The study on X・Y sperm preseparation and the gender detemination by sex-specific DNA diagnosis

目前已有20多名患者接受了植入前性别鉴定，但目前尚需一种更准确、更快速、更简便的方法来进行临床应用。我们开发了一种用于性别鉴定的新的多重PCR方法，即使用X和Y染色体特定重复序列的共同扩增。我们已经评估了该检测的效率和准确性，并测量了该检测所需的时间。确定了该方法的最佳条件，并将50例羊水标本的性别鉴定结果与培养和核型鉴定结果进行了比较。用该方法分析了50个46XX单个羊水细胞和50个46XY单个羊水细胞。对单细胞检测的效率和准确性进行了评价。在装有显微操作器的倒置显微镜下分离单个羊膜细胞。X1-1、X1-2引物组来源于X染色体的着丝粒周围区域(DXZ-1)。Y1-1、Y1-2引物组来源于Y染色体的长臂(DYZ-1)。X和Y来源的扩增片段分别为308和154个碱基对。用该方法对连续50例羊水标本(46XX：17例，46XY：31例，47XY，+18：1例，46XX，t(1：3：16)：1例)进行了胎儿性别的准确诊断。我们分析了50个46XX单个羊水细胞和50个46XY单个羊水细胞。在100个羊水样本中，99个(99%)被准确诊断，只有一个样本没有被扩增。未观察到污染。本实验所需时间为2小时。该方法用于单细胞性别鉴定是一种高效、准确的方法，可用于植入前的诊断。本试验仅需2小时，不会干扰着床率。

More than 20 candidates have undergone preimplantation gender determination by polymerase chain reaction (PCR), but a more accurate, rapid and simple method has been needed for general clinical application. We have developed a new multiplex PCR assay for gender determination, those employing co-amplification of the X and Y chromosome specific repeated sequences. We have evaluated the efficiency and accuracy of this assay and measered the time needed for this assay. The most optimum condition for the PCR assay was determined and the results of sexing by the assay in 50 amniotic fluid samples were compared with that of sexing by culture and karyotyping. Fifty single 46XX amniocytes and 50 single 46XY amniocytes were analyzed by this assay. The efficiency and accuracy of the assay in single cell was evaluated. Single amniocytes were isolated under an inverted microscope equipped with micromanipulators. The X1-1, X1-2 primer set was derived from the pericentric region of the X chromosome (DXZ-1). The Y1-1, Y1-2 primer set was derived from the long arm of the Y chromosome (DYZ-1). The X-and Y-derived amplified fragment ware 308 and 154 base pairs, respectively. Using this multilex PCR assay, we could diagnose fetal sex accurately in 50 consecutive amniotic fluid samples (46XX : 17 cases, 46XY : 31 cases, 47XY,+18 : 1 case, 46XX,t (1 : 3 : 16) : 1 case). We analyzed 50 single 46XX amniocytes and 50 single 46XY amniocytes. Ninety-nine out of 100 amniocyte samples (99%) were accurately diagnosed and only one sample was not amplified. No contamination was observed. The time needed for this assay was 2 hours. This multiplex PCR assay for gender determination in single cell is effeicient and accurate and can be used for preimplantation diagnosis. This assay needs only 2 hours and will not disturb implantation rate.