Non-invasive Fetal Sex Determination Research Articles

Non-invasive Prenatal Diagnosis of Single Gene Disorders by Paternal Mutation Exclusion: 3 Years of Clinical Experience

ABSTRACT Until recently, fetal genetic testing was possible only via invasive sampling (amniocentesis or chorionic villus sampling). Although such testing allows for prenatal diagnosis (PND) of inherited monogenetic disorders, it is also associated with a risk of miscarriage. Identification of cell-free fetal DNA (cfDNA) from maternal circulation allows for noninvasive fetal sex determination and detection of the common aneuploidies. In fact, the entire fetal genome is represented within the circulating cell-free DNA. Clinical services are now available that provide noninvasive PND for monogenetic disorders, although these remain sparse, and most tests of this nature focus on paternally inherited disorders, given that the fetal DNA is diluted by maternal haploidentical sequences. This study reports on an experimental protocol for droplet digital polymerase chain reaction for paternal pathogenic or de novo variants. Since then, this noninvasive PND service has been offered to couples at risk of monogenic disorders in the fetus who have requested PND as a part of standard care. This report summarizes 3 years of clinical experience. Referral indications included the following: those with 50% risk of transmission of an autosomal dominant disorder from the father, those at 25% risk of autosomal recessive disorders and with different parental pathogenic variants such that they could assess for inheritance of the paternal variant, and those at risk for single-gene disorders due to de novo variants based on evident skeletal dysplasia by ultrasound or possible parental germinal mosaicism in a previously affected child. As a matter of practicality and organization, only ongoing pregnancies had new assays designed and ordered. Local French law regarding prenatal genetic testing requires written consent from both parents. Blood from each pregnant woman and blood samples from both parents for extraction of conventional genomic DNA were also collected. Each new variant required creation of a new assay, whose performance was evaluated. All results were reported with summarized interpretations to the clinicians, and invasive diagnostic testing was recommended in cases of autosomal recessive variant identification where inheritance of the paternal variant was detected to identify maternal inheritance. Sampling errors and identity swaps were ruled out by taking a second sample for every negative result. Development of new assays took 7 to 43 days, and overall, these assays were designed to detect 57 individual variants in 16 genes. A result was obtained for all but 2 cases; these were unsuccessful because of a low proportion of fetal DNA. In the autosomal dominant cases, 27% found fetal inheritance of the paternal variant, whereas 73% ruled out such inheritance. In the autosomal recessive cases, the paternal variant was detected in 56% and not detected in the remainder. In cases in which the paternal variant was detected, invasive diagnostic testing was recommended to determine maternal inheritance and whether the fetus was affected. The single inconclusive result was from a pregnancy obtained after egg donation in which ultimately the egg donor and the father both had the same gene variant, making accurate assessment not possible. There were 198 referrals overall, including 22 referrals due to concern for autosomal dominant disorders, 69 due to risk for an autosomal recessive disorder, and 107 due to concern for a de novo variant either because of ultrasound findings or a family history. There were 202 tests performed for 175 different families. All results reported were considered valid, and there were no false-positive results following invasive sampling for maternal inheritance, and no false-negative results reported following delivery. A limitation of this technique is that it addresses only the paternal variant's fetal status. This approach does not allow testing for all types of disease-causing variations such as repetitive elements, copy number variations, or structural variants. Also, maternally transmitted diseases or recessive conditions with both parents as carriers are also not detectable through this method. In the case of multiple pregnancies, there is no method for determining which twin (or whether both twins) will carry the variant. This test allows couples to avoid the risk of miscarriage while being able to make informed decisions about the pregnancy management. In addition, as new treatments are developed for common disorders (CFTR modulators, gene-replacement therapies, uterine genic therapies, and splicing-modifying therapies), the prenatal testing landscape can be expected to drastically evolve to favor a noninvasive approach.

