Endocrine Profile of an Ovariectomized Cynomolgus Monkey (Macaca fascicularis) with a Supernumerary Ovary

Abstract

For 21 mo after a bilateral ovariectomy, a 19-y-old ovariectomized cynomolgus macaque (Macaca fascicularis) continued to have menstrual cycles and measurable premenopausal estradiol and progesterone concentrations. Among these 10 menstrual cycles, 5 cycles were normal in duration and 5 were prolonged. At necropsy, a firm nodule was identified in the omental fat, and histologic evaluation confirmed the presence of ovarian tissue containing various stages of atretic follicles, a regressing corpora lutea, and a degenerating antral follicle. The endometrium and vaginal epithelium were atrophic. The occurrence of ectopic ovarian tissue in any form and location is a rare gynecologic condition in both women and nonhuman primates. Previously reported cases in nonhuman primates have been incidental findings at necropsy; therefore, the steroidogenic capacity and endocrine-related sequelae of such ovarian tissue in any nonhuman primate species is unknown. Based on structure, location, and relationship to normally situated ovaries, the ovarian tissue in this case was classified as a supernumerary ovary. To our knowledge, this is the first case report of a supernumerary ovary in a cynomolgus macaque. This report demonstrates that supernumerary ovaries in nonhuman primates can be biologically active for many years beyond sexual maturity and should be considered as a possible cause for vaginal bleeding and elevated ovarian hormone concentrations after ovariectomy.

The occurrence of ectopic ovarian tissue is a rare gynecologic condition in women. Due to the low prevalence, the clinical profile of this anomaly is largely unknown. Many cases of ovarian ectopia in premenopausal women are asymptomatic and are discovered as incidental findings during autopsy or laparotomy for other gynecologic problems, such as neoplasms and endometriosis. However, some ectopic ovaries have been diagnosed in women with persistent menstruation and morphologic changes of the endometrium after bilateral oophorectomy.^3,8 In light of these observations, several authors have speculated that ectopic ovaries are, at least partially, functional and produce ovarian hormones such as estradiol and progesterone.³

In the human literature, a third ovary is classically categorized as either an accessory or a supernumerary ovary, depending on and relative to the location of the normally situated (eutopic) ovaries.¹¹ Accessory ovaries are located near the eutopic ovaries and commonly have a ligamentous connection through the broad, uteroovarian, or infundibulopelvic ligament. In contrast, supernumerary ovaries are situated remotely in the mesentery, omentum, or retroperitoneal region, with no direct or ligamentous connection with the eutopic ovaries.

According to this terminology, the current case report is the first description of a supernumerary ovary in a cynomolgus macaque (Macaca fascicularis). Ectopic ovarian tissue has previously been described to occur in rhesus (Macaca mulatta)⁴ and cynomolgus macaques;⁵ however, these lesions were located on the broad ligament, on the serosal surface of the uterus, and within the myometrium. Most of these small clusters of ovarian tissue lacked an ovarian medullary region and corpora lutea and did not form discrete, grossly visible nodules. In addition, all previously reported cases of ectopic ovarian tissue in nonhuman primates were incidental findings at necropsy and did not include historical hormone and menstruation data, as presented here. Observations from the current case study likely will provide a better understanding of the clinical profile of this condition in female macaques, perhaps facilitating antemortem diagnosis.

Case Report

The subject of the current case report was a mid- to late-life (age, 19 y), socially housed, female cynomolgus macaque that originally was part of a large randomized study investigating the effect of reproductive life-stage (perimenopausal compared with premenopausal and menopausal [ovariectomized] state) on cardiovascular and bone health.¹ All female cynomolgus macaques used for this study were imported from the Institute Pertanian Bogor (Bogor, Indonesia). Social groups consisted of 2 to 4 monkeys housed in indoor pens equipped with perches and various manipulanda. Husbandry parameters were 12:12-h light:dark cycles; controlled temperature (68 to 84 °F [20.0 to 28.9 °C]), humidity (30% to 70%), and ventilation (10 to 15 air exchanges hourly). All animal manipulations were performed in accordance with state and federal laws, standards of the US Department of Health and Human Services, and guidelines established by the Wake Forest University Animal Care and Use Committee. The animal resources program and facilities of Wake Forest University are fully AAALAC-accredited.

