Invasive Genetic Testing Research Articles

Invasive genetic testing for isolated increased nuchal translucency of 3.0-3.4 mm: Results from cohort analysis with 604 fetuses.

SynopsisIsolated increased nuchal translucency of 3.0–3.4 mm was associated with increased risk of chromosomal anomalies and monogenic syndromes.

Temporal Evaluation of a Minimally Invasive Method of Preimplantation Genetic Testing for Aneuploidy (mi-PGT-A) in Human Embryos

Preimplantation genetic testing for aneuploidy (PGT-A) has become a useful approach for embryo selection following IVF and ICSI. However, the biopsy process associated with PGT-A is expensive, prone to errors in embryo ploidy determination, and potentially damaging, impacting competence and implantation potential. Therefore, a less invasive method of PGT-A would be desirable and more cost-effective. Noninvasive methods for PGT-A (ni-PGT-A) have been well-studied but present limitations in terms of cf-DNA origin and diagnostic accuracy. Minimally invasive pre-implantation genetic testing (mi-PGT-A) for frozen-thawed embryo transfer is a promising, less studied approach that utilizes a combination of spent culture media (SCM) and blastocoelic fluid (BF)-derived cell-free (CF)-DNA for genetic testing. This study aimed to optimize the effectiveness of mi-PGT-A for aneuploidy diagnosis by investigating the optimal temporal sequence for this protocol. SCM+BF was collected at either 48 or 72 h of culture after thawing day 3 preimplantation embryos. cf-DNA in the SCM+BF was amplified, analyzed by next-generation sequencing (NGS) and compared with results from the corresponding whole embryos (WEs) obtained from human embryos donated for research. Fifty-three (42 expanded blastocysts, 9 early blastocysts, and 2 morula) WE and SCM+BF samples were analyzed and compared. The overall concordance rate between SCM+BF and WE was 60%. Gender and ploidy concordance improved with extended culture time from 48 h (73% and 45%) to 72 h (100% and 64%), respectively. These results demonstrate that SCM+BF-derived cf-DNA can be successfully used for mi-PGT-A. Our findings indicate that longer embryo culture time prior to SCM+BF-derived cf-DNA analysis improves DNA detection rate and concordance with WEs and decreases the proportion of false positive results.

Noninvasive twin genotyping for recessive monogenic disorders by relative haplotype dosage.

To establish a haplotype-based noninvasive prenatal testing (NIPT) workflow for single-gene recessive disorders that adapt to dizygotic (DZ) twin pregnancies. Twin pregnancies at risk of Duchenne muscular dystrophy, Becker muscular dystrophy, hemophilia B, spinal muscular atrophy, phenylketonuria, and nonsyndromic hearing loss were recruited. For subsequent analysis, capture sequencing targeting highly heterozygotic single nucleotide polymorphism sites was conducted. Paternal-specific alleles were used to calculate the total and individual fetal fractions and determine zygosity. A two-step Bayes Factor model was applied to clarify the complex genomic landscape in the maternal plasma: the first step involved determining whether the twins inherited the same haplotype, and the second step involved estimating their individual genotypes. NIPT results were subsequently confirmed by invasive diagnosis. Nine twin pregnancies were recruited, including five DZ and four monozygotic (MZ) twins. The earliest gestational age was 8+0 weeks, and the minimum fetal fraction was 4.6%. Three twin pregnancies were reported with one affected fetus, while the remaining six were reported without affected fetuses. Two dichorionic diamniotic twin pregnancies were confirmed to be MZ twins. The NIPT results were 100% consistent with those of invasive procedures or diagnostic genetic testing after birth. This study is the first to perform NIPT for single-gene disorders in twin pregnancies and preliminarily confirm its clinical feasibility. Acknowledging the twins' genotypes in the first trimester is valuable as it empowers obstetric care providers and parents to have adequate time for pregnancy management and decision-making.

