ABSTRACT

Testicular cancer comprises different neoplasms, depending on the cell of origin and the typical age at presentation, but germ cell-derived tumors constitute the vast majority of cases. Testicular germ cell tumors (TGCT) can be diagnosed in every age group, but more than 90% of cases occur in young men. These tumors, comprising seminoma and nonseminoma, are derived from germ cell neoplasia in situ (GCNIS). Pathogenesis of TGCT associated with GCNIS partly overlaps with that of other developmental disorders of the male reproductive system within the testicular dysgenesis syndrome (TDS). Testicular somatic cell neoplasms, known as sex cord-stromal tumors, are relatively rare, but can have endocrine manifestations, such as precocious puberty or gynecomastia. In addition to its malignant features, cancer of the testis represents a developmental, endocrine, and reproductive problem. These issues are the focus of this chapter, and emphasis is given to aspects that are of interest to endocrinologists, including pediatric endocrinologists and andrologists. Management of invasive testicular tumors is largely handled by urologists and oncologists, thus only general information on surgical treatment, radiotherapy, and chemotherapy is presented. Impact of cancer treatment on the endocrine system, co-morbidities, fertility issues, and quality of life issues are also briefly reviewed.

INTRODUCTION

Testicular cancer comprises a number of different neoplasms, depending on the cell of origin and the typical age at presentation (1, 2). Although several cell types in the testis can undergo neoplastic transformation, germ cell-derived tumors constitute the vast majority of cases of testicular neoplasms. The relative distribution depends on age: in young adult men nearly all tumors are germ cell tumors, whereas in patients aged 70 years or older, a large proportion of lymphomas and secondary carcinomas can be expected (2).  In other words, as explained in detail further in the text, germ cell cancer can be diagnosed in every age group, but more than 90% of cases occur in young men, and this subgroup is the main focus of this chapter. Pathogenesis of testicular germ cell tumors (TGCT) of young adults partly overlaps with that of other developmental disorders of the male reproductive system, within the testicular dysgenesis syndrome (TDS) (3, 4, 5). Somatic cell tumors in the testis, known as sex cord-stromal neoplasms are relatively rare, but are also discussed in this chapter. These neoplasms can have endocrine manifestations due to their origin from endocrine cells.

In addition to its malignant features, cancer of the testis represents a developmental, endocrine, and reproductive problem. These issues are the focus of this chapter, and emphasis is given to aspects that are of interest to endocrinologists, including pediatric endocrinologists and andrologists. The contribution of andrologists and endocrinologists to the overall management of patients with testicular cancer is very important, especially concerning early diagnosis, fertility issues, testosterone deficiency, and the impact of treatment on the quality of life of the patients, the majority of whom are young adults. We have to emphasize that only very general information on surgical and oncological treatment options (e.g., chemotherapy) is presented here. However, urologists and oncologists, who are responsible for the clinical management of testicular tumors, would also benefit from learning about the pathogenesis and andrological aspects of testicular cancer summarized in this chapter.

GERM CELL TUMORS (GCT)

Germ cell tumors (GCT) are characterized by extreme phenotypic heterogeneity (2). They display features of pluripotency and about half of them (nonseminomas) can differentiate into virtually any somatic tissue type within the so-called teratomas that recapitulate early embryonic differentiation (6, 7, 8). Although in comparison to other solid tissue cancers, GCTs are relatively rare, they constitute the most common form of solid tissue cancer of young age. The typical localization of GCT is the testis in males and ovary in females, but GCTs may also be found outside the gonads, thus these tumors are named extragonadal GCTs (8, 9).  Extragonadal GCTs occur most often in children of both genders, preferentially along the body midline (intracranial, pineal, mediastinal) but can occur also in adults (8, 9). An increased frequency of extragonadal GCTs, especially in mediastinum, has been associated with Klinefelter syndrome (10). It is important to remember that a large proportion of cases of extragonadal GCT in retroperitoneal locations are associated with pre-malignant changes in testicles, and can be assumed early metastases of testicular neoplasms, although a multi-site development of GCT cannot be completely excluded (8, 9, 11, 12). This chapter focuses on the testicular GCT. For the extragonadal GCT in children and adults, the reader should consult specialized literature.

Classification and Histopathology of Testicular Germ Cell Tumors (TGCT)

Testicular germ cell tumors (TGCT) are by far the most frequent neoplasms of the testis and comprise approximately 90-95% of cases. They may affect infants (rarely), young men (commonly) and elderly men (rarely). The TGCTs of childhood are mentioned only briefly at the end of this section. The diagnosis and management aspects of TGCT in adult men is the focus of this chapter.

The most commonly accepted and currently used classification is the WHO classification of testicular tumors. The main changes occurred in 2016 when the WHO consensus panel proposed a thorough revision of the classification, which for the first time was based on biological evidence (2). The most radical change was the new division of TGCT into two major groups according to the origin from germ cell neoplasia in situ (GCNIS), and coining the term GCNIS, replacing several previously used and confusing names (2, 13). The latest 2022 WHO classification update proposed only minor adjustments; the most important is recognition of gonadoblastoma as a precursor similar to GCNIS and proposing placing seminoma under the major category of germinomas (14).

For use by non-pathologists, a simplified division of TGCT shown in Table 1.

*Footnote: The codes in parentheses are from the International Classification of Diseases for Oncology (ICD-O-3).

Precursor Lesions

The TGCT of young adults originate from a common precursor, germ cell neoplasia in situ (GCNIS), initially termed carcinoma in situ (CIS) testis (13, 15). GCNIS is considered to originate from developmentally arrested immature germ cells (gonocytes) that fail to differentiate to spermatogonia (4, 16, 17). Accordingly, morphology of GCNIS cells resembles closely that of fetal gonocytes, but with more irregular chromatin in the nuclei. GCNIS cells are located inside seminiferous tubules, most frequently in a single row along the basement membrane (Figure 1).

Gonadoblastoma is a preinvasive lesion which occurs almost exclusively in individuals with disorders of sexual development (DSD). This lesion is most often found in female patients with mixed gonadal dysgenesis (45,X/46,XY) or Turner Syndrome but can also occur in males (2, 18, 19). Gonadoblastoma cells and GCNIS cells have a very similar gonocyte/oogonium-like phenotype, but the surrounding somatic cells are different; GCNIS cells are present inside seminiferous tubules, which are usually well developed but may be hypoplastic and contain immature Sertoli cells, while gonadoblastoma consists of groups of germ cells, which are nested in small stromal cells similar to granulosa cells (19, 20, 21, 22). GCNIS and gonadoblastoma can be present in the same gonad and there are also lesions with morphology in between the two entities (18, 19). The clinical course of pure gonadoblastoma may be benign, but it has a potential to transform into a malignant germ cell tumor, especially if accompanied by GCNIS and greater virilization of the patient (19, 22, 23, 24). 

Figure 1. Histology of germ cell neoplasia in situ (GCNIS). The upper panel (hematoxyllin-eosin, HE staining shows a low magnification view of GCNIS in a typical pattern with only GCNIS cells and Sertoli cells present inside tubules. The tubules with neoplasia have a smaller diameter than normal seminiferous tubules. On the right side of this image a few tubules with decreased spermatogenesis are visible. The lower left image shows a fragment of a tubule with GCNIS side-by-side with a tubule with preserved spermatogenesis; note the large GCNIS nuclei. The lower right image displays GCNIS cells visualized by immunohistochemical staining for placental-like alkaline phosphatase (PLAP).

The immunohistochemical profiles of GCNIS and gonadoblastoma cells are virtually identical and resemble very closely those of primordial germ cells and fetal gonocytes (21, 25, 26).  This was subsequently confirmed by comparative studies at the transcriptional level using microarrays (17, 27, 28).  Among many genes highly expressed in GCNIS cells (as well as gonadoblastoma, normal fetal germ cells and several TGCTs) the following should be mentioned because of their interesting biological function (germ cell survival or maintenance of embryonic stem cell pluripotency) and usefulness in histopathological diagnosis (24): OCT4 (29), AP2-gamma/TFAP2C (27, 30), NANOG (27, 31, 32), KIT (25, 33), TP53 (34), and LIN28 (35, 36).

In addition to protein-coding genes, GCNIS cells display a specific profile of embryonic-type micro-RNAs (miRNA); miR-371-3 cluster, miR-302 and miR-367 (37, 38). This miRNA profile is also detectable in overt TGCT, except teratoma (38), see the description in the section on serum markers. 

Testicular Germ Cell Tumors (TGCT) Derived From GCNIS

As mentioned in the introduction, TGCT of young adult men display very variable histology (some examples are shown in Figure 2) and are divided into seminomas (under the main GCT category of germinomas which occur in men and women) and nonseminomas (non-germinomas) (2, 14, 39).

Figure 2. Histology of main types of testicular germ cell tumors. The large images show a general histology pattern of a seminoma (upper panel) and two most often seen types of nonseminoma: undifferentiated embryonal carcinoma (middle panel) and teratoma, a tumor displaying differentiation into various somatic tissues (bottom panel). Small square pictures on the right show cellular characteristics in a greater magnification.  All sections are stained with hematoxylin-eosin (HE).

Seminomas are most often diagnosed in the 25- to 40-year-old age group, whereas nonseminomatous tumors occur in even younger men (adolescence to 30 years). Both types originate from GCNIS (2, 16). Seminoma resembles a mass of immature germ cells; the tumor cells are morphologically very close to GCNIS cells and proliferate as a homogeneous tumor, which retains features of germinal lineage. The gene expression profile of seminoma is similar to that of GCNIS and fetal gonocytes, and virtually identical to the female equivalent of germinoma, called dysgerminoma (26, 39).

Nonseminomatous tumors display a variety of histological forms and contain undifferentiated embryonal carcinoma and somatic components partly differentiated along embryonic lineage of any tissue type (1, 2, 8). Nonseminomas also contain extra-embryonic tissue components (yolk sac tumor and choriocarcinoma). The combined or mixed tumors contain elements of seminoma and nonseminoma but are classified and clinically treated as nonseminoma, which usually has more severe clinical course than seminoma (1, 2).

Testicular Germ Cell Tumors Not Associated with GCNIS

PREPUBERTAL (PEDIATRIC) TGCTs. 

These tumors occur in early childhood (between birth and approximately 5 years of age) and comprise two main histological types: yolk sac tumor of the prepubertal type and mature teratoma (including dermoid cyst) (Table 1). Prepubertal teratomas can contain secondary neuroendocrine elements, which can overgrow the tumor, hence, the latest WHO classification added this entity as the third subtype (14). The histology of the prepubertal tumors does not differ from the adult equivalents, which are components of nonseminoma (2).  Likewise, these tumors display similar characteristic transcriptome and micro-RNA profiles (40, 41). In contrast to the TGCT derived from GCNIS, the tumor genome does not display isochromosome 12p. The etiology and pathogenesis of infantile TGCT remain unknown. These tumors are assumed to originate from primordial germ cells (PGC) but there is no known precursor lesion of GCNIS/gonadoblastoma type, neither are there signs of testicular dysgenesis (2, 42, 43).

SPERMATOCYTIC TUMOR  

This rare tumor occurs primarily in older men (median age at diagnosis around 50-55 years, range 25-94) but occasionally can be diagnosed in men in in the third decade of life (44). Spermatocytic tumor has no extragonadal or ovarian counterpart, and occurs exclusively in post-pubertal testis (1, 2, 8). This tumor is not derived from GCNIS (45) and has the expression profile similar to spermatogonia B or early primary spermatocytes (26, 46, 47, 48). Spermatocytic tumors appear to grow from clonally expanding germ cells, which are committed to but have not yet entered meiosis. The following de novo genetic aberrations causing increased spermatogonial proliferation have been identified; amplifications in chromosome 9p encompassing DMRT1 locus (46), rare gain-of-function mutations in genes encoding FGFR2, FGFR3, HRAS, NRAS, PTPN11, and simultaneous whole chromosome gains of chromosome 9 and chromosome 20 combined with a loss of chromosome 7 (49, 50). Some of these mutations that occur spontaneously in germ cells of aging men have been depicted as “selfish mutations” because - if transmitted to next generation - can cause severe inborn skeletal abnormalities in the offspring, such as achondroplasia, hypochondroplasia, thanatophoric dysplasia or Costello syndrome (51).

Etiology and Pathogenesis of Testicular Germ Cell Cancer of Young Adults (Derived From GCNIS)

INCIDENCE TRENDS AND RISK FACTORS  

Epidemiology of testicular germ cell tumors (TGCT) has attracted the growing attention of researchers, because of the steadily rising incidence (51). The incidence is remarkably variable geographically and dependent on the ethnic background (Figure 3) (52). TGCT is currently the most common malignancy of young men of white European ancestry, while it is relatively rare among Asians and Africans (52, 53, 54). Interestingly, indigenous Maoris of New Zealand have a higher incidence of testicular cancer than the white population (55).

Figure 3. Global incidence rates of testicular cancer.  Age-standardized incidence rates for testicular cancer for men of all ages in selected countries of all regions of the world, extracted from national registries. National reporting systems varied by country, and data quality may have fluctuated between regions. Reprinted from Znaor et al., Eur Urol 2014 (52).

However, the incidence rates are not stable and have been changing over time. Within populations rapid changes have been observed in recent decades. For example, the highest rates of testicular cancer have historically been observed in Norway and Denmark but much lower in the Eastern and Southern Europe. While the incidence in Denmark has been attenuating since the 1970s, an alarming rise has been noted in Finland and in Slavic Balkan countries, especially Slovenia and Croatia (52, 53, 54, 56). Similarly, a rapid increase has been noted among the Hispanic/Latin population in the United States (57). Another interesting feature of epidemiology of testicular cancer is the so-called birth cohort effect; the rise in the incidence correlated with the calendar year of birth rather than with the age at diagnosis (58). For unexplained reasons, cohorts born at wartime had lower incidence than men born just before or after the war (58).

As mentioned before, testicular cancer has an unusual age-specific incidence rate with a small peak in the postnatal period and a major peak in young adult age, starting at puberty. These periods coincide with an activation of gonadal hormones, indicating there may be a possible connection between the hormonal factors and invasive transformation of germ cells. In certain risk groups, the incidence of testicular cancer is much increased. Individuals with developmental abnormalities of the gonads and sex differentiation (DSD) are at high risk of developing germ cell neoplasia. Among these, individuals with the so-called mixed gonadal dysgenesis and the 45,X/46,XY karyotype are at particular risk of harboring GCNIS or gonadoblastoma and developing TGCT in adolescence (18, 19, 20, 23, 59, 60). An association with testicular cancer has been noted in a number of developmental abnormalities, such as cryptorchidism (61, 62), Down syndrome (63) but also low birth weight and unspecific perinatal factors, e.g., premature birth, birth order, high levels of maternal estrogens or bleeding during pregnancy, high maternal age and neonatal jaundice (62, 64). A late age at puberty and tall stature were also associated with lower and higher risk of testicular cancer, respectively (65, 66).

For endocrinologists and andrologists it is essential to know that male infertility (the type linked to TDS) is one of the commonest risk factors for testicular cancer (67). Men with TGCT have poorer semen quality and significantly fewer children than controls prior to development of their tumor (68, 69), see also the section on TDS below.

Patients with a unilateral TGCT are also at an increased risk to develop a new primary testicular tumor of the contralateral testis. Depending on the studied population and the interval from the primary diagnosis, approximately 2 - 5% patients will be diagnosed with a second TGCT during their lifetime (70, 71, 72, 73). The majority of bilateral TGCT occur metachronously (> 6 months after the primary TGCT), some as late as 20-40 years after the primary TGCT (74). The histology of the primary tumor influences the risk only marginally, with the predominance of seminomas among the synchronous TGCTs but primary nonseminomas observed most often in younger patients with metachronous tumors developing after shorter intervals (70, 72). The presence of testicular atrophy increases the risk of bilateral neoplasia considerably (75, 76).

Testicular Dysgenesis Syndrome (TDS)

The association of testicular cancer with poor testicular function, cryptorchidism, hypospadias, and abnormal testicular development led to a hypothesis that poor gonadal development and testicular neoplasia are etiologically linked. A concept of TDS was proposed, in which testicular cancer is one of the symptoms, in addition to other phenotypes, including cryptorchidism, hypospadias, shortened anogenital distance (AGD), reduced Leydig cell function, and decreased spermatogenesis (3, 5). This hypothesis is supported by histological studies showing that dysgenetic features, such as undifferentiated tubules with visibly immature Sertoli cells, clusters of poorly differentiated tubules, or hyaline bodies, often seen in association with testicular cancer, are not uncommon among men referred to andrology clinics because of fertility problems (3, 5, 18, 71). It is important to underline that not all cases of genital malformations and infertility are a part of TDS. Milder phenotypes linked to impaired development of the testis in fetal life due to diverse environmental or lifestyle-related factors can be considered part of TDS (5, 77). Severe genital malformations caused by genetic disorders are clearly a part of DSD but there is a partial overlap between the two syndromes (18). However, TDS severity and prevalence are undoubtedly modulated by the genetic variability (polymorphisms) and epigenetics (5). A schematic representation of the pathogenesis and manifestations of TDS is depicted in Figure 4.

Figure 4. Schematic illustration of aetiology and pathogenesis of disorders grouped within Testicular Dysgenesis Syndrome (TDS). The TDS concept implicates disturbed function of testicular somatic cells (Leydig- and Sertoli cells) caused by inherited genetic variation (gene polymorphisms) in combination with environmental /lifestyle factors acting during early development. Dysfunction of the somatic cells results in disturbed hormonal homeostasis and causes impaired germ cell differentiation. Depending on the severity of the impairment, multiple outcomes or phenotypes may occur, ranging from reduced anogenital distance (AGD), genital malformations to testicular cancer. Note that the most severe forms of TDS (disorders of sex development with gonadoblastoma (GDB) or GCNIS are the least frequently seen, whereas the mildest forms, such as impaired spermatogenesis are quite common. Modified from Skakkebæk et al., Hum Reprod 2001 (3) and Physiol Rev 2016 (5).

Genomics of TGCT and Predisposing Polymorphisms  

Tumors derived from germ cells via GCNIS stage are characterized by the presence of a nearly universal aneuploidy (polyploidization) and amplification of chromosome 12p, often in the form of an isochromosome (8, 78). Additional secondary genomic aberrations in invasive TGCT include rare recurring chromosomal gains or losses (in particular loss of chromosome Y), and fusion transcripts (79, 80). Strikingly few oncogenic mutations have been reported in TGCTs, and the short list of affected genes includes only KIT (predominantly in seminomas) and KRAS (mainly in nonseminomas) (79, 81, 83).

Although testicular cancer in most cases is a sporadic disease, the familial risk of testicular GCT is among the highest when compared to other cancers; brothers and sons of TGCT cases have an 8-10-fold and 4-6-fold increased risk, respectively (84). A twin study from Nordic countries estimated the heritability of testicular cancer as 37%, with 24% attributed to shared environment (85). Recent studies using next generation sequencing technology confirmed the absence of specific oncogenic driver mutations that would explain a high heritable risk of testicular cancer (86). The bulk of inherited risk is instead caused by a constellation of unfavorable gene variants, which jointly account for estimated 44% of testicular cancer heritability (87). Men with a polygenic risk score in the 95^th percentile have a 6.8-fold increased risk of TGCT compared to men with median scores (87). This estimation is based on numerous genome-wide association studies (GWAS) performed in recent decades, which revealed a large number of significant gene variants (markers) associated with TGCT risk (87, 88, 89, 90, 91, 92, 93). The strongest association, which was identified in all GWAS, and most interesting from the biological point of view, is with a cluster of single nucleotide polymorphisms (SNPs) within or near KIT ligand gene (KITLG) and in genes downstream of the KIT/KITLG/MAPK signaling pathway, e.g., SPRY4. This pathway is essential for germ cell migration and survival, is highly active in GCNIS, with the frequent secondary gain-of-function mutations identified in seminomas (33, 81, 82, 83, 89, 94, 95). Among other significant SNPs associated with TGCT risk, the most interesting are DMRT1, a transcription factor involved in sex differentiation and regulation of meiosis (96, 97), PRDM14 and DAZL, factors involved in primordial germ cell specification and differentiation, AMHR2, the receptor for anti-Mullerian hormone (AMH), AR, the androgen receptor, and several genes in the telomerase and DNA repair pathway (87, 90, 91, 92, 93). It is biologically important that several predisposing variants are potentially associated with germ cell differentiation and testis development, including hormone regulation, and there is an overlap with some pathways involved in TDS. 

