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| ESTROGENS AND MALE REPRODUCTION Chapter 17 - Rochira Vincenzo, Madeo Bruno, Diazzi Chiara, Zirilli Lucia, Santi Daniele and Carani Cesare Unit and Chair of Endocrinology & Metabolism, Department of Medicine, Endocrinology & Metabolism, Geriatrics, Azienda Unità Sanitaria Locale of Modena and Department of Biomedical, Metabolic and neural sciences, University of Modena & Reggio Emilia Revised: 6 February 2013 |
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From an historical perspective, the role of estrogen in the human male has been investigated since 1934 when Zondek et al. documented estrogens production in the male, through the discovery of the intratesticular conversion of androgens into estrogens in male stallions (1,2). The demonstration of estrogen production in human male was given only several years later by MacDonald et al. (3). Afterwards, several studies followed year after year in order to expand the comprehension of estrogen's role in men (4,5), leading however, not fully understood the role of estrogens in the physiology of male reproductive tract (6,7).
In the past, a role for estrogen action in the male reproductive system was proposed based on scattered data (8,9). Recently, the acquisition that estrogens regulate several function in men, also including human reproduction (2,10), was given by the development of molecular biology. Thus, allowing detailed characterization of estrogen receptors' structure and function (11), and the discovery of genes involved in estrogens synthesis (12). Furthermore, other contributions to this knowledge were allowed by the development of male transgenic mice lacking of functional estrogen receptors or functional aromatase enzyme (13), and by the discovery of mutations in both the human estrogen receptor alpha (14) and aromatase (15,16) genes. Nowadays, the presence of estrogens in the human testis is well documented (17), and all previous studies definitively changed some classical standpoints in endocrinology, providing evidence that estrogens also exert a wide range of biological effects in men and not only in women (18,19).
In males, estrogens derive from circulating androgens. The key step in estrogen biosynthesis is the aromatization of the C19 androgens, testosterone and androstenedione, to form estradiol and estrone, respectively. This step is under the aromatase enzyme control (Figure 1).
Figure 1. Biochemical pathway of testosterone conversion into estrogen in men
The aromatase enzyme is a P450 mono-oxygenase enzyme complex (12) present in the smooth endoplasmic reticulum, which acts through three consecutive hydroxylation reactions, whose final effect is the aromatization of the A ring of androgens (12,20) (Figure 1). This enzymatic complex is composed of an ubiquitous and non-specific NADPH-cytochrome P450 reductase, together with the regulated form of cytochrome P450 aromatase (17). The conversion of androgens in estrogens takes place mainly in the testes, adipose tissue and muscle tissue (Figure 1).
The P450 aromatase derive from the CYP19 gene: a gene of 123 kb of length, which consists of at least 16 exons and is located on the long arm of chromosome 15 in the q21.2 region in humans (4,12,20) (Figure 2).
Figure 2. Schematic representation of the human aromatase gene.
Circulating estrogens are mainly reversibly bound to sex hormone binding globulin (SHBG), a β-globulin, and, to a lesser degree, to albumin. Estrogen action is mediated by the interaction with specific nuclear estrogen receptors (ERs), which are ligand-inducible transcription factors regulating the expression of target genes after hormone binding (19,20). Two subtypes of ERs have been described: estrogen receptor α (ERα) and the more recently discovered estrogen receptor β (ERβ) (20). The human gene encoding for ERα is located on the long arm of chromosome 6, while the gene encoding for ERβ is located on band q22-24 of chromosome 14. The two ER proteins have a high degree of homology at the amino acid level (Figure 3).
Figure 3: Estrogen receptros and its products
While it is clear that estrogens regulate transcription via nuclear interaction with their receptors, a non-genomic action of estrogens has been recently demonstrated, suggesting a different molecular mechanism accounts for some estrogen actions (20). In vitro studies showed a very short latency time between the administration of estrogens and the appearance of biological effects. These actions are thought to be mediated through cell-surface receptor, a G protein-coupled receptor (GPR30), which are not believed to act through a transcriptional mechanism (17,20). Recently, immunohistochemical analysis in murine tissues found GPR30 in the male reproductive tract, including testes, epididymis, vas deferens, seminal vescicles and prostate (21). The different types of estrogen action are summarized in Table 1.
Table 1. Estrogen actions and related biomolecular pathways and mechanisms.
| Estrogen Actions | Receptors |
Mechanism |
Final effect |
Features |
Genomic |
ERα |
Transcriptional: nuclear interaction with estrogen-responsive elements |
Modulation of estrogen target gene expression |
Slow effects (minutes or hours) |
ERβ |
Transcriptional: nuclear interaction with estrogen-responsive elements |
Modulation of estrogen target gene expression |
Slow effects (minutes or hours) |
|
Non Genomic (cell membranes actions) |
Estrogen receptors on cells membrane (GPR30) |
Cells membrane changes |
Changes in ionic transport through cell surface |
Rapid effects (seconds) |
Aromatase enzyme and ERs are widely expressed in the male reproductive tract both in animals and humans, implying that estrogen biosynthesis occurs at this site and that both locally produced and circulating estrogens may interact with ERs in an intracrine/paracrine and/or endocrine fashion (20). Today, it is clear that not only testicular somatic cells, but also germ cells constitute a source of estrogens in human (17). The concept of a key role for estrogen in the male reproductive tract is strongly supported by the ability of the male reproductive structures to produce and respond to estrogens (22). The distribution of both ERs and aromatase in the male reproductive tract of both animals and humans is summarized below, accounting also for different developmental stages of maturation since both ERs and aromatase are widely expressed at all stages of testicular development, at least in rodents.
Aromatase and ERs are found at a very early stage of development in the rodent testis, thus suggesting a role for estrogens in influencing testicular development (2,23-26).
Leydig cells in fetal rodent testis express ERα before androgen receptor. Moreover, ERα is abundant in the developing efferent ductules, which are the first male reproductive structures to express ERs during fetal development (27,28). Furthermore, also epididymis express ERα in the fetal rodent. By contrast, it is unclear whether ERα is present within the seminiferous tubules of the fetal testis, with variable results reported (22,26).
Also, ERβ is early found in developing testis, particulary in gonocytes, Sertoli cells and Leydig cells, with the gonocytes showing the highest expression suggesting a role for estrogens in their maturation. In addition, ERβ is expressed by rat Wolffian ducts, the structures from which the efferent ductules and epididymis arise (22,26). Actually, ERα is widely expressed in efferent ductules from fetal life to adulthood, implying a crucial role in male reproduction (28); on the other hand, ERβ is mainly expressed during fetal life, suggesting a major role in development of male reproductive structures until birth (22,24).
Table 2. ERs and aromatase distribution in the rodent fetal testis
|
ERα |
ERβ |
Aromatase |
Leyding cells |
++ |
++ |
+ |
Sertoli cells |
- |
++ |
++ |
Gonocytes |
- |
+++ |
- |
Seminiferous tubules |
+/- |
+ |
+ |
Ducts |
+ |
+ |
- |
ERα is expressed in the Leydig cells of both adult rats and mice (29) but not in Sertoli cells, and it is mainly expressed in the proximal (rete testis, efferent ductules, proximal epididymis), rather than in the distal (corpus and cauda of the epididymis, vas deferens) reproductive ducts (22). Furthermore, it has been immunolocalized to ciliated and non-ciliated cell nuclei of the epithelium (30,31). This peculiar distribution explains several important estrogen actions in the proximal ducts, especially the efferent ductules, in which estrogens are involved in fluid reabsorption from the efferent ductules, small and convoluted tubules connetting rete testis to the epididymis (28). Finally, the full-length form of the ERα was recently detected in purified rat germ cells, using a specific antibody directed against the C-terminal region of the protein (32) (Table 3).