Association analysis between chromosomal abnormalities and fetal ultrasonographic soft markers based on 15,263 fetuses

Soft markers are common prenatal ultrasonographic findings that indicate an increased risk for fetal aneuploidy. However, the association between soft markers and pathogenic or likely pathogenic copy number variations is still unclear, and clinicians lack clarity on which soft markers warrant a recommendation for invasive prenatal genetic testing of the fetus. This study aimed to provide guidance on ordering prenatal genetic testing for fetuses with different soft markers and to elucidate the association between specific types of chromosomal abnormalities and specific ultrasonographic soft markers. Low-pass genome sequencing was performed for 15,263 fetuses, including 9123 with ultrasonographic soft markers and 6140 with normal ultrasonographic findings. The detection rate of pathogenic or likely pathogenic copy number variants among fetuses with various ultrasonographic soft markers were compared with that of fetuses with normal ultrasonography. The association of soft markers with aneuploidy and pathogenic or likely pathogenic copy number variants were investigated using Fisher exact tests with Bonferroni correction. The detection rate of aneuploidy and pathogenic or likely pathogenic copy number variants was 3.04% (277/9123) and 3.40% (310/9123), respectively, in fetuses with ultrasonographic soft markers. An absent or a hypoplastic nasal bone was the soft marker in the second trimester with the highest diagnostic rate for aneuploidy of 5.22% (83/1591) among all isolated groups. Four types of isolated ultrasonographic soft markers, namely a thickened nuchal fold, single umbilical artery, mild ventriculomegaly, and absent or hypoplastic nasal bone, had higher diagnostic rates for pathogenic or likely pathogenic copy number variants (P<.05; odds ratio, 1.69-3.31). Furthermore, this study found that the 22q11.2 deletion was associated with an aberrant right subclavian artery, whereas the 16p13.11 deletion, 10q26.13-q26.3 deletion, and 8p23.3-p23.1 deletion were associated with a thickened nuchal fold, and the 16p11.2 deletion and 17p11.2 deletion were associated with mild ventriculomegaly (P<.05). Ultrasonographic phenotype-based genetic testing should be considered in clinical consultations. Copy number variant analysis is recommended for fetuses with an isolated thickened nuchal fold, a single umbilical artery, mild ventriculomegaly, and an absent or a hypoplastic nasal bone. A comprehensive definition of genotype-phenotype correlations in aneuploidy and pathogenic or likely pathogenic copy number variants could provide better information for genetic counseling.

Double aortic arch: implications of antenatal diagnosis, differential growth of arches during pregnancy, associated abnormalities and postnatal outcome.

To evaluate the prenatal characteristics of double aortic arch (DAA), assess the relative size of the arches and their growth during pregnancy, describe associated cardiac, extracardiac and chromosomal/genetic abnormalities and review postnatal presentation and clinical outcome. This was a retrospective cohort study of all fetuses with a confirmed diagnosis of DAA seen in five specialized referral centers in London, UK, between October 2012 and November 2019. Cases were identified from the hospitals' fetal databases. Fetal echocardiographic findings, intracardiac and extracardiac abnormalities, genetic defects, computed tomography (CT) findings and postnatal clinical presentation and outcome were evaluated. A total of 79 fetuses with DAA were included. Of those assessed postnatally, 48.6% had an atretic left aortic arch (LAA), while 5.1% had an atretic LAA at the first fetal scan and were misdiagnosed antenatally with right aortic arch (RAA). The LAA was atretic in 55.8% of those who underwent CT. DAA was an isolated abnormality in 91.1% of cases; 8.9% of patients had an additional intracardiac abnormality and 2.5% had both intra- and extracardiac abnormalities. Among the 52 cases that underwent genetic testing, 11.5% had genetic abnormalities and, specifically, the 22q11 microdeletion was identified in 3.8% of patients. At a median follow-up of 993.5 days, 42.5% of patients had developed symptoms of tracheoesophageal compression (5.5% during the first month after birth) and 56.2% had undergone intervention. Statistical analysis using the χ-square test showed no significant relationship between morphology of DAA (patency of both aortic arches vs atretic LAA) and the need for intervention (P = 0.134), development of vascular ring symptoms (P = 0.350) or evidence of airway compression on CT (P = 0.193). Most cases of DAA can be diagnosed easily at midgestation, as typically both arches are patent with a dominant RAA at this stage. However, we found that the LAA had become atretic in approximately half of the cases postnatally, supporting the theory of differential growth of the arches during pregnancy. DAA is usually an isolated abnormality; however, thorough assessment is required to exclude associated intra- and extracardiac anomalies and to determine the need for invasive prenatal genetic testing. Postnatally, early clinical assessment is needed and CT scan should be considered, irrespective of the presence of symptoms. © 2023 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Embryo ploidy status classification through computer-assisted morphology assessment

