24-chromosome Aneuploidy Screening Research Articles

Performance of noninvasive prenatal screening for fetal sex chromosome aneuploidies in a cohort of 116,862 pregnancies

ABSTRACT Background Noninvasive prenatal screening (NIPS) has shown good performance in screening common aneuploidies. However, its performance in detecting fetal sex chromosome aneuploidies (SCAs) needs to be evaluated in a large cohort. Research design and methods In this retrospective observation, a total of 116,862 women underwent NIPS based on DNA nanoball sequencing from 2015 to 2022. SCAs were diagnosed based on karyotyping or chromosomal microarray analysis (CMA). Among them, 2,084 singleton pregnancies received karyotyping and/or CMA. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of NIPS for fetal SCAs were evaluated. Results The sensitivity was 97.7% (95%CI, 87.7–99.9), 87.3% (95% CI, 76.5–94.4), 96.1% (95%CI, 86.5–99.5), and 95.7% (95% CI, 78.1–99.9), the PPV was 25.8% (95%CI, 19.2–33.2), 80.9% (95%CI, 69.5–89.4), 79.0% (95%CI, 66.8–88.3), and 53.7% (95%CI, 37.4–69.3) for 45,X, 47,XXY, 47,XXX, and 47,XYY, respectively. The specificity was 94.1% (95%CI, 93.0–95.1) for 45,X, and more than 99.0% for sex chromosome trisomy (SCT). The NPV was over 99.0% for all. Conclusions NIPS screening for fetal SCAs has high sensitivity, specificity and NPV. The PPV of SCAs was moderate, but that of 45,X was lower than that of SCTs. Invasive prenatal diagnosis should be recommended for high-risk patients.

Clinical application of noninvasive prenatal testing in twin pregnancies: a single-centre experience

Objectives To evaluate the clinical efficiency of non-invasive prenatal testing (NIPT) for fetal chromosomal aneuploidy screening in twin pregnancies. Methods A total of 1650 women with twin pregnancies were enrolled in the study, which underwent NIPT at the Southwest Hospital, Army Medical University, Chongqing, China from January 2013 to June 2022. Fetal karyotyping analysis was conducted in high-risk patients, with subsequent follow-up on pregnancy outcomes. Results In 1650 pregnancies, NIPT results showed ten cases of the fetal chromosome aneuploidy, of which six cases were true positive and four cases were false positive. The sensitivity, specificity, positive predictive value (PPV), and false-positive rate (FPR) of trisomy 21 were 100%, 99.79%, 57.14%, and 0.18%, respectively. Sensitivity, specificity, PPV, and FPR of trisomy 18 were 100%, 99.94%, 50%, and 0.06%, respectively. The sensitivity, specificity, PPV, and FPR of trisomy 13 were 100%, 100%, 100%, and 0%, respectively. No false negatives were detected and the negative predictive value (NPV) was 100% of the total. Eleven pregnancies failed the NIPT test with no-call due to the low fetal fraction (< 4%). Conclusions NIPT is a high-performing routine primary prenatal screening test in twin pregnancies, with high sensitivity and specificity in screening for fetal aneuploidy.

Clinical application and evaluation of health economics for non-invasive prenatal testing of fetuses in Tianjin

