Moncef Benkhalifa

ANTIOXIDANTS TO REDUCE SPERM DNA FRAGMENTATION: AN UNEXPECTED ADVERSE EFFECT

Reactive oxygen species (ROS) have a negative impact on sperm DNA, leading to the formation of oxidative products such as 8-oxo-7,8-dihydroxyguanosine. This compound causes fragmentation and, thus, has a mutagenic effect. Patient treatment with oral antioxidant vitamins is, therefore, standard practice for male infertility, in an attempt to decrease formation of ROS and improve fertility. In this study, the DNA fragmentation index and the degree of sperm decondensation were measured using the sperm chromatin structure assay before and after 90 days treatment with antioxidant vitamins associated with zinc and selenium. Antioxidant treatment led to a decrease in sperm DNA fragmentation (-19.1%, P < 0.0004), suggesting that at least part of the decay was linked to ROS. However, it also led to an unexpected negative effect: an increase in sperm decondensation with the same order of magnitude (+22.8%, P < 0.0009). The opening of interchain disulphide bridges in protamines may explain this aspect, as antioxidant vitamins, especially vitamin C, are able to open the cystin net, thus interfering with paternal gene activity during preimplantation development. This observation might explain the discrepancy observed concerning the role of these antioxidant treatments in improving male fertility.

Correlation between DNA damage and sperm parameters: a prospective study of 1,633 patients

OBJECTIVE To investigate DNA fragmentation by using terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling in relation to World Health Organization parameters and computer-aided sperm analysis (CASA) in sperm to determine the possibility of obtaining a correlation among CASA parameters, sperm morphology, and DNA fragmentation. DESIGN Sperm analysis according to World Health Organization parameters, terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) for sperm DNA fragmentation, and CASA for sperm movement. Prospective study. SETTING All the patients were under clinical management, consulting for hypofertility at a fertility center in France. PATIENT(S) One thousand six hundred thirty-three men who were referred for infertility investigation, including a complete sperm analysis. INTERVENTION(S) Sperm analysis and DNA damage testing. MAIN OUTCOME MEASURE(S) Sperm morphology, DNA fragmentation, and movement characteristics. RESULT(S) One third of the patients had a TUNEL rate of >30%. Analysis of the 21 semen parameters tested revealed that 7 of them were significantly correlated with the TUNEL results. CONCLUSION(S) World Health Organization sperm parameters and DNA damage are complementary, rather than strongly linked. This should be considered to more fully understand the paternal contribution in assisted reproductive technologies failures.

Expression profile of genes coding for DNA repair in human oocytes using pangenomic microarrays, with a special focus on ROS linked decays

PurposeTo determine the level of expression for mRNAs that regulate DNA repair activity in oocytes at the germinal vesicle (GV) stage. Reactive oxygen species (ROS) have been shown to play a major role in the appearance of deleterious DNA decays, and this study focuses on the repair of damage linked to decay caused by the action of ROS. The oocyte needs a mechanism for repairing DNA decays in the early preimplantation embryo before the onset of genomic activation, since in the absence of repair, residual DNA damage would lead to either apoptosis or tolerance. Tolerance of DNA damage is a source of potential mutations.MethodGV oocytes were selected for this study, both for the ethical reason that they are unsuitable for patient treatment, and because no transcription takes place during the period from GV to MII and then prior to genomic activation. The GV oocyte is therefore a good model for looking at DNA during the first cleavages of early preimplantation development. Six cohorts of GV oocytes were pooled for extraction of mRNA; the DNA was analysed using Affimetrix HG-UG133 Plus 2, containing 54,675 probe sets; spike and housekeeping genes were also added as internal controls.ResultsIn GV oocytes, DNA repair pathways for oxidized bases are redundant. One step repair procedure (OSR), BER (base excision repair), MMR (mismatch repair) and NER (Nucleotide excision repair) are present. All the recognition proteins are also present. The chromatin assembly factors necessary for the maintenance of genomic stability are highly expressed.ConclusionGene expression analysis shows that the oocyte does not allow a high level of tolerance for DNA decays. This regulatory mechanism should avoid transmitting mutations into the next generation.