Current Views on the Environmental Etiology of TGCT

In spite of significant recent inroads into the understanding of the pathogenesis of TGCT, etiology of these tumors, and especially the reason for the rise in incidence, remains obscure. The high incidence of testicular cancer in subjects with congenital errors of gonadal development strongly implicates the involvement of intrauterine factors and perinatal factors. We believe that the neoplastic transformation of male germ cells occurs during their pre-meiotic development, and this happens preferentially in individuals with genetic susceptibility. GCNIS cells and primordial germ cells share some distinct features, such as expression of embryonic pluripotency factors, low DNA methylation and constellation of miRNAs and histone modifications (4, 5, 17, 27, 28, 37, 39, 99, 100).

The mechanisms of neoplastic transformation of early germ cell are not known. There is a growing consensus that there may be multiple mechanisms and testicular cancer is a multifactorial and polygenic disorder. A disturbance in the fetal programming of gonadal development may be a result of an intrauterine hormonal imbalance, which in turn may be caused by a genetic disorder or by an impact of an exogenous factor targeting a key pathway, e.g., androgen signaling, KIT-KITLG signaling, DMRT1 signaling and regulation of meiosis, the TGF-beta superfamily regulation (including Nodal pathway) or the WNT pathway, leading to a delay in the testis development and maturation of fetal gonocytes (3, 4, 91, 98, 94, 101, 102, 103).

As mentioned above, the rising and quickly changing incidence of testicular cancer in well-developed countries suggests a possible adverse influence of environmental or lifestyle-related causative factors. In recent decades a great number of potent natural and synthetic hormones and hormone antagonists have been identified in environment. Observations in wildlife and experiments in laboratory animals exposed to synthetic hormones and a broad range of endocrine disrupters suggest that these substances can cause a disturbance of hormonal milieu of the developing gonad and disturb differentiation of early germ cells (3, 4, 5, 104, 105, 106).  Whether or not the endocrine disrupters have contributed to the rise in testicular cancer remains to be demonstrated. Human exposure studies are difficult, mainly because of the long interval between the vulnerable period of early development and manifestation of cancer development in adulthood. There is also the problem of the plethora of confounding factors, including mixtures of possible contaminating factors present in food, packaging, cosmetics, and other products of daily life. Therefore, the evidence for the role of endocrine disrupters in the etiology of TGCT remains scarce. Among few existing data one can mention a higher serum level of some persistent organic pollutants found in mothers of men with testicular cancer (107), and a greater burden of p,p′-DDE (a DDT metabolite) in serum of testicular cancer patients (108). For more information on the existing evidence and possible mechanisms the reader is referred to recent review articles on the subject (109, 110). It is clear that further studies in large cohorts of patients and controls are needed to identify the causative factors behind the observed testicular cancer epidemics. Few data exist concerning postnatal exposures and risk of TGCT. A heavy use of marijuana (cannabis) has been consistently reported as a risk factor for nonseminoma but the mechanism remains to be elucidated (111, 112).

Diagnosis and Staging of Testicular Germ Cell Neoplasms

PREINVASIVE LESIONS AND TESTICULAR BIOPSY

All attending physicians should consider a possibility of incipient germ cell neoplasia in men with infertility, especially those with small testes, a history of cryptorchidism, or previous testicular tumors, or in individuals from the high-risk group of DSD (5, 68, 69). The preinvasive stage of GCNIS is asymptomatic, so diagnosis at this early stage is sporadic. Ultrasonographic examination can be helpful in some cases. GCNIS or incipient TGCT are frequently associated with an irregular echo pattern or microcalcifications /microlithiasis (113, 114, 115, 116). However, ultrasonic microlithiasis is very common and can be present in normal men or patients with mild disorders, so it alone is not an indication for a testis biopsy. A biopsy is indicated if microlithiasis is accompanied by additional risk factors, e.g., history of cryptorchidism, a family history of testicular cancer, atrophic testis (<12 mL), or poor semen quality, especially azoospermia (117, 118). Surgical testicular biopsy is currently the only sure diagnostic procedure for GCNIS diagnosis (119). Experience in the use of needle biopsies for GCNIS diagnosis is limited and this method is not commonly used in the clinic. Regardless of the method of obtaining the testis tissue, examination for GCNIS by immunohistochemistry in a diagnostic biopsy or any leftover material after testicular sperm extraction in fertility clinics is now considered mandatory (119, 120, 121).

GCNIS is present in the testis contralateral to a testicular tumor in approximately 5% of cases, with a range of 4% to 8%, depending on the studied cohort (71, 75, 121, 122). Screening for the contralateral GCNIS to prevent the appearance of metachronous bilateral TGCT has been practiced in several countries, including nationwide in Denmark and in some centers in other European countries (Germany, Austria, the Netherlands). Most of other centers adopted a recommendation of the European Association of Urology (123) to take contralateral biopsies only in patients at risk for GCNIS presence, such as by a history of cryptorchidism, a small testis volume (<12 ml), or testicular microlithiasis (75, 76). In the USA and Canada, the contralateral biopsy is rarely practiced, and American guidelines recommend this procedure only if the contralateral testis is cryptorchid, shows marked atrophy, or suspicious ultrasonic changes, but not microlithiasis alone (124, 125). Our recommendation is to offer a contralateral biopsy to all patients at the time of surgery for the primary TGCT, perhaps with the exception of men older than 50, because of a minimal risk of GCNIS. Added benefits of a biopsy are a good assessment of the fertility potential of the other testicle, and the peace of mind for the patient concerning the possibility of bilateral cancer (126). Mild post-biopsy complications (3%) are rare and the procedure is well accepted by the patients (127). In young men with normal size testicles, two-site biopsies may be considered to decrease a chance of a false-negative biopsy (126). False-negative cases are relatively rare (128) but the procedure does not completely eliminate the risk of the second TGCT. In the Danish nationwide study, the cumulative incidence of the metachronous TGCT was 1.9% (after median 20-year follow-up) compared to 3.1% in a non-biopsied earlier cohort of patients (121). However, in a sub-cohort of Danish patients, in whom the contralateral biopsy was performed with a quality control and obligatory immunohistochemistry with GCNIS markers, the cumulative rate of the second cancer (after similar follow-up period rate) decreased to 0.95% (121).  

Technical aspects of surgical biopsy are important. The tissue fragment has to be sufficiently large (approx. 3x3x3 mm) and care has to be taken to avoid damage to the specimen, which should be dropped directly to a container with a fixative solution. For morphology evaluation Bouin's or similar solutions are preferred because formalin causes shrinkage artefacts, while for immunohistochemistry buffered formalin is preferred. As for any diagnostic testis biopsy, the contralateral biopsy evaluation must include immunohistochemical staining for at least one (better two) of the known markers for GCNIS, e.g., placental-like alkaline phosphatase (PLAP), OCT-3/4 (POU5F1), PDPN/M2A/D2-40, or AP2γ-TFP2AC (120, 122, 129). An example of immunohistochemical PLAP staining for GCNIS detection is shown in Figure 1.

SEMEN ANALYSIS

Poor spermatogenesis is a good indication that the patient may be at risk of harboring GCNIS (5, 68, 69, 75, 76, 130, 131). Semen analysis cannot as yet be used alone for detection of early stages of testicular cancer. However, it has been known for a long time that GCNIS cells may be occasionally found in semen (132). Over the years, progress has been made towards establishing better quality immunocytochemical detection of GCNIS or early invasive intratubular tumor cells in semen, and an improved automated double-staining assay has been developed (133, 134, 135). Unfortunately, the sensitivity of the cytological method is not high enough for screening, but it can be used as an additional part of semen analysis in experienced andrological centers performing immunohistochemistry (135). It is also not possible to use a micro-RNA (miRNA)-based assay, especially miRNA-371a-3p, a very promising serum marker for invasive TGCT, for detection of GCNIS in seminal fluid, despite that this embryonic-type miRNA is secreted by GCNIS cells (37). Even though some patients with GCNIS can have measurable miR-371a-3p in serum (136), the presence of the same miRNAs in normal germ cells in control patients with non-malignant conditions precludes using this assay in seminal fluid (137, 138, 139).

SERUM TUMOR MARKERS, DIAGNOSIS AND MONITORING OF OVERT TGCT

In the vast majority of cases a scrotal mass is usually the first presentation of testicular cancer, with tenderness reported by very few patients. In a few percent of testicular cancer cases, the presenting symptoms are the result of metastatic disease. They are usually uncharacteristic and may include lumbar pain, palpable abdominal mass, supra-clavicular lymph node enlargement, and in rare cases pulmonary symptoms (1).

The majority of primary and metastatic germ cell tumors secrete proteins and other biochemical products that can be detected in circulating blood. These biochemical serum tumor markers are very helpful in diagnosis and monitoring of testicular cancer (38, 124, 140).

The most important serum markers established in clinical practice are human chorionic gonadotropin (HCG), alpha-fetoprotein (AFP), and lactate dehydrogenase (LDH), the first two mainly useful to detect nonseminomas (1, 140). Among nonseminomatous tumors, choriocarcinoma, which resembles a gestational trophoblast, produces large quantities of HCG, while yolk sac tumor, which is similar in morphology to the embryonic yolk sac, secretes AFP (38, 140, 141). In addition, LDH may be secreted by both seminomas and nonseminomas. LDH levels in serum tend to be higher in patients harboring tumors with an increased copy number of chromosome 12p, consistent with the genomic location of the LDHB gene (142). Increased concentrations of LDH in the absence of AFP and HCG suggests the presence of seminoma. It is important to keep in mind that TGCT rarely occur in pure histological forms, and a subtype of seminoma with syncytiotrophoblastic cells can secrete HCG, which can be measurable in patients’ blood (14, 143). In preinvasive GCNIS and in most cases of pure seminoma, none of the above-mentioned markers are detectable in serum.

The most promising currently emerging serum markers are micro-RNAs (miRNA), among which miRNA 371a-3p is the best studied and most robust marker, and has already been proven valuable for detection of seminomas and nonseminomas, except for differentiated pure teratomas, in children and adults (38, 144, 145). Several large clinical studies clearly demonstrated that miRNA 371a-3p test has a much better specificity than the classical serum markers, and is especially useful for detection and monitoring of HCG- and AFP-negative seminomas (146, 147).

After diagnosis, careful clinical staging is necessary in each patient to decide for the most appropriate treatment strategy, and the measurements of circulating tumor markers are an important part of this process (1, 123, 124). There is a tendency for higher levels of tumor markers to be associated with a poorer prognosis. For example, the presence of syncytiotrophoblastic cells in the subtype of seminoma can lead to a mild increase of serum HCG, but the tumor responds well to treatment regardless of the presence of these cells, so more aggressive management should be avoided.

In addition to serum markers, scrotal ultrasonography is the first line diagnostic procedure. In general, seminoma is a homogenous tumor, whereas nonseminomas usually display heterogeneous patterns, with frequent hyperechoic calcified areas (148, 149). Additional imaging procedures help to evaluate the spread of disease, including CT scan of the chest and abdomen, or MRI (148, 149, 150).

Histopathological evaluation of the orchiectomy specimen is an essential part of the clinical staging. The important risk factors are tumor size, vascular/lymphatic invasion or the presence of tumor cells in rete testis, and the presence of pure embryonal carcinoma (152, 153).

The most commonly used staging and prognostic classification system is the TNM (tumor, node, metastases) System of the International Germ Cell Cancer Collaborative Group (GCCCG) and the American Joint Committee on Cancer (AJCC) (154). The stage grouping updated by the WHO Consensus (2), is shown in Table 2.

Footnote: pT=primary tumor, N=regional lymph nodes, M=distant metastasis, S=serum tumor markers (X=unknown, not available)

Management of GCNIS and Testicular Germ Cell Cancer  

Early diagnosis of testicular neoplasia at the stage of GCNIS, followed by adequate treatment of GCNIS is capable of preventing progression to invasive tumors. Unfortunately, the vast majority of cases progress unnoticed to overt tumors. However, germ cell tumors are extremely radio- and chemo-sensitive and have apparently very high propensity to apoptosis, likely mediated by p53 (155, 156). Because of this sensitivity, and thanks to cisplatin-based chemotherapy regimens, TGCT is a highly curable malignancy, with more than 90% of patients reaching a sustained complete remission (157).

We give here only very general information concerning management of testicular cancer. The reader should consult specialized oncologic and urologic literature for management options pertinent to treatment-resistant tumors and metastatic disease.

TREATMENT OF GCNIS

Spontaneous regression of GCNIS has never been described and it is anticipated that all cases of GCNIS will eventually develop into TCGT. The following management options for GCNIS are available depending on a specific situation (158):

• Orchiectomy - is the curative treatment with the highest assured success rate. It should always be performed on a testis with GCNIS or localized tumor when the second testis is not affected by malignancy (including GCNIS).
• Radiotherapy - low dose radiotherapy is a good alternative to orchiectomy when GCNIS is present bilaterally or in the contralateral testis, so the patient can be spared a bilateral castration and lifelong androgen replacement therapy. The efficacy of radiotherapy with doses as low as 16 Gy was demonstrated in the early studies (159). Even though the lower dose better preserves the function of Leydig cells (160), more recent studies and current EAU guidelines recommend a dose of 18 Gy, given in fractions of 2Gy (123, 161). This dose of radiation will almost always destroy normal germ cells, so radiotherapy may be delayed in patients who wish to secure natural conception of a child.
• Chemotherapy - is not an option to treat GCNIS, because a persistence or relapse has been reported in a high proportion of patients (121, 161). Furthermore, in some cases of extragonadal germ cell tumors treated with chemotherapy, testicular GCNIS progressed to metachronous overt testicular tumors (162). However, in patients with disseminated disease receiving chemotherapy, the risk of metachronous bilateral TGCT is lower (73, 163, 164).
• Surveillance - is potentially hazardous since it increases the risk of metachronous bilateral TGCT (164) and GCNIS may progress to invasive cancer at any time. However, watchful surveillance may be an option after careful informed discussion of risks and monitoring with ultrasound examinations, especially if the patient wishes to defer treatment temporarily for the purpose of paternity.

In men who desire fatherhood, and in all very young men, cryopreservation of semen samples should be offered, if viable spermatozoa are detected.

TREATMENT OF OVERT SEMINOMAS AND NONSEMINOMAS

As far as the treatment of invasive germ cell tumors is concerned, the reader should consult the specialized urology and oncology guidelines.

Prior to surgery, all patients with TGCT should be offered full andrological evaluation and cryopreservation of semen. Endocrinologic evaluation of the patient should also preferably be done before surgery, with the reproductive hormone profile, including serum testosterone, gonadotropins, and inhibin B, if possible. This can be done together with the above-mentioned measurements of serum tumor markers. Pre-operative semen analysis and cryopreservation is especially important in patients with bilateral tumors or only one testicle. In the patients who have azoospermia or cryptozoospermia, and in whom it was impossible to retrieve sperm pre-operatively, testicular sperm extraction (TESE) at the time of orchiectomy (‘onco-TESE’) may be attempted in specialized centers (165). The best predicting factor of sperm retrieval is a small size of the tumor, because a chance of finding tubules with ongoing spermatogenesis increases with the distance from the tumor (166).

Radical orchiectomy remains the primary surgical treatment method of choice. Primary dissection of retroperitoneal lymph nodes (RPLND) was previously commonly practiced, especially in North America. The current consensus is to perform RPLND in experienced high-volume centers, and in selected cases, mainly in patients with nonseminoma and intermediate prognosis (1, 167, 168, 169). Nerve-sparing technique is preferred in order to avoid the loss of antegrade ejaculation, which is the commonest long-term complication of RPLND (169). 

Methods of post-surgical management of overt TGCT are variable, depending upon the histological type of tumor (seminoma vs nonseminoma), levels of serum markers and stage of disease and the presence of residual retroperitoneal masses (1, 115, 151, 167). Adjuvant radiotherapy, e.g., of the retroperitoneal/para-aortic field, which was previously routinely used, is no longer recommended, because of the long-term risk of secondary malignancies (1, 167, 168). 

Pure seminomas have a good prognosis and around 80% of patients with stage I seminoma (tumor confined to the testis) do not require any treatment after the surgery, thus most of the centers practice a surveillance strategy (151, 168, 170). Nonseminomas have a somewhat poorer prognosis (relapse rate is around 30%) and in some centers, patients with nonseminoma and high risk of relapse are treated with one course of adjuvant chemotherapy with cisplatin and etoposide (171, 172), but most centers currently recommend a surveillance strategy (1, 151, 152, 168, 172).

The most common post-surgical management of disseminated disease is systemic chemotherapy with a combination of cytotoxic drugs. The standard first line chemotherapy regimen is BEP (bleomycin, etoposide and cisplatin), administered in 3 or 4 cycles, depending on the patient’s prognosis (173, 174). It is very important to make a dynamic assessment of the progress of treatment through the early stages of chemotherapy, thus monitoring of serum markers is obligatory. Post-chemotherapy retroperitoneal surgery should be performed in patients with a residual tumor after BEP (151). In patients with relapse after first line treatment, salvage regimens and complex surgery for residual tumors need to be performed. Overall, the management is difficult, thus it should be carried out in specialized tertiary centers (1, 167).

The majority of relapses occur within 2 years of the initial treatment but late relapses are observed in some cases, therefore individual management of each patient and lifetime follow-up is advocated in some patients (1, 151, 167). Andrological follow-up should be coordinated with the post-surgery oncological management and controls (see the section on ‘Endocrine problems and late effects’).

SEX CORD-STROMAL TUMORS OF THE TESTIS

In adults, sex cord-stromal tumors of the testis are found in less than 5% of all testicular tumors, whereas in children, these tumors are more common and account for approximately 25% of cases (123, 175, 176, 177, 178). Most of these tumors are benign, only around 5% have malignant characteristics (2). The classification of the tumors by WHO (2016) is shown in a simplified form in Table 3 (2).

Sex cord-stromal tumors are derived from testicular somatic cells; Leydig cells, and Sertoli/granulosa cells. Despite a different cell of origin, some stromal tumors are sometimes misinterpreted as seminoma. A number of features can be used to distinguish sex cord-stromal tumors from germ cell tumors; Inhibin A and B are the best serum markers for this purpose (179, 180, 181). Inhibin A appears to be a common immunohistochemical marker for sex cord-stromal tumors, including Leydig cell, Sertoli cell and juvenile granulosa cell tumors (179, 182). Sertoli cell tumors are positive for anti-Mullerian hormone (183) and GATA-4 (184), while Leydig cell tumors express steroidogenic enzymes and INSL3 (185). Other useful immunohistochemical markers, include SOX9, calretinin, CD99, SF1 (2).

*Footnote to Table 2: The codes in parentheses are from the International Classification of Diseases for Oncology (ICD-O-3).

Leydig Cell Hyperplasia and Tumors

Leydig cells are located in the interstitial compartment of the testis and are involved in the development of secondary male characteristics and maintenance of spermatogenesis by secretion of testosterone. Although Leydig cells in adult men are considered to be a terminally differentiated and mitotically quiescent cell type, in various disorders of testicular function, focal or diffuse Leydig cell hyperplasia is very common.  Micronodules of Leydig cells are frequently seen in certain conditions associated with severe decrease of spermatogenesis or germinal aplasia, such as the Sertoli-cell-only syndrome (Del Castillo syndrome), cryptorchidism, or Klinefelter syndrome, when the nodules can be particularly florid (186, 187). A term 'Leydig cell adenoma' is used when the size of a nodule exceeds several- fold the diameter of a seminiferous tubule. It is unknown whether Leydig cell adenomas can progress further to form overt Leydig cell tumors, but even if it were the case, it is exceedingly rare. Morphological heterogeneity of hyperplastic Leydig cells is noticeable in some cases, and it has been shown that the micronodules contain a large proportion of immature Leydig cells (186, 187).

The mechanism of Leydig cell hyperplasia and larger nodules in the human male is still poorly understood. Leydig cell nodules of variable-size are common in patients with TDS and infertility, and the functional insufficiency of Leydig cells is often reflected by decreased testosterone/LH ratio whereas patients with overt Leydig cell tumors usually have an increased testosterone/LH ratio and high estradiol (186, 188). The disruption of hypothalamo-pituitary-testicular axis leading to an excessive stimulation of Leydig cells by LH can play a central role (186). This in turn leads to an increased renewal of immature, adult-type Leydig cells from their precursors. The immature cells are characterized by low numbers of Reinke crystals, a relatively high expression of a mesenchymal factor DLK1 and low amounts of INSL3 (187, 189, 190). However, Leydig cell hyperplasia is distinct from tumors that are usually solitary. Leydig cell hyperplasia and adenomas can be easily induced in rodents by administration of estrogens, gonadotropins, and a wide range of chemical compounds. Whether or not humans would be similarly susceptible to environmental effects remains to be elucidated.

