Euploid Pregnancies Research Articles

Amniotic fluid gamma-glutamyl transferase for prediction of biliary atresia in cases of non-visualisation of the fetal gallbladder: a retrospective study using a validated analytical platform and local reference range.

The level of amniotic fluid gamma-glutamyl transferase (AFGGT) may help identify biliary atresia (BA) in cases of non-visualisation of the fetal gallbladder (NVFGB). This study aimed to validate a serum/plasma matrix-based gamma-glutamyl transferase (GGT) assay for amniotic fluid (AF) samples, establish a local gestational age-specific AFGGT reference range, and evaluate the efficacy of AFGGT for predicting fetal BA in pregnancies with NVFGB using the constructed reference range. The analytical performance of a serum/plasma matrix-based GGT assay on AF samples was evaluated using a Cobas c502 analyser. Amniotic fluid gamma-glutamyl transferase levels in confirmed euploid singleton pregnancies (16+0 to 22+6 weeks of gestation) were determined using the same analyser to establish a local gestational age-specific reference range (the 2.5th to 97.5th percentiles). This local reference range was used to determine the positive predictive value (PPV) and negative predictive value (NPV) of AFGGT level <2.5th percentile for identifying fetal BA in euploid pregnancies with NVFGB. The serum/plasma matrix-based GGT assay was able to reliably and accurately determine GGT levels in AF samples. Using the constructed local gestational age-specific AFGGT reference range, the NPV and PPV of AFGGT level <2.5th percentile for predicting fetal BA in pregnancies with NVFGB were 100% and 25% (95% confidence interval=0, 53), respectively. In pregnancies with NVFGB, AFGGT level ≥2.5th percentile likely excludes fetal BA. Although AFGGT level <2.5th percentile is not diagnostic of fetal BA, fetuses with AFGGT below this level should be referred for early postnatal investigation.

Rapid non-invasive prenatal screening test for trisomy 21 based on digital droplet PCR

Non-invasive prenatal tests for the detection of fetal aneuploidies are predominantly based on the analysis of cell-free DNA (cfDNA) from the plasma of pregnant women by next-generation sequencing. The development of alternative tests for routine genetic laboratories is therefore desirable. Multiplex digital droplet PCR was used to detect 16 amplicons from chromosome 21 and 16 amplicons from chromosome 18 as the reference. Two fluorescently labeled lock nucleic acid probes were used for the detection of reaction products. The required accuracy was achieved by examining 12 chips from each patient using Stilla technology. The plasma cfDNA of 26 pregnant women with euploid pregnancies and 16 plasma samples from pregnancies with trisomy 21 were analyzed to determine the cutoff value for sample classification. The test was validated in a blind study on 30 plasma samples from pregnant patients with a risk for trisomy 21 ranging from 1:4 to 1:801. The results were in complete agreement with the results of the invasive diagnostic procedure (sensitivity, specificity, PPV, and NPV of 100%). Low cost, and speed of analysis make it a potential screening method for implementation into the clinical workflow to support the combined biochemical and ultrasound results indicating a high risk for trisomy 21.

The Landscape of Point Mutations in Human Protein Coding Genes Leading to Pregnancy Loss.

Pregnancy loss is the most frequent complication of a pregnancy which is devastating for affected families and poses a significant challenge for the health care system. Genetic factors are known to play an important role in the etiology of pregnancy loss; however, despite advances in diagnostics, the causes remain unexplained in more than 30% of cases. In this review, we aggregated the results of the decade-long studies into the genetic risk factors of pregnancy loss (including miscarriage, termination for fetal abnormality, and recurrent pregnancy loss) in euploid pregnancies, focusing on the spectrum of point mutations associated with these conditions. We reviewed the evolution of molecular genetics methods used for the genetic research into causes of pregnancy loss, and collected information about 270 individual genetic variants in 196 unique genes reported as genetic cause of pregnancy loss. Among these, variants in 18 genes have been reported by multiple studies, and two or more variants were reported as causing pregnancy loss for 57 genes. Further analysis of the properties of all known pregnancy loss genes showed that they correspond to broadly expressed, highly evolutionary conserved genes involved in crucial cell differentiation and developmental processes and related signaling pathways. Given the features of known genes, we made an effort to construct a list of candidate genes, variants in which may be expected to contribute to pregnancy loss. We believe that our results may be useful for prediction of pregnancy loss risk in couples, as well as for further investigation and revealing genetic etiology of pregnancy loss.

