Comparative evaluation of the Minimally-Invasive Karyotyping (MINK) algorithm for non-invasive prenatal testing.

Minimally Invasive Karyotyping (MINK) was communicated in 2009 as a novel method for the non-invasive detection of fetal copy number anomalies in maternal plasma DNA. The original manuscript illustrated the potential of MINK using a model system in which fragmented genomic DNA obtained from a trisomy 21 male individual was mixed with that of his karyotypically normal mother at dilutions representing fetal fractions found in maternal plasma. Although it has been previously shown that MINK is able to non-invasively detect fetal microdeletions, its utility for aneuploidy detection in maternal plasma has not previously been demonstrated. The current study illustrates the ability of MINK to detect common aneuploidy in early gestation, compares its performance to other published third party methods (and related software packages) for prenatal aneuploidy detection and evaluates the performance of these methods across a range of sequencing read inputs. Plasma samples were obtained from 416 pregnant women between gestational weeks 8.1 and 34.4. Shotgun DNA sequencing was performed and data analyzed using MINK RAPIDR and WISECONDOR. MINK performed with greater accuracy than RAPIDR and WISECONDOR, correctly identifying 60 out of 61 true trisomy cases, and reporting only one false positive in 355 normal pregnancies. Significantly, MINK achieved accurate detection of trisomy 21 using just 2 million aligned input reads, whereas WISECONDOR required 6 million reads and RAPIDR did not achieve complete accuracy at any read input tested. In conclusion, we demonstrate that MINK provides an analysis pipeline for the detection of fetal aneuploidy in samples of maternal plasma DNA.

Accuracy of prenatal diagnosis of isolated aqueductal stenosis.

OBJECTIVE: The objective of this article is to determine the success rate of prenatally diagnosed isolated aqueductal stenosis (AS) as a first step in an evidence-based reassessment of ventriculoamniotic shunting for isolated AS. METHODS: Cases of ventriculomegaly at Magee-Womens Hospital between 2006 and 2013 were ascertained. AS was suspected when prenatal ultrasound and magnetic resonance imaging (MRI) demonstrated signs of pressure hydrocephalus. The prenatal diagnosis generated by ultrasound and MRI was compared with the postnatal diagnosis based upon neonatal neuroimaging. RESULTS: The initial query of the Magee-Womens Hospital database resulted in 370 cases of ventriculomegaly. After exclusion for associated central nervous system malformations, 110 cases of severe ventriculomegaly remained. The imaging studies on each fetus were reviewed, and cases of prenatally diagnosed AS were identified. The median gestational age of the ultrasound diagnosis and prenatal MRI was 23 weeks 3 days and 33 weeks 3 days, respectively. All cases of prenatally suspected isolated AS (six of six) and cases of AS with other associated central nervous system anomalies (six of six) were correctly identified. CONCLUSION: An accurate prenatal diagnosis of isolated fetal AS is possible. This is an important first step in an evidence-based reassessment of ventriculoamniotic shunting for isolated AS. © 2014 John Wiley & Sons, Ltd.

High resolution non-invasive detection of a fetal microdeletion using the GCREM algorithm.

BACKGROUND/OBJECTIVE: The non-invasive prenatal detection of fetal microdeletions becomes increasingly challenging as the size of the mutation decreases, with current practical lower limits in the range of a few megabases. Our goals were to explore the lower limits of microdeletion size detection via non-invasive prenatal tests using Minimally Invasive Karyotyping (MINK) and introduce/evaluate a novel statistical approach we recently developed called the GC Content Random Effect Model (GCREM). METHODS: Maternal plasma was obtained from a pregnancy affected by a 4.2-Mb fetal microdeletion and three normal controls. Plasma DNA was subjected to capture an 8-Mb sequence spanning the breakpoint region and sequence. Data were analyzed with our published method, MINK, and a new method called GCREM. RESULTS: The 8-Mb capture segment was divided into either 38 or 76 non-overlapping regions of 200 and 100 Kb, respectively. At 200 Kb resolution, using GCREM (but not MINK), we obtained significant adjusted p-values for all 20 regions overlapping the deleted sequence, and non-significant p-values for all 18 reference regions. At 100 Kb resolution, GCREM identified significant adjusted p-values for all but one 100-Kb region located inside the deleted region. CONCLUSION: Targeted sequencing and GCREM analysis may enable cost effective detection of fetal microdeletions and microduplications at high resolution.