Nanoparticle-Enhanced Surface Plasmon Resonance Imaging Enables the Ultrasensitive Detection of Non-Amplified Cell-Free Fetal DNA for Non-Invasive Prenatal Testing

Although many potential applications in early clinical diagnosis have been proposed, the use of a surface plasmon resonance imaging (SPRI) technique for non-invasive prenatal diagnostic approaches based on maternal blood analysis is confined. Here, we report a nanoparticle-enhanced SPRI strategy for a non-invasive prenatal fetal sex determination based on the detection of a Y-chromosome specific sequence (single-gene SRY) in cell-free fetal DNA from maternal plasma. The SPR assay proposed here allows for detection of male DNA in mixtures of 2.5 aM male and female genomic DNAs with no preliminary amplification of the DNA target sequence, thus establishing an analytical protocol that does not require costly, time-consuming, and prone to sample contamination PCR-based procedures. Afterward, the developed protocol was successfully applied to reveal male cell-free fetal DNA in the plasma of pregnant women at different gestational ages, including early gestational ages. This approach would pave the way for the establishment of faster and cost-effective non-invasive prenatal testing.

Noninvasive Fetal Sex Determination by Real-Time PCR and TaqMan Probes.

Noninvasive fetal sex determination by analyzing Y chromosome-specific sequences is very useful in the management of cases related to sex-linked genetic diseases. The aim of this study was to establish a non-invasive fetal sex determination test using Real-Time PCR and specific probes. The study was a prospective observational cohort study conducted from August 2018 to September 2019. Venous blood samples were collected from 25 Iranian pregnant women at weeks 7 to 25 of gestation. Cell-free DNA (cfDNA) was isolated from the plasma of samples and fetal sex was determined by SRY gene analysis using the Real-Time PCR technique. In the absence of SRY detection, the presence of fetal DNA was investigated using cfDNA treated with BstUI enzyme and PCR for the epigenetic marker RASSF1A. Of the total samples analyzed, 48% were male and 52% female. The RASSF1A assay performed on SRY negative cases also confirmed the presence of cell-free fetal DNA. Genotype results were in full agreement with neonate gender, and the accuracy of noninvasive fetal sex determination was 100%. Fetal sex determination using the strategy applied in this study is noninvasive and highly accurate and can be exploited in the management of sex-linked genetic diseases.

Noninvasive fetal sex determination by analysis of cell-free fetal DNA in maternal capillary blood obtained by fingertip puncture.

What's already known about this topic? Noninvasive fetal sex determination searches for Y‐chromosome sequences in maternal plasma isolated from venous blood drawn by venipuncture to indicate fetal sex in the first trimester of pregnancy. Capillary blood collection is much less invasive than venipuncture, thereby offering the possibility of sampling in geographical areas where there is no phlebotomist present. What does this study add? Fetal DNA is present in maternal capillary blood allowing for noninvasive fetal sex determination. Exogenous male DNA may be present, and detectable, on the fingertips of pregnant women. The elimination of the exogenous male DNA is critical for a reliable fetal sex determination using maternal capillary blood. A diluted buffered sodium hypochlorite (0.5%‐1%) can be used to eliminate exogenous male DNA from the fingertips of pregnant women.

The Potential and Accuracy of RNA-based Fetal Sex Determination during Early Pregnancy Using Cell-Free Fetal RNA from Korean Native Cows (Bos taurus coreanae)

Cell-free fetal RNA is useful to determine fetal sex and detect other inherent genetic disorders. However, non-invasive fetal sex determination methods using fetal RNA from maternal plasma is not yet well established in studies pertaining to bovine animals. Thus, the aim of this study was to systematically evaluate the presence of the male-specific ZRSR2Y gene transcript in maternal plasma using Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) assays, and to verify its accuracy, sensitivity, and specificity in determining fetal sex between 30 and 100 days of gestation. Overall accuracy, sensitivity, and specificity of the ZRSR2Y gene transcripts in determining fetal sex were 89.1%, 86.3%, and 100%, respectively. The 30 to 100 days of gestation were further classified into five stages of gestation, and each stage had relatively high accurate, sensitive, and specific results. Overall, these results indicate that the expression of the ZRSR2Y gene can be used for fetal sex determination in bovine animals using circulating cell-free RNA in maternal plasma during early pregnancy.

Cell-Free RNA Is a Reliable Fetoplacental Marker in Noninvasive Fetal Sex Determination.