Performance of prenatal cfDNA screening for sex chromosomes

PurposeThe aim of this study was to assess the performance of cell-free DNA (cfDNA) screening to detect sex chromosome aneuploidies (SCAs) in an unselected obstetrical population with genetic confirmation. MethodsThis was a planned secondary analysis of the multicenter, prospective SNP-based Microdeletion and Aneuploidy RegisTry (SMART) study. Patients receiving cfDNA results for autosomal aneuploidies and who had confirmatory genetic results for the relevant sex chromosomal aneuploidies were included. Screening performance for SCAs, including monosomy X (MX) and the sex chromosome trisomies (SCT: 47,XXX; 47,XXY; 47,XYY) was determined. Fetal sex concordance between cfDNA and genetic screening was also evaluated in euploid pregnancies. ResultsA total of 17,538 cases met inclusion criteria. Performance of cfDNA for MX, SCTs, and fetal sex was determined in 17,297, 10,333, and 14,486 pregnancies, respectively. Sensitivity, specificity, and positive predictive value (PPV) of cfDNA were 83.3%, 99.9%, and 22.7% for MX and 70.4%, 99.9%, and 82.6%, respectively, for the combined SCTs. The accuracy of fetal sex prediction by cfDNA was 100%. ConclusionScreening performance of cfDNA for SCAs is comparable to that reported in other studies. The PPV for the SCTs was similar to the autosomal trisomies, whereas the PPV for MX was substantially lower. No discordance in fetal sex was observed between cfDNA and postnatal genetic screening in euploid pregnancies. These data will assist interpretation and counseling for cfDNA results for sex chromosomes.

The Impact of Isolated Increased Nuchal Translucency ≥95th Centile on Perinatal Outcome: A Prospective Cohort Study from a North Indian Genetic Center

Abstract Objectives The aim of this study was to determine the chromosomal abnormalities and other adverse outcomes like miscarriages, intrauterine deaths, structural defects, and genetic syndromes in fetuses with increased nuchal translucency (NT) more than or equal to 95th centile. This study also compared the outcomes in fetuses with NT between 95th and 99th centile and more than 99th centile. Study Design A prospective cohort of 182 patients with isolated increased NT was evaluated by invasive testing. Fetal chromosomes were examined by fluorescent in situ hybridization and karyotype or chromosomal microarray. Euploid pregnancies were followed-up with level II ultrasound and fetal echocardiography. For pregnancies progressing to delivery, the neonates were followed-up till the age of 3 months. Final outcome was reported as normal or abnormal. Collated data for perinatal outcomes was analyzed and compared between fetuses with NT 95th and 99th centile (group I) and NT more than 99th centile (group II). Results Of the 202 patients recruited, 182 patients consented for invasive testing and chromosomal analysis. Of the 182 patients, group I (NT 95–99th centile) included 92 patients and group II 90 patients. Chromosomal abnormalities were present in 50 (27.4%), 14 (7.6%) in group I, and 36 (19.4%) in group II. Of the 132 euploid pregnancies, adverse outcomes were present in 22 (16%) fetuses, 7 (5.3%) in group I, and 15(11.7%) in group II. A normal outcome was present in 110 (60.4%) pregnancies of the 182 fetuses with NT more than or equal to 95th centile. Normal outcome observed in group I was 77.1% and in group II, it was 43.4%. Conclusion An increased NT is associated with poor perinatal outcomes in 39.6% patients. Chromosomal analysis and follow-up for adverse outcome in fetuses with NT more than or equal to 95th centile is important to enable a take home neonatal rate of 60.4%. No pregnancy with increased NT should be discontinued without detailed fetal evaluation for genetic disorders, structural malformation, and fetal growth.

Previously reported and here added cases demonstrate euploid pregnancies followed by PGT-A as “mosaic” as well as “aneuploid” designated embryos

BackgroundAfter the longest time opposing all transfers of embryos by preimplantation genetic testing for aneuploidy (PGT-A) diagnosed as “chromosomal-abnormal,” the field has over recent years slowly been moving toward selective transfers of by PGT-A as “mosaic” diagnosed embryos, but is still rejecting transfers of embryos by PGT-A defined as “aneuploid.”MethodsUpon review of the literature, we report published cases of euploid pregnancies following transfers of PGT-A as “aneuploid” diagnosed embryos and add several additional, ongoing cases at our center.ResultsAmong the published cases from our center, we identified seven euploid pregnancies from “aneuploid” embryos, four of which preceded the PGT-A industry’s 2016 switch from binary “euploid” – “aneuploid” reporting to “euploid,” “mosaic,” and “aneuploid” reporting. That those four cases post 2016 PGT-A definition involving “mosaic” embryos, therefore, cannot be ruled out. Since then, we recently established three additional ongoing pregnancies from transfers of “aneuploid” embryos which still await confirmation of euploidy after delivery. A recent fourth pregnancy from the transfer of a trisomy 9 embryo miscarried before a fetal heart. Outside our own center’s experience, the literature revealed only one additional such transfer, involving PGT-A as a “chaotic-aneuploid” diagnosed embryo with six abnormalities, leading to normal euploid delivery. In reviewing the literature, we furthermore demonstrate why current PGT-A reporting that differentiates between “mosaic” and “aneuploid” embryos based on relative percentages of euploid and aneuploid DNA in a single trophectoderm biopsy of on average 5-6 cells, is biologically non-sensical.ConclusionBasic biological evidence and a clinically still very limited experience with transfers of PGT-A as “aneuploid” labeled embryos demonstrate beyond reasonable doubt that at least some “aneuploid” embryos can lead to healthy euploid births. Therefore, this observation establishes beyond reasonable doubt that the rejection of all “aneuploid” embryos from transfer reduces pregnancy and live birth chances for IVF patients. Whether (and to what possible degree) pregnancy and live birth chances differ between “mosaic” and “aneuploid” embryos, remains to be determined. The answer will likely depend on the aneuploidy(ies) of an embryo and to what degree percentages of “mosaicism” in a single, on average 5/6-cell trophectoderm biopsy can reflect the ploidy-status of a complete embryo.