To assess the clinical efficacy and health economic value of non-invasive prenatal testing (NIPT) for the prenatal screening of common fetal chromosomal aneuploidies. 10 612 pregnant women from October 2017 to December 2019 presented at the antenatal screening clinic of the General Hospital of Tianjin Medical University were selected as the study subjects. Results of NIPT and invasive prenatal diagnosis and follow-up outcome for the 10 612 pregnant women were retrospectively analyzed and compared. Meanwhile, NIPT data for two periods were analyzed for assessing the health economic value of NIPT as the second- or first-tier screening strategy for the prenatal diagnosis of fetal trisomies 21, 18 and 13. The NIPT was successful in 10 528 (99.72%) subjects, with the sensitivity for fetal trisomies 21, 18 and 13 being 100%, 92.86% and 100%, and the positive predictive value (PPV) being 89.74%, 61.90% and 44.44%, respectively. The PPV of NIPT for sex chromosome aneuploidies was 34.21%. Except for one false negative case of trisomy 18, the negative predictive value for trisomy 21, trisomy 13 and other chromosomal abnormalities were 100%. For pregnant women with high risk by serological screening, advanced maternal age or abnormal ultrasound soft markers, NIPT has yielded a significantly increased high risk ratio. There was no statistical difference in the PPV of NIPT among pregnant women from each subgroup. NIPT would have higher health economic value as a second-tier screening until 2019, while compared to 2015 ~ 2017, its incremental cost-effectiveness ratio as a first-tier screening had declined clearly. The screening efficacy of NIPT for trisomies 21, 18 and 13 for a mixed population is significantly better than conventional serological screening, but it is relatively low for sex chromosomal abnormalities. NIPT can also be recommended for populations with relatively high risks along with detailed pre- and post-test genetic counselling. From the perspective of health economics, except for open neural tube defects, it is possible for NIPT to replace the conventional serological screening in the future as its cost continues to decrease.

Retrospective analysis for 424 330 first-line screening results of non-invasive prenatal testing in Hebei province

Objective: To evaluate the effect of noninvasive prenatal testing (NIPT) as first-line screening in fetal chromosome aneuploidy screening practice, and to provide evidence for the prevention and control strategy of birth defects. Methods: Since July 2019, Hebei province had carried out the NIPT project providing first-line screening for eligible pregnant women in the area (except for those who were not applicable). Pregnant women with high risk received genetic counseling, prenatal diagnosis and intervention guidance. Low risk and false-positive ones received continuous detection and moved to prenatal diagnosis center for counseling and diagnosis if abnormities were discovered. All pregnant women were followed up to learn about pregnancy outcomes and newborn health status. Detection results and clinical data of pregnant women participating the NIPT project from July 2019 to July 2020 were collected. The detection results and effect of NIPT were analyzed. Results: (1) Basic information of the screened population: A total of 424 330 pregnant women were screened, and 423 596 were successfully detected, with a success rate of 99.83% (423 596/424 330). The age of pregnant women was (28.8±4.5) years old; the gestational age of screening was (16.6±2.3) weeks; the proportion of advanced-age pregnant women (≥35 years old) was 10.18% (43 132/423 596); in vitro fertilization-embryo transfer (IVF-ET) rate was 1.58% (6 713/423 596); the twin rate was 1.38% (5 849/423 596); the proportion of primipara was 34.23% (144 977/423 596). (2) Screening results and detection performance: totally, 325, 73 and 20 pregnant women were diagnosed with trisomy 21, 18 and 13; the sensitivity were 99.39%, 100.00% and 100.00%; the specificity were 99.98%, 99.99% and 99.98%; the positive predictive value were 75.76%, 68.87% and 21.51%, respectively. Besides, 249 190 pregnant women were received supplementary reports as well, and 255, 10 and 9 were confirmed for sex chromosome aneuploidy, other autosomal aneuploidy and deletion/duplication syndrome; the positive predictive value were 37.78%, 6.06% and 32.14%, respectively. The sensitivity of NIPT for target trisomy (trisomy 21, 18 and 13) screening in advanced-age, IVF-ET and twin pregnant women were 99.29%, 100.00% and 90.00%, respectively; the specificity were 99.93% for all; the positive predictive value were 82.25%, 61.54% and 69.23%, respectively. Conclusions: NIPT has a significant effect and good performance in the first-line screening of fetal chromosome aneuploidy in the whole population, which might provide reference for the improvement of birth defect prevention and control strategy.

