Florence Eustache

Chronic Dietary Exposure to a Low-Dose Mixture of Genistein and Vinclozolin Modifies the Reproductive Axis, Testis Transcriptome, and Fertility

Background The reproductive consequences and mechanisms of action of chronic exposure to low-dose endocrine disruptors are poorly understood. Objective We assessed the effects of a continuous, low-dose exposure to a phytoestrogen (genistein) and/or an antiandrogenic food contaminant (vinclozolin) on the male reproductive tract and fertility. Methods Male rats were exposed by gavage to genistein and vinclozolin from conception to adulthood, alone or in combination, at low doses (1 mg/kg/day) or higher doses (10 and 30 mg/kg/day). We studied a number of standard reproductive toxicology end points and also assessed testicular mRNA expression profiles using long-oligonucleotide microarrays. Results The low-dose mixture and high-dose vinclozolin produced the most significant alterations in adults: decreased sperm counts, reduced sperm motion parameters, decreased litter sizes, and increased post implantation loss. Testicular mRNA expression profiles for these exposure conditions were strongly correlated. Functional clustering indicated that many of the genes induced belong to the “neuroactive ligand-receptor interactions” family encompassing several hormonally related actors (e.g., follicle-stimulating hormone and its receptor). All exposure conditions decreased the levels of mRNAs involved in ribosome function, indicating probable decreased protein production. Conclusions Our study shows that chronic exposure to a mixture of a dose of a phytoestrogen equivalent to that in the human diet and a low dose—albeit not environmental—of a common anti-androgenic food contaminant may seriously affect the male reproductive tract and fertility.

Standardisation de la classification morphologique des spermatozoïdes humains selon la méthode de David modifiée

RésuméIl est maintenant bien établi que le pourcentage de spermatozoïdes normaux et de certaines anomalies spécifiques des spermatozoïdes a une valeur pronostiquein vivo etin vitro. Aussi, le spermocytogramme représente un temps indispensable de l’analyse du sperme humain. Malheureusement, cette analyse, simple à première vue, présente de réelles difficultés avec pour conséquence une fiabilité très relative des résultats d’un laboratoire à l’autre. Aussi, les procédures employées, de la réalisation des frottis au rendu des résultats, doivent faire l’objet d’une standardisation. La lecture des lames colorées de préférence au Shorr est faite à l’objectif ×100 à immersion. La méthode de David modifiée pour le classement des anomalies morphologiques des spermatozoïdes humains a été adoptée en France par la grande majorité des laboratoires publics et privés. Elle permet le classement de 1°) sept anomalies de la tête: têtes allongées, amincies, microcéphales, macrocéphales, multiples, présentant un acrosome anormal ou absent, présentant une base (région post-acrosomique) anormale, 2°) trois anomalies de la pièce intermédiaire: reste cytoplasmique, grêle, angulée et 3°) cinq anomalies de la pièce principale: absente, écourtée, de calibre irrégulier, enroulée et multiple. La grande originalité de la méthode est de recenser l’ensemble des anomalies associées par spermatozoïde anormal grâce à un système à entrées multiples. Comme il n’y a aucune raison objective de considérer plus une anomalie qu’une autre, il est impératif de décrire toutes les anomalies observées pour chaque spermatozoïde anormal. L’index d’anomalies multiples ou IAM, application directe de ce système n’est autre que le rapport du nombre total d’anomalies recensées au nombre total de spermatozoïdes anormaux. L’IAM, indicateur du nombre moyen d’anomalies associées par spermatozoïde anormal, présente une valeur pronostique de la fertilité et I’OMS recommande son utilisation. La présente mise au point s’inscrivant dans la démarche nécessaire d’assurance de qualité en biologie de la reproduction est focalisée sur une redéfinition précise des anomalies, les justifications ultrastructurales et fonctionnelles et la pratique du classement des spermatozoïdes humains typiques et atypiques selon la méthode de David modifiée.AbstractIt has now been clearly established that the percentage of morphologically normal sperm and the level of several specific anomalies of spermatozoa have a prognostic valuein vivo andin vitro. Assessment of human sperm morphology therefore represents an essential step in routine semen analysis. Smears, preferably stained by the Shorr technique, are examined at a final magnification of ×1000 (×100 objective with oil immersion and ×10 ocular lens). David’s method for classification of morphological anomalies of human spermatozoa was proposed in the mid-seventies and was modified in the early nineties to account for all of the defects known to interfere with normal sperm functions. This method has been adopted in France by the vast majority of public and private laboratories. It allows classification of seven anomalies of the head: tapered, thin, microcephalous, macrocephalous, multiple, abnormal or absent acrosome, abnormal postacrosome, three anomalies of the midpiece: cytoplasmic droplet, thin, bent, and five anomalies of the principal piece: absent, short, irregular, coiled and multiple. The originality of the method is to account for all combinations of anomalies found for each abnormal sperm by means of a multiple entry system. Since there is no objective reason to favour certain anomalies over others, all anomalies found for each abnormal sperm should be recorded using a systematic procedure: description of the head, followed by the midpiece, and lastly the principal piece. The multiple anomalies index or MAI is calculated. This index, which is a direct application of the multiple entry system, corresponds to the mean number of anomalies per abnormal sperm (the ratio of the total number of anomalies to the number of abnormal sperm). Several studies have indicated the prognostic value of MAI, and calculation of the MAI is recommended in the WHO manual on semen analysis. Unfortunately, the morphological assessment of human spermatozoa which appears simple at a first glance, raises a number of real difficulties: this microscopic analysis is highly subjective by nature and no uniform recommendations for staining technique and classification have yet been defined. Internal and external quality control schemes have revealed the marked intra- and interindividual variability of the results, which in turn have important consequences on the reliability of the analysis and the management of infertile couples. All steps of the procedure, from preparing the smear to writing the examination report must therefore be standardised. The present article focuses on standardisation of the classification step using David’s method. Precise descriptions of the anomalies found by conventional microscopy and their ultrastructural substratum are presented, together with practical recommendations for the classification of sperm defects and calculation of the MAI.