Integrative analyses of maternal plasma cell-free DNA nucleosome footprint differences reveal chromosomal aneuploidy fetuses gene expression profile

BackgroundChromosomal aneuploidy is the most common birth defect. However, the developmental mechanism and gene expression profile of fetuses with chromosomal aneuploidy are relatively unknown, and the maternal immune changes induced by fetal aneuploidy remain unclear. The inability to obtain the placenta multiple times in real-time is a bottleneck in research on aneuploid pregnancies. Plasma cell-free DNA (cfDNA) carries the gene expression profile information of its source cells and may be used to evaluate the development of fetuses with aneuploidy and the immune changes induced in the mother owing to fetal aneuploidy.MethodsHere, we carried out whole-genome sequencing of the plasma cfDNA of 101 pregnant women carrying a fetus with trisomy (trisomy 21, n = 42; trisomy 18, n = 28; trisomy 13, n = 31) based on non-invasive prenatal testing (NIPT) screening and 140 normal pregnant women to identify differential genes according to the cfDNA nucleosome profile in the region around the transcription start sites (TSSs).ResultsThe plasma cfDNA promoter profiles were found to differ between aneuploid and euploid pregnancies. A total of 158 genes with significant differences were identified, of which 43 genes were upregulated and 98 genes were downregulated. Functional enrichment and signaling pathway analysis were performed based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases found that these signal pathways were mainly related to the coordination of developmental signals during embryonic development, the control of cell growth and development, regulation of neuronal survival, and immune regulation, such as the MAPK, Hippo, TGF-β, and Rap1 signaling pathways, which play important roles in the development of embryonic tissues and organs. Furthermore, based on the results of differential gene analysis, a total of 14 immune-related genes with significant differences from the ImmPort database were collected and analyzed. These significantly different immune genes were mainly associated with the maintenance of embryonic homeostasis and normal development.ConclusionsThese results suggest that the distribution characteristics of cfDNA nucleosomes in maternal plasma can be used to reflect the status of fetal development and changes of the immune responses in trisomic pregnancies. Overall, our findings may provide research ideas for non-invasive detection of the physiological and pathological states of other diseases.

P-646 IVF outcomes of embryos with abnormal PGT-A biopsy previously refused transfer – a prospective cohort study

Abstract Study question Will patients who have embryos diagnosed as abnormal by preimplantation genetic testing for aneuploidy (PGT-A) choose to have those embryos transferred after those embryos were refused transfer at other institutions and what are the potential clinical outcomes of those transfers? Summary answer Many patients have selected embryos with PGT-A abnormal trophectoderm biopsies transferred since these embryos can still offer a chance of live birth. What is known already PGT-A is a widely practiced method of selecting embryos for transfer based on biopsy of a few cells. Many clinical practices refuse to transfer PGT-A abnormal embryos even when there are no other “normal” embryos available. Study design, size, duration This is a prospective cohort of 69 couples who, since 2014, moved a total of 444 PGT-A abnormal embryos previously refused transfer at their parent institutions to our practice. Among these, 50 patients have, thus far, undergone 57 transfer cycles of 141 embryos. Participants/materials, setting, methods Embryos diagnosed at other institutions by PGT-A as abnormal (mostly using next generation sequencing) were moved to our academically affiliated private fertility and research center in New York City. Female age at retrieval was 41.35 ± 3.98 years, 74% were Caucasian, 12% Asian and 10% were of African descent. All embryos identified as PGT-A abnormal among prospectively identified couples were recorded in our center’s registry. Main results and the role of chance Among the 144 embryos transferred 102 (72.3%) had only 1 or 2 chromosomal abnormalities, 30 (21.3%) had 3 or more and 9 (6.4%) were “undiagnosed” because of degraded DNA, yet still had been refused transfer. Transfer of PGT-A abnormal embryos resulted in 8 live births, 11 miscarriages and no voluntary terminations. One child was born with a segmental duplication and required repair of coarctation of the aorta as a newborn. Many couples with only PGT-A abnormal embryos are willing to have their PGT-A abnormal embryos transferred and such transfers can result in establishment of ongoing euploid pregnancies and live births. Limitations, reasons for caution Findings in this case series may not be representative of the wider population of couples undergoing IVF with PGT-A in general. Not all abnormal phenotypes present in the immediate postnatal period so it will be important to continue to follow the development of these children. Wider implications of the findings PGT-A can result in a clinics refusal to transfer embryos with abnormal PGT-A biopsies, even those with mosaic findings, consequently large numbers of infertile women are prematurely advised that their only chance of motherhood is through third-party egg-donation. Trial registration number Not applicable

IVF outcomes of embryos with abnormal PGT-A biopsy previously refused transfer: a prospective cohort study.

