Esmaeili Mahnaz, Bazrgar Masood, Gourabi Hamid, Ebrahimi Bita, Boroujeni Parnaz Borjian, Fakhri Mostafa
Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran; Department of Genetics, Faculty of Basic Sciences and Advanced Technologies in Biology, Department of Genetics, University of Science and Culture, ACECR, Tehran, Iran.
Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Eur J Obstet Gynecol Reprod Biol. 2020 Sep;252:89-93. doi: 10.1016/j.ejogrb.2020.06.023. Epub 2020 Jun 12.
Preimplantation genetic testing (PGT) routinely requires biopsy which is an invasive approach. The aim of this study was to examine a noninvasive approach for sexing of preimplantation embryos using polymerase chain reaction (PCR)/reverse transcriptase-PCR (RT-PCR) based on the presence of SRY DNA/RNA in the spent culture medium. Two groups were evaluated: in group 1, 40 embryos of routine PGT volunteers were cultured individually after biopsy and in group 2, 31 embryos were cultured individually until Day-5 post-fertilization. Each group was further divided into three subgroups: RNA extraction (RE), nucleic acid (NA) and DNase treated (DT). In the NA and DT subgroups, cDNA synthesis was performed directly on culture medium with or without DNase treatment in DT and NA subgroups, respectively. The results of sexing based on the PCR/RT-PCR in the culture medium, were compared with the results of sexing by fluorescence in situ hybridization (FISH) technique. In group 1, all samples were correctly diagnosed. In group 2, five female samples were misdiagnosed. Test's sensitivity, specificity and accuracy were 100 %, 94.44 % and 96.88 %, in RE, 100 %, 81.82 % and 93.55 % in DT and 100 %, 71.43 % and 85.71 % in NA, respectively. Preimplantation sexing without embryo biopsy, in the spent embryo culture media using RNA amplification based methods including RE and DT seem to be more reliable while nucleic acid based method, NA, led to the highest misdiagnoses probably due to DNA contamination. Since all male samples were correctly diagnosed in all subgroups of this preliminary study, preimplantation noninvasive sexing on culture medium seems feasible, however all sources of nucleic acid contamination must be carefully avoided.
植入前基因检测（PGT）通常需要活检，这是一种侵入性方法。本研究的目的是基于废弃培养基中SRY DNA/RNA的存在，研究一种使用聚合酶链反应（PCR）/逆转录聚合酶链反应（RT-PCR）对植入前胚胎进行性别鉴定的非侵入性方法。评估了两组：第1组，40例常规PGT志愿者的胚胎在活检后单独培养，第2组，31例胚胎单独培养至受精后第5天。每组进一步分为三个亚组：RNA提取（RE）、核酸（NA）和脱氧核糖核酸酶处理（DT）。在NA和DT亚组中，分别在有或无脱氧核糖核酸酶处理的培养基上直接进行cDNA合成。将基于培养基中PCR/RT-PCR的性别鉴定结果与荧光原位杂交（FISH）技术的性别鉴定结果进行比较。在第1组中，所有样本均被正确诊断。在第2组中，5个女性样本被误诊。在RE组中，检测的灵敏度、特异性和准确性分别为100%、94.44%和96.88%，在DT组中分别为100%、81.82%和93.55%，在NA组中分别为100%、71.43%和85.71%。在废弃胚胎培养基中使用包括RE和DT在内的基于RNA扩增的方法进行无胚胎活检的植入前性别鉴定似乎更可靠，而基于核酸的方法NA导致误诊率最高，可能是由于DNA污染。由于在这项初步研究的所有亚组中所有男性样本均被正确诊断，因此在培养基上进行植入前非侵入性性别鉴定似乎是可行的，然而必须小心避免所有核酸污染来源。
