Application of risk score analysis to low-coverage whole genome sequencing data for the noninvasive detection of trisomy 21, trisomy 18, and trisomy 13.

OBJECTIVES: Clinical performance of a low coverage, low cost, massively parallel sequencing (MPS)-based assay to stratify risk of trisomy 21, 18, and 13 pregnancies was determined. METHODS: The study included 1100 samples with birth outcome or karyotype results, comprising low-risk patients (84.2%) negative for risk indications from maternal age, serum screening, ultrasound, or family history, and high-risk patients (15.8%) with at least one of the aforementioned indications. Cell free DNA (cfDNA) was extracted from maternal plasma. Library preparation incorporated 96 index barcodes to enable sequencing on a HiSeq 2000 or 2500. Risk scores were calculated using chromosomal representation, fetal fraction, and maternal age at the estimated date of delivery. A risk score greater than or equal to 1 in 100 was used to stratify samples as high risk for trisomy 21, trisomy 18, or trisomy 13. RESULTS: Sensitivity and specificity were calculated based on risk group stratification. Trisomy 21, trisomy 18, and trisomy 13 were detected with greater than 99% sensitivity and 99.9% specificity. Fetal sex classification accuracy was 99.3%. CONCLUSIONS: We conclude that simplified MPS can be used to stratify the risk of pregnancies for trisomy 21, trisomy 18, and trisomy 13 and accurately determine fetal sex. © 2015 John Wiley & Sons, Ltd.

Circulating cell free DNA testing: are some test failures informative?

OBJECTIVE: The proportion of circulating cell free DNA derived from the feto-placental unit (fetal fraction or FF) correlates with test success and interpretation reliability. Some fetal disorders are associated with systematically lower FF, sometimes resulting in noninformative results. METHODS: We analyzed results from pregnancies tested in a nested case/control study derived from a cohort of 4664 high-risk pregnancies. Low FF was defined before and after adjusting for maternal weight and gestational age. RESULTS: Compared with euploid pregnancies, the median FF was significantly higher in Down syndrome pregnancies (ratio 1.17) and significantly lower in trisomy 18 and triploid pregnancies (ratios 0.71 and 0.19, respectively). Among 2157 pregnancies tested, 13 (0.6%) had FF <3.0% (all noninformative), including three trisomy 18 and three triploidy fetuses. After adjustment, 16 pregnancies (0.7%) had FF <0.3 multiples of the median (six informative), including one trisomy 18 and three triploidy fetuses. Modeled positive predictive values for low and high-risk populations were 7% and 30%, respectively. CONCLUSION: Among women with noninformative results attributable to low FF, trisomy 18 and/or triploidy risk are sufficiently high to warrant offering additional assessments (e.g. ultrasound). If the testing indication is ultrasound abnormality, amniocentesis and karyotype/microarray should be considered. © 2014 John Wiley & Sons, Ltd.

Screening for the aneuploid fetus.

Advances in ultrasound technology have dramatically improved the detection of fetal defects. Although only an invasive test can provide a diagnosis, the incorporation of sonography into current biochemically based screening programs should significantly improve the detection of a host of other physically based fetal abnormalities. This article provides an overview and discussion of the prenatal sonographic features that may suggest the presence of a significant chromosomal abnormality.

Noninvasive first-trimester screening for fetal aneuploidy.

We reviewed all studies concerning noninvasive first trimester screening for fetal aneuploidy obtained from a MEDLINE search through June 1996 with additional sources identified through cross-referencing. Three screening and diagnostic modalities are of potential application in noninvasive first trimester testing for fetal aneuploidy: ultrasound, maternal biochemical markers, and analysis of fetal cells retrieved from maternal sources. Sensitivities of the sonographic finding of nuchal translucency thickness in combination with maternal age for trisomy 21, performed between 10 and 14 weeks of gestation in experienced hands, and maternal biochemical markers independently may be as high as 86 percent and 60 percent, respectively. Sensitivity, specificity, and predictive values of these diagnostic modalities alone, in combination with each other, or in conjunction with other predisposing factors such as maternal age, in large low risk populations have not currently been established. Analysis of fetal cells retrieved from maternal sources, although more complex, may offer definitive noninvasive prenatal diagnosis yet is not currently available in clinical practice. We conclude that noninvasive first trimester screening for fetal aneuploidy modalities including sonographic examination for nuchal translucency thickness and maternal biochemical markers, is feasible. Clinical feasibility; and all-encompassing clinical management paradigms of these and other early noninvasive first trimester screening methods for fetal aneuploidy, are not yet available.