What are the outcomes for patients who choose to move embryos diagnosed as abnormal by preimplantation genetic testing for aneuploidy (PGT-A) to a new institution for transfer after the diagnosing institution refused to transfer them? Many patients seek to have selected embryos with PGT-A abnormal trophectoderm biopsies transferred recognizing that these embryos can still offer a chance of pregnancy and live birth. : PGT-A is a widely practiced method of selecting embryos for transfer based on biopsy of a few cells. Many clinical practices refuse to transfer PGT-A abnormal embryos even when there are no other 'normal' embryos available. This is a prospective cohort of 69 couples who, since 2014, moved a total of 444 PGT-A abnormal embryos previously refused transfer at their parent institutions to our practice. Among these, 50 patients have, thus far, undergone 57 transfer cycles of 141 embryos. Embryos diagnosed at other institutions by PGT-A as abnormal (mostly using next generation sequencing) were moved to our academically affiliated private fertility and research center in New York City. Female age at retrieval was 41.35 ± 3.98 years, 74% were Caucasian, 12% Asian and 10% were of African descent. All embryos identified as PGT-A abnormal among prospectively identified couples were recorded in our center's registry. Among the 144 embryos transferred 102 (72.3%) had only 1 or 2 chromosomal abnormalities, 30 (21.3%) had 3 or more and 9 (6.4%) were 'undiagnosed' because of degraded DNA, yet still had been refused transfer. Transfer of PGT-A abnormal embryos resulted in 8 live births, 11 miscarriages and no voluntary terminations. One child was born with a segmental duplication and required repair of coarctation of the aorta as a newborn. Many couples with only PGT-A abnormal embryos are willing to have their PGT-A abnormal embryos transferred and such transfers can result in the establishment of ongoing euploid pregnancies and live births. Findings in this case series represent couples who chose to have their embryos transferred after having been refused transfer elsewhere and may not be representative of the wider population of couples undergoing IVF with PGT-A in general. Not all abnormal phenotypes present in the immediate postnatal period so it will be important to continue to follow the development of these children. PGT-A can result in a clinics refusal to transfer embryos with abnormal PGT-A biopsies, even those with mosaic findings, consequently large numbers of infertile women are prematurely advised that their only chance of motherhood is through third-party egg-donation. This work was supported by intramural funds from the Center for Human Reproduction and the not-for-profit research Foundation for Reproductive Medicine, both in New York, NY, USA. N.G. and D.H.B. are listed as co-inventors on several U.S. patents. One of these patents (US Patent# 7,615,544) relates to pre-supplementation of hypo-androgenic infertile women with androgens, such as DHEA and testosterone and, therefore, at least peripherally related to the subject of this manuscript. N.G. and D.F.A. also received travel funds and speaker honoraria from several pharmaceutical and medical device companies, though none related to the here presented subject and manuscript. N.G. is a shareholder in Fertility Nutraceuticals and he and D.H.B. receive royalty payments from Fertility Nutraceuticals LLC. N/A.

