A Multi-centre Experience of Trans-abdominal Chorionic Villus Sampling in Pakistan.

OBJECTIVE: To determine the safety and outcomes of trans-abdominal chorionic villus sampling technique. STUDY DESIGN: Observational study. PLACE AND DURATION OF STUDY: Departments of Obstetrics and Gynaecology, PNS Shifa Karachi, Pak-Emirates Military Hospital Rawalpindi and CMH Lahore, from 2005-2020.   Methodology: A total of 1530 consecutive chorionic villus samplings (CVS) were performed on pregnant females between 10-20 weeks of gestation using the transabdominal approach. Patients were subjected to integrated, stepwise sequential screening. Analysis of data was based on demographic features, indications for sampling, gestational age, attempts of CVS, needle aspiration time, assessment, placental location, sample yield, complications, pain estimation by visual analogue scale (VAS), CVS culture results and pregnancy outcomes. RESULTS: The most common indication for CVS was couple having thalassemia traits and history of having a thalassemia major child previously (55.2%). Pain was the most common complication (64.1%). Procedure-related pregnancy loss (considered to be till 20 weeks of gestation) was observed in two cases (0.1%) only. The most common abnormal karyotype was found to be ß-Thalassemia trait (23.6%) followed by ß-Thalassemia major (22.1%) and Trisomy 21(16.8%). No abnormality was detected in 33.5% of the cases. Five hundred and eighty-nine (38.4%) interruptions of pregnancies were done on the basis of CVS results. CONCLUSION: CVS is a safe and useful technique for sampling in prenatal diagnosis of genetic disorders, markedly affecting the management. KEY WORDS: Chorionic villus sampling, Pre-natal diagnosis, Karyotype.

Prenatal genetic testing 2: diagnostic tests.

PURPOSE OF REVIEW: Prenatal genetic testing can be divided into two categories: screening and diagnosis. Prenatal genetic screening tests are used to assess carrier status or as a fetal risk assessment for a particular genetic disorder [1]. Prenatal genetic diagnostic testing is used to diagnose particular genetic conditions with as much certainty as possible [1,2]. This review will focus on the diagnostic side of prenatal genetic testing. RECENT FINDINGS: Next generation sequencing (NGS) has revolutionized prenatal genetic diagnostic testing. NGS methods are becoming more advanced and accurate as more genetic information is being linked to genetic conditions. SUMMARY: Prenatal genetic diagnostic testing involves clinicians invasively obtaining tissue via amniocentesis or chorionic villus sampling to identify if a fetus has a genetic condition. This testing has traditionally been done through fluorescence in-situ hybridization, karyotype, or chromosomal microarray analysis. However, genetic testing is in a time of rapid technologic expansion and new methods like NGS, which includes targeted gene panels, whole exome sequencing, and whole genome sequencing are being used too. In this time of growth, it is important that providers educate themselves on the research support and indication behind each type of genetic diagnostic test.

Fetal reduction and twins.

Infertility treatments have allowed millions of couples to have their own children, but resultant multiple pregnancies with their increased morbidity and mortality have been a significant complication. Fetal reduction was developed to ameliorate this issue. Over 30 years of publications show that fetal reduction has been highly successful in substantially reducing both mortality and morbidity related to multiple pregnancies. As with most radically new techniques, initial cases were in the "nothing to lose" category. With experience, indications liberalize, and quality of life issues gain relevance. The overall risks of twin pregnancy are not twice that of singleton pregnancy; they are about 4 to 5 times higher. In experienced hands, the combination of genetic testing by chorionic villus sampling followed by fetal reduction has made the outcomes of most multiple pregnancies statistically equivalent to those of pregnancies with lower fetal numbers. Use of microarray analysis to better determine fetal genetic health before deciding on which fetus(es) to keep or reduce further improves pediatric outcomes. With increasing experience and lower average starting numbers, the proportion of fetal reductions to a singleton has increased considerably. Twins to a singleton fetal reductions now constitute an increasing proportion of cases performed. Data on such cases show improved outcomes, and we believe fetal reduction should be at least discussed and offered to all patients with a dichorionic twin pregnancy or higher. With the increasing reliance on elective single-embryo transfers, monochorionic twins, which have much higher complication rates than dichorionic twins, have increased substantially. Furthermore, monochorionic twins cannot be readily and safely reduced, so the adverse perinatal statistics of elective single-embryo transfer are a major setback for good outcomes. Although elective single-embryo transfer is appropriate for some, we believe that for many couples, the transfer of 2 embryos is generally a more rational approach.

