Fertility regulation in the male

Bulletin of the World Health Organization, 56 (3): 353-360 (1978) Fertility regulation in the male D. M. DE KRETSER' The current state ofresearch into new methods ofmale contraception is reviewed, with special emphasis on the efforts of the WHO Special Programme of Research, Development and Research Training in Human Reproduction. The article concen- trates mainly on the development of orally administered or injectable substances capable of either (a) interfering with the hormonal control of testicular function, (b) disrupting spermatogenesis by direct influence on testis function, or (c) inter- fering with the fertilizing ability ofthe sperm and their transport. It is concluded that, despite the numerous areas of research currently being pursued, the availability ofa new male contraceptive remains several years away. Considerable attention has recently been given to the subject of contraception by scientists, politicians, and the public. In the past, emphasis has been placed on the develop- ment and use of contraceptive methods for women but, with increasing publicity on the problems associated with the use of oral estrogen-gestagen contraceptives, the role of the male in contraceptive practice is being re-examined. From the numbers of vasectomies performed throughout the world it is evident that there exist men who are willing to accept responsibility for contraception. Consequently, over the past decade increasing effort has been devoted to a re-examination of the male role in contraception and research into new methods is being stimulated. What then are the possibilities for alternative methods of male contraception? What are the associated problems? How widespread is their use likely to be? Today, as was the case 10 years ago, a man willing to take the responsibility for con- traception has three methods from which to choose: coitus interruptus, condoms, and vasec- tomy. Coitus interruptus is widely practised butit is subject to error and is often less psycholo- gically rewarding, especially for the female. Condoms are effective if used properly but lack of care in use can result in an unacceptably high pregnancy rate. Nevertheless, distribu- tion is easy, no medical personnel are required, no toxic effects have been noted, and the method is cheap. Vasectomy, though very effective, is assumed to be a permanent method of sterilization rather than reversible contraception. It is against this background that agencies interested in contraceptive development have been attempting to stimulate research in the development of contraceptives for males. Through its Special Programme of Research, Development and Research Training in Human Reproduction, the World Health Organiza- tion has been able to increase significantly the research effort in male contraceptive develop- ment, by initiating a task force to develop methods of regulating male fertility.a The major aim of research on male contraceptive development is to produce a safe, effective, and acceptable method ofcontraception that is reversible. The theoretical prospects for controlling male fertility are numerous, b ca but can basically be considered under 1 Professor of Anatomy, Monash University, Clayton, Victoria, 3168, Australia. a WORLD HEALTH ORGANIZATION. Sixth annual report of the Special Programme of Research, Development and Research Training in Human Reproduction. Unpublished document HRP/77.3, 1977. b DE KRETSER, D. M. Contraception, 9: 561-600 (1974). C BREMNER, W. J. & DE KRETSER, D. M. New Englandjournal ofmedicine, 295: 1111-1117 (1976). d SHORT, R. V. & BAIRD, D. T., ED. Proceedings of the Royal Society ofLondon, B, 195: 3-224 (1976). 3698 -353- D. M. DE KRETSER three major headings: (a) interference with the hormonal control of the testis; (b) disruption of spermatogenesis by direct influence on testis function; and (c) post-testicular interference with the fertilizing ability of sperm and their transport. INTERFERENCE WITH THE HORMONAL CONTROL OF TESTICULAR FUNCTION It is well known that the testis is controlled by the pituitary gland through the secretion of the gonadotrophic hormones follicle stimulating hormone (FSH) and luteinizing hormone (LH). FSH acts on the seminiferous tubules, probably influencing spermatogenesis indirectly by modifying the function of the Sertoli or supporting cells. LH stimulates testoterone production by the interstitial cells and, by maintaining high levels of intratesticular testo- sterone, it indirectly stimulates spermatogenesis. Though both FSH and LH stimulate spermatogenesis they cannot influence the rate of sperm production, which remains a biological constant for each species; in man it is approximately 70 days. Furthermore, through their action on the testis, FSH and LH stimulate the secretion of substances that inhibit secretion of these hormones by the pituitary gland, i.e., there is negative feedback. For LH, testosterone exerts this effect directly or through conversion to estradiol 17k, but the signal for FSH control