Impact of fetal trisomy 21 on umbilical artery Doppler indices

Objectives Umbilical artery (UA) Doppler indices are surrogate measures of placental function, most commonly used to assess fetal wellbeing in pregnancies with fetal growth restriction. Fetuses with trisomy 21 (t21) are reported to have elevated UA Doppler indices, but reference percentiles are currently lacking for this population. We hypothesized that gestational age-specific values of UA Doppler indices in pregnancies complicated by t21 will be elevated compared to established percentiles based on euploid pregnancies. We aimed to assess UA Doppler indices longitudinally in fetuses with t21 in order to demonstrate Doppler patterns across gestation in this population, compare them with euploid fetuses, and investigate their association with pregnancy outcomes. Methods We conducted a retrospective cohort study of singleton pregnancies with confirmed fetal t21 who underwent UA Doppler surveillance antenatally from January 2012 to August 2019. UA Doppler indices, including systolic/diastolic (S/D) ratio, pulsatility index (PI), and resistance index (RI) were extracted from ultrasound reports or directly from ultrasound images. UA S/D, PI, and RI percentiles by gestational week were created from available observations from our cohort via a data-driven approach using a generalized additive model. A secondary analysis was run to statistically compare t21 values to established percentiles based on observations from a historical population of euploid fetuses. Results UA Doppler measurements from 86 t21 fetuses and 130 euploid fetuses were included in our analysis. Median (IQR) maternal age in t21 pregnancies and euploid pregnancies were 35 years (29–38) and 30 years (27–33), respectively. As in euploid fetuses, we found a negative association between Doppler indices and gestational age in the t21 fetuses. Maternal tobacco use, obesity, or chronic hypertension had no significant effect on UA Doppler indices. As hypothesized, values for UA S/D ratio, PI, and RI at the 2.5th, 5th, 10th, 25th, 50th, 75th, 90th, 95th, and 97.5th percentiles by gestational week were significantly higher in t21 fetuses compared to euploid fetuses (p<.001). Overall, 55.8% (48/86) of the t21 fetuses demonstrated at least one Doppler value above the 95th percentile for gestational age based on euploid reference standard. At birth, eight (9.3%) of the t21 fetuses were small for gestational age. When these pregnancies were removed from analysis, UA Doppler indices remained significantly higher than established percentiles at each week of gestation (p < .001). Only three pregnancies ended in fetal demise in the t21 population, two of which had persistently elevated Dopplers above the 95th percentile per established reference percentiles. Conclusions At each week of gestation, UA Doppler indices in t21 fetuses were significantly higher than established percentiles from a euploid population. Reference intervals based on euploid fetuses may therefore not be appropriate for antenatal surveillance of fetuses with t21. Prospective studies are needed to investigate the role and impact of serial UA Doppler velocimetry in the surveillance of pregnancies complicated by fetal t21.

SY10-3. Complement Activation and Regulation in Preeclampsia and HELLP Syndrome

To evaluate the potential of maternal serum placental growth factor (PlGF) as a marker for Trisomy 21 in Chinese pregnant women.Serum samples were collected and stored from 600 women who have a viable singleton pregnancy and underwent first trimester screening for Trisomy 21 between 2011 and 2014. Serum concentration of PlGF was measured using the automated time-solved immunofluorometric assay and expressed as Multiples of the expected median (MoM) from the 600 women available stored serum samples (558 Euploidy and 42 Trisomy 21). The levels of PlGF MoM were compared between Trisomy 21 cases and Euploid pregnancies. Expected median PlGF levels in Chinese were also compared to that published for Caucasians. Multivariate Gaussian modeling was performed to predict detection and false-positive rates.In euploid pregnancies the concentrations of PlGF increased with Crown Rump Length(CRL) and decreased with maternal weight. The overall median MoM of PlGF in Chinese was higher than that of Caucasian. The median PlGF level was 0.63 MoM in the cases and 1.00 MoM in the controls (p < 0.0001). The prediction of Trisomy 21 has been slightly improved by the addition of PlGF to the standard screening test in China. The detection rate of screening after adding PlGF data was increased from 93.4% to 94.6% at a false-positive rate of 3% and the false positive rate decreased from 0.23% to 0.17% for a detection rate of 80%.The median PlGF concentrations in Chinese is higher than those of Caucasians. Results suggest adding PlGF may substantially improve the performance of current first trimester screening strategy for Trisomy 21 in Chinese pregnant women.

The Placenta in Down Syndrome Provides Key Insights for Euploid and Aneuploid Pregnancies

The Placenta in Down Syndrome Provides Key Insights for Euploid and Aneuploid Pregnancies

Application of an individualized nomogram in first-trimester screening for trisomy 21.