OP50 Health Equity Considerations In HTA: A Case Example Of Prenatal Screening

IntroductionThe Washington Health Technology Assessment (HTA) program has been guiding coverage decisions in the state since 2007. The Center for Evidence-based Policy works with the program to develop evidence-based HTA reports. In 2020, we presented an HTA on cell-free DNA prenatal screening for chromosomal aneuploidies. In the committee’s discussion, questions around access to screening tests and to prenatal care more generally were raised. We present a case study of how health equities were considered in the development of coverage criteria.MethodsWe conducted an HTA using standard systematic review methodologies. Outcomes focused on test accuracy and the impact of screening. We did not look for evidence on access to prenatal screening tests or people’s experience of prenatal screening. We reviewed the meeting transcript to identify issues of health equity and how they influenced the final decision.ResultsDuring the discussion of the evidence, the committee raised concerns around equitable access to cell-free DNA tests and prenatal screening, including: direct costs to the person; access to the full range of prenatal screening, including ultrasound; and, uptake of prenatal screening. Based on the findings from the evidence report, expert testimony, and public comment, the committee voted to cover cell-free DNA prenatal screening for chromosomal aneuploidies unconditionally.ConclusionsHealth equity is increasingly important in healthcare decision-making. Decision makers should consider how a decision may reduce health inequities and how it may inadvertently increase existing health inequities. Decision makers also need to understand the context within which the decision will be implemented. Consideration of health equity can be addressed in a number of ways, for example through systematic review of equity issues or patient experience or through the use of contextual knowledge from expert and public testimony. Regardless of the method, decision makers should remain transparent in how health equity considerations influenced their final determination.

Cell-Free Fetal Deoxyribonucleic Acid (cffDNA) Analysis as a Remarkable Method of Non-Invasive Prenatal Screening

The cell-free fetal DNA (cffDNA) analysis for screening fetal genetic anomalies has increased dramatically since its commercialization in 2011 worldwide. In the early weeks of pregnancy, it offers a hassle-free, non-invasive procedure of antenatal screening. It guides and protects mothers from undergoing unwanted risk-laden invasive prenatal testing. cffDNA testing is accurate at detecting the abnormal fetus chromosome among a large pool population. Patau syndrome, Edward syndrome, and Down syndrome are currently being accurately screened by this method. Due to their sensitivity and specificity, they now have become the screening method of choice, approaching almost 100% in various studies with a large sample pool. The latest procedures to analyze cffDNA, like the new digital droplet polymerase chain reaction (ddPCR) and sophisticated next-generation sequencing (NGS), haveincreased detection rates with decreased analyzing time. The latest techniques make it possible to screen large numbers of the population with faster report generation. Screening for Rh incompatibility and its timely prevention is now more accessible and more accurate with the help of cffDNA analysis. The problem arises when we deviate from the primary disease and start testing for anomalies not intended to be screened by cffDNA in the first place. Fetal sex chromosome aneuploidy screening by cffDNA is one area where the test gives mixed results either due to differences in machinery, laboratory parameters, or human error. Other rare occurrences like trisomes, such as trisomy 7, trisomy 16, trisomy 22, and a few microdeletion syndromes are also being screened but with less accuracy. Like every technology, cffDNA analysis is not entirely free of criticism. Its high testing cost, potential to accurately prognosticate the gender of the developing fetus and absence of standard testing practices will become an issue as the test becomes routine worldwide.

Multisite assessment of the impact of cell-free DNA-based screening for rare autosomal aneuploidies on pregnancy management and outcomes.

Cell-free (cf) DNA screening is a noninvasive prenatal screening approach that is typically used to screen for common fetal trisomies, with optional screening for sex chromosomal aneuploidies and fetal sex. Genome-wide cfDNA screening can screen for a wide variety of additional anomalies, including rare autosomal aneuploidies (RAAs) and copy number variants. Here, we describe a multi-cohort, global retrospective study that looked at the clinical outcomes of cases with a high-risk cfDNA screening result for a RAA. Our study cohort included a total of 109 cases from five different sites, with diagnostic outcome information available for 68% (74/109) of patients. Based on confirmatory diagnostic testing, we found a concordance rate of 20.3% for presence of a RAA (15/74) in our study population. Pregnancy outcome was also available for 77% (84/109) of cases in our cohort. Many of the patients experienced adverse pregnancy outcomes, including intrauterine fetal demise, fetal growth restriction, and preterm birth. These adverse outcomes were observed both in patients with fetal or placental confirmation of the presence of a RAA, as well as patients that did not undergo fetal and/or placental diagnostic testing. In addition, we have proposed some suggestions for pregnancy management and counseling considerations for situations where a RAA is noted on a cfDNA screen. In conclusion, our study has shown that genome-wide cfDNA screening for the presence of rare autosomal aneuploidies can be beneficial for both patients and their healthcare practitioners. This can provide a possible explanation for an adverse pregnancy outcome or result in a change in pregnancy management, such as increased monitoring for adverse outcomes.