Leydig cell tumors account for up to 5% of testicular neoplasms and occur in all age groups (2, 178, 191). Approximately 20% are found in boys, most often between five and ten years of age. There is perception that the prevalence of benign Leydig cell tumors among the adults is greater than previously thought, possibly thanks to improved detection of small nonpalpable nodules by modern imaging methods. Higher incidence numbers have also been reported in fertility centers that actively scan the testes of infertile patients (192).

In a subset of cases of Leydig cell tumors, activating mutations of the LH receptor (193, 194, 195) or G proteins (196, 197) can be detected. Constitutively activating mutations of LH receptor cause early Leydig cell hyperplasia and precocious puberty (193, 195, 197). Similarly, constitutively activating mutations of Gs-protein in Leydig cells or inactivation of PKAR1A (protein kinase cyclic adenosine monophosphate-dependent regulatory type 1 alpha) lead to hyperplasia and endocrine hyperactivity (178, 197). In adult Leydig cell tumors, germline fumarate hydratase mutations have been identified in a few cases which also had hereditary leiomyomatosis and renal cell cancer (199). 

Precocious puberty (so-called testotoxicosis) is the presenting symptom in most of cases of Leydig cell adenomas or tumors in children, due to the excessive androgen production, mainly testosterone that causes growth of penis, pubic hair, accelerated skeletal and muscle growth, advancement of bone age, skin changes (acne, comedos, hair greasing), and adult-type odor of sweat. Androgen secretion in the pediatric cases is gonadotropin independent, and therefore LH and FSH remain low in spite of external signs of puberty (195). Approximately 10% of the boys also have gynecomastia that is caused by estrogens produced in excess due to aromatase activity in some of the tumors or peripheral aromatization of testosterone. It is important to keep in mind that transient gynecomastia or pseudo-gynecomastia can occur in newborns, in obese boys, and at puberty as a non-pathological condition (200). In adults, gynecomastia is a frequent condition with a reported prevalence of 32–65%, depending on the age and the criteria used for definition (summarized in 201). Gynecomastia is sometimes associated with loss of libido, impotence, and infertility (201). However, Leydig cell tumor is the cause in only 1-2% of cases, and the excessive androgen secretion by these tumors rarely causes notable effects in adults (202). Malignant tumors are hormonally active only in exceptional cases (2, 178).

In children, Leydig cell tumors are always benign and can be treated with surgical enucleation when the tumor is encapsulated (203). In adults malignant Leydig cell tumors have been found in 10-15 % of patients, and inguinal orchiectomy is often used (204), while testis-sparing enucleation remains an option, if there are no signs of malignancy in the frozen preparation (205). The presence of cytologic atypia, necrosis, angiolymphatic invasion, increased mitotic activity, atypical mitotic figures, infiltrative margins, extension beyond testicular parenchyma, and DNA aneuploidy are associated with metastatic behavior in Leydig cell tumors (178, 202, 206) (see Figure 5).

Figure 5. Histology of a Leydig cell tumor.  The appearance of tumor cells resembles normal Leydig cells. A section stained with hematoxylin-eosin (HE).

Malignant Leydig cell tumors do not respond favorably to conventional chemotherapy and irradiation (204). Survival time has ranged from 2 months to 17 years (median, 2 years), and metastases have been detected as late as nine years after the diagnosis (178, 202). Metastatic Leydig cell tumors require salvage surgery, radiotherapy and intensive chemotherapy regimens but in most cases the outcome is poor (188, 207). Therefore, follow-up of patients with malignant Leydig cell tumors has to be life-long. The remaining testis may be irreversibly damaged by longstanding high estrogen levels, resulting in both permanent infertility and hypoandrogenism (202, 206).

Testicular Adrenal Rest Tumors (TART)

Excessive secretion of adrenocorticotropin (ACTH) in 21-hydroxylase deficiency (congenital adrenal hyperplasia, CAH) or Nelson syndrome (post adrenalectomy status) may lead to development of hyperplastic interstitial nodules called testicular adrenal rests tumors (TART) resembling Leydig cell tumor or hyperplasia (191, 208, 209). These cells are hormonally active in secreting androgens. It is important to remember that the adrenal rests are almost invariably bilateral, whereas the Leydig cell tumors are usually unilateral. Adrenal rests can be treated by appropriate glucocorticoid substitution of the patient, which leads to gradual regression of the 'tumor' in 75 percent of cases (208, 209). TART have to be distinguished from benign hyperplasia, adenomas and malignant Leydig cell tumors (185).

Histopathological and endocrine evaluation covering both testicular and adrenal steroids and pituitary gonadotropins and ACTH are important in the differential diagnosis (185, 208). It would be an error to orchidectomize the CAH patients with TART, since the tumors are always benign and only some of them continue to be active after appropriate glucocorticoid substitution. In the patients who do not respond well to glucocorticoid replacement, or develop fibrosis, testis sparing surgery with enucleation of the larger nodules can be considered, if the testicles have become so large that they cause discomfort for the patient (209).

Sertoli Cell Tumors

Sertoli cells are the somatic cells in the seminiferous epithelium giving structural, metabolic, and hormonal support to spermatogenic cells. Sertoli cells cease their proliferation at puberty. In rare infantile cases, multiple foci of proliferating Sertoli cells have been described and proposed to be early intratubular forms of Sertoli cell tumors (210). The classification of these tumors according to the WHO (2, 178), is shown in Table 3.

Sertoli cell tumors often occur as a part of multiple neoplasia syndromes (see below). Rare Sertoli cell tumors which do not belong to a syndrome are called ‘not otherwise specified’ (NOS). About 5% of these tumors are malignant and can metastasize. The age of patients ranges from 18 to 80 years, but most of them are young adults (median age 30). Out of 60 patients, only four were younger than 20 years old in the series reviewed by Young et al. (211). The tumors typically are composed of sex cord cells with tubular differentiation, with a subset of tumors hyalinized, previously classified as sclerosing variant (2).  Some tumors often contain lipid droplets but do not show any endocrine activity. The molecular origin is known only in a small proportion of tumors with sclerosing appearance, in which CTNNB1mutations causing nuclear accumulation of β-catenin were identified (212). The tumors occurred in descended testes and were always unilateral. An infiltrative margin was found in four cases, but most of the tumors were well demarcated. The tumors were hormonally inactive, and only two patients with alcoholic cirrhosis also had gynecomastia. Eighteen pediatric cases were reported from the Kiel Pediatric Tumor Registry (176), but perhaps the histopathologic pattern was somewhat different, because the age of the children was very young, ranging from 0 to 14 months (median 4 months). Juvenile Sertoli cell tumors often showed infiltrative growth into adjacent tissue, dense cellularity and considerable proliferative activity. However, after surgical excision no local recurrences and no metastases occurred. Thus, these patients have a good prognosis. These Sertoli cell tumors can be treated by orchiectomy, and retroperitoneal lymphadenectomy is indicated only when there is radiographically detected retroperitoneal involvement (213).

Calcifying Sertoli cell tumors, are frequently found in association with two distinct multiple neoplasm syndromes, Carney complex and Peutz-Jeghers syndrome, however in the latter syndrome, the tumors belong to a distinct ‘intratubular’ morphological group (2, 178, 214). Association of large-cell calcifying Sertoli cell tumors with other neoplasms, particularly heart myxomas in Carney complex and gastrointestinal tumors in Peutz-Jeghers syndrome, should be kept in mind to reach an early diagnosis of these potentially fatal diseases.

The Carney complex is characterized by skin myxomas, heart myxomas, skin pigmentations, adrenal and testicular tumors, but other tumors can also occur (215). The testicular tumors are large-cell calcifying Sertoli cell tumors that are multifocal and bilateral, and should be distinguished from teratomas (Figure 6) (178, 216, 217). The tumors appear usually during the second decade of life (218). Only one malignant case has been reported in association with Carney complex (in an adult patient), whereas seven malignant tumors were reported in other patients with large-cell calcifying Sertoli cell tumors (218). These patients were older than 25 years. The malignant cases were unilateral and solitary in contrast to bilateral and multifocal occurrence of testicular tumors in Carney complex. Large-cell calcifying Sertoli cell tumors are usually not hormonally active, although elevated levels of serum inhibin B or testosterone have been reported, but other tumors of Carney complex, including Leydig cell tumors, can cause endocrine manifestations (181, 213, 214).

Figure 6. Large cell calcifying Sertoli cell tumor isolated from a 12-year-old boy. The neoplastic tubules contain only large pale Sertoli cells and visible calcifications in the lumen (stained with PAS). Adjacent normal tubules show advanced spermatogenesis.

Two genetic loci for Carney complex have been identified on chromosome 2p16 (196) and 17q23-24 (219, 220). Germline mutations identified in Carney complex most often occur in type I-alpha regulatory subunit of protein kinase A, PRKAR1A (221, 222). Inactivating mutations of phosphodiesterase 11A212 and phosphodiesterase 8B213 are associated with bilateral adrenocortical hyperplasia in Carney complex patients. Genetic variation of the phosphodiesterase 11A gene can modify the development of the testicular tumors (223). Molecular genetic diagnosis is available to many of these patients. However, only a part of the mutations occur in the germ line, and therefore genetic analysis should be performed on affected tissues, such as the tumors, in cases of somatic cell line mutations.

In Peutz-Jeghers syndrome Sertoli cell tumors are similar in appearance to those found in Carney complex patients, but can be distinguished by typical intratubular proliferation of lightly eosinophilic cells with prominent basement membrane deposits, and occasionally, features of ovarian sex-cord tumors with annular tubules (178). Thus, the tumors are described as intratubular large-cell hyalinizing Sertoli cell neoplasia. These tumors may have strong aromatase activity and therefore be associated with gynecomastia and advanced bone age (224). No malignant testicular tumors have been reported in Peutz-Jeghers patients, but they have a highly increased risk of other neoplasms, especially colorectal, breast, pancreatic and ovarian cancers (214). Germline loss-of-function mutations in the STK11/LKB1 gene that encodes for a serine-threonine kinase causes Peutz-Jeghers syndrome in the majority of patients, allowing molecular genetic diagnostics (214, 224, 225).

Patients with the Sertoli cell tumors should be treated conservatively during childhood to give them a possibility for sperm banking before orchiectomy comes necessary (226).  Autosomal dominant inheritance should be considered in genetic counselling. The patients should be frequently controlled with ultrasound examinations to follow changes in the tumors size, and reproductive hormone measurements. If precocious puberty and/or gynecomastia appear, aromatase inhibitors and antiandrogens can be used to prevent estrogen formation and androgen action (219, 226). There are currently no clear guidelines for the treatment of metastatic Sertoli cell cancers, but orchiectomy and retroperitoneal lymph node dissection are usually performed combined with individualized chemotherapy and radiotherapy (227). Without surgery, the metastatic disease progresses and 20-month median survival time of patients was reported (IQR: 6–30 months) (227).

 Granulosa Cell Tumors

Juvenile-type granulosa cell tumors are rare but are the most common somatic testicular tumors in infants and occur during the first 6 months after birth (2, 176, 178, 228). These tumors were described in patients with undescended testes with abnormal sex chromosomes and ambiguous genitalia (176). Prominent differentiation into follicles and immature nuclei distinguishes juvenile granulosa cell tumors from other Sertoli cell tumors that express tubular differentiation (178, 229). Most of the immunohistochemical markers in these tumor types are similar, e.g., inhibin, calretinin, but also distinct, e.g., expression of FOXL2 (229). Juvenile granulosa cell tumors always have a good prognosis (230). Testicular tumors do not show endocrine hyperactivity, in contrast to ovarian juvenile granulosa cell tumors. Aberrant WNT signaling (231) and stimulatory G-protein mediated signaling (232) have been linked with juvenile granulosa cell tumors. The patients should be managed comprehensively by a multidisciplinary DSD team, and the treatment of tumors may require orchiectomy.

Adult-type granulosa cell tumors are comparable to the ovarian tumors, but are extremely rare (2, 178, 233). These tumors occur in adults at an average age of 42 years. Twenty percent of the patients have shown gynecomastia due to the hormonal activity of the tumor. Most of the tumors are benign, but some malignant cases have also been reported (230). Mutations in FOXL2 have been reported in adult ovarian granulosa cell tumors but only few secondary mutations have been found in testicular granulosa cell tumors (234, 235). The treatment is primarily surgical.

Fibroma-Thecoma Tumors

These exceedingly rare neoplasms are composed of fibroblastic cells of testicular stroma or tunica albuginea (2). These tumors are reported to be benign.

Mixed and Unclassified Sex Cord-Stromal Tumors

Tumors consisting of more than one stromal or tubular component or have indeterminate morphology are classified as mixed and unclassified sex cord-stromal tumors (2). Sex cord-stromal tumors can contain combinations of Leydig, Sertoli, granulosa, and theca cells, and are therefore called mixed tumors (178). Leydig cells can be difficult to recognize in these tumors. These tumors are rare and can occur at any age. Depending on the predominant cell type the tumors may behave differently. Gynecomastia, as a sign of endocrine activity, can be found in 10% of patients (2). These tumors are always benign in children, but in adults, malignancy can be found (232). Thus, most of the patients can be treated by orchiectomy, and lymph node dissection is indicated only in cases with overt malignant features on microscopic examination.

OTHER TESTICULAR TUMORS

Other tumors occurring in the testis are divided into miscellaneous and hematolymphoid tumors (2, 178). The first group includes ovarian epithelial-type tumors, serous or mucinous cystadenomas, adenocarcinomas, Brenner tumor, xantogranuloma, and hemangioma. The hematolymphoid tumors comprise malignant lymphomas (B-cell, NK/T-cell or follicular), plasmacytoma, myeloid sarcoma and Rosai-Dorfman disease (2, 178). In addition, testicular spread of malignant acute leukemia is common in young boys, and metastases from other solid tumors including the prostate gland, colon, kidney, stomach, pancreas, and malignant melanoma can be found in the testis of adults.

ENDOCRINE PROBLEMS AND LATE EFFECTS IN TESTICULAR CANCER PATIENTS

Testis Dysfunction and Fertility Issues

Relative imbalance of androgen signaling (excess or deficiency) causes the most pronounced secondary endocrine symptoms associated with testicular tumors. Testosterone is produced by tumors, such as Leydig cell tumors, or by normal Leydig cells stimulated by large amounts of hCG from some germ cell tumors. Excess of androgens would lead to precocious puberty in children (193, 194, 195). In addition, aromatization of androgens leads to a relative excess of estrogens, which causes impairment of spermatogenesis in adults and gynecomastia at any age (224, 236).

Testicular dysfunction in young adult patients with testicular cancer, who usually are in their best reproductive age, is a serious clinical problem. Patients with TGCT have poor spermatogenesis and decreased fertility even before the overt tumor has developed (67, 68, 69, 130) and before cytotoxic treatment (237, 238). Pathological features can include oligozoospermia or azoospermia, moderately decreased testosterone and elevated LH levels. If testicular biopsies are taken, a variable degree of testicular dysgenesis or atrophy is often seen, and in some cases further complicated by the presence of GCNIS. Examination of a contralateral biopsy in a patient with a unilateral tumor may show a similar picture; with at least 5-7% risk of the presence of GCNIS cells (71, 126).

Testicular function is further disturbed by treatment of malignancy. In recent years there is growing concern about adverse late effects of irradiation and chemotherapy, which induce severe impairment of spermatogenesis and DNA damage (1, 167, 168, 239, 240).  Refinement of the dosage must be considered in each patient individually, to eradicate the neoplasm with least possible damage to the endocrine function. The eradication of GCNIS or a tumor by irradiation in bilateral cancer cases leads also invariably to the disappearance of all germ cells and sterility. This underlines the importance of semen cryopreservation before treatment, which will allow assisted reproduction treatment, if needed (238, 241).

All patients treated for testicular cancer require assessment of their reproductive hormones and spermatogenic capacity by semen analysis with respect to their future fertility. If semen banking or pre-operative sperm retrieval is not possible or if the patient has azoospermia or cryptozoospermia, testicular sperm extraction (TESE) at the time of orchiectomy (‘onco-TESE’) may be the only chance of fertility, and can be attempted in specialized centers (165, 166). TESE and subsequent intracytoplasmic sperm injection (ICSI) may be also an option for fertility treatment in some cases of post-chemotherapy azoospermia (242).

Fertility preservation is a serious challenge in children and young adolescents. In the adolescent boys who are unable to ejaculate, penile vibratory stimulation or electroejaculation can be considered (243). However, there is currently no treatment for prepubertal boys. There are ongoing studies attempting to optimize protocols of cryopreservation of immature testis tissue before cytotoxic treatment or orchiectomy (244). This approach would require in vitro or in vivoinduction of spermatogenesis and gametogenesis, which has not yet been achieved satisfactorily in humans, but studies in experimental animals, including nonhuman primates are promising (245).

Long-Term Sequelae and Quality of Life

All patients treated for testicular cancer require monitoring of their reproductive hormone profiles not only with respect to their fertility, if paternity is desired, but also risk of testosterone deficiency and ensuing symptoms (246, 247, 248).

An increased risk of cardiovascular disease is present in TGCT survivors who have been treated with radio- or chemotherapy (249, 250) but a recent Danish study found that the risk decreases during long-term follow-up, although it remains elevated (251). Additional problems in survivors of TGCT treated with radiotherapy or chemotherapy are increased risk of peripheral neuropathy or ototoxicity (after chemotherapy) and sexual dysfunction (after chemotherapy and radiotherapy) (239, 240, 250, 251, 252). 

Survivors of metastatic TGCT have increased mortality rates when compared to the general male population. A study from Norway reported that most of the excess deaths occurred within a decade from diagnosis due to the testicular cancer, but during a longer follow-up, additional excess deaths appeared in patients treated with radiotherapy or cisplatin-based chemotherapy, mainly due to non-TGCT second cancers; primarily gastric, pancreatic or bladder tumors (253, 254). A population-based large study from the US found an increased risk of leukemia after chemotherapy and a marked excess of solid tumors in patients treated with radiotherapy, which can appear after long follow-up (255). This study confirmed previously published worrying reports from several centers, which called for reducing the use of radiotherapy and radiologic imaging procedures during the follow-up (256).

Of importance for the testicular cancer survivors are also quality of life issues related to prolonged anxiety, depression and psychological stress, and loss in socioeconomic status or unemployment, leading to excess of suicide among the patients (167, 252, 254, 257, 258, 255, 259, 260). Hence, a psychologist/social advisor should be added to the multidisciplinary team of experts caring for survivors of TGCT. Clearly, the focus of the current comprehensive care has moved from survival to survivorship of the patients after their recovery from testicular cancer.

CONCLUSION

Key take home points are shown in figure 7.

Figure 7. Key take home points.

ACKNOWLEDGEMENTS  

The authors thank Prof. Niels E. Skakkebæk for his mentorship, and the research and clinical teams at their departments for the contribution to the studies summarized in this review. The work was supported by grants from numerous foundations, with the biggest contributions from the Danish Cancer Society, the Lundbeck Foundation, the Svend Andersen Foundation, the Danish Advanced Technology Foundation, Sigrid Juselius Foundation and Novo Nordisk Foundation.

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ABSTRACT

In males, estrogens exert pleiotropic effects by acting on several tissue and organs, including the male reproductive system. The action of estrogens is manifest from prenatal life during which the exposure to estrogen excess might influence the development of some structures of the male reproductive tract. Male fertility is under the control of estrogens, especially in rodents. The loss of function of estrogen receptor alpha and/or of the aromatase enzyme leads to infertility in mice. In men, estrogens are able to exert their actions at several levels through the reproductive tract and on several different reproductive cells. However, the regulation of human male reproduction is complex, and the role of estrogens is less clear compared to mice. During fetal and perinatal life, estrogen acts on the central nervous system by modulating the development of some areas within the brain that are committed to controlling male sexual behavior in terms of setting gender identity, sexual orientation development and the evolution of normal adult male sexual behavior. This organizational, central effect of estrogens is of particular significance in other species (especially rodents and rams), but probably less important in men where psychosocial factors become more determining. Other relevant, non-reproductive physiological events, such as bone maturation and mineralization and glucose metabolism, depend on estrogen in men and an increasing body of evidence is disclosing new non-reproductive estrogen function. In this chapter we provide an update of estrogen’s role by reviewing the physiological actions of estrogen on male reproduction and the pathophysiology related to estrogen deficiency and estrogen excess. Phenotypes associated with estrogen deficiency and excess in rodents and in man have shed new light on the mechanisms involved in male reproduction, challenging the perception of the predominant importance of androgens in men. It is now clear that the imbalance between estrogen and androgen in men might affect male reproductive function even in presence of normal circulating androgens. Some uncertainties still remain, especially regarding the impact of abnormal serum estrogen levels on male health, particularly due to the fact that estrogen is not routinely measured in men in clinical practice. Advancements in methods to precisely measure estrogens in men, together with a reduction of their costs, should provide better evidence on this issue and inform clinical practice. In parallel, new basic, genetic, and clinical research is required to improve our knowledge on the role of estrogen in male reproductive function and men’s health in general.

