The regulation of male fertility the state of the art and future possibilities

THE REGULATION

OF MALE FERTILITY

THE STATE OF THE ART AND FUTURE POSSIBILITIES

D.M. de Kretser Department of Medicine Monash University Prince Henry's Hospital Melbourne, Australia

ABSTRACT

This review presents the current state-of-knowledge with respect to Furthermore,areas methods for the regulation of fertility in the male. where future studies may be profitably encouraged are indicated. It was written for the World Health Organization Task Force on Methods for the Regulation of Male Fertility. This is one of several collaborative efforts in WHO's Expanded Programme of Research, Development and Research Training in Human Reproduction. This Programme aims to develop a variety of new, safe, effective and acceptable methods of fertility regulation. The constructive criticism and co-operation of scientific colleagues on the Task Force has made possible the compilation of this review.

Accepted

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1.

INTRODUCTION

The ultimate aim in the regulation of fertility in the male is the development of suitable methods which reversibly interfere with his fertilizing capacity without compromising libido and potency. While the theoretical sites of interference are numerous, the close association, both morphological and functional, of the spermatogenic and androgenic compartments of the testis render many prospective methods unacceptable. The additional specification of reversibility further restricts the field. The sites where control of male fertility may be possible, extend from the disruption of the spermatogenic process to interference with While available knowledge permits transport of ejaculated spermatozoa. the formulation of suitable experimental procedures to assess some areas, in many instances,ignorance of fundamental reproductive processes has prevented exploration of theoretical avenues of control. It is the purpose of this review to examine the physiological data available in several areas of male reproductive biology and its relationship to the available methods The need for extension of of fertility regulation and their limitations. basic knowledge in several areas of reproductive physiology that could conceivably lead to suitable methods of fertility regulation, is discussed. The scope of this review is necessarily broad and it is possible to subdivide the field into three major methods by which the interruption of male fertility may be achieved. These include: 1.

Disruption

2.

Interference with the sperm maturation

of sperm formation

3.

Interruption

2.

2.1

Physiological

after spermiation

of sperm transport.

INTERRUPTION

OF SPERMATOGENESIS

Considerations.

The results of investigations in a number of 2.1.1 Spermatogenesis: mammalian species have established the cytological sequence of changes The kinetics resulting in the production of mature spermatozoa (1, 2, 3). of mammalian spermatogenesis has been the subject of detailed studies by Clermont (1, 4, 5), Huckins (6, 7) and Oakberg (8) but some controversy Most still exists regarding the kinetics of spermatogonial division. investigators agree that spermatogonia divide by mitosis and,after a period of replication,undergo differentiation leading by a sequence of changes to Following meiosis which involves both the primary and secondary meiosis. spermatocyte stages,qermatids are formed containing the haploid chromosomal The spermatids undergo a metamorphic process which involves no complement.

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further cell division but a complex differentiation leading to the release The latter metamorphic process is of spermatozoa into the tubule lumen. termed spermiogenesis and ends with the process of spermiation or release of sperm from the seminiferous epithelium. The cytological features of spermatogenesis have been studied in the human by Clermont (9) who identified six cell associations and established This process was difficult to discern a seminiferous cycle in man (10). in man where more than one cell association could be found in a single seminiferous tubule cross-section and contrasted to the well defined cycle which was easily identifiable in other mammalian species (2, 11). The ultrastructural features of human spermatogenesis have also been carefully studied and the cytological details of spermatogonia, primary spermatocytes and the details of spermiogenesis are now available as well as data regarding the interstitial cells (12 - 21). Studies of the kinetics of spermatogenesis in mammals have established that the time taken for this process in each species 1s unique and a biological constant (1, 2, 10). The time required varies between species and for man is 74 ? 4 days (10). The studies by Heller and Clermont (10) demonstrated in man that this rate was not affected by hormones which were supposedly suppressive or stimulatory to spermatogenesis nor was it affected by such agents as irradiation. It appears that once spermatogonia have begun the spermatogenic process, they progress through the timed sequence of development or degenerate. This fact is of importance in assessing the response of the testis to agents that supposedly stimulate spermatogenesis as they should be used for at least 70-80 days before conclusions can be drawn. It is also important to recognize this factor in assessing the activity of agents capable of interrupting spermatogenesis for fertility control, as those acting at a spermatogonial level may not influence fertility initially. Despite knowledge of the kinetics of spermatogenesis, little is known of the factors responsible for maintaining the cycle of the seminiferous epithelium. The intra-tubular factors responsible for co-ordination of germinal cell differentiation remain difficult to resolve. The arborizatlon of Sertoli cell cytoplasm and the intercellular bridges that link considerable numbers of germinal cells at all stages of development may aid in co-ordination (22, 23). Identification of the co-ordinating mechanism could lead eventually to the development of methods of disrupting spermatogenesis. 2.1.2 Hormonal Requirements for Spermatogenesis: The results of many studies have demonstrated the dependence of testicular function on an intact, normal hypothalamo-hypophyseal unit (24). While the glycoprotein hormones, follicle stimulating hormone(hFSH)and luteinizing hormone(hLH), have been implicated in the regulation of testicular function for some years, it is only recently that the releasing factor controlling hypophyseal function has been characterized (25). Luteinizing hormone releasing hormone(LHRH) has been shown to be a decapeptide and small doses stimulate the release of hLH (26, 27). In addition smaller amounts of hFSH are released by LHRH and