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)).

Soy, phyto-oestrogens and male reproductive function: a review.

There is growing interest in the possible health threat posed by the effects of endocrine disruptors on reproduction. Soy and soy-derived products contain isoflavones that mimic the actions of oestrogens and may exert adverse effects on male fertility. The purpose of this review was to examine the evidence regarding the potential detrimental effects of soy and phyto-oestrogens on male reproductive function and fertility in humans and animals. Overall, there are some indications that phyto-oestrogens, alone or in combination with other endocrine disruptors, may alter reproductive hormones, spermatogenesis, sperm capacitation and fertility. However, these results must be interpreted with care, as a result of the paucity of human studies and as numerous reports did not reveal any adverse effects on male reproductive physiology. Further investigation is needed before a firm conclusion can be drawn. In the meantime, caution would suggest that perinatal phyto-oestrogen exposure, such as that found in infants feeding on soy-based formula, should be avoided.

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.

Modified Expression of Several Sperm Proteins after Chronic Exposure to the Antiandrogenic Compound Vinclozolin

Little is known about the molecular impact of in vivo exposure to endocrine disruptors (EDs) on sperm structures and functions. We recently reported that the lifelong exposure of rats to the antiandrogenic compound vinclozolin results in low epididymal weight, changes in sperm kinematic parameters, and immature sperm chromatin condensation, together with the impairment of several fertility end points. These results led us to focus specifically on possible molecular abnormalities in sperm. Sperm samples were recovered from the frozen epididymides of rats exposed during the previous study. The proteins present in the samples from six exposed and six control rats were analyzed in pairs, by two-dimensional fluorescence difference gel electrophoresis, to investigate possible exposure-induced changes to sperm protein profiles. Twelve proteins, from the 380 matched spots observed in at least five gels, were present in larger or smaller amounts after vinclozolin exposure. These proteins were identified by mass spectrometry, and several are known to play a crucial role in the sperm fertilizing ability, among which, two mitochondrial enzymes, malate dehydrogenase 2 and aldehyde dehydrogenase (both of which were present in smaller amounts after treatment) and A-kinase anchor protein 4 (larger amounts of precursor after treatment). Finally, Ingenuity Pathway Analysis revealed highly significant interactions between proteins over- and underexpressed after treatment. This is the first study to show an association between in vivo exposure to an ED and changes to the sperm protein profile. These modifications may be at least partly responsible for the reproductive abnormalities and impaired fertility recently reported in this rat model of vinclozolin exposure.