Mannose binding lectin genotypes are not associated with increased risk of unexplained recurrent pregnancy loss.

PURPOSE: Immune response to infections has been associated with recurrent pregnancy loss (RPL). Low plasma mannose binding lectin (MBL) levels, an innate immunity factor in infections, has been related to RPL. In this study, we tested the hypothesis that MBL genotypes that are known to cause reduced plasma MBL levels are significantly more frequent among women experiencing unexplained RPL. METHODS: This study included 219 Caucasian women diagnosed with unexplained RPL and 236 control women. All participants were genotyped for two promoter (-550 C > G and -221 G > C) and three missense (R52C, G54D and G57E) mutations in exon 1. These mutations are known to be associated with variations in plasma MBL levels. Genotype frequencies were estimated by gene counting and were compared to the expectation of Hardy-Weinberg equilibrium by chi-squared (X(2)) analysis and Fisher's exact test. Allele and genotype frequencies were compared in cases and controls using X(2) contingency table analysis. RESULTS: There was no difference in demographics between cases and controls. The number of miscarriages in the participants with RPL ranged from 2 to 10 spontaneous abortions (SAB's) per participant. Populations genotyped were in Hardy-Weinberg equilibrium. There was no association between a history of RPL and multi-SNP genotypes at the MBL locus. In unexplained RPL, the number of SAB's and live birth rates were unaffected by MBL genotype. There was no association between MBL genotype and the risk of unexplained RPL. The occurrence of live birth was not associated with MBL genotype. CONCLUSION: Genotypes known to cause low MBL plasma levels are not associated with an increased risk of unexplained RPL.

Prenatal detection of del(10)(q11.2) mosaicism in chorionic villus specimens likely caused by a common chromosomal fragile site FRA10G is associated with a normal phenotype.

OBJECTIVE: To summarize the pregnancy outcomes of cases with mosaicism for chromosome 10q11.2 deletion detected by chorionic villus sampling (CVS) and determine whether extensive cytogenetic work-up and follow-up amniocentesis are necessary in such cases. METHODS: CVS was performed at 10-12 weeks of gestation. Chromosome analysis of chorionic villi was performed by standard G-banding techniques. RESULTS: Mosaicism of chromosome 10q11.2 deletion was observed in 24 out of 6063 CVS cases (0.39%). A common fragile site, FRA10G is located at the breakpoint region. The level of mosaicism ranged from 4% to 25%. No evidence of mosaic 10q11.2 deletion was found in follow-up amniocentesis, maternal peripheral blood cells, or from cytogenetic studies of other pregnancies from the same group of patients. All these cases resulted in the live birth of normal healthy infants. CONCLUSION: The presence of del(10)(q11.2) mosaicism in chorionic villus specimens most likely represents an in vitro culture artifact due to FRA10G fragile site in this region without any clinical consequences. If ultrasound results are normal, it is not necessary to perform follow-up amniocenteses and additional laboratory work-up for such cases.

Structural and regulatory characterization of the placental epigenome at its maternal interface.

Epigenetics can be loosely defined as the study of cellular "traits" that influence biological phenotype in a fashion that is not dependent on the underlying primary DNA sequence. One setting in which epigenetics is likely to have a profound influence on biological phenotype is during intrauterine development. In this context there is a defined and critical window during which balanced homeostasis is essential for normal fetal growth and development. We have carried out a detailed structural and functional analysis of the placental epigenome at its maternal interface. Specifically, we performed genome wide analysis of DNA methylation in samples of chorionic villus (CVS) and maternal blood cells (MBC) using both commercially available and custom designed microarrays. We then compared these data with genome wide transcription data for the same tissues. In addition to the discovery that CVS genomes are significantly more hypomethylated than their MBC counterparts, we identified numerous tissue-specific differentially methylated regions (T-DMRs). We further discovered that these T-DMRs are clustered spatially along the genome and are enriched for genes with tissue-specific biological functions. We identified unique patterns of DNA methylation associated with distinct genomic structures such as gene bodies, promoters and CpG islands and identified both direct and inverse relationships between DNA methylation levels and gene expression levels in gene bodies and promoters respectively. Furthermore, we found that these relationships were significantly associated with CpG content. We conclude that the early gestational placental DNA methylome is highly organized and is significantly and globally associated with transcription. These data provide a unique insight into the structural and regulatory characteristics of the placental epigenome at its maternal interface and will drive future analyses of the role of placental dysfunction in gestational disease.