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to date no factor capable of exclusively releasing hFSH specifically has been characterized (28, 29, 30). A considerable number of chemical analogues have been synthesized, some with considerably less activity while in others accentuated potency in terms of hLH release has been observed (31, 32, 33). The specific role assigned to hFSH in the control of spermatogenesis has yet to be identified. Some investigators suggest that early phases of spermatogenesis are dependent on hFSH (34, 35) whereas others suggest that spermiogenesis is the dependent process (24, 36). Part of the difficulty in defining the role of hFSH is due to the lack of hFSH preparations which are pure and free from hLH activity and also due to the possibility that species may differ as to their hormonal requirements for spermatogenesis. In addition, it is possible that the role of hFSH may differ during the initiation of testicular function from its role In the maintenance of testicular function (37). The treatment of hypogonadotrophic hypogonadism with human pituitary gonadotrophin preparations has demonstrated the necessity for hFSH activity to initiate full spermatogenesis (35, 38). Studies in the rat have identified specific intratubular receptors for FSH (39) and following large doses, labelled FSH can be shown, by histochemical and electromicroscopic methods, to be localized in the Sertoli cells (40). The action of hLH in the control of testicular function is less controversial and it is generally agreed that it stimulates testosterone and estradiol production by the interstitial cells (41, 42). Specific receptors for hLH have been demonstrated on the interstitial cells (43) and no intratubular site of localization could be identified using 125I-LH. However recent studies using 3H-human chorionic gonadotrophin(hCG)have suggested that localization occur.s in spermatogonia (44). Nevertheless, it is likely that the major influence of LH on spermatogenesis is exerted by stimulation of the interstitial cells to produce testosterone (24). Some investigators have suggested that in the toad (45, 46, 47) and more recently in rodents that LH influences spermiation but other studies have Some evidence is also failed to confirm this intratubular effect (48). available that FSH may act in a synergistic role, potentiating the action of LH on the interstitial cells (49). The feedback control of LH is exerted by testosterone but the failure of the episodic peaks of LH and testosterone to coincide in man in any specific manner may indicate a relatively loose relationship (50, 51). There is little doubt The feedback control of FSH remains an enigma. that the testis exerts a feedback influence on FSH secretion and that severely damaged seminiferous tubules are usually associated with high FSH levels (52). There is evidence from the early studies of McCullagh and Others that aqueous testicular extracts contain a substance capable of exerting an inhibitory influence on pituitary FSH excretion and termed Unfortunately,this work has not been this compound Inhibin (53, 54, 55). For example, non-specific explored further using more modern techniques. assays that measured both FSH and LH were utilized to generate this concept. The source of this material also remains obscure and some investigators

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argue that its production is related directly to specific stages of spermatogenesis whilst others cannot demonstrate this relationship While the source of the compound is uncertain, (52, 56, 57, 58, 59). Setchell (48) has identified an antigonadotrophic factor in rete testis fluid and Franchimont (60) has reported similar findings on a compound from seminal plasma. The possibility of a "steroid feedback" on FSH secretion has not However,in a recent report by Swerdloff et al been entirely excluded. (61). no steroid compound was identified that could exert a more profound Testosterone and estrogens are each suppressive effect on FSH than LH. capable of suppressing FSH but this universally occurs in conjunction with While both FSH and LH were suppressed following suppression of LH (62, 63). infusions of testosterone at rates equivalent to twice the daily production rate, rebound of LH was noted on ceasing the infusion. The demonstration that the rare occurrence of interstitial cell tumours III childhood is often accompanied by spermatogenesis in the seminiferous tubules nearest to the adenoma, is suggestive evidence that steroid substances, possibly testosterone, are capable of influencing spermatogenesis (24). Similarly the demonstration that high local intra-testicular concentrations of testosterone released from Silastic implants can maintain spermatogenesis also suggests an important role for testosterone It has also been shown that the seminiferous tubules are capable (64). of stero.id metabolism (65, 66) and these studies in rodents and man have Rivarola & Podesta (68) been extended by Lacy and his colleagues (67). have shown the metabolism of testosterone by seminiferous tubules to androstanediol and that the capacity for this conversion increases with age. Further studies are necessary to define the influence of the steroid biosynthetic capabilities of the seminiferous tubule on spermatogenesis. 2.1.3 Requirements for a Specialized Environment: It is apparent from the peculiar composition of the fluid in the seminiferous tubules and rete testis (see Section 3.1.5) that all substances do not exchange freely between these fluids and blood ; otherwise,the concentrations would equalize. Indeed, some substances pass only slowly from blood into the seminiferous tubules (236). Proteins, such as albumin, enter the tubules very slowly (237, 2381, but gonadotrophins seem to enter more quickly than other proteins (48, 237). The site of this blood-testis barrier has been located by electron microscopy using electron-opaque markers. In rodents, but not in the monkey,there is some restriction at the level of the myoid cells, and in all species, markers do not penetrate beyond the specialized junctions between adjacent pairs of Sertoli cells, and thus gain direct access only to the spermatogonia and not the other germinal cells (69, 70, The reason for this specialized and protected environment is not 239). known but it develops at about the time at which the first meiotic divisions occur (71, 240, 241). One substance that readily penetrates the blood-testis barrier is testosterone (238) and the arrangement of the Leydig cells around the

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blood vessels, or in clumps in the interstitial spaces,means that the walls of the seminiferous tubules are bathed in fluid with a much higher testosterone concentration than structures elsewhere in the body. Spermatogenesis is dependent on a sufficient concentration of testosterone, although testosterone can be replaced by other non-androgenic steroids. Other steroids penetrate the barrier at variable rates and cholesterol does not penetrate at all (238). a(-Chlorohydrin, like glycerol,enters quite rapidly (242). Another aspect of the specialized environment required for normal spermatogenesis is the dependence of the process on the maintenance of It testicular temperature several degrees below body temperature (72). is well known that externally applied heat is capable of interrupting spermatogenesis both in animals and man (72, 73, 74).

2.2

Currently Available

Methods

of Spermatogenic

Interruption.

Several methods are available to cause disruption of spermatogenesis and employ a variety of compounds. The mode of action of some of these compounds is understood but in many, their antispermatogenic activity was unsuspected. These compounds can be grouped into three categories based on their suspected site of action and include:(i) interference with cell division during spermatogenesis,(ii) compounds with activity involving the vascular system of the testes,and (iii) interference with the hormonal milieu. The scope of this review does not permit a detailed analysis of each of these groups of compounds but the results of such investigations are available from reviews by Jackson (75) and Patanelli (76). 2.2.1

Interference

with Cell Division:

Colchicine. The ability of this compound and its derivatives (aa to inhibit mitosis at metaphase has been recognized and widely Until recently, no report of the utilized in chromosomal studies. effect of this compound on spermatogenesis in man was available. However,the recently reported observation of a depression of sperm counts in a man treated with colchicine for gouty arthritis (77) suggests that further investigations of this drug and its derivatives may prove useful in the search for compounds capable of interrupting spermatogenesis. The action of these agents on bone marrow Alkylating Agents. (b) and neoplastic tissue has been extensively studied and many of the drugs are used as cytotoxic agents for the treatment of neoplasia. This group of compounds includes such agents as ethyleneimine derivatives, nitrogen mustards and diesters of methanesulphonic acid. The disruption of spermatogenesis by these compounds has been studied extensively in the rat by Jackson and his co-workers and was reviewed recently (75). The general action of such compounds involves the interruption of spermatogonial multiplication when low doses are used.

