When Gonads have both Sexes!
Ovotesticular Syndrome, Ovotesticular Cords and Follicles
During normal development, DNA from the Y Chromosome causes the undifferentiated gonad to form a testicle by cascade of well define genes (i.e. SRY, SOX9, WT1). Ovarian formation is equally complex requiring its own unique set of genes (i.e. DAX-1, RSPO1 and β-Catenin). The testicle is defined by the presence of four major cell types each with specific protein markers and morphologic characteristics: (1) Sertoli cells located within the seminiferous cords, (2) germ cells located within the seminiferous cords (3) peritubular myoid cells surrounding the seminiferous cords, and (4) Leydig cells located in the interstitial space between the seminiferous cords[1]. The ovary is defined by germ cells nests, as well as primordial, primary, secondary and tertiary follicles containing oocytes surrounded by granulosa cells [2]. The ovarian space between the follicles contains stromal cells .
In rare instances the gonad differentiates into both testicular and ovarian tissue forming an ovotestis. characterized by the presence of both ovarian and testicular tissue. These patients with “ovotesticular syndrome” are often born with atypical genitalia with an atypical appearance, intermediate between male and female.
The majority of patients with ovotesticular syndrome have a 46,XX karyotype (~ 60%) with the next most common karyotype being a mosaic (i.e. 46,XX/46,XY, 46XX/46 XXY, 46,XX /47,XXY)(~ 30%). Only ~ 10% of patients have a 46XY karyotype [3, 4]. Fertility has been reported in female patients but not in males. The production of both testosterone and estrogen can lead to both male and female secondary sexual characteristic such as breast development in males with masculinized external genitalia. Post pubertal monthly cyclic abdominal pain or scrotal/labial pain depending on the location of the ovotestis signifies the presence of ovulating follicles [5, 6].
Recently, we have re-analyzed 20 human “ovotestis” in 17 patients revealing that only 14 of the gonads met the strict definition of an ovotestis with the presence of both seminiferous tubules and ovarian follicles. This illustrates the difficulty in diagnosing ovotestis and hence ovotesticular syndrome [7].
In this analysis we described two new structural entities that may be a useful adjunct to facilitate the diagnose of ovotesticular syndrome [7, 8]. The first is an ovotesticular cord, a unique structure that has not been previously described in ovotesticular pathologic specimens. We recently described ovotesticular cords in our experimental model of minced human ovotesticular syndrome . In both our human experimental model and patients with ovotestis, FOXL2-positive XX granulosa cells can co-exist within ovotesticular cords along with SOX9 positive XY Sertoli cell. It is unclear why in our bipolar model ovotesticular cords were not seen [9]. It is possible that in the minced experimental model, as the human fetal testis and ovary recombine, the Sertoli, and granuloma cells have greater chance of co-mingling to form ovotesticular cords. This is consistent with possible reprogramming of granulosa and theca cells which normally support oocytes in the ovary and Sertoli cells that support testosterone production and spermatogenesis in the testis [10]. It is also possible that our minced experimental xenografts trigger trans-differentiation of granulosa cells to Sertoli cells and/or vice-versa.
The second novel structure observed in patients with ovotestis, 4 of 11 ovotestis [36%]) were ovotesticular follicles. Ovotesticular follicles were typically observed in close association with normal testicular cords or ovarian follicles of normal morphology. Interestingly the germ cells, Sertoli cells, oocytes and granulosa cells all appeared to have normal morphology and express their characteristic immunohistochemical markers. Ovotesticular follicles were also seen in our minced model of ovotestis [8].
In our re-review of human specimens diagnosed with ovotestis [7] , two experimental models of human ovotestis:1) bipolar [9] and 2) minced and a comprehensive immunohistochemical ontogeny of the human testis [1] and ovary [2] has led to increased clarity in diagnosis of ovotestis and hence ovotesticular syndrome. Our recommendation is that gonads undergoing pathologic review for the diagnosis of ovotestis be assessed by morphology for the presence of seminiferous cords/tubules and associated interstitium to document the presence of testis tissue and primordial and primary follicles to document the presence of ovarian tissue. Specific markers to document the presence of ovarian stroma remains elusive.
