A double-blinded comparison of in situ TUNEL and aniline blue versus flow cytometry acridine orange for the determination of sperm DNA fragmentation and nucleus decondensation state index.

The impact of sperm DNA fragmentation on assisted reproductive technology (ART) successes, in terms of outcome, is now established. High levels of DNA strand breaks severely affect the probability of pregnancy. The importance of sperm nucleus condensation in early embryogenesis and, subsequently, on the quality of the conceptus is now emerging. In this article we have compared in situ analyses with terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labelling (TUNEL) (for DNA fragmentation) with aniline blue (AB) (for nucleus decondensation), versus flow cytometry (FC) after acridine orange staining, in a double-blinded analysis. In our hands, TUNEL and acridine orange give perfectly comparable results. For decondensation the results are also comparable, but the double-stranded green fluorescence obtained with acridine orange seems to slightly underestimate the decondensation status obtained with AB.

RHOXF2 gene, a new candidate gene for spermatogenesis failure.

INTRODUCTION: Genes involved in testicular differentiation, spermatogenesis, proliferation and apoptosis of germ cells have been shown to evolve rapidly and display rapid DNA changes. These genes are therefore good candidates for explaining impairments in spermatogenesis. Initial studies of some of these genes appear to confirm this hypothesis. The RHOXF2 candidate gene belongs to the RHOX family clustered in Xq24 and is specifically expressed in the testis. It contains four exons and codes for a 288 amino acid (aa) transcription factor. It has a high degree of homology (>99.9%) with its paralogue RHOXF2B, which is also preferentially expressed in the testis. OBJECTIVES: To sequence RHOXF2 and RHOXF2B in intracytoplasmic sperm injection (ICSI) patients and identify any single-nucleotide polymorphisms (SNPs) associated with impaired spermatogenesis. MATERIALS: A cohort of 327 patients in ICSI programmes at Poissy and Bichat hospitals. All patients gave their written, informed consent to participation. One hundred patients had unaffected spermatogenesis and 227 displayed impaired spermatogenesis. METHODS: The four exons in each of RHOXF2 and RHOXF2B were sequenced in 47 patients with oligospermia or non-obstructive azoospermia. Given that exons 2 and 3 were found to harbour most of the SNPs, only these two exons were sequenced in the remaining 280 subjects. RESULTS: Due to the extremely high degree of sequence identity between RHOXF2 and RHOXF2B, we were not able to distinguish between the sequences of these two genes. Although 9 SNPs were identified, there were no significant frequency differences between ICSI patients with normal vs. impaired spermatogenesis. Two insertions were identified: a 21-nucleotide insertion was retrieved in both groups and a guanine insertion (inducing a premature stop codon) only found in two patients with impaired spermatogenesis. CONCLUSION/OUTLOOK: RHOXF2 is a good candidate for rapid evolution by positive selection. Analysis of the polymorphism frequency in exons 2 and 3 did not allow us to correlate the identified SNPs with male infertility. However, a single nucleotide insertion was identified only in men with impaired spermatogenesis. Further work will be needed to establish whether genetic changes in RHOXF2 can give rise to defects in spermatogenesis.

INTRODUCTION: Les gènes impliqués dans la différenciation des testicules, la spermatogenèse, la prolifération et l'apoptose des cellules germinales ont été montrés comme ayant une évolution rapide de la séquence d'ADN. Ces gènes sont donc de bons candidats pour expliquer les déficiences de la spermatogenèse. Les premières études semblent confirmer cette hypothèse. Le gène RHOXF2, appartenant à la famille des gènes RHOX avec un cluster dans Xq24, est un bon candidat car spécifiquement exprimé dans les testicules. Ce gène a un degré élevé d'homologie (> 99,9%), avec son paralogue RHOXF2B , qui est également exprimé préférentiellement dans les testicules. OBJECTIFS: Séquencer RHOXF2 chez des patients infertiles bénéficiant d'une injection intracytoplasmique de spermatozoïdes (ICSI) afin d'identifier des polymorphismes associés à une déficience de la spermatogenèse. MATÉRIELS: Une cohorte de 327 patients inclus dans un programme d'ICSI. Tous les patients ont donné leur consentement écrit et éclairé à la participation de cette étude. Cent patients n'avaient pas d'altération de la spermatogenèse et 227 avaient une déficience. MÉTHODES: Les quatre exons de RHOXF2 ont été séquencés chez 47 patients présentant une oligospermie ou une azoospermie non obstructive. Étant donné que les exons 2 et 3 ont été trouvés comme ayant le plus de SNPs, seuls ces deux exons ont été séquencés dans les 280 sujets restants. RÉSULTATS: Bien que 9 SNPs aient été identifiés, il n'y avait pas de différence de fréquences significatives entre les patients ayant une altération, ou non de la spermatogenèse. Deux insertions ont été identifiées: une insertion de 21 nucléotides retrouvées dans les deux groupes et une insertion d'une guanine (induisant un codon stop prématuré) chez deux patients présentant une altération de la spermatogenèse. CONCLUSION: RHOXF2 est un bon candidat pour une évolution rapide par sélection positive. L'analyse de la fréquence des polymorphismes dans les exons 2 et 3 ne nous permet pas actuellement de corréler les SNP identifiés avec l'infertilité masculine. Cependant, une insertion d'un seul nucléotide a été identifiée uniquement chez des hommes avec une déficience de la spermatogenèse. Des travaux complémentaires seront nécessaires pour déterminer l'impact du gène RHOXF2 sur la spermatogenèse.

Impact of freezing/thawing technique on sperm DNA integrity in HIV-1 patients.

INTRODUCTION: According french legislation, sperm freezing/thawing procedures are used to prevent ART contaminations in couple with HIV-1 infected men. We determined sperm nuclear fragmentation rate before and after selection and freezing/thawing in HIV-1 14 patients. METHODS: Two groups of patients were studied: 20 control patients with normal sperm (group 1) and without viral infection and 20 fertile treated HIV-1 patients (group 2). DNA fragmentation was evaluated using terminal uridine nick end labeling, before and after gradient selection, and after cryopreservation and thawing procedures. RESULTS: DNA fragmentation rates in fresh semen were increased in HIV patients (6.38% vs 3.39%) (p < 0.05) compared with control patients. After sperm migration, fragmentation rates were significantly lower (p < 0.0001) in the two groups compared with fresh sperm rates. After freezing/thawing, values were similar to those of fresh semen with an increased rate (p < 0.01) for HIV-1 patients, with respectively 3.40% and 5.18% rates in control and infected patients. HIV-1-infected patients treated by antiretroviral therapy showed a significant increase in sperm DNA fragmentation in fresh sperm and also after freezing/thawing procedures, but these two fragmentation rates were not significantly different. CONCLUSION: So, freezing/thawing procedures do not seem to impair sperm DNA and preserve probability of conception for couples with HIV-1 infected men.