¹Unit of Reproductive Endocrinology, First Department of Obstetrics and Gynecology, Papageorgiou General Hospital, Aristotle University of Thessaloniki, Thessaloniki, Greece, ²Laboratory of Genetics, Second Department of Obstetrics and Gynecology, University of Ioannina, Ioannina, Greece

An 18-year old, phenotypically female individual was examined for primary amenorrhea. Three months before her referral, the patient underwent surgery and a pelvic mass was removed. The physical examination revealed normal female external genitalia, normal breast develop­ment, sparse pubic hair and absence of axillary hair. The gynecological examination revealed a short blind vagina pouch and absence of cervix and uterus. Serum testosterone and dihy­drotestosterone levels were very high. Karyotype was that of a normal male (46,XY). The trans­abdominal ultrasound, computed tomography (CT) and Magnetic resonance imaging (MRI) showed absence of uterus and fallopian tubes and revealed testis-like gonads located at the internal opening of the inguinal canal bilaterally. Bilateral gonadectomy was subsequently performed. The pathology report was that of .hamartomatous testes. and associated parates­ticular leiomyoma. The clinical, laboratory, imaging, genetic and histological findings con­firmed the diagnosis of complete androgen insensitivity syndrome. DNA analysis revealed a R831X mutation in exon 7 of the androgen receptor gene. A Sertoli-cell dynamic test showed elevated basal serum inhibin-B and anti-Müllerian hormone levels without further rise fol­lowing FSH stimulation. The patient was started on hormone replacement therapy with conju­gated estrogens. Complete androgen insensitivity syndrome must be considered in any case of primary amenorrhea. Gonadectomy must be planned to eliminate the risk of gonadal malig­nancy.

Androgen insensitivity, Androgen receptor, Inhibin-B, Testicular hamartoma, Tes­ticular leiomyoma

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CASE REPORT

An 18-year old, phenotypically female individu­al was referred to the Unit of Reproductive Endo­crinology due to primary amenorrhea. A year be­fore her referral, a progestogen challenge test and a combined administration of estrogen and progesto­gen failed to induce menstrual bleeding. Three months before her referral, a transabdominal ultra­sound scan revealed a pelvic mass. The patient un­derwent surgical removal of the mass (operation­1), which was described as an .ovarian cystadenofi­broma. in the pathology report.

Basal hormonal levels before the operation were: follicle-stimulating hormo
evels of prolactin, thyroid-stimulating hormone, free triidothyronine and free thyroxine were within the normal range. Karyotype was that of a normal male (46,XY). At this time she was referred to our Unit for further investigation.

The patient was born after an uncomplicated pregnancy with normal female external genitalia. Except for the primary amenorrhea, there were no other major events in her personal medical history. The physical examination revealed normal female external genitalia, normal breast development (Tan­ner stage IV . V), sparse pubic hair and absence of axillary hair. The gynecological examination re­vealed a short blind vagina pouch and absence of cervix and uterus. No hernias or palpable masses were found in either of the inguinal areas. The se­rum dihydrotestosterone (DHT) level was 1.9 nmol/ L (normal range: 0.17-0.76 nmol/L). A Sertoli-cell test (measurement of serum inhibin-B and anti-Mullerian hormone levels before and 24h and 48h after administration of 300 IU rhFSH subcutane­ously) showed inhibin-B levels of 238 pg/ml at base­line, 219 pg/ml at 24h and 157 pg/ml at 48 h (normal range of basal values, as estimated in our Unit on a group of 20 normal men: 80-180 pg/ml at baseline). Serum anti-Müllerian hormone (AMH) levels were 10.27 ng/ml at baseline, 11.53 ng/ml at 24h and 11.95 ng/ml at 48h (normal range of basal values, as esti­mated in our Unit on a group of 10 normal men: 5.10-13.30 ng/ml at baseline). The transabdominal ultrasound, CT and MRI scans showed absence of uterus and fallopian tubes and revealed testis-like gonads (solid tissue with no apparent follicles) lo­cated at the internal opening of the inguinal canals bilaterally.

At this stage the provisional clinical diagnosis of Complete Androgen Insensitivity Syndrome (CAIS; former nomenclature: testicular feminization) was made based on the female phenotype, high levels of T and DHT, absence of Müllerian duct derivatives and 46,XY karyotype. The patient was referred for bilateral gonadectomy which was carried out by lap­arotomy. The pathology report of operation-1 was re-evaluated following the histopathologic examina­tion of the removed gonads. The new combined pa­thology report was that of "hamartomatous testi­cles". Testicular parenchyma was replaced by hama­rtomatous nodules, which were separated by fibrous tissue (Figure 1). Microscopically the gonads con­sisted of immature seminiferous tubules lined by im­mature Sertoli cells, spindle-cell stroma resembling ovarian stroma and prominent Leydig cells. The immature tubules contained rare spermatogonia.

