A study on bisulfite sequencing method for methylation status of imprinted genes in single human oocytes / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish and improve the method of bisulfite sequencing for methylation status of imprinted genes in single human oocytes.</p><p><b>METHODS</b>Single superovulated immature human oocyte was embedded into low melting point agarose, followed by bisulfite treatment and polymerase chain reaction (PCR) amplification of the H19 and MEST genes. The PCR products were then subjected to TA cloning and sequencing to determine the methylation status.</p><p><b>RESULTS</b>With the modified methods of embedding and bisulfite treatment, we achieved a high PCR success rate of 82.46%, with the somatic cell contamination rate as low as 7.14%. The sequencing results showed no non-CpG cytosine and exact conformity to the theoretical sequences.</p><p><b>CONCLUSION</b>The bisulfite sequencing method we used to determine the methylation status of imprinted genes at the single-cell level was highly efficient and reliable, which can serve as a foundation for the further study of the influences of human assisted reproductive technology on genomic imprinting.</p>

Meiotic segregation results of male reciprocal chromosome translocations / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze the meiotic segregation results of male reciprocal chromosome translocation by fluorescence in situ hybridization (FISH).</p><p><b>METHODS</b>Multi-color FISH using 3 combined probes located in any 3 chromosome segments on both sides of two breakpoints was performed on the de-condensed sperm head to analyze the sperm chromosomal contents and segregation patterns.</p><p><b>RESULTS</b>Four male reciprocal translocation carriers were included in the study, with the karyotypes of 46, XY, t(2;18) (p16; q23); 46, XY, t(4;6) (q34;q21); 46, XY, t(8;13) (q23;q21) and 46, XY, t(4;5) (4q31;5q13), respectively. The results showed that 4 carriers had different proportions of various segregated spermatozoa. The spermatozoa of alternate, adjacent-1, adjacent-2, 3:1, non-disjunction in meiosis II, and 4:0 or diploidy accounted for 27.1%-49.4%, 26.9%-37.6%, 2.7%-15.7%, 8.6%-32.7%, 0.2%-1.9%, and 0.1%-0.4%, respectively.</p><p><b>CONCLUSION</b>For each-reciprocal translocation carrier seems to have a particular meiotic segregation results, FISH analysis on sperm head should be done for each carrier in order to provide an accurate genetic counseling.</p>