Detection of Cryptic Fragile X Full Mutation Alleles by Southern Blot in a Female and Her Foetal DNA via Chorionic Villus Sampling, Complicated by Mosaicism for 45,X0/46,XX/47,XXX.

We describe a female with a 72 CGG FMR1 premutation (PM) (CGG 55-199) and family history of fragile X syndrome (FXS), referred for prenatal testing. The proband had a high risk of having an affected pregnancy with a full mutation allele (FM) (CGG > 200), that causes FXS through hypermethylation of the FMR1 promoter. The CGG sizing analysis in this study used AmplideX triplet repeat primed polymerase chain reaction (TP-PCR) and long-range methylation sensitive PCR (mPCR). These methods detected a 73 CGG PM allele in the proband's blood, and a 164 CGG PM allele in her male cultured chorionic villus sample (CVS). In contrast, the Southern blot analysis showed mosaicism for: (i) a PM (71 CGG) and an FM (285-768 CGG) in the proband's blood, and (ii) a PM (165 CGG) and an FM (408-625 CGG) in the male CVS. The FMR1 methylation analysis, using an EpiTYPER system in the proband, showed levels in the range observed for mosaic Turner syndrome. This was confirmed by molecular and cytogenetic karyotyping, identifying 45,X0/46,XX/47,XXX lines. In conclusion, this case highlights the importance of Southern blot in pre- and postnatal testing for presence of an FM, which was not detected using AmplideX TP-PCR or mPCR in the proband and her CVS.

Performance of conventional cytogenetic analysis on chorionic villi when only one cell layer, cytotrophoblast or mesenchyme alone, is analyzed.

OBJECTIVE: To provide an estimation of the probability of error when chorionic villi (CV) cytogenetic analysis is limited to a single placental layer; either a direct preparation (Dir) or long-term culture (LTC). METHODS: We retrospectively reviewed cytogenetic studies on 81,593 consecutive CV samples in which both Dir and LTC were analyzed. All mosaic cases received amniocentesis. The false omission and false discovery rates were calculated by assessing the results that would have been reported when analysis was limited to either Dir or LTC. RESULTS: For all abnormalities combined, the proportion of normal Dir or LTC only reports that would have been inconsistent with a subsequent amniocentesis was 0.09% and 0.03%, respectively (false omissions). Among abnormal reports based on Dir or LTC alone, 8.01% and 3.17%, respectively, would be inconsistent with a subsequent amniocentesis result (false discoveries). Differences are present for individual abnormalities. CONCLUSIONS: From the perspective of identifying all abnormalities of potential clinical significance, the analysis of both placental layers is optimal. LTC alone is the preferred approach if only one layer of placenta is to be analyzed. Although rare, it is important to acknowledge that one cell layer analysis alone can cause misdiagnosis due to undetected mosaicism.

Chromosomal mosaicism: Origins and clinical implications in preimplantation and prenatal diagnosis.

The diagnosis of chromosomal mosaicism in the preimplantation and prenatal stage is fraught with uncertainty and multiple factors need to be considered in order to gauge the likely impact. The clinical effects of chromosomal mosaicism are directly linked to the type of the imbalance (size, gene content, and copy number), the timing of the initial event leading to mosaicism during embryogenesis/fetal development, the distribution of the abnormal cells throughout the various tissues within the body as well as the ratio of normal/abnormal cells within each of those tissues. Additional factors such as assay noise and culture artifacts also have an impact on the significance and management of mosaic cases. Genetic counseling is an important part of educating patients about the likelihood of having a liveborn with a chromosome abnormality and these risks differ according to the time of ascertainment and the tissue where the mosaic cells were initially discovered. Each situation needs to be assessed on a case-by-case basis and counseled accordingly. This review will discuss the clinical impact of finding mosaicism through: embryo biopsy, chorionic villus sampling, amniocentesis, and noninvasive prenatal testing using cell-free DNA.