Figure 1. "Hamartomatous testicles" consist of all components of normal testicular parenchyma (seminiferous tubules and in­terstitial tissue) with disrupted architecture. In the upper left part of the picture the seminiferous tubules are normal but pre­pubertal; in the middle there is excess fibrous tissue and in the lower right part the architecture of the seminiferous tubules is disrupted.

Sertoli and Leydig cells were immunoreactive for inhibin (Figure 2) and calretinin (Figure 3), respec­tively. The investigation for atypical germ cells us­ing the immunohistochemical staining of placental alkaline phosphatase was negative. A serous cyst and a tumor with the microscopic characteristics of a leiomyoma were adjacent to the left gonad. It be­came evident that the majority of the testicular tis­sue of the left gonad had been removed during op­eration-1.

Figure 2. Inhibin immunohistochemical staining for Sertoli cells.Only Sertoli cells are positive for inhibin.

After operation-2, the patient.s serum FSH was 36.8 IU/L, LH 58.7 IU/L, O <0.7 nmol/L, estradiol (E2) 114 pmol/L, basal inhibin-B 14 pg/ml and AMH 1.41 ng/ml.

DNA was extracted from peripheral blood lym­phocytes according to the standard salt extraction procedure. All eight exons of the Androgen Recep­tor (AR) gene were amplified using specific oligo­nucleotide primers. Polymerase chain reaction - sin­gle strand conformation polymorphism (PCR ­SSCP) analysis was used as the screening method for genetic variants. The unusual SSCP pattern found in exon 7 of the AR gene led to sequencing by ABI 3700 DNA Automated Sequencer. On se­quencing, the AR gene was found to have a single nucleotide substitution, a C to T transition in codon 831 (exon 7 of the AR gene), resulting in a missing arginine and a stop codon (R831X).

Figure 3. Calretinin immunohistochemical staining for Leydigcells. Leydig cells are strongly positive, whereas the Sertoli cells in the seminiferous tubules are weakly positive for calretinin.

The genetic and histological findings confirmed the diagnosis of CAIS. The patient was started on Hormone Replacement Therapy with conjugated estrogen (Premarin®) 0.625 mg daily.
DISCUSSION
The androgen insensitivity syndrome (AIS) is a rare (approximately 1:20,000 male births),¹ X-linked disorder characterized by 46,XY karyotype, pres­ence of normal testes and normal androgen produc­tion and metabolism. It is caused by a defect in the androgen receptor (AR) gene, which results in com­plete (CAIS) or partial (PAIS) androgen insensiti­vity with variable phenotypic expression. While in­dividuals with CAIS have female external genitalia, PAIS cases have variable genitalia ambiguity. In the case of PAIS, if the gonads have not descended into the scrotum, the patients must undergo gonadecto­my to eliminate the risk of gonadal malignancy.²

The AR gene is located on chromosomal locus Xq11-q12. More than 300 mutations have been de­scribed in patients with AIS, which include complete and partial gene deletions, point mutations and small insertions/deletions.³ It is, however, unclear wheth­er there is a relationship between the site and type of mutation and the abnormality in androgen bind­ing.⁴ Prenatal diagnosis as well as decisions about sex of rearing are hindered because of the clinical heterogeneity of phenotype for a specific mutation.

In our patient a previously reported mutation (R831X) of the androgen receptor gene was identi­fied. SSCP analysis permits detection of mutations including single-base substitutions⁴ and is followed by direct DNA sequencing in the event the prod­ucts show abnormal conformation. The AR gene is composed of eight exons, of which exon 1 encodes the N-terminal transcriptional activation domain, exons 2 and 3 encode the DNA-binding domain and exons 4-8 encode the C-terminal ligand-binding do­main. In our patient a point mutation (R831X) in the ligand-binding domain resulted in a stop codon and premature termination. In codon 831 mutations R831X, R831Q and R831L have been found in CAIS (Androgen Receptor Gene Mutations Data­base, website: http://www.mcgill.ca/androgendb/, accessed on 18/3/2006). It is of interest to mention that, as in our patient, R831X mutation has also been described in another pubertal patient with early development of testicular hamartoma.⁵

Individuals with CAIS present at various stages of life, the timing of presentation having changed over the past 50 years as the index of suspicion for the diagnosis has increased. A very common pre­sentation is the development of inguinal hernia, of­ten bilateral, during infancy.⁴ A proportion of indi­viduals, undiagnosed throughout childhood, present after puberty with primary amenorrhea, as was the case in this report. Karyotypic male individuals with CAIS have unambiguously female external genita­lia, sparse or absent axillary and pubic hair, a blind­ly-ended vagina (which may be shortened or, less often, of normal depth), no uterus and normal breast development at puberty. The testes may be located at any site along the normal course of testicular de­scent: abdominal, inguinal or in the labia majora. In some cases remnants of the Mullerian or Wolffi­an ducts or their derivatives may be found.⁶

Classically, although not universally, postpuber­tal individuals with CAIS have increased serum con­centrations of LH, normal or elevated serum con­centrations of FSH, T similar to those of normal males and upper limit of normal or elevated blood or urinary estrogens.⁷ The LH-dependent stimula­tion of Leydig cells increases both the testicular an­drogens and estrogens and simultaneously the in­creased testosterone is aromatized to estradiol.