The subject was assigned randomly to the menopausal study group and underwent bilateral ovariectomy of the 2 eutopic ovaries (surgical menopause). Histologic evaluation of both ovaries confirmed that the ovaries were removed intact, and no gross or histologic abnormalities were present in either ovary. During a 21-mo postoperative period, persistent menstruation and elevated ovarian hormone concentrations were observed. Based on daily vaginal swabbing, the subject experienced 5 menstrual cycles of normal duration (mean ± SEM, 31.6 ± 0.75 d) and 5 prolonged cycles (93.2 ± 22.13 d). Table 1 depicts plasma 17β-estradiol (E2), progesterone, antiMüllerian hormone (AMH), and follicle-stimulating hormone (FSH) concentrations measured intermittently across the 21-mo period. Postoperative E2 and progesterone concentrations were similar to preovariectomy levels for the subject (Table 2) and to those observed for the ovary-intact (premenopausal) group; however, postovariectomy FSH and AMH concentrations for the subject were comparable to those of the bilaterally ovariectomized (menopausal) group.

Table 1.

Cyclicity and hormone data of an ovariectomized monkey with a supernumerary ovary

Cycle		Sample obtained on cycle day
No.	Duration (d)		Estradiol (pg/mL)	Progesterone (ng/mL)	AMH (ng/mL)	FSH (ng/mL)
1	163	61	NS	1.08	0.13	NS
1	163	88	2.43	0.58	NS	NS
1	163	116	NS	NS	0.13	NS
4	32	26	17.32	9.99	NS	NS
5	29	1	9.64	1.50	NS	NS
5	29	15	6.08	3.70	NS	NS
5	29	25	NS	NS	NS	26.29
7	121	3	18.46	0.89	NS	NS
8	33	24	8.02	12.65	NS	NS

NS, not sampled.

Due to limited serum availability, postovariectomy hormone concentrations were not measured during all 10 menstrual cycles observed postoperatively.

Table 2.

Hormone levels in a monkey with a supernumerary ovary (subject) compared with those of bilaterally ovariectomized (surgically menopausal) and ovary-intact monkeys

	Subject		Bilaterally ovariectomized (n = 15)	Ovary-intact (n = 18)
Hormone	Before ovariectomy	After ovariectomy
Early follicular phase estradiol (pg/mL)	15.35 (15.08–15.62)	14.05 (9.64–18.46)a	1.83 ± 0.98 (1.78)e	14.80 ± 9.91 (13.74)
Mean luteal progesterone peak (ng/mL)	18.53 (11.01–26.04)	11.32 (9.99–12.65)b	<1.0e	8.81 ± 5.39 (7.70)
AMH (ng/mL)	16.87 (15.31–18.87)	0.13 (0.13–0.13)ce	0.20 ± 0.16 (0.15)e	11.54 ± 10.32 (8.27)
FSH (ng/mL)	1.45 ± 0.90 (1.31)	26.29d	15.16 ± 7.31 (15.01)	1.50 ± 0.97 (1.30)

Data represented as mean ± 1 SD (median) or, if 3 or fewer samples were available, as mean (range).

^aEarly follicular phase estradiol measurements based on blood samples obtained 9 and 12 mo prior to necropsy (cycles 5 and 7 in Table 1).

^bMean luteal progesterone peak measurements based on blood samples obtained 5 and 12 mo prior to necropsy (cycles 4 and 8 in Table 1).

^cAMH measurements based on blood samples obtained 17 and 19 mo prior to necropsy (cycle 1 in Table 1).

^dFSH measurements based on a single blood sample obtained 11 mo prior to necropsy (cycles 5 in Table 1).

^eBelow the level of detection.