Gene expression in first trimester preeclampsia placenta.

BACKGROUND: The goal of this study was to further validate eight candidate genes identified in a microarray analysis of first trimester placentas in preeclampsia. MATERIAL AND METHOD: Surplus chorionic villus sampling (CVS) specimens of 4 women subsequently diagnosed with preeclampsia (PE) and 8 control women (C) without preeclampsia analyzed previously by microarray and 24 independent additional control samples (AS) were submitted for confirmatory studies by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: Downregulation was significant in FSTL3 in PE as compared to C and AS (p = .04). PAEP was downregulated, but the difference was only significant between C and AS (p = .002) rather than between PE and either of the control groups. Expression levels for CFH, EPAS1, IGFBP1, MMP12, and SEMA3C were not statistically different among groups, but trends were consistent with microarray results; there was no anti-correlation. S100A8 was not measurable in all samples, probably because different probes and primers were needed. CONCLUSIONS: This study corroborates reduced FSTL3 expression in the first trimester of preeclampsia. Nonsignificant trends in the other genes may require follow-up in studies powered for medium or medium/large effect sizes. qRT-PCR verification of the prior microarray of CVS may support the placental origins of preeclampsia hypothesis. Replication is needed for the candidate genes as potential biomarkers of susceptibility, early detection, and/or individualized care of maternal-infant preeclampsia.

Statistical considerations for digital approaches to non-invasive fetal genotyping.

MOTIVATION: A growing body of literature has demonstrated the potential for non-invasive diagnosis of a variety of human genetic diseases using cell-free DNA extracted from maternal plasma samples in early gestation. Such methods are of great significance to the obstetrics community because of their potential use as clinical standard of care. Proof of concept for such approaches has been established for aneuploidy and paternally inherited dominant traits. Although significant progress has recently been made, the non-invasive diagnosis of monogenic diseases that segregate in a recessive mendelian fashion is more problematic. Recent developments in microfluidic digital PCR and DNA sequencing have resulted in a number of recent advances in this field. These have largely, although not exclusively, been used for the development of diagnostic methods for aneuploidy. However, given their prevalence, it is likely that such methods will be utilized towards the development of non-invasive methods for diagnosing monogenetic disorders. RESULTS: With this in mind, we have undertaken a statistical modeling of three contemporary (digital) analytical methods in the context of prenatal diagnosis using cell free DNA for monogenic diseases that segregate in a recessive mendelian fashion. We provide an experimental framework for the future development of diagnostic methods in this context that should be considered when designing molecular assays that seek to establish proof of concept in this field.

Comprehensive analysis of HLA-G: implications for recurrent spontaneous abortion.

Miscarriage is one of the most common pregnancy complications. Recurrent spontaneous abortion is defined as 2 or more pregnancy losses and may be associated with genetic variation. Human leukocyte antigen-G (HLA-G) is a ligand for natural killer (NK) cell receptors and has the ability to block NK cell activity, which if not blocked can potentially harm a fetus. Consequently a deletion or mutation of the HLA-G gene could lead to miscarriage. Our cases (n = 238) include Caucasian women experiencing 2 or more spontaneous abortions, and controls (n = 233) include women with at least 1 live birth and no history of SA. We sequenced approximately 1400 base pairs (bp) of the HLA-G promoter region, genotyped the 14 bp exon 8 insertion/deletion and single nucleotide polymorphism (SNP) in the coding region of HLA-G. Promoter haplotypes were constructed from sequence information. Twenty-three SNPs were observed in the promoter region with minor allele frequency >0.02. Twelve SNPs differed significantly in frequency between cases and controls. Two haplotypes incorporating these 12 SNPs accounted for 90% of haplotypes and differed significantly in frequency between the 2 populations. Cases were more likely to carry haplotype 2 (P = .0078) and controls to have haplotype 6 (P = .0004). Cases also had a higher frequency of individuals homozygous for the 14 bp insertion. Among the 12 alleles carried on haplotype 2, 5 are predicted to disrupt transcription factor binding sites. The HLA-G promoter is highly associated with the risk of spontaneous abortion, but high linkage disequilibrium in the promoter prevents assignment of the causal variant.

