model for future evaluations of similar patient educational materials.

Maternal plasma DNA testing: experience of women counseled at a prenatal diagnosis center.

AIMS: To evaluate the early introduction of circulating cell-free (ccf) DNA testing in a prenatal diagnosis center serving a statewide population. RESULTS: A retrospective chart review of patients at high aneuploidy risk counseled during the two 10-week periods that documents indication, risk, maternal age, insurance coverage, decisions, and reasoning behind that decision. Among the 299 included women, indication was advanced maternal age (17% with and 56% without an additional indication), positive serum screen (15%), and abnormal ultrasound (12%). Uptake increased from 10% to 17%, as did patient awareness of the test (4% to 14%). Women with lower copayments were more likely to complete testing (23% vs. 5%, p<0.001). Most women completing testing (75%) wanted to avoid an invasive procedure, while those declining cited testing would not change anything (47%), preferred diagnostic testing (16%), negative follow-up testing (20%), and cost/insurance issues (9%). One of 42 tests was positive (trisomy 21). CONCLUSIONS: Individual patient follow-up allows us to document ccfDNA-related patient decision-making. Nearly half of the women did not want further testing and one in seven preferred immediate diagnostic testing. Patient costs were a barrier to testing that, if avoided, could increase test uptake by 50% or more.

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.

OBJECTIVE: 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. METHOD: Among a cohort of 4664 high-risk pregnancies, maternal plasma samples were tested from 25 twin pregnancies (17 euploid, five discordant and two concordant for Down syndrome; one discordant for trisomy 13) and two euploid triplet pregnancies [Correction made here after initial online publication.]. Results were corrected for GC content bias. For each target chromosome (21, 18, and 13), z-scores of 3 or higher were considered consistent with trisomy. RESULTS: Seven twin pregnancies with Down syndrome, one with trisomy 13, and all 17 twin euploid pregnancies were correctly classified [detection rate 100%, 95% confidence interval (CI) 59%-100%, false positive rate 0%, 95% CI 0%-19.5%], as were the two triplet euploid pregnancies. CONCLUSION: Although study size is limited, the underlying biology combined with the present data provide evidence that MPSS testing can be reliably used as a secondary screening test for Down syndrome in women with high-risk twin gestations.

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.