Intratubular Germ Cell Neoplasia

Background

IGCNU or intratubular germ cell neoplasia are malignant germ cells within the seminiferous tubules. It is considered a precursor lesion of most germ cell tumors.

OUTLINE

Epidemiology

Disease Associations

Pathogenesis

Laboratory/Radiologic/
Other Diagnostic Testing

Gross Appearance
and Clinical Variants

Histopathological Features
and Variants

Special Stains/
Immunohistochemistry/
Electron Microscopy

Differential Diagnosis

Prognosis

Treatment

Commonly Used Terms

Internet Links

EPIDEMIOLOGY CHARACTERIZATION

SYNONYMS Carcinoma in situ of the testis
Gonocytoma in situ

INCIDENCE Rare but may be underreported

DISEASE ASSOCIATIONS CHARACTERIZATION

Cryptorchidism Frequency of IGCNU is 2-8%

Prior germ cell tumor 5% in contralateral testis

Infertility 0.4-1%

Gonadal dysgenesis 4/4 patients

Androgen insensitivity (Testicular feminization) 3/12 patients

Invasive germ cell tumor 98% had associated
More common in nonseminomatous tumors

PATHOGENESIS CHARACTERIZATION

CHROMOSOMAL ABNORMALITIES

Isochromosome 12p Identified
Also found in several type of germ cell tumors

Transition from In Situ to Invasive Testicular Germ Cell Neoplasia is Associated with the Loss of p21 and Gain of mdm-2 Expression

Milton W. Datta, etal.

Mod Pathol 2001;14:437-442 Abstract quote

Introduction: The tumor suppressor gene p53 has been shown to transcriptionally regulate expression of the cell cycle dependent kinase inhibitor p21. p53 is in turn regulated by the ubiquitin ligase mouse double minute-2 (mdm-2).

We have set out to examine p21 expression in testicular germ cell tumors and its relationship with p53 and mdm-2 expression.

Methods: Immunohistochemical analysis was performed on formalin-fixed paraffin-embedded tissue for p53, p21, and mdm-2 in 31 testicular germ cell tumors, which included 17 pure seminomas and 14 mixed germ cell tumors composed predominantly of embryonal carcinoma. Twenty-seven cases contained adjacent areas of intratubular germ cell neoplasia (ITGCN).

Results: 17 out of 17 seminomas and 14 out of 14 embryonal carcinomas expressed p53 in both ITGCN and the invasive tumor. In contrast, none of the 17 seminomas and only 2 of 14 embryonal carcinomas revealed positive staining for p21 protein. p21 expression was noted in 18 of 27 cases (67%) of ITGCN, and in 16 of these cases (89%) the corresponding invasive tumor had lost p21 expression. In nine additional cases p21 expression was absent in both the invasive and intratubular tumor. mdm-2 expression was present in 8 out of 17 (47%) seminomas and 13 out of 14 (93%) embryonal carcinomas but was present in only 2 out of 27 (7%) cases of ITGCN. Statistically significant associations for loss of p21 and gain of mdm-2 expression in invasive tumors were present (P < .0001).

Conclusions: The co-expression of p53 and p21 in ITGCN is consistent with preservation of p53-directed induction of p21. The loss of p21 expression in invasive tumors suggests a disruption of the p53 regulatory pathway. The inverse correlation of p21 and mdm-2 expression in both ITGCN and invasive tumors could indicate that loss of the functional p53 regulatory pathway may be correlated with the onset of mdm-2 expression. These results raise the possibility that the loss of p21 expression may be associated with the development of invasive germ cell tumors from ITGCN. Persistent p53 expression in the presence of mdm-2 suggests that in testicular germ cell tumors, while mdm-2 can block the transactivation potential of p53, it can no longer target p53 for degradation.

Fhit EXPRESION

Loss of fhit expression in testicular germ cell tumors and intratubular germ cell neoplasia.

Eyzaguirre E, Gatalica Z.

Department of Pathology, Division of Surgical Pathology, The University of Texas Medical Branch, Galveston, Texas.
Mod Pathol 2002 Oct;15(10):1068-72 Abstract quote
The FHIT gene, located at human chromosome 3p14.2, is frequently deleted in a number of human cancers, and interstitial deletions at this site were recently described in a significant proportion (41%) of testicular germ cell tumors.

