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

目前已有20多例胚胎植入前性别鉴定的研究，但尚需一种更为准确、快速、简便的方法来指导临床应用。我们已经开发了一种新的多重PCR检测性别确定，那些采用共扩增的X和Y染色体特异性重复序列。我们已经评估了该测定的效率和准确度，并测量了该测定所需的时间。确定了PCR法的最佳条件，并对50例羊水标本进行了性别鉴定，与培养法和染色体核型法进行了比较。通过该测定分析了50个单个46 XX巨噬细胞和50个单个46 XY巨噬细胞。评价了单细胞检测的效率和准确性。在配备显微操作器的倒置显微镜下分离单个巨噬细胞。X1-1、X1-2引物来自X染色体臂间区（DXZ-1）。Y1-1、Y1-2引物来自Y染色体长臂（DYZ-1）。X和Y扩增片段分别为308和154个碱基对。应用该方法对50例连续羊水标本（46 XX：17例，46 XY：31例，47 XY，+18：1例，46 XX，t（1：3：16）：1例）进行了胎儿性别诊断。我们分析了50个46 XX单核细胞和50个46 XY单核细胞。100个巨噬细胞样本中有99个（99%）被准确诊断，只有一个样本没有扩增。未观察到污染。该测定所需的时间为2小时。该方法可用于单细胞胚胎性别鉴定，具有快速、准确的特点，可用于胚胎植入前诊断。该试验仅需2小时，不会干扰植入率。

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.