Cell-free DNA test for fetal chromosomal abnormalities in multiple pregnancies.

INTRODUCTION: This study aimed to report the screening performance of cell-free DNA (cfDNA) testing for chromosomal abnormalities in twins, triplets, and vanishing twin pregnancies. MATERIAL AND METHODS: Data were obtained from pregnant women with a multiple pregnancy or a vanishing twin pregnancy at ≥10 weeks' gestation who requested self-financed cfDNA testing between May 2015 and December 2021. Those that had positive screening results had diagnostic confirmatory procedures after counseling and consent. The performance of screening of the cfDNA test was determined by calculating confirmation rate and combined false-positive rate (cFPR). RESULTS: Data from 292 women were included after exclusion of those lost to follow-up, with no-result on cfDNA testing, or had reductions. Of the 292 pregnancies, 10 (3.4%) were triplets, including no cases of trisomy 21 and trisomy 18; 249 (85.3%) were twins, including 3 cases of trisomy 21 and no cases of trisomy 18 and 13; and 33 (11.3%) were vanishing twins, including 3 cases of trisomy 21 and 1 case of trisomy 18. The median (IQR) maternal age was 34 years (31-37). For triplet pregnancies, the initial no-result rate was 10.3% (95% confidence interval [CI] 3.6-26.4), all with results after redraw. For twin pregnancies, the initial no-result rate was 12.9% (95% CI 9.6-17.0), and the no-result rate after redraw was 1.6% (95% CI 0.7-3.6). For vanishing twins, there were no cases with no-result. All triplets had low-risk cfDNA results. The confirmation rate for trisomy 21 was 100% with a FPR at 0% due to the small number of positive cases for twins. For vanishing twins, one high-risk case for trisomy 21 and the only high-risk case for trisomy 18 were confirmed with a cFPR of 8.3% (n = 2/24; 95% CI 2.3-25.9). CONCLUSIONS: cfDNA testing in twin pregnancies has sufficient screening performance for trisomy 21 but the number of affected cases for other conditions is limited to draw any meaningful conclusion. The use of cfDNA testing in triplet pregnancies and vanishing twins remains an area for further research.

Genome-Wide Cell-Free DNA Test for Fetal Chromosomal Abnormalities and Variants: Unrestricted Versus Restricted Reporting.

This study aimed to compare the screening performance of genome-wide cfDNA testing for chromosomal abnormalities between two periods where additional findings were reported and not reported. Data were obtained from consecutive pregnant women with a singleton pregnancy at ≥10 weeks who requested cfDNA testing during 2015-2019. The performance of screening of the cfDNA test was determined by calculating the concordance rate, detection rate, and false-positive rate. Data from 3981 women were included. The no-result rates were similar between the two reporting periods (2.04% vs. 2.08%). Concordance rates for trisomy 21 and 18 were 100% and 100%, respectively. There were two cases tested high risk for trisomy 13, with a concordance rate of 0%. In total, 12 cases were high risk for any sex chromosome aneuploidy with an overall concordance of 75%, and 15 cases tested high risk for any rare autosomal trisomy, with a 13.3% concordance rate. The detection rates for trisomy 21 and 18 were 100% and 100%, respectively. For any SCA, the detection rate was 90%. For the two reporting periods, the combined false-positive rates were 0.93% and 0.17%, which were significantly different (p = 0.002). Restricting the reporting of additional findings from genome-wide cfDNA analysis has reduced the false-positive rate but without a reduction in the no-result rate.

A prospective study of non-invasive preimplantation genetic testing for aneuploidies (NiPGT-A) using next-generation sequencing (NGS) on spent culture media (SCM).