We studied the expression of Fhit protein in the progression and differentiation of testicular germ cell tumors to further elucidate its role in this type of malignancy. Forty-five patients with testicular germ cell tumors and intratubular germ cell neoplasia (identified in 42/45 cases) were included in the study. Immunohistochemical staining with polyclonal rabbit IgG antibody to Fhit (ZR44, Zymed Laboratories) on formalin-fixed, paraffin-embedded tissues was used. Fhit was constitutively expressed in germ cells, Sertoli cells, and Leydig cells.

All 42 cases of intratubular germ cell neoplasia revealed no expression of this protein. No expression of Fhit was observed in any case of pure seminoma or in the seminomatous component of mixed germ cell tumors. Unexpectedly, Fhit expression was frequently (16/18) observed in the glandular tissue of mature teratomatous component of mixed germ cell tumors, despite the absence of Fhit in the intratubular germ cell neoplasia, the presumed precursor lesion.

The loss of Fhit expression is a consistent characteristic of intratubular germ cell neoplasia, which suggests a potential role in a maturation/differentiation defect early in the development of testicular germ cell tumors. Likewise, the lack of expression in seminomas is supportive of this view. However, re-expression of Fhit in well-differentiated glandular epithelium of teratomatous component of mixed germ cell tumors suggests that there is no loss of FHIT gene in this subset of neoplasia but rather that Fhit protein expression is differently regulated through the phases of germ cell tumor progression.

LABORATORY/
RADIOLOGIC/
OTHER TESTS
CHARACTERIZATION

Flow cytometry Aneuploid

HISTOLOGICAL TYPES CHARACTERIZATION

General
If IGCNU involves >2% of the testicular volume, a testicular biopsy will detect more than 50% of cases
Bilateral biopsy recommended

Enlarged cells with clear cytoplasm aligned along the basal portion of the seminiferous tubules
Thickened irregular nuclear membranes
Mitoses frequent
Seminiferous tubules have decreased tubular diameter and thickened peritubular basement membrane

Intratubular germ cell neoplasia, unclassified

Intratubular germ cell neoplasia, unclassified, with extratubular extension (early seminoma)

Intratubular seminoma Classic
Spermatocytic

Intratubular embryonal carcinoma

Intratubular yolk sac tumor

Intratubular germ cell neoplasia, other forms

OTHER VARIANTS

Sertoli Cell Proliferations of the Infantile Testis An Intratubular Form of Sertoli Cell Tumor?

Marcela Venara, M.D.; Rodolfo Rey, M.D., Ph.D.; Ignacio Bergad�, M.D.; Hern�n Mendilaharzu, M.D.; Stella Campo, M.Sc.; H�ctor Chemes, M.D., Ph.D.

From the Laboratory of Testicular Physiology and Pathology, Endocrinology Division (M.V., R.R., I.B., S.C., H.C.), Children's Hospital �Ricardo Guti�rrez,� CONICET. Buenos Aires, Argentina; and the Department of Endocrinology (H.M.), Pediatric Hospital �J.P. Garrahan,� Buenos Aires, Argentina.

Am J Surg Pathol 2001;25:1237-1244 Abstract quote

We report on six boys with intratubular Sertoli cell proliferations (ISCPs), studied by routine histologic methods, electron microscopy, and immunohistochemistry of anti-m�llerian hormone (AMH), inhibin -subunit, 3-hydroxysteroid dehydrogenase (3-HSD), proliferative cellular nuclear antigen, and p53, and carefully followed for extended periods with periodic clinical examinations, testicular ultrasonographies, and determinations of serum levels of AMH and inhibin B.

Peutz�Jeghers syndrome was found in four of six patients, and gynecomastia occurred in five of six patients. One boy had isosexual pseudoprecocity. ISCPs were observed as multiple foci of seminiferous tubules with large and proliferated Sertoli cells replacing germ cells and limited by the basement membrane. Mitotic figures, atypia, and/or interstitial invasion were not observed. Bilateral ISCPs were the only pathologic finding in three patients (patient nos. 1�3) and were associated with a microscopic tumor that resembled a large-cell calcifying Sertoli cell tumor (LCCSCT) in a fourth patient (patient no. 4). In the two remaining patients (patient nos. 5 and 6) ISCPs and LCCSCT were found in both testes.