remains a subject for controversy. It is likely that the testis secretes a specific inhibitor of FSH secretion termed inhibin, which probably synergizes with testosterone in controlling the output of FSH. Superimposed on the interrelationship of the pituitary and testis is the hypothalamic control of FSH and LH, exerted through the secretion of a releasing hormone, gonadotro- phin releasing hormone (GnRH). This decapeptide stimulates the secretion of both LH and FSH, reaching the pituitary by the hypophyseal portal vessels. The relatively simple chemical structure of GnRH has led to a number of synthetic modifications, some of which have demonstrated augmented activity while others appear to interfere with the action of endogenous GnRH, thereby lowering the levels of FSH and LH. Unfortunately, the dose of these inhibitory analogues of GnRH required to decrease FSH and LH is very large and a search for more active inhibitors is being pursued. As prospective compounds for regulating male fertility these substances currently suffer from three major disadvantages -their short duration of action, their inactivation when given orally, and their suppression of both LH and FSH. A number of studies performed some 25 years ago showed that treatment of normal males with progestagens or androgens could reduce sperm counts to zero and that there was full recovery on cessation of therapy. These studies thus demonstrated the feasibility of inducing spermatogenic suppression by depressing gonadotrophic secretion by the pituitary gland. During treatment with the progestagens then in use, gynaecomastia and loss of libido occurred, the former probably being due to conversion of the progestagen to estrogenic metabolites. The loss of libido and potency experienced by these men high- lights the problems of suppressing both FSH and LH secretion for, though spermatogenesis is disrupted, testosterone production by the interstitial cells is also impaired. The loss of libido and potency was not noted during spermatogenic suppression using testosterone treatment, since this effectively replaced interstitial cell function. Despite the pilot studies demonstrating the efficacy of testosterone as a suppressor of sperm production, no further work was reported until 1972, when it was again shown 354 FERTILITY REGULATION IN THE MALE 355 that testosterone propionate at a dose of 25 mg/day by intramuscular injection was effective in causing azoospermia in man. More recently, further investigations have established that long-acting esters of testosterone (200-250 mg) given intramuscularly every 10-12 days are equally effective as the daily injections in suppressing spermatogenesis. Currently, larger-scale trials sponsored by several agencies are under way to determine the efficacy of this regimen in a greater number of men and, more important, to determine whether the dose required to suppress spermatogenesis results in side effects such as a raised haemato- crit, excessive weight gain, or changes in lipoprotein concentration. Since spermatogenic suppression with large doses of progestagens led to a reduction in testosterone and thereby loss of libido and potency, the possibility of androgen replace- ment in such men has led to trials of gestagen-androgen mixtures. The possibility also exists that the dose of both the androgenic and progestagenic agents used in these combina- tions may be considerably reduced in comparison to their use as single agents without loss of efficacy in terms of spermatogenic suppression. The effectiveness of gestagen- androgen combinations in suppressing sperm production has been demonstrated, with recovery following cessation of treatment. Through a network of centres throughout the world, the WHO Task Force on methods of regulating male fertility is sponsoring trials designed to evaluate the efficacy of combinations of long-acting testosterone esters with the gestagens medroxyprogesterone acetate, + -norgestrel, and 17, hydroxyprogesterone caproate with a view to selecting the most effective and least toxic. With the completion of these trials, information will be available concerning the lowest effective doses and the most appropriate method of administering the gestagenic component. In all of the above trials, the androgen has been administered parenterally, since the oral testosterone compounds currently on the market were relatively less effective and were associated with the risk of hepatic side effects.a Monthly injections of testosterone esters may well prove to be unacceptable for long-term use and alternative methods of delivery are being explored. In trials organized by The Population Council, testosterone has been successfully delivered by silastic implants, which have remained effective for periods of up to 12 months. A programme initiated by the task force is attempting to produce new androgens that may be delivered by alternative routes. In recently completed trials, Paulsen et al.b demonstrated reversible spermatogenic suppression with combinations of an impeded