Laboratory Studies
Cytogenetic investigation
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46,XX peripheral karyotype: Diagnostic possibilities include adrenogenital syndrome (ie, CAH), maternal androgen ingestion, excess maternal androgen production (eg, from maternal adrenocortical tumor), multiple congenital anomalies, and ovotesticular disorder of sexual development. This is the most common karyotype, constituting anywhere from 60-80% of patients.
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46,XY peripheral karyotype: Diagnostic possibilities include enzymatic defects in testosterone synthesis, such as 5-alpha reductase deficiency, complete or partial androgen insensitivity syndrome, gonadotropin deficiency, structural testicular abnormalities such as gonadal dysgenesis, and ovotesticular disorder of sexual development. This karyotype is found in about 10-15% of patients.
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46,XX/46,XY peripheral karyotype: Diagnostic possibilities include ovotesticular disorder of sexual development in a chimeric individual.
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45,X/46,XY, other mosaic patterns in peripheral karyotype: Diagnostic possibilities include mixed gonadal dysgenesis or ovotesticular disorder of sexual development.
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Molecular studies: Fluorescent in situ hybridization for occult Y-DNA is used clinically today to enhance cytogenetic results. Probing for evidence of the Y-centromere (Y97 probe) and SRY is currently used on an investigational basis. Both peripheral lymphocytes and gonadal tissue samples can be studied. Several cases have been published showing discordance between the peripheral karyotype and the gonadal tissue.
Hormonal evaluation
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Serum 17-hydroxyprogesterone: CAH must be ruled out with the use of serum 17-hydroxyprogesterone measurements. Individuals with ovotesticular disorder of sexual development have levels of this hormone that are within the reference range. In the newborn period, supportive management must be provided for those suspected of having the salt-wasting forms of CAH and pituitary insufficiency.
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Basal and stimulated serum androgens: The presence of functional testicular tissue can be determined with the use of a human chorionic gonadotropin (HCG) stimulation test. In this test, basal levels of testosterone, dehydroepiandrosterone sulfate, androstenedione, and dihydrotestosterone (DHT) should be obtained. HCG (3000-5000 IU/m2/d IM) is then administered for 5 days. On day 6, the serum hormone level tests are repeated. A rise in serum testosterone demonstrates the presence of functioning Leydig cells. Elevated testosterone precursors may suggest a specific defect of testosterone synthesis. Failure of testosterone to reduce to DHT may suggest a 5-alpha hydroxylase deficiency.
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Basal and stimulated estrogen levels: The presence of functional ovarian tissue can be determined with the use of gonadotropin or clomiphene citrate administration. An estradiol response to gonadotropin stimulation is a reliable test to differentiate ovotesticular disorder of sexual development from other disorders.
Study of androgen target cells: Defects in peripheral sensitivity to androgens may be responsible for genital ambiguity in male individuals with partial androgen insensitivity. Androgen receptor activity can be determined in fibroblasts grown from a genital skin biopsy sample; 5-alpha reductase activity also may be determined by this method.
Imaging Studies
Ultrasonography, genitogram, and intravenous pyelogram studies have traditionally been used prior to definitive surgery.
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A scrotal ultrasonography may be used to detect occult gonads.
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A genitogram is used to evaluate the structure of the urethra and to confirm the presence of a vagina.
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An intravenous pyelogram is important to rule out any associated urinary tract anomalies.
Today, pelvic or abdominal ultrasonography, CT scan, or MRI may aid in the identification of gonads and duct structures.
MRI demonstrating uterus and vagina in a 46XY infant. Courtesy of BMC Springer Nature [Scarpa MG, Lesma A, Di Grazia M, et al. Ovotesticular differences of sex development: male or female? Case series. Italian Journal of Pediatrics. 30 May 2019; 45;66. Online at https://ijponline.biomedcentral.com/articles/10.1186/s13052-019-0660-8].
Other Tests
The diagnosis of ovotesticular disorder of sexual development can sometimes be made prenatally. An amniocentesis following prenatal ultrasonography showing ambiguous genitalia may reveal a 46,XX, 46,XX/46,XY or 46,XX/47,XXY karyotype, providing a hint to the diagnosis. [16]
Procedures
Laparoscopy or laparotomy with gonadal biopsy/gonadectomy allows for histologic confirmation of both ovarian and testicular tissue.
Conservative gonadal surgery is the procedure of choice for individuals with ovotesticular disorder of sexual development. [17] This approach entails partial resection of ovotestes guided by intraoperative histologic examination, preservation of gonadal tissue concordant with sex of rearing, and removal of all discordant tissue.
Cystoscopy may be used to determine the position of entry of the vagina into the urethra or urogenital sinus.
Prophylactic gonadectomy should be considered in those who manifest signs of virilization or are at an increased risk of gonadal malignancy.
Histologic Findings
Ovotestis, ovary and/or testis, and associated duct structures may be recovered on surgical exploration.
Ovotestes contain both ovarian follicles and testicular tubular elements. They usually are compartmentalized, with connective tissue separating the ovarian from the testicular components. However, on rare occasions, an intermixture of these elements may occur.
Ovaries and ovarian portions of ovotestes appear normal and demonstrate follicular growth capable of estradiol production.
In true hermaphroditism, testicular elements show degeneration and hyalinization of the seminiferous tubules with poor germ cell development. Leydig cell hyperplasia also may occur. Spermatogenesis in the testes is rare.
Internal duct development usually corresponds to the adjacent gonad, therefore Wolffian duct structures are observed on the gonad side(s) containing functioning testicular tissue. Müllerian duct structures are observed on the gonad side not containing testicular tissue.
H&E stain of ovotestis demonstrating ovarian parenchyma (left) with multiple primary follicles and testicular parenchyma (right). Courtesy of Cureus [De Jesus Escano MR, Mejia Sang ME, Reyes-Mugica M, Colaco M, Fox J. Ovotesticular Disorder of Sex Development: Approach and Management of an Index Case in the Dominican Republic. Case Report. Cureus. Published 5 Oct 2021. Online at https://www.cureus.com/articles/70052-ovotesticular-disorder-of-sex-development-approach-and-management-of-an-index-case-in-the-dominican-republic].
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MRI demonstrating uterus and vagina in a 46XY infant. Courtesy of BMC Springer Nature [Scarpa MG, Lesma A, Di Grazia M, et al. Ovotesticular differences of sex development: male or female? Case series. Italian Journal of Pediatrics. 30 May 2019; 45;66. Online at https://ijponline.biomedcentral.com/articles/10.1186/s13052-019-0660-8].
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Intraoperative finding of uterus, fallopian tube, and streak gonad in 46XY infant. Courtesy of BMC Springer Nature [Scarpa MG, Lesma A, Di Grazia M, et al. Ovotesticular differences of sex development: male or female? Case series. Italian Journal of Pediatrics. 30 May 2019; 45;66. Online at https://ijponline.biomedcentral.com/articles/10.1186/s13052-019-0660-8].
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H&E stain of ovotestis demonstrating ovarian parenchyma (left) with multiple primary follicles and testicular parenchyma (right). Courtesy of Cureus [De Jesus Escano MR, Mejia Sang ME, Reyes-Mugica M, Colaco M, Fox J. Ovotesticular Disorder of Sex Development: Approach and Management of an Index Case in the Dominican Republic. Case Report. Cureus. Published 5 Oct 2021. Online at https://www.cureus.com/articles/70052-ovotesticular-disorder-of-sex-development-approach-and-management-of-an-index-case-in-the-dominican-republic].