Ultrastructural examination showed large Sertoli cells, with round nuclei, sparse organelles, and some glycogen. Inhibin -subunit immunolocalization was positive in the five patients in whom it was determined (patient nos. 2�6), AMH was positive in those ISCPs associated with tumors (patient nos. 4�6) and negative in isolated ISCPs (patient nos. 2 and 3); 3-HSD and PCNA were variable, and p53 was negative in all ISCPs.

Patient nos. 1�4 have been followed for 2�19 years. One of them is currently entering puberty, the other two have already completed puberty and have testes of normal size, and the remaining one is an adult with clinically normal testes and sperm production. None of these patients had evidence of tumor development during follow-up as shown by serial ultrasonographies and serum levels of AMH and inhibin B. Patient nos. 5 and 6 who had bilateral ISCPs and LCCSCT were orchidectomized and evolved for 2�10 years after surgery without tumor recurrence.

he prognostic significance of ISCPs, particularly when they are the only pathologic finding in a testicular biopsy, is a matter of controversy. Based on the long normal evolution, we recommend a conservative approach to therapy. The bilateral and multicentric character of ISCPs and their association with Sertoli tumors and Peutz�Jeghers syndrome suggest that they represent either proliferative lesions with tumorigenic potential or the intraepithelial stage in the evolution of some testicular Sertoli cell tumors.

SPECIAL STAINS/
IMMUNOPEROXIDASE/
OTHER CHARACTERIZATION

Special stains Ferritin positive in 83%
Glycogen positive

Immunoperoxidase PLAP positive in high percentage of cases
20/24 (83%)
154/155 (99%)
52/53 (98%)

PROGNOSIS AND TREATMENT CHARACTERIZATION

Prognostic Factors 50% of patients developed invasive tumors by 5 years

Recurrence Estimated risk following chemotherapy of 21 and 42% at 5 and 10 years

Treatment Orchiectomy is treatment of choice with unilateral disease
Low dose radiation in patients with bilateral disease

Radiotherapy of testicular intraepithelial neoplasia (TIN): a novel treatment regimen for a rare disease.

Sedlmayer F, Holtl W, Kozak W, Hawliczek R, Gebhart F, Gerber E, Joos H, Albrecht W, Pummer K, Kogelnik HD;

The Australian Uro-Oncology Group (AUO). Department of Radiotherapy and Radio-Oncology, Landeskliniken Salzburg, Salzburg, Austria.

Int J Radiat Oncol Biol Phys 2001 Jul 15;50(4):909-13 Abstract quote

PURPOSE: Testicular intraepithelial neoplasia (TIN) is a consistent precursor of most invasive germ cell tumors, currently treated by radiotherapy with 20 Gy, which destroys TIN but preserves Leydig cells. Nevertheless, analysis has shown dose-dependent dysfunction even with low therapeutic doses of 20 Gy in some cases. Therefore, we tested a dose reduction regimen by delivering smaller fractional doses to enhance the tolerance of Leydig cells.

METHODS AND MATERIALS: Between 1993 and 1999, 9 patients were treated for TIN in a prospective multicenter trial. A total dose of 13 Gy was administered in 10 fractions of 1.3 Gy. Hormonal levels of follicle-stimulating hormone, luteinizing hormone, and testosterone were assayed serially.

RESULTS: During a median follow-up time of 36 months, no patient showed evidence of local disease. A first postradiation biopsy was obtained 3-12 months after radiotherapy; 5 patients underwent a second biopsy 2-3 years after treatment. All biopsies showed a Sertoli cell-only pattern. Follicle-stimulating hormone levels continued to increase 1 year after radiotherapy, signaling eradicated spermiogenesis. Luteinizing hormone and testosterone remained within the normal range 2 years after radiotherapy.

CONCLUSIONS: In the treatment of TIN, there seems to be a dose reduction potential to 13 Gy by lowering single fractional doses, which enhances the therapeutic ratio in favor of the Leydig cells.