Levels of urinary beta-core fragment, total oestriol, and the ratio of the two in second-trimester screening for Down syndrome.

Levels of beta-core fragment and total oestriol in second-trimester maternal urine samples were measured in 32 Down syndrome pregnancies and 206 control pregnancies. Beta-core fragment and total oestriol values were corrected for the urinary creatinine level and expressed as multiples of the control medians (MOM). In addition, the ratio of the beta-core fragment level to the total oestriol level, without creatinine correction, was calculated, and expressed as MOM values. The median beta-core fragment, total oestriol, and ratio levels in Down syndrome cases were 5.42, 0.64, and 9.32 MOM, respectively. In the Down syndrome pregnancies, 66 per cent of the beta-core fragment levels were above the 95th centile of control levels, while 22 per cent of the total oestriol levels were below the fifth centile of control levels. In combination with maternal age, measurement of beta-core fragment and total oestriol levels in Down syndrome pregnancy resulted in an 80 per cent detection rate at a 5 per cent false-positive rate. Use of the ratio resulted in a univariate detection rate of 72 per cent. In combination with maternal age, the ratio resulted in a detection rate of 81 per cent at a 5 per cent false-positive rate. Based on this unmatched study, the measurement of a ratio of beta-core fragment to total oestriol levels, without the need for creatinine correction, may be useful in screening for fetal Down syndrome in second-trimester urine.

Prenatal diagnosis of type 2 Pfeiffer syndrome.

Pfeiffer syndrome is an autosomal dominantly inherited disorder consisting of craniosynostosis, a flattened midface with a beaked nose and ocular proptosis, and broad and medially deviated thumbs and great toes. Recently, based on clinical findings, the disorder has been divided into three subtypes: type 1, characterized by mild expression; type 2, in which clover leaf skull deformity and multiple congenital anomalies are present at birth; and type 3, which is similar to type 2, but lacks the presence of the clover leaf skull at birth. We describe a fetus in whom sonographic findings of clover leaf skull deformity, ocular hypertelorism, and varus deformity of the great toe led to the prenatal diagnosis of Pfeiffer syndrome type 2. We believe this is the second prenatal diagnosis of Pfeiffer syndrome, and the first time type 2 has been definitely identified in the second trimester of pregnancy.

The genetics of ovarian cancer: an assessment of current screening protocols and recommendations for counseling families at risk.

Although the need for effective ovarian cancer screening is apparent, a highly sensitive and specific screening methodology has yet to be elucidated. 42-44 Given that there are more than 43 million women in the United States older than 45 years of age and that the average cost of a pelvic sonogram is $ 275 (and $ 45 for CA125 screening), the screening of this population is estimated to increase health care costs by $ 14 billion per year. 45 The additional cost of BRCA1 screening varies according to the level of diagnostic effort required to establish BRCA1 gene mutations in a particular family and ranges from $ 295 to $ 1,200 per sample. Assuming an average cost of $ 600 per sample, initial screening of these same women would likely increased costs in excess of $ 25 billion. Current knowledge and technology in ovarian cancer screening has not yet proved beneficial for the general population or for women with fewer than two affected family members. For women with two or more affected family members, there is a 3% chance of that patient being a proband in a hereditary cancer syndrome family. 11,46 In this group, who may be at increased risk for developing a malignancy, heightened surveillance is warranted, although there are still no data to confirm that screening even these high-risk women will reduce mortality. Nevertheless, annual bimanual examination, serum CA125, and transvaginal sonography are recommended among this particular subgroup of women at risk, and are likely to be recommended for young, asymptomatic, at-risk women who screen positive for the 185delAG BRCA1 deletion commonly found in persons of Ashkenazi Jewish ancestry. It is only through prospective, randomized trials that reliable data regarding the risk/benefit ratio of ovarian cancer screening among various populations at risk will be determined. The results of the prospective/randomized PLCO trial and the mature data from ongoing prospective, nonrandomized screening trials for women with a family history of cancer may provide this information and are eagerly awaited.

Placental echolucencies: does their presence influence outcome following genetic amniocentesis?