INTRODUCTION

From an historical perspective, estrogens were identified about 85 years ago and estradiol was identified in 1940, reviewed in (1,2). The first evidence of estrogen production in the male was provided in 1934 by Zondek (3), who documented that male stallions excrete high levels of estrogens in the urine and hypothesized that estrogen production in the male occurs via the intratesticular conversion of androgens into estrogens (1,3,4).

In men, the conversion of androgens into estrogens was first demonstrated a few years later, when an increase in urinary estrogens after the administration of exogenous testosterone was recorded in normal men (5) (Figure 1).

Figure 1. Milestones in the advancement of research in the area of estrogens in men.
[E2: 17β-estradiol; ER: estrogen receptor; ERKO: Estrogen Receptor Knock out; ARKO: Aromatase Knock out]

A more detailed demonstration of estrogen production in the human male was provided several years later in 1979 by MacDonald et al. who showed that the aromatization of androgens to estrogens can occur also peripherally in several tissues other than in the testes (2,6).

Prior to the demonstration of estrogen production in males, the effects of estrogen excess on the development of male reproductive organs had been evident since the 1930s (7). Thus, the concept that male tissues are responsive to estrogens was not new, but it was thought that only a great amount of estrogens was able to induce such changes in males (2).

Notwithstanding the large amount of data accumulated in the last eighty years, research in the field of estrogen excess and its role on male reproductive system is still ongoing (8) (Figure 1).

The pioneering studies of Zondek and MacDonald opened the way for an appreciation of the physiological roles of estrogens in the male beyond their effects during embryogenesis (2). Several studies, year after year, provided further data on estrogen’s role in men (9-12), since the first pilot studies on estrogens and male reproductive function (13,14).

The progressive development of immunohistochemical studies and the subsequent progress in the field of molecular biology highlighted the actions of estrogens in the male [for further details see (4)] and opened the way for the creation of estrogen null mice, a useful animal model to study the physiological role of estrogens in vivo (15) (Figure 1).

The detailed characterization of estrogen receptors’ structure and function (15,16) together with the discovery and the characterization of genes involved in estrogens synthesis (17) disclosed the biomolecular mechanisms and related pathways involved in estrogen function and dysfunction. It is now clear that estrogen effects in the male are not confined to reproductive organs but are pleiotropic (18).

In addition, the development of male transgenic mice lacking functional estrogen receptors or the aromatase enzyme (responsible for estrogen biosynthesis) further contributed to advancements in this field (15,16). Finally, the discovery of mutations in both the human estrogen receptor alpha (19) and aromatase (20,21) genes contributed to an understanding of estrogen’s role in human male physiology and pathophysiology (11,12,22-25) (Figure 1).

Nowadays, notwithstanding this long history of studies, reviewed in (26-28), the role of estrogens in the physiology of the male reproductive tract is still not fully understood. The presence of estrogens in the human testis is well documented (29,30), and there is clear evidence that estrogens exert a wide range of biological effects in both men and women (10,12,18,24,25,31).

PHYSIOLOGY

Estrogen Biosynthesis in Males

The term estrogen refers to any substance, natural or synthetic, able to interact with the estrogen receptor (ER) (32,33). 17β-estradiol (estradiol) is the prevalent endogenous estrogen form in mammals, although many of its metabolites could be detected with several degrees of estrogenic activity (34). In humans, the three major endogenous estrogens are estrone (E1), estradiol (E2), and estriol (E3) (33) (Figure 2). In males, estrogens mainly 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 (32). This step is under the control of the aromatase enzyme (32,35) (Figure 2).

Figure 2. Biochemical pathway of testosterone conversion into estrogen.

However, a wide number of other endogenous products belongs to the category of estrogenic compounds, such as 27-hydroxycholesterol, dehydroepiandrosterone (DHEA), 7-oxo-DHEA, 7α-hydroxy-DHEA, 16α-hydroxy-DHEA, 7β-hydroxyepiandrosterone, Δ4-androstenedione, Δ5-androstenediol, 3α-androstanediol (3α-Adiol), 3β-androstanediol (3β-Adiol), 2-hydroxyestradiol, 2-hydroxyestrone, 4-hydroxyestradiol, and 4-hydroxyestrone and 16α-hydroxyestrone(33). In particular, dihydrotestosterone (DHT), an androgenic metabolite of testosterone that is synthesized by the enzyme 5 alpha reductase, can be metabolized into 3 β-Adiol, an intermediate metabolite with estrogenic activity (32,36). All these molecules differ in terms of ER affinity (33). Various exogenous substances also show estrogenic activity, such as bisphenol A, metalloestrogens, phytoestrogens (e.g., coumestrol, daidzein, genistein, miroestrol) and mycoestrogens (e.g., zeranol) (37). These exogenous estrogens can influence human physiology via environmental exposure or ingestion, however the real impact in vivo as well as critical thresholds and cumulative amount of exposure remain to be fully elucidated (38).

The aromatase enzyme is a P450 mono-oxygenase enzyme complex (17) present in the smooth endoplasmic reticulum, which acts through three consecutive hydroxylation reactions, with the final reaction being the aromatization of the A ring of androgens (17,34) (Figure 2). This enzymatic complex is composed of a ubiquitous and non-specific NADPH-cytochrome P450 reductase, together with the regulated form of cytochrome P450 aromatase (17,29). The latter is highly specific for androgens (39,40). The conversion of androgens into estrogens takes place mainly in the testes, adipose tissue and muscle tissue, even though other male tissues are also involved to a lesser extent (17,34,35) (Figure 2).

The P450 aromatase enzyme is encoded by the CYP19A1 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 (9,17,34) (Figure 3). This gene belongs to the cytochrome P450 superfamily, similar to other enzymes involved in steroidogenesis (32).

Figure 3. Schematic representation of the human aromatase (CYP19) gene.
[Red bars: first exons associated with upstream alternative, tissue-specific promoters; yellow bars: coding exons; black bar: heme-binding region].

Circulating estrogens are mainly reversibly bound to sex hormone binding globulin (SHBG), a β-globulin, and, to a lesser degree, to albumin (41). The amount of circulating free estradiol depends on several factors, of which the concentrations of albumin and SHBG are the most important (41). Serum free estradiol may be calculated by a complicated formula using total estradiol, SHBG, and albumin levels or may be measured by means of equilibrium dialysis or centrifugal ultrafiltration methodology; both, however, are too time consuming and expensive to be employed in routine clinical practice (41). When calculating free estradiol, the reliability of the value of total serum estradiol should be considered, since assays commonly used for estradiol in clinical laboratories have poor accuracy when measuring the low serum estrogen characteristic in males (42-44).

Estrogen Actions in Males

Estrogen action is mediated by interaction with specific nuclear estrogen receptors (ERs), which are ligand-inducible transcription factors regulating the expression of target genes after hormone binding (10,34,45). Two subtypes of ERs have been described: estrogen receptor α (ERα) and the more recently discovered estrogen receptor β (ERβ) (34,45). These two ER subtypes show different ligand specificity and transcriptional activity, and mediate the classical, direct, ligand-dependent pathway involving estrogen response elements in the promoters of targets genes and protein-protein interactions with several transcription factors (45). These two different ERs have different transcriptional activity (46). In particular, ERβ shows a weaker transcriptional activity compared to ERα (45). This difference is due to the presence of different ERβ isoforms, which can modulate estrogen signaling using different pathways and lead to different impacts on the regulation of target genes (45,46). In addition, it should be remarked that the co-expression of both ERα and ERβ in the same cell determines a complex cross-talk finally resulting in the antagonistic effect exerted by ERβ on ERα-dependent transcription (45,46). Thus, the presence/absence of ER subtypes together with their cross-talk determines a cell’s ability to respond to different ligands as well as the regulation of transcription of different target genes (45).

ERα in humans is encoded by the ESR1 gene located on the long arm of chromosome 6, while the ESR2 gene encodes ERβ and is located on band q22-24 of human chromosome 14 (45,46). The two ER proteins have a high degree of homology at the amino acid level (45) (Figure 4).

Figure 4. Estrogen receptor gene structure showing the 9 exons (lower panel), cDNA domains (indicating exons), and protein structures of both ERα and ERβ (upper panels: colored boxes denote the different functional domains of the protein).

ERs are nuclear receptors in which structurally and functionally distinct domains are recognized. Estrogens bind the COOH-terminal multifunctional ligand-binding domain (LBD), whereas the DNA-binding domain recognizes and binds DNA (45,46). The NH2-terminal domain, the most variable domain, is involved in the transcriptional activation (45). This domain recruits a range of coregulatory protein complexes to the DNA-bound receptor (45). The two ER forms share a high degree of sequence homology except in their NH2-terminal domains. This specificity accounts for different transcriptional effects on different target genes (45,46). The ER genomic pathway begins with the binding of estrogen to ER (45). This interaction induces conformational changes in the ER, allowing receptor dimerization and subsequent nuclear translocation prior to binding to estrogen response elements or to other regulatory sites within target genes (45,46). Thereafter, the availability of several coregulatory proteins influences the transcriptional response to estrogen (45,46).

While it is clear that estrogens regulate transcription via nuclear interaction with their receptors, a non-genomic action of estrogens has been also demonstrated, suggesting a different molecular mechanism involved in some estrogen actions (34,45-48). In vitro studies show a very short latency time between the administration of estrogens and the appearance of its biological effects. These actions seem to be mediated by a cell-surface G protein-coupled receptor, known as GPR30, that does not act through a transcriptional mechanism (34,47,48). Rapid effects of estrogens result from the actions of specific receptors localized most often to the plasma membrane; in particular it seems that a monomeric portion of the ERα is translocated from the nucleus to the plasma membrane (47,48).

Recently, immunohistochemical analysis of murine tissues reported the presence of GPR30 in the male reproductive tract, including testes, epididymis, vas deferens, seminal vesicles and prostate (49). Furthermore, a rapid response to estradiol suggests that non-genomic estrogen actions are involved also in human spermatozoa (50,51). The different intracellular pathways of estrogen action are summarized in Table 1.

[ERα: estrogen receptor alpha; ERβ: estrogen receptor beta].

Aromatase enzyme and ERs are widely expressed in the male reproductive tract both in animals and humans (52,53), 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 (34). Today, it is clear that not only testicular somatic cells, but also germ cells constitute a source of estrogens in human (29,54). Thus, 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 (26,52). In men, the aromatase enzyme and ERs are expressed in several tissues including those involved in male reproduction. The distribution and expression of aromatase and ERs described below concerns the male reproductive organs.

Aromatase and ERs in the Male Reproductive Tract

The distribution of ERs and aromatase in both the developing and adult male reproductive tract of rodents and humans is summarized below.

DISTRIBUTION OF ERs AND AROMATASE IN FETAL 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 (4,26,55-57).

Leydig cells in fetal rodent testis express ERα before the androgen receptor. Moreover, ERα is abundant in the developing efferent ductules, which are the first male reproductive structures to express ERs during fetal development (58-60). Furthermore, the epididymis also expresses ERα in the fetal rodent. By contrast, it is unclear whether ERα is present within the seminiferous tubules of the fetal testis since conflicting results have been reported in literature (26,29,57).

ERβ is found early in fetal testis, particularly in gonocytes, Sertoli and Leydig cells, with the gonocytes showing the highest expression between 10-16 days post coitum (61). This suggests 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 (26,57). ERα is widely expressed in efferent ductules from fetal life to adulthood, implying a crucial role in male reproduction that has been well documented in adult rodents (27,52,60). On the other hand, ERβ is mainly expressed during fetal life, suggesting a major role in the development of male reproductive structures until birth (26).

A recent study suggests that estradiol is also able to increase the production of stem cell factors by fetal human Sertoli cells, finally resulting in the proliferation and growth of spermatogonial stem cells (62). With this in view, estrogen deficiency during fetal life may conditioning the total amount of spermatogonia available in the future for the spermatogenetic maturation.

Aromatase is expressed in both Leydig and Sertoli cells in the fetal rodent testis, but not in gonocytes and immature structures of the seminal tract. ER and aromatase distribution in the fetal testes as summarized in Table 2. The presence of both aromatase and ERs in the developing fetal testis implies a possible involvement of estrogens in the process of differentiation and maturation of developing rodent testis just starting from an early stage of embryogenesis, with ERβ possibly playing a greater role than ERα (53,55,56).

Both ERα and ERβ are expressed in the fetal penile tissue and estrogens seem to be important for penile growth as well as for the normal differentiation of the terminal part of the urethra (63,64). In particular estrogen takes part together with androgens in the final fusion of the penile urethra (64) and estrogen deficiency due to both ERs disruption and aromatase deficiency may cause hypospadias in rodents (63-65).

The proposed distribution is based on information from various studies including immunohistochemical and mRNA expression studies.

[ERα: estrogen receptor alpha; ERβ: estrogen receptor beta].

DISTRIBUTION OF ERs AND AROMATASE IN ADULT RODENT REPRODUCTIVE TRACT     

ERα is expressed (both in terms of mRNA and protein) in the Leydig cells of both adult rats and mice (66) but not in Sertoli cells, and 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 (26). However, in neonatal and prepubertal rats, estradiol increases the expression of proteins involved in the proliferation and differentiation of Sertoli cells and of proteins involved in the adhesion of germ cells to Sertoli cells (67). Furthermore, ERα has been immunolocalized in ciliated and non-ciliated cell nuclei of the epididymal epithelium (59,68). This peculiar distribution explains several important estrogen actions in the proximal ducts, especially within the efferent ductules that are small and convoluted tubules connecting the rete testis (an anastomosing network of intricate and tenuous tubules located in the hilum of the testis) to the epididymis (60). In the efferent ductules, estrogens promote fluid reabsorption (52,60,69). Finally, the full-length form of ERα has been detected in purified rat germ cells, using a specific antibody directed against the C-terminal region of the protein (70) (Table 3).

ERβ is expressed (both in terms of mRNA and protein) in Leydig and Sertoli cells in adult rodents (26,57,60) and in monkey germ cells (71); furthermore, it is expressed also in epithelial and peritubular cells of efferent ducts (59,68). For many years the presence of ERβ in rodent germ cells has been the subject of some debate due to discrepancies in the results of different immunohistochemical studies (72). Immunolocalization of ERβ in differentiated germ cells of adult rodents has been revealed in various studies (61,73). Conversely, no ERβ immunoreactivity was found in rodent germ cells in other studies (74), while mRNA expression seems to decline from fetal life to adulthood in the rat (72). Nevertheless, ERβ seem to be involved in the regulation of gonocyte multiplication, which is under the influence of growth factors and estradiol (16), suggesting a functional role for ERβ at least in immature male germ cells. In addition, several studies have recently identified several pathways involving the ERβ in germ cells confirming both its presence and activity of these cells (75,76).

The proposed distribution is based on information from various studies including immunohistochemical and mRNA expression studies.

[ER α: estrogen receptor alpha; ERβ: estrogen receptor beta; GPR30: G protein-coupled receptor].

GPR30 is widely expressed (both in terms of mRNA and protein) in rodent testis (77). In particular, this receptor is expressed in rat Leydig cells (78) and Sertoli cells (79,80), in the spermatogonia GC-1 cell line (81), in rat pachytene spermatocytes (70), and in round spermatids (82).

Rodent Leydig cells show higher aromatase expression than Sertoli cells (83). Aromatase is also expressed at high levels in germ cells throughout all stages of maturation, with its expression increasing as germ cells mature into spermatids. Aromatase mRNA expression and enzyme activity  are present in both rat and mouse germ cells from the pachytene spermatocyte stage, and during their subsequent maturation into round spermatids (57,60,83) (Table 3). Carreau et al. demonstrated that aromatase activity in germ cells was more than 50% of that of the whole testis (29). This intensive activity suggests that germ cells may be a major source of estrogen in adult rodents (57,60,83) (Table 3). Specifically, when fully developed spermatids are released from the epithelium, aromatase is present in the residual body (the remains of the spermatid cytoplasm that is removed during spermiation) and is subsequently phagocytosed by the Sertoli cell. Aromatase activity also remains detectable in the cytoplasmic droplet attached to the flagellum when sperm passes through the epididymis, suggesting that mature spermatozoa are able to synthesize their own estrogen as they pass through the efferent ducts (29,84). The ability to synthesize estrogen gradually decreases as the droplet slowly moves to the end of the tail during epididymal transit until it is 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, and might directly modulate some functions such as the reabsorption of fluid from the efferent ductules (60).

DISTRIBUTION OF ERs AND AROMATASE IN THE HUMAN MALE REPRODUCTIVE SYSTEM

ERs are present in human testis and reproductive tract (29,60,85,86). 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 (86,87). Furthermore, ERβ immunoreactivity in the epididymis suggests a putative role for locally produced estrogens, the actions of which are likely mediated by ERβ in this site. This supports the importance of estrogens for the prenatal development and function of male reproductive structures, which is well documented in literature (87). In particular, estrogens play an important role in the development of the rete testis, efferent ductules, epididymis, and vas deferens (88).

Aromatase and ERβ, but not ERα, continue to be expressed (both in terms of mRNA and protein) during the prepubertal period in men, but their function during infancy remains unclear, especially if the very low  levels of both circulating and locally produced sex steroids in this period of life is taken into account (89).

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 (74) (Table 4). The presence of ERs in the human epididymis is still a matter of debate (27), even though ERα has been detected in the nuclei of epithelial cells of the caput of the epididymis (90), and recent data confirms its presence in the epididymis (86). Both ERs (ERα and β) have been identified in isolated immature germ cells (29). Furthermore, they were localized in mature spermatozoa (91) and in ejaculated spermatozoa (92). Luconi et al. first described an estrogen receptor-related protein in the sperm membrane (50,51). This protein is able to bind steroid hormones and may act through a calcium-calmodulin dependent pathway, accounting for a well-documented rapid non-genomic action (50,51). Subsequently, the expression (both in terms of mRNA and protein) of both ERs in human ejaculated spermatozoa (92,93) reinforced the concept that estrogens are able to modulate the spermatogenic process from its onset within the testes through to the final process of sperm maturation after ejaculation (4,29,92,93). The ERα and ERβ localize to different regions in human sperm, with ERα present in the 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 (57). This confirms that each type of receptor probably has a distinct role in sperm physiology and in the process of fertilization (75,94).

The proposed distribution is based on information from various studies including immunohistochemical and mRNA expression studies.

[ERα: estrogen receptor alpha; ERβ: estrogen receptor beta; GPR30: G protein-coupled receptor].

Of particular interest is the demonstration of differential expression in the human testis of wild type ERβ (ERβ1) and of a human variant form of ERβ, the latter arising from alternate splicing (known as ERβcx, or ERβ2), (95,96). ERβ2 expression seems to be associated with prevalent, negative inhibition of ER action by inhibiting ERα–induced transactivation (97); it is highest in spermatogonia and Sertoli cells in adult men, suggesting that these cells may be "protected" from estrogen action (95,96). Wild type ERβ1 was mostly present in pachytene spermatocytes and round spermatids, which have been proposed to be more estrogen sensitive (26), yet ERβ1 was low in less mature germ cells (95). 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 (96).

Besides, the cellular distribution of non-genomic GPR30 estrogen receptor in human testicular biopsies was examined (98). Immunohistochemical analysis of testicular sections identified the GPR30 receptor in the cytoplasm of Leydig cells, Sertoli cells and spermatogonia (98). This pattern of localization was further demonstrated by the analysis of GPR30 expression (both in terms of mRNA and protein) in isolated germ cells and in Sertoli cell culture (98). This peculiar distribution suggests that GPR30 may be involved in germ cell differentiation (98). Furthermore, the presence of GPR30 in human spermatozoa has been confirmed at both the mRNA and protein level, with this receptor being localized in the sperm mid-piece (99). The co-expression of the two classic ERs and of the GPR30 receptor in the same area within the spermatozoa (mid-piece and acrosome region) suggests a complex cross-talk among all these receptors able to influence physiological processes and pathological implications, such as tumorigenesis (100).