Statistical model for whole genome sequencing and its application to minimally invasive diagnosis of fetal genetic disease.

There is currently great interest in the development of methods for the minimally invasive diagnosis of fetal genetic disease using cell-free DNA from maternal plasma samples obtained in the first trimester of pregnancy. With the rapid development of high-throughput sequencing technology, the possibility of detecting the presence of trisomy fetal genomes in the maternal plasma DNA sample has recently been explored. The major concern of this whole genome sequencing approach is that, while detecting the karyotype of the fetal genome from the maternal plasma requires extremely high accuracy of copy number estimation, the majority of the available high-throughput sequencing technologies require polymerase chain reaction (PCR) and are subject to the substantial bias that is inherent to the PCR process. We introduce a novel and sophisticated statistical model for the whole genome sequencing data, and based on this model, develop a highly sensitive method of Minimally Invasive Karyotyping (MINK) for the diagnosis of the fetal genetic disease. Specifically we demonstrate, by applying our statistical method to ultra high-throughput whole sequencing data, that trisomy 21 can be detected in a minor ('fetal') genome when it is mixed into a major ('maternal') background genome at frequencies as low as 5%. This observation provides additional proof of concept and justification for the further development of this method towards its eventual clinical application. Here, we describe the statistical and experimental methods that illustrate this approach and discuss future directions for technical development and potential clinical applications.

Altered global gene expression in first trimester placentas of women destined to develop preeclampsia.

BACKGROUND: Preeclampsia is a pregnancy-specific disorder that remains a leading cause of maternal, fetal and neonatal morbidity and mortality, and is associated with risk for future cardiovascular disease. There are no reliable predictors, specific preventative measures or treatments other than delivery. A widely held view is that the antecedents of preeclampsia lie with impaired placentation in early pregnancy. Accordingly, we hypothesized dysregulation of global gene expression in first trimester placentas of women who later manifested preeclampsia. METHODS: Surplus chorionic villus sampling (CVS) tissues were collected at 10-12 weeks gestation in 160 patients with singleton fetuses. Four patients developed preeclampsia, and their banked CVS specimens were matched to 8 control samples from patients with unaffected pregnancies. Affymetrix HG-U133 Plus 2.0 GeneChips were utilized for microarray analysis. Naïve Bayes prediction modeling and pathway analysis were conducted. qRT-PCR examined three of the dysregulated genes. RESULTS: Thirty-six differentially expressed genes were identified in the preeclampsia placentas. qRT-PCR verified the microarray analysis. Thirty-one genes were down-regulated. Many were related to inflammation/immunoregulation and cell motility. Decidual gene dysregulation was prominent. No evidence was found for alterations in hypoxia and oxidative stress regulated genes. CONCLUSIONS: To our knowledge, this is the first study to show dysregulation of gene expression in the early placentas of women approximately 6 months before developing preeclampsia, thereby reinforcing a placental origin of the disorder. We hypothesize that placentation in preeclampsia is compromised in the first trimester by maternal and fetal immune dysregulation, abnormal decidualization, or both, thereby impairing trophoblast invasion. Several of the genes provide potential targets for the development of clinical biomarkers in maternal blood during the first trimester. Supplementary materials are available for this article via the publisher's online edition.

Recurrent spontaneous abortion and skewed X-inactivation: is there an association?

OBJECTIVE: The purpose of this study was to determine whether there is an association between skewed X-inactivation and recurrent spontaneous abortion in a large, well-defined sample of women with recurrent loss. STUDY DESIGN: X-chromosome inactivation patterns were compared in 5 groups of women. Group 1 (recurrent spontaneous abortion) consisted of 357 women with 2 or more spontaneous losses. In group 2 (infertility), there were 349 subjects from infertility practices recruited at the time of a positive serum beta-human chorionic gonadotropin. Group 3 (spontaneous abortion) women (n = 81) were recruited at the time of an ultrasound diagnosis of an embryonic demise or an anembryonic gestation. Groups 4 (primiparous) and 5 (multiparous) were healthy pregnant subjects previously enrolled in another study to determine the incidence and cause of pregnancy complications, such as preeclampsia and intrauterine growth restriction. The Primiparous group included 114 women in their first pregnancy, whereas the Multiparous group consisted of 79 women with 2 or more pregnancies but without pregnancy loss. RESULTS: The rate of extreme skewing (90% or greater) in the recurrent spontaneous abortion population was 8.6%, and not statistically different from any of the other groups, except the Primiparous group (1.0%, P < .01). The incidence of X-inactivation skewing of 90% or greater was no different whether there had been at least 1 live birth (9.9%), or no previous live births and at least 3 losses (5.6%, P > .05). When age and skewing of 90% or greater are compared, subjects with extreme skewing have a mean age of 2 years older than those without extreme skewing (P < .05). CONCLUSION: Skewed X-inactivation is not associated with recurrent spontaneous abortion but is associated with increasing maternal age.