Noninvasive genetic tests that use cell-free fetal DNA (cffDNA) are used increasingly in prenatal care. A low amount of cffDNA can have detrimental effects on the reliability of these tests. A marker to confirm the presence of fetal nucleic acids is therefore required that is universally applicable and easy to incorporate. We developed a novel multiplex, single-tube, noninvasive fetal sex determination assay by combining amplification of AMELY cffDNA with one-step reverse transcription (RT)-PCR of trophoblast-derived cell-free RNA (cfRNA), which functions as a sex-independent fetoplacental marker. We tested plasma samples from 75 pregnant women in duplicate in a blinded fashion. The fetus was considered to be male in the case of a positive result for AMELY and cfRNA amplification in both RT-PCRs. The fetus was considered to be female in the case of negative AMELY and positive cfRNA result in both RT-PCRs. In other cases, the test was repeated. We compared the results with invasive prenatal testing and pregnancy outcomes. The AMELY cffDNA amplification and cfRNA result was unambiguous and identical in duplicate in 71 of 75 plasma samples (95%). Four samples (5%) required an extra replicate because of an absent fetoplacental marker. Thereafter, fetal sex was correctly determined in all 75 plasma samples. Amplification of trophoblast-derived cfRNA is a reliable marker for the confirmation of the presence of fetoplacentally derived nucleic acids in noninvasive fetal sex determination.

Pitfalls in noninvasive fetal RhD and sex determination due to a vanishing twin

What's already known about this topic? Risk of misdiagnosis in NIPT carried out by cfDNA analysis in presence of vanishing twins. Importance of ultrasound monitoring since early gestational age in case of NIPT.What does this study add? Precise monitoring of the cfDNA released from a vanishing twin in a noninvasive fetal RhD and sex determination. Exhaustive follow‐up of pregnancy since delivery.

Vanishing twin as a potential source of bias in non-invasive fetal sex determination: A case report

Detection of fetal sex based on fetal DNA present in maternal plasma is already in clinical use. Here we present a case of false positivity during the first trimester which may be attributable to a vanishing twin. The presence of Y-chromosome-specific sequences is used as a marker to indicate a male fetus and the absence of a female fetus. Fetal sex determination was conducted in a pregnant woman at gestational week 10. The sample was positive in all triplicates. Ultrasonography at gestational week 20 revealed female sex. Analysis of sample taken at gestational week 22 indicated a female fetus. According to recently published meta-analyses, non-invasive prenatal diagnosis of fetal sex has high sensitivity and specificity values. Nevertheless, false negative and false positive cases occur. Future studies focusing on the dynamics of fetal DNA are needed. Vanishing twin might be one of the possible causes of false positivity in fetal sex determination.

Non-invasive fetal sex determination by maternal plasma sequencing and application in X-linked disorder counseling

Objective: To develop a fetal sex determination method based on maternal plasma sequencing (MPS), assess its performance and potential use in X-linked disorder counseling.Methods: 900 cases of MPS data from a previous study were reviewed, in which 100 and 800 cases were used as training and validation set, respectively. The percentage of uniquely mapped sequencing reads on Y chromosome was calculated and used to classify male and female cases. Eight pregnant women who are carriers of Duchenne muscular dystrophy (DMD) mutations were recruited, whose plasma were subjected to multiplex sequencing and fetal sex determination analysis.Results: In the training set, a sensitivity of 96% and false positive rate of 0% for male cases detection were reached in our method. The blinded validation results showed 421 in 423 male cases and 374 in 377 female cases were successfully identified, revealing sensitivity and specificity of 99.53% and 99.20% for fetal sex determination, at as early as 12 gestational weeks. Fetal sex for all eight DMD genetic counseling cases were correctly identified, which were confirmed by amniocentesis.Conclusions: Based on MPS, high accuracy of non-invasive fetal sex determination can be achieved. This method can potentially be used for prenatal genetic counseling.

Overview of Five-Years of Experience Performing Non-Invasive Fetal Sex Assessment in Maternal Blood.

