Debbie Montjean

Human Male Infertility Associated with Mutations in NR5A1 Encoding Steroidogenic Factor 1

One in seven couples worldwide are infertile, and male factor infertility accounts for approximately 30%-50% of these cases. Although many genes are known to be essential for gametogenesis, there are surprisingly few monogenic mutations that have been conclusively demonstrated to cause human spermatogenic failure. A nuclear receptor, NR5A1 (also called steroidogenic factor 1), is a key transcriptional regulator of genes involved in the hypothalamic-pituitary-steroidogenic axis, and it is expressed in the steroidogenic tissue of the developing and adult human gonad. Mutations of NR5A1 have been reported in 46,XY disorders of sex development and in 46,XX primary ovarian insufficiency. To test the hypothesis that mutations in NR5A1 cause male infertility, we sequenced NR5A1 in 315 men with idiopathic spermatogenic failure. We identified seven men with severe spermatogenic failure who carried missense mutations in NR5A1. Functional studies indicated that these mutations impaired NR5A1 transactivational activity. We did not observe these mutations in more than 4000 control alleles, including the entire coding sequence of 359 normospermic men and 370 fertile male controls. NR5A1 mutations are found in approximately 4% of men with otherwise unexplained severe spermatogenic failure.

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.

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.

Methylation changes in mature sperm deoxyribonucleic acid from oligozoospermic men: assessment of genetic variants and assisted reproductive technology outcome

OBJECTIVE To characterize a potential genetic cause for methylation errors described in oligozoospermia. DESIGN Analysis of PEG1/MEST-DMR and H19-DMR methylation level in sperm, in parallel with the study of several genes on the Y chromosome, DNMT3A, and DNMT3L. Clinical outcome was also looked at regarding PEG1/MEST-DMR and H19-DMR methylation level in sperm. SETTING Research and diagnostic laboratories. PATIENT(S) One hundred nineteen normospermic and 175 oligozoospermic men consulting for couple infertility. INTERVENTION(S) We studied PEG1/MEST-DMR and H19-DMR methylation profiles in 294 men. We searched for Y chromosome gene aberrations and for mutations in both DNMT3A and DNMT3L genes in men showing epimutations. Assisted reproductive technology (ART) outcomes were also investigated. MAIN OUTCOME MEASURE(S) Sperm samples were collected from 294 volunteers for genomic DNA isolation that was used to study methylation profiles in imprinted loci and Y chromosome SMCY, DNMT3A, and DNMT3L genes. Pregnancy rate was also studied after ART treatment using sperm showing epimutations. RESULT(S) Epimutations in H19-DMR and PEG1/MEST-DMR were found in 20% and 3% of oligozoospermic men, respectively. We identified an amino acid change in DNMT3A in one case and in DNMT3L in eight men with altered methylation profiles. No mutations were detected in SMCY or in selected Y chromsome genes. No correlation between ART outcome and epimutations was found. CONCLUSION(S) We observed epimethylations in spermatozoa of oligozoospermic individuals, but no association was found with genetic variants or in the ART outcome.

Polymorphisms in MTHFR and MTRR genes associated with blood plasma homocysteine concentration and sperm counts

OBJECTIVE To investigate the relationship between MTHFR and MTRR genetic variants with respect to both blood plasma homocysteine concentration and sperm counts. DESIGN Polymerase chain reaction followed by specific enzymatic digestion to determine the genotype of the individuals and blood plasma homocysteine quantification by high-performance liquid chromatography. SETTING Research laboratory. PATIENT(S) Two hundred sixty-eight men seeking infertility counseling and 254 partners of infertile women. INTERVENTION(S) We studied three MTHFR (c.1286A → C, c.665C → T and c.203G → A) and two MTRR (c.66A → G and c.524C → T) single-nucleotide polymorphisms and characterized sperm parameters in both oligozoospermic and normospermic men. A cohort of 522 men was examined for this study. A subgroup of 103 men was constituted for quantification of Hcy levels. MAIN OUTCOME MEASURE(S) Semen samples were collected for determinations of sperm concentration, motility, and morphology according to World Health Organization guidelines as well as for DNA isolation. Blood samples of the corresponding individuals were obtained to quantify plasma homocysteine levels. RESULT(S) We did not observe a relationship between homocysteinemia and sperm counts. The MTHFR c.665C → T variant is associated with mild hyperhomocysteinemia in blood plasma in the TT homozygous state. CONCLUSION(S) No association was found between MTHFR/MTRR genetic variants and sperm counts. Although no association was observed with reduced sperm counts, the MTHFR 665TT genotype is associated with a significant increase in blood plasma homocysteine levels.

