Isabelle Berthaut

Influence of sickle cell disease and treatment with hydroxyurea on sperm parameters and fertility of human males

The use of hydroxyurea has considerably modified the prognosis of sickle cell disease and many more patients now reach reproductive age. This study shows alterations of semen parameters due to sickle cell disease that seem to be exacerbated by hydroxyurea treatment. The authors suggest that a pre-treatment sperm analysis be performed and sperm cryopreservation be offered to patients before hydroxyurea treatment. Background Recent progress in the treatment of sickle cell disease, in particular the use of hydroxyurea, has considerably modified the prognosis of this disease. Many more patients now reach reproductive age. The objective of this study was to assess the potential impact of hydroxyurea on the semen of patients. Design and Methods In this retrospective multicenter study, we evaluated the sperm parameters and fertility of 44 patients and analyzed the potential impact of hydroxyurea. Results We report data from the largest series so far of semen analyses in patients with sickle cell disease: 108 samples were analyzed, of which 76 were collected before treatment. We found that at least one sperm parameter was abnormal in 91% of the patients before treatment, in agreement with published literature. All sperm parameters seemed to be affected in semen samples collected during hydroxyurea treatment, and this impairment occurred in less than 6 months, later reaching a plateau. Furthermore, after hydroxyurea cessation, while global results in 30 patients were not statistically different before and after hydroxyurea treatment, in four individuals follow-up sperm parameters did not seem to recover quickly and the total number of spermatozoa per ejaculate fell below the normal range in about half the cases. Conclusions The observed alterations of semen parameters due to sickle cell disease seem to be exacerbated by hydroxyurea treatment. Until prospective studies reveal reassuring findings, we suggest that a pre-treatment sperm analysis be performed and sperm cryopreservation be offered to patients before hydroxyurea treatment.

Obesity leads to higher risk of sperm DNA damage in infertile patients

There has been a growing interest over the past few years in the impact of male nutrition on fertility. Infertility has been linked to male overweight or obesity, and conventional semen parameter values seem to be altered in case of high body mass index (BMI). A few studies assessing the impact of BMI on sperm DNA integrity have been published, but they did not lead to a strong consensus. Our objective was to explore further the relationship between sperm DNA integrity and BMI, through a 3-year multicentre study. Three hundred and thirty male partners in subfertile couples were included. Using the terminal uridine nick-end labelling (TUNEL) assay, we observed an increased rate of sperm DNA damage in obese men (odds ratio (95% confidence interval): 2.5 (1.2-5.1)).

Impact of chemotherapy and radiotherapy for testicular germ cell tumors on spermatogenesis and sperm DNA: a multicenter prospective study from the CECOS network

OBJECTIVE To determine the consequences of adjuvant testicular germ cell tumor treatment (TGCT) on sperm characteristics and sperm DNA, and to evaluate the predictors of sperm recovery. DESIGN Multicenter prospective longitudinal study of patients analyzed before treatment and after 3, 6, 12, and 24 months. SETTING University hospitals. PATIENT(S) One hundred twenty-nine volunteer TGCT patients and a control group of 257 fertile men. INTERVENTION(S) Routine semen analyses, sperm DNA, and chromatin assessments. MAIN OUTCOME MEASURE(S) Comparisons of mean sperm characteristics before and after treatment, with sperm recovery analyzed by the Kaplan-Meier method. RESULT(S) The quantitative and qualitative sperm characteristics decreased after treatment, with lowest values at 3 and 6 months and with variations according to treatment type. The mean total sperm count recovered to pretreatment values at 12 months after treatment after two or fewer bleomycin, etoposide, and cisplatin (BEP) cycles, but not after radiotherapy or more than two BEP cycles. Only the treatment modalities and pretreatment sperm production were related to recovery of the World Health Organization reference sperm values. An increased proportion of patients had elevated high sperm DNA stainability at 6 months after radiotherapy. CONCLUSION(S) Adjuvant treatments for testicular germ cell tumor have drastic effects on spermatogenesis and sperm chromatin quality. These new data on both the recovery period according to treatment modalities and the post-treatment chromatin status of sperm are useful tools for counseling patients wishing to conceive.

