Evaluating sex chromosome content of sorted human sperm samples with use of dual-color fluorescence in situ hybridization

OBJECTIVE: Although most methods for selecting the sex of offspring by sorting spermatozoa are ineffective at shifting the ratio of Y- to X-containing cells, some commercial sources continue to offer such services. Our objective was to evaluate commercially “sorted” samples with use of dual-color fluorescence in situ hybridization and to identify variations in assessment by comparing motile and total sperm populations, donors, observers, and fluorescence in situ hybridization probes.STUDY DESIGN: Cryopreserved sperm from seven anonymous donors were processed as for insemination. Sperm cells from each total sample or motile subfraction were prepared for fluorescence in situ hybridization by incubation with disulfide-reducing agents to expand sperm nuclei. Two sets of X and Y chromosome–specific, fluorophore-labeled deoxyribonucleic acid probes were used. At least 400 nuclei from each preparation were classified independently by three blinded observers. Hybridization efficiency, aneuploidy, and sex chromosome content were evaluated in subsets of five unsorted, five female-oriented, and five male-oriented samples. Total and motile subfractions were compared with eight samples. Fluorescence in situ hybridization probes were compared in five paired unsorted samples.RESULTS: No differences were detected between washed samples and paired motile subfractions. No differences in hybridization and aneuploidy were detected between groups of sorted samples. The Y/X ratio was significantly different between the sorted groups. However, male-oriented samples had a lower Y/X ratio than female-oriented samples did. Observer and probe choice accounted for small but significant variations that did not alter conclusions about the Y/X ratio for sorted samples.CONCLUSION: In a series of 10 sorted samples from one commercial source, dual-color fluorescence in situ hybridization demonstrated a small but significant shift in the sex chromosome ratios among samples. However, this shift was opposite to that expected by the orientation of the sorted samples. (Am J Obstet Gynecol 1997;176:1172-80.)     

海藻糖在气管低温保存中的保护机制

目的探讨海藻糖在气管低温保存中的保护作用和机制。方法新鲜配制4组保存液，其中：Ⅰ组LPD；Ⅱ组LPD 10％DMSO；Ⅲ组LPD 0．15M海藻糖；Ⅳ组LPD 10％DMSO 0．15M海藻糖。切取SD大鼠气管后立即分别放入含上述4种溶液的冻存管，在程序降温仪降至-80℃后投入液氮中保存。20d后对气管标本进行HE染色和Bcl-2、Pax蛋白检测(免疫组化)，并将其异位移植到Wistar大鼠腹腔，15d后取出移植气管，观察组织学变化。结果4种保存液低温保存气管20d后，Ⅲ组和Ⅳ组气管结构完整，软骨细胞Pax蛋白表达较低，尤其Ⅳ组保存效果最好，同种异体异位移植后生长良好。各组Bcl-2蛋白表达无明显区别。结论在气管低温保存中海藻糖的保护作用主要是通过对软骨细胞的保护实现的，抑制软骨细胞Pax基因的表达是其中的保护机制之一。海藻糖和DMSO合用保护作用更好。