Background: The presence of an ovotestis is a rare difference of sex development. Histologically, ovotestis come in two forms: 1) Mixed or 2) Bipolar. We hypothesize that it is not surgically possible to separate and preserve either the testicular or ovarian component of an ovotestis. Methods: Twenty human gonads with a previous diagnosis of ovotestis were re-sectioned in entirety and re-stained with markers for testicular (SOX9, TSPY, SALL4, DDX4, cP450, AR, α-actin) and ovarian tissue (FOXL2, SALL4, DDX4). Histologic sections were photographed at low power to confirm the presence of the entire cross section of the ovotestis. High power was used to confirm an ovotestis based on the presence of both seminiferous cords (testis) and follicles (ovary). Results: Six of twenty ovotestis did not meet our criterion for the diagnosis of ovotestis lacking the histologic evidence of both testicular and ovarian tissue (presence of ovarian stroma but lacking follicles). The remaining 13 patients in which 14 separate specimens were evaluated, contained ovotestis defined by the presence of both seminiferous cords and ovarian follicles. Seven of these specimens had low power confirmational histologic images that included the entire ovotestis and could be completely evaluated for a potential surgical plane of separation. The other seven specimens were consistent with an ovotestis biopsy without complete borders. For the seven specimen that included the entire ovotestis, 6 of the 7 had ovotesticular cords and 3 had the presence of ovotesticular follicles. None of the seven complete specimens had a clear surgical plane where testicular or ovarian components could be isolated. Often the ovarian component was composed of a thin layer of follicles, surrounded the testicular component with a mixed layer of both seminiferous cords and follicles, in-between (Figure 1 and 2). Note the presence of with ovotesticular cords and follicles (Figure 3). The remaining seven ovotestis biopsy specimens also did not have a clear surgical plane for isolating either the testicular or ovarian compartment. Conclusion: Based on the histologic evaluation of the entire ovotestis as well as ovotestis biopsy specimens it does not appear possible to surgically separate the testicular and ovarian component. We have reservations with the concept in both mixed and bipolar ovotestis that it is possible to surgically preserve either the ovarian or testicular component without leaving incongruent tissue.