Ethical issues associated with prenatal screening using non-invasive prenatal testing for sex chromosome aneuploidy.

Prenatal screening for sex chromosome aneuploidies (SCAs) is increasingly available through expanded non-invasive prenatal testing (NIPT). NIPT for SCAs raises complex ethical issues for clinical providers, prospective parents and future children. This paper discusses the ethical issues that arise around NIPT for SCAs and current guidelines and protocols for management. The first section outlines current practice and the limitations of NIPT for SCAs. It then outlines key guidelines before discussing the ethical issues raised by this use of NIPT. We conclude that while screening for SCAs should be made available for people seeking to use NIPT, its implementation requires careful consideration of what, when and how information is provided to users.

Detection of maternal carriers of common α-thalassemia deletions from cell-free DNA

α-Thalassemia is a common inherited blood disorder manifested mainly by the deletions of α-globin genes. In geographical areas with high carrier frequencies, screening of α-thalassemia carrier state is therefore of vital importance. This study presents a novel method for identifying female carriers of common α-thalassemia deletions using samples routinely taken for non-invasive prenatal tests for screening of fetal chromosomal aneuploidies. A total of 68,885 Vietnamese pregnant women were recruited and α-thalassemia statuses were determined by gap-PCR, revealing 5344 women (7.76%) carried deletions including αα/−−SEA (4.066%), αα/−α3.7 (2.934%), αα/−α4.2 (0.656%), and rare genotypes (0.102%). A two-stage model was built to predict these α-thalassemia deletions from targeted sequencing of the HBA gene cluster on maternal cfDNA. Our method achieved F1-scores of 97.14–99.55% for detecting the three common genotypes and 94.74% for detecting rare genotypes (−α3.7/−α4.2, αα/−−THAI, −α3.7/−−SEA, −α4.2/−−SEA). Additionally, the positive predictive values were 100.00% for αα/αα, 99.29% for αα/−−SEA, 94.87% for αα/−α3.7, and 96.51% for αα/−α4.2; and the negative predictive values were 97.63%, 99.99%, 99.99%, and 100.00%, respectively. As NIPT is increasingly adopted for pregnant women, utilizing cfDNA from NIPT to detect maternal carriers of common α-thalassemia deletions will be cost-effective and expand the benefits of NIPT.

Noninvasive prenatal screening in southeast China: clinical application and accuracy evaluation

ABSTRACT Objective To assess the clinical performance of noninvasive prenatal screening (NIPS) for both common trisomy and sex chromosome aneuploidy (SCA). Methods We recruited 71,888 pregnant women to undergo NIPS testing from December 2015 to June 2021. Demographic characteristics, diagnostic results, and follow-up outcomes were collected. Results There were a total of 381 high-risk cases for common trisomy and 343 positive screens for SCA. Invasive prenatal diagnosis (IPD) was performed in 507 (70.0%) participants. The positive predictive value (PPV) was 83.7% for T21, 72.5% for T18, 14.3% for T13, 31.9% for 45,X, 72.0% for 47,XXX, 89.8% for 47,XXY, and 72.2% for 47,XYY, respectively. Logistic regression analysis presented a significant association between Z-score and PPV in common trisomy (P < 0.05) while not in SCA (P > 0.05). PPV in the high-risk group (Z-score ≥ cutoff) was superior to that in the intermediate risk group (3 ≤ Z-score < cutoff) for T21/T18/T13. PPV for 45,X, 47,XXY, and 47,XYY tended to be higher with the increasing Z-score, except for 47,XXX. Conclusions NIPS would be a valuable strategy in prenatal screening, while cautions should be kept in mind for subsequent genetic consulting about the risk of Z-score.

15. Clinical validation of non-invasive prenatal screening using synthetic plasmas

Non-invasive prenatal screening (NIPS) is a highly sensitive first trimester screening for chromosome aneuploidies and certain other abnormalities in pregnancy. Implementation of NIPS in a clinical laboratory may encounter technical difficulties, such as lack of established validation guidelines, bioinformatic pipeline, and positive validation plasmas. In particular, it is difficult to collect sufficient positive specimens in a short period. Here, we describe an effective way to synthesize positive plasmas.