Tumors of the Testis, Adnexa, Spermatic cord, and scrotum in Atlas of Tumor Pathology. Third Series. Fascicle 25. 1999.
Henry JB. Clinical Diagnosis and Management by Laboratory Methods. Twentieth Edition. WB Saunders. 2001.
Rosai J. Ackerman's Surgical Pathology. Ninth Edition. Mosby 2004.
Sternberg S. Diagnostic Surgical Pathology. Fourth Edition. Lipincott Williams and Wilkins 2004.
Robbins Pathologic Basis of Disease. Sixth Edition. WB Saunders 1999.
DeMay RM. The Art and Science of Cytopathology. Volume 1 and 2. ASCP Press. 1996.
Weedon D. Weedon's Skin Pathology Second Edition. Churchill Livingstone. 2002
Fitzpatrick's Dermatology in General Medicine. 5th Edition. McGraw-Hill. 1999.
Weiss SW and Goldblum JR. Enzinger and Weiss's Soft Tissue Tumors. Fourth Edition. Mosby 2001.

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Last Updated 1/5/2004

Epidemiology
Disease Associations
Pathogenesis
Laboratory/Radiologic/ Other Diagnostic Testing
Gross Appearance and Clinical Variants
Histopathological Features and Variants
Special Stains/ Immunohistochemistry/ Electron Microscopy
Differential Diagnosis
Prognosis
Treatment
Commonly Used Terms
Internet Links

EPIDEMIOLOGY	CHARACTERIZATION
SYNONYMS	Carcinoma in situ of the testis Gonocytoma in situ
INCIDENCE	Rare but may be underreported

DISEASE ASSOCIATIONS	CHARACTERIZATION
Cryptorchidism	Frequency of IGCNU is 2-8%
Prior germ cell tumor	5% in contralateral testis
Infertility	0.4-1%
Gonadal dysgenesis	4/4 patients
Androgen insensitivity (Testicular feminization)	3/12 patients
Invasive germ cell tumor	98% had associated More common in nonseminomatous tumors

PATHOGENESIS	CHARACTERIZATION
CHROMOSOMAL ABNORMALITIES
Isochromosome 12p	Identified Also found in several type of germ cell tumors
Transition from In Situ to Invasive Testicular Germ Cell Neoplasia is Associated with the Loss of p21 and Gain of mdm-2 Expression Milton W. Datta, etal.	Mod Pathol 2001;14:437-442 Abstract quote Introduction: The tumor suppressor gene p53 has been shown to transcriptionally regulate expression of the cell cycle dependent kinase inhibitor p21. p53 is in turn regulated by the ubiquitin ligase mouse double minute-2 (mdm-2). We have set out to examine p21 expression in testicular germ cell tumors and its relationship with p53 and mdm-2 expression. Methods: Immunohistochemical analysis was performed on formalin-fixed paraffin-embedded tissue for p53, p21, and mdm-2 in 31 testicular germ cell tumors, which included 17 pure seminomas and 14 mixed germ cell tumors composed predominantly of embryonal carcinoma. Twenty-seven cases contained adjacent areas of intratubular germ cell neoplasia (ITGCN). Results: 17 out of 17 seminomas and 14 out of 14 embryonal carcinomas expressed p53 in both ITGCN and the invasive tumor. In contrast, none of the 17 seminomas and only 2 of 14 embryonal carcinomas revealed positive staining for p21 protein. p21 expression was noted in 18 of 27 cases (67%) of ITGCN, and in 16 of these cases (89%) the corresponding invasive tumor had lost p21 expression. In nine additional cases p21 expression was absent in both the invasive and intratubular tumor. mdm-2 expression was present in 8 out of 17 (47%) seminomas and 13 out of 14 (93%) embryonal carcinomas but was present in only 2 out of 27 (7%) cases of ITGCN. Statistically significant associations for loss of p21 and gain of mdm-2 expression in invasive tumors were present (P < .0001). Conclusions: The co-expression of p53 and p21 in ITGCN is consistent with preservation of p53-directed induction of p21. The loss of p21 expression in invasive tumors suggests a disruption of the p53 regulatory pathway. The inverse correlation of p21 and mdm-2 expression in both ITGCN and invasive tumors could indicate that loss of the functional p53 regulatory pathway may be correlated with the onset of mdm-2 expression. These results raise the possibility that the loss of p21 expression may be associated with the development of invasive germ cell tumors from ITGCN. Persistent p53 expression in the presence of mdm-2 suggests that in testicular germ cell tumors, while mdm-2 can block the transactivation potential of p53, it can no longer target p53 for degradation.
Fhit EXPRESION
Loss of fhit expression in testicular germ cell tumors and intratubular germ cell neoplasia. Eyzaguirre E, Gatalica Z. Department of Pathology, Division of Surgical Pathology, The University of Texas Medical Branch, Galveston, Texas.	Mod Pathol 2002 Oct;15(10):1068-72 Abstract quote The FHIT gene, located at human chromosome 3p14.2, is frequently deleted in a number of human cancers, and interstitial deletions at this site were recently described in a significant proportion (41%) of testicular germ cell tumors. We studied the expression of Fhit protein in the progression and differentiation of testicular germ cell tumors to further elucidate its role in this type of malignancy. Forty-five patients with testicular germ cell tumors and intratubular germ cell neoplasia (identified in 42/45 cases) were included in the study. Immunohistochemical staining with polyclonal rabbit IgG antibody to Fhit (ZR44, Zymed Laboratories) on formalin-fixed, paraffin-embedded tissues was used. Fhit was constitutively expressed in germ cells, Sertoli cells, and Leydig cells. All 42 cases of intratubular germ cell neoplasia revealed no expression of this protein. No expression of Fhit was observed in any case of pure seminoma or in the seminomatous component of mixed germ cell tumors. Unexpectedly, Fhit expression was frequently (16/18) observed in the glandular tissue of mature teratomatous component of mixed germ cell tumors, despite the absence of Fhit in the intratubular germ cell neoplasia, the presumed precursor lesion. The loss of Fhit expression is a consistent characteristic of intratubular germ cell neoplasia, which suggests a potential role in a maturation/differentiation defect early in the development of testicular germ cell tumors. Likewise, the lack of expression in seminomas is supportive of this view. However, re-expression of Fhit in well-differentiated glandular epithelium of teratomatous component of mixed germ cell tumors suggests that there is no loss of FHIT gene in this subset of neoplasia but rather that Fhit protein expression is differently regulated through the phases of germ cell tumor progression.

