Meiotic segregation and sperm DNA fragmentation in Tunisian men with dysplasia of the fibrous sheath (DFS) associated with head abnormalities.

PURPOSE: Dysplasia of the Fibrous Sheath (DFS) is a primitive flagellar pathology for which a broad spectrum of ultrastructural flagellar abnormalities has been described responsible for a severe to total asthenozoospermia. To this phenotype other morphological abnormalities including cephalic and abnormalities in nuclear structure can be associated that could compromise embryonic development in case of use of Assisted Reproductive Technology (ART). The aim of this study was to evaluate the level of DNA fragmentation and aneuploidy rate in ejaculated spermatozoa of Tunisian men presented with DFS sperm defect associated to high percentage of head abnormalities and to compare the results with those from fertile men. METHODS: Sperm DNA fragmentation was evaluated by the terminal desoxynucleotidyl transferase mediated deoxyuridine triphosphate biotin nick-end labelling (TUNEL) assay. The study of meiotic segregation was performed by Fluorescence in situ hybridization (FISH) for chromosomes X, Y and 18. RESULTS: The mean DNA fragmentation index was significantly higher in patients compared to the control group. FISH revealed a significantly higher incidence of sperm aneuploidies compared with controls. All patients showed elevated frequencies of sex chromosomes disomy, disomy 18 and diploidy. CONCLUSIONS: In some cases of syndromic teratozoospermia due to sperm tail structural abnormalities, such as DFS, other morphological cephalic abnormalities may be associated. In these cases we have demonstrated impaired sperm nuclear quality which will affect the results in ICSI. Hence the interest of a thorough study of the sperm nucleus in these forms of infertility in order to predict the chances of success in ART.

Efficacy of the density gradient centrifugation method in eliminating sperm with aneuploidy.

The aim of this study was to evaluate the efficacy of the PureSperm density gradient centrifugation on the selecting sperm with less chromosomal aneuploidy. Semen samples were obtained from 30 infertile men with teratozoospermia and 15 fertile men with normal semen parameters. The frequencies of numerical chromosomes aberrations were simultaneously identified in neat semen and in the different fractions of the density gradient centrifugation from the same samples. Using a triple colour FISH, we show that patients with severe teratozoospermia have a significantly increased frequency of chromosomal abnormalities in their neat semen compared with normozoospermic men (P < 0.001). The mean sperm motility and sperm morphology were improved significantly after semen processing with three layers PureSperm gradient compared with whole semen (P < 0.001). In addition, aneuploidy frequencies were lower in specimens enriched by the gradient centrifugation compared with unprocessed semen. Significant differences were observed in the disomy rates for the autosome and for either sex chromosome between the neat semen and the different PureSperm fractions (P < 0.001). In conclusion, our study shows that semen processing by density gradient centrifugation is very efficient in reducing sperm with aneuploidy and diploidy.

Study of aneuploidy rate and sperm DNA fragmentation in large-headed, multiple-tailed spermatozoa.

The aim of this study was to analyse the meiotic segregation and DNA fragmentation rates in ejaculated spermatozoa of Tunisian men who presented the macrocephalic sperm head syndrome and to compare the results with those from 20 fertile men with normal semen profiles. Sperm DNA fragmentation was evaluated by the terminal desoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labelling assay. Fluorescence in situ hybridisation for chromosomes X, Y and 18 was performed for the study of meiotic segregation. Despite a normal blood karyotype, patients with large-headed spermatozoa showed a significantly higher incidence of sperm chromosomal abnormalities compared with the control group. For all the patients, tetraploidy, triploidy and diploidy were the most observed abnormalities. A very high level of DNA fragmentation was shown for these patients. In conclusion, our results demonstrated that patients with large-headed, multiple-tailed spermatozoa had significantly higher incidence of sperm chromosomal abnormalities and very high level of DNA fragmentation. So intracytoplasmic sperm injection should not be recommended to these patients, not only because of its low chances of success rate but also because of its high genetic risk.

Aneuploidy rate in spermatozoa of selected men with severe teratozoospermia.

The aim of this study was to evaluate the incidence of spermatic aneuploidies in men with severe teratozoospermia and to determine an eventual relation between aneuploidies and a specific morphology of spermatozoa. Fluorescence in situ hybridisation (FISH) using a probe cocktail containing the alpha satellite for the centromeric region of chromosome X, Y and 18 was performed on decondensed spermatozoa from fresh ejaculates of thirty patients with severe teratozoospermia (abnormal forms >80%) and 15 fertile men with normal semen profiles. The mean frequency of teratozoospermia in patients was 91 ± 6.99%. There was statistically a significantly increased frequency of 1818, XY, XX and YY disomies in sperm with severe teratozoospermia compared with normal sperm (1.24% versus 0.08%, 1.42% versus 0.31%, 1.13% versus 0.19% and 1.11% versus 0.24%, respectively, P < 0.001 in all comparisons). The rate of total diploidy was significantly increased in patients compared with controls (1.46% versus 0.16%, P < 0.001). There was a correlation between macrocephalic spermatozoa and diploidy (r = 0.37, P < 0.05). Our data add further evidence that patients with severe teratozoospermia have an increased sperm aneuploidy rate and that this is particularly high in macrocephalic spermatozoa; FISH analysis on sperm could help to improve risk assessment and reproductive counselling in these individuals who are frequently candidates for intracytoplasmic sperm injection (ICSI) as a treatment of their infertility, as the use of ICSI has created consequential debate concerning the genetic risk for the offspring.

Semen processing by density gradient centrifugation is useful in selecting sperm with higher double-strand DNA integrity.

The objective of this study was to determine the effects of density gradient centrifugation on sperm cell DNA integrity and to correlate any detected DNA damage with semen analysis parameter. A total of 40 semen samples were collected from nonazoospermic men presenting for infertility evaluation at our department. Individual samples were divided into two parts: one part of the semen was washed and the remainder was prepared using the PureSperm density gradient centrifugation. Sperm DNA fragmentation as evaluated by the terminal desoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labelling assay, was monitored in the initially washed sample and in the different layers of the density gradient centrifugation. No significant correlations were observed between sperm DNA fragmentation, age of patient, concentration and motility. However, a significant correlation existed with strict spermatic morphology. Following density gradient centrifugation, the proportion of spermatozoa with DNA fragmentation decreased significantly when compared with whole semen. In addition, we found that spermatozoa isolated in the 90% layer possessed a significantly lower percentage of DNA damage when compared with those remaining in the 70% and 50% layers. These results demonstrate that semen processing by the PureSperm gradient is useful in selecting sperm with higher double-strand DNA integrity.