Summary: SRY gene amplifications and genotypings revealed the occurrence of the hidden maternal decidual cells in 46,XX karyotyped spontaneous abortions: Reports of abnormal karyotypes or normal 46,XY karyotypes of the abortion materials derivateci from tissue cultures are mostly addressing the pregnancy loss tissues. The accuracy of the cytogenetic reports of normal 46,XX karyotypes is obscure, as the results may reflect the normal karyotyped female pregnancy losses or the hidden maternal decidual cells covering the cytogenetically normal or abnormal male or female products of conception. In the present study, thirty-eight 46,XX normal karyotyped abortion materials cultivated from villi were re-analysed for excluding maternal cell contamination by using molecular approaches in an accurate algorithm. Abortion materials DNAs were amplified by polymerase chain reaction (PCR) technique in order to search the products of the sex determinating region gene of chromosome Y (SRY). Sixteen out of 38 abortion materials revealed Y-chromosome component (42.1%). Amplification negative DNAs and their parental DNAs were genotyped by using high-polymorphic microsatellite DNA markers to identify the origin of the components of the chromosome X. Maternal chromosome X components were detected in 18 (81.8%). As a result, SRY amplifications and genotypings ascertained the high rate of maternal decidual cells in 46,XX products of conception.
Key-words: Abortion materials - Maternal cell contamination - SRY gene - PCR - Genotyping
Conventional cytogenetic analysis of abortion material is still a valuable tool as the approximately 20-50% of the abortions are due to chromosomal abnormalities and at least 80% of these aberrations are numerical (3-8, 11, 14, 17, 19). There is no doubt the abnormal reports and the normal 46,XY reports of the abortion materials, which are derivated from the cultivation of the villi, are reflecting the cytogenetic analysis of the pregnancy loss tissues. There can be misdiagnosis of cytogenetic reports of "normal 46,XX" karyotypes because of the probability of maternal cell contamination (MCC) of a normal and/ or an abnormal female or male product of conception (1, 12, 14, 18). The study was aimed to present the efficient way of distinguishing the maternal cells of these materials by structuring an accurate algorithm with the usage of the molecular techniques. To address this, in the first step, sex determination region gene of chromosome Y (SRY) amplifications of the abortion materials were performed by polymerase chain reaction (PCR) technique and SRY positive materials were evaluated as maternal cell contaminated. The remaining SRY negative materials were genotyped with their maternal and paternal DNAs by using highpolymorphic microsatellite DNA markers in order to detect the origin of the components of the chromosome X.
MATERIALS AND METHODS
Thirty-eight normal 46,XX karyotyped abortion material DNAs (from singleton pregnancies confirmed by ultrasonography) and their related maternal and paternal DNAs were analysed by using molecular techniques. No abnormal karyotyped and no 46,XY karyotyped spontaneous abortion materials were included in the study. Bioethics Review Board of the Faculty of Medicine, University of Gazi approved the study. With the routine informed consent for karyotyping, the parents also gave consent for advanced molecular analyses of the specimens.
TISSUE CULTURE AND CHROMOSOME ANALYSIS
Tissue samples which are obtained by curetting of the abortus were transferred immediately to the laboratory into the sterile culture medium. The materials were washed from blood in Hanks Balanced Solution (Biological Industries, Israel) and Bio-amf 1 Medium Solution (Biological Industries, Israel), respectively and were separated from decidual tissues. Some parts of the minced materials were used to initiate two or three separate cell cultures containing Bio-amf 1 Medium Solution, whereas the rest of the materials were stored at -2O0C for DNA isolations. …