Preimplantation genetic testing for aneuploidy has been proven to be effective in determining the embryo's chromosomal or ploidy status. The test requires a biopsy of embryonic cells on day 3, 5, or 6 from which complete information on the chromosomes would be obtained. The main drawbacks of preimplantation genetic testing for aneuploidy include its relatively invasive approach and the lack of research studies on the long-term effects of preimplantation genetic testing for aneuploidy. Computer-assisted predictive modeling through machine learning and deep learning algorithms has been proposed to minimize the use of invasive preimplantation genetic testing for aneuploidy. The capability to predict morphologic characteristics of embryo ploidy status creates a meaningful support system for decision-making before further treatment. Image processing is a component in developing a predictive model specialized in image classification through which a model is able to differentiate images based on unique features. Image processing is obtained through image augmentation to capture segmented embryos and perform feature extraction. Furthermore, multiple machine learning and deep learning algorithms were used to create prediction-based modeling, and all of the prediction models undergo similar model performance assessments to determine the best model prediction algorithm. An efficient artificial intelligence model that can predict embryo ploidy status was developed using image processing through a histogram of oriented gradient and then followed by principal component analysis. The gradient boosting algorithm showed an advantage against other algorithms and yielded an accuracy of 0.74, an aneuploid precision of 0.83, and an aneuploid predictive value (recall) of 0.84. This research study proved that machine-assisted technology perceives the embryo differently than human observation and determined that further research on in vitro fertilization is needed. The study finding serves as a basis for developing a better computer-assisted prediction model.

Pregnancy outcomes and prenatal traditional karyotype analysis with fetal omphalocele

Omphalocele is associated with many aneuploidies, deletions and congenital anomalies. This study evaluates pregnancies diagnosed with omphalocele and its relevance to concomitant genetic disorders. The data of patients with the intrauterine diagnosis of omphalocele who had invasive diagnostic testing performed between January 2017 and January 2020 were evaluated retrospectively. The traditional karyotype analysis was performed to prenatal diagnosis for all fetuses. During the study period, all patients were scanned via ultrasonography by an experienced perinatologist, prenatally. We evaluated 22 cases of omphalocele whose genetic testing results were available. The mean maternal age was 25 (18-41) years. The median gestational week at diagnosis was 13 (11-22). Invasive genetic testing revealed aneuploidy in 7 patients (31.8%), 2 with trisomy 13 (9.1%), and 5 with trisomy 18 (22.8%). There were 5 fetuses (22.7%) that had extracorporeal liver: 1 had trisomy 18 (20%), 1 had trisomy 13 (20%), and the other 3 fetuses had a normal karyotype (60%). Further, 14 (63.6%) pregnancies were terminated: 4 had trisomy 18 (28.6%), 1 had trisomy 13 (7.1%), and 9 of the terminated pregnancies (64.3%) had additional congenital anomalies. There were 4 infants who died (50%) born from 8 patients who decided to continue with their pregnancy. The omphalocele sac of 1 infant spontaneously regressed in the ensuing weeks of pregnancy who is now 1 year old. The chromosomal abnormalities presented in up to 31.8% of cases diagnosed with omphalocele. Moreover, for cases with normal genetic testing results, the propensity for additional structural defects was high and the prognosis remains poor. Counseling parents to consider their option of terminating the pregnancy is appropriate.

Fetal genetic findings by chromosomal microarray analysis and karyotyping for fetal growth restriction without structural malformations at a territory referral center: 10-year experience

