Imprinting analysis in spermatozoa prepared for intracytoplasmic sperm injection (ICSI)

Genetic imprinting is a mechanism of gene regulation by which only one of the parental copies of a gene is expressed. This process is mediated by the methylation of DNA. As spermatozoa represent exclusively the paternal contribution to a future individual, they are expected to carry the paternal imprint only. For intracytoplasmic sperm injection (ICSI), spermatozoa mostly have to be selected from samples with pathological semen parameters. Correct establishment of the paternal imprint in these spermatozoa has not yet been demonstrated. In the present study, imprinting analysis was undertaken using DNA extracted from spermatozoa from men with normal semen analysis (group A: n=30 patients) and from men with an abnormal sperm count (B: n=30 patients with 5--20 million spermatozoa/mL and C: n=30 patients with < or =5 million spermatozoa/mL) from the ICSI program. It was performed using firstly a conventional methylation-specific polymerase-chain-reaction (M-PCR) and secondly a more sensitive modified hemi-nested M-PCR technique. In addition, a single cell PCR was performed on a total of 88 single spermatozoa (collected from nine males) and on 25 leucocytes (control group). With the conventional M-PCR, exclusively paternal imprints were found in all groups. Using the more sensitive hemi-nested M-PCR, additional maternal imprints were found in 63% of the samples in A, 57% in B and 60% in C. In the single cell PCR, exclusively paternal imprints were detected. Because of the very small amount of DNA (3 pg), a complete amplification failure occurred in 43% of spermatozoa. The correct paternal and maternal imprints were found in 56% of the analysed leucocytes (complete amplification failure in the other 44%). In conclusion, ejaculated spermatozoa from males with medium or high-grade semen pathology proved to have the same imprinting status as those from males with normal semen parameters. As the additional maternal imprints were never found at the single cell level, they were classified as contamination by diploid cells such as leucocytes or immature germ cells in the processed and purified semen samples, which can be detected by a more sensitive PCR method in contrast to the conventional standard PCR.