Variable outcomes in mosaic trisomy 16: five case reports and literature analysis.

OBJECTIVES: To report five cases of mosaic trisomy 16 with variable outcomes in the context of the literature on mosaic trisomy 16. Complications in these cases include preeclampsia, IUGR, fetal anomalies, and death, with no predictable pattern. METHODS: Observation of five new cases and statistical analysis of 125 reported cases of mosaic trisomy 16 with prenatal detection and outcome data. RESULTS: (1) IUGR, premature delivery, and/or physical anomalies are observed commonly, even when the trisomy is thought to be confined to the placenta; (2) Level II mosaicism for trisomy 16 in amniotic fluid may reflect a true mosaic state with phenotypic consequences; (3) FISH is more sensitive than traditional cytogenetics in detecting mosaicism in all tissue types examined; (4) hCG levels can be extremely elevated, and MS-AFP levels are often elevated; and (5) Uniparental disomy (UPD) increases the rates of IUGR and physical anomalies in CPM cases. CONCLUSION: While there is no obvious mosaic trisomy 16 syndrome, IUGR and heart defects commonly occur, even if the mosaicism appears to be confined to the placenta. A completely normal outcome occurs only in about 20% of the cases; however, complications can often be limited to prematurity, small-for-gestational-age infants, and/or minor or surgically reparable birth defects.

Elevated first-trimester nuchal translucency increases the risk of congenital heart defects.

OBJECTIVE: We sought to evaluate the association between first trimester nuchal translucency measurement and the risk for major congenital heart defect in chromosomally normal fetuses. STUDY DESIGN: First trimester (10 weeks 4 days of gestation to 13 weeks 6 days of gestation) nuchal translucency was obtained in a large prospective multicenter National Institute of Child Health and Human Development study for Down syndrome prediction. The study, which was conducted between May 1998 and December 2000, was restricted to singleton pregnancies. Gestational age was determined by crown rump length measurements. Perinatal outcomes were determined and included the frequency of major congenital heart defect, which was defined as those cases that potentially could require surgery, intensive medical therapy, or prolonged follow-up time. Logistic regression analysis was used to determine whether nuchal translucency was a significant predictor of congenital heart defect. RESULTS: There were 8167 chromosomally normal pregnancies, of which 21 cases of major congenital heart defect were identified at follow-up examination (incidence, 2.6/1000 pregnancies). The risk of congenital heart defect rose with increasing nuchal translucency measurements. The mean nuchal translucency value for the normal and congenital heart defect groups were 1.5 mm and 1.9 mm, respectively (P = .05). With a nuchal translucency measurement of < 2.0 mm, the incidence of congenital heart defect was 13 of 6757 pregnancies (1.9 of every 1000 pregnancies). At 2.0 to 2.4 mm, the incidence was 5 of 1032 pregnancies (4.8 of every 1000 pregnancies). At 2.5 to 3.4 mm, the incidence was 2 of 335 pregnancies (6.0 of every 1000 pregnancies). At > or = 3.5 mm, the incidence was 1 of 43 pregnancies (23 of every 1000 pregnancies). Logistic regression analysis confirmed that nuchal translucency was associated significantly with congenital heart defect (odds ratio, 2.1; 95% CI, 1.4-3.1; P = .0004). CONCLUSION: Increased first trimester nuchal translucency measurement was associated with a higher risk of major congenital heart defect in chromosomally normal pregnancies. The practical implications of our findings are that patients with unexplained elevations of nuchal translucency may need referral for a fetal echocardiogram.

Sequential pathways of testing after first-trimester screening for trisomy 21.