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Fertility is still possible for sometime as spermatozoa continue to be produced from the unaffected spermatocytes and spermatids (75). With larger doses,more widespread interference with spermatogenesis occurs (76). Disruption of spermatogenesis has been demonstrated 1n me* treated with chlorambucil (78) for neoplastic disease and cycloIn the maJority of patients phosphamide (79, 80) for renal disease. in the series, azoospermia occurred and was associated with the histological appearance of "Sertoli-cell-only" syndrome in the The effects of such therapy on the pituitary gonadal testis. (81) who noted moderately axis have been studied by Van Thiel et. elevated levels of serum FSH and minor rises in serum LII. It would appear that these agents are capable of causing prolonged interruption of spermatogenesis with relatively minor changes in In a few patients treated so far, LH and testosterone levels. spermatogenesis has recovered and this recovery may be related to a shorter duration of exposure to the agent employed. To date no details are avaIlable concerning the possibility of genetic changes in germinal cells following recovery. Heterocyclic Anti-Spermatogenlc Agents. This group of Cc) compounds is capable of interrupting spermatogenesls without interfering with the endocrine function of the testis, although this aspect has not been studied extensively with modern assay The group include the mitrofurans, thiophenes, bis techniques. (dxchloroacetyl)diamines and dinitropyroles. These agents act principally at the primary spermatocyte stage by mechanisms unknown (75). The nitrofurans have been studied in mammals (82, 83, 84, 85, 86) and are also effective in man (87, 88). However undesirable effects were noted during clinical trials and further investigations have not been performed (76). The bis(dichloroacety1) diamines are well tolerated in man and produce azoospermia within 8-11 weeks of treatment. This action principally affects spermatlds leaving the spermatogonia intact and thereby allowing reversal of the spermatogenic inhibition However, the use of these compounds prevents the oxidation (89). of ingested alcohol; thus an "antabuse-type" reaction occurs. This potential for serious adverse reactions forced discontinuation of further trials. The more recently synthesized group of compounds, the dlnitropyroles, have been shown to suppress spermatogenesis at the primary spermatocyte in the rats.

stage

(90) and

result

in complete

infertllity

in 21 days

However,toxic effects noted in dogs have prevented trials in man (76). The nitroimidazoles also act principally at the primary spermatocyte and early spermatid level but leave resting

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spermatocytes intact in the rat (76). Fertility in the rat is present for 2 to 3 weeks following treatment and is then followed by a 3-4 week period of infertility. Subsequently spermatogenesis is restored and no mutagenic effects were noted in offsprings (76). To date no reports of studies in ma" are available. Fluoroacetamide. Early studies demonstrated that doses of Cd) 50 mg/kg or 4 mg kg body weight caused almost complete disappearance of germinal cells from the seminiferous epithelium in the rat However, studies by Sud and Steinberger (93) showed (91, 92). that 250 pg orally interfered with the division of secondary spermatocytes to form spermatids. No studies have been performed in ma" due to the toxicity of this compound. 2.2.2 Interference with Testicular Vasculature: The effects of experimental ligation of the internal spermatic artery seem to be very variable (243, 244). Nevertheless,it is very probable that most of the very specific effects of cadmium on the testis (94, 95, 96, 97, 98, 245) are due to interference with the testicular vasculature (99, 100, 101). Cadmium does appear to have some direct action on the seminiferous tubules, possibly causing a breakdown of the blood-testis barrier (246). The effects of cadmium can be blocked with zinc, selenium and BAL (British Anti-Lewisite) (94, 99, 102, 103, 104, 105). Although some studies in monkeys have suggested that recovery of the germinal cells may occur (1061, it is agreed in most species that destruction of the germinal epithelium by cadmium is permanent. From a toxicological point of view (renal toxicity, hypertension),the use of cadmium salts as such in ma" are precluded. 2.2.3

Interference

with the Hormonal Milieu:

Hypothalamic Level. The identification and synthesis of LHRH (al has enabled investigation of the role of this factor in the control of reproductive function. Recently, evidence suggests that receptors for LHRH exist in the gonadotrophin secreting cells of the anterior pituitary, similar to those demonstrated for TRH (234). The possibility exists, therefore, of identifying compounds capable of interfering with this hormone-receptor interaction leading to disruption of reproductive function. While many analogues of LHRH have been synthesized, testing to date has been confined to assessing their activity in terms of LHRH and no information is available regarding their ability to competitively block the release of hLH Unfortunately, a specific releasing hormone for and hFSH by LHRH. hFSH has not yet been characterized and attempts to competitively inhibit LHRH with a view to suppressing spermatogenesis are highly likely to be associated with loss of libido and potency due to This objection is also cessation of testosterone production. applicable to future attempts to interfere with LHRH activity by immunization procedures against LHRH.

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Interference

(b) -

Inhibition

with Gonadotrophin

Release and Action.

of Release by Steroids.

(i) Oestrogens: It has been known for many years that oestrogens have the capability of inhibiting pituitary gonadotrophin release (107, 108) and the recent studies using radioimmunoassay techniques have established that oestrogens have the ability to suppress both LH and FSH. In general, the suppression of LH is more rapid and profound but the effect on FSH is slower in onset but The combined suppression somewhat more prolonged (109). of FSH and LH produces inhibition of spermatogenesis as well as atrophy of interstitial cells and accessory sex glands in animals (76). In man, Heller et al. (110) demonstrated azoospermia following treatment with 450 pg per day of ethynylestradiol-3-methyl ether but this was accompanied by decreased libido and potentia together with the universal occurrence of painful gynaecomastia. While it is possible that the diminished libido and sexual potency could be improved by concomitant androgen replacement, the occurrence of symptomatic gynaecomastia and other metabolic side effects such as those demonstrated in women receiving oral contraceptive preparations, make it unlikely that oestrogens will be accepted in formulations designed to interrupt male fertility. (ii) Progestagens: Several investigators have studied the anti-spermatogenic activity of a group of 19-nor-progestagens and progesterone in rats and in man (76, 111, 112). In the study reported by Heller et al. (110, 113),relatively high doses of progesterone (50 mg daily), 170(-ethyl-19-nortestosterone, 17ðynyl-19-nortestosterone and 170C-ethynyl-17-hydroxyestren-3-one (15 mg twice daily) were used and produced azoospermia in 4 to 10 weeks. In the men who received the "19-nor" preparation,a loss of libido and sexual potency in association with the appearance of painful gynaecomastia occurred. Recovery of spermatogenesis took place on cessation of these agents and semen analyses returned to normal within 6 months. While specific techniques for measurement of hFSH and hLH were not available at that time, the loss of libido and spermatogenic inhibition indicates suppression of both gonadotrophic hormone secretion. More recently, MacLeod (114) has reported that B-medroxyprogesterone acetate (Depo-Provera) is capable of causing spermatogenic suppression without any feminizing effect or loss of libido. In the second study (76) using this agent, azoospermia was not achieved and the modest spermatogenic suppression was not prolonged.

