Cell-free fetal DNA in maternal blood: evolving clinical applications.
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Blood Specimen Collection↗
Biomedical subjects
Joe Leigh Simpson
Publications and source records attributed to Joe Leigh Simpson.
Interlaboratory comparison of fetal male DNA detection from common maternal plasma samples by real-time PCR.
BACKGROUND: Analysis of fetal DNA from maternal plasma by PCR offers great potential for noninvasive prenatal genetic diagnosis. To further evaluate this potential, we developed and validated a standard protocol to determine whether fetal DNA sequences could be reproducibly amplified and measured across multiple laboratories in a common set of specimens. METHODS: Each of five participating centers in a National Institute of Child Health and Human Development consortium collected 20 mL of peripheral blood from 20 pregnant women between 10 and 20 weeks of gestation. The plasma fraction was separated according to a common protocol, divided, and frozen in five aliquots. One aliquot was shipped to each participating laboratory, where DNA was extracted according to a standard protocol. All plasma samples (n = 100) were then analyzed blindly for the presence and quantity of total DNA (GAPDH) and male fetal DNA (SRY) by real-time PCR. Genomic DNA was isolated from female and male cells at one center, quantified, and shipped to the others to serve as calibrators for GAPDH and SRY, respectively. RESULTS: The amplification of known quantities of DNA was consistent among all centers. The mean quantity of male DNA amplified from maternal plasma when the fetus was male ranged from 51 to 228 genome equivalents (GE)/mL. Qualitative concordance was found overall among centers. The sensitivity of the assay for detection of male DNA when the fetus was male varied from 31% to 97% among centers. Specificity was more consistent (93-100%) with only four false-positive results obtained across the entire study. CONCLUSIONS: All centers were able to consistently amplify frozen and shipped DNA. The PCR procedure used here is reliable and reproducible. Centers that extracted and amplified more DNA per milliliter of maternal plasma had superior sensitivities of Y chromosome sequence detection. The specificity of the assay was more consistent among centers. A robust and thoroughly optimized protocol for the extraction of DNA from maternal plasma is needed to make testing of fetal DNA in maternal plasma a clinically relevant analytical tool.
Clinical Laboratory Techniques↗
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.
Chorionic Gonadotropin, beta Subunit, Human↗
Genetics of endometriosis: heritability and candidate genes.
Endometriosis is a complex gynecologic disorder that affects as many as 10-15% of premenopausal women. Epidemiologic studies have confirmed that this disease is a genetic disorder of polygenic/multifactorial inheritance. The disorder has long been recognized to show heritable tendencies with recurrence risks of 5-7% for first-degree relatives. The current investigational goal is to determine the number and location of causative genes, a process that has been made possible by recent advances in molecular technology. This chapter discusses heritability studies supporting polygenic/multifactorial inheritance, the scientific basis of genome-wide strategies for identifying causative genes and potential candidate genes.
Endometriosis↗
The genetics of polycystic ovary syndrome.
Polycystic ovary syndrome (PCOS) is a heterogeneous disorder. There is evidence for a genetic component in PCOS based on familial clustering of cases. The majority of the evidence supports an autosomal dominant form of inheritance. Steroidogenesis has been shown to be upregulated in PCOS theca cells, suggesting that the genetic abnormality in PCOS affects signal transduction pathways controlling the expression of a family of genes. Although a number of candidate genes have been proposed, the putative PCOS gene(s) has yet to be identified. Linkage and association studies implicate a region near the insulin receptor gene at chr 19p13.3. New genetic approaches, such as microarray technology, hold promise for elucidation of the pathophysiology underlying PCOS.
Cytochrome P-450 Enzyme System↗
Over a decade of experience with preimplantation genetic diagnosis: a multicenter report.
OBJECTIVE: To review a 12-year experience of the world's three largest preimplantation genetic diagnosis (PGD) centers. DESIGN: Multicenter analysis of the clinical outcome of PGD. SETTING: In vitro fertilization programs at the Reproductive Genetics Institute, Chicago, Illinois; Saint Barnabas Medical Center, West Orange, New Jersey; and SISMER, Bologna, Italy. PATIENT(S): Poor-prognosis IVF patients, patients carrying balanced chromosomal translocations, and couples at risk for producing children with Mendelian disorders. INTERVENTION(S): In vitro fertilization, intracytoplasmic sperm injection, polar body removal, blastomere biopsy, and ET. MAIN OUTCOME MEASURE(S): DNA or chromosomal analysis of biopsied polar bodies or blastomeres, implantation and clinical pregnancy rates, and live-born pregnancy outcome. RESULT(S): A total of 754 babies have been born as a result of 4,748 PGD attempts, which shows the expanded application and the practical relevance of PGD for single-gene disorders, chromosomal aneuploidies and translocations, late-onset diseases with genetic predisposition, and nondisease testing in couples at need for human leukocyte antigens-matched offspring for treatment of affected siblings. CONCLUSION(S): Preimplantation genetic diagnosis is evolving to become a clinical option for couples at risk for producing offspring with Mendelian diseases, has a positive numerical impact in standard assisted reproduction practices through aneuploidy testing, and reduces by at least fourfold the spontaneous abortion rate in couples carrying translocations.