Women undergoing genetic amniocentesis procedures were evaluated for the presence of ultrasonographic echolucencies within the placenta. Non-calcified sonolucencies < 4 to 5 cm were classified as subchorionic hematomas (SH). Of the 1,000 pregnancies evaluated, 153 (15%) pregnancies manifested SH prior to amniocentesis (study group). The indications for referral were similar in the study and control groups. There were 13 (1.3%) losses: 3 and 10 in the study and control groups, respectively, with no statistical difference in fetal losses between the groups: RR 1.52 (95% confidence interval 0.56-4.14; p = 0.32). Placental sonolucencies < 4 to 5 cm in diameter appear to be incidental ultrasound findings not associated with increased fetal loss following genetic amniocentesis. Complications following invasive prenatal diagnosis cannot be attributed to the presence of these common ultrasound findings.

Management of immune thrombocytopenia purpura at a military medical center.

OBJECTIVE: To investigate whether fetal platelets in immune thrombocytopenia purpura (ITP) may be predicted by antepartum assessment. METHODS: A prospective analysis was conducted of 28 pregnant women with ITP out of 8,056 deliveries. Of the 28 patients, 13 were evaluted by fetal scalp sampling and 11 were evaluated by percutaneous umbilical blood sampling (PUBS). RESULTS: The mean fetal and maternal platelet counts prior to delivery were 146,000 and 176,000, respectively. The mean fetal and maternal platelet counts after delivery were 245,000 and 149,000, respectively. Fetal cord platelet counts could not be predicted by maternal platelet count, the presence/absence of maternal direct/indirect anti-platelet antibodies, steroid therapy, or history of splenectomy. PUBS for fetal platelet assessment correlated well with fetal postdelivery counts. CONCLUSIONS: Patients with ITP rarely exhibit complications. No antepartum characteristic enhances reliable prediction of neonatal outcome. Method of delivery should be based on obstetric guidelines.

Current maternal age recommendations for prenatal diagnosis: a reappraisal using the expected utility theory.

The expected utility theory suggests eliminating an age-specific criterion for recommending prenatal diagnosis to patients. We isolate the factors which patients and physicians need to consider intelligently in prenatal diagnosis, and show that the sole use of a threshold age as a screening device is inadequate. Such a threshold fails to consider adequately patients' attitudes regarding many of the possible outcomes of prenatal diagnosis; in particular, the birth of a chromosomally abnormal child and procedural-related miscarriages. It also precludes testing younger women and encourages testing in patients who do not necessarily require or desire it. All pregnant women should be informed about their prenatal diagnosis options, screening techniques, and diagnostic procedures, including their respective limitations, risks, and benefits.

Procedure-related fetal losses in transplacental versus nontransplacental genetic amniocentesis.

OBJECTIVE: We hypothesize that loss rates after amniocentesis do not differ in transplacental and nontransplacental taps performed by experienced operators. STUDY DESIGN: Subjects were 1000 women undergoing second-trimester amniocentesis: 745 were referred for maternal age; 132 for positive maternal serum alpha-fetoprotein screens, 41 indicating a risk for fetal neural tube defect, 91 indicating a risk for fetal chromosome abnormality; and 123 were referred for other reasons. All procedures were videotaped. The placenta was anterior in 518 cases; in 306 of these the needle went through the placenta. All pregnancies were prospectively evaluated through delivery. RESULTS: There were 13 losses among the 1000 procedures (1.3%). The transplacental losses occurred from 4 to 71 days after procedure, median 26.5 days; the nontransplacental losses from 12 days after procedure to term, median 25 days. The loss rate was essentially similar in the two categories: six transplacental (1.96%) and seven nontransplacental (1%) (relative risk 1.52 [95% confidence limits 0.84 to 2.75], p = 0.23). If the three patients with elevated maternal serum alpha-fetoprotein values were excluded from data analysis, the loss rates in the two groups were virtually identical (relative risk 0.98 [95% confidence limits 0.38 to 2.54], p = 1.0000). CONCLUSION: Transplacental amniocentesis does not appear to increase the fetal loss rate in the hands of experienced surgeons. Moreover, in view of the time span between amniocentesis and loss in both groups, a procedural cause seems questionable.

Molecular analysis of genetic diseases: an overview for clinicians.