PURPOSE: This study was to evaluate if spent culture media (SCM) of embryos could be used as a non-invasive tool to achieve aneuploidy screening. Ploidy calls, as well as concordance rates between PGT-A results from trophectoderm (TE) and SCM, were compared. Clinical outcomes of single euploid transfers were also evaluated. METHODS: The study was conducted from March 2017 to June 2018 in a university-based ART center. SCM of day 3 to the day(s) of TE biopsy of all biopsied blastocysts were collected for testing. PGT-A results of SCM were compared with the standard results of TE, with clinical relevance and outcomes examined. RESULTS: NiPGT-A using SCM gave a sensitivity of 81.6%, specificity of 48.3%, positive predictive value of 82.6%, and negative predictive value of 46.7% in ploidy calling. The concordance rates for autosomes and sex determination were 62.1% and 82.4%, respectively. There were 14 single embryo transfer cycles of euploids as determined by TE biopsy. Clinical outcomes not only confirmed 3 false positive results from SCM but also reflected the true ploidy status of the transferred embryo in one case. If ploidy calls were dichotomized without mosaic embryos, the sensitivity and NPV would increase to 91.0% and 66.7% (p = 0.60 and p = 0.25), respectively. CONCLUSIONS: Cell-free DNA found in SCM could provide ploidy information of an embryo as in PGT-A from its TE. Given its potential to reflect the comprehensive chromosomal profile of the whole embryo, more research based on clinical outcomes is required to determine if SCM could be a reliable selection tool in PGT-A.

Semi-quantitative fluorescent PCR analysis identifies PRKAA1 on chromosome 5 as a potential candidate cancer gene of cervical cancer.

OBJECTIVE: Comparative genomic hybridization has frequently detected amplification of chromosome 5p in cervical cancer, but candidate cancer genes within the region are rarely known. Therefore, we pursued to identify potential candidate gene related to cervical cancer development. METHODS: A series of 128 cervical tumor samples were examined by semi-quantitative fluorescent differential PCR for copy number changes on three candidate genes (PRKAA1, CTNND2 and POLS) mapped to chromosome 5p and one gene (ERBIN) mapped to chromosome 5q12.3. The impact of gene copy number was later analyzed in relation to HPV infection, tumor stage or tumor radiosensitivity. RESULTS: DNA copy numbers of PRKAA1, CTNND2 and ERBIN were significantly different from normal controls (P < 0.05). DNA copy number changes did not correlate with HPV infection, tumor stages or tumor radiosensitivity. Using RT-PCR, PRKAA1 mRNA expression in seven tumor samples with known 5p amplification was amplified from 3- to 15-fold. Over-expression of PRKAA1 was further confirmed by immunohistochemical staining on 125 paraffin-embedded cervical cancer tissues. The expression level in cervical tumor was significantly higher than that in normal epithelium (P < 0.001). CONCLUSIONS: PRKAA1 gene codes for the catalytic alpha 1 subunit of the AMP-activated protein kinase which is an important cellular metabolic stress regulator. It might assist tumor cells growth under stress. Thus, PRKAA1 may be one of the potential candidate genes for cervical carcinogenesis.

Genetic abnormalities and HPV status in cervical and vulvar squamous cell carcinomas.

Cervical and vulvar cancers are diseases of the female lower genital tract, and high-risk human papillomavirus (HPV) infection is the most important risk factor for the development of both cancers. However, it is clear that additional genetic events are necessary for tumor progression, particularly in HPV-negative cases. We detected the presence of high-risk HPV16 and HPV18 genomes by gene-specific polymerase chain reaction and searched for common genetic imbalances by comparative genomic hybridization (CGH) in 28 cervical and 8 vulvar tumor samples and 7 cancer cell lines. The presence of the HPV genome was detected in 25/28 (89%) cervical tumors and 6/8 (75%) vulvar tumors. CGH of cervical and vulvar tumor samples revealed a consistent pattern of genetic changes in both cancers. Frequent gains were found in 1q, 3q, 5p, and 8q, and less consistent losses were detected in 2q, 3p, 4p, and 11p. Notably, a high-level amplification of 3q was found in 9/28 (32%) cervical tumors and 1/8 (12.5%) vulvar tumors, indicating a pivotal role of gain of 3q in cervical and vulvar carcinogenesis. Furthermore, gains of 5p identified in 9/28 (32%) cervical tumors and 3/8 (37.5%) vulvar tumors were seldom described, particularly in vulvar tumors. Our findings suggest that cervical and vulvar carcinomas bear similar chromosomal alteration hot spots that largely coincide with common genomic lesions during tumor progression, besides the initiation by infection and integration of oncogenic HPV.