Table 3. ERs and Aromatase distribution in the adult rodent testis.
|
ERα |
ERβ |
Aromatase |
Leyding cells |
+ |
+ / - |
+++ |
Sertoli cells |
- |
+ |
+ |
Germ cells |
+ |
++ |
++++ |
Efferent ductules |
++++ |
+ |
+ |
ERβ is expressed in Leydig and Sertoli cells in adult rodents (22,26,28) and in primate germ cells (33); furthermore, it is expressed also in epithelial and peritubular cells (30,31). However, up to now the ERβ presence in germ cells remains debated. Accordingly, there are some controversies about ERβ; localization, with immunohistochemical studies showing some discrepancies, possibly due to methodological differences. Finally, ERβ; seems to be involved in the regulation of gonocyte multiplication, which is under the influence of growth factors and estradiol (13).
Rodent Leyding cells showed an aromatase activity higher than Aromatase activity in Sertoli cells (34). Aromatase is also expressed at high levels in germ cells throughout all stages of maturation, and its expression seems to increase with germ cell maturation to spermatid. Aromatase mRNA expression and activity, in fact, are proven in both rats and mice germ cells from the pachytene spermatocyte stage, and during their subsequent maturation into round spermatids (17,26,28,34) (Table 3). Carreau et al. had demonstrated that aromatase activity in germ cells were more than 50% of that of the whole testis (17). This intensive activity suggests that germ cells, when compared with Leydig cells, may be a major source of estrogen in adult rodents (17,26,28,34) (Table 3). Specifically, when fully developed spermatids are released from the epithelium, aromatase remains in the residual body and it is subsequently phagocyted by the Sertoli cell. Some aromatase activity is present in the cytoplasmic droplet attached to the flagellum as the sperm make its way through the epididymis, suggesting that mature spermatozoa are able to synthesize their own estrogen as they pass through the efferent ducts (17). The ability to synthesize estrogen gradually decreases as the droplet slowly moves to the end of the tail during epididymal transit until it's finally lost. The demonstration of aromatase in sperm is important as it suggests that the sperm itself could control the levels of estrogen present in the luminal fluid, directly modulating functions such as the reabsorption of fluid from the efferent ductules (28).
Finally, for what concerns etrogens non genomic activities, it was demonstrated the presence of GPR30 expression in a mouse spermatogonial cell line GC-1 (35) and in rat pachytene spermatocytes (32).
Both ERs are present in human testis and reproductive tract (17,28). In the male fetus both ERβ and aromatase are expressed in Sertoli, Leydig and germ cells from 13 to 24 weeks, whereas ERα expression is absent (36). Furthermore, ERβ immunoreactivity has been shown in the epididymi,s thus suggesting a role for locally produced estrogens mediated by ERβ, probably involving both autocrine and paracrine mechanisms. The importance of estrogens for the prenatal development and function of male reproductive structures is certain (36).
Aromatase and ERβ, but not ERα are expressed already during the prepubertal period in men (37).
In adult men ERα is expressed only in Leydig cells, while ERβ has been documented in both Leydig and Sertoli cells and in the efferent ducts (38) (Table 4). The presence of ERs in the human epididymis is still a matter of debate (6), even though ERα has been detected in the nuclei of epithelial cells of the caput of the epididymis (39). Both ERs have been identified in isolated immature germ cells (17). Furthermore, they were discovered in mature spermatozoa (40) and in human ejaculated spermatozoa (Aquila et al. 2004). In particular, Luconi et al firstly described (41) an estrogen receptor-related protein in the sperm membrane. This protein is able to bind steroid hormones that may act through a calcium-calmodulin dependent pathway, accounting for a well documented rapid non-genomic action (41). Subsequently, the expression of both ERs in human ejaculated spermatozoa (42) reinforced the concept that estrogens are able to modulate the spermatogenetic process from its onset within the testes, through all the reproductive structures as far as the final process of sperm maturation after ejaculation (2,17,42). The relative localization in human sperm of ERα and ERβ is different, being ERα in the form of a compact zone in the equatorial segment of the upper post-acrosomal region of the sperm head, and ERβ in the mid-piece, at the site of the mitochondria (17). Thus, confirming that probably each type of receptor has a distinct role on the physiology of sperm and in the process of fertilization (43).
Table 4. ERs and Aromatase distribution in the human testis.
|
ERα |
ERβ |
Aromatase |
Leyding cells |
+ |
+ / - |
+ |
Sertoli cells |
- |
+ |
+ |
Germ cells |
- |
+ |
ND |
Efferent ductules |
+ |
+ |
+ |
Of particular interest is the demonstration of differential expression of wild type ERβ (ERβ1) and a novel human variant form of ERβ, arising from alternate splicing (ERβcx, or ERβ2), in the human testis (44,45). ERβ2 expression, which may act as a prevalent negative inhibitor of ER action, was highest in spermatogonia and Sertoli cells in adult men, suggesting that these cells may be "protected" from estrogen action. While, wild type ERβ1 was mostly present in pachytene spermatocytes and round spermatids, which have been proposed to be estrogen sensitive (22), yet ERβ1 was low in less mature germ cells (44). In addition, the discovery of several splice variants of ERβ (including ERβ4) in human testicular cells suggests a specific and more complex estrogen action on spermatogenesis (45).
Aromatase expression in the human testis is present in both somatic and germ cells. Specifically, it is expressed in Leyding and Setrtoli cells (46,47), in immature germ cells, from pachytene spermatocytes through elongated spermatids (26,46), and ejaculated sperm cells (48). Estrogens in sperm locally produced seems to exert a protective action on sperm DNA by preventing sperm DNA damage (49), thus accounting for the estrogen role as survival factor during sperm transit in the seminal way (50). Unlike rodents, aromatase expression in human gametes persists during transit through the genital tracts since P450 aromatase was demonstrated in human ejaculated spermatozoa at three different functional levels: mRNA expression, protein production and activity (42). With this in view, sperm have to be considered a potential site of estrogen biosynthesis (2,42,47,49). The presence of functionally aromatase in human spermatozoa allows the conversion of androgens into estrogens throughout the whole transit of reproductive tract, providing free estrogens in the seminal fluid able to act on the cells of the reproductive ducts. Thus, human spermatozoa should be considered a mobile endocrine unit.
In summary, the testes are able to synthesize and respond to estrogens throughout their development. The localization of ERα, ERβ and aromatase suggests that estrogen action is likely to be important for testicular and efferent ductules function. Differences among various polymorphisms of ERs genes may account for different responses to estrogens in term of sperm count and sperm quality (51,52). The role of estrogens in the male reproductive system become clearer in animals, and the mapping of ERs and aromatase distribution in the human male reproductive system has led to the suggestion that estrogen plays a role in human male reproduction. As a consequence, a new field of research has evolved, aimed at improving our knowledge on estrogen action on male reproduction, and the molecular mechanisms involved in both animals and men.
