Morphologie des spermatozoïdes

Spermatozoa morphology is one of the qualitative characteristics of spermatogenesis. However, because of both the variations in the definition of normal morphology and the existence of different kinds of sperm abnormalities as well as the use of various techniques of morphology assessment, such a parameter is poorly used in usual laboratory work. Morphological sperm anomalies can be from testicular or post-testicular origines, while the latter is still unproved. The causes of such anomalies are either from genetic origines, but in these cases any spermatozoa demonstrate this anomaly, or due to an endogenous factor with varicocele the most usually quoted but unproved pathology, But exogenous factors, either chemical such as drugs and pesticides or physical such as heat, are also responsible for morphological sperm anomalies. Analysis of sperm morphology is indicative of both the testicular health status (in cases of occupational exposure to chemical or physical toxics) and the fertility potential since morphology is correlated to sperm motility and involved in fertilization through the acrosome reaction.     

Radicaux libres et spermatozoïdes humains: physiologie et physiopathologie

The role of reactive oxygen species in the physiopathology of human sperm function has been emphasized in recent years. Their production in semen has been associated with loss of motility, decreased capacity for spermoocyte fusion and loss of fertility. In semen preparations, there are two major sources of reactive oxygen species: leucocytes and spermatozoa themselve. It has been proposed that reactive oxygen species production by human spermatozoa was dependent upon a membrane-bound NADPH oxidase or a mitochondrial diaphorase. Hydrogen peroxide produced by the dismutation of superoxide anion has been recognized as the most toxic oxidizing species for human spermatozoa. Owing to their high content of polyunsaturated fatty acids, it has been proposed that lipid peroxidation of the sperm plasma membrane is largely responsible for defective sperm function. Reactive oxygen species also affect the sperm axoneme as a result of ATP depletion, inhibit mitochondrial functions, and synthesis of DNA, RNA and proteins, produce cytoskeletal modifications and inhibit sperm-oocyte fusion. Human spermatozoa possess enzymatic defence systems such as superoxide dismutase, glutathion peroxidas/reductase and catalase to counteract the toxic effects induced by reactive oxygen species. Correlations have been reported between their effectiveness and the duration of sperm motility. If the excessive production of reactive oxygen species is detrimental for human spermatozoa, they could also participate in the physiological function of the spermatozoa when present at low concentrations. Indeed, reactive oxygen species have been shown to be involved in the activation of several enzymes. Furthermore, sperm capacitation, acrosome reaction and sperm-zona interaction would be enhanced by reactive oxygen species.     

Evaluation morphologique des spermatozoïdes

Semen analysis, as defined by World Health Organisation (WHO), is a fundamental step in the work-up of infertile couples. Spermatozoa morphology has been recognised as the best predictive factor in natural fertility, and in intrauterine insemination and classicalin vitro fertilisation. Ultrastructural spermatozoa abnormalities are the only sperm alterations likely to influence the outcome of ICSI. However, these abnormalities cannot be detected by conventional microscopy (¥100 or ¥200–400). Bartoov et al. (2002) developed a real-time spermatozoa observation system using a ¥6600 magnification called MSOME (Magnification Motile Sperm Morphology Examination). Spermatozoa abnormalities detected with this technique are vacuoles localised on spermatozoa heads with variable number, size and site (nucleus or acrosome). Spermatozoa evaluation using MSOME could be performed to predict the probability of fertilisation by these spermatozoa either spontaneously or after assisted reproductive technology.     

Mise au point Estrogènes et reproduction chez le mâle

Aromatase is the terminal enzyme responsible for estrogen biosynthesis. Besides somatic cells, the aromatase gene expression and its transduction in a fully active protein in germ cells of rodent testes in one hand, the widespread distribution of estrogen receptors (ERα and ERβ) in the genital tract of the male in an other hand, are clearly in favour of a physiological role for estrogens in the regulation of mammalian testicular functions. In mouse and man, the aromatase deficiency is associated with severe bone maturation problems and sterility. Therefore, together with gonadotrophins and androgens, estrogens (or the balance androgens/estrogens) likely play a physiological role (either directly or via testicular somatic cells) in maintenance of male gonadal functions and obviously, several steps are concerned especially the spermatid production (both in terms of quality and number) and epididymal sperm maturation.     