First-trimester diagnosis of congenital cytomegalovirus infection after maternal primary infection in early pregnancy: feasibility study of viral genome amplification by PCR on chorionic villi obtained by CVS.

OBJECTIVE: To evaluate the feasibility of amplification of the viral genome by polymerase chain reaction (PCR) analysis of trophoblast samples obtained by chorionic villus sampling (CVS) in cases of maternal primary infection (MPI) with cytomegalovirus (CMV) in early pregnancy. METHODS: This was a prospective study carried out at the Department of Obstetrics and Fetal Medicine, Hopital Necker-E.M., between October 2019 and October 2020. Following CMV serology screening in early pregnancy, CVS was offered to women at 11-14 weeks' gestation after CMV-MPI ≤ 10 weeks. Array-comparative genomic hybridization and amplification of the viral genome by PCR were performed on the trophoblasts obtained by CVS. All cases also underwent amniocentesis from 17 weeks onwards and PCR was performed on the amniotic fluid. Secondary prevention with valacyclovir was initiated as soon as MPI was diagnosed, to decrease the risk of vertical transmission. We evaluated the diagnostic performance of CMV-PCR of trophoblast obtained by CVS, using as the reference standard PCR of amniotic fluid obtained by amniocentesis. RESULTS: CVS was performed in 37 pregnancies, at a median (range) gestational age of 12.7 (11.3-14.4) weeks. CMV-PCR in chorionic villi was positive in three and negative in 34 cases. CMV-PCR following amniocentesis, performed at a median (range) gestational age of 17.6 (16.7-29.9) weeks, was positive for the three cases which were positive following CVS and, of the 34 patients with a negative finding following CVS, amniocentesis was negative in 31 and positive in three. The sensitivity of CMV-PCR analysis of trophoblast obtained by CVS for the diagnosis of CMV, using as the reference standard PCR analysis of amniotic fluid obtained by amniocentesis, was 50% (95% CI, 19-81%), specificity was 100% (95% CI, 89-100%), positive predictive value was 100% (95% CI, 44-100%) and negative predictive value was 91% (95% CI, 77-97%). CONCLUSIONS: Diagnosis of placental infection following MPI in early pregnancy can be achieved by PCR amplification of the CMV genome in chorionic villi. We propose that negative CMV-PCR in the trophoblast after 12 weeks could be used to exclude CMV-related embryopathy leading to sequelae. However, this needs to be confirmed through long-term follow-up evaluation. These findings could help to establish CVS as the diagnostic test of choice following maternal serology screening in early pregnancy. © 2021 International Society of Ultrasound in Obstetrics and Gynecology.

A report on the impact of rapid prenatal exome sequencing on the clinical management of 52 ongoing pregnancies: a retrospective review.

OBJECTIVE: Studies have shown that prenatal exome sequencing (PES) improves diagnostic yield in cases of fetal structural malformation. We have retrospectively analysed PES cases from two of the largest fetal medicine centres in the UK to determine the impact of results on management of a pregnancy. DESIGN: A retrospective review of clinical case notes. SETTING: Two tertiary fetal medicine centres. POPULATION: Pregnancies with fetal structural abnormalities referred to clinical genetics via a multidisciplinary team. METHODS: We retrospectively reviewed the notes of all patients who had undergone PES. DNA samples were obtained via chorionic villus sampling or amniocentesis. Variants were filtered using patient-specific panels and interpreted using American College of Medical Genetics guidelines. RESULTS: A molecular diagnosis was made in 42% (18/43) ongoing pregnancies; of this group, there was a significant management implication in 44% (8/18). A positive result contributed to the decision to terminate a pregnancy in 16% (7/43) of cases. A negative result had a significant impact on management in two cases by affirming the decision to continue pregnancy. CONCLUSIONS: We demonstrate that the results of PES can inform pregnancy management. Challenges include variant interpretation with limited phenotype information. These results emphasise the importance of the MDT and collecting phenotype and variant data. As this testing is soon to be widely available, we should look to move beyond diagnostic yield as a measure of the value of PES. TWEETABLE ABSTRACT: Prenatal exome sequencing can aid decision-making in pregnancy management; review ahead of routine implementation in NHS.