Consequently, the increased estrogens induce breast growth. In addition, our patient had received a com­bination of estrogen and progestogen for four months in the past. Hence, as expected, the breast development was normal. Gonadectomy results in a marked increase in LH and FSH levels indicating that some testis-mediated negative feedback sup­pression of hypothalamic GnRH secretion is present in individuals with AIS. In our patient the baseline FSH levels were low before operation-1, probably on account of the combined therapy with estradiol valerate and norgestrel. A significant increase in LH and FSH levels was observed after operation-1, dur­ing which a large part of the left gonad was removed. Additional increase was observed after complete bilateral gonadectomy (operation-2).

To the best of our knowledge, this is the first re­port of a Sertoli-cell dynamic test⁸ in a case of CAIS. As expected, the basal levels of inhibin-B exceed that of normal men in a mechanism similar to gonadal steroid levels.⁹ These results indicate that Sertoli cells are probably fully functional despite the hama­rtomatous nature of the testicles and that they are at maximal stimulation, as the administration of rhF-SH did not result in further increase of serum in­hibin-B. According to some researchers the chronic gonadotropin stimulation may play a role in the in­duction of carcinogenesis in CAIS.¹⁰ Serum AMH levels have also been found elevated during puber­ty in individuals with CAIS, probably due to lack of T action, which is a major AMH inhibitor.¹¹

Testicular tumors of germ cell and non-germ cell precursors occur with increased frequency in indi­viduals with AIS, although series are small and it is unclear whether the incidence is any greater than that seen in simple cryptorchidism.² Germ cell neo­plasia is usually low grade, such as intratubular germ cell neoplasia (former nomenclature: carcinoma in situ), but seminomas (also called germinomas or dysgerminomas) have occasionally been reported, especially in postpubertal patients.¹² The youngest such patient was a 14-year old girl.¹³ These individ­uals tend to be more than 30 years old and the risk of malignant germ cell tumors increases with age, ranging from about 3% at 20 years to 30% at 50 years.¹² Tumors of non-germ cell origin also tend to be low grade, the most frequent being adenomas of Sertoli or Leydig cells. Sertoli-cell adenomas are common, occurring in 23% of the cases.¹⁴ Although Leydig-cell adenomas are less common, Leydig-cell hyperplasia is well recognized, as chronic gonadot­ropin stimulation may play a role in induction of Leydig-cell hyperplasia and subsequent neoplasia.¹⁰ Benign lesions, such as hamartomatous nodules, consisting of all components of normal testicular parenchyma but with a disrupted architecture, can also be found, as occurred in our patient.¹⁴ Inhibin and calretinin¹⁵ were used for immunohistochemi­cal staining of Sertoli and Leydig cells, respectively, suggesting that they constitute valuable markers of normal and neoplastic testicular tissue. Finally, the co-existence of testicular hamartoma with parates­ticular leiomyoma, as in our case, seems to be an extremely rare finding.¹⁶

In conclusion, in an 18-year old phenotypically female individual with primary amenorrhea, CAIS caused by R831X mutation of the AR gene was de­tected. In addition to displaying the well-described features of CAIS, basal serum inhibin-B levels were increased without further rise past FSH stimulation. Bilateral testicular hamartomas and a paratesticu­lar leiomyoma were present. CAIS must be consid­ered in any case of primary amenorrhea. Gonadec­tomy must be carried out to eliminate the risk of malignancy.

ACKNOWLEDGMENTS
We would like to thank Dr. John Tsitourides, Dr. Paris Polychronou and Ms. Vassiliki Pavlidou for their invaluable help during the clinical management of the case.

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15. Augusto D, Leteurtre E, De La Taille A, Gosselin B, Leroy X, 2002 Calretinin: A valuable marker of nor­mal and neoplastic Leydig cells of the testis. Appl Im­munohistochem Mol Morph 10: 159-162.
16. Krichen Makni S, Mnif Hachicha L, Ellouze S, et al, 2005 Feminizing testicular syndrome with multiple hamartomas and bilateral paratesticular leiomyomas. Rev Med Interne 26: 980.983.

Address correspondence and requests for reprints to:
Dimitrios G. Goulis, Unit of Reproductive Endocrinology, First Department of Obstetrics and Gynecology,
Papageorgiou General Hospital, Ring Road, Nea Efkarpia, GR-546 03, Thessaloniki, Greece,
FAX: +30-2310-991.510, e-mail: dgg30@otenet.gr

Received 05-04-06, Revised 05-06-06, Accepted 20-06-06