Title: Prospective Cytogenetic and Y Chromosome Microdeletion Screening in Male Fetuses Pregnant After Intracytoplasmic Sperm Injection
Abstract: Objectives: Intracytoplasmic sperm injection (ICSI) is a new technology incorporated in the field of assisted reproductive technology. ICSI is a more invasive procedure, because one sperm cell is injected through the oocyte's cytoplasm and fertilization can be obtained from spermatozoa. However, this procedure has not been previously applied in fertility treatment. Therefore, the safety of this assisted fertilization should be carefully assessed. The purpose of this study was to screen chromosomal aberrations and DAZ deletions in cultured amniocytes of confirmed male fetuses pregnant after intracytoplasmic sperm injection. Design: Prospective clinical study. Materials and Methods: The subjects consisted of 94 male fetuses that confirmed by cultured amniocytes. Chromosomal preparations were obtained from cultured amniocytes according to modified technique of Verma and Babu (1989). Sequence-tagged sites (STSs)-based PCR analyzes were performed on DNA isolated from amniocytes derived from amniotic fluid of 15–23 weeks of gestation that had previously undergone the ICSI procedure. Ten primer pairs namely, sY134, sY138, MK5, sY152, sY147, sY254, sY255, SPGY1, sY269 and sY158 were used. The identified STS primer pairs were tested by two independent amplification reactions to confirm the deletion. DNA from non-ICSI male and female amniocytes was used as a control. The samples with deletions were verified at least three times. Results: Three out of 94 (3.2%) subjects showed chromosomal aberrations. Two out of total 94 (2.1%) subjects were familial structural aberrations: 46,XY,t(4;6)(q31.3;q25) and 46,X,der(Y)t(Y;18)(p12;p11.2). All these structural aberrations were inherited from the father. Abnormal fetal karyotypes was found in only one case out of 94 fetuses tested: 46,XY,t(9;12)(p13;q24). On the other hand, deletion frequency for each category of semen profile of the father of male fetus was as follows: Sixteen (16.8%) of the 94 male fetuses had deletions at the DAZ region of the Y chromosome. Three of the 20 azoospermic (15%), four of the 24 oligozoospermic (16.7%), five of the 26 asthenozoospermic (19.3%), three of the 20 OATS (oligo-astheno-teratozoospermia) (15%) and one of the 5 normozoospermic (20%) fathers' sperm profile of male fetus had such deletions. In this study, deletions were not detected in patients with abnormal chromosomal aberrations. Conclusions: Cytogenetic analysis determined a total of 94 karyotypes, of which one was de novo (1.1%) and two (2.1%) were familial structural aberrations, all transmitted from the father and were already present before conception. Those de novo and familial structural aberrations are probably directly linked to the characteristics of the infertile man treated rather than to the ICSI procedure itself. Surprisingly, deletion was found even in the male fetus of normozoospermic father. According to fathers' sperm profile, there were no differences in deletion frequency. In this respect, chromosome test and analysis of microdeletions should also be performed in all patients who are candidates for microassisted fertilization technique. However, one should be cautious in analyzing these data, as the study group was relatively small.