Effect of maternal and paternal age on pregnancy and miscarriage rates after intrauterine insemination

More than 17,000 intrauterine insemination (lUI) cycles were analysed retrospectively with respect to outcome according to differing aetiologies of infertility. The quantity and motility of spermatozoa in the final preparation used for insemination had a positive effect on the outcome, as classically observed in the past. It was found that advanced maternal age had a negative effect on the pregnancy rate and was associated with increased miscarriage rate. More interestingly, an exactly parallel effect was found for paternal age. The impact of increased age on necrospermia and sperm DNA structure is discussed as a probable direct cause of this paternal effect.

Sperm transcriptome profiling in oligozoospermia

PurposeInvestigate in what extent sperm transcriptome of infertile men is different from that of fertile individuals.MethodsSemen samples were collected for determination of sperm parameters as well as for RNA isolation. Gene expression profile was investigated in spermatozoa of 8 infertile and 3 fertile men by microarray analysis using the Affymetrix Chip HG-U133 Plus 2.0.Result(s)We observed up to 33-fold reduction expression of genes involved in spermatogenesis and sperm motility. Furthermore, there is an important decrease in expression of genes involved in DNA repair as well as oxidative stress regulation. In this study, we also show a striking drop in expression of histone modification genes.Conclusion(s)We found that transcription profile in germ cells of men with idiopathic infertility is different from that of fertile individuals. Interestingly, about 15% of the regulated genes (Eddy Rev Reprod 4:23–30, 1999) play a role in spermatogenesis.

Globozoospermia is mainly due to DPY19L2 deletion via non-allelic homologous recombination involving two recombination hotspots

To date, mutations in two genes, SPATA16 and DPY19L2, have been identified as responsible for a severe teratozoospermia, namely globozoospermia. The two initial descriptions of the DPY19L2 deletion lead to a very different rate of occurrence of this mutation among globospermic patients. In order to better estimate the contribution of DPY19L2 in globozoospermia, we screened a larger cohort including 64 globozoospermic patients. Twenty of the new patients were homozygous for the DPY19L2 deletion, and 7 were compound heterozygous for both this deletion and a point mutation. We also identified four additional mutated patients. The final mutation load in our cohort is 66.7% (36 out of 54). Out of 36 mutated patients, 69.4% are homozygous deleted, 19.4% heterozygous composite and 11.1% showed a homozygous point mutation. The mechanism underlying the deletion is a non-allelic homologous recombination (NAHR) between the flanking low-copy repeats. Here, we characterized a total of nine breakpoints for the DPY19L2 NAHR-driven deletion that clustered in two recombination hotspots, both containing direct repeat elements (AluSq2 in hotspot 1, THE1B in hotspot 2). Globozoospermia can be considered as a new genomic disorder. This study confirms that DPY19L2 is the major gene responsible for globozoospermia and enlarges the spectrum of possible mutations in the gene. This is a major finding and should contribute to the development of an efficient molecular diagnosis strategy for globozoospermia.

Cytogenetics of uncleaved oocytes and arrested zygotes in IVF programs

AbstractPurpose: Cytogenetic studies of arrested oocytes and zygotes were used to understand in vitro fertilization (IVF) failures. Methods: We investigated the cytogenetics (Giemsa banding and FISH) of 710 uncleaved oocytes and 94 arrested zygotes from 208 patients undergoing IVF procedures. Results and Conclusions: Of uncleaved oocytes without a polar body, 39% were judged cytogenetically abnormal (17% unbalanced predivision and 21.5% diploid). Of 575 oocytes with a polar body, 124 (21.5%) showed numerical or structural chromosome aberrations. In arrested zygotes, approximately equal cases were found with separate condensed haploid complements (no syngamy), nuclear asynchrony and pulverized DNA, and apparently cytogenetically normal zygotes arrested at mitosis. These data on chromosome abnormalities were also analyzed with respect to two ovarian stimulation protocols and to maternal age. Both ovarian stimulation protocols showed the same levels of chromosome abnormalities. Overall chromosome abnormalities and premature chromosome condensation were also unchanged with maternal age. These data illustrate the significance of chromosome aberrations in IVF failures.