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The possibility of combining progestagens with androgens to achieve synergism and to prevent diminished libido has also been proposed. Studies in rats by Terner and MacLaughlin (115) suggest that treatment with such combinations produces depression of spermatogenesis without suppression of libido. In this regard, 100 or 500 pg of d-l norgestrel is capable of suppressing spermatogenesis in the rat and at higher doses inconsistently in the rhesus monkey (116). To date studies of this compound alone or in combination with androgens has not been explored. Alternatively,it may be possible to combine long-acting progestagens such as 6-medroxyprogesterone acetate or 17-hydroxyprogesterone caproate with long-acting esters of testosterone for use as injectable depot compounds capable of suppressing spermatogenesis. During the preparation of this report, preliminary results of the suppression of spermatogenesis in man with gestagen-testosterone combinations has been reported and will serve as a guide to further investigations (247, 248, 249). (iii) Androgens: The capacity of testosterone to suppress FSH and LH levels in plasma has been amply documented in recent studies using radioimmunoassay techniques to measure the gonadotrophic hormones (62, 63). As with oestrogens, the suppression of LH with testosterone is more rapid and profound than that of FSH. Unfortunately the effects of testosterone on spermatogenesis are somewhat confused because many of the early experiments were performed in the rat. Thus,spermatogenesis in the hypophysectomized rat can be maintained with testosterone alone (24, 117), a phenomenon not known to occur in man. It is likely that the effective local peritubular concentration of testosterone achieved is the important factor determining whether testosterone will support spermatogenesis. In man, Heller et al. (118, 119) demonstrated that 25 mg testosterone propionate daily, administered intramuscularly, would suppress spermatogenesis without altering libido and sexual potency. Recovery of spermatogenesis occurred within More recently,200 mg 5 to 6 months after ceasing treatment. of testosterone enanthate given weekly to normal men has been Similar shown by Heller et al. (120) to cause azoospermia. results have been obtained by MacLeod(ll4) using 250 mg of testosterone enanthate weekly. The high doses of testosterone that are necessary may interfere with lipoprotein metabolism and in certain men cause erythrocythaemia. These effects may prevent their use in male contraception but if synergism can be demonstrated with progestagens,then lower doses may be effective in combination contraceptive agents in the male.

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A derivative of ethinyl testosterone, Danazol (170(pregn-4-en-2@yno-(2,3-d) isoxazol-17-ol),has been shown to produce decreases in plasma testosterone levels when Despite the fall in administered to normal men (121). testosterone levels at low doses (200 mg per day),plasma LH levels remain unchanged, but at higher dosage (600 mg Although no per day),suppression of LH resulted. consistent change in sperm count was noted, 2 men receiving 600 mg per day of this compound showed a decrease in sperm count. Further studies of this compound in combination with testosterone are currently underway to determine the effectiveness of this compound in suppressing spermatogenesis (122). -

Non-steroidal

Gonadotrophin

Inhibitors.

Several non-steroidal compounds are known to be able to inhibit either the secretion of gonadotrophins or to decrease These compounds include: (i) l-Co
Interference

with Gonadotrophins

by Immunization.

A number of investigators have studied the effect of active immunization of males from a number of mammallan species with an heterologous source of LH and noted disruption of spermatogenesis and a variable effect on libido (128, 129, 130). Talaat and Laurence (131) found that a remarkable loss of libido occurred following immunization of rabbits with bovine LH and that this was accompanied by exfoliation of Immature germinal cells from the seminiferous epithellum. However,their studies (131, 132, 133) did not demonstrate any effect on spermatogenesis following immunization with FSH and raised the question of the role of FSH in testicular function. Furthermore, Turner and Johnson (134) demonstrated the onset of sterility in rats l-2 weeks after treatment with antisera to FSH but could show no effect on spermatogenesis. They claim that this effect may result from changes observed in the cells of the cauda epididymis. It seems clear that further studies are necessary in this area "sing highly purified FSH and LH as immunogens to define their role in testicular function and to establish whether interruption of spermatogenesis without interference with libido is possible using immunological methods.

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(cl

Interference

testosterone

and

interstitial

cells

undefined.

The

with

Steroid

possibly

other

influence

Action. steroid

As

spermatogenesis

possibility

discussed

compounds

of interfering

earlier,

secreted

by the

by mechanisms

as yet

with

this

steroidogenic

action as a means of limiting or disrupting spermatogenesis needs to be explored. Two methods are currently available for inhibiting this action: (i) the use of antisera to testosterone developed either

passively

interfere

with

or actively; the binding

(ii) the use of antiandrogens that of testosterone to specific receptor

sites. -

Antisera

to testosterone.

Neri et al. (135) have shown that the administration antisera to testosterone can block the activity of testosterone

given

to castrated

have shown that sterone-protein

rats.

Nieschlag

et al.

of

(136)

active immunization of rabbits with testoconjugates can result in interstitial cell

hyperplasia and grossly elevated, presumably protein bound, testosterone levels. It seems likely that the development of testosterone antibodies resulted in greater testosterone binding,

thus

reducing

the interstitial

cells

free

testosterone

via LH secretion.

levels

and

No defect

stimulating was

Further studies using noted in the seminiferous tubules. passive immunization are necessary as it is doubtful whether a significant intratesticular decrease in testosterone levels can be achieved -

by active

immunization.

Antiandrogens. The use of antiandrogens

to inhibit

spermatogenesis

by

interference with the action of testosterone has been achieved by the use of cyproterone. Rats, given 40 mg per day over a S-week period showed atrophy of the necessary sex glands, hyperstimulation of interstitial cells and spermatogenic Similar changes impairment associated with infertility. can be produced in rats with cyproterone acetate in doses of 10 mg per day but some regression in the interstitial cells was noted (137, 138). However, in man, 200 mg of cyproterone for 90 days

causes

no alteration

in spermatogenesis

(139).