BackgroundPrenatal invasive genetic testing is commonly recommended to pregnancies of early-onset FGR or FGR combined with a structural defect. Our study aimed to explore the genetic findings for FGR without structural malformations according to cytogenetic karyotyping and single nucleotide polymorphism array (SNP array) technology over a 10-year period.MethodsA total of 488 pregnancies diagnosed with FGR without structural malformation were retrospectively reviewed. Cytogenetic karyotyping was performed on all the subjects, and SNP array was available from 272 of them. Based on the gestational age at onset, the cohort was classified into four groups: ≤ 24, 25–28, 29–32, and > 32 weeks of gestation. According to the ultrasound findings, they were grouped into isolated FGR, FGR with soft markers, and FGR with non-structural anomalies. In pregnancies of young maternal age, based on the results of maternal serum screening (MSS), they were categorized into high-risk and low-risk MSS groups.ResultsNineteen (3.9%) cases of chromosomal abnormalities were detected by cytogenetic karyotyping, including 11 cases of numerical abnormalities, 5 cases of structural abnormalities, and 3 cases of mosaicism. Trisomy 21 was the most frequent abnormality. Abnormal karyotypes were more frequently observed in cases diagnosed at ≤ 24 weeks (7.2%) than those in any other group. Among pregnancies with normal karyotype, an incremental yield of 4.2% were revealed by SNP array technology regarding clinically relevant aberrations. The additional detection rates by SNP array in cases diagnosed at ≤ 24 weeks (6.5%), cases with soft markers (9.5%), and cases with high-risk MSS (12.0%) were higher than those in other groups within each classification. All the cases with abnormal karyotypes and 7 out of 11 pregnancies with clinically relevant anomalies revealed by SNP array alone resulted in pregnancy terminations.ConclusionChromosome abnormality is an important etiology for FGR with no associated structural malformations, and plays a crucial role in pregnancies decision-making. SNP array improves the detection of genetic anomalies especially in FGR diagnosed at ≤ 24 weeks, FGR combined with soft makers, and FGR combined with high-risk MSS.

Prediction of live birth - selection of embryos using morphokinetic parameters.

The goal of assisted reproduction is for a couple treated with IVF techniques to end the treatment by giving birth to a healthy baby. A neccessary presumption for success is the identification of the best embryo with high implantation and developmental potential. One option is to select an euploid embryo by invasive preimplantaion genetic testing for aneuploidy (PGT-A) or it is possible to select the best embryo by non-invasive time-lapse monitoring (TLM), specifically based on morphokinetic parameters and morphological markers that are able to identify an embryo with high developmental potential. The study involved a total of 1060 embryos (585 euploid and 475 aneuploid embryos after PGT-A) with good morphology from 329 patients in the period 01/2016-10/2021. All embryos were cultured in a time-lapse incubator, trophectoderm (TE) cells biopsies for PGT-A examination were performed on day 5 (D5) or day 6 (D6) of culture. During the study period, 225 frozen embryo transfers (FET) of one euploid embryo were performed. Based on the treatment outcome, the embryos were divided into 2 groups - euploid embryos, which led to the birth of a healthy child, and euploid embryos that did not show fetal heartbeat (FHB) after FET. Based on the statistical analysis of the embryos without implantation and the embryos with live birth, it is clear that the morphokinetic parameters t5 (time of division into 5 cells) and tSB (time of start of blastulation) are significantly different. The results suggest that of the morphokinetic parameters tSB and t5 are predictive indicators for selecting an embryo with high developmental potential and with a high probability of achieving the birth of a healthy child.

Fetal cerebral ventriculomegaly: What do we tell the prospective parents?

Fetal cerebral ventriculomegaly is a relatively common finding, observed during approximately 1% of obstetric ultrasounds. In the second and third trimester, mild (≥10mm) and severe ventriculomegaly (≥15mm) are defined according to the measurement of distal lateral ventricles that is included in the routine sonographic examination of central nervous system. A detailed neurosonography and anatomy ultrasound should be performed to detect other associated anomalies in the central nervous system and in other systems, respectively. Fetal MRI might be useful when neurosonography is unavailable or suboptimal. The risk of chromosomal and non-chromosomal genetic disorders associated with ventriculomegaly is high, therefore invasive genetic testing, including microarray, is recommended. Screening for prenatal infections, in particular cytomegalovirus and toxoplasmosis, should also be carried out at diagnosis. The prognosis is determined by the severity of ventriculomegaly and/or by the presence of co-existing abnormalities. Fetal ventriculoamniotic shunting in progressive isolated severe ventriculomegaly is an experimental procedure. After delivery, ventricular-peritoneal shunting or ventriculostomy are the two available options to treat hydrocephalus in specific conditions with similar long-term outcomes. A multidisciplinary fetal neurology team, including perinatologists, geneticists, pediatric neurologists, neuroradiologists and neurosurgeons, can provide parents with the most thorough prenatal counseling. This review outlines the latest evidence on diagnosis and management of pregnancies complicated by fetal cerebral ventriculomegaly.