La vitalité des spermatozoïdes

Most of the numerous techniques used to assess sperm viability only have research applications, while only two classical tests, i.e. eosin-Y and hypo-osmotic swelling test (HOST), are currently used in routine sperm analysis to determine the percentage of viable sperm. A viability rate below 50% of living sperm defines necrozoospermia, a condition whose clinical significance is fairly difficult to assess as the mechanisms of sperm cell death are still poorly understood. However, even when a precise cause for necrozoospermia cannot be identified, abnormal viability requires further andrological investigations with particular emphasis on clinical and laboratory signs of chronic infection of the male reproductive tract. Intracytoplasmic sperm injection (ICSI) can yield very good pregnancy rates, even in couples with the most severe forms of male infertility. However, when no motile sperm are available after sperm preparation, the outcome of ICSI is seriously impaired, probably because of a high risk of injecting dead sperm. In these patients, sperm viability could therefore be assessed by the hypo-osmotic swelling test in order to select only viable sperm for ICSI. However, the long incubation time of sperm in the hypo-osmotic solution, as recommended in the classical HOST procedure, has been shown to be detrimental to the spermatozoa. A single sperm test able to assess the viability of each individual spermatozoon within microdroplets covered by mineral oil therefore seems to be preferable. This selection procedure is less suitable in the case of immotile frozen-thawed sperm, as viability does not appear to be reliably predicted by HOST in cryopreserved sperm. Examination of sperm viability now also evaluates programmed cell death or apoptosis, as apoptotic alterations can be detected in spermatozoa by several techniques. The percentage of apoptotic sperm is correlated with deficient sperm parameters and poor outcome of assisted reproductive techniques. More effective selection procedures are therefore needed in order to identify spermatozoa not only with intact membranes but also with an intact genome to be used for ICSI.     

Oestrogènes et reproduction masculine

The role of estrogen on male reproductive function has become clearer in the last decade. During these years the study of the effect of testosterone, estrogen or an aromatase inhibitor in hypogonadal men provided a first evidence of the effects of estrogens in the regulation of gonadotropin secretion. At the same time, the development of a line of transgenic male mice lacking estrogen receptor α, estrogen receptor β or aromatase gene provided further evidence about the role of estrogens not only in the regulation of gonadotropin secretion, but also on the effects of estrogens on testicular function and development. A confirmation of these actions of estrogens came from the observation of naturally occurring mutations of the estrogen receptor and of the aromatase gene in human males. Based on these data it has been demonstrated that estrogens are major regulators of gonadotropin secretion acting both at pituitary and hypotalamic level. The presence in the human reproductive structures of estrogen receptor α, estrogen receptor β and the aromatase enzyme indicates the existence of receptor α, estrogen receptor β or aromatase estrogen actions at this level. Anyway, the precise role of estrogens in testicular development and function and on the regulation of human spermatogenesis has not yet been precisely clarified.     

Principes de préparation et de congélation des spermatozoïdes testiculaires humains

The advantages and feasibility of human testicular spermatozoa cryoconservation for intracytoplasmic sperm injection (ICSI) have now been clearly demonstrated. However, the freezing protocol is based on empirical knowledge obtained from freezing of ejaculated spermatozoa. Testicular spermatozoa may not be fully mature gametes and may also be retrieved in only limited quantities. Little research has been conducted to determine whether they have the same cryobiological requirements as ejaculated spermatozoa. A better understanding of their cryobiological features and assessment of possible subcellular changes after thawing would help to optimize testicular preparations for cryopreservation (whole biopsies, seminiferous tubules, shredded suspension, single spermatozoa, etc.), freezing-thawing procedure, freezing media, and storage. Finally, there is a growing need for welldefined criteria (nuclear quality, etc.) to evaluate the tolerance of testicular spermatozoa to freezing-thawing procedure for ICSI     