Since the discovery of the presence of fetal DNA in maternal blood, non-invasive fetal sex determination has been the test most widely translated into clinical practice. To date there is no agreement between the different laboratories performing such tests in relation to which is the best protocol. As a consequence there are almost as many protocols as laboratories offering the service, using different methodologies and thus obtaining different diagnostic accuracies. By the end of 2007, after a validation study performed in 316 maternal samples collected between the 5th and 12th week of gestation, the fetal sex determination was incorporated into clinical practice in our Service. The test is performed in the first trimester of pregnancy, and it is offered as part of the genetic counseling process for couples at risk of X-linked disorders. As a general rule and in order to avoid misdiagnosis, two samples at different gestational ages are tested per patient. The analysis is performed by the study of the SRY gene by RT-PCR. Two hundred and twenty six pregnancies have been tested so far in these 5 years. Neither false positives nor false negatives diagnoses have been registered, thus giving a diagnostic accuracy of 100%.

Early non-invasive fetal RHD genotyping and sex determination by conventional and multiplex PCR performing a rapid and low-cost cell-free fetal DNA extraction method

Early non-invasive fetal RHD genotyping and sex determination by conventional and multiplex PCR performing a rapid and low-cost cell-free fetal DNA extraction method

Current Resources for Evidence‐Based Practice January/February 2012

The Institute of Medicine Report on Clinical Preventive Services for Women: From Evidence to Policy As a central component of the Affordable Care Act (ACA), new health insurance plans will be required to cover preventive health services without cost sharing to plan members beginning on or after August 1, 2012. In response to this directive within the ACA, the United States Department of Health and Human Services (HHS) charged the Institute of Medicine (IOM) with conducting a comprehensive review of women’s preventive health services and recommending those services deemed necessary for the health of women in the United States (Gee et al, 2011).

Noninvasive fetal sex determination in maternal plasma: a prospective feasibility study

PurposeTo prospectively validate a protocol for noninvasive fetal sex determination in maternal plasma and demonstrate its applicability to clinical practice. MethodsPeripheral blood from 404 pregnant women undergoing prenatal invasive testing was collected from 6 to 23 weeks of gestation. Real-time PCR was performed for the SRY gene and multicopy DYS14 marker sequence located within the TSPY gene by the TaqMan minor groove binder probe assay as a first-line test. Owing to a false-positive result, amplification of repetitive motifs of the DAZ gene region was also tested as a second-line test performed in the last 232 patients enrolled in our series. A diagnostic algorithm was designed using a combination of these three markers. Fetal gender determined by noninvasive prenatal diagnosis (NIPD) was compared with that diagnosed by quantitative fluorescent PCR after invasive testing or ultrasound. ResultsA single false-positive result was obtained in the first 172 pregnancies. Reporting criteria were modified in the subsequent 232 pregnancies, giving an overall sensitivity and specificity of 100% (95% CI 99.8–100%) and 99.5% (95% CI 98.1–100%), respectively. Pregnancy outcome was obtained in all cases, including 221 male-bearing and 183 female-bearing pregnancies. ConclusionNIPD for fetal sex determination in maternal plasma is highly accurate and clinically applicable if robust reporting criteria are applied.

805: Utilization of maternal blood on Guthrie card for first trimester screen for noninvasive fetal sex determination and RhD genotyping

805: Utilization of maternal blood on Guthrie card for first trimester screen for noninvasive fetal sex determination and RhD genotyping

Noninvasive Fetal Sex Determination Using Cell-Free Fetal DNA: A Systematic Review and Meta-Analysis