Contribution of chromosomal microarray analysis and next-generation sequencing to genetic diagnosis in fetuses with normal karyotype.

The aim of this study was to investigate the contribution of chromosomal microarray analysis (CMA) and next-generation sequencing (NGS) to genetic diagnosis in fetuses with normal karyotype who underwent invasive testing for different indications. The results of invasive genetic testing performed at a tertiary center between September 2020 and March 2022 were retrospectively analyzed. Indications for invasive tests were classified as fetal structural malformation, presence of soft markers, and high risk in screening tests. CMA results were classified as pathogenic or likely pathogenic (pCNVs), benign (bCNVs), and variants of unknown clinical significance (VOUS). A total of 830 invasive tests were performed and aneuploidy was detected in 11.2% of the fetuses. CMA was performed in 465 fetuses with normal karyotype, and pCNVs were detected in 6.9%. pCNVs were detected in 8.2% of fetuses with structural malformations, 6.5% in soft markers, and 4.7% in high risk in screening tests. Pathogenic variants were detected by NGS in 33.8% of fetuses with bCNVs. pCNVs can be significantly detected not only in fetuses with structural malformations, but also in invasive testing with other indications. NGS significantly contributes to genetic diagnosis in fetuses with structural malformations.

Aneuploidy and mosaicism concordance between cell-free embryonic DNA in spent embryo culture medium combined with blastocoel fluid and trophectoderm biopsy DNA

Background and Aim: Some mosaic blastocysts are capable of forming viable normal pregnancies. However, the association between mosaicism in trophectoderm (TE) and the inner cell mass (ICM) is far from being established. Unlike trophectoderm biopsy, non-invasive or minimally invasive preimplantation genetic testing for aneuploidy (miPGT-A) may better represent the entire blastocyst since cell-free embryonic DNA is most likely released into blastocoel fluid (BF) and spent embryo culture medium (SEM) from both TE and ICM.

P-563 The effect of embryo culture time on concordance rates between invasive and non-invasive preimplantation genetic testing for aneuploidies (niPGT-A) in spent culture media (SCM) analysis

Abstract Study question To evaluate different embryo culture times (day5 versus day6) to understand the ideal time-frame for the performance of concordance analysis between invasive and non-invasive PGT-A(niPGT-A) Summary answer Different levels of ploidy concordance rates were observed between day5 and day6 groups: 72,6% versus 84,8%, respectively What is known already The recent data supports that a non-invasive approach for evaluating embryo ploidy status may be an alternative to standard invasive methods. Embryo cell-free DNA(cfDNA) released into culture media during in-vitro embryo development represents the potential source for this analysis. The release of cfDNA from embryos is expected to be directly proportional to embryo culture time and late-stage released cfDNA may be more representative of the embryo. Therefore, it is important to estimate the most effective time frame of the culture that will provide the most conclusive data from spent-culture-media(SCM) without adversely affecting the development of the embryo Study design, size, duration A total of 334 SCM from blastocyst stage embryos have been included in this study. The SCM samples were divided in 2 subgroups according to the embryo culture time as day5 (n = 154) and day6 (n = 180). The cfDNA of SCM samples were amplified by whole genome amplification(WGA) and analyzed by next generation sequencing(NGS) in parallel to day5 or day6 trophectodermal(TE) biopsied samples of their corresponding embryos. Ploidy status and concordance were compared between two groups Participants/materials, setting, methods Day3 embryos were washed and transferred in 20µl fresh culture media until the biopsy. SCM samples were collected in PCR tubes and stored at -20 °C while embryos were biopsied for standard PGT-A analysis. Both SCM and corresponding TE biopsy samples were amplified by Sureplex(Vitrolife). Then TE and SCM samples were analyzed by next-generation sequencing(NGS) using MiSeq® System (Illumina). Data analysis has been done by Bluefuse Multi Software 4.5(Illumina) for all SCM and TE samples Main results and the role of chance A successful DNA amplification rate (&amp;gt;30ng/µl) was obtained in both day5 and day6 groups with 95,5% (147/154) and 99,4% (179/180) respectively. The DNA concentration after WGA was ranging between 30.1-122.5ng/µl and 38.5-123.2ng/µl in day5 and day6 groups respectively. Not conclusive (NC) results including a noisy NGS profile and chaotic chromosome aneuploidies (&amp;gt;5 chromosomes) were excluded in both groups. Therefore, 64,6% (95/147) of SCM samples in day5 group and 81,0% (145/179) of SCM samples in day6 group were conclusive for NGS analysis. The ploidy concordance rate between SCM and TE samples (euploid vs euploid, aneuploid vs aneuploid) was 72,6% (69/95) in day5 and 84,8% (123/145) in day6 group. In day5 group, the false-negative rate was 10.5% (10/95), and false-positive rate was 16.8% (16/95) while in day6 group the false-negative rate was 6,8% (10/145), and false-positive rate was 8,2% (12/145). Sensitivity and specificity were calculated as 83,6% and 52,9% in day5 group, and 90,9% and 65,7 in day6 group respectively Limitations, reasons for caution We have high number of samples in our study, but larger prospective studies may change the significance of ploidy concordance. One of the important issues in SCM analysis is the maternal DNA contamination risk which cannot be revealed always. Therefore the use of molecular markers would increase the reliability Wider implications of the findings A non-invasive approach for aneuploidy screening may be an alternative to standard PGT-A procedure with invasive embryo biopsy but the evaluation of ideal culture time is crucial for the performance of niPGT-A method. Our study demonstrates that the late collection time of SCM provides significantly better ploidy concordance rates Trial registration number Not applicable