Effets secondaires des androgènes

Androgens, when used in medical replacement dosages, are well tolerated with no major side effects. In contrast, the illegal use by athletes of doses of androgens 10 to 100 fold higher than therapeutical doses can be responsible for a number of health hazards, all the more so since they are not medically controled. In addition, new usages of androgens at high doses are now emerging such as male contraception and hormone replacement therapy for andropause. Androgen side effects can be classified in 4 categories: il all cases they depend on the dose and the molecule used. 1) Androgenic side-effects are constant. In men, they include testicular atrophy and azoospermia leading to sterility. They are readily reversible in 2 to 3 months after cessation of treatment. However, potential long term effects on prostate should be evaluated. In women and adolescents, high doses of androgens will also produce androgenic side effects (increase of body mass, acne, deepening of the voice, hirsutism, balding) and in adolescents, premature epiphyseal fusion. These effects are often irreversible and should preclude high dose androgen administration in these subjects. 2) Toxic side-effects, especially due to the use of 17α-alkylated derivatives, bear mainly on liver function; they involves pathological liver function tests and jaundice but can also include more serious hepatic complications such aspeliosis hepatitis or hepatoma. 3) Effects on the cardiovascular system: blood lipids and lipoproteins change toward values promoting atherosclerosis are constant and an increased thrombogenic risk has also been reported. 4) Behavioural side-effects: Androgens are supposed to develop aggressivity and mental strength; psychotic manifestations have been reported, but in the human these effects are poorly documented and there seemingly exists a strongplacebo effect. In conclusion, in adult man at least, high doses of androgens seem to result in essentially reversible effects and androgen abuse do not involve major risks. However further studies are necessary to evaluate potential longterm effects such as liver toxic effects, significance of the cardiovascular changes and moreover, the potential development of an androgen-dependent prostate pathology.     

Androgènes et sexualité masculine

Testosteron is known to be critical for the right maintenance of masculin sexuality. It acts on all components of sexuality: libido, erection and ejaculation. As far as erection is concerned, one has to emphasize on the fact that only spontaneous nocturnal erections are androgen-dependent unlike erectile responses to visual erotic stimuliThe T can act, at the level of target organes, either as unchanged or as more often observed after reduction to DHT or aromatisation to E2. It is essencially as DHT that the male hormone seems to act on the sexuality at both the central and peripherical levels. The oestrogens even if now shown to have no peripherical action, are still subject to controversy as far as their central action is concerned. Therefore an action at the cerebral level of the oestrogens produced locally can not be definitively eliminated.The concentrations of T required for the re-establishment of the sexual function in case of hypogonadism are closer to the lower limits of normal values. The upper treshold of the action of T on the sexuality, if any, would be at supra-physiological levels.Androgenotherapy of sexual dysfonctionnalities is wittnissing a constant evolution. New and more performing products, as well as novel and less astreignant ways of administration, are already disponible or under evaluation. Moreover, the improvement of plasmic androgen exploration (dosage of free and non-sex hormone-binding globulin bound testosterone) associated to a better understanding of side effects of the androgenic therapeutics may allow in the near futur to reach a consensus on the androgenotherapy indications. As a matter of fact among the population of patients considered eugonadic (based on normal levels of T) and showing a sexual dysfonctionality, two subgroups might be interested: That of elderly patients displaying either an increase of the hormone-binding globulin (SHBG) or a decrease of the non-sex hormone-binding globulin bound testosterone levels and that of young patients suffering from idiopathic impotence. It should be, however, pointed at that the recent character of the different physiopathologic and therapeutic acquisitions requires a validation through larger series and long term studies.     

Observation des spermatozoïdes au fort grossissement (MSOME): intérêt et perspectives

Motile Sperm Organelle Morphology Examination (MSOME) constitutes a real improvement in ART management and outcome, as it allows detection of specific sperm anomalies on living cells, which cannot be detected by routine analysis. MSOME applied to the selection of sperm injected into the oocyte is called IMSI (Intracytoplasmic Morphologically Selected sperm Injection) and is associated with a considerable improvement of implantation, clinical pregnancy and delivery rates. A high-power microscope (X 2,000 to X 10,000) and video enhancement system are necessary and technical limitations are related to cryptozoospermia and/or severe teratozoospermia. Compared to routine sperm morphology assessment, MSOME allows the detection of subtle cephalic anomalies, such as vacuoles. These vacuoles seem to have a deleterious effect on fertilization and embryo developmentin vitro. These observations have led to a detailed classification of anomalies and this morphological diagnosis on living sperm demonstrates that most sperm selected for conventional ICSI at X 400 are actually abnormal on MSOME at X 6,000 or more. In addition to the very good results obtained in IMSI, this new approach opens up interesting prospects concerning the relationship between the phenotype of the injected sperm and its fertilization capacity and embryo development. In terms of diagnosis, MSOME could be used to select and study homogeneous groups of normal sperm or homogeneous groups of sperm exhibiting the same well defined anomaly. Such studies, associated with fine analysis of injected sperm and follow-up of each oocyte and each embryo, should provide more information about the relationship between sperm structure and function and should help to define the relevant indications for IMSI and the choice of spermatozoa to be injected.     