Aromatase expression in the human testis is present in both somatic and germ cells (53,88). Specifically, it is expressed in Leydig and Sertoli cells (101,102), in immature germ cells, from pachytene spermatocytes through elongated spermatids (57,101), and ejaculated sperm cells (103). Locally produced estrogens in sperm are proposed to exert a protective action on sperm DNA by preventing sperm DNA damage (104), thus accounting for estrogen’s potential role as a survival factor during sperm transit through the seminal vesicles (105). Unlike rodents, aromatase expression in human gametes persists during the transit through the genital tracts, since P450 aromatase has been demonstrated in human ejaculated spermatozoa at three different functional levels: mRNA expression, protein production and activity (92). Therefore, as in rodents, human sperm are considered a potential site of estrogen biosynthesis (4,92,101,102,104). The presence of functional aromatase in human spermatozoa allows the conversion of androgens into estrogens as they transit the 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 (53,54,88,106).

In summary, the testes are able to synthesize and respond to estrogens throughout their development (53,88). The localization of ERα, ERβ and aromatase suggests that estrogen action is likely to be important for testicular and efferent ductule function. Differences among various polymorphisms of ER genes may account for different responses to estrogens in term of sperm count and sperm quality (107,108). The role of estrogens in the male reproductive system is clearer in rodents (see below), 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 (4,53,55). 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 animal models and in men.

ROLE OF ESTROGENS IN MALE REPRODUCTION

Estrogens in Animal Male Reproduction: Effects of Estrogen Deficiency

Estrogen-deficient knockout mice are useful models to investigate estrogen action in rodents (16,26). At present, four different lines of estrogen receptor-deficient knockout mice have been generated: 1) ERα knockout (α-ERKO) mice with disrupted ERα gene (109-111); 2) ERβ knockout (β-ERKO) mice, with an inactivated Erβ (112), 3) double ERα and ERβ knockout (αβ-ERKO) mice with non-functioning ERα and ERβ (16), and 4) GPR30 knockout mice (113-115). The αERKO, βERKO and αβERKO mice provide very helpful information on the loss of ER function, leading to estrogen resistance. The knockout of the aromatase gene in aromatase knockout (ArKO) mice is an experimental model useful for investigating the congenital lack of both circulating and locally produced estradiol (16,26,116,117). Estrogen-resistant mice (αERKO, βERKO, and αβERKO) have high levels of circulating estrogens with the non-genomic pathway still likely functional. Aromatase-deficient mice have no circulating estradiol however estrogen receptors could be activated by other estrogenic compounds produced outside the aromatase pathway (e.g. 3β-Adiol) or introduced by diet (e.g. phytoestrogens) (26). Furthermore, in 2009, Sinkevicius et al. created transgenic mice with a G525L point mutation in the ligand-binding domain of ERα (ENERKI mice) (118). This allows differentiation of ligand-dependent vs ligand-independent ER actions since these two different pathways could lead to different actions in vivo. The study of fertility of the ENERKI mouse shows that the efferent ductule fluid reabsorption is regulated by ligand-independent actions of ERα, whereas germ cell production and/or viability requires ligand-dependent ERα actions (118). Recently, Yao et al. mapped the Era-binding sites in the efferent ductules of male mice and they found 12105 peaks, of which about 50% were shared by the androgen receptor (119).

Recently, the creation of the knockout mice lacking GPR30 estrogen receptor (113,115) allowed an investigation of the reproductive phenotype of mice lacking a functional GPR30, with the results suggesting a minor role of this receptor in male fertility. GPR30 knockout mice did not show abnormalities of endocrine organs, alterations of spermatogenesis and mating behavior, or decreased fertility (114,120). A detailed study of spermatogenesis in this mouse model is, however, still lacking.

Studies on transgenic mice lacking ERs or the aromatase enzyme demonstrate that the lack of estrogen action is compatible with life (22,121). 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 worsens during aging (16,26,60,69,109-112,116,122). Reproductive characteristics of male mouse models are summarized in Table 5.

The G protein-coupled receptor (GPR30) knockout mice have normal reproductive phenotype.

[ERKO: estrogen receptor knockout mice; α: estrogen receptor alpha; β: estrogen receptor beta; ArKO: Aromatase knockout mice].

Male αERKO mice are infertile as the seminiferous epithelium is atrophic and degenerated, and seminiferous tubules and rete testis are dilated (60,69,111), even though the development of male reproductive tract is largely unaffected (16,109,111,123). The disruption of spermatogenesis is progressive as the testicular histology is normal at postnatal day 10, but starts to degenerate at twenty-thirty days of age (69,111). From 40 to 60 days, tubules are markedly dilated with a corresponding significant increase in testicular volume, while the seminiferous epithelium becomes atrophic (16,69,111). A severe impairment in tubule fluid absorption at efferent ducts level is 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 (59,60,69). In the male genital tract, the highest concentration of ERα is in the efferent ducts (69) 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) (59,69). This mechanism appears to be the consequence of the ligand-independent ERα activation (118). 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 (124,125). 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 (27,52,60,69,111,126). 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 age (16,52,60,69). When germ cells from αERKO mice are transplanted in wild type mice, they show normal development (127). Recently, it has been demonstrated that some genes playing important role in the efferent ductules are regulated by Er both independently by estrogens or in combination with androgens where estrogen responsive elements colocalize with androgen responsive elements (119).

The αERKO mouse is also characterized by a reduced number, motility, and fertilizing capacity of the sperm levels (Table 5). In addition, αERKO male mice show increased serum luteinizing hormone (LH) and testosterone as well as Leydig cells hyperplasia, together with normal serum follicle-stimulating hormone (FSH) levels (Table 5) (16).

The production of both ArKO (122) and βERKO (112) mice added further insights in this field, supporting the idea that estrogen actions on the male reproductive tract are more complex than previously suggested on the basis of the studies performed on αERKO mice (16). In fact, unlike αERKO mice, male ArKO mice are initially fully fertile (122), but fertility decreases with advancing age (Table 5) (26,116,117). Furthermore, βERKO mice are fully fertile and apparently reproductively normal in adulthood (Table 5) (112).

The mechanism involved in the development of infertility in ArKO male mice therefore differs from that of the αERKO mice (26). Transgenic mice models suggest that ligand-independent ERα signaling is essential for concentrating epididymal sperm via regulation of efferent ductule fluid reabsorption, while ligand-dependent ERαis involved in germ cell production and/or viability (118). Thus, the lack of estrogen action at the level of the seminiferous epithelium rather than a problem due to impaired fluid reabsorption probably explains infertility in ArKO male mice (26,50). Accordingly, estradiol seems to be necessary for round spermatid survival and estrogen deficiency seems to promote apoptosis before differentiation into elongated spermatid (26,92,105).

Studies of αβERKO mice showed a male phenotype very close to that of αERKO mice characterized by infertility and dilated seminiferous tubules (16,26). On the contrary, βERKO male mice were fully fertile (112). 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 behavior in male mice. For this reason, infertility in αERKO, αβERKO and ArKO mice is at least in part due to the reduction of various components of mating behavior from an early age (Table 5) (16,26).

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 (26). Thus, negative effects on male reproduction are the direct result of estrogen deprivation in the reproductive structures or of indirect changes in the regulation of sex steroid secretion.

Taken together, all these studies support the concept that a functional ERα, but not ERβ and GPR30, is needed for the development and maintenance of a normal fertility in male mice (15,16,52,55,59,60,69,111,112). Anyhow, it should be remarked that estrogens are also able to self-regulate all these estrogen-related pathways in the male reproductive tract since estrogen receptor expression is regulated by estradiol in rats. In particular ERα positively regulates the expression of both ERα and ERβ while androgen receptor and ERβ negatively regulates ERs expression (128).

Estrogens in Human Male Reproduction: Effects of Estrogen Deficiency

The demonstration of wide expression of the aromatase enzyme, ERα and ERβ throughout the male reproductive system and within human sperm underlines the role of estrogens in human male reproductive function (4,29,55,106,129). Accordingly, estrogens seem to modulate sperm maturation (50,129), since spermatozoa express ERα and ERβ, and are responsive to estrogens throughout their journey from the testes to the urethra.

The characterization of human diseases leading to estrogen deficiency have increased our knowledge about the role of estrogen in male reproductive function as well as on other important physiological functions ranging from longitudinal growth, bone mass acquisition, and metabolic alterations (24,130-132).

Data from human subjects with congenital estrogen deficiency have provided conflicting and confusing results. Fertility has been investigated in only one man with estrogen resistance who exhibited a mutation in ERα, rendering him unable to respond to estrogen, thus he could be considered as a human equivalent of the αERKO mouse. However, this man had normal testicular volumes and normal sperm count but with slightly reduced motility (19) (Table 6). At present only one other patient has been diagnosed with estrogen resistance, but a semen analysis was not available for this 18 years-old boy, a possible impairment of fertility being hypothesized on the basis of low inhibin serum levels, reduced testis volume, and cryptorchidism (133). The human reproductive phenotype seems different from that observed in αERKO mice (16,26,52,69,111) since there was no clinical evidence of obstruction of the efferent ductules in the man with estrogen resistance, different to that observed in the rodent model (19). However, no data on the histology of the testis and efferent ductules is available from these two men with estrogen-resistance (19,133,134).

The other human model of estrogen deficiency is congenital aromatase deficiency (135). At present, sixteen men with aromatase deficiency have been described (Table 6 and Table 7) (20,136-149). For most of them the genetic diagnosis (143,144) and/or the clinical description (21,150-152), as well as the following clinical studies (153-160)were performed by our research group. These patients showed a variable degree of impaired spermatogenesis (4,11,143,144,161). The hormonal pattern of the patients affected by aromatase deficiency is summarized in Table 6(4,55,135,162). Testicular size in aromatase-deficient men is normal except for three cases having a smaller testes volume (Table 6), while data on testes volume are lacking in some reports (148). Among the eight patients with semen analysis available, six had normal sperm count (139,140,143,146,148,152) and the remaining two had oligospermia (21,22,138) from moderate (138) to severe (21) (Table 6). Anyhow, moderate to severe asthenospermia without teratospermia was also reported independently from the sperm count (21,22,138-140,152) (Table 6). Sperm count was unavailable in the other eight men with aromatase deficiency due to lack of data, diagnosis made at birth, and cases described in prepubertal age (20,136,137,141,143-145,147,149). Moreover, a variable degree of germ cell arrest, ranging from complete depletion of germ cells to arrest at the stage of primary spermatocytes, was described in three aromatase-deficient men who underwent biopsy of the testes (21,22,150,151) (Table 6).

Furthermore, a history of cryptorchidism was present in four patients (22,2%) being bilateral in two cases (145,150)and unilateral in the remaining two (139,140,152). These data suggest a possible role of estrogen in testis descent, although this was not seen in the transgenic mouse models. The small number of cases 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. Besides, hypospadias has been reported in one case (145) and preliminary data speak in favor of estrogen role on penile tissue development during fetal growth (63-65), but this aspect needs to be confirmed by more robust studies.

In addition, a clinical condition of aromatase deficiency may be also caused by mutation in the cytochrome P450 oxidoreductase (POR) (163), as previously reviewed (162).

It should be remarked, however, that a clear cause-effect relationship between infertility and aromatase deficiency was not demonstrated in all these patients (4,135). For this reason, the different degree of fertility impairment found in men with congenital estrogen deficiency does not allow us to establish with certainty whether sperm abnormalities are a consequence of the lack of estrogen action or are an epiphenomenon. Again, this spermatogenetic pattern is different from that observed in ArKO mice (16,26,52,116,117,122).

[LH: luteinizing hormone; FSH: follicle-stimulating hormone]

The frequency of sperm abnormalities in these patients together with the results from rodent studies suggests a possible role for estrogen in human spermatogenesis, however this requires further elucidation (4,12,24,30). Our knowledge on estrogen’s role in human male reproduction in vivo remains far from complete. The data available in the literature suggests that the action exerted by estrogens on male reproductive organs is more complex than that seen in mice and that estrogen alone does not directly control spermatogenesis to the same extent than in rodents, but are involved in a more complex and evolved network (26,118,164)

In addition to human models of congenital estrogen deficiency, other experimental settings have provided information on the role of estrogens on human male fertility.

Studies on the association between ER polymorphisms and infertility in men showed that two polymorphisms of ERα (XbaI and PvuII) are associated with azoospermia, severe oligospermia and impaired sperm motility (165-168) and the multiallele (TA)n polymorphism with male infertility (108). However, data available in literature provides conflicting results since Pvull resulted strongly associated with infertility (166), but also a strong protective factor of male fertility (169), depending on the research setting. The RsaI polymorphism of the ERβ has been associated with male infertility in one study (107), but not confirmed in another study (167). The ERβ (RsaI) polymorphism AluI was also associated with sperm motility, while no association with motility was found for the RsaI polymorphism (167). Thus, the association of polymorphisms of estrogen-related genes with both sperm concentration and motility, but not with sperm morphology, further supports a putative role of estrogen in controlling sperm production and quality (170).

Furthermore, the investigation of ERs in the nuclear matrix of human spermatozoa showed a reduction of ER levels in the nucleus of idiopathic infertile men compared to normospermic fertile men (171).

Interventional research studies show that the administration of aromatase inhibitors to infertile men with documented impaired testosterone-to-estradiol ratio may result in an improvement of their fertility rate, but further evidence is needed to verify their efficacy and safety (see paragraph below ‘Anti-estrogen treatment in men’ for further details) (172,173). These results suggest that such modulation of estrogen metabolism will influence sex hormone balance and the HPT axis while dissecting out direct effects of estrogen on spermatogenesis in vivo is extremely challenging.

It seems that exposure to increasing estradiol concentrations might influence glucose metabolism in spermatozoa and that the increase of aromatase activity and estradiol enhances glucose metabolism in capacitated, but not in non-capacitated sperm (93). Recently, intratesticular T and E2 were strongly correlated to aromatase expression in Leydig cells in infertile men; intratesticular T was higher and E2 lower in men with obstructive azoospermia compared to those with nonobstructive azoospermia, suggesting that an imbalance in aromatase expression and thus in the intratesticular T to E2 ratio might play a role in the pathogenesis of male infertility (174). In addition, serum estradiol was directly correlated with motility, sperm count and sperm morphology in male partners of infertile couples enrolled prospectively and low estradiol entered among risk factors for decreased semen quality in multivariate analysis (175).

It seems probable that most of estrogen actions operating in mice, such as regulation of sperm motility, sperm capacitation, acrosome reaction, and sperm metabolism also occur in men, but the contribution of estrogens to these processes is quantitatively less important in humans. It seems likely that most of these processes in humans are also regulated by other factors in a complex crosstalk system involving also estrogens. This could also explain why high amounts of estrogens or the exposure to an excess of environmental estrogens (or to xenoestrogens with high estrogenic potency) could negatively impact on male fertility. For these reasons, it is apparently difficult to reconcile existing data about effects of both estrogen deficiency and excess on male reproductive function (13,31,176-178).

Regulation of Gonadotropin Feedback

The 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, a role for estrogens was recently clarified in studies performed in normal and GnRH-deficient men.  We now know that ERs are expressed both in the hypothalamus and the pituitary. In particular, GnRH neurons express ERβ but not ERα (179,180), thus the inhibitory effects of estrogens on these cells is mediated through other neuromediators (e.g. kisspeptin, neurokinin B) released by other neurons expressing also the ERα (181). ERs, especially ERα are expressed in gonadotropes cells (182).

The response of the 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 (183). However, the discovery of men with congenital estrogen deficiency has provided further evidence for a relationship between estrogens and gonadotropin secretion also in men (22). In fact, serum gonadotropins are high in all adult patients with aromatase deficiency, notwithstanding normal to increased serum testosterone levels (135), thus implying that estrogens are also important for the regulation of circulating gonadotropins levels in men.

The effects of estrogens on gonadotropin secretion have been investigated in GnRH-deficient men whose gonadotropin secretion was normalized by pulsatile GnRH administration. Moreover, in order to determine the precise role of sex steroids on the hypothalamo-pituitary-testicular axis, several studies characterized by the administration of testosterone, testosterone plus testolactone (an aromatase inhibitor), or estradiol have been performed (184,185). Testosterone alone induced a significant decrease in mean basal LH and FSH levels as well as of LH pulse amplitude, demonstrating a direct suppressive effect on the pituitary of testosterone and its metabolites. In general, mean LH levels and LH pulse frequency are suppressed to a greater extent in normal control subjects under testosterone administration, suggesting the involvement of a hypothalamic site of action of testosterone (or its metabolites) 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 this aromatase inhibitor completely inhibited the testosterone effect on gonadotropin secretion both in normal and GnRH-deficient men, thus leading to a significant increase in mean LH levels in both groups. The latter was greater in normal men who received testolactone alone than in normal men who received testosterone plus testolactone, thus confirming a direct effect of androgens on gonadotropin secretion in normal men. On the basis of the results of these studies, it is clear that the aromatization of testosterone to estradiol is, at least in part, required for normal gonadotropin feedback at the pituitary level (185). In fact, when the same experimental model was applied using estradiol administration instead of testolactone, mean LH and FSH levels as well as LH pulse amplitude decreased significantly during the treatment (184). These studies have demonstrated an important direct inhibitory effect of estradiol on gonadotropin secretion in both GnRH-deficient and normal men (184,185) and support the concept that, at least in part, the inhibitory effect on gonadotropin secretion is mediated by the conversion of testosterone to estradiol (4,186). Accordingly, the administration of the aromatase inhibitor letrozole to healthy adult males is able to suppress aromatase activity and serum estradiol levels leading to an increase of gonadotropins (187). Only the restoration of normal circulating estrogens, by means of transdermal estrogen administration, normalized gonadotropin secretion in this setting (187). In contrast, it seems that the 5α-reduction of testosterone to DHT does not play a very important role in pituitary secretion of gonadotropins (188); DHT, in fact, slightly decreases LH and FSH only after long-term administration (183).

All these studies suggest possible estrogen action at the level of hypothalamus. In order to clarify the role of estrogen on the feedback regulation of gonadotropin secretion at hypothalamic level, Hayes et al. (189) conducted a study involving men affected by idiopathic hypogonadotropic hypogonadism (IHH), whose gonadotropin secretion was normalized by long-term pulsatile GnRH therapy, followed by treatment with the aromatase inhibitor anastrozole. They observed that the inhibition of estradiol synthesis led to an increase in mean gonadotropin levels that was greater in normal subjects than in IIH men, suggesting a hypothalamic involvement. The rise in mean LH concentrations in normal subjects due to anastrozole was due to increased LH pulse frequency and amplitude. The authors concluded that estrogen acts at the hypothalamic level by decreasing GnRH pulse frequency and pituitary responsiveness to GnRH (189). Subsequently, the same group (190) 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. These authors went on to report that the sex steroid component to FSH negative feedback was not androgenic but rather was mediated by estradiol effects on the frequency of GnRH stimulation (190,191).

For many years another important unresolved issue has been the relative role of circulating vs. locally produced estrogens in the control of gonadotropin secretion. Now we know that the effects of circulating estrogen are more relevant than that of locally produced, at both the hypothalamic and pituitary level (187) (Figure 5). Accordingly, the administration of both the aromatase inhibitor letrozole and estradiol at different dosages showed that serum testosterone and gonadotropins were inversely related to circulating estradiol, depending on the dose of exogenous estradiol (187). The serum estradiol required to obtain the same levels of gonadotropins were not different compared to that at baseline, suggesting that aromatase inhibition and the blockade of locally produced estrogens are less important than previously thought (187). In the same year, our group reached the same conclusions using a different approach. In men with aromatase deficiency, we demonstrated that circulating rather than locally produced estrogens are the main inhibitors of LH secretion (157). This implies that the role of locally produced estradiol on gonadotropin feedback at hypothalamic and pituitary levels is relatively modest in vivo (Figure 5). However, the role of locally produced estrogens has been poorly investigated since evaluating the effects of locally produced estrogens in vivo is challenging (186).

Data available in the literature demonstrate that (i) circulating estrogens are involved in gonadotropin suppression both at pituitary (187) and hypothalamic level (157,190), and (ii) estrogen effects on hypothalamus are independent from central aromatization, but requires adequate amounts of circulating estrogens in normal healthy men (187), in men with IHH (190,191), and in men with aromatase deficiency (157).