With cyproterone acetate,however,azoospermia has resulted probably due to the antigonadotrophic activity of this compound in the doses used (140). However, Morse et al. (141) recently reported that cyproterone acetate in doses of 200 mg per day caused decreased spermatogenesis without altering FSH and LH It is doubtful whether these compounds will be of levels. use in man due to alterations in libido and sexual potency. The identification and definition of intratubular steroid requirements together with a characterization of receptors for these compounds may lead to a more specific blockade which did not interfere with the action of testosterone on libido.

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2.3

Future Possibilities

There is little doubt that further investigation of the areas already reviewed may yield methods of interfering with spermatogenesis which will be suitable for clinical use. In considering the recent advances in male reproductive physiology and the fields where further knowledge is required, several theoretical methods of disrupting spermatogenesis suggest themselves. 2.3.1 Specific inhibitors of meiosis: Considerable work is necessary to understand the environmental requirements to permit or induce meiosis. It seems likely that the gonads offer a unique environment,perhaps created in the testis by the presence of the blood-testicular barrier,which places the meiotic cells beyond the normal milieu bathing somatic cells. It is possible that unique metabolic requirements of cells in meiosis may allow specific blockade of spermatogenesis at this stage. 2.3.2 Specific inhibitors of FSH secretion: The method by which the testis influences FSH secretion remains one of the most important unsolved problems of reproductive physiology. There is little doubt that testosterone, estradiol and their derivatives can suppress FSH secretion, but this does not occur without concomitant suppression of LH. There is also evidence from both human and animal studies that increased FSH secretion can occur independently of LH secretion indicating a separate control system. The possibility of identifying a compound capable of exerting an independent suppressive influence on FSH secretion should be pursued from any likely source. The earlier reports of McCullagh (53) suggest that testicular extracts may contain this material, while more recently,rete testis fluid andseminalplasma have been suggested as alternative sources (48, 60). The advantage of such a compound would be the possibility of selectively inhibiting FSH secretion thereby interfering with spermatogenesis without alteration of libido and sexual potency. 2.3.3 Inhibition of binding of FSH and IN to receptors: The identification of specific receptors in the testis for FSH and LH (39, 43) have led to studies of the kinetics of their interaction. The specificity and site of the receptors for each hormone again demonstrates the intratubular role of FSH. Further developments may identify methods and compounds capable of interfering with the binding of FSH to its receptor and permit interruption of spermatogenesis.

3.

3.1

Physiological

INTERFERENCE WITH SPERM MATURATION

Considerations

Introduction: 3.1.1 Spermatozoa released from the seminiferous epithelium at spermiation differ significantly from ejaculated sperm in many parameters such as their morphology, metabolism and fertilizing capacity (for review see Bedford and Hamilton 142, 143). These changes

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are acquired during their passage through the excurrent duct system connecting the testis and the eJaculatory ducts. Knowledge of the detailed sequence of changes that occur during this process of sperm maturation iS not available for any single mammalian species and available data clearly indicate species differences in the rate and site at which these maturational changes occur. 3.1.2 Development of fertilizing capacity of spermatozoa: Studies XI many mammalian species indicate that sperm recovered from the caput epididymls are unable to fertilize ova wheras this ability is developed by the time sperm reach the cauda (for review see Orgebin-Crist and Bedford 142, 250). These findings indicate that maturational changes in sperm continue during their epididymal transit,and due to variations in the rate of passage of sperm, the profile of fertilizing ability at any one region of the epididymis may fluctuate. Interruption of the passage of sperm through the proximal segment of the epididymis does not halt the acquisition of fertilizing ability but is clearly important as many of the ova fertilized by such sperm do not develop normally (142, 146, 147, 148, 250). It IS likely that sc~me aspects of sperm maturation may be intrinsic to the cell but it is clear that others require the epididymal environment (143, 250). A number of 3.1.3 Identifiable changes during sperm maturation: changes in the morphology, motility and metabolism of maturing sperm have been found but no specific feature has been correlated with the development of fertilizing capacity. These changes have been extensively reviewed by Bedford (142) and only pertinent details are considered herein. It has been clearly demonstrated that passage through the epididymis results in the onset of purposeful, directed motile activity by sperm (143 -145, 147, 149, 251). No morphological features have been identified in association with the change in motile status which also has been shown to occur in the human epididymis (142). Significant differences have been noted in the metabolism of sperm obtained from the rete testis, epididymis, vas and ejaculate. The changes in oxygen uptake, gluconeogenesis, lipid differences include The nature metabolism and inositol synthesis (for review see 142, 143). of these differences reflects alterations in the synthetic and energy producing pathways of sperm and the changes are probably associated with the onset of motility and the development of fertilizing capacity. Identifiable morphological changes also occur during maturation but Migration of the the degree of change varies remarkably between species. cytoplasmic droplet and acrosomal changes are the most prominent (150, 151, More subtle changes in the surface of the plasmalemma have been 152, 153). identified by Bedford and others (see 142) who have demonstrated differences in electrophoretic mobility, surface charge patterns, swelling of the plasma membrane and changes in agglutination pattern of spermatozoa as they pass Furthermore,differences in the quality and through the epididymis (142). the stability of the chromatin,to agents capable of disrupting weak and S-S bonds, have been demonstrated (154, 155).

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It thus seems clear that demonstrable changes occur in spermatozoa during their transit through the excurrent duct system and it is pertinent to review some aspects of the environment in which the maturation occurs. 3.1.4 Regional differences in the excurrent duct system: Following release from the seminiferous epithelium,spermatozoa are transported to the rete testis partly by contractile activity of the seminiferous tubules (156, 157), and partly by movement of the fluid secreted by the seminiferous epithelium (48). From the rete, several ducts, the ductuli efferentes, convey spermatozoa to a single, highly convoluted ductile organ, the epididymis. In some mammals the ductuli efferentes are convoluted and comprise a portion of the epididymis in conjunction with the duct of the eprdidymis, whereas in others these ducts can be identified separately. These species differences,therefore,make it difficult to equate functionally the major subdivisions of the epididymis, the caput, corpus and cauda epididymis, from one species to another (158). Several investigators, on the basis of histological changes, have attempted to subdivide the epididymis further but inter-species comparison of these areas is extremely difficult (143, 159). Of these subdivisionsythe initial segment, that portion into which the ductuli efferentes empty,ls the most easily identifiable region and appears to have a higher blood flow than anywhere else along the epididymis (160). 'The cauda epididymidis merges and continues as the "as deferens and from there via the ampulla of the "as to the ejaculatory duct. Recent ultrastructural studies have extended the earlier light microscopic investigations and have defined regional differences in the rete testis and epididymis. These cytological details have been extensively reviewed by Hamilton (143, 158) for mammalian species and by Holstein (161) for man. It is clear that several cell types can be identified and their distribution and form, contribute to the regional cytological differences in the epididymis. While the ultrastructural features of these cells are indicative of considerable metabolic activity, investigations to date have not correlated specific metabolic actions with any single cell type. It seems likely that the principal cells may be responsible for the fluid transport that occurs across the epididymal epithelium (158). The microvilli, present throughout the epididymis and "as, seem likely to aid in resorption,and the transport of spermatozoa is probably largely dependent on the smooth muscle layers which are best developed around the "as. 3.1.5