ABSTRACTObjectivesTo develop and validate a nomogram based on fetal nuchal translucency thickness (NT) and ultrasonographic facial markers for screening for trisomy 21 in the first trimester of pregnancy.MethodsThis was a retrospective case–control study using stored two‐dimensional midsagittal fetal profile images captured at 11 + 0 to 13 + 6 weeks' gestation in singleton pregnancies. We included images from 302 trisomy‐21 pregnancies and 322 euploid pregnancies. Cases were divided into a training set (200 euploid + 200 with trisomy 21) and a validation set (122 euploid + 102 with trisomy 21) at a ratio of approximately 2:1. For each, the maternal age, gestational age, fetal NT and karyotype were noted, and 12 ultrasonographic fetal facial markers were measured. The least absolute shrinkage and selection operator (LASSO) method and multivariable analysis were used to select automatically the discriminative markers. Logistic regression was used to develop a LASSO model, based on the selected markers, to screen for trisomy 21 in the first trimester of pregnancy. Furthermore, 60 of the 624 images were selected randomly as a retest set to evaluate the model's robustness. The predictive performance of screening for trisomy 21 of a model based on fetal NT and maternal age and of the LASSO model was assessed using the area under the receiver‐operating‐characteristics curve (AUC). A nomogram was developed as an individualized tool to predict patient‐specific probability for trisomy 21, which is a more visual presentation of the LASSO model. The performance of the nomogram was assessed using the C‐index and calibration curve.ResultsInto the LASSO model were incorporated eight markers, including fetal NT, prenasal‐thickness‐to‐nasal‐bone‐length ratio, facial profile line, frontomaxillary facial angle, frontonasal facial angle, mandibulomaxillary facial angle, maxilla‐nasion‐mandible angle and d2 (distance between the anterior edge of the prefrontal skin and the mandibulomaxillary line) (all P < 0.05). The AUCs of the LASSO model for screening for trisomy 21 were 0.983 (95% CI, 0.971–0.994) in the training set and 0.979 (95% CI, 0.966–0.993) in the validation set, and these were higher than the AUCs of all eight individual ultrasonographic markers included in the model. The AUC of the LASSO model in the retest set was 0.997 (95% CI, 0.990–1.000), indicating good robustness of the LASSO model. The AUC of the LASSO model was significantly higher than that of the model based on fetal NT and maternal age in both training and validation sets (P < 0.001 for both). The nomogram of the LASSO model showed good discrimination of trisomy 21, with C‐indices of 0.983 in the training set and 0.981 in the validation set.ConclusionsWe present an individualized nomogram which incorporates fetal NT and a series of ultrasonographic facial profile markers selected by the LASSO method and multivariable analysis. This nomogram can potentially be utilized as a convenient and effective tool in screening for trisomy 21 in the first trimester of pregnancy. © 2020 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Prenatal Screening of Trisomy 21: Could Oxidative Stress Markers Play a Role?

Despite significant progress in trisomy 21 (T21) diagnostic tools, amniocentesis is still used for the confirmation of an abnormal fetal karyotype. Invasive tests carry the potential risk of miscarriage; thus, screening biomarkers are commonly used before undergoing invasive procedures. In our study, we investigated the possible application of oxidative stress markers in the prenatal screening of trisomy 21. The DNA/RNA oxidative stress damage products (OSDPs), advanced glycation end (AGE) products, ischemia-modified albumin (IMA), alfa-1-antitrypsin (A1AT), asprosin, and vitamin D concentrations were measured in both maternal plasma and amniotic fluid in trisomy 21 (T21) and euploid pregnancies. The obtained results indicated increased levels of DNA/RNA OSDPs and asprosin with simultaneous decreased levels of vitamin D and A1AT in the study group. The diagnostic utility of the plasma measurement based on the area under the received operative characteristic (ROC) curve (AUC) calculation of asprosin (AUC = 0.965), IMA (AUC = 0.880), AGE (AUC = 0.846) and DNA/RNA OSDPs (AUC = 0.506) in T21 screening was demonstrated. The obtained results indicate a potential role for the application of oxidative stress markers in the prenatal screening of T21 with the highest screening utility of plasma asprosin.

5 Perinatal and genetic outcomes associated with no call cfDNA results in 18,496 pregnancies

Failed or no call cell free DNA (cfDNA) testing confers an increased risk of aneuploidy, but few series include complete obstetric, infant, and genetic outcomes. We hypothesized that failed tests would be associated with aneuploidy and adverse perinatal outcomes. Secondary analysis of a multicenter prospective study of SNP-based cfDNA; confirmatory genetic testing was performed in all cases during pregnancy or after birth. Demographics, pregnancy outcomes, and confirmatory genetic results were compared between no call and resulted cases. Univariate analysis compared differences between groups; odds ratios (OR) were calculated after adjusting for BMI, gestational age at draw, and black race. Results applying an updated algorithm after study completion were also compared. 18,496 women had both cfDNA screening and genetic confirmation. A first draw result was reported in 17,885 while 611 (3.3%) were no calls; 320/435 (73.6%) redraws gave a result, leaving 291 as no calls. No calls were associated with higher BMI, later gestational age, lower fetal fraction, and black race. T13, 18, or 21 was confirmed in 1.6% of no calls vs 0.7% with a result (p=.013). After adjustment for confounders, the aOR for aneuploidy was 2.2 (95% CI 1.1,4.5) after a first no call; this increased to 3.8 (95% CI 1.7, 8.4) after the second. Livebirths occurred in 94.9% with a no call vs 98.8% with a result (p< .001; aOR for livebirth: 0.17 [95% CI 0.10, 0.28]). PTB < 28, 34, and 37wks and preeclampsia were all higher after a no call. All risks were higher after a second no call result (Table). With the updated algorithm, the no call rate decreased to 0.6% and the adverse associations were further increased. The association with perinatal outcomes persisted in euploid pregnancies. Patients with no call cfDNA results are at increased risk for aneuploidy; this risk is further increased with a second no call. The risk of other adverse perinatal outcomes is also increased, including in euploid pregnancies.

Study on amniotic fluid metabolism in the second trimester of Trisomy 21.