Context Noninvasive prenatal determination of fetal sex using cell-free fetal DNA provides an alternative to invasive techniques for some heritable disorders. In some countries this testing has transitioned to clinical care, despite the absence of a formal assessment of performance. Objective To document overall test performance of noninvasive fetal sex determination using cell-free fetal DNA and to identify variables that affect performance. Data Sources Systematic review and meta-analysis with search of PubMed (January 1, 1997-April 17, 2011) to identify English-language human studies reporting primary data. References from review articles were also searched. Study Selection and Data Extraction Abstracts were read independently to identify studies reporting primary data suitable for analysis. Covariates included publication year, sample type, DNA amplification methodology, Y chromosome sequence, and gestational age. Data were independently extracted by 2 reviewers. Results From 57 selected studies, 80 data sets (representing 3524 male-bearing pregnancies and 3017 female-bearing pregnancies) were analyzed. Overall performance of the test to detect Y chromosome sequences had the following characteristics: sensitivity, 95.4% (95% confidence interval [CI], 94.7%-96.1%) and specificity, 98.6% (95% CI, 98.1%-99.0%); diagnostic odds ratio (OR), 885; positive predictive value, 98.8%; negative predictive value, 94.8%; area under curve (AUC), 0.993 (95% CI, 0.989-0.995), with significant interstudy heterogeneity. DNA methodology and gestational age had the largest effects on test performance. Methodology test characteristics were AUC, 0.988 (95% CI, 0.979-0.993) for polymerase chain reaction (PCR) and AUC, 0.996 (95% CI, 0.993-0.998) for real-time quantitative PCR (RTQ-PCR) (P = .02). Gestational age test characteristics were AUC, 0.989 (95% CI, 0.965-0.998) ( 20 weeks) (P = .02 for comparison of diagnostic ORs across age ranges). RTQ-PCR (sensitivity, 96.0%; specificity, 99.0%) outperformed conventional PCR (sensitivity, 94.0%; specificity, 97.3%). Testing after 20 weeks (sensitivity, 99.0%; specificity, 99.6%) outperformed testing prior to 7 weeks (sensitivity, 74.5%; specificity, 99.1%), testing at 7 through 12 weeks (sensitivity, 94.8%; specificity, 98.9%), and 13 through 20 weeks (sensitivity, 95.5%; specificity, 99.1%). Conclusions Despite interstudy variability, performance was high using maternal blood. Sensitivity and specificity for detection of Y chromosome sequences was greatest using RTQ-PCR after 20 weeks' gestation. Tests using urine and tests performed before 7 weeks' gestation were unreliable.

Noninvasive Fetal Sex Determination Using Cell-Free Fetal DNA

Noninvasive prenatal determination of fetal sex using cell-free fetal DNA provides an alternative to invasive techniques for some heritable disorders. In some countries this testing has transitioned to clinical care, despite the absence of a formal assessment of performance. To document overall test performance of noninvasive fetal sex determination using cell-free fetal DNA and to identify variables that affect performance. Systematic review and meta-analysis with search of PubMed (January 1, 1997-April 17, 2011) to identify English-language human studies reporting primary data. References from review articles were also searched. Abstracts were read independently to identify studies reporting primary data suitable for analysis. Covariates included publication year, sample type, DNA amplification methodology, Y chromosome sequence, and gestational age. Data were independently extracted by 2 reviewers. From 57 selected studies, 80 data sets (representing 3524 male-bearing pregnancies and 3017 female-bearing pregnancies) were analyzed. Overall performance of the test to detect Y chromosome sequences had the following characteristics: sensitivity, 95.4% (95% confidence interval [CI], 94.7%-96.1%) and specificity, 98.6% (95% CI, 98.1%-99.0%); diagnostic odds ratio (OR), 885; positive predictive value, 98.8%; negative predictive value, 94.8%; area under curve (AUC), 0.993 (95% CI, 0.989-0.995), with significant interstudy heterogeneity. DNA methodology and gestational age had the largest effects on test performance. Methodology test characteristics were AUC, 0.988 (95% CI, 0.979-0.993) for polymerase chain reaction (PCR) and AUC, 0.996 (95% CI, 0.993-0.998) for real-time quantitative PCR (RTQ-PCR) (P = .02). Gestational age test characteristics were AUC, 0.989 (95% CI, 0.965-0.998) (<7 weeks); AUC, 0.994 (95% CI, 0.987-0.997) (7-12 weeks); AUC, 0.992 (95% CI, 0.983-0.996) (13-20 weeks); and AUC, 0.998 (95% CI, 0.990-0.999) (>20 weeks) (P = .02 for comparison of diagnostic ORs across age ranges). RTQ-PCR (sensitivity, 96.0%; specificity, 99.0%) outperformed conventional PCR (sensitivity, 94.0%; specificity, 97.3%). Testing after 20 weeks (sensitivity, 99.0%; specificity, 99.6%) outperformed testing prior to 7 weeks (sensitivity, 74.5%; specificity, 99.1%), testing at 7 through 12 weeks (sensitivity, 94.8%; specificity, 98.9%), and 13 through 20 weeks (sensitivity, 95.5%; specificity, 99.1%). Despite interstudy variability, performance was high using maternal blood. Sensitivity and specificity for detection of Y chromosome sequences was greatest using RTQ-PCR after 20 weeks' gestation. Tests using urine and tests performed before 7 weeks' gestation were unreliable.

