Fetal Nucleated Red Blood Cells Preferable Than Cell-Free Fetal DNA for Early Determination of Gender Among Invasive and Non-Invasive Source Using Novel Four Genes Multiplex PCR

Abstract

Background Deoxyribonucleic acid from invasive, non-invasive and 9th week embryo can be a resource for the determination of fetal sex using highly sensitive and specific multiplex PCR. Methods A total of 402 DNA samples were used to test the newly developed novel multiplex PCR including male specific (3 genes: SRY, DAZ2 and TSPY1 ) Y-biomarkers and internal control, ACTB . The study isolated cffDNA (Cell-free fetal DNA; n = 73) from mother’s plasma, serum and urine, fetal DNA from 9th week embryo and cord blood, and fetal DNA from CD71 +ve nucleated red blood cells (fNRBC; n = 73). Paternal and maternal DNA from buccal cells (n = 20) and blood (n = 232) used for male and female confirmation. Results The study observed that SRY alone cannot be a suitable Y-biomarker. Confirmation from any two Y-biomarkers is mandatory for male fetus identification. Direct sequencing of the gel eluted multiplex and single amplicons confirmed the specific sequences. Presence of two out of 3 Y-biomarkers OR single Y-biomarker with >1,000,000 intensity is considered positive for male. The multiplex PCR is suitable for determining sex from all source of fetal DNA including highly degraded cffDNA and can detect the sex using 0.5ng DNA. Individual marker-based real-time qPCR followed by combined melt curve analysis showed distinguished melt curve peaks for the markers. Conclusion The multiplex PCR achieved 100% accuracy on fetal DNA from fNRBC for early determinations (<13 weeks) of gender. The developed novel and simple multiplex PCR and individual qPCR can be adopted in all types of laboratories for determining human fetal gender using fetal DNA from fNRBC. Early identification of gender can support to prepare for possible X-linked analysis, reduce anxiety in mother, strengthen a bond between mother and fetus, and effective decision making. Non-invasive source of fetal DNA from fNRBC preferred for identifying gender to reduce the risk of invasive procedures in early (8–13 weeks) pregnancy.

Aptamer-Mediated Enrichment of Rare Circulating Fetal Nucleated Red Blood Cells for Noninvasive Prenatal Diagnosis

“…In addition to high-throughput sequencing of cffDNA fragments, circulating fetal cells containing intact genome information with high purity provide another promising target for noninvasive genetic diagnosis. − So far, different kinds of fetal cells, including trophoblast cells, hematopoietic progenitor cells, fetal lymphocytes, and fetal NRBCs, have been identified in maternal circulation. ,, Among them, fNRBCs have advantages such as fetal rather than placental origin and short lifetime during maternal circulation, making them an optimal target for cell-based NIPT. − With the advancement of single-cell genetic analysis, the development of reliable strategies for rare circulating fetal cell enrichment from maternal peripheral blood remains one of the biggest obstacles to the clinical translation of cfNRBC-based NIPT. In past decades, different fetal cell enrichment technologies have been developed based on their biochemical or biophysical properties, such as magnetic- or fluorescence-activated cell sorting, , microfluidics, − dielectrophoresis, and cell size-based isolation .…”