Abstract
Spontaneous ectopic uterine ovarian tissue was found in 9 of 118 cynomolgus monkeys (Macaca fascicularis), an incidence of 7.6%. These ectopic tissues were minute and were incidentally observed in the parametrium by microscopic examination. They were composed of primordial or primary follicles and ovarian stroma. Ectopic ovarian tissue may result from disturbed migration of the ovarian primordium during embryogenesis.
Introduction
Cynomolgus monkeys (Macaca fascicularis) are widely used as a non-rodent species for toxicity studies, so background data have accumulated (Shimoi et al., 1998). However, to our knowledge, spontaneous normal ectopic uterine ovarian tissue in the monkey has not been recorded. Ectopic ovarian tissue has been rarely reported (Litos et al., 2003) and is usually discovered when a neoplasm or cyst has arisen in humans (Heller et al., 1990; Kuga et al., 1999) or monkeys (Lim et al., 2004). Since normal ectopic ovarian tissues are likely to be asymptomatic, their occurrence may have been underestimated. The present paper describes histopathological findings of ectopic uterine ovarian tissue found in cynomolgus monkeys.
Materials and Methods
One hundred and eighteen (118) cynomolgus monkeys ages 3 to 7 years old were used in toxicity studies. They were purchased from Guangxi Research Center of Primate Laboratory Animal (China), Yulin Hongfeng Experimental Animals’s Domesticating and Breeding Center (China), Guangxi Guidong Quadramana Development and Laboratory Co., Ltd. (China) and Pt. Prestasi Fauna Nusantara (Indonesia). No abnormality was observed clinically. At the end of the dosing of the toxicity studies, the monkeys were euthanized by exsanguination under anesthesia from an intravenous injection of sodium pentobarbital solution and necropsied. Removed organs were fixed in 10% neutral-buffered formalin. The uterine corpus was sliced at the center of and embedded in paraffin. The sliced sections were stained with hematoxylin and eosin (H&E). Selected sections were processed for estradiol (Nichirei, Japan), inhibin alpha (abcam, UK, 1:100) and proliferating cell nuclear antigen (PCNA, Dako, Denmark, 1:100) immunohistochemistry. The deparaffinized sections were treated with 0.3% hydrogen peroxide and masking was carried out with 5% skimmed milk. The sections were incubated with the primary antibodies and then processed for labeled streptavidin biotin peroxidase procedure (Dako LSAB kit, Dako). Immunoreactivities were visualized by diaminobenzidine tetrachloride (DAB).
Results
Ectopic ovarian tissue, which was not grossly apparent, was found incidentally by histological examination of the uterus. The uterus with ectopic tissue was separate from the eutopic ovaries. On the cut surface of the uterus, there were no grossly visible nodules. Histologically, we found ectopic ovarian tissues in 9 of 118 monkeys (7.6%). These tissues were observed in the parametrium, which is the junctional area of the broad ligament and the uterus (Figure 1). The ectopic tissue was located in the parametrium of the perimetrium and was composed of primordial and/or primary follicles and ovarian stroma (Figure 2). Some atretic immature follicles were observed, and grown atretic follicles that were suspected of having progressed to maturation were also observed (Figure 3). The ectopic ovarian tissues differed from eutopic ovaries in having defects of the ovarian medulla and corpus luteum. Immunohistochemical staining for anti-inhibin alpha exclusively stained granulosa cells positive (Figure 4). Although estradiol-positive follicles were not detected, some granulosa cells were positive to anti-PCNA. These results and the morphologic findings indicated that most follicles remained immature, but granulosa cells in some follicles showed proliferative activity. Neither degenerative nor inflammatory lesions were found in the uterus.
Discussion
Histopathological examinations showed the ectopic tissue to be ovarian tissue with immature follicles located in the parametrium, which is the junctional area of broad ligament and the uterus. The location may indicate the pathogenesis of the ectopic ovarian tissue. The uterus is supported by broad ligaments, which are involved with primary ovaries during embryogenesis (Sadler, 2004). It was suggested that the ectopic ovarian tissue was accidentally implanted in the broad ligament and settled in the parametrium of the uterus during embryogenesis. Since degenerative/inflammatory lesions were not observed, the implanted ovarian tissue was suspected to be of embryologic origin, and not due to post-surgical or post-inflammatory implantation (Payan and Gilbert, 1987). This hypothesis suggests that it is likely that ectopic ovarian tissue occurred in the broad ligament, including the mesovarium and mesosalpinx. The ectopic ovarian tissues have been reported in the uterine wall (Lachman and Berman, 1991), broad ligament (Vendeland and Shehadeh, 2000), suspensionary ligament (Cruikshank, 1991) and the mesosalpinx (Wallace, 1991) of humans. However, the precise pathogenesis of ectopic ovary remains to be elucidated (Cruikshank, 1991).
‘Ovarian remnant syndrome’ is a well-documented entity in veterinary medicine (Sangter, 2005) and has also been diagnosed in women (Vavilis et al., 2000) in whom estrogen-dependent disease conditions have persisted after ovariectomy. The cause of this syndrome is ovarian tissue remaining in the abdomen following ovariectomy (Wallace, 1991). Ectopic ovarian tissues have not been documented in monkeys.
Most follicles in the ectopic ovarian tissue were considered immature, because they were negative to anti-estradiol. However, some granulosa cells showed proliferative activity and some follicles that were positive to anti-inhibin alpha were suspected to be developing. These findings suggested that some follicles in the ectopic ovarian tissue were in the process of maturing, and may have produced estradiol. Cynomolgus monkeys are widely used in pharmacological examinations after ovariectomy. It is recommended that estradiol levels be accessed after ovariectomy.