Estrogen-deficient knock-out mice are useful models to investigate estrogen action in rodents (13,22). At present, four different lines of estrogen-deficient knock-out mice have been generated: 1) ERα knock-out (α-ERKO) mice with disrupted ERα gene; 2) ERβ knock-out (β-ERKO) mice, with an inactivated ERβ, and 3) ERα and ERβ knock-out (αβ-ERKO) mice with non-functioning ERα and ERβ (13). The αERKO, βERKO and αβERKO mice provide helpful information on the loss of ER function, leding to estrogen resistance. The knock-out of aromatase gene leads to aromatase knock-out mice (ArKO), an experimental animal model useful for investigating the congenital lack of both circulating and locally produced estrogens since birth (13,22,53). Estrogen-resistant mice have high levels of circulating estrogens with the non-genomic pathway still functioning, while aromatase-deficient mice have no circulating estrogens and none of the estrogen pathway are activated (22).
Male αERKO mice are infertile since the seminiferous epithelium is atrophic and degenerated and both tubules and rete testis are dilated (28,54), even though the development of male reproductive tract is largely unaffected (13). The disruption of spermatogenesis is progressive as the testicular histology is normal at ten days of age, but starts to degenerate at twenty-thirty days. From 40 to 60 days tubules are markedly dilated with a corresponding significant increase in testicular volume, while the seminiferous epithelium becomes atrophic (13). A severe impairment in tubule fluid absorption at efferent ducts level was the cause of infertility in αERKO male mice, and this defect is partially mimicked also by the administration of an anti-estrogen drug in wild-type mice (28). In the male genital tract the highest concentration of ERα is in the efferent ducts (30) and the estrogen-dependent fluid reabsorption at this site probably results from estrogen interaction with the ERα that seems to regulate the expression of the Na(+)/H(+) exchanger-3 (NHE3). In fact, the disruption of ERα, or the use of anti-estrogens, results in a decreased expression of NHE3 mRNA, as well as in a decrease of other proteins involved in water reabsorption, such as aquaporin I (55,56). The lack of fluid reabsorption in the efferent ductules of αERKO male mice and the consequent dilatation induces a retroactive progressive swelling of the seminiferous tubules. The seminiferous tubule damage results from the increased fluid pressure and severely impaired spermatogenesis coupled with testicular atrophy as seen at the age of 150 days of mice life (13,28). In addition αERKO male mice show serum LH significantly increased, with a consequent increased serum testosterone and Leydig cells hyperplasia, together with normal serum FSH levels (13).
The production of both ArKO (57) and βERKO (58) mice supports the idea that in mice estrogen actions on the male reproductive tract are more complex than previously suggested on the basis of the αERKO mice (13). In fact, unlike αERKO mice, male ArKO mice are initially fully fertile (57), but fertility decreases with advancing age. Furthermore, βERKO mice are fully fertile and apparently reproductively normal in adulthood (58). Thus, histology of the testes of one-year-old ArKO mice shows a disruption of spermatogenesis at the early spermatid stage without significant changes in the volume of seminiferous tubule lumen, together with Leydig cell hyperplasia (59). Despite the phenotype of αERKO male mice, the mechanism involved in the development of infertility is different in ArKO male mice, since the early arrest of spermatogenesis suggests a failure of germ cell differentiation probably caused by the lack of estrogen action at the seminiferous epithelium level rather than a problem ascribable to impaired fluid reabsorption (41). Estradiol seems to be a survival factor for round spermatids and its lack may promote apoptosis resulting with a failure in elongated spermatid differentiation.
Studies in αβERKO mice showed a male phenotype very close to αERKO mice characterized by infertility and dilated seminiferous tubules (13). On the contrary, βERKO male mice were fully fertile (58). These findings lead to the hypothesis that estrogen activity in the male reproductive tract depends on both the type of estrogen receptor involved, and the site of action through the male reproductive tract. Interestingly, results from mice lacking functional ERs or aromatase, point to an important role for estrogen in the maintenance of mating behaviour in male mice, and that infertility in αERKO, αβERKO and ArKO mice are at least in part due to reductions in various aspects of mating behavior from an early age (13,22). These studies supported the concept that a functional ERα, but not ERβ, is needed for the development and maintenance of a normal fertility in male mice (13,24,28,54,58).
Estrogens in human male reproductionThe demonstration of wide expression of aromatase enzyme, ERα and ERβ throughout both the male reproductive system and the human sperm, underlines the role of estrogens in male reproductive function (2,17,24). Thus, estrogens seems to modulate sperm maturation (17,41), since spermatozoa also express ERα and ERβ, and they are responsive to estrogens throughout their run from the testes to the urethra.
Data from human subjects with congenital estrogen deficiency provided conflicting and confusing results. The only man with estrogen resistance discovered up till now, a human equivalent of the ERKO mouse, had normal testicular volumes and normal sperm count but with slightly reduced motility (14) (Table 5). Another human model of estrogen deficiency is represented by patients with congenital aromatase deficiency (24). The eight men affected by this congenital disease showed a variable degree of impaired spermatogenesis (2,5,60). Of the four patients with available semen analysis, two had normal sperm density (61-63) and the remaining two had oligospermia (10,15,64) from moderate (64) to severe (10,15) (Table 5). In all the four patients, however, a moderate to severe asthenospermia without teratospermia was reported (10,15,61-64) (Table 5). Data on sperm analysis are not available from another men with aromatase deficiency (16,65) as well as in the unique aromatase-deficient boy (66,67). Moreover, a variable degree of germ cell arrest was described in three cases that underwent biopsy of the testes (10,15,68,69) (Table 5).
Table 5. Reproductive phenotypes of men with congenital estrogen deficiency.
|
Estrogen resistance |
Aromatase deficiency |
Total subjects |
1 |
8 |
Subjects diagnosed during adulthood |
1 |
7 |
Age (mean + DS) |
28 years |
27.7 + 4.8 years |
REPRODUCTIVE HORMONES |
|
|
LH |
High |
Normal to high |
FSH |
High |
High |
Testosterone |
Normal |
Normal to high |
Estradiol |
High |
Undetectable |
EXTERNAL GENITALIA |
|
|
Size testis |
Normal |
Small to normal to increase |
Cryptorchidism |
Absent |
3 cases |
SEMEN ANALYSIS |
|
|
Sperm count |
Normozoospermia |
Oligo to normozoospermia |
Viability |
Asthenozoospermia |
Asthenozoospermia |
Testis biopsy |
Not performed |
Depletion or germ cell arrest at primary spematocyte level |
It should be remarked that a clear cause-effect relationship between infertility and aromatase deficiency is not demonstrable in these patients (2,15). Accordingly, differences on fertility impairment degree in men with congenital estrogen deficiency does not allow to distinguish whether these features are a consequence of a lack of estrogen action, or are only epiphenomena, even though a possible estrogen role on human spermatogenesis is suggested by rodent studies (2).
Actually, our knowledge on the estrogen role in human male reproduction is far from complete. The exposure to the excess of environmental estrogens has been proposed as a possible cause of impaired fertility. Thus, administration of aromatase inhibitors to infertile men with impaired testosterone-to-estradiol ratio resulted in an improvement of their fertility rate (70,71). Furthermore, it is difficult to reconcile existing data about effects of both estrogen deficiency and excess on male reproductive function (8,18,72,73). However, the role of estrogen in controlling sperm production and quality has been recently confirmed by the association of polymorphisms of estrogen-related genes with both sperm concentration and motility, but not with sperm morphology (74).