Blastula↗
Over a decade of experience with preimplantation genetic diagnosis.
The three respondents provide additional support for preimplantation genetic diagnosis (PGD) having the pivotal place it now has in prenatal genetic diagnosis: chromosomal abnormalities (e.g., unbalanced translocations), Mendelian disorders, and HLA typing for transfer of compatible, genetically normal, embryos. Transferring euploid embryos has decreased the clinical abortion rate and increased the implantation rate in assisted reproductive technologies (ART), but it has not necessarily improved the live-birth rate. Safer embryo biopsy, more extensive diagnostic efforts (i.e., microarray analysis), and more refined patient selection may be required before shifting from preselection of embryos based solely on morphological parameters to transfer of only aneuploidy-free embryos.
Abortion, Spontaneous↗
Protein expression profiling of endometriosis: validation of 2-mm tissue microarrays.
Tissue microarrays (TMAs) are useful tools for studying protein expression in endometriosis. Tissue microarray analyses of immunohistochemical profiles of estrogen receptor-alpha and P receptor corroborate previously published results from the use of conventional immunohistochemistry, thus validating TMA use in endometriosis.
Endometriosis↗
Paternal gonadal mosaicism detected in a couple with recurrent abortions undergoing PGD: FISH analysis of sperm nuclei proves valuable.
Many couples are now seeking preimplantation genetic diagnosis (PGD) and fluorescence in-situ hybridization (FISH) as an alternative approach to avoid spontaneous abortion by ensuring transfer of presumed chromosomally normal embryos. This case report describes unexpected findings in a couple having three spontaneous abortions and two failed IVF cycles. In two IVF PGD cycles, four of 13 (30.8%) embryos (blastomeres) demonstrated duplication involving the Down syndrome critical region, detectable by a locus specific chromosome 21 probe. The same duplication was subsequently detected by FISH in 66 of 1002 (6.6%) sperm nuclei, demonstrating paternal gonadal mosaicism. Cytogenetic studies of peripheral blood revealed normal karyotypes in both the male and female partners. This identification of paternal germ cell or gonadal mosaicism suggests that analysis of sperm nuclei prior to undergoing IVF with PGD may be of value in patients with recurrent spontaneous abortions or multiple failed IVF.
Abortion, Habitual↗
Pregnancy-associated plasma protein A, free beta-hCG, nuchal translucency, and risk of pregnancy loss.
OBJECTIVE: To estimate the likelihood of clinical early and late pregnancy loss as a function of first-trimester maternal serum analytes and fetal nuchal translucency measurements. METHODS: Study subjects were recruited for a National Institute of Child Health and Human Development-sponsored multicenter cohort study initially designed to study the detection of Down syndrome during the first trimester of pregnancy. The cohort consisted of women who had a live fetus between 10 and 14 weeks of gestation and had no significant vaginal bleeding. Women with prior fetal trisomy (T21/18) and those with structural or chromosomal abnormalities in the index pregnancy were excluded. First-trimester screening consisted of pregnancy-associated plasma protein A (PAPP-A), free beta-hCG, and nuchal translucency. Pregnancy loss rates in women with various levels of PAPP-A, free beta-hCG, or nuchal translucency (less than 1st, less than 5th, more than 95th, and more than 99th percentile) were compared with losses in women with normal values (5th to 95th percentile). RESULTS: The mean gestational age at screening of 7,932 women meeting study criteria was 12.1 weeks. Loss rates were only 0.36% at less than 20 weeks after normal free beta-hCG, PAPP-A, and nuchal translucency. Conversely, low levels of PAPP-A and free beta-hCG as well as increased nuchal translucency were individually associated with increased early loss. These associations persisted after controlling for maternal age and race using logistic regression analysis. CONCLUSION: Normal values of PAPP-A, free beta-hCG, and nuchal translucency are associated with a very low risk of pregnancy loss at less than 20 weeks.
Abortion, Spontaneous↗
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
Adult↗
Genetic basis of endometriosis.
Endometriosis is a complex gynecologic disorder that has long been recognized as showing heritable tendencies, with recurrence risks of 5-7% for first-degree relatives. Familial and epidemiologic studies support that this disease is a genetic disorder of polygenic/multifactorial inheritance. The current investigational challenge is to determine the number and location of causative genes. Recent advances in molecular technology make identification and elucidation of these genes now possible. In this review, we update previous communications in which we also reviewed heritability studies supporting polygenic/multifactorial inheritance, discuss the scientific basis of genomewide strategies for identifying causative genes, and identify potential candidate genes.