BackgroundTrisomy 21 is a common aneuploid condition in humans and accounts for approximately one quarter of all aneuploid live births. To date, early diagnosis of Trisomy 21 remains a challenging task. Metabolomics may prove an innovative tool to study the early pathophysiology of Trisomy 21 at a functional level.MethodsUltra‐performance liquid chromatography coupled with mass spectrometer (UPLC‐MS) was used for untargeted metabolomic analysis of amniotic fluid samples from women having normal and trisomy 21 fetuses.ResultsMany significantly changed metabolites were identified between amniotic fluid samples from Trisomy 21 pregnancies and normal euploid pregnancies, such as generally lower levels of several steroid hormones and their derivatives, higher levels of glutathione catabolites coupled with lower levels of gamma‐glutamyl amino acids, and increased levels of phospholipid catabolites, sugars, and dicarboxylic acids. The identification of a human milk oligosaccharide in amniotic fluid may worth further investigation, since confirmation of this observation may have significant implications for regulation of fetal development.ConclusionsThe metabolisms in amniotic fluid from Trisomy 21 and normal pregnancies are quite different, and some of the significantly changed metabolites may be considered as candidates of early diagnostic biomarkers for Trisomy 21.

Mature miR-99a Upregulation in the Amniotic Fluid Samples from Female Fetus Down Syndrome Pregnancies: A Pilot Study.

Background and Objective: Although Down syndrome is the most frequent aneuploidy, its pathogenic molecular mechanisms are not yet fully understood. The aim of our study is to quantify—by qRT-PCR—the expression levels of both the mature forms and the pri-miRNAs of the microRNAs resident on chromosome 21 (miR(21)) in the amniotic fluid samples from Down syndrome singleton pregnancies and to estimate the impact of the differentially expressed microRNAs on Down syndrome fetal heart and amniocytes transcriptomes. Materials and methods: We collected amniotic fluid samples harvested by trained obstetricians as part of the second trimester screening/diagnostic procedure for aneuploidies to assess the trisomy 21 status by QF-PCR and karyotyping. Next, we evaluated—by Taqman qRT-PCR—the expression levels of both the mature forms and the pri-miRNA precursors of the microRNAs resident on chromosome 21 in amniotic fluid samples from singleton Down syndrome and euploid pregnancies. Further, we combined miRWalk 3.0 microRNA target prediction with GEO DataSets analysis to estimate the impact of hsa-miR-99a abnormal expression on Down syndrome heart and amniocytes transcriptome. Results: We found a statistically significant up-regulation of the mature form of miR-99a, but not pri-miR-99a, in the amniotic fluid samples from Down syndrome pregnancies with female fetuses. GATHER functional enrichment analysis of miRWalk3.0-predicted targets from Down syndrome amniocytes and fetal hearts transcriptome GEODataSets outlined both focal adhesion and cytokine–cytokine receptor interaction signaling as novel signaling pathways impacted by miR-99a and associated with cardiac defects in female Down syndrome patients. Conclusions: The significant overexpression of miR-99a, but not pri-miR-99a, points towards an alteration of the post-transcriptional mechanisms of hsa-miR-99a maturation and/or stability in the female trisomic milieu, with a potential impact on signaling pathways important for proper development of the heart.

Detection of fetal trisomy and single gene disease by massively parallel sequencing of extracellular vesicle DNA in maternal plasma: a proof-of-concept validation

BackgroundDuring human pregnancy, placental trophectoderm cells release extracellular vesicles (EVs) into maternal circulation. Trophoblasts also give rise to cell-free DNA (cfDNA) in maternal blood, and has been used for noninvasive prenatal screening for chromosomal aneuploidy. We intended to prove the existence of DNA in the EVs (evDNA) of maternal blood, and compared evDNA with plasma cfDNA in terms of genome distribution, fragment length, and the possibility of detecting genetic diseases.MethodsMaternal blood from 20 euploid pregnancies, 9 T21 pregnancies, 3 T18 pregnancies, 1 T13 pregnancy, and 2 pregnancies with FGFR3 mutations were obtained. EVs were separated from maternal plasma, and confirmed by transmission electronic microscopy (TEM), western blotting, and flow cytometry (FACS). evDNA was extracted and its fetal origin was confirmed by quantitative PCR (qPCR). Pair-end (PE) whole genome sequencing was performed to characterize evDNA, and the results were compared with that of cfDNA. The fetal risk of aneuploidy and monogenic diseases was analyzed using the evDNA sequencing data.ResultsEVs separated from maternal plasma were confirmed with morphology by TEM, and protein markers of CD9, CD63, CD81 as well as the placental specific protein placental alkaline phosphatase (PLAP) were confirmed by western blotting or flow cytometry. EvDNA could be successfully extracted for qPCR and sequencing from the plasma EVs. Sequencing data showed that evDNA span on all 23 pairs of chromosomes and mitochondria, sharing a similar distribution pattern and higher GC content comparing with cfDNA. EvDNA showed shorter fragments yet lower fetal fraction than cfDNA. EvDNA could be used to correctly determine fetal gender, trisomies, and de novo FGFR3 mutations.ConclusionsWe proved that fetal DNA could be detected in EVs separated from maternal plasma. EvDNA shared some similar features to plasma cfDNA, and could potentially be used to detect genetic diseases in fetus.