Aneuploidy in first trimester chorionic villi and spontaneous abortions: Windows into the origin and fate of aneuploidy through embryonic and fetal development.

OBJECTIVE: To review the mosaic autosomal trisomies in chorionic villi sample (CVS) trophoblasts, mesenchyme, and both cell lineages and to compare them with trisomies in spontaneous abortions. METHODS: Mosaic autosomal trisomies from 76 102 diagnostic CVS tests were classified as involving trophoblasts, involving mesenchyme, or present in both. Autosomal trisomies in products of conception were based on 18 published studies. We evaluated correlates between trisomy frequency with chromosome size or number of protein coding genes in the imbalance. RESULTS: Distinctly different patterns of trisomy were found in trophoblasts, mesenchyme, or both. In trisomic spontaneous abortions, there was a weak, borderline significant, inverse association between frequency and trisomic chromosome size and also with the number of protein coding genes involved (r = 0.43, P = 0.04 and r = 0.39, P = 0.07, respectively). These associations became stronger after excluding trisomy 16 (r = 0.52, P = 0.01 and r = 0.64, P = 0.001, respectively). Only CVS trisomies in both trophoblasts and mesenchyme resembled the trisomies found in spontaneous abortions and these were also associated with chromosome size and protein coding genes (r = 0.42, P = 0.05 and r = 0.57, P = 0.006, respectively). CONCLUSION: The abnormalities seen in CVS differ from those reported in early embryos. From conception through birth, there are lineage-specific, evolving spectrums of aneuploidy in trophoblasts, mesenchyme, and fetus.

Trisomy 8 mosaicism in the placenta: A Danish cohort study of 37 cases and a literature review.

OBJECTIVE: To evaluate the risk of fetal involvement when trisomy 8 mosaicism (T8M) is detected in chorionic villus samples (CVS). METHODS: A retrospective descriptive study of registered pregnancies in Denmark with T8M in CVS identified through a database search and a review of published cases of T8M found through a systematic literature search and inclusion of cross references. Pregnancies with T8M in CVS and no additional numerical chromosomal aberrations were included. RESULTS: A total of 37 Danish cases and 60 published cases were included. T8M detected in a CVS was associated with fetal involvement in 18 out of 97 pregnancies (18.6% [95%CI: 11.4-27.7]). Eight out of 70 (11.4% [95%CI: 5.1-21.3]) interpreted prenatally to be confined placental mosaicism (CPM) were subsequently found to be true fetal mosaicisms (TFM). CONCLUSION: T8M detected in CVS poses a significant risk of fetal involvement, and examination of amniotic fluid (AF) and/or fetal tissue should be offered. However, a normal result of AF still has a considerable residual risk of fetal involvement. Genetic counselling at an early gestational age is essential, and follow-up ultrasonography should be performed to predict fetal involvement if possible.

Prenatal array comparative genomic hybridization in a well-defined cohort of high-risk pregnancies. A 3-year implementation results in a public tertiary academic referral hospital.

OBJECTIVE: To find out whether the diagnostic yield of prenatal array comparative genomic hybridization (aCGH) can be improved by targeting preselected high-risk pregnancies. METHOD: All the in-house arrays ordered by the Fetomaternal Medical Center from February 2016 until December 2018 were retrospectively analyzed. The indications for array analysis included fetal structural abnormalities, increased nuchal translucency ≥3.5 mm and a chromosomal abnormality in a parent or a sibling. Common aneuploidies were excluded. RESULTS: Diagnostic yield was 15.1% in the entire patient cohort and as high as 20% in fetuses with multiple structural anomalies. The diagnostic yield was lowest in the group with isolated growth retardation. A total of 76 copy number variants (CNVs) were reported from a total of 65 samples, including 16 CNVs associated with a well-described microdeletion/microduplication syndrome, six autosomal trisomies in mosaic form, and three pathogenic single-gene deletions with dominant inheritance and 12 CNVs known to be risk factors for eg developmental delay. CONCLUSION: The diagnostic yield of aCGH was higher than what has previously been reported in less defined patient cohorts. However, the number of CNVs with unclear correlation to the fetal ultrasound findings was still relatively high. The importance of adequate pre- and posttest counseling must therefore be emphasized.