Regional

physiology

of the excurrent duct system:

Rete Testis. It is now thought that primary fluid formed (a) in the tubules mixes with other fluid secreted into the ret@ testis, and then flows as rete testis fluid through the ductuli (vasa) efferentia into the epididymis (162, 163, 164, 48). Analysis of this fluid collected from conscious rams and bulls and anaesthetized rats revealed that it flows at the rate of l-2 ml/testis/hr (ram) or 0.05-0.1 ml/testis/hr (rat). It contains about 1 x lo8 spermatozoa/ ml (ram) or 0.3 x 108/ml (rat) with certain enzyme inhibitors (170) which may serve to "stabilize" testicular spermatozoa during their passage through the efferent and epididymal ducts. The sperm-free

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fluid is potassium-rich, but with less sodium and bicarbonate than plasma, with wh'ich it is isosmotic. It is a low protein fluid, containing certain specific proteins (167, 168, 169),and also contains high levels of free myoinositol (48, 162). It seems likely that the secretion of this fluid is not influenced by gonadotrophins (162, 165, 166) but there is some evidence that FSH, LH and growth hormone may enter rete testis fluid (162). Preliminary investigations by Setchell (48) suggest that an "antigonadotrophic factor" is present in rete testis fluid and the significance of this finding has been discussed earlier in this review. R&e testis fluid contains testosterone in varying concentrations, always greater than in arterial blood but less than in testicular venous blood. The varying levels recorded may be natural, due to fluctuating rates of secretion, or associated with the techniques of collection and analysis. Nevertheless, together with the presence of testosterone binding protein, this fluid represents a rich source for androgen to gain access to the caput epididymis through the ductuli efferentia (252, 253, 254, 255). Epididymis. The results of a number of investigators support (b) the conclusion that approximately 90% of fluid produced in the testis is reabsorbed in the proximal portion of the epididymis (171, 172). The results of micropuncture studies by Levine and Marsh (164) have established a progressive fall in sodium concentration during progression down the epididymal duct and their measurements of osmotic pressures indicate that an anion gap develops which is suggestive of epididymal secretion. In addition to fluid exchange, particular matter introduced into the epididymal lumen is absorbed by the epithelium of the ductule efferentes and all subsequent segments of epididymis and vas deferens (173, 174, 175). However,the sequestration of degenerating sperm has only been demonstrated following use of the drug U5897 (143). While several unusual metabolites are known to be secreted by the epididymis, little is known of their cellular source or function. Dawson & Rowlands (176) demonstrated the secretion of glycerylphosphorylcholine (GPC) by the epididymis and further studies using 32P identified The role of this site as the source of GPC found in semen (177). GPC remains obscure although suggestions have been made that it maintains the luminal osmotic pressure as sodium is absorbed (143). Similarly high concentrations of carnitine are found in the rat epididymis and these levels fall following castration but recover with testosterone replacement (178, 179). Again,the cellular source and specific role of carnitine remain obscure.

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There 1s good evidence which demonstrates the secretion of glycoproteins such as sialic acid by the epididymis (180, 181, 182). There is some evidence that the Golgi region of the principal cells forms the carbohydrate components and by analogy with other tissues, the protein complex is probably synthesized by the rough endoplasmic reticulum (183, 184). The function of sialic acid 1s still unknown and several hypotheses have been advanced suggesting it is a compound lowering friction between millions of spermatozoa, as representing sperm coating antigens and that it may be responsible for altering the electrophoretic mobility of sperm during their epididymal passage (143). More recently, the epididymis has been shown to be capable of steroid biosynthesis, producing testosterone from various precursors This work has been confirmed by other investigators (185, (143). 186), but the extent of its contribution to steroids found in It is also of interest in view epididymal fluid remains unclear. receptors of the demonstration of specific 5-0(_dlhydrotestosterone in the epididymis (187, 256). However, it seems paradoxical that the steroid biosynthetic activity of the epidldymis is dependent on activity 1s lost testicular steroid synthesis as the epididymal following castration and regained after replacement with testosterone (190, 191, 192). The steroid metabolic activity of the epididymis is highest proximally and declines in the cauda and vas (143) but the relationship of this activity to sperm maturation remains unknown. The dependence of the epididymis on testosterone secretion by the testis has been clearly established (188, 189) and recent studies indicate that sperm fertilizing ability is rapidly lost following hypophysectomy or castration and is x-e-established after androgen replacement (193, 257). Comparable effects have been produced with antiandrogens, fertility disappearing within 18 days of commencing therapy (258). Similarly,the fertilizing ability of sperm is dependent on the maintenance of a temperature below that of body temperature but it is uncertain in most mammalian species how this effect is mediated (142). Isotopic labelling has enabled study of the rate of passage of sperm through the epididymis and although some variation is present, there is remarkable inter-species similarity in the duration of epididymal transit (reviewed by Bedford 142). In man, the mean time is approximately 12 days (194). While considerable ranges are noted for the duration of epididymal transit, frequent ejaculation can only reduce the total time by lo-20% (142). The passage of sperm is facilitated by the peristaltic contractions of the epididymal musculature but this activity decreases in the cauda (189). Evidence reviewed by Bedford (142) indicates that sperm can be stored in this region for several weeks and still retain their fertilizing ability.