OBJECTIVE: To evaluate the performance and use of second-trimester multiple-marker maternal serum screening for trisomy 21 by women who had previously undergone first-trimester combined screening (nuchal translucency, pregnancy-associated plasma protein A, and free beta-hCG), with disclosure of risk estimates. METHODS: In a multicenter, first-trimester screening study sponsored by the National Institute of Child Health and Human Development, multiple-marker maternal serum screening with alpha-fetoprotein, unconjugated estriol, and total hCG was performed in 4,145 (7 with trisomy 21) of 7,392 (9 with trisomy 21) women who were first-trimester screen-negative and 180 (7 with trisomy 21) of 813 (52 with trisomy 21) who were first-trimester screen-positive. Second-trimester risks were calculated using multiples of the median and a standardized risk algorithm with a cutoff risk of 1:270. RESULTS: Among the first-trimester screen-negative cohort, 6 of 7 (86%) trisomy 21 cases were detected by second-trimester multiple-marker maternal serum screening with a false-positive rate of 8.9%. Among the first-trimester screen-positive cohort, all 7 trisomy 21 cases were also detected in the second trimester, albeit with a 38.7% false-positive rate. CONCLUSION: Our data demonstrate that a sequential screening program that provides patients with first-trimester results and offers the option for early invasive testing or additional serum screening in the second trimester can detect 98% of trisomy 21-affected pregnancies. However, such an approach will result in 17% of patients being considered at risk and, hence, potentially having an invasive test. LEVEL OF EVIDENCE: II-2

Association of extreme first-trimester free human chorionic gonadotropin-beta, pregnancy-associated plasma protein A, and nuchal translucency with intrauterine growth restriction and other adverse pregnancy outcomes.

OBJECTIVE: The purpose of this study was to determine the association between first-trimester trisomy 21 screening markers (free human chorionic gonadotropin-beta [hCG], pregnancy-associated plasma protein A [PAPP-A], and nuchal translucency) and adverse pregnancy outcome. STUDY DESIGN: This was a cohort study of 8012 patients enrolled in a National Institute of Child Health and Human Development-sponsored study of first-trimester trisomy 21 and 18 screening. Trisomy 21 and 18 risk results and individual marker levels in unaffected pregnancies and pregnancies with adverse outcomes were evaluated. RESULTS: PAPP-A <1st percentile (OR 5.4, 95% CI 2.8-10.3) and PAPP-A <5th percentile (OR 2.7, 95% CI 1.9-3.9) and free beta-hCG <1st percentile (OR 2.7, 95% CI 1.3-5.9) were associated with increased risk of intrauterine growth restriction (IUGR) with positive predictive values of 24.1%, 14.1%, and 14.3%, respectively. PAPP-A <5th percentile (OR 2.3 95% CI 1.1-4.7) and nuchal translucency >99th percentile (OR 3.5, 95% CI 1.1-11.3) were associated with increased risk of preterm delivery before 34 weeks. Increased risk at screening for trisomy 21 and 18 identified 16 of the 29 other chromosomal abnormalities (55%). Low free beta-hCG, low PAPP-A, and increased nuchal translucency were all associated with an increased rate of fetal abnormality. CONCLUSION: Extreme values of first-trimester free beta-hCG, PAPP-A, and nuchal translucency are all associated with adverse outcomes. The especially high predictive value for IUGR of PAPP-A levels below the 1st percentile suggests that patients within this group may benefit from increased surveillance for this condition.

Association of undetectable unconjugated estriol on multiple marker screening with steroid sulfatase deficiency.

OBJECTIVE: To determine whether all patients with undetectable unconjugated estriol (uE3) on multiple marker screening (MMS) are carriers for steroid sulfatase (STS) deficiency. METHODS: This is a retrospective review of 65 pregnancies with undetectable uE3 on MMS. RESULTS: Of the 65 pregnancies, there were 21 that continued, 40 spontaneous losses, 2 lost to follow-up and 2 elective terminations. Of the 21 continuing pregnancies, 15 were determined to be carriers of the STS deletion. Twenty-seven of the 40 pregnancy losses were associated with elevated alpha-fetoprotein (AFP); about half of the losses were shown to have occurred prior to sampling. CONCLUSION: Patients with undetectable uE3 are likely to be carriers of the STS deletion, except those with associated elevated AFP. Elevated AFP with undetectable uE3 on MMS is a marker of preexisting or impending fetal demise.