Regulation of gonadotropin feedbackThe regulation of gonadotropin feedback is an important and well-documented action of estrogen in males. While testosterone has been classically considered the key hormone for the control of gonadotropin feedback in the male (Figure 4), a role for estrogens was recently clarified from studies performed in normal and GnRH-deficient men. The response of hypothalamic-pituitary gonadal axis to androgens is confirmed by the administration of dihydrotestosterone (DHT), which is able to partially decrease LH and FSH with a concomitant reduction in serum testosterone and estradiol (75). However, the discovery of men with congenital estrogen deficiency has provided further evidence for a relationship between estrogens and gonadotropin secretion also in men (10). In fact, serum gonadotropins are high in all adult patients with aromatase deficiency, notwithstanding normal to increased serum testosterone levels (24), thus implying that also estrogens are important for the regulation of circulating gonadotropins levels in men.
Figure 4: Traditional knowledge concerning sex steroids on the control of gonadotropin secretion.
The effects of estrogens on gonadotropin secretion have been investigated in GnRH deficient males whose gonadotropin secretion was normalized by pulsatile GnRH administration. Moreover, in order to determine the precise role of sex steroids in the hypothalamo-pituitary-testicular axis, several studies characterized by the administration of testosterone, testosterone plus testolactone (an aromatase inhibitor), or estradiol were performed (76,77). Testosterone alone, caused a significant decrease in mean basal LH and FSH levels as well as LH pulse amplitude, demonstrating a direct suppressive effect on the pituitary of testosterone and its metabolites. While, mean LH levels and LH pulses frequency suppression to a greater extent in normal control subjects suggests also a hypothalamic site of action of testosterone in suppressing GnRH secretion. In order to discriminate the impact of testosterone from its aromatized products, both groups of subjects were treated with testosterone plus testolactone. The addition of the aromatase inhibitor completely exclude testosterone effects on gonadotropin secretion both in normal and GnRH deficient men leading to a significant increase in mean LH levels in both groups. The latter being greater in normal men who received testolactone alone than normal men who received testosterone plus testolactone, thus confirming also a direct effect of androgens in normal men. Consequently, it is clear that the aromatization of testosterone to estradiol is required for normal gonadotropin feedback at the pituitary level (76). In fact, when the same experimental model was applied using estradiol administration, mean LH and FSH levels as well as LH pulse amplitude decreased significantly during the treatment (77). These studies demonstrate an important direct inhibitory effect of estradiol on gonadotropin secretion in both GnRH-deficient and normal men (76,77) and support the concept that at least in part the inhibitory effect on gonadotropin secretion is mediated by the conversion of testosterone to estradiol. Accordingly, the administration of the aromatase inhibitor letrozole to healthy adult males suppressed aromatase activity and serum estradiol levels leading to an increase of gonadotropins (78). Thus, only the restoration of normal circulating estrogens, by means of transdermal estrogen administration, normalized gonadotropin secretion in this setting (78). In contrast, it seems that the 5α-reduction of testosterone to DHT does not play an important role in pituitary secretion of gonadotropins (79).
All these studies confirmed the presence of a hypothalamic site of action for estrogens also in men. The effect of circulating estrogen seems to be more relevant than that of locally produced, both at hypothalamic and pituitary level (78). In order to clarify the role of estrogen on the feedback regulation of gonadotropin secretion at hypothalamic level, Hayes et al (80) conducted a study involving men affected by idiopathic hypogonadotropic hypogonadism (IHH), whose gonadotropin secretion was normalized by long term pulsatile GnRH therapy, treated with aromatase inhibitor (anastrozole). They observed that inhibition of estradiol synthesis led to a greater increase in mean gonadotropin levels in normal subjects than in IIH men, suggesting a hypothalamic involvement. The rise in mean LH levels in normal subjects related to anastrozole effect was the result of an increase in LH pulse frequency and amplitude. The authors concluded that estrogen acts at the hypothalamic level decreasing GnRH pulse frequency and pituitary responsiveness to GnRH (80). Recently, the same group (81) demonstrated that the administration of estradiol in normal subjects, whose endogenous testosterone and estradiol synthesis was inhibited through the use of ketoconazole, reduced mean LH levels by lowering LH pulse frequency, but not amplitude. The authors suggested that estradiol feedback occurs predominantly at the hypothalamus (81). Data available in literature, demonstrate that (i) circulating estrogens are involved in gonadotropin suppression both at pituitary (81) and hypothalamic level (81,82), and (ii) estrogen effects on hypothalamus are independent from central aromatization, but requires adequate amounts of circulating estrogens in normal healthy men (78), in men with IHH (81,83), and in men with aromatase deficiency (82).
Accordingly, the effects of estrogen on gonadotropin secretion at pituitary level has been demonstrated to operate from early- to mid-puberty (84,85) into old age in men (86). The administration of an aromatase inhibitor (anastrozole 1 mg daily for 10 weeks) to young men aged 15-22 years (84) resulted in a 50% decrease in serum estradiol concentrations, increase in testosterone concentrations and increase in both LH and FSH values during the whole study protocol. These hormonal parameters were restored after the discontinuation of anastrozole treatment. The administration of letrozole increased serum LH levels, LH pulse frequency and amplitude and the response of LH to GnRH administration in boys during early and mid-pubertal phases, confirming that estrogens acts at pituitary level during early phases of puberty (85). The same mechanism is still present during adulthood and early senescence, as shown in fifteen eugonadal men, aged 65 years treated with 2 mg anastrozole for 9 weeks, in which serum FSH and LH levels increased significantly, in spite of an increase in serum testosterone levels (71,86,87).
Previous data suggest that estradiol may modulate GnRH receptor number and function at hypothalamic-pituitary level (88), since ERs were detected in GnRH secreting neurons (89). Moreover, both genomic and non-genomic estrogen actions seems to be involved in the regulation of the gonadotropin feedback in males (89,90), even if the precise mechanism remains unclear (91). Nevertheless, it is now well established that androgens need to be converted to estrogens in order to ensure the integrity of the gonadotropin feedback mechanism in men, testosterone itself having a minor role than previously thought (Figure 5), and circulating estrogen having a major role than locally produced estrogen at the hypothalamic pituitary level (78,81,82).
Figure 5: Sex steroids control of gonadotropin secretion after recent advances.
Furthermore, our knowledge on the role of estrogens in gonadotropin feedback has been enhanced through studies of men with congenital estrogen deficiency. The description of a man lacking a functional ERα (14) revealed a remarkable hormonal pattern consisting of a normal serum testosterone, high estradiol and estrone levels, but increased serum FSH and LH concentrations (Table 4). Other important information about the estrogens role in human male came from the discovery of naturally occurring mutations in the aromatase gene. To date eight different cases of human male aromatase deficiency have been described, seven of these males were discovered to be aromatase deficient during adulthood and one as a child (Table 4). The eight patients had increased basal FSH concentrations, except for the subject diagnosed during childhood having normal FSH in infancy (66) and high to normal FSH levels at puberty (67). LH was normal except for one subject with elevated (16,65) and two subjects with high to normal LH levels (60,92) (Table 4). Serum testosterone concentrations were generally normal or high to normal except for the first case described with elevated serum levels (16,65), and two other aromatase-deficient men with testosterone slightly above the normal range (61,64). Conversely, another man with aromatase deficiency presented with low to normal serum testosterone levels (68,93). In all eight patients estradiol concentrations were undetectable (24) (Table 4). The detection of increased gonadotropin levels despite of normal to increased serum testosterone levels, in these men, further highlights the key role for estrogen in regulating circulating gonadotropins in men (82) particularly, FSH (2,23). Accordingly, in normal men with pharmacologically induced sex steroid deprivation, estradiol but not testosterone, was able to restore normal FSH serum levels (83). Due to the concomitant impairment of the patient's spermatogenesis, complete normalization of serum FSH was not achieved in all aromatase-deficient men during estradiol treatment, even in the presence of physiological levels of circulating estradiol (24), only supraphysiological levels of estrogens being necessary to obtain FSH normalization (15,24,92).