The effects of estrogen on gonadotropin secretion at the pituitary level operate from early- to mid-puberty (186,192,193) into old age in men (194). The administration of an aromatase inhibitor (anastrozole 1 mg daily for 10 weeks) to young men aged 15-22 years (192) resulted in a 50% decrease in serum estradiol concentrations, an increase in testosterone concentrations and an increase in both LH and FSH values during the study protocol. These hormonal parameters were restored after the discontinuation of anastrozole treatment (192). In addition, 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 act at the pituitary level during early phases of puberty (193), the role of estrogens in infancy and at the beginning of puberty remaining less known (186). The same mechanism continues to operate during adulthood and early senescence (195), 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 (194). Similar results were replicated by using letrozole in older men (173). For these reasons, the use of aromatase inhibitors as blockers of the negative feedback on gonadotropin has been tested as a possible strategy useful for the treatment of late-onset male hypogonadism (196). The rationale was that increasing endogenous serum testosterone through the inhibition of the rate of conversion of testosterone into estradiol led to the consequent LH and FSH increase (196). After the first encouraging results (197-199), this kind of treatment seems to be not effective, especially on large-scale clinical trials and for long periods of time (195,196,200,201).

Figure 5. Sex steroid control of gonadotropin secretion after recent advances: estrogens, but not androgens, are the main regulator of gonadotropins and the action of circulating estradiol prevails with respect to that of locally produced estradiol.
[T: testosterone; DHT: dihydrotestosterone; E2: estradiol; GnRH: gonadotropin releasing hormone; LH: luteinizing hormone; FSH: follicle-stimulating hormone]

Previous data suggest that estradiol may modulate GnRH receptor number and function at hypothalamic-pituitary level (202), since ERs were detected in GnRH secreting neurons (203). Moreover, both genomic and non-genomic estrogen actions seems to be involved in the regulation of the gonadotropin feedback in males (203,204), although the precise mechanism remains unclear (205). 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 lesser role than previously thought (Figure 5), and circulating estrogen, rather than locally produced estrogen, having a major role at the hypothalamic pituitary level (157,187,191).

In a complementary way, 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 (19)revealed a remarkable hormonal pattern consisting of normal serum testosterone, high estradiol and estrone levels, but increased serum FSH and LH concentrations; the serum testosterone remained in the normal range because of increased aromatization of testosterone to estradiol (Table 6). Other important information about estrogen’s role in the human male came from the discovery of naturally occurring mutations in the aromatase gene. To date, of the sixteen different cases of human male aromatase deficiency that have been described, all were discovered to be aromatase-deficient as adults, except one who was diagnosed as a child (137,141) and another one who was diagnosed at 15 months of age (145) (Table 6). Eight out of fourteen adult patients with aromatase deficiency had increased basal FSH concentrations (20,21,135,138-140,144,150-152,154), two had serum FSH in the upper normal range (143,144), and the remaining four had normal FSH (144,146-148). The subject diagnosed during childhood had normal FSH in infancy (137) and high to normal FSH levels at puberty (141). The unique patient diagnosed early at 15 months had normal serum T, LH, and FSH (145). LH was normal in all aromatase adult patients (138-140,144,146,147,150-152), except for one subject with elevated serum LH (20,136) and two subjects with high to normal LH levels (21,143,154)(Table 6). Serum testosterone concentrations were generally normal or high-to-normal except for the first case described with elevated serum levels (20,136), and two other aromatase-deficient men with testosterone slightly above the normal range (138,139). Conversely, another man with aromatase deficiency presented with low to normal serum testosterone levels (150,156). In all sixteen patients estradiol concentrations were undetectable (20,136-149).  (Table 6). The detection of increased gonadotropin levels despite normal-to-increased serum testosterone levels, in these men, further highlights the key role for estrogen in regulating circulating gonadotropins in men (155,157), In normal men with pharmacologically induced sex steroid deprivation, estradiol but not testosterone, was able to restore normal FSH serum levels (191). 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 (135), only supraphysiological levels of estrogens were able to normalize FSH (21,135,154,155).

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 (138,155,157). In these studies, 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 (138,155,157). In 2006, Rochira et al. (157), demonstrated that estrogen’s 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. In normal physiology, these data provide evidence that the negative feedback effects of circulating estrogens is more important than estrogen locally produced at the hypothalamic level (157). As previously explained, these data confirm data from healthy men (186).

Notwithstanding recent advances in the study of estrogen’s role in males, some difficulties remain when data from men with congenital estrogen deficiency are interpreted, particularly if phenotype heterogeneity is considered (161,186). No abnormalities were found in either gonadotropin secretion or in testis position and size in the patient with congenital aromatase deficiency diagnosed in childhood (137), unlike female newborns (206). For these reasons, 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 smaller than expected increase in FSH levels (given the prevailing serum testosterone levels and impaired spermatogenesis) in two estrogen-deficient men (157), suggests a possible role of estrogens in priming and maturation of hypothalamus-pituitary-gonadal axis in men (155,156). Thus, the control of gonadotropin feedback exerted by sex steroids during early infancy and childhood remains a matter of debate in the human male (186).

In conclusion, estrogens are the main sex steroids involved in the control of gonadotropin secretion in men, testosterone having a minor but determinant role as demonstrated by evidence coming from complete androgen insensitivity (CAIS) syndrome in which serum LH is above normal as a consequence of androgen resistance notwithstanding elevated circulating estradiol (207).

Estrogens and Prostate

Androgens regulate prostate gland growth and differentiation, particularly during its development. Estrogens also act on prostate growth and differentiation through both ERα, and ERβ (208,209). In rodents, the prostate is sensitive to estrogen exposure during development (210).

Studies on animals have helped to better understand estrogen’s role in prostate growth. Studies in mice overexpressing aromatase (AROM+) demonstrated that prostate lobes are significantly reduced as a consequence of estrogen excess (211). On the other hand, aromatase-deficient mice presented a hyperplastic prostate gland probably due to the excess of circulating androgens (212) and consistent with hyperplasia of the epithelial, interstitial and luminal compartments (210). 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 (212). Moreover, studies on ArKO mice demonstrated that the administration of an ERβ-specific agonist reverted the existing hyperplastic epithelial pathology (212).

In terms of prostate carcinogenesis, it is generally assumed that androgenic hormones play a major role in tumor development, since the prostate gland is an androgen-dependent tissue, as is prostate cancer (213). However, considering the fact that testosterone can be converted to estradiol, and that ERs are present in the prostate epithelium (214), theoretically estrogen might also be involved in the induction of prostate cancer. Some polymorphisms (rs2470152, rs10459592, and rs4775936) of the CYP19A1 aromatase gene were associated with an increased risk of prostate cancer (215,216). Besides, patients with prostate cancer who are carriers of the rs4775936 polymorphism of the CYP19A1 aromatase gene show a significantly shorter time of cancer-specific survival compared to patients who do not carry this polymorphism (215). In line with this Bosland et al. found that combined treatment of rats with estradiol and testosterone lead to an increased incidence of prostate cancer from 35-40% with androgen alone to 90-100% (217). The estrogen pathways that may be involved at the molecular level in the process of prostate carcinogenesis are very complex (209). Several studies demonstrate that both ERα and β are involved in the transduction of estrogen signaling in prostate cancer such as cell proliferation pathways (209). Furthermore, ERβ seems mainly involved in pro-apoptotic pathways (e.g. FOXO3 and p-53), while ERα is involved in chronic inflammation, and the two ERs seem to act differently on oncogenes playing suppressive (ERβ) and oncogenic (ERα) roles (209). Proliferation of prostatic cells seems to be promoted by the activation of the ER while ER and GPER seems to exert an antiproliferative action (218,219). The different effects of each ER on the proliferation of prostate cells may accounts for the contrasting results (proliferative/antiproliferative) available in literature, depending on the prevailing activated pathway. However, estrogens also display a biphasic effects in vitro on prostate cells growth, which is enhanced by low estradiol and inhibited by high dose of estradiol (220). Probably, different pathways are activated in presence of estrogen excess, thus leading to a shift in the final effect on cell growth (218,220). At present studies on estrogen signaling in prostate cancer tissue are also providing promising results in term of the utilization of this signature as biomarker useful to tailor hormonal treatment (218).

Prostate was normal in aromatase-deficient men and did not change in volume during estrogen replacement therapy (Carani & Rochira; data not published data). Besides, the administration of selective inhibitors of aromatase are helpful for the evaluation of estrogen in vivo effects on prostate. Recently, the combined therapy with transdermal testosterone and the aromatase inhibitor anastrozole in older men with low or low-to-normal serum testosterone (< 350 ng/dL) prevented the increase of prostate volume, but not that of prostate-specific antigen seen in patients treated with testosterone alone (221). Similarly, high serum estradiol resulted directly related to prostate volume in 239 Chinese men with benign prostatic hyperplasia (222) even though these data are limited by the poor accuracy of estradiol measured by immunometric assay.

This is in line with the above-mentioned experimental results suggesting an active role of estrogens in prostate cell proliferation in prostate carcinogenesis. Traditionally, exogenous estrogens have been used for the treatment of prostate cancer since the 1940s thanks to their potent inhibitory effect on the HPT axis resulting in the suppression of circulating testosterone (223). However, diethylstilbestrol (DES) used in the past for prostate cancer was strongly associated with thromboembolic side effects(223). Recently, the use of exogenous estrogens for the therapy of prostate cancer is being reconsidered (224). Transdermal estradiol (patch) seems to be effective in inhibiting gonadotropins and in reducing serum testosterone in men with prostate cancer without increasing cardiovascular events (224).In the near future, if estrogen’s role in the prostate will be further elucidated, new treatment strategies will become available for benign prostate hypertrophy and cancer, especially in men with concomitant hypogonadism (225).

Estrogens and Male Sexual Behavior

Sex steroids act on several aspects of male sexual behavior (226). Sex steroids, mainly testosterone, modulate adult male sexual behavior in mammals (227). In men, sexual behavior is more complex than in other species since it results also from cognitive processes, cultural environment, and an individual system of beliefs (226,228). Thus, sexual behavior does not depend only on hormonal and genetic prerequisites in men (226,228). Traditionally, it was thought that only testosterone, the male hormone, is responsible for the control of male sexual behavior (229). In the last two decades, the possibility that estrogens may be involved in the control of male sexual behavior has received more attention, and an impact of estradiol on male sexuality has become evident (199).

Testosterone is mainly involved in the control of sexual desire and sexual drive and in the facilitation and maintenance of a normal sexual genital response (226). Erections, especially nocturnal erections, are also under the control of androgens (230,231). The role of estrogen on male sexual behavior has been poorly investigated and knowledge derives mainly from studies performed on animals or from rare models of human estrogen deficiency. The increasing interest on the treatment of transgender people (232) and on the cross actions of male and female hormones on both sexual behavior (233) and other physiological functions (234) probably have contributed to a better focus on this area of research. In recent years, however, several in vivo experimental settings have addressed this issue. As a result, nowadays all studies on steroid sex hormones action on male sexual behavior tend to investigate androgens and estrogens separately (199,235-238). Furthermore, steroid sex hormones may influence both gender-identity and sexual orientation (239,240), even though in humans this action is mitigated by the strong influence of psychosocial factors.

Estrogens and Gender Identity and Sexual Orientation

Testosterone aromatization to estradiol in the brain was traditionally considered the key step in the development of a male brain and in determining sexual dimorphism of the central nervous system in non-primate mammals (241-243). According to Dörner’s hypothesis (244), prenatal and perinatal brain exposure to estrogens may be responsible for the establishment of a male brain (240,245), an event occurring only in the male, but not female, brain. Accordingly, ovaries release a very small amount of estrogen, soon inactivated in rodents (4,245), while the testes produce a greater amount of androgen that is converted into estrogen. Thus, circulating estrogens are paradoxically greater in males than in females during fetal life (240,246) and this accounts for the sexual dimorphism of hypothalamic structures in rodents and other species like sheep (245-247).

The same mechanism seems to be also involved in the differentiation in hypothalamic structures between men and women (244,246,248). Prenatal hormonal exposure is classically considered to be involved in determining sexual orientation, on the basis of some differences in hypothalamic structures between heterosexual and homosexual men (243,248). 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 (240,241,243,245). Accordingly, the intrinsic pattern of mammalian brain development is female, and estrogen is required for the development of a male brain (240,243,244), thus emphasizing the role of locally produced estrogen (245). Permanent changes in the organization of different neural circuits, fundamental for sex-specific regulation of reproductive and sexual behavior, probably also occurs under the effects of estrogen (240,242,243,245,249). Considering all the above mentioned aspects, the lack of estrogen action on the developing brain in males should be considered strictly related to the direction of future development of sexual orientation, and of dimorphism of hypothalamic structures (240,241,243,245,248). Most of the data supporting this evidence, however, came from studies performed in rodents or other species, but not in humans (240,242,243,245).

The role of hypothalamic aromatase activity and expression in partner preference has been elegantly confirmed in rams (250). In this study, the choice of sexual partner was associated with both the volume of the ovine sexually dimorphic nucleus and different patterns of aromatase expression (250). This provides the first demonstration that differences in aromatase expression within the brain are related to partner choice and to the determination of adult sexual behavior (245,247,250). However, in humans, a clear cause-effect relationship between prenatal exposure to sex steroids (especially estrogens) and the differences in volume of some dimorphic brain areas (e.g. sexually dimorphic nucleus of the preoptic area and the intermediate nucleus) has not been demonstrated (240).

Aromatase-deficient men represent an interesting model to investigate the role of estradiol on human male sexual development and behavior from fetal life through adulthood (4,55,135). All men with aromatase deficiency who underwent a comprehensive evaluation of sexual behavior had male gender-identity and heterosexual orientation (4,20-22,135,138,143,144,150,152,153,156) (Table 7). The fact that congenital aromatase deficiency does not affect psychosexual orientation and gender-identity in humans suggests that estrogens do not mediate the organizational effects on male sexuality induced by early exposure to androgens. Differently from animals, psychological and social factors are the most relevant determinants of gender role behavior in men, with hormones probably having a minor role compared to other species (4,135,228,247). Accordingly, the prenatal exposure to DES, a potent estrogenic compound, is able to modify partner preference in animal studies, but not in humans (251).

In conclusion, aromatase plays a key role in controlling male reproductive behavior especially in animals (rodents and rams), by modulating organizational effects on the developing brain during fetal life (249,252); the latter are 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, rams, and monkeys (247,252,253) and its poor or minor effect in humans (4,135,153,156) and other primates, respectively (253). Thus the debate about nurture (254) versus nature (246) remains still open in humans.

Estrogens and Sexual Behavior

In adult men sexual behavior is partially dependent on testosterone, the main hormone involved in male sexuality (226,229,230). Accordingly, testosterone deficiency frequently causes loss of libido and erectile dysfunction (226,230,255). These are restored by testosterone replacement therapy, which is effective in increasing sexual interest and improving sexual function (226,227,255,256). Other hormones, however, are involved in the control of male sexual behavior, including estrogens (257,258).

In experimental animal models, the knockout of estrogen pathways or a pharmacologically induced estrogen deficiency results in severe impairment of sexual behavior (4,16,26). Accordingly, ArKO mice (259), αβERKO male mice (260) and αERKO mice (16,109) all exhibit a significant reduction in mounting frequency and prolonged latency to mount when compared with wild-type animals (16,26). On the contrary, βERKO mice did not show abnormalities of sexual behavior (16,112). These findings suggest that androgen receptor activation alone is not sufficient for fully normal sexual behavior in rodents and that a normal functioning ERα together with adequate levels of circulating or locally produced estrogen are required for mounting behavior in male mice (4,55).

Less is known about the role of estrogens in sexual behavior in men since the relative importance of testosterone and its metabolite estradiol on male sexual behavior is still not known. In the past five years, only a few studies have investigated the direct effect of estrogen on male sexual behavior (261,262), indirect evidence being available only from rare cases of men with congenital estrogen deficiency (4,55,130,135,143,144) (Table 7). A detailed sexual investigation of aromatase-deficient men documented an increase in all the parameters of sexual activity during estrogen treatment (153,156), with the best outcome in terms of sexual behavior obtained only when a concomitant normalization of both serum testosterone and estradiol was reached (156). These results support the concept that both sex steroids are required for normal sexual behavior 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 behavior because of the inadequacy of studies and the conflicting results reported in the literature.

* Only these two patients underwent an extensive, well-designed study of sexual behavior in terms of psychosexual issues (gender identity and sexual orientation) and sexual function (desire and erectile function), while for other patients the information was obtained by patients’ interview and medical history. No data on sexual behavior are available for the other patient with estrogen resistance (133) and for the other aromatase deficient-men (139,140,145,148).

Recently, a very elegant study provided evidence-based information on the relative role of testosterone and estradiol on male sexual function in men (199). In this study, a considerable number of healthy men (n= 400) underwent gonadotropin suppression by the administration of a GnRH analogue (goserelin acetate), resulting in testosterone and estradiol suppression (199). In order to investigate the placebo effect and the effects of testosterone and estradiol, subjects were assigned to receive i) placebo, ii) testosterone treatment at different dosages, and iii) testosterone at different dosages plus the aromatase inhibitor anastrozole (199).

In the testosterone group, serum testosterone and estradiol varied from physiological levels to low levels according to the different doses of exogenous testosterone in each group and the estradiol to testosterone ratio remaining substantially unchanged in all groups (199). This pharmacologic scheme allowed testing the effects of lowering both serum testosterone and estradiol in a similar way on several physiological functions; the result was a decline of both sexual desire and erectile function in parallel with the decrease of both sex steroids (199). In the testosterone plus anastrozole group, the decline of serum testosterone paralleled that obtained in the testosterone group, while serum estradiol was quite suppressed and changed to a lesser degree in each testosterone dose group, thus fluctuating across very low values (199). In this group, both sexual desire and erectile function were severely affected in patients with low serum levels of estradiol despite normal serum testosterone in patients taking the higher doses of testosterone (199). Conversely, goserelin treatment resulted in the maximum reduction of both sexual desire and erectile function in the placebo group (199). These results confirm observations in aromatase-deficient men (135,153,156) and suggest that estrogen deficiency is largely responsible for the impairment in sexual function occurring when serum testosterone is suppressed in hypogonadal men (199). A possible role of estrogen on male sexual function is also provided by further studies showing that testosterone therapy is more effective on libido when the treatment produces serum estradiol levels greater than 5 ng/dL (235) and that this serum estradiol is directly related to sexual function in men (236). In particular, serum estradiol is associated with sexual activity and desire, but not with erectile function (263). In addition, exogenous estradiol improves sexual desire in men with low testosterone and prostate cancer (264).

Notwithstanding these studies, the role of estrogen in male sexual behavior remains controversial (257) since several studies reached opposite conclusions. In particular, Sartorius et al. found that DHT was effective in maintaining male sexual function in healthy, older men, despite its suppressive effect on both testosterone and estradiol, suggesting that male sexual function can be ensured without aromatization (237). Furthermore, other cross-sectional studies failed to demonstrate a clear association between serum estradiol and male sexual function (238,265).

To add further complexity, estrogen action on erectile function seems to be biphasic, since estrogen deficiency may affect the ability to achieve an erection, yet estrogen excess and an increased estradiol to testosterone ratio is associated with an impaired erectile function. A Chinese group reported that serum estradiol is higher in a large sample of adult men with erectile dysfunction compared to men with normal erectile function, while it was not different among men with and without premature ejaculation (266,267). The same group has proposed that high serum estradiol and reduced estradiol to testosterone ratio may be independent risk factor for organic erectile dysfunction (268). Similar results have been obtained by other authors in hypogonadal men where high serum estradiol was associated to more severe erectile dysfunction (269). Also in an experimental rabbit model of the metabolic syndrome (a model used to investigate erectile function), erectile dysfunction is associated with high serum estradiol rather than low testosterone in line with the above mentioned clinical data (270). Conversely, other authors did not find any correlation between testosterone to estradiol ratio and erectile dysfunction (271). All these data, however, needs to be confirmed by further studies since the strength of evidence is weak due to many flaws such as the lack of data on the cause of sexual dysfunction, the poor accuracy of estradiol assay, and the retrospective collection of data.

A possible explanation for these results is that a serum estradiol in the normal male range is required for a fully normal male sexual function in addition to testosterone, while both estrogen deficiency and estrogen excess have a negative impact on male sexual activity (156,236,238).

