FSH Injections and Ultrasonography Determine Presence of Ovarian Components in the Evaluation of Ovotesticular Disorders of Sex Development

Ovotesticular disorder of sex development (DSD) [1], formerly known as true hermaphroditism, is characterized by the presence of both ovarian and testicular tissue in the gonads of an individual. The traditional approach to establishing a diagnosis of ovotesticular DSD has included history, physical examination, evaluation of chromosomes, sex steroid secretion, imaging of internal anatomy and laparoscopy as the gold standard of establishing the diagnosis. Differentiating the neonatal gonads (testess and ovaries) by sonographic appearance alone is difficult. In the setting of ambiguous genitalia when no clear diagnosis has been established, even with elevated testosterone, ovotestis must be suspected. Performing laparoscopy and biopsy to establish a diagnosis is important for management decisions.

Exogenous FSH has been used for some time in fertility treatments to induce multifollicular development [2]. Pharmacological doses of FSH stimulate the growth of ovarian follicles of various sizes. We report the use of FSH injections in three infants with suspected ovotestes to induce ovarian follicular development, both to determine if the patients would have a responsive ovarian component to their gonads and to enhance the use of sonography in assessing the presence and location of gonads.

In North America, ovotesticular DSD is a rare occurrence. The highest incidence is found in Africa [6].

Human menopausal gonadotropins (hMG) or ultrasonography has been used in the past to diagnose ovotesticular DSD, but both tests have limitations. Mendez et al. [7] demonstrated that an hMG challenge was able to identify ovarian tissue based on a specified rise in estradiol; however, the hMG challenge required waiting 6 to 8 weeks after a 4-day hCG stimulation test was performed and monitoring estradiol concentrations for up to 14 days. Sonographic evaluation of ovotestes also has been reported, but not all cases of ovotestes are detected with ultrasound [8].

Because of the duration of time needed with the hMG challenge and the inadequate sensitivity of ultrasonography, our Gender Medicine Team used an innovative approach, FSH stimulation testing, in three cases (two Hispanic infants and one African American/Hispanic infant) in an attempt to overcome these limitations. Using adult studies, a safe dose was extrapolated, adjusting for body surface area.

The FSH method was applied as an innovative tool to increase the sensitivity of ultrasound in the evaluation of ovotestes and to determine the presence of ovarian tissue. The use of rhFSH in these cases was intended as a diagnostic tool and was considered a nonexperimental innovation, in which the outcome of an innovation can be reliably predicted [9, 10]. Non-experimental innovation is a common practice in pediatrics, and consent to use it should be obtained from the patient or the patient's surrogate [10, 11].

The presence of follicles in portions of the gonadal tissue after FSH stimulation suggested the diagnosis of ovotestis by ultrasound in two of the three infants. All three cases later were confirmed by gonadal biopsy. In one case, follicles in the gonads developed only after FSH stimulation. In a second case, follicles increased in size and number after FSH stimulation, providing a more conclusive sonographic diagnosis of ovotestes. In the third case, no follicles were seen before or after FSH stimulation. No abdominal findings were manifested, and there was no pathologic evidence of the ovaries when biopsy results were available. In retrospect, it may have been helpful to have pre-FSH estradiol concentrations. It appears that a longer duration of FSH therapy, or delayed estradiol follow-up, is required to appreciate an increase in estradiol. No side effects were reported or observed, including breast development, clitoromegaly, vaginal bleeding, vomiting, diarrhea or fluid retention.

The FSH stimulation test, when compared with the previously reported hMG challenge, was of shorter duration and utilized imaging of stimulated gonadal tissue in place of estradiol concentrations. In each of our cases, the FSH stimulation testing provided for earlier diagnosis and earlier visualization of the gonads, thereby allowing the parents to consider the therapeutic options at an earlier date than otherwise was possible [12]. Identifying ovotestes facilitates the discussion of sex assignment at an earlier date than otherwise possible. When these cases demonstrated that FSH stimulation could be a useful diagnostic tool, we submitted a proposal to our IRB to test the sensitivity and specificity of this hypothesis, which was recently approved. There are no previous reports of FSH being used for this specific indication.

A recent report from Steinmetz et al. examined the use of combined LH and FSH injections to evaluate testosterone, estrogen, and inhibin A response in a cohort of patients with DSD [13]. The study cohort included patients with DSD (mean age: 8.4 years) and females with congenital adrenal hyperplasia (CAH) as the positive-ovarian tissue controls [13]. The subjects received the adult recommended dose of LH/FSH, 150 IU, subcutaneously daily for a total of 3 days [13]. No imaging studies were obtained after the stimulation was performed. A robust estradiol and inhibin A response occurred in the DSD group, similar to the one observed in the CAH females, demonstrating the effectiveness of combined LH/FSH injections to identify functional ovarian tissue by measuring in blood samples specific secretory products of ovarian tissue [13].

In contrast to that study, our approach aimed to increase the yield of ultrasonography by stimulating follicle formation from ovarian tissue in infants with DSD. Hence, we opted to use FSH, the gonadotropin with a predominant stimulatory role in follicle formation. The approach of measuring pre- and poststimulation concentrations of estradiol and inhibin A has the advantage of providing biochemical identification of ovarian tissue, and, in combination with the ultrasanography, should provide an ideal substitute for laparoscopy, which is considered the gold standard. Our approach with FSH stimulation relies on the principle of inducing follicle formation to enhance the yield of ultrasonography and, therefore, does not require a significant amount of blood to be drawn. As a safety precaution, we adjusted the FSH dose for body surface area, which significantly decreased the concentration of the FSH delivered to our infant subjects and may have limited the formation of follicles in our third case. This infant was the youngest patient, and the FSH stimulation test was performed when the infant was 2 weeks old. We speculate that a higher dose of FSH may be necessary in neonates as they undergo a physiological "mini puberty" period in which LH and FSH are known to be the highest in the life cycle [5].

Using recombinant FSH to stimulate the development of sonographically identifiable follicles in ovotestes allowed us to establish the diagnosis of ovotesticular DSD earlier in two of our three cases. In conclusion, the use of FSH stimulation followed by ultrasonography and the measurements of estradiol and inhibin A may help preclude the need for laparoscopy and biopsy in this population and thereby contribute to the attempt to provide less invasive interventions in the treatment of ambiguous genitalia. Nonetheless, as more studies are needed in this area, laparoscopy remains the gold standard. The confirmation of our hypothesis of the advantage of combining FSH stimulation and ultrasonography will require further studies to establish sensitivity and specificity. Furthermore, additional research is required to address the optimal recombinant FSH dosing, the timing of followup evaluation, and the addition of hormonal assays to confirm the early diagnosis of ovotesticular DSD. We, therefore, are continuing our studies under the recently approved IRB, and our study is open for recruitment.