A detailed study of the effects of different doses of transdermal estradiol on pituitary function in two men with congenital aromatase deficiency demonstrated that estrogens might control not only basal secretion of gonadotropins but also their responsiveness to GnRH administration (60,82). In this study, estrogen administration to three male patients with aromatase deficiency caused a decrease in both basal and GnRH-stimulated LH, FSH and α-subunit secretion with a dose-dependent response to GnRH administration (60,64,82). Recently, Rochira et al (82), demonstrated that estrogens effects on LH secretion are exerted both at pituitary and hypothalamic level, as shown by the decrease of basal and GnRH-stimulated secretion of LH and the LH pulse amplitude, and the reduction of the frequency of LH pulses respectively, during estrogen treatment to normalize estradiol serum levels in two aromatase-deficient men. Moreover these data provide evidence that feed-back action exerted by circulating estrogens is more relevant than that of locally produced at the hypothalamic level (82). As previously explained, these data confirm data from healthy men.
Notwithstanding, recent advances in the study of estrogen role on male subjects, some difficulties remain with interpreting data from men with congenital estrogen deficiency, particularly considering phenotype heterogeneity. Accordingly, in the patient with congenital aromatase deficiency diagnosed in childhood, no abnormalities were found in either gonadotropin secretion nor in testis position and size (66), unlike female newborns. Thus, suggesting that the role of estrogens in the hypothalamo-pituitary-testicular axis should become relevant in a later stage of life than infancy in men. Furthermore, the lesser than expected increase of FSH levels (given the prevailing serum testosterone levels and impaired spermatogenesis) in two estrogen deficient men (82), suggests a possible role of estrogens in priming and maturation of hypothalamus-pituitary-gonadal axis in men (64,93). Thus, the control of gonadotropin feedback exerted by sex steroids during early infancy and childhood remains a matter of debate in the human male.
The same mechanism seems to be also involved in the differentiation in hypothalamic structures between men and women (99,102). Prenatal hormonal exposure is classically considered involved in determining sexual orientation, on the basis of some differences found in hypothalamic structures between heterosexual and homosexual men (98,102). This hypothesis is supported by the concept that brain sexual differentiation during fetal life occurs in parallel with the peak of testosterone secretion from the testis and the consequent increase in serum estradiol (96,98,100). Accordingly, the intrinsic pattern of mammalian brain development is female, and estrogen is required for the development of a male brain (98,99), thus emphasizing the role of locally produced estrogen (100). Permanent changes in the organization of different neural circuits, fundamental for sex-specific regulation of reproductive and sexual behaviour, probably also occurs under the effects of estrogen (97,98,100). Considering all the above mentioned aspects, the lack of estrogen action on the developing brain in males, should be considered strictly related to the impairment of future development of sexual orientation, and of dimorphism of hypothalamic structures (96,98,100,102), even though most of the data came from studies performed in rodents (97,98,100).
Recently, the role of hypothalamic aromatase activity and expression in partner preference has been confirmed in rams (103). In this study, the choice of sexual partner were associated with both the volume of the ovine sexually dimorphic nucleus and different patterns of aromatase expression, providing the first demonstration that differences in aromatase expression within the brain are related to partner choice and to the determination of adult sexual behaviour (103).
Aromatase-deficient men represent an interesting model to investigate the role of estradiol on human male sexual development and behaviour from fetal life through adulthood (2,24). All men with aromatase deficiency had male gender-identity and heterosexual orientation (2,16,24,63,64,69,93,104). The fact that congenital aromatase deficiency does not affect psychosexual orientation and gender-identity in humans allows the hypothesis that, differently from animals, psychological and social factors may be the most relevant determinants of gender role behaviour in men, with hormones probably having a minor role. (2,24,101).
In conclusion, aromatase plays a key role in controlling male reproductive behaviour especially in animals (rodents and rams), by the introduction of organizational effects on the developing brain during fetal life (105), mediated by estrogen production within the brain and exposure to circulating estrogens. However, differences among species could explain the essential role of aromatization in rodents (101,105) and its poor effect in humans (24,93,104).
Sexual behaviourIn adult men sexual behaviour is partially dependent on testosterone, the main hormone involved in male sexuality (95,106,107). Accordingly, testosterone deficiency frequently causes loss of libido and erectile dysfunction (95,107). Thus, testosterone replacement therapy increases sexual interest and improves sexual behaviour (94,95).
In experimental animal models, the knock-out of estrogen pathways or a pharmacologically induced estrogen deficiency result in severe impairment of sexual behaviour (24,95). Accordingly, ArKO mice (108), αβERKO male mice and αERKO mice (13), all exhibit significant reduction in mounting frequency and prolonged latency to mount when compared with wild-type animals (13,22). On the contrary, βERKO mice did not show of sexual behavior impairment (13). These findings suggest that androgen receptor activation alone is not sufficient for fully normal sexual behaviour in rodents and also normal ERα is required for mounting behaviour in male mice (24.95).
Less is known about the role of estrogens in sexual behaviour in men since the percentage of testosterone effects, through aromatization to estradiol, on male sexual behaviour is still not known. A detailed sexual investigation of aromatase-deficient men documented an increase of all the parameters of sexual activity during estrogen treatment (93,104), with the best outcome in terms of sexual behaviour obtained only when a concomitant normalization of both serum testosterone and estradiol was reached (93), supporting the concept that both sex steroids are required for normal sexual behaviour in men. Outside the context of congenital lack of estrogens it is difficult to reach conclusive information on the role of estrogen on male sexual behaviour because of inadequacy of studies and the conflicting results available in literature. (Table 6).
Table 6. Sexual behavior in men with congenital estrogen and aromatase deficiency.
Subjects |
Authors |
Sexual function |
Gender identity |
Psychosexual orientation |
Estrogen resistace (Age:28 years) |
Smith et al. 1994 (14) |
Libido: normal. |
Male |
Heterosexual |
Aromatase deficiency (Age 24 years) |
Morishima et al. 1995 (16) |
Libido: modest. Morning erections: normal. |
Male |
Heterosexual |
Aromatase deficiency (Age 38 years) |
Carani et al. 1997 (15) Carani et al. 1999 (104) |
Libido: normal. |
Male |
Heterosexual |
Aromatase deficiency (Age 28 years) |
Maffei et al. 2004 (68) |
Morning erections: normal. |
Male |
Heterosexual |
Aromatase deficiency (Age 27 years) |
Herrman et al. 2002 (64) Herrman et al. 2005 (149) |
Libido: normal. |
Male |
Heterosexual |
Aromatase deficiency (Age 25 years) |
Maffei et al. 2007 (69) |
Libido: normal. |
Male |
Heterosexual |
Aromatase deficiency (Age 27 years) |
Lanfranco et al. 2008 (63) |
Libido: normal. |
Male |
Heterosexual |
Finally, estrogen receptors and the aromatase enzyme have been identified in the penile tissue of a large number of species, including humans (109-111) suggesting direct estrogenic activity within the penis. At present, knowledge on estrogen action within the penis derives from the observation that: i) male off spring exposure to estrogen-like endocrine disruptors in utero induces micropenis and hypospadia (72), and ii) in animals penile development and function is estrogen-dependent (112).