The Table lists recommendations for immunohistochemical staining to facilitate documentation of human testicular and ovarian tissue. We recommend SOX9, which is specific for Sertoli cells at all ages that reside within the seminiferous cords/tubules, and FOXL2 staining that is specific for the granulosa cells in the ovary which will facilitate the identification of primordial and primary follicles [1, 2]. In addition, TSPY is extremely useful to detect male germ cells in the seminiferous cords/tubules to define testicular tissue but only in patients that have a Y chromosome (~ 40 of patients with ovotestis (XY karyotype + mosaic karyotype)) [1, 11]. We also recommend SALL4 and/or DDX4, that will stain the germ cells in the seminiferous cords as well as the oocytes in the ovarian follicles [1, 2, 12]. OCT4 stains germ cells during the fetal stage but does not stain in the postnatal stage except in the setting of carcinoma in-situ [13] and is therefore not particular useful. AMH and inhibin will stain Sertoli cells prenatally but have poor post-natal staining capacity [1]. Cytochrome P450 is useful marker for Leydig cell staining at all ages, but not specific to the interstitial area of the testis with staining also seen within the ovary. Calretinin does stain Leydig cells but also stains cells within the seminiferous cords/tubules. AR although not specific to either the testis or ovary does preferentially stain the interstitial cells outlining the seminiferous cords/tubules. Finally, smooth muscle a-actin is a characteristic marker of myoid cells that surround the seminiferous cords [1]. Thus, we recommend using eight immunohistochemical markers to diagnose an ovotestis: 1) SOX9, TSPY, SALL4, DDX4, cytochrome P450, AR, smooth muscle a-actin for the testicular component and FOXL2 and SALL4, DDX4 for the ovarian component. SOX9 and TSPY (useful only in the presence of a Y karyotype) are specific testicular markers and FOXL2 the only specific ovarian marker.
Recommended Immunohistochemical Panel to Diagnose Ovotestis
We found ovotesticular cords and ovotesticular follicles in both human bipolar and mixed ovotestis specimens both with and without the presence of the Y chromosome. The clinical significance of ovotesticular cords and follicles remains unknown. We did not observe any obvious abnormalities in cellular architecture with the juxtaposition of testicular cells and ovarian cells. Further studies are in progress to better characterize the potentially unique molecular signals involved in the development of ovotesticular cords and ovotesticular follicles.
- Li, Y., et al., Development of the human fetal testis: Morphology and expression of cellular differentiation markers. Differentiation, 2023. 129: p. 17-36.
- Overland, M.R., et al., Development of the human ovary: Fetal through pubertal ovarian morphology, folliculogenesis and expression of cellular differentiation markers. Differentiation, 2023. 129: p. 37-59.
- Yu, R. and D. Diamond, Disorders of Sexual Development, in Campbell - Walsh -Wein Urology. 2021, Elsevier. p. 1006.
- Kim, H.I., et al., Ovotesticular Disorder of Sex Development in Korean Children: A Single-Center Analysis over a 30-Year Period. J Pediatr Adolesc Gynecol, 2021.
- Fernandez, N., S. Rodriguez, and J. Perez, Acute labio-scrotal pain in a patient with ovotesticular syndrome. Case report. J Pediatr Urol, 2013. 9(3): p. e123-6.
- Mirshahvalad, S.A., et al., Ovotesticular Disorder of Sex Development Presenting as an Acute Scrotum. Urology, 2018. 116: p. 176-179.
- Baskin, L.S., et al., Ovotesticular cords and ovotesticular follicles: New histologic markers for human ovotesticular syndrome. J Pediatr Urol, 2024.
- Baskin, L., et al., Ovotesticular cords and ovotesticular follicles: New markers in a model of human mixed ovotestis. Differentiation, 2023: p. 100739.
- Baskin, L., et al., A model to study human ovotesticular syndrome. Differentiation, 2023. 129: p. 60-78.
- Uhlenhaut, N.H., et al., Somatic sex reprogramming of adult ovaries to testes by FOXL2 ablation. Cell, 2009. 139(6): p. 1130-42.
- Kido, T. and Y.F. Lau, A Cre gene directed by a human TSPY promoter is specific for germ cells and neurons. Genesis, 2005. 42(4): p. 263-75.
- Boellaard, W.P.A., et al., VASA mRNA (DDX4) detection is more specific than immunohistochemistry using poly- or monoclonal antibodies for germ cells in the male urogenital tract. Medicine (Baltimore), 2017. 96(30): p. e7489.
- Cheng, L., et al., OCT4: biological functions and clinical applications as a marker of germ cell neoplasia. J Pathol, 2007. 211(1): p. 1-9.