Finally, estrogen receptors and the aromatase enzyme have been identified in the penile tissue of a large number of species, including humans (272-274) suggesting direct estrogenic activity within the penis. At present, knowledge on estrogen action within the penis derives from the observation that: i) male offspring exposure to estrogen-like endocrine disruptors in utero induces micropenis and hypospadias (176), and that ii) penile development and function is estrogen-dependent in animals (275).

OTHER NON-REPRODUCTIVE PHYSIOLOGICAL ESTROGEN ACTIONS IN MEN

Estrogens, Metabolism, and Cardiovascular Diseases in Men

The role of estrogen on glucose and insulin metabolism in men is difficult to establish since it is challenging to differentiate androgen from estrogen actions in vivo. In estrogen-deficient men, both insulin resistance and fasting glucose are increased and improve during estrogen treatment (150,158,161), confirming data from mice models (16). Thus, severe estrogen to testosterone ratio imbalance (increased androgens and decreased estrogens) seems to favor  the development of insulin resistance in men (150,151,158), and not only in estrogen-deficient men (276). In healthy men, the administration of an aromatase inhibitor in a double blind, randomized, controlled, crossover study led to a decrease of insulin sensitivity (277). Several other clinical studies confirmed that aromatase inhibition worsens insulin sensitivity in older men (278), and in obese men, independently from the increase in serum testosterone (279). The same results come from studies comparing exogenous estradiol to GnRH analogues for the treatment of prostate cancer showing that fasting both fasting glucose and cholesterol decreased in men treated with estradiol (224).

In accordance with this findings, relative estrogen deficiency is found in men with type 2 diabetes mellitus and low serum testosterone who display also low serum estradiol (280).

Furthermore, congenital estrogen deficiency is associated with an altered lipid profile (22,161), mainly characterized by higher total cholesterol and triglycerides serum levels, higher low-density lipoprotein (LDL) cholesterol and very low high-density lipoprotein (HDL) cholesterol (11,135). In these patients, estradiol treatment induces a moderate increase of HDL-cholesterol together with a small reduction of triglycerides, total cholesterol, and LDL cholesterol (21,135,138,150), resembling the effects of estrogen on lipid metabolism exerted in females (10). The administration of aromatase inhibitors leads to no change in serum lipids after 12 months of therapy (278). Outside the context of rare congenital diseases, the effects of estrogens and antiestrogens on serum lipids remains not well established due to conflicting results, and additional studies are needed (24).

In community-based men aged 20-70 years, high serum E2 is associated with reduced carotid plaque and serum E2 is directly related to carotid intima media thickness (281). The T to E2 ratio was associated with increased atheromatous plaque inflammation and increased risk of subsequent major adverse cardiovascular events (MACE) in men with documented atherosclerotic disease (282). In an aromatase-deficient man the normalization of serum E2 induced by estradiol replacement was able to reduce carotid atherosclerotic plaque volume on ultrasonography (150). While serum E2 may exert protective effects against atherosclerosis, the serum T to E2 ratio maybe a marker of low serum T and increased adiposity, especially in overweight and obese men and may be associated with increased cardiovascular (CV) risk (282,283). However, these clinical studies suffer from serum E2 measurement with commercially available immunoassay that are unreliable for E2 values in the normal male serum range (41,42,131). An association between coronary artery calcification (a surrogate radiological markers of coronary atherosclerosis) and both low serum testosterone and low serum estradiol measured by liquid chromatography/tandem mass spectrometry (LC-MS/MS) has been found in men participating to the Framingham Heart Study (284).

In hypogonadal men, estrogen deficiency, but not testosterone deficiency is responsible for vasomotor symptoms (i.e., hot flushes). In men treated with an LHRH analogue and replaced with placebo, different dosages of T alone or T plus aromatase inhibitors, only estrogen deficiency resulted in the occurrence of vasomotor symptoms (285). The main role of estrogen deficiency in the occurrence of hot flushes has been also confirmed in men with prostate cancer treated with exogenous estradiol or LHRH analogue, being hot flushes significantly more prevalent in men with estrogen deficiency due to LHRH therapy. Taken together these results reinforce the concept that in men with hypogonadism several clinical manifestations are due to relative estrogen deficiency rather than to testosterone deficiency per se (285).

Estrogens and the Male Bone

There is increasing evidence suggesting that circulating estrogens plays a key role in bone health in men, as in women (286). The relative contribution of androgen versus estrogens in the regulation of the male skeleton, however, is complex and relatively unclear (287). Some estrogen actions on male bone, such as bone maturation and the acceleration of growth arrest, are now well defined (286,288). 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 (135,152) and by several epidemiological studies (289,290). All patients with congenital estrogen deficiency due to estrogen resistance (19,133,134) or aromatase deficiency (20,136-149). have unfused epiphyses in adulthood and fail to reach their closure and complete bone maturation (11,18,25,130,286). Estrogen replacement therapy led to skeletal maturation and improvement of bone mineral density in all aromatase-deficient men described so far (11,135,144) in a dose-dependent way (147,154) while testosterone treatment did not (21,22,159). During puberty and late adolescence, epiphyseal closure, growth arrest, the achievement of peak bone mass, and final bone maturation are mainly under the control of estrogens and all these processes do not progress in the case of severe estrogen deficiency leading to tall stature and osteoporosis (21,22,135,286). The eunuchoid body proportions of the skeleton typical of hypogonadal men are the effect of estrogen deficiency during late adolescence and of the disproportional growth between long bones and the appendicular skeleton (11,286). During adulthood, both normal circulating estradiol and testosterone are required for maintaining bone mineral density in aromatase-deficient men (136,159,160) as well as in the general male population (132,286,287,289-292). Estrogen action on bone seems to be possible only when circulating estradiol is above a threshold between approximately 15 and 25 pg/mL (55-92 pmol/L) (286,289). This mechanism has been suggested both for growth arrest and bone maturation (152) and for bone mineral density (BMD) (132,289,292) and suggest that circulating estrogens above this threshold are required for optimal skeletal maturation and mineralization in men (130,286). Relative estrogen deficiency, rather than testosterone deficiency, is responsible for bone loss in hypogonadal men as clearly demonstrated when using different doses of exogenous testosterone alone or in combination with a potent aromatase inhibitor in men treated with GnRH analogues (293). In this study, BMD decreased and indices of bone resorption increased only in the group of men treated with both testosterone and anastrozole independently from the dose of exogenous testosterone administered to men with pharmacologically-induced hypogonadism (293). Furthermore, this study confirms that serum estradiol below 10 pg/mL is particularly harmful for bone health (293).

Similarly, in men with prostate cancer, estrogen deficiency induced by Androgen Deprivation Therapy (ADT) is the main factor involved in bone loss, increase of bone fragility, and the occurrence of osteoporotic fractures since serum estradiol falls below 5 pg/mL in men receiving ADT (294-296) similar to what happens in women taking antiestrogens for breast cancer (132,292,297). Accordingly, new strategies for the treatment of prostate cancer by using blockers of the androgen receptor and exogenous estradiol are being investigated in order to mitigate the risk of bone fractures and other side effects of ADT (224,295,296). In particular, transdermal estradiol resulted effective in preventing bone loss compared to LHRH analogue in men with prostate cancer (298).

Figure 6. Genetically determined factors influencing the amount of serum estrogens in men starting from a determined amount of circulating serum T.
[SNPs: single-nucleotide polymorphisms; T: testosterone; DHT: dihydrotestosterone; E2: estradiol; GnRH: gonadotropin releasing hormone; LH: luteinizing hormone; FSH: follicle-stimulating hormone]

Except for rare cases of congenital estrogen deficiency (162), clinical experimental models of estrogen deficiency (199,285,293), and ADT (295,296), the most common condition in men is relative estrogen deficiency induced by hypogonadism and low serum levels of T, the precursor for estrogen production (Figure 1) (131,290,299,300). Relative estrogen deficiency may occur in hypogonadal men (299,300) especially in those with a less functioning aromatase enzyme (290,301), whose function is mainly under genetic determination (Figure 6) (131,132,292). The association between some polymorphisms of both ERα (302,303) and aromatase (304,305) genes and low bone mineral density (BMD) have clearly pointed out that the estrogen pathway is crucial for bone health in men confirming epidemiological and clinical data. These data have been recently confirmed by studies based on genome-wide association studies. These studies provided evidence on the role of aromatase enzyme in relative estrogen deficiency occurring in men with hypogonadism and its impact on BMD (Figure 6). Accordingly, a genome-wide association study demonstrated how some genetic variants of the aromatase enzyme are correlated with circulating serum E2 and BMD and that genetically determined values of circulating estradiol are linked to BMD by estimating that every 1 pg/mL of serum E2 corresponds to a genetically determined increase of BMD of 0.048 standard deviation (306). These results have been recently replicated by another GWAS study (307). Similarly, serum estradiol but not testosterone was identified as a causal factor in bone osteoporotic fractures in 175,583 men studied using a Mendelian randomization approach (308). Thus, the lower the serum estradiol the greater the extent of bone loss in hypogonadal men, the decrease of serum T exerting a direct, minor role (131,132,157,292,293). As a matter of fact, in male to female transexuals, high serum E2 induced by estrogen therapy maintains and increases bone mass despite low serum T (309,310). The degree or relative estrogen deficiency in hypogonadal men depends on several factors, including aromatase functioning (Figure6).

Estrogens, Body Composition, and Obesity in Men

The role of sex steroids action on body composition and adipose tissue metabolism in humans is long been recognized (311). Testosterone has been largely considered the main sex steroid involved in fat pathophysiology in men (311,312). However, it has become clear that estrogen also plays an important role in adipose tissue physiology in men since the first description of human cases of congenital estrogen deficiency (11,135,151) and the generation of knock-out mice models of estrogen deficiency (16,117,313,314) showing a phenotype of increased adiposity in spite of normal to increased serum testosterone.

Accordingly, both aromatase enzyme and ERs are expressed in the adipose tissue and in the skeletal muscle (12,18,24,25).

BODY COMPOSITION

Testosterone increases muscle mass and reduces fat mass in vivo (312). However, it is now clear that much these effects are due to its conversion into estradiol.

In the context of an experimental in vivo setting, healthy younger men treated with GnRH analogues and then substituted with different doses of exogenous testosterone alone together with placebo or in combination with an aromatase inhibitor, showed an increase in both subcutaneous and visceral fat mass mainly in presence of estrogen deficiency induced by the aromatase inhibitor (199). Similar results demonstrate that fat mass increases only in the group of healthy men with relative estrogen deficiency (below 15 pg/mL) induced pharmacologically by the short-term administration of GnRH analogue and aromatase inhibitors, but not in men with estradiol in the normal range (315). In accordance obese men treated with supraphysiological doses of transdermal testosterone or dutasteride showed an increase in fat mass only in the group taking an aromatase inhibitor compared to placebo (279).

Similarly, men treated with androgen deprivation therapy for prostate cancer increase their fat mass and weight and loose muscle mass (316-318). However, a double-blind randomized study tested the effects of 6 months of transdermal estradiol therapy in men on androgen deprivation therapy, and this study showed no beneficial effect of estradiol to prevent the increase of fat mass (319). This study was prematurely stopped due to Covid 19 pandemic, and the premature discontinuation might have resulted in insufficient power to detect a benefit (319).  Estradiol therapy also seems to not influence fat-free mass and muscle size (199).

Congenital human models of sex steroids deficiency also support the importance of estrogen deficiency in the body fat mass increase since men with congenital estrogen deficiency show increased adiposity (11,135,150,151) and patients with complete androgen insensitivity syndrome do not accumulate fat mass even in  absence of testosterone action thanks to a normal production of estrogens (320).  The mechanisms through which estrogens modulate fat mass in men are not fully understood and several, different actions are involved and interlinked in a complex network.

ESTROGENS AND THE GH/IGF-1 AXIS

Estrogens modulate the GH/IGF-1 axis by enhancing both the GH and IGF-1 secretion in men (321). Thus, estrogen deficiency indirectly leads to body composition changes related to the inhibition of the GH-IGF-1 axis (321).

ESTROGEN ACTION ON ADIPOSE TISSUE

It is well known that the expression of the ER within adipose tissue is decreased in obese women (322) and that the deletion of the ERS1 gene encoding for the ER in adipocytes increases the adipocytes volume and total fat mass in both male and female rodents (323), but the underlying mechanisms needs to be fully ascertained.

Recently, the study performed in approximately 700 women and 800 men demonstrates that the expression of the estrogen receptor ESR1 within adipose tissue is inversely associated with abdominal fat mass and insulin insensitivity (324). Therefore, men with relative estrogen deficiency due to lower ESR1 expression in adipose tissue or low circulating estradiol, or both, tend to have higher fat mass and insulin resistance, thus pointing out on a main role of estrogens on weight and body composition in men.  The reduction of ESR1 expression was associated with mitochondrial dysfunction of adipocytes, and these effects seem to be mediated by the reduction of expression of Polg1, a subunit of the polymerase enzyme involved in mitochondrial DNA replication and transcription (324). Reduced ESR1 expression in both white and brown adipocytes of ERKO mice (with deleted ESR1 gene) leads to increased fat in the former and reduced energy burning in the latter (324). Accordingly, in healthy adults of both sexes, serum E2 regulates brown adipose tissue thermogenesis, the latter being increased in women compared to men and associated with serum E2 (325,326).

ESTROGENS AND FEEDING BEHAVIOR

Sex hormones are able to influence adiposity in both men and women not only through peripheral mechanisms but also through a direct action on the brain (326,327) where they may modulate both energy balance and feeding behavior (328,329). Several data suggest that estrogens modulate energy balance at the hypothalamic level where they exert an anorexigenic action (326).

In addition to the energy expenditure, increasing evidence suggests that estrogens modulate appetitive behavior and food intake through their action on hedonic pathways operating at central level. Estrogens seem to lessen appetite and to reduce food intake. Recent studies have focused on the role of locally produced estrogens within the amygdala where its amount seems to be crucial for the regulation of feeding behavior. The amygdala is involved in the control of food intake both in humans and animals. In particular, functional imaging studies have demonstrated the activation of amygdala when images/videos of food or eating behavior are administered to volunteers and that functional dysregulation of this kind of activation is present in obese men and women, especially in hunger conditions (330). By measuring aromatase availability in the amygdala using positron emission tomography (PET) with the aromatase inhibitor [11^C]vorozole in normal- weight, overweight, or obese men, Biegon et al. demonstrated that aromatase availability in the amygdala, but not circulating sex steroids, was inversely correlated to BMI (331). In particular, the aromatase inhibitor [11^C]vorozole (a surrogate marker of aromatase expression/activity in vivo and of locally produced estrogens) was less available within the amygdala of obese men compared to healthy and normal-weight men (331). This suggests that locally produced estrogens within the amygdala can suppress eating behavior thus contributing to the modulation of weight gain since they facilitate the individual control of impulsive eating through cognitive/hedonic central effects within the brain (331).

Several actions of estrogens on food intake, energy homeostasis and body fat mass have been well characterized mainly in women (326) and till now the current knowledge was that estrogens are less important in men than in women in the control of these physiological functions, but recent evidence is changing this paradigm. In fact, all of the above findings point to an important role of estrogens on body composition, energy expenditure and control of feeding behavior in men and suggest that estrogen may represent a possible target to prevent and/or reverse weight gain  in men (332)

Other Non-Reproductive Estrogen Functions in Men

During the last five years an increasing body of evidence suggest that estrogen may play a role on several other non-reproductive function in men. Among them the most investigated are cognitive function (333) and aging (334,335). However, at present more evidence is needed to confirm the putative role of estrogens on these functions in men.

EFFECTS OF ESTROGEN EXCESS

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, potent estrogenic compound (336). The period between 13 to 24 weeks of human fetal life corresponds with the highest susceptibility of male reproductive organs to endocrine disruptors (4,53,55,336). Many studies in rodents suggest that the inappropriate exposure to estrogen in utero and/or during the neonatal period impairs the hypothalamic-pituitary-gonadal axis, testicular descent, efferent ductule function and testicular function (26,31,176,178). The latter effect is a direct consequence of the exposure to estrogen excess, of the indirect effect of perturbations in circulating hormones, and of the ability of the efferent ductules to reabsorb fluid. It seems that ERβ may mediate the process through which excess estrogens produce negative effects on male reproduction (26,31,57). The effects of estrogen excess during the neonatal period can induce irreversible alterations of the testis that become manifest in adulthood, consisting of permanent changes in both testis function and spermatogenesis (26,31).

AROMATASE OVER EXPRESSION IN RODENTS

The transgenic model of mice overexpressing the aromatase enzyme (AROM+) exhibits highly elevated serum estradiol concentrations together with a decrease of serum testosterone levels due to gonadotropin suppression (211,337). The phenotypic abnormalities of AROM+ males are like those developed by mice that are perinatally exposed to estrogens. The most frequent abnormalities include: undescended testes, testicular interstitial cell hyperplasia, hypoandrogenism, and growth inhibition of accessory sex glands (211). The impairment of spermatogenesis observed in AROM+ may be due to multiple factors, including cryptorchidism, abnormal Leydig cell function, testosterone deficiency or hyperestrogenemia (211). Thus, estrogens are thought to inhibit Leydig cell development, growth and function, resulting in the final suppression of androgen production (26). Furthermore, the observation of numerous degenerating germ cells and the absence of spermatids within the seminiferous tubules of AROM+ mice suggest that germ cell development arrests at the pachytene spermatocyte stage (26). However, a possible role of cryptorchidism per se on germ cell arrest cannot be excluded since cryptorchidism is known to induce germ cell arrest in rodents (338). Interestingly, the spermatogenic arrest occurred at a stage where P450arom expression is generally high. The spermatogenic arrest found in the AROM+ mice could be explained, at least in part, by the suppression of FSH action (211,337). In fact, the reduced serum FSH levels associated with normal LH levels provide further evidence of the inhibiting actions of estrogens on FSH secretion in in AROM+ males (211,337).

Effects of Exposure to Excess Estrogens in Men

The observation that the clinical use of DES by pregnant women to prevent miscarriage is associated with a dramatic increase in the incidence of genital malformations in their sons represents the first evidence in humans on the potential for estrogen excess to provoke urogenital malformations (339). The most frequent structural and functional abnormalities include epididymal cysts, meatal stenosis, hypospadias, cryptorchidism and microphallus (339-341). The frequency of abnormalities is dependent on the timing of estrogen exposure; in fact, men who were exposed to DES before the 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 later (339,341). These data support the hypothesis that the asynchrony between formation and regression of embryonic reproductive structures is probably strongly influenced by estrogen exposure.

Various reports demonstrated that semen quality of men exposed to DES in utero is significantly worse than in unexposed controls (342), even though sperm concentrations of most of these patients was average, with normal fertility (14). The implications for human spermatogenesis in terms of exposure to environmental estrogens remain 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 testicular cancer risk, together with increased incidences of cryptorchidism, hypospadias, and impaired spermatogenesis (343). However, more direct evidence will be necessary in order to fully understand this issue and particularly to identify the exact estrogenic mechanism of action (343). It is clear that exogenous estrogens could interfere with the development of genital structures if administered during early organogenesis (341). The main effect is an impairment of gonadotropin secretion and the imbalance of estrogen to androgen ratio, which may account for impaired androgen receptor stimulation or inhibition according to the dose, the cell type and the timing of exposure (339,341). Furthermore, it seems that an excess of environmental estrogens could be a possible cause of impaired fertility in humans (176,177,341) since environmental estrogens are associated with an increased risk of subfertility in several studies (344). Although controversial, a proposed progressive decline in sperm count has been reported in some Western countries during the past 50 years, and has been suggested to involve  negative effects of environmental contaminants, especially xenoestrogens, on male reproductive function(13,176,339,344).

In adult men the effects of estrogen excess are limited to rare causes of congenital aromatase overexpression and other rare conditions such as male to female transexuals taking exogenous estrogens.

