Decrease of spermatozoa with an unbalanced chromosome content after cell sorting in men carrying a structural chromosomal abnormality

Abstract

Background: We showed that in men with a constitutional chromosomal abnormality, DNA fragmentation was significantly higher in chromosomally unbalanced spermatozoa than in spermatozoa with a normal or balanced chromosomal content. These results could be explained by a phenomenon already described in infertile men: abortive apoptosis. Objectives: To determine if magnetic-activated cell separation could select spermatozoa with lower levels of DNA fragmentation and unbalanced chromosome content in men carrying a structural chromosomal abnormality. Materials and methods: The spermatozoa of ten males with a chromosomal rearrangement were separated into two populations using MACS (annexin V (-) and annexin V (+) fractions), in order to study meiotic segregation by FISH, the percentage of spermatozoa with an externalization of phosphatidylserine (EPS) by annexin V staining and DNA fragmentation by TUNEL on the whole ejaculate and on selected spermatozoa in the same patient. Results: For all patients, the percentage of spermatozoa with EPS decreased in the annexin V (-) fraction and increased in the annexin V (+) fraction as compared to the frozen-thawed semen sample. The rates of DNA fragmentation were statistically much lower in the annexin V (-) fraction when compared to the rate before MACS for all but one patient. Conversely, we observed a statistically significant higher rate of DNA fragmentation in the annexin V (+) fraction for 6 patients. After MACS, there was a significant increase of normal/balanced spermatozoa in the fraction of annexin V (-) for all patients. Conversely, we observed a significant decrease in the fraction of annexin V (+) for seven patients. Discussion and Conclusions: MACS is a promising tool for increasing the selection of healthy spermatozoa, with a decrease in the number of spermatozoa with EPS, DNA fragmentation and chromosome unbalance, for use in assisted reproductive technologies such as ICSI for males with a chromosomal structural abnormality.

MACS-annexin V cell sorting of semen samples with high TUNEL values decreases the concentration of cells with abnormal chromosomal content: a pilot study

“…On the contrary, the externalization of phosphatidylserine significantly increased in the apoptotic fraction compared to the frozen-thawed semen samples. Those trends were also identified in our previously published study in men with a structural chromosomal abnormality 35 and were similar to that found by other teams. 35 38 39 40 Moreover, our results confirm that MACS technique can effectively eliminate spermatozoa with EPS caused by apoptotic processes or caused by the premature acrosomal reaction.…”

“…Those trends were also identified in our previously published study in men with a structural chromosomal abnormality 35 and were similar to that found by other teams. 35 38 39 40 Moreover, our results confirm that MACS technique can effectively eliminate spermatozoa with EPS caused by apoptotic processes or caused by the premature acrosomal reaction. 35 38 39 40 However, in previous studies, we hypothesized that the number of beads or unspecific binding could be limited and these parameters could explain the presence of spermatozoa with EPS in the nonapoptotic fraction and/or the presence of spermatozoa negative for EPS in the apoptotic fraction.…”

“…The procedure has been previously published. 35 For each sample, five hundred spermatozoa were analyzed. The spermatozoa without EPS showed blue fluorescence and those with EPS showed green fluorescence.…”

“…The TUNEL assay has been described previously. 35 36 For each sample, five hundred spermatozoa were analyzed. Spermatozoa were considered to contain either fragmented (red fluorescence) or normal (blue fluorescence) DNA.…”

“…The procedure has been previously published. 35 37 For each sample, a minimum of 1000 spermatozoa was examined using a Zeiss Axioplan microscope (Zeiss, Le Pecq, France).…”