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summary: It is clear that sperm acquire fertilizing ability during passage from the testis to the caudal pole of the epididymis. This is accompanied by the development of motility and certain morphological modifications. There is some evidence that the epididymal environment is necessary for optimum normal maturation of sperm but the nature of this influence is obscure. Further studies are clearly necessary to establish the role played by the epididymis in sperm maturation especially in man. It is also necessary to investigate further the regional changes in the epididymal environment and the processes by which any differences are maintained. 3.2

Currently Available

Agents

is available currently for "se in man that is capable of No agent modifying the epididymal environment to render sperm infertile. The theoretical requirements of such a compound are that it should induce infertility rapidly yet fail to disrupt spermatogenesis and interstitial cell function. There are two compounds which are possibilities and they are briefly considered.

for use in this manner

3.2.1 Antiandrogens: Cyproterone and cyproterone acetate: These compounds have been investigated in mammalian species and have been shown to have antiandrogenic properties, i.e. they are capable of directly inhibiting androgen action at a cellular level (137). However,in considering the action of cyproterone acetate,it is important to realize that it has progestational activity and is thus capable of inhibiting gonadoThe results of many investigations trophic secretion by the pituitary gland. have established that cyproterone is capable of inhibiting the action of testosterone both centrally and peripherally,leading to interstitial cell stimulation and atrophy of the accessory sex glands (137, 138). This was associated with regression of spermatogenesis and a reduction of pituitary gonadotrophic content (137). In its application to fertility control, Prasad et al. (195) have capsules, suggested that,at low dose (230 pg/day) released fromSilastic cyproterone acetate is capable of interfering with the epididymal environment resulting in loss of sperm fertilizing ability within 10 days. This was associated with no change in testis, prostate and seminal vesicle weight. They demonstrated that sperm were non-motile and that the epididymal secretion of sialic acid was reduced (196). thus RauschConflicting results are available from studies in man; Strooman et al. (139) could demonstrate no changes in spermatogenesis However, in normal men treated for 90 days with 200 mg day of cyproterone. using cyproterone acetate in doses of 200 mg / day,Laschet & Laschet (140) reported that azoospermia occurred in the first few months of treatment but The difference in the this was associated with impairment of libido. results "sing these compounds may be presumably attributed to the progestaIt seems clear that further studies tional activity of cyproterone acetate.

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are necessary to document whether the differential epididymal demonstrated by Prasad et al. (195) in the rat, is applicable

action to man.

This simple 3.2.2 0(-chlorohydrin (3-chloropropane-1, 2-dial): monochloro-derivative of glycerol has been shown to produce sterility in This effect is due to an rats, guinea pigs and monkeys (197, 198, 199). The activity of the compound unidentified action on epididymal sperm. is determined by the chlorohydrin groupifig as bromhydrin derivatives were Furthermore,they demonstrated that the chlorohydrin group inactive (200). should be adJacent to a carbon bearing a hydroxy group. Most of the tichlorohydrin is excreted unchanged in urine and high doses were shown to have an antispermatogenic acxion (197). It has been demonstrated that this compound produces obstruction and spermatocoele formation in the epididymis when given at 2-3 times the minimal effective dose (198). However,at the lower dose,lt is proposed that vascular changes occur in the epididymis which interfere with sperm maturation. The selective epididymal action may be mediated by altered lipid metabolism or by the formation of chlorinated phospholipids (197, 199). Jackson (75) proposes that c&hlorohydrin acts as an alkylating agent but in contrast to another compound with similar activity, methylene dimethane-sulphonate, does not induce dominant lethal mutations (201). During trial of the efficiency of b(-chlorohydrin in rhesus monkeys, side effects due to bone marrow depresslon were noted in 5% of animals at the dose used (30 mg,/kg/day orally). This resulted in death of 2 of the 6 animals in the trial; however,it should be noted that the dose used was close to the lethal dose in rats and lower doses (2.5 mg/kg/day) did not result in toxicity (199). While bone marrow toxicity limits the use of these specific compounds in primates, the simplicity of this group of compounds and the site of their antifertility activity requires further intensive investigation. 3.3

Future Possibilities

No agents other than those discussed appear to be available for immediate application in the post-testicular control of sperm fertilizing capacity. However,the theoretical advantages of such a method of control make it advisable to stimulate further research designed to understand the intricaclev of post-testicular sperm maturation and the effects of the epididymal environment on this process. Though difficult to execute, studies in man should be pursued to document the interaction of spermatozoa and the epldidymis in relation to the acquisition of fertilizing ability.

4.

4.1

INTERFERENCE WITH THE TMNSPORT

Physiological

OF SPERMATOZOA

Considerations

The interruption of sperm transport presents a loglcal means of preventing fertilization of the ovum. This can clearly be prevented

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following eJaculation by the use of barriers such as the condom in the male or cervical devices in the female. It can also be achieved by interruption of the excurrent duct system. The structure and physiology of the epididymis has already been briefly reviewed. The transport of spermatozoa continues along the "as deferens which shows many features in common with the epididymal epithelium. The principal cells seen in the epididymis are also present in the "as but are increased in height and studded by microvilli. The clear cells are not present but a further cell type, the pencil cell,is seen (143). The "as deferens differs from the epididymis by the presence of a thick,well organized and richly innervated smooth muscle coat acquired by the "as deferens. In comparison to the proximal portion of the epididymls where adrenergic terminals are sparsely distributed and principally found around blood vessels, the cauda epididymis and "as deferens exhibit rich networks of sympathetic fibres terminetlng directly in muscle layers (202, 203, 204, 205). The terminals contain norepinephrine and are associated with short postganglionic adrenergic neurones (202, 205),an arrangement which Bedford (142) suggests may allow more precise regional control of tubular contraction at orgasm and ejaculation. The fate of uneJaculated spermatozoa has been studied in a number of species and conflicting results have been obtained. The results of some studies have led to suggestions that various regions of the epididymis and "as are capable of phagocytosing UneJacUlated spermatozoa (206, 207, 208). The ampullary region of the "as was Implicated in studies using 32P-labelled spermatozoa in the bull (209). Ilowever, very few spermatozoa or their organelles have been ldentlfied within phagocytic vacuoles in the epithelium Recent of the excurrent duct system using the electron microscope (142). studies in the monkey after long-term vasectomy have explained this puzzling fact by showing that sperm are removed by macrophages in the ductuli efferentes (259). The opposite view point suggests that spermatozoa escape following nonorgasmic contractions of the duct either into urine In the ram or in association with preputial gland secretion in the rat, during sleep (210, 211, 212). Spontaneous ejaculations have also been noted in the guinea pig (213) and occur nocturnally in the continent human male. The studies by Line Aet al (210) indicate that in the ram the recovery of spermatozoa from urine accounts for the daily sperm output from the testis. 4.2

Available

Methods

The srmplest, effective method of interrupting sperm transport III the male consists of the use of mechanical barriers such as the condom which will not be considered further. The surgical interruption of the "as deferens forms a commonly used and increasingly popular method of sterilization in the male (214). The prlnciPa1 disadvantage of the method is that of permanency as the success of 'There is no reversibility varies markedly and cannot be guaranteed.