LABORATORY/ RADIOLOGIC/ OTHER TESTS	CHARACTERIZATION
Flow cytometry	Aneuploid

HISTOLOGICAL TYPES	CHARACTERIZATION
General	If IGCNU involves >2% of the testicular volume, a testicular biopsy will detect more than 50% of cases Bilateral biopsy recommended Enlarged cells with clear cytoplasm aligned along the basal portion of the seminiferous tubules Thickened irregular nuclear membranes Mitoses frequent Seminiferous tubules have decreased tubular diameter and thickened peritubular basement membrane
Intratubular germ cell neoplasia, unclassified
Intratubular germ cell neoplasia, unclassified, with extratubular extension (early seminoma)
Intratubular seminoma	Classic Spermatocytic
Intratubular embryonal carcinoma
Intratubular yolk sac tumor
Intratubular germ cell neoplasia, other forms
OTHER VARIANTS
Sertoli Cell Proliferations of the Infantile Testis An Intratubular Form of Sertoli Cell Tumor? Marcela Venara, M.D.; Rodolfo Rey, M.D., Ph.D.; Ignacio Bergad�, M.D.; Hern�n Mendilaharzu, M.D.; Stella Campo, M.Sc.; H�ctor Chemes, M.D., Ph.D. From the Laboratory of Testicular Physiology and Pathology, Endocrinology Division (M.V., R.R., I.B., S.C., H.C.), Children's Hospital �Ricardo Guti�rrez,� CONICET. Buenos Aires, Argentina; and the Department of Endocrinology (H.M.), Pediatric Hospital �J.P. Garrahan,� Buenos Aires, Argentina.	Am J Surg Pathol 2001;25:1237-1244 Abstract quote We report on six boys with intratubular Sertoli cell proliferations (ISCPs), studied by routine histologic methods, electron microscopy, and immunohistochemistry of anti-m�llerian hormone (AMH), inhibin -subunit, 3-hydroxysteroid dehydrogenase (3-HSD), proliferative cellular nuclear antigen, and p53, and carefully followed for extended periods with periodic clinical examinations, testicular ultrasonographies, and determinations of serum levels of AMH and inhibin B. Peutz�Jeghers syndrome was found in four of six patients, and gynecomastia occurred in five of six patients. One boy had isosexual pseudoprecocity. ISCPs were observed as multiple foci of seminiferous tubules with large and proliferated Sertoli cells replacing germ cells and limited by the basement membrane. Mitotic figures, atypia, and/or interstitial invasion were not observed. Bilateral ISCPs were the only pathologic finding in three patients (patient nos. 1�3) and were associated with a microscopic tumor that resembled a large-cell calcifying Sertoli cell tumor (LCCSCT) in a fourth patient (patient no. 4). In the two remaining patients (patient nos. 5 and 6) ISCPs and LCCSCT were found in both testes. Ultrastructural examination showed large Sertoli cells, with round nuclei, sparse organelles, and some glycogen. Inhibin -subunit immunolocalization was positive in the five patients in whom it was determined (patient nos. 2�6), AMH was positive in those ISCPs associated with tumors (patient nos. 4�6) and negative in isolated ISCPs (patient nos. 2 and 3); 3-HSD and PCNA were variable, and p53 was negative in all ISCPs. Patient nos. 1�4 have been followed for 2�19 years. One of them is currently entering puberty, the other two have already completed puberty and have testes of normal size, and the remaining one is an adult with clinically normal testes and sperm production. None of these patients had evidence of tumor development during follow-up as shown by serial ultrasonographies and serum levels of AMH and inhibin B. Patient nos. 5 and 6 who had bilateral ISCPs and LCCSCT were orchidectomized and evolved for 2�10 years after surgery without tumor recurrence. he prognostic significance of ISCPs, particularly when they are the only pathologic finding in a testicular biopsy, is a matter of controversy. Based on the long normal evolution, we recommend a conservative approach to therapy. The bilateral and multicentric character of ISCPs and their association with Sertoli tumors and Peutz�Jeghers syndrome suggest that they represent either proliferative lesions with tumorigenic potential or the intraepithelial stage in the evolution of some testicular Sertoli cell tumors.