Sperm cryopreservation in adolescents and young adults with cancer: results of the French national sperm banking network (CECOS)

OBJECTIVE To determine the feasibility of fertility preservation in adolescent males with cancer. DESIGN Large multicenter retrospective study of male patients ≤20 years from 23 centers of a national network of sperm banks over a 34-year period. SETTING Sperm banks. PATIENT(S) A total of 4,345 boys and young men aged 11 to 20 years. INTERVENTION(S) Age, cancer diagnosis, feasibility of sperm banking, and sperm parameters. MAIN OUTCOME MEASURE(S) Description of patients, and success of their fertility preservation. RESULT(S) We observed a mean yearly increase in referred patients of 9.5% (95% confidence interval, 9.1%-9.8%) between 1973 and 2007. Over the study period, the percentage of younger cancer patients who banked their sperm increased, especially in the 11-14 year age group, rising from 1% in 1986 to 9% in 2006. We found that 4,314 patients attempted to produce a semen sample, 4,004 succeeded, and sperm was banked for 3,616. The mean total sperm count was 61.75 × 10(6) for the 11-14 year age group, and 138.81 × 10(6) for the 18-20 year age group. It was noteworthy that intercenter variations in practices involving young patients seeking to preserve their fertility before cancer therapy were observed within this national network. CONCLUSION(S) Our results emphasize the need for decisive changes in public health policy to facilitate the access to reproductive health-care for young cancer patients.

Lack of Association between Genetic Polymorphisms in Enzymes Associated with Folate Metabolism and Unexplained Reduced Sperm Counts

Background The metabolic pathway of folate is thought to influence DNA stability either by inducing single/double stranded breaks or by producing low levels of S-adenosyl-methionine leading to abnormal gene expression and chromosome segregation. Polymorphisms in the genes encoding enzymes in the folate metabolism pathway show distinct geographic and/or ethnic variations and in some cases have been linked to disease. Notably, the gene Methylenetetrahydrofolate reductase (MTHFR) in which the homozygous (TT) state of the polymorphism c.665C>T (p.A222V) is associated with reduced specific activity and increased thermolability of the enzyme causing mild hyperhomocysteinemia. Recently several studies have suggested that men carrying this polymorphism may be at increased risk to develop infertility. Methodology/Principal Findings We have tested this hypothesis in a case/control study of ethnic French individuals. We examined the incidence of polymorphisms in the genes MTHFR (R68Q, A222V and E429A), Methionine synthase reductase MTRR; (I22M and S175L) and Cystathionine beta-synthase (CBS; G307S). The case population consisted of DNA samples from men with unexplained azoospermia (n = 70) or oligozoospermia (n = 182) and the control population consisted of normospermic and fertile men (n = 114). We found no evidence of an association between the incidence of any of these variants and reduced sperm counts. In addition haplotype analysis did not reveal differences between the case and control populations. Conclusions/Significance We could find no evidence for an association between reduced sperm counts and polymorphisms in enzymes involved in folate metabolism in the French population.

Impact of lymphoma treatments on spermatogenesis and sperm deoxyribonucleic acid: a multicenter prospective study from the CECOS network

OBJECTIVE To determine consequences of lymphoma treatments on sperm characteristics and sperm DNA, and to evaluate predictors of sperm recovery. DESIGN Multicenter prospective longitudinal study of patients analyzed before treatment and after 3, 6, 12, and 24 months. SETTING University hospitals. PATIENT(S) Seventy-five Hodgkin lymphoma and non-Hodgkin lymphoma patients and a control group of 257 fertile men. INTERVENTION(S) Semen analyses, and sperm DNA and chromatin assessments. MAIN OUTCOME MEASURE(S) Comparisons of sperm characteristics before and after treatment. RESULT(S) Patients already had altered sperm characteristics before lymphoma treatment, with no identified risk factor. Sperm count, total sperm count, motility, and vitality decreased after treatment, with lowest values at 3 and 6 months. Twelve months after treatment, mean sperm count recovered to pretreatment values after doxorubicin, bleomycin, vinblastine, darcarbacine (ABVD) or ABVD+radiotherapy, but not after doxorubicin, cyclophosphamide, vincristine, prednisone (CHOP) or mechlorethamine, oncovin, procarbazine, prednisone (MOPP) chemotherapies. It was noteworthy that 7% of patients remained azoospermic at 24 months. After 24 months, Kaplan-Meier estimates showed that more than 90% of patients will recover normal sperm count after ABVD or ABVD+radiotherapy vs. 61% for CHOP chemotherapies. In multivariate analyses including diagnosis and treatment protocol, only pretreatment total sperm count was related to recovery. Compared with a control group, lymphoma patients had higher sperm chromatin alterations and DNA fragmentation before any treatment. After treatment, DNA fragmentation assessed by TUNEL assay and sperm chromatin structure assay decreased from 3 and 6 months, respectively, while remaining higher than in the control group during follow-up. CONCLUSION(S) Lymphoma patients had altered sperm DNA and chromatin before treatment. Lymphoma treatment had damaging effects on spermatogenesis. These data on both the recovery period according to treatment modalities and the pre- and post-treatment chromatin status of sperm are useful tools for counseling patients wishing to conceive.