A Nonimmune Hydrops Case.

A Nonimmune Hydrops Case.

Prenatal diagnosis of fetuses with Emanuel syndrome: Results of ultrasound examination and invasive genetic testing.

To investigate prenatal manifestations of Emanuel syndrome (ES) by retrospectively analyzing the results of prenatal diagnosis. Thirteen fetuses were collected from five hospitals, of which six were confirmed with 47,der(22)t(11;22; ES) by karyotype and chromosomal microarray analysis (CMA). Seven were diagnosed with 46,t(11;22) balanced translocations by karyotype, including one de novo mosaic 46,XX,t(11;22). In 3/7, CMA was performed but did not identify chromosomal imbalances. The results of prenatal diagnoses were reviewed, including ultrasound examinations and genetic testing. In ES fetuses, the derivative 22 was consistently inherited from the mother, while in the balanced translocation group, the t(11;22) chromosome was of paternal origin in 3/6 cases, All ES fetuses presented with multiple abnormalities by ultrasound examinations. Diaphragm hernia (3/6), Dandy-Walker complex (3/6), and kidney aplasia (3/6), were the most common ultrasound findings. Sonographic soft markers such as increased nuchal translucency, increased nuchal fold thickness appeared in 3 cases and all of these were associated with other anomalies. However, none of the ultrasound findings differentiated ES from other genetic syndromes during fetal period. In this series, in fetuses with a der(22), the derivative chromosome was consistently of maternal origin. In contrast, 46,t(11;22) balanced translocations were of maternal or paternal origin. The results contribute to the literature regarding the fetal phenotype of ES. Due to the absence of specific features distinguishing ES from other genetic syndromes, confirming the diagnosis through invasive genetic testing is necessary.

Ethical issues in assisted reproductive technology and prenatal care – Non–invasive prenatal genetic testing and preimplantation genetic testing –

Ethical issues in assisted reproductive technology and prenatal care – Non–invasive prenatal genetic testing and preimplantation genetic testing –

The role of first-trimester ultrasound screening for women with positive noninvasive prenatal testing results.