The Next Frontier in Noninvasive Prenatal Diagnostics: Cell-Free Fetal DNA Analysis for Monogenic Disease Assessment.

With the widespread clinical adoption of noninvasive screening for fetal chromosomal aneuploidies based on cell-free DNA analysis from maternal plasma, more researchers are turning their attention to noninvasive prenatal assessment for single-gene disorders. The development of a spectrum of approaches to analyze cell-free DNA in maternal circulation, including relative mutation dosage, relative haplotype dosage, and size-based methods, has expanded the scope of noninvasive prenatal testing to sex-linked and autosomal recessive disorders. Cell-free fetal DNA analysis for several of the more prevalent single-gene disorders has recently been introduced into clinical service. This article reviews the analytical approaches currently available and discusses the extent of the clinical implementation of noninvasive prenatal testing for single-gene disorders.

EP450: Effectiveness and utility of noninvasive prenatal screening (NIPS) for sex chromosome aneuploidies

Noninvasive prenatal screening (NIPS) has high sensitivity and specificity for the common autosomal aneuploidies. However, compared to these autosomal trisomies, the few studies assessing NIPS for detecting sex chromosome aneuploidy (SCA) have shown higher false positive rates; particularly for monosomy X. The primary goal of this study was to determine the effectiveness and utility of NIPS as a tool for recognizing SCA; we also discuss uptake of diagnostic testing. Over 39 months (August 2018-October 2021), we performed NIPS for 1438 specimens. Overall, the proportion of specimens with any high-risk result was 2.6% (37/1438). Of those, autosomal aneuploidy was prognosticated in 2.09% (30/1438), while SCA was suspected in 0.49% (7/1438) of patients, with similar frequencies of abnormal results being published among other laboratories performing this test. Of the seven pregnancies at high risk for SCA, three were high risk for Turner syndrome (45,X); three for Klinefelter syndrome (47,XXY); and one for Triple X (47,XXX). None of the patients with high-risk results for SCA elected to have additional prenatal diagnostic testing. Also, no patients elected to terminate pregnancies based on NIPS SCA results. Follow-up karyotype analysis was available for four of these seven cases: three newborns and one product of conception (POC) specimen. Of the three pregnancies at high risk for monosomy X, two resulted in miscarriage; with one having follow-up genetic testing that confirmed monosomy X (true positive). The third high-risk monosomy X case resulted in a liveborn. Subsequent testing of the newborn including FISH, karyotype and microarray yielded results consistent with a normal female complement (false positive). Of the three pregnancies at high risk for 47,XXY, all resulted in live births, with two confirmed as true positives in the newborns (the third did not pursue testing). The case at high risk for 47,XXX was reported as phenotypically normal but no diagnostic testing was performed. Seven patients received high-risk results for SCA; however, none elected to pursue diagnostic testing prenatally via amniocentesis or CVS. Follow-up testing was available in four of the seven cases. Three were true positive (two 47,XXY and one 45,X) and one was a false positive (45,X). Calculation of the test specifications (sensitivity, specificity, PPV, NPV) for SCAs is limited due to lack of confirmatory genetic testing and limited phenotypes observed in the newborn period (and beyond) for some conditions. NIPS allows for earlier diagnosis of SCA, which could aid in behavioral interventions or fertility concerns/management for people with SCAs.