The identification of fetal genetic disease has, for the most part, relied on examination of an end product, such as analysis of factor VIII levels obtained from cord blood in fetuses at risk for hemophilia. Advances in molecular genetics have shifted our focus in prenatal diagnosis away from protein product analysis toward etiology, making new discoveries gleaned from the Human Genome Project relevant to clinicians. This review discusses the basic principles involved in gene-based diagnosis, highlighting the complexities of current approaches to molecular diagnosis of fetal genetic disease. Given an understanding of both the theory and practice of genetic analysis, the review covers the fundamental principles of molecular biology (structure, function, packaging, and regulation) and discusses recombinant DNA techniques presently used for the analysis of mutations. Clinical examples are presented to introduce the techniques most commonly employed in service laboratories: direct detection assays, where the specific mutation is recognized, and indirect detection assays, useful for the deduction of an inheritance pattern where the actual mutation or its gene is not known but may be closely linked to known DNA polymorphisms.

Early genetic amniocentesis and its relationship to respiratory difficulties in paediatric patients: a report of findings in patients and matched controls 3-5 years post-procedure.

This study evaluates the long-term pulmonary complications of 25 children from a prospective, matched-control, pilot study evaluating short-term complications of early (11-14 weeks' gestation) versus traditional (15 weeks' gestation and later) genetic amniocentesis. Five children in the early amniocentesis group were found to have various respiratory difficulties, a morbidity rate comparable to that of paediatric patients in the general population. These data identify the need for larger, multicentre trials.

Reproductive genetics for couples older than 40 years of age.

The counseling process, which involves genetic screening and prenatal diagnosis, is presented. Also addressed are the recent advances in assisted reproductive technologies, which may prove to be particularly useful for these patients.

Unexplained positive/elevated maternal serum alpha-fetoprotein associated with placenta increta. A case report.

Maternal serum alpha-fetoprotein (MSAFP) is a regularly utilized antenatal screening test for the identification of pregnancies at increased risk for a variety of genetic and nongenetic abnormalities. Complete mid-trimester evaluation of the patient with a positive screening test may fail to reveal an etiology for a positive MSAFP value. This case report concerns an unexplained positive/elevated MSAFP screening test for a patient found at delivery to have abnormal placentation.

Molecular analysis and chorionic villus sampling (CVS): opportunities for rapid prenatal diagnosis in the military.

Prior to recent advances in the field of molecular biology, prenatal diagnosis of hemoglobinopathies was accomplished by direct analysis of the gene product(s) in samples obtained by fetoscopy or, more recently, percutaneous umbilical blood sampling. The use of the polymerase chain reaction enables quicker diagnosis than with indirect or cumbersome Southern blot techniques. This case, reporting a couple at risk for fetal sickle cell disease, is a model for rapid prenatal diagnosis and demonstrates the capabilities which exist in the military health care referral system. Within 24 hours of a couple's presentation for consultation and accomplishment of chorionic villus sampling (CVS), preliminary results of both cytogenetic and sickle cell status of an at-risk fetus were determined. Final analysis was accomplished by 48 hours. The combination of early referral, early sampling by CVS, and application of advances in DNA laboratory technologies offers tremendous improvements for care heretofore unavailable as a complete "package" in the military. The implications and limitations for prenatal testing for single gene disorders within the military medical health care system will be discussed.

The modulation of prostaglandin synthesis by peritoneal fluids.

Biological fluids from several sources (e.g. blood, fetal urine, amniotic fluid) have been shown to contain factors that modulate prostaglandin (PG) synthesis. In this study, we investigated the possibility that peritoneal fluid contains substances that may regulate PG synthesis. Peritoneal fluids were obtained from women undergoing diagnostic laparoscopy for infertility. Fluids from women without evident pelvic pathology were incubated with prostaglandin synthase prepared from bull seminal vesicles in the presence of excess arachidonic acid, and the production of PGE2, PGF2 alpha, and 6-keto-PGF1 alpha was quantified by specific radioimmunoassay. The untreated fluids inhibited potently the synthesis of PGE2 but such inhibitory activity was not extractable by chloroform:methanol. An ultrafiltrate of the fluid containing molecules smaller than 10,000 Daltons stimulated PGF2 alpha synthesis but this activity was also lost after extraction. The extracted fluid did, however, stimulate the synthesis of prostacyclin (as reflected by 6-keto-PGF1 alpha).