Malonaldehyde formation and DNA fragmentation: two independent sperm decays linked to reactive oxygen species

Malondialdehyde (MDA), a product involved in membrane lipid peroxidation, was dosed in the sperm of 163 patients who had consulted the clinic regarding hypofertility. We attempted to determine if there was correlation between MDA content, sperm World Health Organization parameters and DNA fragmentation that results mainly from reactive oxygen species assaults. We found that no correlation could be established; however MDA and sperm decondensation were shown to be significantly linked. The impact of membrane polyunsaturated fatty acids and the role of phospholipid hydroperoxide glutathione peroxidase are discussed.

How to overcome male infertility after 40: Influence of paternal age on fertility

The recent trend toward delayed parenthood raises major safety concerns because of the adverse effects of aging on couple fertility. Studies have demonstrated that aging clearly affects female fertility, but can also affect male fertility. Although several theories have been proposed, the exact mechanisms responsible for the observed age-related decline in male fertility remain to be elucidated. It has been shown that advanced paternal age (PA) is associated with reduced semen volume as well as, reduced sperm count, motility and morphology. Recent studies have also reported that paternal aging is associated with a significant increase in the prevalence of both genomic and epigenomic sperm defects. In the context of natural and intrauterine insemination (IUI) conception, advanced paternal age has been associated with lower pregnancy rates and increased rates of spontaneous abortion (independent of maternal age). In IVF and oocyte donation programs, a significant decrease in late blastocyst development has been seen in those cycles using spermatozoa of men older than 55. However, no significant relationship between paternal age and IVF or ICSI pregnancy rates has been observed. Although there are no treatments that can fully restore the age-related decline in male fertility, various measures have been shown to optimize male fertility potential. Specific therapies (e.g. varicocelectomy) and lifestyle changes (e.g. dietary antioxidant supplements) may help minimize some of the age-related deleterious effects on spermatogenesis, such as, oxidative stress and endocrine abnormalities.

High body mass index has a deleterious effect on semen parameters except morphology: results from a large cohort study

OBJECTIVE To evaluate the influence of body mass index (BMI) on semen characteristics. DESIGN Cohort study. SETTING Single private andrology laboratory. PATIENT(S) All patients (n=10,665) consulting for a semen analysis from October 9, 2010, to October 8, 2011. When analyses were repeated on the same patient, only the first was included. INTERVENTION(S) Recording of self-reported weight and height and of semen analysis. MAIN OUTCOME MEASURE(S) All parameters of standard semen analysis: pH, volume, sperm concentration per mL, total sperm count per ejaculate, motility (%) within 1 hour after ejaculation (overall and progressive), viability (%), and normal sperm morphology (%). Parametric and nonparametric statistical methods were applied, and results are given either with mean±SD, or 10th, 50th, and 90th percentiles. RESULT(S) Semen volume decreased from 3.3±1.6 to 2.7±1.6 mL when BMI increased from normal (20-25 kg/m2) to extreme obesity (>40 kg/m2). The same was true for semen concentration (56.4±54.9 to 39.4±51.0 million/mL), total sperm count (171±170 to 92±95 million), and progressive motility (36.9±16.8% to 34.7±17.1%). The percentage of cases with azoospermia and cryptozoospermia increased from 1.9% to 9.1% and from 4.7% to 15.2%, respectively. The other semen characteristics were not affected. Multivariate models including age and abstinence duration confirmed these results. CONCLUSION(S) In this study, on a large patient sample size, increased BMI was associated with decreased semen quality, affecting volume, concentration, and motility. The percentage of normal forms was not decreased.