This study aimed to assess the utility of ultrasound screening for pregnancies with positive noninvasive prenatal testing results for trisomy 21, trisomy 18, and trisomy 13. We performed a retrospective analysis of positive noninvasive prenatal testing results and first-trimester ultrasound screening at our department between 2013 and 2019. Invasive genetic testing was performed if the patient had positive noninvasive prenatal testing results. Fetal ultrasound and cytogenetic data were collected. Noninvasive prenatal testing was performed in the women for advanced maternal age, nuchal translucency thickness, or history of abnormality in the previous child or relative. Forty-one pregnant women had positive noninvasive prenatal testing results for trisomy 21, trisomy 18, and trisomy 13. Twenty-three women had positive results for trisomy 21, 13 had positive results for trisomy 18, and 5 had positive results for trisomy 13 at 11 to 14 weeks of gestation. The positive predictive value of noninvasive prenatal testing was 100% for trisomy 21, 84.6% for trisomy 18, and 100% for trisomy 13. The positive predictive value of positive noninvasive prenatal testing results and fetal morphological abnormalities was 100% for trisomy 21, trisomy 18, and trisomy 13. Combining an ultrasound examination with noninvasive prenatal testing resulted in a higher positive predictive value for trisomy 18. Normal ultrasound examination results can help alleviate stress caused by false-positive noninvasive prenatal testing results. In contrast, the positive predictive value and negative predictive value for trisomy 21 were not altered by adding an ultrasound examination to noninvasive prenatal testing.

A Non-invasive Chromosome Screening Strategy for Prioritizing in vitro Fertilization Embryos for Implantation.

Preimplantation genetic testing for aneuploidy (PGT-A) is widely used to select embryos having normal ploidy for transfer, but they require an invasive embryo biopsy procedure that may cause harm to the embryos and offspring. Therefore, a non-invasive approach to select embryos with normal ploidy for implantation is highly demanded. Non-invasive chromosome screening (NICS) methods have been proposed and applied in clinical practices, but a large-scale validation versus invasive preimplantation genetic testing (PGT) and the whole embryo ploidy has not yet been reported. In this study, by using the whole embryo as a gold standard, we validated NICS assay in a total of 265 donated human embryos and compared its performance with conventional trophectoderm (TE) biopsy PGT. The NICS assay showed promising performance, which is comparable to PGT-TE [sensitivity: 87.36 versus 89.66%; specificity: 80.28 versus 82.39%; negative predictive value (NPV): 91.2 versus 92.86%; positive predictive value (PPV): 73.08 versus 75.73%]. Additionally, NICS provides a scoring system for prioritizing embryo: embryos can be categorized into three groups with euploid prediction probabilities of 90.0, 27.8, and 72.2% for group euploid (A), aneuploid (B), and multiple abnormal chromosomes (MAC) (C), respectively. When an addition of TE assay is provided as a secondary validation, the accuracy significantly increases from 72.2 to 84.3% for group B and from 27.8 to 83.3% for group C. Our results suggest that NICS is a good rule in assay for identifying chromosomal normal embryos for transfer and might serve as a non-invasive approach for prioritizing embryos instead of preventing transfer of aneuploid and MAC embryos. It will help to ensure the safety of offspring and efficient utilization of embryos.

P–582 High level of concordance between invasive and non-invasive preimplantation genetic testing for aneuploidies (niPGT-A) at day5 and day6–7