AROMATASE OVER EXPRESSION IN HUMANS

Aromatase over-expression causes an increased conversion of androgens to estrogens with a consequent excess of estrogen. Excess estrogen in boys causes gynecomastia, a premature growth spurt, early fusion of epiphyses, and decreased adult height (162,345). Increased extraglandular aromatization was firstly reported in an adopted boy with prepubertal gynecomastia in 1977 (346). Four families were then described, in which several members had estrogen excess (manifested as gynecomastia in boys and men and premature thelarche in girls) due to increased extraglandular aromatization (347-349), and one case with a gain-of-function mutation of the aromatase gene (345). The latter seemed to be an autosomal dominant inherited disease (345,348). In adult men, elevated serum estradiol levels induce mild hypogonadotropic hypogonadism due to enhanced negative feedback on pituitary gonadotropins exerted by estrogens (162,345,348). 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 having estrogen-secreting tumors, probably because serum estradiol and/or estrone levels are lower in the former (348). External genitalia in adult men with aromatase excess syndrome are characterized by normal penile and testicular size (162,345,348). This clinical reproductive phenotype has been observed also in other patients with aromatase excess syndrome due to gain-of-function mutations of the aromatase enzyme (162,350-352). Even though spermatogenesis and sexual behavior were not specifically studied, the adult men described were fertile and reported normal libido (345,348) and sperm count was normal in other studies (350). In these patients, treatment with an aromatase inhibitor reduces estrogen levels and normalized testosterone, LH and FSH serum levels (345,353), confirming a crucial role of estrogen in the suppression of both gonadotropins in men.

ESTROGEN EXCESS IN ADULT MEN

Klinefelter’s Syndrome has been classically considered a feminizing syndrome on the basis of signs (gynecomastia) and the observation of circulating estradiol higher than normal (354,355). In the literature, however, the data concerning hyperestrogenism in Klinefelter patients are not solid since they come from single case reports or studies using old assays for the measurement of serum estradiol. Data from mouse models of Klinefelter’s are not conclusive about the real increase of circulating estrogens and aromatase expression and activity (356). Infertility in these patients is mainly due to the genetic abnormalities rather than to the hormonal status (357). However, preliminary results from a recent meta-analysis does not confirm that is higher serum estradiol in Klinefelter’s patients compared with non-Klinefelter’s men, but show a condition of relative hyperestrogenism consisting with a slightly elevated estradiol to testosterone ratio in Klinefelter’s (358).

Most male to female transexuals who undergo exogenous estrogen therapy continue to have sperm production and spermatogenesis progresses even after a long period of therapy with estrogens. Histological analysis of the testes removed as part of gender affirmation procedures in 72 male to female transexuals on long -term estrogen therapy (>1 year) shows the presence of germ cells and spermatids in about 80% and 40% of cases, respectively; these percentages being inversely related to testes volume (359). In particular, a reduced diameter of seminiferous tubules, Sertoli and Leydig cells abnormalities consisting with glycoproteins accumulation, germ cells and Leydig cells hypoplasia, and down regulation of Era expression in the seminiferous tubules have all been found in testes of male to female transexuals under long-term estrogen therapy (360,361). All these changes may be associated to impaired spermatogenesis ranging from the absence of spermatozoa production (362) to various degrees of reduction in number of spermatozoa and spermatozoa precursors (spermatids) in the seminiferous tubules (360,361,363). Serum T suppression below 50 ng/dL results almost constantly in a complete suppression of spermatogenesis (362), thus incomplete suppression of spermatogenesis may be considered a marker of inadequate hormonal treatment due to underdosage or lack of patients’ adherence to therapy (360,363).

CLINICAL IMPLICATIONS OF ESTROGENS IN MALES

Diagnostic Aspects: Significance of Serum Estradiol in Men

Approximately 50 μg of estradiol are produced daily: about 5-10μg 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 (4,130,131). In adult men, the normal range of serum estradiol is around 14-43 pg/mL (51-157 pmol/L), accordingly to different studies (300,364). Based on chromatography techniques, such as liquid chromatography-tandem mass spectrometry, progress has been made in the measurement of serum estrogens within the low and low-normal range of men thereby overcoming the unreliability of immunometric commercially available assays (44). In clinical practice, the measurement of serum E2 in men is mandatory when a congenital condition of estrogen deficiency is suspected (135,162). In particular, the clinical work-up for the evaluation of male infertility may involve the serum estradiol assay when clinical aspects suggestive for aromatase deficiency, coupled with normal to high testosterone and gonadotropins levels and/or history of cryptorchidism are present (Table 6) (365). Outside this clinical context, the measurement of serum E2 could be helpful to identify a condition of relative estrogen deficiency in men with hypogonadism and osteoporosis and hot flushes. However,  the accuracy of most of the major commercially available kits for the detection of serum estradiol remains poor, especially for low serum levels of estradiol typical of the male range (1,41,42,366,367) leaving the measurement of serum E2 in men substantially not indicated in the clinic (132,292,297,358). Therefore, the assay of serum estradiol is suggested only if the method used in clinical laboratories has a very high sensitivity and specificity (e.g. 3rd generation RIA or some immunometric assays with an acceptable accuracy) (290,301,368-370). At present, the gold standard test for E2 measurement remains the gas chromatography/tandem mass spectrometry (41-44,366,367) and its progressive introduction in laboratories for clinical routine evaluations of sex steroids in recent years (1,42-44,371,372) allows precise and accurate sex steroids measurement in a clinical setting (371,372) allowing ruling in/ruling out relative estrogen deficiency in men (131,132,292) and keeping serum E2 in the normal range in hypogonadal men treated with testosterone (373,374). Recently, the results from the Testosterone Trials have pointed out the importance of serum estradiol for the outcomes of testosterone replacement therapy (375). Accordingly, changes in serum estradiol  best predicted not only BMD increase (an expected result) but also other classical outcomes of testosterone therapy in hypogonadal men such as sexual desire, hemoglobin, and HDL cholesterol suggesting that serum estradiol assayed by LC-MS/MS may be a good clinical marker of adequate testosterone substitution (375).

Estrogens and Male Infertility: Clinical and Therapeutic Implications

Estrogens are involved in male fertility and could represent a potential factor involved in the pathogenesis of infertility as well as a possible pathway to explore new therapies for human male infertility.

ESTROGEN TREATMENT

At present there is no indication to prescribe estrogen compounds to men, except for the treatment of rare diseases such as congenital estrogen deficiency (130,135,365) or in the management of transgender patients. The increasing evidence of the existence of several testosterone actions that are mainly mediated by estrogens theoretically support the concept that tailoring estrogens in the treatment of hypogonadal men may improve the outcome in terms of benefits for patients (197,198). However, at present, there is no evidence on the effectiveness and safety of such therapeutic strategy. In the future, advances in the field of routine clinical measurement of very low amounts of circulating estrogens (1,42-44,371) will open new frontiers for testing the effect of estrogen compound or of SERMs alone or combined to androgens in men with documented mild estrogen deficiency.

ESTROGEN TREATMENT OF AROMATASE DEFICIENT MEN

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 (11,131,135,286). 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 (131,135,365). 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) (135,141). The main target of estrogen replacement therapy in these patients is the skeleton in order to promote epiphyseal closure, bone maturation and mineralization and the completion of these physiological processes on time. Accordingly, high doses of estrogen in adult men with aromatase deficiency might be used to lead a rapid completion of skeletal maturation within 6-9 months in adults with epiphyseal cartilages still open, through rapid bone elongation and an increase in height followed by quick epiphyseal closure and growth arrest (131,135,147,154,365). Once epiphyseal closure has been achieved, estrogen replacement treatment should be continued lifelong. The main goal is to prevent bone loss and to reduce the risk of cardiovascular disease. In this case, the dose of estradiol should be reduced to ensure serum estradiol within the normal range for adult men (131,135,147,154,365). Moreover, estrogen treatment in aromatase deficient men is effective in normalizing or improving other aspects such as gonadotropin secretion, bone mineral density, glucose metabolism, insulin sensitivity, liver function, and circulating lipids (131,143,146-148,150,151,157-159). When estrogen treatment is started at puberty, the effects of estrogen treatment on spermatogenesis are unknown, but the administration of estrogens in a more physiological way could theoretically be associated with normal spermatogenesis in adulthood. Conversely in adult patients, impaired spermatogenesis is irreversible even when estradiol treatment is administered (135). Other aspects related to estrogen deficiency cannot be modified by estrogen treatment when the treatment is started during adulthood (e.g. eunuchoid body proportions, genu valgum, failure in reaching the bone peak mass, normal body weight restoration) (135,151).

Finally, the real impact of estrogen treatment on sexual behavior in adult aromatase-deficient men remains to be determined (135).

ANTI-ESTROGEN TREATMENT IN MEN

As estrogens act on gonadotropic feedback inhibition (157,187,190), they could be a good target in the clinical management of male infertility. The rationale is to employ anti-estrogen drugs in order to modulate gonadotropin feedback by blocking the inhibitory effect exerted by estrogen on gonadotropins and to increase both LH and FSH. This will result in increased testosterone and FSH with potential benefits on spermatogenesis (376,377). However, the real effectiveness of this approach in treating male infertility remains to be established, since conflicting results are available (4,129,172,173,200,377-381) and this kind of treatment remains empirical and ‘off label‘ (200,376,378). Thus, the real efficacy of anti-estrogens is far from being elucidated and whether the increase of sperm density induced by anti-estrogens is actually related to a real improvement of both sperm fertility and pregnancy rates is a matter of debate (4,129,200,377) (Table 8).

Since the 1960s, anti-estrogen agents have been used as an empirical treatment of male infertility (378,382) based on their modulation of the hypothalamic-pituitary testicular axis. The main classes of drug that have been tested are aromatase inhibitors. They are the most potent blockers of the estrogen-mediated negative feedback on gonadotropins and excites LH and FSH secretion aiming to stimulate spermatogenesis (383). However, no clear evidence of direct effects of anti-estrogens on spermatogenesis exists (200,376,383), but LH and FSH serum levels generally increase during aromatase inhibitor administration in infertile men (384).

Clomiphene at a dosage of 25-50 mg daily for 3-12 months, or tamoxifen at dosage of 20-30 mg daily for 3-6 months, represent the most frequently used anti-estrogen agents for the treatment of male infertility (385) (Table 8); on the contrary the new generation of selective estrogen receptor modulators does not result in significant changes in male fertility (386) (Table 8).

The use of these drugs is still off-label.

Clomiphene (25-50 mg/day) has been recently studied in a cohort of 86 men with hypogonadism for six months (387). This treatment represented an effective and apparently safe alternative to testosterone supplementation in hypogonadal men wishing to preserve their fertility (387). Furthermore, Ghanem et al. have recently found that combined treatment with clomiphene (25 mg/day) and an antioxidant drug (vitamin E) increased the pregnancy rate and improved sperm count and progressive motility in men with idiopathic oligoasthenozoospermia (388). More or less these data have been confirmed by several studies, most of them being retrospective or observational (389-391), with few RTCs studies available (392,393). Notwithstanding the improvement of sperm parameters in a variable percentage of men with infertility (393), a recent systematic review points out a possible impairment of sperm parameters (a decrease in semen count, concentration, motility, morphology and total motile sperm count) in up to 20% of patients treated with clomiphene citrate, this impairment of sperm remaining irreversible in 17% among men who had a decline in semen parameters after therapy discontinuation (394). In men with secondary hypogonadism treated with testosterone, enclomiphene (the transisomer of clomiphene) was able to prevent gonadotropin suppression and the related oligospermia compared to placebo (395) and these preliminary data have been confirmed by other studies (396)(Table 8). Clomiphene may be used off-label, but enclomiphene has not been approved by regulatory agencies and its use is limited to experimental trials (396).

Tamoxifen (20 mg/day) has been also used in combination 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 (397). In 2012, Moein et al. studied thirty-two azoospermic infertile men with proven non-obstructive azoospermia, administrating Tamoxifen for 3 months (398). Tamoxifen treatment led to the recovery of spermatozoa in the ejaculates of six patients (398). These studies showed that treatment of patients with non-obstructive 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. Also other non-controlled trials suggest improvements in sperm quality or sperm concentration(399,400), however, no well-performed clinical trial has confirmed these results (376), except for one RCT comparing tamoxifen alone and tamoxifen plus folate with placebo confirming that tamoxifen increased sperm concentration in men with sperm abnormalities (399) (Table 8). A recent meta-analysis including a very small number of studies supports the empirical use of the estrogen antagonists clomiphene and tamoxifen at the dose of 50 mg and 20 to 30 mg daily based on the finding of the detection of a doubling rate of pregnancy outcome among men with idiopathic infertility (401). 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 anti-estrogen therapy. However, studies have failed to identify the characteristics of this subgroup and thus physicians cannot distinguish potential responders and non-responders (376).

Few data are available on the effect of aromatase inhibitors in male infertility (Table 8). An old study failed to demonstrate the efficacy of testolactone in the treatment of idiopathic oligozoospermic infertility (384). However, when aromatase inhibitors (testolactone or anastrozole) were administered in a selected group of infertile men with abnormal baseline testosterone-to-estradiol ratio, an improvement of fertility rate was generally obtained (172). In particular, letrozole treatment improved semen parameters and estradiol to testosterone imbalance in patients with low testosterone and increased estradiol to testosterone ratio (381). In 2011, Saylam et al. treated 27 infertile, hypogonadotropic men with 2.5 mg daily of letrozole for six months, finding an improvement of both testosterone serum levels and semen parameters after treatment (173). Thus, it seems that letrozole may facilitate some improvement in infertile men with azoospermia by improving the number of sperm in the ejaculate (173). Accordingly, a further study on the effects of letrozole on sperm parameters showed that letrozole but not placebo was effective in increasing sperm count and improving sperm motility after 6 months of treatment in a small group of 46 patients (22 on letrozole; 24 on placebo) who were azoospermic or cryptozoospermic at baseline (402) (Table 8). These results have been also replicated by not controlled studies (403-407). However, positive effects of aromatase inhibitors on sperm concentration and quality comes mainly from case series, retrospective, and cross-sectional studies, thus leaving the strength of evidence concerning the aromatase inhibitors efficacy on male fertility of low grade (200,201,377,408).

In men with hypogonadism, antiestrogens have the advantage to be effective in increasing serum T without suppressing gonadotropins if compared with testosterone replacement therapy, thus preserving spermatogenesis (200,201,377).

Data concerning the safety of anti-estrogens for treatment of male infertility are scant, especially as far as long-term treatment is concerned (195,196,376,377,409-411). Safety data regarding the use of clomiphene and tamoxifen for male infertility is limited, but information available supports their safety (410,411), the latter  might be also derived indirectly from small groups of men with breast cancer (412). Conversely, more data are available on aromatase inhibitors (195,196,377). Six months of therapy with letrozole seems to not affect psychometric tests, glucose tolerance, serum circulating lipids, markers of bone turnover, and body composition, including BMD, in obese, hypogonadal men(413). In this study, however, moderate aromatase inhibition resulted in serum estradiol still within the normal male range and all the outcomes were obtained after a short period of treatment (413). In the literature, opposite results are available and suggest possible undesired effects of aromatase inhibitors, especially on metabolism and bone. Evidence exists that high-dose aromatase inhibition might lead to several side effects, especially when patients are treated for more than 12 months with an aromatase inhibitor (195,196). Both very short-term and short-term treatment with aromatase inhibitors had deleterious metabolic effects: one study demonstrated a prompt worsening of both insulin sensitivity and lipid profile in young and older men after 28 days of treatment with letrozole (414), while anastrozole reduced insulin sensitivity in healthy men after 6 weeks of treatment (338). In the case of longer treatments (with outcomes obtained after 1 year) vertebral deformities (415) and decreased BMD (173,197,416) were found in young and older men, respectively. In addition, treatment with aromatase inhibitors lowered HDL-cholesterol in peripubertal boys (417) and in both adult and older men (195,414) while data on total and LDL-cholesterol are conflicting (277,338,414). Data available from a very small subset of male patients operated on for male breast cancer and treated with anti-estrogens (most of them with tamoxifen) provides data on long-term effects and major adverse events (412). The authors concluded that side effects and major adverse events did not differ between men and women taking anti-estrogens and that cerebrovascular or coronary events, thromboembolic events (deep venous thrombosis)(418,419), depression, muscle cramps, and hot flashes might occur also in men during anti-estrogens treatment, hot flashes being the most frequent (411,412). These data, however, should be regarded with caution due to the small sample size, the lack of a control group, and the difficulties in proving a cause-effect relationship between major adverse events and the use of anti-estrogens in men. Besides, most of the studies on antiestrogens in men are based on short-term therapy, thus safety data for long-term therapy are not available.

As a result, it should be remarked that none of the drugs belonging to the category of anti-estrogens (i.e. clomiphene, tamoxifen, aromatase inhibitors) is approved for the indication of the treatment of male infertility by regulatory drug agencies (e.g. FDA, EMEA, TPD and TGA Regulations) nor is recommended by guidelines provided by Scientific Societies (e.g. NICE, ASA, EAA, SIAMS etc.) for use in idiopathic infertility (420,421). At present, all the data available on anti-estrogens in male infertility comes from the use of these drugs for research purposes outside the context of clinical practice. In addition, none of the studies are of adequate design, strength, and power. For all these reasons, their clinical use remains anecdotal and is off label (195,196,200,201).

In conclusion anti-estrogens, alone or in combination with testosterone, may represent a potential therapy for idiopathic oligozoospermia, however this remains an empirical off-label treatment (376,401). The data set does not yet provide sufficient evidence for these applications, but there is suggestive evidence that encourages further study (401). Further well-designed studies on adequate sample size (and homogeneous groups of men with infertility) are needed to detect their true efficacy in improving the pregnancy rate, or to identify the features of the responders.

FUTURE DIRECTIONS

Notwithstanding consistent advancements in the comprehension of estrogen role in men the pathophysiology of estrogens in males remains not fully explored and further studies are advocated. Preliminary data suggest that the decline in serum estradiol is associated with all-cause mortality in community-dwelling older men, but further investigations are needed to prove this relationship (422). In addition, the knowledge of the role of estrogen-related genetic determinants in male pathophysiology is still in its infancy. In recent years some research  based on genome-wide association studies (GWAS) have provided new insights to this field (76,306-308,423). GWAS are useful in examining unexplored areas concerning physiological and pathological actions and related genetic determinants (Figure 6). Concerning estrogens in men a recent GWAS allowed disclosing more details on the association between low serum estradiol and increased adiposity and showed that higher estradiol levels were associated with lower adiposity (423). Thus, GWAS are promising for disclosing new important aspects related to estrogen pathophysiology in men and for improving the knowledge of genetic determinants of estrogens in health and disease. Advancement in the comprehension of individual genetically determined estrogen actions (Figure 6) together with the diffusion of LC-MS/MS in clinical laboratories allowing precise measurement of estrogens in men will pave the way to better tailor the management and therapy of both estrogen deficiency and excess in the human male.

CONCLUSIONS

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. 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 biological importance of estrogen action in males. Thus, estrogens, not only androgens, are responsible for some crucial physiological functions in men like fertility, reproduction, and bone health. In particular, the balance of serum estradiol to testosterone ratio is likely crucial for maintaining all these functions, thus suggesting that the homeostatic equilibrium between estrogens and androgens is important for the correct functioning of several physiological systems in men (11,22,34,121,158,172,381). From an evolutionary perspective, this relevance of estrogen actions in males 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 7).

This chapter has addressed the reproductive effects of estrogens in males but there are emerging roles for estrogens in non-reproductive tissues. In particular, even though testosterone has traditionally been considered the sex hormone involved in bone maturation and growth arrest in men the key role of estrogens on growth has recently been revealed.

Figure 7. Direct and indirect (estrogen mediated) testosterone actions.
[DHT: dihydrotestosterone, AR: androgen receptor; ERs: estrogen receptors]

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 (130).

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 (Figure 7). The progress made in the last thirty years in this field have clarified the importance of estrogen in men but leaves some issues still unsolved. In particular, estrogen actions on bone and on gonadotropin secretion are now well characterized and part of the estrogen action on spermatogenesis is known, but further evidence is needed to clarify several aspects still under debate.

ACKNOWLEDGMENTS

We are indebted to Kenneth Korach S. (National Institutes of Health, Research Triangle Park, NC, United States), Evan Simpson (Hudson Institute of Medical Research, Clayton, Australia), Laura Maffei (Buenos Aires, Argentina) for their collaboration with our group in this research field.

A special thanks to Marco Faustini-Fustini (Ospedale Bellaria, Bologna, Italy), Antonio Balestrieri (Ospedale Bufalini, Cesena, Italy), Antonio R.M. Granata (University of Modena and Reggio Emilia), Elisa Pignatti (University of Modena and Reggio Emilia), Fabio Lanfranco (University of Turin, Italy), and Paolo Beck-Peccoz (Univesity of Milan, Italy) for fruitful discussion and collaboration in the research area of estrogen role in the human male.

We are grateful to Bruno Madeo, MD, PhD, Chiara Diazzi, MD, PhD Lucia Zirilli, MD, PhD Daniele Santi MD, PhD (University of Modena and Reggio Emilia) for their contribution in revising some parts of the previous version of this chapter (November 24, 2016).

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