Effects of exposure to excess estrogens in animals
In order to evaluate the effect of estrogen excess on the reproductive tract, several studies have been performed in various animal species treated with diethylstilbestrol (DES), a synthetic estrogenic compound (113). From 13 to 24 weeks of fetal life coincides with the highest susceptibility to endocrine disruptors (2). Many studies in rodents suggest that inappropriate exposure to estrogen in utero and during the neonatal period impair the hypothalamic-pituitary-gonadal axis, the testicular descent, the efferent ductules function and the testicular function (18,22). The latter effect being a direct consequence of exposure to excess estrogen, as well as a secondary effect of perturbations in circulating hormones or the ability of the efferent ductules to reabsorb fluid. ERβ may mediate the process through which estrogens produce negative effects on male reproduction. The effects of excess estrogen in the neonatal period can affect also testis in adulthood, with permanent changes in their function and spermatogenesis (18, 22).
Aromatase over-expression in rodents
The transgenic model of mice overexpressing aromatase enzyme (AROM+) shows highly elevated serum estradiol concentrations, with a concurrent decrease in testosterone levels (114,115). The AROM+ males display several changes observed also in males perinatally exposed to estrogens, such as undescended testes, testicular interstitial cell hyperplasia, hypoandrogenism, and growth inhibition of accessory sex glands. The impairment of spermatogenesis observed, could be a consequence of multiple factors, including cryptorchidism, abnormal Leydig cell function, hypoandrogenemia or hyperestrogenemia. Thus, estrogens are thought to inhibit Leydig cell development, growth and function, resulting in the suppression of androgen production (22). Furthermore, the observation of numerous degenerating germ cells and the absence of spermatids within the seminiferous tubules of AROM+ mice suggest that germ cells development was arrested at the pachytene spermatocyte stage in the cryptorchid testes (22). Interestingly, the spermatogenic arrest occurred at a stage where P450arom is typically expressed. The spermatogenic arrest found in the AROM+ mice could be explained, at least in part, by the suppression of FSH action. In fact, the reduced serum FSH levels, associated with normal LH levels,, in AROM+ males are further evidence of the inhibiting actions of estrogens on FSH secretion in males (114,115).
Effects of exposure to excess estrogens in humans
The clinical use of DES by pregnant women in order to prevent miscarriage resulted in an increased incidence of genital malformations in their sons (116). The most frequent structural and functional abnormalities are: epididymal cysts, meatal stenosis, hypospadias, cryptorchidism and microphallus (116). The frequency of abnormalities was dependent on the timing of estrogen exposure: in fact, men who were exposed to DES before 11th week of gestation (i.e. the time of Műllerian ducts formation) had a two fold higher rate of abnormalities than those who were exposed only later (116). These data support the hypothesis that the asynchrony between formation and regression of embryonal reproductive structures is determined by estrogen exposure.
Various reports demonstrated that semen quality of men exposed to DES in utero is significantly worse than in unexposed controls (117), even though sperm concentrations of most of the DES exposed men were above the lower limit, with normal fertility (9). The implications for human spermatogenesis in environmental estrogens remains less clear. The risk of testicular cancer among men exposed to DES in utero has been a controversial issue and several meta-analyses showed a doubling of cancer risk of the testes, together with cryptorchidism, hypospadias, and impaired spermatogenesis (118). However, more direct evidence will be necessary in order to fully understand this issue and particularly to identify the exact estrogenic mechanism of action (118). Up to now, it is clear that exogenous estrogens could interfere with the development of genital structures if administered during early organogenesis. The main effect are an impairment of gonadotropin secretion and the imbalance in the androgen to estrogen ratio, which may account for impaired androgen receptor stimulation or inhibition according to the dose, the cell type and age (116). Furthermore, it seems that an excess of environmental estrogens could be a possible cause of impaired fertility in humans (72,73). In fact, a progressive decline in sperm count has been reported in some Western countries during the past 50 years, suggesting a possible negative effect of environmental contaminants on male reproductive function (8,72,73).
Aromatase over-expression in humans
Aromatase over-expression causes an increased conversion of androgens to estrogens with a consequent excess of the hormone. Excess estrogen in boys causes gynecomastia, a premature growth spurt, early fusion of epiphyses, and decreased adult height. Increased extraglandular aromatization was firstly reported in an adopted boy with prepubertal gynecomastia in 1977 (119). Then, four families in which several members had estrogen excess (manifested as gynecomastia in boys and men and premature thelarche in girls) as a result of increased extraglandular aromatization (120-122), and one case with a mutation of the aromatase gene (123) were described. The latter seems an autosomal dominant inherited disease (121,123). In adult men elevated serum estradiol levels are associated with a sex hormonal pattern such as a mild hypogonadotropic hypogonadism probably due to a direct estrogen negative feedback effect on pituitary gonadotropins (121,123). This inhibitory effect of estrogen on reproductive function appears to be milder in males with aromatase excess syndrome than in patients receiving exogenous estrogens or with estrogen-secreting tumors, probably because serum estradiol and/or estrone levels are lower in the former (121). External genitalia in adult men are characterized by normal penile and testicular size (121,123). Even if spermatogenesis and sexual behavior were not specifically studied, the adult men described were fertile and reported normal libido (121,123). Treatment with an aromatase inhibitor reduces estrogen levels and normalizes testosterone, LH and FSH serum levels (123), confirming a crucial role of estrogen in the suppression of both gonadotropins in men.
Estrogen deficiency in animal models
The study of transgenic mice lacking ERs or the aromatase enzyme demonstrated that a lack of estrogen was compatible with life and represented an interesting model to evaluate the physiology of estrogen in males as previously described. Congenital estrogen deficiency in mice leads to an impairment of male reproductive function, ranging from normal fertility with a fully male phenotype in βERKO mice, to complete infertility in both αERKO and αβERKO mice. An intermediate pattern exists for the ArKO mice in which spermatogenesis is normal in young mice, but progressively worsen during aging (13,22,28,53,54,57-59,124). Characteristics of male mouse models are summarized in table 7.
Table 7. Male mouse models of estrogen deficiency.
αERKO |
βERKO |
αβERKO |
ArKO |
Infertility |
Fully fertile |
Similar to αERKO mice |
Normal fertility in young mice, infertility with advancing age |
Normal FSH |
- |
- |
Normal FSH |
Germ cell deprivation with dilated seminiferous tubules |
Normal testicular histology |
Testicular histology similar to αERKO mice |
Histology of the testis is disrupted with advancing age |
Impairment of sexual behavior |
Normal sexual behavior |
Complete suppression of sexual behavior |
Impairment of sexual behavior |
The testes of infertile αERKO male mice show significant atrophy of the seminiferous epithelium and severe dilation of the tubule lumen. Interestingly these defects are not present at birth, but they progressively become evident with the worsening of testicular phenotype (13). When germ cells from αERKO mice are transplanted in wild type mice, they show a normal development (125). The αERKO mouse is also characterized by a reduced number, motility and fertilizing capacity of the sperm. On the other hand, the βERKO mice have normal testes and normal sperm count and they are fertile (13). Furthermore, the development of ArKO mice allowed to clarify the effects of the lack of estrogens on male reproductive function. Morphological studies revealed a progressive disruption of spermatogenesis at one year of age, with a concomitant impairment of sperm count and motility (57,58).