Imprinting: RNA expression for homocysteine recycling in the human oocyte

OBJECTIVE To investigate whether homocysteine, a well known inhibitor of methylation, which is produced after imprinting and other methylation processes, can be recycled to methionine in the oocyte, at least until the stage of maternal to zygotic transition (i.e., four- to eight-cell stage); before this stage, most of the biochemical processes are carried out with the use of maternal stores of protein and mRNA. DESIGN A first approach using microarrays and then reverse-transcription polymerase chain reaction (RT-PCR) for methionine synthase (5-methyltetrahydrofolate-homocysteine methyltransferase [MTR]), betaine-homocysteine methyltransferase (BHMT), and cystathionine-beta synthase (CBS). SETTING Two private hospitals. PATIENT(S) Patients involved in IVF/ICSI procedures. INTERVENTION(S) Germinal vesicle oocytes collected at the time of oocyte retrieval, RNA extraction amplification, RT-PCR, microarrays. MAIN OUTCOME MEASURE(S) mRNA expression of all the enzymes involved in the chain of methylation and recycling of homocysteine to methionine. RESULT(S) All of the enzymes required for methylation are present in the oocyte. Homocysteine can be recycled with BHMT and MTR. CONCLUSION(S) The human oocyte is able to regulate its Hcy level via remethylation using MTR and BHMT but not CBS. This aspect is important, because recent studies have shown that controlled ovarian hyperstimulation affects the homocysteine concentration in follicular fluid. This may regulate, at least in part, the risk of imprinting problems during IVF procedures.

Role of sperm αvβ3 integrin in mouse fertilization

Oocyte integrins have been described as essential for fertilization. But this concept has been challenged by deletion experiments. Recently, we have shown that sperm integrin α6β1 plays a determinant role in mouse gamete interaction. In this study, we demonstrate the presence of αvβ3 integrin by Western blot and immunofluorescence on the sperm membrane. Oocytes and/or sperm preincubations with anti‐αv or anti‐β3 antibodies were performed before in vitro fertilization on cumulus‐intact and zona‐free egg assays. We observed inhibitory effects on the fusion process mostly by means of sperm function. An antibody directed against vitronectin inhibited gametes fusion, whereas the presence of exogenous vitronectin increased its efficiency. We suggest that vitronectin (on multimeric forms) can play a first nonspecific link corresponding to loosely bound spermatozoa to oocyte and that this link could be mediated by means of oocyte proteoglycans or integrins, and sperm αvβ3 integrin. Developmental Dynamics 239:773–783, 2010.

Carnitine content in the follicular fluid and expression of the enzymes involved in beta oxidation in oocytes and cumulus cells

PurposeThe purpose of this study is to study lipid metabolism in oocytes and embryos that is a neglected parameter in human IVF.MethodsWe have tested the total carnitine content (TC) in the follicular fluid of 278 patients (217 non pregnant, 61 pregnant) undergoing IVF.ResultsThe follicular fluid TC is neither correlated with the circulating estradiol content in serum nor with the outcome the IVF attempt. Carnitine, through the carnitine shuttle, is a major partner in lipid beta oxidation, metabolic pathway involved in the acquisition of oocyte competence. The expression of carnitine synthesis enzymes and lipid beta oxidation was studied in cumulus cells collected at the time of ovum retrieval and in oocyte. Surprisingly the expression for carnitine synthesis is not detectable in oocytes whereas the enzymes involved in lipid beta oxidation are rather strongly expressed.ConclusionsThe addition of carnitine in oocyte maturation and embryo culture media should not be overlooked.

Sequential (hFSH + recFSH) vs homogenous (hFSH or recFSH alone) stimulation: clinical and biochemical (cumulus cell gene expression) aspects

FSH is a key hormone in the regulation of follicular development. Together with the EGF network, these molecules mediate oocyte maturation and competence in preparation for the action of LH. FSH isoforms regulate distinct biological pathways and have specific effects on granulosa cell function and maturation of the ovarian follicle. Their dynamic interactions occur during the follicular cycle; short-living forms are predominant in the pre-ovulatory phase, whereas long-acting molecules characterize the luteal-follicular transition. Recombinant FSH (rFSH) molecules have a reduced number of isoforms and are less acidic, with a shorter half-life. We have investigated sequential stimulation, comparing hFSH + rFSH, vs. rFSH alone and hFSH alone for the entire stimulation phase. Sequential stimulation leads to an E2 per MII oocyte ratio that is much lower than is seen during treatment with the two drugs individually. Although there is a positive tendency in favor of the sequential treatment, there was no significant difference in pregnancy rates, even taking frozen embryos into consideration. The cumulus cell transcriptome varies considerably between the treatments, although with no clear significance. When comparing pregnant vs. non-pregnant patients, in general a decrease in mRNA expression can be observed in the pregnant patients, especially in expression of folic acid receptor 1 and ovostatin 2. This indicates that material has been transferred from CC to the oocyte. However, a common observation in the literature is that variations in the transcriptome of the cumulus cells are highly dependent upon the patient genotype; the potential for applying this strategy as a basis for selecting embryos is, at the very least, questionable.

Effect of temozolomide on male gametes: an epigenetic risk to the offspring?

IntroductionTemozolomide is an oral alkylating agent with proven efficacy in recurrent high-grade glioma. The antitumour activity of this molecule is attributed to the inhibition of replication through DNA methylation. However, this methylation may also perturb other DNA-dependent processes, such as spermatogenesis. The ability to father a child may be affected by having this treatment. Here we report a pregnancy and a baby born after 6 cures of temozolomide.MethodsThe quality of gametes of the father has been studied through these cures and after the cessation of treatment. Sperm parameters, chromosomal content and epigenetic profiles of H19, MEST and MGMT have been analysed.ResultsSperm counts decrease significantly and hypomethylation of the H19 locus increase with time even staying in the normal range.ConclusionThis is the first report of an epigenetic modification in sperm after temozolomide treatment suggesting a potential risk for the offspring. A sperm cryopreservation before the initiation of temozolomide treatment should be recommended.