Biochemical Investigation Limits in Recognizing Some Abnormal Hemoglobin Types: Hemoglobins O Arab And S

Hemoglobinopathies are a major health problem worldwide. These disorders are characterized by a variable clinical and hematological situation related to a phenotypic heterogeneity. Moreover, in order to have an exact correlation between the biochemical picture and the genetic defect associated with it, it is useful and often indispensable the molecular study of alpha and beta globin genes. The present case report concerns a pregnant woman of Moroccan ethnicity who came to our observation to undergo combined first-trimester testing useful for screening of major chromosomal aneuploidies. Study of hemoglobins A, A2, and F by High performance liquid chromatography (HPLC) revealed the presence of a proportion of abnormal hemoglobin associated with hemoglobin S. Molecular investigation of globin genes excluded that the biochemical variant in question was related to hemoglobin S. In fact, molecular investigation of beta globin genes, found that the observed variant was due to the presence of a genetic defect leading to hemoglobin O Arab synthesis. Evidence suggests that molecular analysis of globin genes provides the most effective and correct way to correlate the detected biochemical picture with its associated genetic defect. The only biochemical study in the presented case determines an incorrect clinical evaluation with consequent inaccurate prognosis. The mutation detected in this work can be identified using a simple and inexpensive kit. This would generate, in economic terms, significant savings associated with a correct diagnosis

Reply

We thank Drs Jing and Li for their thoughtful comments on our study1 and their reflections on prenatal screening for 22q11.2 deletion. They raise important points on which we would like to comment further and provide clarification. Drs Jing and Li suggest that ultrasound is more sensitive than cell-free DNA (cfDNA) testing in screening for 22q11.2 deletion, citing a study by Schindewolf et al.2. However, that study was intended to document the ultrasound findings seen most frequently in pregnancies with a 22q11.2 deletion in the second and third trimesters, not to determine the sensitivity of ultrasound for fetal 22q11.2 deletion. To our knowledge, this has not been evaluated in any study. Nonetheless, we agree that prenatal ultrasound findings have been well described by Schindewolf et al. and others and suggest that cfDNA screening in the first trimester can complement, not replace, second-trimester ultrasound as a screening tool for 22q11.2 deletion. As Drs Jing and Li note, the collective prevalence of other fetal microduplications and microdeletions is not insignificant, even when ultrasound is normal, and there is broad consensus that chorionic villus sampling or amniocentesis with microarray analysis should be offered whenever evaluation of a pregnancy for submicroscopic genomic alterations is desired. Similar to Drs Jing and Li, we would also welcome a method for accurate, genome-wide non-invasive prenatal screening with demonstrated clinical utility for detecting chromosomal imbalances at a resolution comparable to that of microarray, but currently available technologies fall short of this goal. In a prospective head-to-head comparison of genome-wide cfDNA testing and chromosomal microarray testing in 198 pregnancies, Guseh and colleagues showed that expanded non-invasive genome-wide screening has a lower detection rate for clinically significant chromosomal abnormalities when compared with that of chromosomal microarray analysis, and that pathogenic subchromosomal imbalances (< 7 Mb) contribute substantially to the missed abnormalities3. A clinical trial from The Eunice Kennedy Shriver National Institute of Child Health and Human Development showed that > 50% of (likely) pathogenic subchromosomal imbalances identified by chromosomal microarray analysis in anatomically normal fetuses are < 5 Mb in size4. We would also like to clarify a minor point. The cfDNA test evaluated in our study is described as ‘targeted’ in order to differentiate it from ‘genome-wide’ cfDNA testing because of the different technological design of the two approaches. It is not a standalone test for 22q11.2 deletion, as might have been misinterpreted from the phrasing. The Harmony® test screens for trisomy 21, trisomy 18 and trisomy 13, with the option of screening for sex chromosome aneuploidy and/or 22q11.2 deletion from the same blood collection. In many respects, we agree with Drs Jing and Li and support an approach to prenatal screening in which the benefits of cfDNA testing, ultrasound and diagnostic testing are incorporated to guide pregnancy management and improve outcome in 22q11.2 deletion syndrome and other prenatally diagnosable conditions.