Accurate fetal variant calling in the presence of maternal cell contamination.

High-throughput sequencing of fetal DNA is a promising and increasingly common method for the discovery of all (or all coding) genetic variants in the fetus, either as part of prenatal screening or diagnosis, or for genetic diagnosis of spontaneous abortions. In many cases, the fetal DNA (from chorionic villi, amniotic fluid, or abortive tissue) can be contaminated with maternal cells, resulting in the mixture of fetal and maternal DNA. This maternal cell contamination (MCC) undermines the assumption, made by traditional variant callers, that each allele in a heterozygous site is covered, on average, by 50% of the reads, and therefore can lead to erroneous genotype calls. We present a panel of methods for reducing the genotyping error in the presence of MCC. All methods start with the output of GATK HaplotypeCaller on the sequencing data for the (contaminated) fetal sample and both of its parents, and additionally rely on information about the MCC fraction (which itself is readily estimated from the high-throughput sequencing data). The first of these methods uses a Bayesian probabilistic model to correct the fetal genotype calls produced by MCC-unaware HaplotypeCaller. The other two methods "learn" the genotype-correction model from examples. We use simulated contaminated fetal data to train and test the models. Using the test sets, we show that all three methods lead to substantially improved accuracy when compared with the original MCC-unaware HaplotypeCaller calls. We then apply the best-performing method to three chorionic villus samples from spontaneously terminated pregnancies.

Determination of morphologic and immunohistochemical stain (p57 kip2) discrepancy of complete and partial hydatidiform mole by using microsatellite genotyping.

OBJECTIVE: to evaluate the role of microsatellite genotyping in discordant results between morphologic examination and p57Kip2 staining in hydatidiform mole. MATERIALS AND METHODS: 127 cases of hydatidiform mole who had morphologic examination and p57Kip2immunohistochemical staining were evaluated. Six discrepant cases between morphologic examination and p57Kip2 staining were recruited. DNA was extracted from chorionic villi and paired maternal decidual tissue in Formalin fixed paraffin embedded tissue sections. The STR DNA genotyping was performed by Applied Biosystems 3500 Genetic Analyzer. Genetic data analysis was performed by Gene mapper ID-X software. Three concordant cases were used as control. Results were compared to histopathology, p57Kip2 stain and development of post-molar GTN. RESULTS: All controlled cases were confirmed PHM. Two cases of histologic CHM with positive p57Kip2and 2 cases of PHM with negative p57Kip2 were reported as PHM from microsatellite. Other 2 cases of histologic diagnosis PHM with negative p57Kip2 reported as CHM from microsatellite test and both of them developed post-molar GTN. CONCLUSION: Microsatellite genotyping is a high accuracy method for differential diagnosis from complete and partial hydatidiform moles. However, cost of microsatellite genotyping is still too high to use routinely. Therefore, selected use in discrepancy cases may be suitable.

Comparison of indications and results of prenatal invasive diagnostic tests before and after the implementation of the use of cell-free fetal DNA: a tertiary referral center experience.

PURPOSE: In this study, we aimed to compare the changes in the number, yield, and the percentage of karyotyping indications of the invasive prenatal diagnostic tests between the periods before and after cell-free fetal DNA was introduced to clinical use. METHOD: The number of invasive prenatal diagnostic procedures such as amniocentesis and chorionic villus sampling, indication percentages and karyotype results in the periods before (January 1, 2009-December 31, 2010), (n = 1412) and after (January 1, 2016-December 31, 2017), and (n = 593) the introduction of cell-free fetal DNA was retrospectively evaluated. RESULTS: When compared with the period before cell-free fetal DNA came into clinical use, the number of invasive prenatal diagnostic tests decreased by 58% while their yield was found to have increased (4.4% vs. 10.3%) in the period after cell-free DNA began to be used (p < 0.001). While there was a decrease in the indications due to advanced maternal age, an increase was found in ultrasonography indications for structural anomaly and the risk of a single-gene disorder (p < 0.001). Amniocentesis rate was found to have decreased in invasive prenatal diagnostic procedure types, while an increase was reported in CVS rates (p < 0.001). CONCLUSIONS: Invasive prenatal diagnosis gradually decreases over the years, but the yield of invasive prenatal diagnostic tests increases. In parallel with the rapid development of modern molecular technologies and cheaper and easier access to the tests, we think that the number of invasive prenatal diagnostic tests will experience a more dramatic decrease in the following years.