First trimester uterine artery pulsatility index levels in euploid and aneuploid pregnancies.

To examine whether the uterine artery PI is different in aneuploid and euploid pregnancies. Retrospective case-matched study at the department of prenatal medicine at the University of Tuebingen, Germany. The study involved patients with complete data on first trimester screening for trisomies and preeclampsia except PlGF. For each case with trisomy 21 we randomly selected 50 cases with a euploid fetus where complete data on screening for aneuploidy and preeclampsia were also available. The uterine artery pulsatility index and the corresponding MoM values of euploid and the aneuploid population were compared with a Man-Whitney U test. The dataset consisted of 4591 singleton pregnancies. The karyotype was normal in 4500 cases and was abnormal in the remaining 91 pregnancies. There were 50 pregnancies with trisomy 21, 31 with trisomy 18 and 13, and 10 with triploidy. In the group with euploid fetuses, median uterine artery PI was 1.55 (0.99 MoM). In the group with trisomy 21, the median PI (1.42) and MoM (0.89) levels were both significantly lower than in the euploid (p < 0.001). However, the measurements in the trisomy 18 and 13 [1.61 (0.93 MoM)] and in the triploidy [1.99 (1.13 MoM)] groups were not significantly different from those in the euploid group (p = 0.468 and p = 0.632, respectively). In conclusion, uterine artery PI levels in the first trimester are slightly lower in pregnancies with trisomy 21. This knowledge may prove to be useful in cases where a low PAPP-A level is seen on the first trimester maternal serum biochemical evaluation to differentiate whether the more likely cause for this finding is placental dysfunction or aneuploidy, specifically trisomy 21.

54. IMPLEMENTATION OF FIRST NIPT SERVICE LABORATORY IN AN INDIAN HOSPITAL

Introduction Non-Invasive Prenatal Testing (NIPT) for common fetal aneuploidies is rapidly increasing in India for both naturally conceived pregnancies and pregnancies conceived by assisted reproductive technologies (ART). This study aims to establish the very first next generation sequencing (NGS) laboratory for NIPT in an Indian hospital using the Ion Torrent platform to provide a complete in-house solution for pregnant women. Materials and Methods In brief, all plasma DNA samples (both control and test groups) were converted to genomic DNA libraries using Fragment Library Kit (Thermo Fisher Scientific, Cat. No. 4471252) with Ion Xpress Barcode-Adapters mix (Thermo Fisher Scientific, Cat. No. 4471250) using our laboratory developed protocol. The libraries were then quantified using Qubit dsDNA HS (high sensitivity) Assay Kit, (Thermo Fisher Scientific) and diluted to equimolar amounts before pooling. The Ion Chef System (Thermo Fisher Scientific), as part of the workflow, was used for automated template preparation and loading the sequencing chip. Thereafter, low-pass whole-genome sequencing was done using the ion semiconductor sequencer Ion S5 System (Thermo Fisher Scientific,) to achieve at least 5 million sequence reads. In the dry-lab, the sequenced reads were aligned to the reference human genome (UCSC hg19) locally and their BAM files were generated. The euploid BAM files from the training set of samples were assigned to NIPT reference group and their aligned reads were partitioned into 50,000 bp bins. The number of reads in each bin becomes the unit for comparison with the test samples. GC corrections were performed based on the average coverage of genomic regions having a similar GC-content to correct the coverage differences between bins having a different GC percentage. In this validation study, the standard Z-score prediction models returned a value between -3 and +3 for all the chromosomes, indicating that there is no trisomy present. The scores for aneuploidy samples all showed a value above 4. The z-score refers to the number of standard deviations from the mean of a reference data set and in all molecular counting approaches, a z-score of more than 4 is considered a positive result. Results The algorithm was blindly tested on a cohort of 100 known samples, of which the 12 were T21, eight were T18, four were T13 and 76 were from euploid pregnancies. Our algorithm correctly called all euploid and aneuploid cases. For all aneuploidies, z scores were above a value of 4. Interestingly, accurate detection of aneuploidy was also noted at foetal fraction as low as 3.2%. Therefore, based on our findings, our algorithm is expected to be sensitive and specific for up to 99.9% of pregnancies with one of the three common trisomies. DISCUSSION Based on the high performance of our NIPT test in validation studies, testing has commenced at Lifeline Hospital, including pregnancies conceived by ART. Clinical performance is currently under evaluation to derive positive predictive value (PPV) and negative predictive value (NPV) for each trisomy.