O-237 A non-invasive approach for aneuploidy analysis in clinical miscarriages

Abstract Study question Is maternal cell-free DNA (cfDNA) testing a feasible alternative to the analysis of the product of conception (POC) in clinical miscarriages? Summary answer This study demonstrates that genome-wide cfDNA testing in the maternal bloodstream constitutes a reliable tool to analyse chromosome aneuploidies in clinical miscarriages. What is known already It is well established that 50-70% of clinical miscarriages are caused by numerical chromosomal anomalies (aneuploidies), mostly trisomies. To date, conventional cytogenetic and advanced molecular techniques are used for the analysis of POC to identify the genetic cause of miscarriage, providing valuable information for genetic counselling. However, both approaches are based in the direct analysis of the abortive tissue, which entails several limitations due to the risk of culture failure and/or maternal cell contamination. To solve these drawbacks, maternal cfDNA testing emerges as a promising alternative due to the accumulated evidence. Study design, size, duration This was a retrospective study conducted in a reference genetic laboratory from January to December 2020. Before carrying out the foetal tissues collection that precludes the POC analysis, a blood sample was drawn to evaluate possible aneuploidies by cfDNA testing. Using NGS+STR POC results as the gold standard, results derived from both studies were compared to assess the percentage of concordance and the cases of non-informativeness (foetal fraction (FF) &amp;lt;2%), false positives, and false negatives. Participants/materials, setting, methods A total of 12 cases were included in the study. cfDNA testing in the mother’s blood was performed by using Illumina’s technology platform. Genetic testing for POC was done using an NGS technology (Thermo Fisher Scientific, USA) for 24 chromosome aneuploidy screening. Short-tandem repeat (STR) analysis allowed us to detect or rule out maternal cell contamination (MCC) and some types of polyploidies. Main results and the role of chance The non-informative rate for both analysis techniques was 9.1% (1 out of 12 cases: 1 low FF for cfDNA testing and maternal cell contamination for POC analysis). The median cfDNA FF was 9.0%. Using the molecular POC analysis as gold standard, the concordance rate between both studies was 90.0% (9 out of 10 cases;1 monosomy X, 1 trisomy (T) 21, 1 T22, 1 T11 and 5 patients with no alteration detected). No mosaics or structural rearrangements were identified by either of the two analysis techniques. The only discordant result was a case in which cytogenetics of POCs identified a triploidy. This discordancy is expected since triploidies are outside the scope of cfDNA testing. Also, foetal sex was correctly assigned in all informative cases. The sensibility and specificity of the study were estimated at 80.0 (4/5) and 100.0% (6/6), respectively. Statistics analysis suggested that no significant difference was found between both techniques regarding the aneuploidy detection ability (P=0.5). These promising results indicate that genome-wide cfDNA-based screening provides a non-invasive approach for determining whether foetal aneuploidy could explain the loss in patients experiencing early o recurrent pregnancy loss (RPL). Limitations, reasons for caution The sample size prevents drawing more significant conclusions regarding the diagnosis power similarity between both testing techniques. Therefore, a larger cohort will be essential to improve confirm the cfDNA testing performance. Current cfDNA testing technology fails in polyploidy identification, which is a potential cause of pregnancy loss. Wider implications of the findings CfDNA testing could be an alternative to POC analysis in clinical miscarriage. If optimized, cfDNA testing could be used contingently with the molecular POC analysis in cases where maternal cell contamination is present. As a result, the overall success rate in the POC program could be substantially improved. Trial registration number NA

Results of non-invasive prenatal testing for 2473 women with twin pregnancy

To assess the value of non-invasive prenatal testing (NIPT) for the detection of fetal chromosomal aneuploidies in women with twin pregnancy. A total of 2473 women with twin pregnancy underwent the NIPT test to assess the risk for fetal chromosomal aneuploidies from January 2016 to September 2019. Those with a high risk by NIPT were confirmed by amniocentesis or chorionic villus sampling. All cases were followed up to evaluate the positive prediction value of NIPT for twin pregnancies. Among the 2473 women, the NIPT test has identified 31 cases (1.25%) with a high risk for fetal chromosomal aneuploidies, which included 5 cases of trisomy 21, 1 case of chromosome 21 deletion, 4 cases of trisomy 18, 7 cases of sex chromosome abnormality and 14 cases of microdeletion and microduplication. By invasive prenatal diagnosis or chromosomal karyotyping analysis of neonates, 5 cases of trisomy 21, 3 cases of trisomy 18, 1 case of sex chromosome abnormality, and 2 cases of microdeletion and microduplication were confirmed, which yielded a positive predictive value of 100%, 75%, 25% and 25%, respectively. NIPT can be used for the screening of fetal chromosomal aneuploidies in women with twin pregnancy with high accuracy. The method is non-invasive, safe and effective for the screening of fetal chromosomal aneuploidies, in particular trisomy 21.