The Critical Role of Prenatal Genetic Study in Prevention of Primary Immunodeficiency in High-risk Families: The Largest Report of 107 Cases.

This study aims to investigate the role of prenatal diagnosis (PND) in Iranian couples with a previous history of primary immunodeficiency disorders (PIDD) in their family. All referred couples with a family history of PIDD and a tendency for PND were included in this project. Based on gestational age, chorionic villus sampling (CVS) was performed to analyze the molecular defect of the fetus according to the previous gene defect of the affected case in the family. Postnatal confirmation was performed by immunological screening tests. In a total of 100 cases, CVS was not evaluated in 19 patients due to unwillingness (n=5), late prenatal referral (n=7), miscarriage before CVS (n=3), and female fetus with x-linked diseases in previous children (n=4). In the remaining 81 patients, heterozygous and homozygous mutations were found in 33 and 23 cases, respectively. The hemizygous mutation was obtained in 6 and no pathogenic mutations were found in 19 individuals. Postnatal evaluations revealed that a total of 65 babies were healthy, 32 fetuses were aborted (3 cases before CVS, 2 spontaneous abortions of a healthy and as affected fetus in the CVS subgroup, and 27 cases were aborted due to therapeutic causes). One fetus from the heterozygous subgroup was spontaneously aborted with severe combined immunodeficiency (SCID) and one fetus from the homozygous subgroup that was supposed to be healthy was affected by the autosomal dominant-chronic granulomatous disease (AR-CGD). The diagnostic error was 1.2%. PND is highly recommended in families with a history of PID in their previous child to prevent an affected baby being born and to reduce the government, family, and personal burden of these diseases.

Placental mosaicism in the era of chromosomal microarrays.

OBJECTIVE: Placental mosaicism for a subset of a chromosome, a structural chromosomal aberration, is thought to be a very rare finding in chorionic villus samples. Here, we present clinical and laboratory data on five cases with such mosaicism for structural chromosomal aberrations. METHODS: During a period of 6 months, chromosomal microarray was carried out on DNA extracted from 100 uncultured chorion villous samples from high-risk pregnancies. RESULTS: In five of 100 consecutively collected samples (5/100), mosaicism for a structural chromosomal aberration was detected. The mosaic aberration was subsequently detected in fetal tissue in three of the five cases. CONCLUSION: Chromosomal microarray can detect placental mosaicism for structural chromosomal aberrations. This kind of mosaicism may be more frequent than previously anticipated, and the fetal involvement seems difficult to predict. These findings highlight the complexity of mosaicism for structural chromosomal aberrations in prenatal samples in the chromosomal microarray era.

The current and future impact of genome-wide sequencing on fetal precision medicine.

Next-generation sequencing and other genomic technologies are transforming prenatal and reproductive screening and testing for fetal genetic disorders at an unprecedented pace. Original approaches of screening and testing for fetal genetic and genomic disorders were focused on a few more prevalent conditions that were easily diagnosable with pre-genomic era diagnostic tools. First, chromosomal microarray analysis and then next-generation sequencing brought technology capable of more detailed genomic evaluation to prenatal genetic screening and diagnosis. This has facilitated parallel introduction of a variety of new tests on maternal blood samples, including expanded carrier screening and cell-free DNA-based non-invasive screening for fetal aneuploidy, selected copy number variants, and single-gene disorders. Genomic tests on fetal DNA samples, obtained primarily through amniocentesis or chorionic villus sampling, include chromosomal microarray analysis and gene panel and exome sequencing. All these form the diagnostic pillar of the emerging field of fetal precision medicine, but their implementation is associated with ethical, counseling and healthcare resource utilization challenges. We discuss where in the reproductive and prenatal care continuum these exciting new technologies are integrated, along with associated challenges. We propose areas of priority for research to gain the data in support of their responsible implementation into clinical reproductive and prenatal care.