androgen, danazol (600 mg orally per day) with monthly injections of testosterone enantate. With this combination, oligozoospermia (less than 5 million spermatozoa per ml) or azoospermia was found in 11 of 13 volunteers associated with suppression of serum LH and to a lesser degree serum FSH. It is of interest that suppression took up to 16 weeks to occur and recovery often required 3-6 months, reflecting the long duration of the seminiferous cycle in man. The administration of estrogens clearly suppresses pituitary secretion of both FSH and LH, resulting in the depression of sperm and androgen production. Side effects resulting therefrom comprise loss of libido and potency and the occurrence of gynaecomastia; these are clearly not compatible with a successful contraceptive. However, limited studies with an estrogen-androgen combination resulted in the suppression of sperm production without side effects. Furthermore, experimental studies on rats have demonstrated that the dose of estradiol needed to suppress fertility is small when combined with testosterone, a BOYER, J. L. ET AL. Contraception, 13: 461-468 (1976). b PAULSEN, C. A. & LEONARD, M. In: Spilman, C. H., ed. Regulatory mechanisms of male reproductive phys- iology. Amsterdam, Elsevier, 1976, P. 197. 356 D. M. DE KRETSER and synergistic effects were obtained.a For use in man, the lowest dose compatible with spermatogenic suppression would need to be found to reduce the recognized risks of thromboembolic phenomena associated with the use of estrogens. Although it has been included in this section ofthe review, the possible use of cyproterone acetate as a male antifertility agent was prompted by the suggestion that its antiandrogenic activity enabled it to influence the epididymal maturation of sperm at a dose that did not interfere with testicular function. Consequently, trials with cyproterone acetate at doses of 5, 10, and 20 mg/day were initiated by the Task Force; the results showed that at the higher doses a decrease in sperm count occurred in the majority of men but azoospermia was not achieved. This change was associated with a 50-80% reduction in plasma testo- sterone levels and a depression of serum LH levels by 35%, both of which suggest that the antifertility action is more likely to be due to the progestagenic than to the antiandrogenic properties of cyproterone acetate. More recent studies have confirmed these findings and have indicated that, although sperm counts considered to represent mild oligospermia (10-20 million/ml) had been achieved, some unplanned pregnancies had resulted. Further- more fatigue, weakness, and depression occurred in a few men during therapy but, despite the fall in plasma testosterone levels, loss of libido and potency was not experienced. The failure to achieve azoospermia with cyproterone acetate precludes its use alone as an antifertility agent, but owing to its potent progestagenic properties it may be suitable if combined with androgens. The use of this agent must be viewed cautiously since at higher doses used in the treatment of women with hirsuitism, interference with adrenocortical function has been found and the results of similar studies at doses of 10 and 20 mg/day in males must be awaited. All the agents mentioned to this point interfere with the pituitary secretion of both LH and FSH and consequently result in suppression of both sperm and androgen production. The concept that a specific feedback signal for FSH exists has gained support from numerous studies in male and female reproductive physiology that have demonstrated that FSH levels can vary independently of LH levels. Though its existence is suspected, isolation of the feedback material for FSH, termed inhibin, has not been achieved. However, its potential use in suppressing FSH and thereby spermatogenesis is recognized and conse- quently, studies were initiated by the Task Force in an attempt to isolate inhibin and thereby assess its potential in suppressing sperm production. The results of these studies in Belgium, India, England, and Australia demonstrated that inhibin activity is present in extracts of testes, rete testis fluid, testicular lymph, and seminal plasma and that the material is most probably a protein. The interest generated by these studies has led to other studies, which have demonstrated inhibin activity in culture media from Sertoli cells and in ovarian follicular fluid. In parallel with the attempts to isolate inhibin, Task Force-sponsored studies have been made in an attempt to elucidate the role of FSH in maintaining spermatogenesis, using passive immunization with specific antisera to FSH. The results of these studies in rats and monkeys, together with further investigations into the chemical nature of inhibin, will determine the usefulness of this approach. Should the molecular weight of inhibin be low enough to permit its synthesis and should FSH be shown to be essential in main- taining spermatogenesis in the adult animal, then the prospects for this approach will remain