Abstract Study question To explore ploidy concordance between invasive and non-invasive PGTA (niPGT-A) at different embryo culture time. Summary answer High level (&amp;gt;84%) of concordance rate for ploidy and sex, sensitivity (&amp;gt;88%), and specificity (76%) were obtained for both day6/7 samples and day5 samples. What is known already The analysis of embryo cell free DNA (cfDNA) that are released into culture media during in vitro embryo development has the potential to evaluate embryo ploidy status. However, obtaining sufficient quality and quantity of cfDNA is essential to achieve interpretable results for niPGT-A. More culture time is expected to be directly proportional to the release of more cfDNA. But embryo culture time is limited due to in-vitro embryo survival potential. Therefore, it is important to estimate the duration of the culture that will provide the maximum cfDNA that can be obtained without adversely affecting the development of the embryo. Study design, size, duration A total of 105 spent culture media (SCM) from day5-day7 blastocyst stage embryos have been included in this cohort study. The cfDNA of SCM samples were amplified and analyzed for niPGT-A by NGS analysis. The SCM samples were divided into 2 subgroups according the embryo culture hours (Day5 and Day6/7 group). The DNA concentration, informativity and euploidy results have then been compared with their corresponding embryos after trophectoderm biopsy (TE) and PGT-A analysis by NGS Participants/materials, setting, methods Embryos cultured until Day3 washed and cultured again in 20µl fresh culture media until embryo biopsy on Day5, 6, or 7. After biopsy SCM samples were immediately collected in PCR tubes and conserved at –20 °C until whole genome amplification by MALBAC® (Yicon Genomics). The TE and SCM samples were analyzed by next-generation sequencing (NGS) using Illumina MiSeq® System. NGS data analysis has been done by Bluefuse Multi Software 4.5 (Illumina) for SCM and TE samples Main results and the role of chance Only the SCM samples which have an embryo with a conclusive result were included in this cohort (n = 105). Overall 97.1% (102/105) of SCM samples gave a successful DNA amplification with a concentration ranging 32.4–128.5ng/µl. Non-informative (NI) results including a chaotic profile (&amp;gt;5 chromosome aneuploidies) were observed in 17 samples, so 83.3%(85/102) of SCM samples were informative for NGS data analysis. Ploidy concordance rate with the corresponding TE biopsies (euploid vs euploid, aneuploid vs aneuploid) was 84.7% (72/85). Sensitivity and specificity were 92,8% and 76,7%, respectively with no significant difference for all parameters for day 6/7 samples compared with day 5 samples. The false-negative rate was 3.5% (3/85), and false-positive rate was 11.7% (10/85). Limitations, reasons for caution The sample size is relatively small. Larger prospective studies are needed. As this is a single-center study, the impact of the variations in embryo culture conditions can be underestimated. Maternal DNA contamination risk cannot be revealed in SCM, therefore the use of molecular markers would increase the reliability. Wider implications of the findings: Non-invasive analysis of embryo cfDNA analyzed in spent culture media demonstrates high concordance with TE biopsy results in both early and late culture time. A non-invasive approach for aneuploidy screening offers important advantages such as avoiding invasive embryo biopsy and decreased cost, potentially increasing accessibility for a wider patient population. Trial registration number Not applicable

Should prenatal chromosomal microarray analysis be offered for isolated fetal growth restriction? A French multicenter study

Compared with standard karyotype, chromosomal microarray analysis improves the detection of genetic anomalies and is thus recommended in many prenatal indications. However, evidence is still lacking on the clinical utility of chromosomal microarray analysis in cases of isolated fetal growth restriction. This study aimed to estimate the proportion of copy number variants detected by chromosomal microarray analysis and the incremental yield of chromosomal microarray analysis compared with karyotype in the detection of genetic abnormalities in fetuses with isolated fetal growth restriction. This retrospective study included all singleton fetuses diagnosed with fetal growth restriction and no structural ultrasound anomalies and referred to 13 French fetal medicine centers over 1 year in 2016. Fetal growth restriction was defined as an estimated fetal weight of <tenth percentile for gestational age identified in ultrasound reports. For this analysis, we selected fetuses who underwent invasive genetic testing with karyotype and chromosomal microarray analysis results. Data were obtained from medical records and ultrasound databases and postmortem and placental examination reports in case of spontaneous stillbirths and terminations of pregnancy. Following the American College of Medical Genetics and Genomics guidelines, copy number variants were classified into 5 groups as following: pathogenic, likely pathogenic, variant of unknown significance, likely benign, and benign. Of 682 referred fetuses diagnosed with isolated fetal growth restriction, both karyotype and chromosomal microarray analysis were performed in 146 fetuses. Overall, the detection rate of genetic anomalies found by chromosomal microarray analysis was estimated to be 7.5% (11 of 146 [95% confidence interval, 3.3-11.8]), including 10 copy number variants classified as pathogenic and 1 copy number variant classified as likely pathogenic. Among the 139 fetuses with normal karyotype, 5 were detected with pathogenic and likely pathogenic copy number variants, resulting in an incremental yield of 3.6% (95% confidence interval, 0.5-6.6) in chromosomal microarray analysis compared with karyotype. All fetuses detected with pathogenic or likely pathogenic copy number variants resulted in terminations of pregnancy. In addition, 3 fetuses with normal karyotype were detected with a variant of unknown significance (2.1%). Among the 7 fetuses with abnormal karyotype, chromosomal microarray analysis did not detect trisomy 18 mosaicism in all fetuses. Our study found that compared with karyotype, chromosomal microarray analysis improves the detection of genetic anomalies in fetuses diagnosed with isolated fetal growth restriction. These results support the use of chromosomal microarray analysis in addition to karyotype for isolated fetal growth restriction.