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.

Progressive alcohol-induced sperm alterations leading to spermatogenic arrest, which was reversed after alcohol withdrawal.

This is a report of a 6-year follow-up of a male patients semen parameters during heavy chronic alcohol intoxication and after withdrawal. A slowly progressive negative impact of alcohol could be observed: isolated moderate teratozoospermia was firstly noted followed by oligoasthenoteratospermia. Then a severe worsening resulted in cryptozoospermia and ultimately in azoospermia. At this moment, the histological analysis of a testicular biopsy revealed a maturation arrest of the germinal cells at the pachytene stage with no mature sperm cells. Alcohol withdrawal was then obtained, allowing a very fast and drastic improvement of semen characteristics; strictly normal semen parameters were observed after no more than 3 months. Taking into consideration these data, patients should be questioned about their alcohol intake before assisted reproductive technology and should be informed about this adverse effect. Moreover, this case report emphasizes how quickly benefits can be obtained after withdrawal, even in the case of heavy chronic alcohol intake.

Infertilité masculine chez les patients normospermiques : analyse protéomique des spermes normaux non fécondants en fécondation in vitro classique

OBJECTIVE Despite normal sperm parameters, 5% of in vitro fertilization (IVF) attempts result in an unpredictable failure of fertilization. In 56% of the cases, there is no obvious oocyte anomaly, but lack of sperm binding to the zona pellucida. This study aims to contribute to clarify the male molecular causes of failures in IVF, which are undetected by classical sperm analysis. PATIENTS AND METHODS The spermatic proteomic profiles of patients, with a complete failure of fertilization and no spermatozoa bound to the zona pellucida, is compared to controls (patients with normal fertilization and cleavage rates after a classical IVF for tubal indication). All samples are analysed by 2 Dimensional Electrophoresis-Differential In Gel Electrophoresis (2DE-DIGE) after being divided into three fractions according to their isoelectric point (acid, intermediate and basic). RESULTS Fourteen proteins differentially expressed between all the cases and all the controls were highlighted. Twelve of these proteins were identified by mass spectrometry (six from the acid fraction and six from the basic fraction). Two of these proteins may have an interest in gametic interaction: the laminin receptor LR67 and the L-xylulose reductase. DISCUSSION AND CONCLUSION More investigation is needed to understand the involvement of the identified proteins in the IVF fertilization failure of the infertile patients in this study.

Proteomic identification of target proteins in normal but nonfertilizing sperm

OBJECTIVE To identify the male molecular causes of failures of IVF (with a deficient binding of spermatozoa to the zona pellucida, without any obvious oocyte anomaly), which are undetected by classical sperm analysis. DESIGN Case-control prospective study. SETTING University hospital. PATIENT(S) Proteomic profiles of spermatozoa in patients with a complete failure of fertilization and no spermatozoa bound to the zona pellucida were compared with those of controls (men with normal fertilization and cleavage rates after classical IVF for tubal indication). INTERVENTION(S) All samples were analyzed by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) after being divided into three fractions according to their isoelectric point. MAIN OUTCOME MEASURE(S) Differentially expressed proteins between infertile men and controls were identified by mass spectrometry. RESULT(S) Seventeen proteins differentially expressed between cases and controls were found. Twelve of these proteins were identified by mass spectrometry, and two may influence gametes interaction: laminin receptor LR67 and L-xylulose reductase (P34H). CONCLUSION(S) This study shows that 2D-DIGE might be useful in finding potential targets for diagnosis and prognosis of idiopathic infertility in IVF.