Perspectives de l’analyse de la mobilité des spermatozoïdes

Several studies have shown a good correlation between sperm motility and fertility though the microscopic evaluation of the percentage of motile sperm is highly subjective by nature. Therefore in the last decade, various objectives methods have been proposed to overcome this problem. Two types of methods were developed: The methods based on the analysis of images obtained by microphotography, microcinematography and microvideography and the global, undirect methods based on physical principles. Several systems based on video and image analysis (Computer Aided Sperm Analysis, CASA) have been developed and are used in numerous laboratories of reproductive biology. CASA technology offers the possibility to analyse some characteristics of sperm motion which are related to the fertilization potential and to develop new parameters related to some important aspects of sperm behavior such as hyperactivation. However, there is a large amount of interactions between the operator and the CASA machine. CASA instruments are not “ready-to-use” robots: the reliability of CASA depends largely on the expertise and training of the user and the application of standardized procedures and quality control schemes. By contrast, there is only minimal interaction between the operator and the Sperm Quality Anlyser which is a new device measuring and index of sperm motility highly correlated to the concentration of progressively motile sperm. The device uses light passed through a small sample of semen introduced in a capillary tube to detect variations in optical density that result from moving particles. The reproducibility of the measurements is excellent, the device is easy to use and this is a potentially useful tool for field-work studies. Further investigations of this device in the managment of male infertility is warranted. Finally, both types of objectives approaches are complementary to the conventional analysis of sperm motility and they will not replace it. Standardized procedures have been proposed by the World Health Organization for the subjective evaluation of sperm motility. Such procedures are very useful to reduce significantly the intra- and interlaboratory variations but internal and external quality controls schemes indicate that they are not sufficient to achieve acceptable levels of variation and regular quality controls followed by the definition and the application of corrective procedures are required.     

Polymorphisme du gène du récepteur des androgènes: recherche de mosaïques tissulaires et relation avec l'infertilité masculine

Androgens are male steroid hormones necessary for initiation and maintenance of spermatogenesis. They act via a specific receptor which belongs to the nuclear receptor superfamily. The transactivation N-terminal domain of this receptor is characterized by a CAG repeat polymorphism in the first exon of the gene coding for polyglutamines. An increased CAG repeat length is involved in Kennedy's disease, a neurodegenerative disease associated with infertility and impaired virilization. Some recent studies have shown a correlation between the number of CAG repeats and male fertility. The aim of this study was to define this correlation and to determine whether or not infertile men presented a tissue mosaicism between blood and sperm, as described in several diseases involving CAG repeats. The length of CAG repeats of blood and testicular cells was measured in 36 oligospermic or azoospermic patients and 15 controls. An inverse correlation was found between CAG repeat length and total number of ejaculated spermatozoa. However, no tissue mosaicism between blood and sperm was observed in our population.     

Etude enzymatique comparée de champignons entomopathogènes des genres Beauveria et Metarhizium

Résumé Les extraits antigéniques de trois champignons entomopathogènes (Beauveria bassiana, Beauveria brongniartii et Metarhizium anisopliae), soumis à l'analyse éléctrophorétique en gel d'agar révèlent au total 23 activités enzymatiques différentes (5 oxydo-réductases et 18 hydrolases), dont 18 ont été retrouvées à l'analyse immunoélectrophorétique. La carte enzymatique de ces germes complète leur analyse immunoélectrophorétique; la comparaison des isoenzymes des divers isolats permet de mettre en évidence des différences interspécifiques et intraspécifiques.Chaque souche cryptogamique se caractérise par des profils enzymatiques particuliers. Ces résultats montrent donc que l'étude des enzymogrammes est un complément utile à l'analyse immunoélectrophorétique appliquée à la caractérisation des hyphomycètes entomopathogènes. Enfin, les souches n 44 et 51 de M. anisopliae paraissent être suffisamment apparentées pour être réunies au sein d'un même biotype.