SPECIAL STAINS/ IMMUNOPEROXIDASE/ OTHER	CHARACTERIZATION
Special stains	Ferritin positive in 83% Glycogen positive
Immunoperoxidase	PLAP positive in high percentage of cases 20/24 (83%) 154/155 (99%) 52/53 (98%)

PROGNOSIS AND TREATMENT	CHARACTERIZATION
Prognostic Factors	50% of patients developed invasive tumors by 5 years
Recurrence	Estimated risk following chemotherapy of 21 and 42% at 5 and 10 years
Treatment	Orchiectomy is treatment of choice with unilateral disease Low dose radiation in patients with bilateral disease
Radiotherapy of testicular intraepithelial neoplasia (TIN): a novel treatment regimen for a rare disease. Sedlmayer F, Holtl W, Kozak W, Hawliczek R, Gebhart F, Gerber E, Joos H, Albrecht W, Pummer K, Kogelnik HD; The Australian Uro-Oncology Group (AUO). Department of Radiotherapy and Radio-Oncology, Landeskliniken Salzburg, Salzburg, Austria.	Int J Radiat Oncol Biol Phys 2001 Jul 15;50(4):909-13 Abstract quote PURPOSE: Testicular intraepithelial neoplasia (TIN) is a consistent precursor of most invasive germ cell tumors, currently treated by radiotherapy with 20 Gy, which destroys TIN but preserves Leydig cells. Nevertheless, analysis has shown dose-dependent dysfunction even with low therapeutic doses of 20 Gy in some cases. Therefore, we tested a dose reduction regimen by delivering smaller fractional doses to enhance the tolerance of Leydig cells. METHODS AND MATERIALS: Between 1993 and 1999, 9 patients were treated for TIN in a prospective multicenter trial. A total dose of 13 Gy was administered in 10 fractions of 1.3 Gy. Hormonal levels of follicle-stimulating hormone, luteinizing hormone, and testosterone were assayed serially. RESULTS: During a median follow-up time of 36 months, no patient showed evidence of local disease. A first postradiation biopsy was obtained 3-12 months after radiotherapy; 5 patients underwent a second biopsy 2-3 years after treatment. All biopsies showed a Sertoli cell-only pattern. Follicle-stimulating hormone levels continued to increase 1 year after radiotherapy, signaling eradicated spermiogenesis. Luteinizing hormone and testosterone remained within the normal range 2 years after radiotherapy. CONCLUSIONS: In the treatment of TIN, there seems to be a dose reduction potential to 13 Gy by lowering single fractional doses, which enhances the therapeutic ratio in favor of the Leydig cells.