The function of the hypothalamo-pituitary-testicular axis is impaired in both αERKO and ArKO male mice, leading to elevated serum LH levels in the presence of normal values of FSH, while, as expected, testosterone is augmented and estrogens are higher than normal or undetectable in αERKO and ArKO mice, respectively (5,22). Thus, negative effects on male reproduction are the result of estrogen deprivation directly in the reproductive structures or indirectly through changes in the regulation of sex steroid secretion.
Estrogen deficiency in human models
Important information about the role of estrogens in the human male came from the discovery of naturally occurring mutations in the aromatase gene. To date only eight different cases of human male aromatase deficiency have been found (Table 5). The hormonal pattern of the patients affected by aromatase deficiency is summarized in Table 5 (24). The study of men with aromatase deficiency shows that estrogens modulate gonadotropin feedback by regulating both basal secretion of gonadotropins and their responsiveness to GnRH (24).
Testicular size in aromatase-deficient men is normal except for three cases having a smaller testes volume (Table 5). Sperm analysis was performed in four patients (15,61-64) and documented an asthenozoospermia in all cases coupled with a moderate (64) to severe (15) oligozoospermia in two patients (Table 5). The histological study of the testes was performed in only three patients (15,68,69,93) and showed profound alterations in germ cell development, characterized by a complete depletion of germ cells to a germ cells arrest at primary spermatocytes. However, it remains unclear the estrogen role for the spermatogenic defects.
Furthermore, a history of cryptorchidism was present in three of the nine patients (33,3%) being bilateral in one case (68) and unilateral in the remaining two (61,62). These data suggest a possible role of estrogen also in testis descent, although this was not seen in the transgenic mice models. The small number of case of cryptorchidism among men with aromatase deficiency does not allow any conclusion concerning a possible relationship between estrogen deficiency and the occurrence of abnormalities in testis development and descent.
In normal adult men the normal range of serum estradiol levels is 18-40 pg/mL (66-147 pmol/L). Approximately 50 μg of estradiol are daily produced: about 5-10 μg in the testis (10 to 20%) and the remaining 40-45 μg (80 to 90%) in peripheral tissues (adipose tissue, muscle, breast, brain liver and bone) in which the aromatase enzyme is expressed (82).
In particular circumstances the clinical approach to investigate male infertility may involve also the assay of serum estradiol when clinical aspects suggestive for aromatase deficiency, coupled with normal to high testosterone and gonadotropins levels and/or history of cryptorchidism are present (Table 5). However, it has to be remarked that the accuracy of the major commercially available kits for the detection of serum estradiol is poor, especially for low serum levels of estradiol (82). Therefore, the assay of serum estradiol is recommended only if high sensitivity and specificity 3rd generation RIA is available (82). The gold standard test is the gas chromatography/tandem mass spectrometry (126), but a good result in term of sensitivity could be obtained also by ultrasensitive recombinant cell bioassay. The latter being approximately 100-fold more sensitive than previous estradiol assays with a sensitivity of < 0.02 pg/ml estradiol equivalents (127), which is adequate for values of serum estradiol within the normal male range. The limits of these two methods are their costs and time consuming procedures that do not allow a routinary use in clinical practice, limiting their usefulness only for research purposes.
The role of estrogen on glucose and insulin metabolism in men is difficult to establish since differentiate androgen and estrogen action in vivo remains challenging. In estrogen-deficient men insulin resistance and fasting glucose are increased and improve during estrogen treatment (60,68,82), confirming data from mice models (13). Thus, severe impairment of the estrogen to testosterone ratio (increased androgens and decreased estrogens) seems to represent a condition for the development of insulin resistance in men (68,82), not only in estrogen deficient men (128).
Furthermore, congenital estrogen deficiency is associated with an altered lipid profile (10,60), mainly characterized by higher total cholesterol and triglycerides serum levels, higher low-density lipoprotein (LDL) cholesterol and very low high-density lipoprotein (HDL) cholesterol (5,24). In these patients, estradiol treatment determined a moderate increase of HDL cholesterol together with a small reduction of triglycerides, total cholesterol, and LDL cholesterol (15,60,66,68), resembling the effects of estrogen on lipid metabolism exerted in females (19).
There is increasing evidence suggesting that circulating estrogens plays a key role for bone health also in men. The relative contribution of androgen versus estrogens in the regulation of the male skeleton, however, is complex and partially unclear; however, some estrogen actions on male bone, such as the bone maturation and the acceleration of growth arrest are now well defined. The important role of estrogen in bone metabolism in men has been characterized in the last 15 years by means of the description of rare case reports of estrogen-deficient men (24,63) and by several epidemiological studies (82,129). The estrogen replacement therapy led to skeletal maturation and improvement of bone quality in aromatase-deficient men (5).
Androgens are known regulators of prostate gland growth and differentiation particularly during its development. On the other hand, the role of estrogens is less characterized, but dual direct and indirect actions on prostate growth and differentiation have been demonstrated, throughout both ERα, and ERβ (130,131). Furthermore, it is now clear that in rodents, during development, prostate is sensitive to estrogen exposure (132).
Also in this field, studies on animals can help to better understand the estrogen role on prostate function. Studies in AROM+ mice demonstrated that prostate lobes were significantly reduced (114). On the other hand, aromatase deficient mice presented a hyperplastic prostate gland (133), due to an hyperplasia of the epithelial, interstitial and luminal compartments (132). Furthermore, McPherson et al., using tissue recombination and an ERβ-specific agonist, demonstrated that ERβ activation results in an anti-proliferative response not influenced by systemic androgen levels, or activation of ERα (133). Moreover, studies on ArKO mice, demonstrated that the administration of an ERβ-specific agonist reverted the existing hyperplastic epithelial pathology (133).
For what concern prostate carcinogenesis, it is generally assumed that androgenic hormones play a major role in tumor development, since prostate gland is an androgen-dependent tissue as prostate cancer (134,135). However, considering the fact that testosterone can be converted in to estradiol, and ERs were identified in prostate epithelium (136), also estrogen may be involved in the induction of prostate cancer. In fact, Bosland et al. found that the combined treatment with estradiol and testosterone on rats, lead to an increased prostate cancer incidence from 35-40% with androgen alone, to 90-100% (135,137). The development of selective inhibitors of aromatase may be helpful for the evaluation of estrogen effects on prostate. Furthermore, if estrogen role will be demonstrated, new treatment strategies will be available for benign prostate hypertrophy and cancer (138).
Finally, as regard aromatase-deficient men, prostate was normal without any change after introduction of estrogen replacement therapy (Carani 2004 − not published data).
On the basis of the certain role of estrogens on gonadotropic feedback inhibition (78,81,82), some clinical insights on the management of male infertility have been made. However, it remains to be established if estrogen in men could be a good target for improving or modulating male fertility, since conflicting results are available in the area of aromatase inhibitors used for the treatment of male infertility (17,24,70,71). Thus, the real efficacy of antiestrogens is far from being elucidated and it is a matter of debate whether the increase of sperm density induced by antiestrogens is actually related to a real improvement of both sperm fertility and pregnancy rates (17,24) (Table 8).