CfDNA deconvolution via NIPT of a pregnant woman after bone marrow transplant and donor egg IVF

Cell-free DNA is known to be a mixture of DNA fragments originating from various tissue types and organs of the human body and can be utilized for several clinical applications and potentially more to be created. Non-invasive prenatal testing (NIPT), by high throughput sequencing of cell-free DNA (cfDNA), has been successfully applied in the clinical screening of fetal chromosomal aneuploidies, with more extended coverage under active research.In this study, via a quite unique and rare NIPT sample, who has undergone both bone marrow transplant and donor egg IVF, we investigated the sources of oddness observed in the NIPT result using a combination of molecular genetics and genomic methods and eventually had the case fully resolved. Along the process, we devised a clinically viable process to dissect the sample mixture.Eventually, we used the proposed scheme to evaluate the relatedness of individuals and the demultiplexed sample components following modified population genetics concepts, exemplifying a noninvasive prenatal paternity test prototype. For NIPT specific applicational concern, more thorough and detailed clinical information should therefore be collected prior to cfDNA-based screening procedure like NIPT and systematically reviewed when an abnormal report is obtained to improve genetic counseling and overall patient care.

Non-invasive Prenatal Test - A Pilot Pan-India Experience of an Indian Laboratory

Background: Serum-based tests are considered to be a very affordable screening option for pregnant women. The non-invasive prenatal test (NIPT), among prenatal screening for chromosomal aneuploidies has a sensitivity of >99%. The focus of this report is to present an overview of NIPT outcome from samples tested in our national laboratory over a period of six months. Materials and Methods: NIPT was done by whole genome shot gun sequencing and a total of 513 pregnant women were tested in our laboratory. The samples were analyzed for reporting on risk of trisomy 21, 18 and 13 as well as sex chromosome aneuploidies. Results: A total of 513 blood samples were received for NIPT testing and 9.3% were cancelled at the pre-analytical stage. Frequency of routine and IVF pregnancies was detected to be 83% and 12% respectively. Frequency of high risk including trisomy 21, trisomy 18 and sex chromosome aneuploidies was detected to be at 3.0% respectively. A total of 7% of cases which were reported high risk by serum screening were detected to be low risk by NIPT. Conclusion: NIPT is an efficient aneuploidy screening tool as it studies fetal DNA and our analysis indicates that the same can be beneficial in screening for fetal chromosomal risk with high sensitivity to identify cases which need confirmatory tests.

Evolution and Utility of Preimplantation Genetic Testing for Monogenic Disorders in Assisted Reproduction - A Narrative Review.

Preimplantation genetic testing (PGT) for monogenic disorders and assisted reproductive technology have evolved and progressed in tandem. PGT started with single-cell polymerase chain reaction (PCR) followed by fluorescent in situ hybridisation for a limited number of chromosomes, later called ‘preimplantation genetic diagnosis (PGD) version 1’. This review highlights the various molecular genetic techniques that have evolved to detect specific inherited monogenic disorders in the preimplantation embryo. Literature review in English was performed in PubMed from 1990 to 2021, using the term ‘preimplantation genetic diagnosis’. With whole-genome amplification, multiple copies of embryonic DNA were created. This helped in avoiding misdiagnosis caused by allele dropout. Multiplex fluorescent PCR analysed informative short tandem repeats (STR) and detected mutations simultaneously on automated capillary electrophoresis sequencers by mini-sequencing. Comparative genomic hybridisation (CGH) and array CGH were used for 24 chromosome aneuploidy screening. Subsequently, aneuploidies were detected by next-generation sequencing using single-nucleotide polymorphism arrays, while STR markers were used for haplotyping. ‘PGD version 2’ included accurate marker-based diagnosis of most monogenic disorders and detection of aneuploidy of all chromosomes. Human leukocyte antigen matching of embryos has important implications in diagnosis and cure of haemoglobinopathies and immunodeficiencies in children by means of matched related haematopoietic stem cell transplantation from an unaffected ‘saviour sibling’ obtained by PGT.