Endometriosis↗
First-trimester screening for trisomies 21 and 18.
BACKGROUND: Screening for aneuploid pregnancies is routinely performed after 15 weeks of gestation and has a sensitivity of approximately 65 percent, with a false positive rate of 5 percent. First-trimester markers of aneuploidy have been developed, but their use in combination has not been adequately evaluated in clinical practice. METHODS: We conducted a multicenter study of screening for trisomies 21 and 18 among patients with pregnancies between 74 and 97 days of gestation, based on maternal age, maternal levels of free beta human chorionic gonadotropin and pregnancy-associated plasma protein A, and ultrasonographic measurement of fetal nuchal translucency. A screening result was considered to be positive for trisomy 21 if the calculated risk was at least 1 in 270 pregnancies and positive for trisomy 18 if the risk was at least 1 in 150. RESULTS: Screening was completed in 8514 patients with singleton pregnancies. This approach to screening identified 85.2 percent of the 61 cases of Down's syndrome (95 percent confidence interval, 73.8 to 93.0), with a false positive rate of 9.4 percent (95 percent confidence interval, 8.8 to 10.1). At a false positive rate of 5 percent, the detection rate was 78.7 percent (95 percent confidence interval, 66.3 to 88.1). Screening identified 90.9 percent of the 11 cases of trisomy 18 (95 percent confidence interval, 58.7 to 99.8), with a 2 percent false positive rate. Among women 35 years of age or older, screening identified 89.8 percent of fetuses with trisomy 21, with a false positive rate of 15.2 percent, and 100 percent of fetuses with trisomy 18. CONCLUSIONS: First-trimester screening for trisomies 21 and 18 on the basis of maternal age, maternal levels of free beta human chorionic gonadotropin and pregnancy-associated plasma protein A, and measurement of fetal nuchal translucency has good sensitivity at an acceptable false positive rate.
Chorionic Gonadotropin, beta Subunit, Human↗
Intact fetal cells in maternal plasma: are they really there?
Rare fetal cells can be recovered from maternal blood, which suggests that non-invasive prenatal diagnosis is possible. However, recovery and analysis of fetal cells from blood is complex, and sensitivity is low because of the rarity of these cells in the maternal circulation. An alternative strategy, which suggested that intact fetal cells can be found in maternal plasma by use of simple enrichment methods, has been reported. We aimed to replicate this technique. However, five independent laboratories were unable to identify any intact male cells from the plasma of 38 women known to be carrying male fetuses. Although apoptotic intact fetal cells could contribute to the detection of fetal DNA in maternal plasma, we believe that recovery of these cells is difficult and not clinically practical.
Aneuploidy↗
Genetics of endometriosis.
Endometriosis long has been recognized as showing heritable tendencies, with recurrence risks of 5% to 7% for first-degree relatives. The risk indicates that polygenic and multifactorial etiology is far more likely to be the cause than mendelian inheritance. The current task is to determine the number and location of genes responsible for endometriosis. Molecular advances of the past decade make identification and elucidation of these genes a reality. The authors review the basis for concluding that endometriosis is a genetic disorder of polygenic/multifactorial inheritance. Genome-wide strategies for identifying causative genes are considered and available data on association or linkage to putative candidate genes systematically reviewed.
Endometriosis↗
Public objections to designer babies and cloning in USA: not quite what was expected.
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Cloning, Organism↗
Heritability and candidate genes for endometriosis.
Endometriosis is inherited in polygenic/multifactorial fashion, with recurrence risks of 5-7% for first-degree relatives. The current task is to determine the number and location of causative genes. This review initially supports the basis for polygenic/multifactorial inheritance and then outlines genome-wide strategies for identifying causative genes. Potential candidate genes are surveyed, and discrepant results emphasized. Finally, a hypothesis for endometriosis, having a multi-step pathogenesis analogous to the pathogenesis of neoplasia, is considered.
Endometriosis↗
Fertilisation involving ageing gametes, major birth defects, and Down's syndrome.
For many years, ageing of gametes as a result of prolonged retention in the female reproductive tract before fertilisation has been circumstantially associated with major birth defects. To assess this association, we studied pregnant women who had recorded the timing, with regard to presumed ovulation, of the coital event leading to conception. We found major anomalies in 11 (2.7%) of 400 infants born to women with optimally timed conceptions (on the day of or 1 day before ovulation), compared with 14 (2.5%) of 538 infants of women with non-optimally timed conceptions (odds ratio 0.94, 95% CI 0.43-2.06). The numbers of infants with Down's syndrome were two (0.5%) of 400, and four (0.7%) of 538, respectively (1.48, 0.27-8.06). There is no association between ageing gametes and major birth defects, including Down's syndrome.
Abortion, Spontaneous↗