Regulatory identification of BPA as an endocrine disruptor: Context and methodology

BPA is one of the most investigated substances for its endocrine disruptor (ED) properties and it is at the same time in the center of many ED-related controversies. The analysis on how BPA fits to the regulatory identification as an ED is a challenge in terms of methodology. It is also a great opportunity to test the regulatory framework with a uniquely data-rich substance and learn valuable lessons for future cases. From this extensive database, it was considered important to engage in a detailed analysis so as to provide specific and strong evidences of ED while reflecting accurately the complexity of the response as well the multiplicity of adverse effects. An appropriate delineation of the scope of the analysis was therefore critical. Four effects namely, alterations of estrous cyclicity, mammary gland development, brain development and memory function, and metabolism, were considered to provide solid evidence of ED-mediated effects of BPA.

Adverse effect of hydroxyurea on spermatogenesis in patients with sickle cell anemia after six months of treatment

To the editor: The prognosis of sickle cell anemia (SCA), initially disastrous in the severe forms of the disease, has greatly improved, leading to increased life expectancy. More than 95% to 99% of the children diagnosed with SCA in developed countries will become young adults, reaching

Sperm FISH analysis in two healthy infertile brothers with t(15;18) unbalanced translocation: Implications for genetic counselling and reproductive management.

Numerous studies have shown that balanced reciprocal or Robertsonian translocations and inversions are associated with reduced or absent sperm production. In contrast, a similar association has been rarely reported for unbalanced translocations. An unbalanced translocation, 45,XY,-15,der(18)t(15;18)(q11.2;q23), was found in two healthy infertile brothers who were referred to our hospital together with their partners for infertility. At least two routine semen analyses and karyotyping were done for each of the brothers. Sperm meiotic segregation was studied for both with a three-color FISH assay using locus-specific probes. Semen analyses showed a severe oligo-astheno-teratozoospermia with remarkably similar profiles in the two brothers. The unbalanced translocation had a deletion of 15pter-15q11.2 as well as a deletion of 18q23-18qter. The meiotic segregation was similar in the two brothers with a prevalence of alternate segregation mode. However, no phenotypic effect in the offspring can be expected only if the normal chromosomes 15 and 18 are transmitted to progeny. According to the sperm FISH results, the theoretical probability of this happening is about 25%. Based on the overall results, genetic and reproductive counselling was offered to both couples. Finally, both couples chose the alternative of donor insemination rather than preimplantation genetic diagnosis. The present study helps delineating a phenotypically silent CNV at the distal part of chromosome 18 long arm and illustrates the advantages of an integrated multidisciplinary genetic, reproductive and psychological approach to give the best possible assistance to couples who are faced with a complex and distressing genetic cause of infertility.

Human sperm quality and lipid content after migration into normal ovulatory human cervical mucus containing low numbers of leukocytes.

The aim of this study was to investigate whether a relationship exists between the presence of low numbers of leukocytes in normal ovulatory cervical mucus and sperm quality and lipid content after migration. The percentages of live, motile and morphologically normal spermatozoa, movement parameters assessed by computer-aided sperm analysis (CASA), and ionophore-induced acrosome reaction measured by flow cytometry were determined before and after migration. High-performance liquid chromatography with ultraviolet detection was used to measure the sperm lipid content, including the various diacyl subspecies. The number of leukocytes found in solubilized mucus samples was counted using a haemocytometric method. Overall, the presence of leukocytes in the cervical mucus samples did not significantly influence sperm motility and morphology, sperm kinematic parameters, or the sperm content in sphingomyelin or cholesterol. In contrast, after migration, the decrease in various sperm diacyls and the level of induced acrosome reaction was significantly less pronounced in mucus samples containing>or=10(4) leukocytes than in mucus samples with no or rare leukocytes whereas the level of induced acrosome reaction was higher. The present data suggest that the low level of leukocytes found in normal ovulatory cervical mucus could influence the process of sperm lipid remodelling/capacitation.