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doubt about the effectiveness of vasectomy if it is realized that sterility is not instantaneous and first requires the dissipation of stored spermThe frequency of coital activity following vasectomy increases atozoa. the rate of development of azoospermia (215) which can be speeded up by irrigation of the distal end of the duct at the time of the operation (216). The operative procedure 1s simple, it can be carried out under local anaesthesia and the operative procedure can vary from excision of several centimetres of the duct, to simple incision, fulguration and oversewing The operative complications of the fascial sheaths (217, 218, 219, 220). occasionally include haematoma formation or infection but are rarely serious. The long-term effects of vasectomy have been consldered recently with emphasis on the fate of spermatozoa, changes in the immune status of the patient and the possible psychological sequelae. Until recently little was known concerning the state of the testis and the fate of sperm produced therein. The recent ultrastructural study of the testis in vasectomized rats demonstrated no significant detectable alterations in the cytology of While no data to this effect are the seminiferous epithelium (221). available in man, examination of testicular biopsies from men with epididymal obstruction show an apparently normal seminiferous epithelium. It is likely therefore that sperm production continues normally following vasectomy. Experimental studies in the ram have demonstrated that following ligation of the vas, the epididymal duct became distended and Invariably ruptured forming several granulomata at these sites (222). Similar results are found with ligation of the vas in hamsters, rabbits and rhesus monkeys (142) and the sites of granulomata formation allow Ingress of leucocytes which are capable of phagocytosing sperm organelles. Studies In the rhesus monkey suggest that the "as efferentia are a major site of sperm reabsorption (259). Flickinger (223) demonstrated that granulomata formation occurred in most rats following vasectomy and his ultrastructural studies provide evidence of phagocytosis of sperm by epithelial cells of the cauda epididymis. The formation of granulomata and ingress of phagocytic cells presumably accounts for the immunological reaction noted in some men following vasectomy. This reaction consists of the development of circulating auto-agglutinins to spermatozoa and was noted in men following vasectomy and in patients with obstructive azoospermia (224, 225). It has been shown that about 2% of normal men showcirculating autoantibodies to sperm and this rises to between 3%-6% following vasectomy (226, 235). The significance of these autoantibodies remains unclear as fertility has been reported in man exhibiting this phenomenon in whom obstructive azoospermia had been surgically corrected (227). Whether the presence of these antibodies influences fertility in men following reanastomosis of the vasa must remain in question. Autoantibodies have more recently been shown to occur in the rat following excurrent duct obstruction (228). It seems clear that further studies, preferably in man or other primates, are necessary to define the significance of these immune phenomena and to establish the fate of unejaculated spermatozoa. The psychological response to vasectomy has been retrospectively analysed in a number of different countries by differing methods. Using questionnaires,

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such studies indicate acceptance in 90-9% of subjects while l-3% reported deterioration in their sexual performance and would not have the operation again (226, 229, 230). However,more recent prospective studies have noted some adverse changes in marital satisfaction several years after vasectomy (231, 232, 233). The study used interview, questionnaire and testing procedures to follow 42 couples for 4 years following vasectomy, and compared their responses to 39 couples using oral contraceptives. The reactions were interpreted as indicating that the couples assume that vasectomy had a demasculinizing potential and that the male was scrutinized for such evidence. It was noted that the frequency of coitus increased and temporary impotence occurred in some men in this high-frequency coitus The investigators inferred that this represented overreaching of group. sexual capacity to confirm masculinity. Clearly further prospective studies are necessary before these findings can be considered conclusive. Furthermore,methods need to be improved so that identification of couples likely to react in this fashion can be achieved before vasectomy is recommended. The lack of other suitable methods for long-term control of fertility in the male has led to a wide acceptance of vasectomy in developed and developing countries. The relative permanence of the procedure has been emphasized and the success of attempts to reverse the procedure depend on the original technique used. 4.3

Future Possibilities

In this field,future possibilities may result in the development of The possibility silastic barriers which may be removed at a later time. of some type of specific neurological blockade has also been suggested but would require some unique property in the innervation of the vas to be demonstrated.

5.

CONCLUSIONS

It is evident from this review that many details concerning fundamental Many processes in male reproductive physiology remain to be elucidated. sites have been identified which may logically provide means of interrupting male fertility,but in many of these areas,considerable research is necessary In considering endocrine to more critically assess the possibilities. manipulations designed to interrupt fertility, the known close physiological association of the gonadotrophic hormones, hFSH and hLH, makes disruption of spermatogenesis difficult without effects on libido and sexual potency. Consequently,research should actively be supported to identify specific inhibitors of FSH secretion, specific FSH receptor sites and specific actions of FSH on spermatogenesis. The seventyday developmental cycle of spermatogenesis in man means that failure to take an effective agent for a few days is unlikely to lead to It is thus equally likely that antispermatogenic restoration of fertility. agents,when developed,will provide exceptionally effective methods of contraception.

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Logically, post-testicular interruption of sperm maturation has many advantages and is less likely to interfere with libido and sexual However,detailed understanding of this process is poor, potency. The identiespecially in man, and requires further intensive research. fication of compounds such as the chlorohydrins may lead to less toxic but equally effective agents. In the short-term,it seems that the condom and vasectomy will remain However,the possibility the major methods of controlling male fertility. of using androgen-gestagen combinations to disrupt spermatogenesis has possibilities for short-term development. New pharmacological compounds should be sought diligently and should Promising compounds must be assessed be exhaustively tested and evaluated. fully, including the use of specific radioimmunoassay techniques for evaluating their effects on the pituitary-gonadal axis.

ACKNOWLEDGEMENTS

The author wishes to thank Drs J.M. Bedford, D.W. Hamilton, D.J. Patanelli and B.P. Setchell for providing reviews, some of which are still "In Press", in their areas of expertise. These reviews provide valuable perspectives and greatly facilitated the preparation of this review. A grant-in-aid was made available by the World Health Organization to provide the secretarial assistance necessary in compiling this review.

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