Fetal Sex Determination of Macaque Monkeys by a Nested PCR Using Maternal Plasma

Non-invasive fetal sex determination is required for biomedical studies, in which some sexual difference would be expected in fetal events, in order to make a choice of male or female fetus. To detect male fetal DNA of the sex-determining region Y gene (SRY) in maternal macaque plasma, nested real-time PCR using the SYBR Green system was developed. In all cases of pregnant macaques with male fetuses, a nested PCR product of SRY was amplified from the mother's plasma, while no amplicon was detected in any case of pregnancy with a female fetus. Interestingly, fetal SRY DNA appeared to be cleared rapidly from the maternal blood after parturition. The current method is sensitive and can be performed with a regular PCR machine.

Reliability of Fetal Sex Determination Using Maternal Plasma

To determine the diagnostic accuracy of noninvasive fetal sex determination in maternal plasma. All consecutive patients for whom fetal sex determination in maternal plasma was performed in our laboratory from 2003 up to 2009 were included in the study. Real-time polymerase chain reaction was performed for the SRY gene and multicopy DYS14 marker sequence. A stringent diagnostic algorithm was applied. In the case of a positive result for both Y chromosome-specific assays, a male-bearing pregnancy was reported. In the case of a negative result, the presence of fetal DNA was ascertained through the use of 24 biallelic insertion/deletion polymorphisms or paternally inherited blood group antigens. Only if the presence of fetal DNA was confirmed was a female-bearing pregnancy reported. Results were compared with the pregnancy outcomes. A total of 201 women were tested. The median gestational age was 9 0/7 weeks (interquartile range 8 0/7 to 10 0/7 weeks). In 189 of 201 cases (94%), a test result was issued; in 10 cases, the presence of fetal DNA could not be confirmed; in two cases, an early miscarriage was observed. Pregnancy outcome was obtained in 197 cases, including 105 male-bearing and 81 female-bearing pregnancies and 11 miscarriages. Sensitivity and specificity of the test were 100% (95% confidence intervals 96.6-100% and 95.6-100%, respectively). In all 10 cases in which the presence of fetal DNA could not be confirmed, a female was born. Noninvasive fetal sex determination in maternal plasma is highly reliable and clinically applicable. III.

Molecular Genetic Testing of Hemophilia A

Genetic testing in hemophilia A continues to diversify. This article describes recent advances in several aspects of genetic analysis and its interpretation and reporting. The intron 1 and 22 inversions responsible for 50% of severe hemophilia A cases can be sought using long and inverse polymerase chain reaction (PCR) techniques. Point mutations are analyzed in remaining patients by PCR amplification of the F8 protein-coding region followed by either mutation screening to identify the mutated amplicon and subsequent DNA sequencing or by directly sequencing amplified DNA. Many technique modifications and sequence analysis software packages are available to reduce time and effort required to identify a mutation. Dosage analysis and gap PCR have been described to identify carriers of large F8 deletions. Noninvasive prenatal fetal sex determination and preimplantation genetic diagnosis extend the reproductive options available to hemophilia carriers. Guidelines on undertaking and reporting the testing plus external quality assessment are now available to help ensure that genetic analysis yields accurate and well-interpreted results.

Non-invasive fetal sex determination: Impact on clinical practice

Prenatal fetal sex determination is undertaken in women at high risk of serious genetic disorders affecting a specific sex. Traditionally, this is undertaken by invasive testing, usually chorionic villus sampling, which carries a risk of miscarriage of around 1%. The identification of cell-free fetal DNA in the maternal circulation has allowed the development of 'non-invasive prenatal diagnostic tests', which permit fetal sex determination without risk to the pregnancy.