viable. a EWING, L. L. ET AL. Nature, 269: 409-411 (1977). FERTILITY REGULATION IN THE MALE 357 DISRUPTION OF SPERMATOGENESIS BY DIRECT ACTION ON THE TESTIS The fact that spermatogenesis involves a series of cell divisions whereas the interstitial cells by comparison are quiescent opens the possibility that agents could be developed to disrupt seminiferous tubule function without affecting androgen production by the inter- stitial cells. Clearly, for the process to be reversible, these agents must be capable of sparing the spermatogonial cells. A large number of compounds have been described that can disrupt spermatogenesis through their action on replicating cells and these have been reviewed recently.a b Unfortunately, the effects of these agents on other tissues prevent their use in man. Furthermore, some of them deplete the stem cell population so severely that recovery does not occur on cessation of treatment. This irreversible sterility has now been noted with drugs such as cyclophosphamide and chlorambucil, which are used in the treatment of neoplastic disorders and certain types of renal disease. While there is no doubt that the initial action of such agents is on the spermatogenic process, there is some evidence that long-standing severe damage to the spermatogenic process is associated with elevated LH levels and low or low-to-normal testosterone levels indicative of inter- stitial cell failure. Recent investigations in rats have demonstrated the important regulatory role played by the Sertoli cells in the spermatogenic process. By producing a protein that binds testo- sterone (androgen binding protein, ABP) the Sertoli cell ensures that the centrally placed germ cells and the epididymis are provided with supplies of testosterone. The possibility that interrupting the production of ABP would disrupt spermatogenesis has been pursued by Task Force scientists in Norway, Sweden, and the USA. By purifying ABP they have been able to produce an antiserum against it in rabbits. This antiserum, injected into rats prior to puberty, caused infertility. The possible uses of this type of approach in man may well be limited by the fact that the structure of ABP appears to be very similar to that of the circulating sex steroid binding globulin and any antiserum is likely to cross-react significantly, perhaps interfering with testosterone transport in man. It has been demonstrated recently that 5-thio-D-glucose given orally causes disruption of spermatogenesis and infertility in male mice without interfering with mating behaviour. The substance was nontoxic in doses required to inhibit spermatogenesis and recovery was rapid when the treatment was ceased. The mechanism by which this compound acts is unknown and its activity in other species is under investigation. These results will enable its potential as a widespread antifertility agent to be assessed. INTERFERENCE WITH EPIDIDYMAL SPERM MATURATION AND SUBSEQUENT TRANSPORT The mechanisms by which sperm acquire fertilizing ability during their passage through the epididymis still remain unclear. While it has been shown that the capacity for progressive- motility is acquired during transit through the epididymis, other changes occur such as alterations in morphology and metabolism. Which of these modifications results in the a JACKSON, H. British medical bulletin, 26: 78-86 (1970). b PATANELLI, D. J. In: Hamilton, D. W. & Greep, R. O., ed. Handbook of physiology. Baltimore, Williams & Wilkins, Section 7, 1975. 358 D. M. DE KRETSER acquisition of fertilizing capacity remains obscure and only further investigations will clarify these issues. Nevertheless, the potential of interfering with this process and thereby causing infertility without altering testicular function remains attractive. Despite promising results with an amino derivative of a-chlorohydrin (DL-l-amino-3- dichloro-2-propanol hydrochloride), Task Force-stimulated trials demonstrated toxicity in primates. The simplicity of the chemical structure of a-chlorohydrin has stimulated studies of its metabolism and mode of action in the hope that a nontoxic but active metabo- lite might be identified. Furthermore, investigations of related synthetic compounds have demonstrated antifertility activity in certain halogenated sugars. This activity appears to be exerted by interference with the metabolic activity of sperm within the cauda epididymis. The early promise of these substances needs confirmation in other species and the results of chronic toxicity testing will be awaited with interest. In the past, couples contemplating the use of vasectomy as a contraceptive method were usually advised that it should be considered as irreversible. This concept requires some modification since microsurgical anastomosis of the vas results in anatomical continuity and patency in over 80% of cases. However, the actual pregnancy rate following reanas- tomosis is somewhat lower owing to unidentifiable factors, one of which may be the development of antisperm antibodies following vasectomy. Investigations