Table 8. Range dosing and time duration of oral antiestrogen and aromatase inibitors and their different effect on semen analisys in clinical trials reported in literature
Treatment |
Dose (mg/daily) |
Duration (months) |
Effects on semen analysis |
Antiestrogens |
|
|
|
Clomiphene |
25-50 |
3-12 |
Semen volume: No effect or ↑ |
Tamoxifen |
20-30 |
3-6 |
Semen volume: No effect |
Tamoxifene |
20 |
6 |
Semen volume: No effect |
Aromatase inhibitors |
|
|
|
Testolactone |
2000 |
8 |
No effect |
Testolactone or Anastrozole |
100-200 |
6 |
Semen volume: ↑ |
Letrozolo |
2,5 |
6 |
Semen volume: No effect |
Since 1960s antiestrogen agents have been used as an empirical treatment of male infertility (139), based on their modulation of the hypothalamic-pituitary testicular axis. The blockade of the estrogen-mediated negative feedback on gonadotropins, in fact, excites LH and FSH secretion with a proposed consequent stimulatory effect on spermatogenesis, in the absence of clear evidence of direct effects of antiestrogens on spermatogenesis (140). Accordingly, LH and FSH serum levels increase after aromatase inhibitor administration in infertile men (141).
Clomiphene at the dosage of 25-50 mg daily for 3-12 month or tamoxifen at dosage of 20-30 mg daily for 3-6 month represent the most used antiestrogen agents for the treatment of male infertility (142) (Table 8); on the contrary the new generation of selective estrogen receptor modulators does not show significant changes in male fertility (143).
Clomiphene (25-50 mg/day) has been recently studied in a cohort of 86 men with hypogonadism for six months (144). This treatment represented an effective and safe alternative to testosterone supplemention in hypogonadic men wishing to preserve their fertility (144). Furthermore, Ghanem et al. have recently found that the combined treatment with clomiphene (25 mg/day) and antioxidant drug (vitamin E) increased the pregnancy rate and improved sperm count and progressive motility in men with idiopathic oligoasthenozoospermia (145).
Tamoxifen (20 mg/day) has been also used with oral testosterone undecanoate (120 mg/day) in men affected by idiopathic oligozoospermia. This combined treatment was effective in improving not only the sperm parameters (total sperm number, sperm morphology and motility), but also the pregnancy rate (146). Recently, Moein et al. studied thirty-two azoospermic infertile men with proved nonobstructive azoospermia, administrating Tamoxifen for 3 months (147). The tamoxifen treatment led to the recovery of spermatozoa in ejaculates of six patients (147). These studies showed that treatment of patients with nonobstructive azoospermia with anti-estrogenic drugs like tamoxifen can improve the results of sperm recovery in testis samples and also increase the chance of pregnancy by microinjection.
The uncertain role of these therapies on male fertility, may be related to the fact that idiopathic oligozoospermia constitutes a group of heterogeneous disorders of which only a subgroup might respond to antiestrogen therapy. However, till now all the studies failed to identify the characteristics of this subgroup and thus physicians cannot distinguish potential responders and non-responders.
Few data are available about the effect of aromatase inhibitors in male infertility. A previous study failed in demonstrating the efficacy of testolactone in the treatment of idiopathic oligozoospermic infertility (141). However, when aromatase inhibitors (testolactone or anastrazole) are administered in a selected group of infertile men with abnormal baseline testosterone-to-estradiol ratio an improvement of fertility rate is obtained (70). Recently, Saylam et al. treated 27 infertile, hypogonadic men with 2,5 mg daily of letrozolo for six months, finding an improvement of both testosterone serum levels and semen parameters after treatment (71). Thus, it seems that letrozole may facilitate some improvement in infertile men with azoospermia by providing sperm in the ejaculate (71).
In conclusion antiestrogens, alone or in combination with testosterone, may represent a potential therapy for idiopathic oligozoospermia, however, further studies are needed to detect their true efficacy in improving the pregnancy rate, or to identify the features of the responders.
The clinical features common to all aromatase deficient men are: tall stature, delayed bone maturation, osteopenia/osteoporosis, eunuchoid skeleton, bone pain and progressive genu valgum (24,148). Estrogen replacement treatment, at the daily dose of 0.22 to 0.35 μg/kg of transdermal estradiol in adult men, should be started as soon as the diagnosis of estrogen deficiency has been reached. When the diagnosis is available at birth, or is achieved during infancy, low dosages of exogenous estradiol should be administered at the beginning of puberty (0.8 to 0.12 μg/kg daily). The major target of estrogen replacement therapy in these patients is the skeleton: epiphyseal closure, bone maturation and mineralization. In fact, high doses of estrogen in adult men with aromatase deficiency leads to a rapid completion of skeletal maturation within 6-9 months, through rapid bone elongation and an increase in height followed by quick epiphyseal closure and growth arrest (24). Once epiphyseal closure has been achieved, estrogen treatment should be continued lifelong with the main goal to prevent bone loss and to reduce risk of cardiovascular disease by reducing the dose of estradiol in order to maintain serum estradiol within the normal range for adult men (24). Moreover, estrogen treatment in aromatase deficient men is effective in normalizing or improving other aspects such as gonadotropin secretion, glucose metabolism, insulin sensitivity, liver function, circulating lipids. When estrogen treatment is started at puberty, the effects of estrogen treatment on spermatogenesis are actually unknown, but the administration of estrogens in a more physiological way probably will be associated with normal spermatogenesis in adulthood. Conversely, in adult patients impaired spermatogenesis is irreversible even when estradiol treatment is administered (24).
Finally, the real impact of estrogen treatment on sexual behaviour in adult aromatase deficient men remains still to be determined (24).
Sex steroids account for sexual dimorphism because they are responsible for the establishment of primary and secondary sexual characteristics, which are under the control of androgens and estrogens in male and female, respectively (Figure 6). Advances in the understanding of the role of estrogens in animal and human models suggest a role for this sex steroid in the reproductive function of both sexes. The fact that both estrogen excess and estrogen deficiency influence male sexual development, testis function, the hypothalamic-pituitary-testis axis, spermatogenesis and ultimately male fertility, highlight the importance of estrogen action in male. From an evolutionary perspective this provides an example of the parsimony operating in biological events that are crucial for the evolution of the human species such as growth and reproduction.
Figure 6. Direct and indirect (estrogen.mediated) testosterone action.
This chapter has been concerned with the reproductive effects of estrogens in males but there are emerging roles for estrogens in non-reproductive tissues. In particular, while traditionally, testosterone has been considered the sex hormone involved in bone maturation and growth arrest in men, but recently the key role of estrogens on growth has been emphasized.
A major area of uncertainty is the possible role of estrogen in boys before puberty. It is known that low levels of circulating estradiol are detected in infancy when using ultrasensitive assays, but their significance is not known.
Several lines of evidence support the view that estrogens are required for and in part mediate androgen actions on several tissues and organs in men. The progress made in the last twenty years in this field clarified the importance of estrogen in men but leaves some issues still unsolved. In particular, estrogen action on bone and on gonadotropin secretion are now well characterized and part of the estrogen action on spermatogenesis is known but further evidence are needed to clarify the aspects still under debate.