into temporary vas occlusive mechanisms a are also being explored and include the injection of poly- merizing silicone rubber and copper or Silastic implants. Devices such as Y-valves are also being tested that can direct sperm through a long length of tubing when fertility is not desired. GENERAL COMMENTS Two problems common to all avenues of investigation for male fertility control remain to be resolved-acceptability and methods of assessing efficacy. It is evident from the number of men undergoing vasectomy that a proportion of the male population is willing to accept contraceptive responsibility. Whether this group would be willing to undertake courses of medication administered orally or intramuscularly over prolonged periods instead of a single period of limited discomfort at vasectomy is still unknown. The availa- bility of reversible contraceptives for men might, however, recruit other men who had been unwilling to accept the irreversibility of vasectomy. Of the methods under investigation, either androgens alone or in combination appear to be the most promising and advanced, but they carry with them the likelihood of parenteral administration of at least the andro- genic component. The method of delivery would appear to be a further factor influencing acceptability and such influences are currently under study. The question ofwhich methods can be used to test the efficacy ofnew male contraceptives remains unanswered. This problem is clearly less important if the technique results in azoospermia but with some substances, such as cyproterone acetate, oligozoospermia is created and with others, e.g., a-chlorohydrin, sperm numbers are unaltered but are rendered infertile. Ethical issues arise when the prospects of open trials with abortion availability are considered as test procedures, but such trials may yet have to be used. The assessment of fertilizing ability in vitro remains one avenue open to study and the use of oocytes recovered after death may defuse some of the ethical issues that could be raised to this a LUBELL, I. & FRISCHER, R. Proceedings of the Royal Society of London, B, 195: 93-114 (1976). FERTILITY REGULATION IN THE MALE type of procedure. Alternatively, the penetration of ova from other animal species by human sperm offers the possibility of an in vitro test if the capacity for penetration can be equated with fertilizing ability in vivo. Despite the numerous areas of research currently being pursued, the prospect of a marketable male contraceptive remains several years away. On the results available to date, the most promising method is the use of androgens alone or in combination with gestagens. This type of methodology has been shown to lower sperm counts severely with reversibility on cessation of treatment. Furthermore, no significant toxicity problems have been en- countered in the limited trials carried out to date. Before such investigations can be ex- panded, a definitive combination and dosage must be chosen, teratogenicity studies com- menced, and further toxicology initiated. It is only when these are completed and more extensive clinical trials concluded that a decision concerning marketability can be taken subject to the approval of drug regulatory agencies in each country. The pathway is long and expensive but unchecked population growth carries with it misery and demands far more expensive than any contraceptive development programme. RtSUMIt Regulation de la fecondite chez 1'homme Les milieux scientifiques ont ete amenes, au cours des dix dernieres annees, a reconsiderer la question de la contraception a la suite des problemes souvent poses par l'administration aux femmes de contraceptifs oraux a base d'estrogenes/progestogenes, et de la volonte manifestee par de nom- breux hommes d'assumer leur part de responsabilite en se soumettant notamment ai la vasectomie. Les diverses institutions interessees s'efforcent en consequence d'encourager les recherches sur la contraception masculine et l'OMS a cree, dans le cadre de son programme special de recherche, de developpement et de formation a la recherche en reproduction humaine, un groupe d'action pour la mise au point de methodes de regulation de la fecondite chez l'homme. Cet article examine les trois principaux types de methodes permettant d'agir de maniere non irreversible sur la fecondite masculine. 1) Action au niveau du controle hormonal de la fonction testiculaire Le contr6le hormonal de la fonction testiculaire est assure par 1'hypophyse, qui secrete les gonadotrophines, soit l'hormone folliculostimulante (FSH) et l'hormone luteinisante (LH). La FSH et la LH stimulent la spermatogenese, mais leur secretion est freinee par une retroaction nega- tive, due a la testosterone pour LH et sans doute a un inhibiteur specifique pour FSH. La secretion hypophysaire de FSH et de LH est elle-meme stimulee par un facteur de liberation des gonado- trophines (GRF) elabore par l'hypothalamus. Toutefois, l'emploi d'analogues de ce facteur en concurrence avec le GRF endogene n'a pas paru pouvoir inhiber assez activement ni assez specifi- quement la secretion des deux gonadotrophines. Lors d'experiences deja anciennes, l'administra- tion d'androgenes avait permis la suppression complete de la spermatogenese par retroaction nega- tive sur la secretion hypophysaire de gonadotrophines. Mais le traitement par des progestog6nes entrainait des effets indesirables sur la virilite, effets qui n'etaient pas constates avec 1'emploi de testosterone. Des experiences plus recentes ont confirme 1'efficacite de la testosterone - en parti- culier d'esters de testosterone a effet prolonge administres par voie parenterale a raison de 200 a 250 mg tous les 10-12 jours - pour inhiber la spermatogenese. L'essai a plus grande echelle de ce traitement se poursuit sous 1'e'gide de plusieurs institutions, en vue notamment de determiner les effets secondaires possibles. Le groupe d'action de I'OMS patronne, pour sa part, des essais menes par un reseau de centres pour evaluer I'efficacite de combinaisons de ces esters avec divers proges- 359 360 D. M. DE KRETSER togenes en vue de laselection du plus efficace et dumoin s toxique de ceux-ci. On pense en effetqu'une telle combinaison pourrait permettrea la fois d'6viter la perte de libido et d'administer une dose beaucoup plus faible de chacun des deux agents. L'administration par voieparenterale ae t prpffr6e dans tous ces essais pour des raisons d'efficacite et desecurite. Le groupe d'action alancee galement un programme visanta produire de nouveaux androgenes pouvantet re administr6s sous diverses formes. L'administration journaliere d'un androgenea effetprolong6, le danazol,associe e 'a une injection mensuelled' nantate de testosterone, ae etexperi- mentee avec de bonsresultats. La suppression de LH dans leserum et celle, momns marquee, de FSH, ne sont toutefois intervenues qu'au bout de 16 semaines et le retour 'a la normaleapres cessation du traitement a pris de 3 'a 6 mois. L'emploi d'estrogenes parait beaucoup plus al6atoire en raison de leurs effets secondaires.Quelquesetudes ont cependantete faites avec une combinaison estrogene/androgene et l'on a pu, chez le rat, provoquer la st6rilite en associant une faible dose d'estradiol'a la testosterone, les deux substances agissant en synergie. Des experiences doiventetre faites chezl'homme pourdeterminer la dose d'estrogene la plus faible qui pourraite re employee, de manire'aduire le risque de throm-boembolie. Les travaux concernant la cyprot6rone ont mis en evidence ses propri6t6s progestogenes, mais le groupe d'action a estim6 que de nouvelles recherches sur la possibilite del'associer'a dessteroides androgenes 6taient n6cessaires. Le groupe soutient, d'autre part, des etudes visant'a isoler le fac- teur inhibiteur qui limite lasecr6tion de FSH (il s'agit vraisemblablement d'une proteine). On cherche aussia preciser lerole de la FSH dans le maintien de la spermatogenese chez des animauxd'experience. 2) Interruption de la spermatogenese par une action directe sur le testis Les composes agissant sur la division des spermatogonies - comme le chlorambucil - ontete r6cemment passes en revue. Mais ils peuvent endommager durablement non seulement les cellules souches dans les tubesseminiferes, mais aussi les cellules interstitielles. Lerole des cellules de Sertoli aete approfondi, et notamment celui de I'ABP (proteine de liaison del'androgene), et l'injection d'un serum anti-ABP'a des rats a entraine lasterilite; il est cependant'a craindre qu'un telserum affecte aussi la testoterone circulant dans le sang chezl'homme. L'administration orale de 5-thio-D-glucose pour inhiber la spermatogenese a donn6 de bonsresultats chez le rat, mais son mode d'action n'a pu encoreetreelucid6. 3) Action sur la maturation dansl'e'pididyme et le transport du sperme La motilit6 du spermatozoide s'accroit au cours de son passage dansl'epididymeet il subit d'autres modifications encore mal connues, notamment metaboliques. Certaines substances pou- vant entrainerla st6rilit6 par une action surl'activite metabolique du spermatozoide dansl'epidi- dyme font l'objet d'experiences sur diverses esp6ces d'animaux. Enfin, des proc6d6s susceptibles de remplacer la vasectomie, consid6ree comme irreversible, sonta l'etude (anastomose microchirurgicale, implants, derivation). Toutes les methodes envisag6es posent un probleme d'acceptabilite. La possibilite - qu'offre notamment l'administration d'androg6nes - d'un retour 'a la normale apres cessation du traite- ment est un 6l6ment important, mais ces st6roldes doiventetre injectes par voie parenterale. L'eva- luation pose aussi un probleme, en particulier lors de l'emploi de substances qui agissent sur la capacit6 de fecondation du sperme et non sur la quantite de spermatozoides. Aucun produit n'a encore ete suffisamment mis 'a l'epreuve, sur le plan de la formulation, du dosage, de la toxicite et de la teratog6nicite, pour pouvoir etre propose comme contraceptif mascu- lin et on ne peut guere s'attendre 'a des r6sultats concluants avant plusieurs annees. 360