Twenty-one months after ovariectomy, the subject was euthanized due to acute onset of lethargy, weight loss, and signs of impaired kidney and liver function (for example, marked azotemia, hyponatremia, metabolic acidosis, hypoalbuminemia, hypertriglyceridemia, and elevated alkaline phosphatase). Severe hepatic and renal lipidosis (an abnormal accumulation of lipid) were confirmed at necropsy as the cause of the morbidity and clinical chemistry abnormalities.

An encapsulated nodule (4 × 4 × 5 mm), resembling an intact ovary, was found attached to the omental adipose tissue (Figure 1 A). Histologic evaluation of the nodule confirmed the presence of ovarian stroma containing various stages of regressing corpora lutea, atretic follicles, and a degenerating antral follicle (Figure 1 B through D). Although several sections of the supernumerary ovary were obtained, no primordial, primary, or secondary follicles were observed. Despite a history of persistent menstruation and intermittent measures of E2 and progesterone concentrations indicative of an ovarian source, the endometrium and vaginal epithelium at the time of necropsy were thin and atrophic. The endometrium had dense stroma with sparse glandular tissue, and the vaginal epithelial layer had a thin to absent keratin layer, suggesting low peripheral estrogen concentrations at the time of necropsy. No other histopathologic abnormalities of the uterus, cervix, or vagina were noted.

Figure 1.

Supernumerary ovary in a 19-y-old cynomolgus monkey (Macaca fascicularis). (A) Gross appearance of the ectopic ovary with a visible (regressing) corpus luteum found within the omental fat. (B) Microphotograph image of the supernumerary ovary showing a 2.5- to 3-mm regressing corpus luteum (CL) and 2 smaller corpora albicans (ca) likely from previous cycles. Hematoxylin and eosin stain; bar, 400 µm. (C) A regressing antral follicle found in the ovarian cortex with a few apoptotic granulosa cells (arrowheads). Hematoxylin and eosin stain; bar, 80 µm. (D) An advanced atretic follicle with a collapsed antrum. Hematoxylin and eosin stain; bar, 15 µm.

Discussion

Due to a close phylogenetic relationship, female macaques develop many of the same reproductive abnormalities as women, including dysfunctional uterine bleeding, polycystic ovarian syndrome, endometriosis (adenomyosis), mesonephric duct remnants, and neoplasms (ovarian granulosa cell tumors and teratomas).² The present case report provides evidence that supernumerary ovaries can occur in female macaques and have clinical signs similar to those in women, including persistent menstruation and measurable levels of E2 and progesterone after ovariectomy.

Postmenopausal FSH and AMH concentrations and an atrophic reproductive tract were observed in the case reported here. In contrast, women with ectopic ovaries are reported to have premenopausal concentrations of FSH and active endometrial tissue (obtained by biopsy).^3,8 The supernumerary ovary in the present case was at least partially active based on multiple samples of elevated E2 after ovariectomy; however, a reason for the observed high FSH and low AMH concentrations is unclear. A thorough evaluation of the effect of this supernumerary ovary on FSH release from the anterior pituitary was not possible due to limited serum and data availability from the parent study. Important hormones to have measured at or near the day of FSH sampling include estradiol and inhibin B, produced by the granulosa cells of growing ovarian follicles. Inhibin B provides a major negative feedback effect on FSH release; however, inhibin B was not measured in the parent study at the time of this case report. Estradiol concentrations were measured within the same menstrual cycle as FSH (Table 1, cycle 5); however, extra serum samples and data were only available 10 and 24 d prior to available FSH data.

Based on histologic findings of the supernumerary ovary at necropsy, it appears that this ectopic ovary was near follicle depletion (senescence). No small growing follicles, which would produce AMH and estradiol, were observed histologically. In women, AMH concentrations have been reported to fall below the level of detection as long as 5 y before the final menstrual period.⁷ Hormone data were not available at the time of death of the current subject, but low peripheral estradiol levels would be expected given the atrophic appearance of the endometrial and vaginal tissue. In regard to clinical importance, these findings demonstrate that quiescent endometrial and vaginal tissue detected on ultrasound or cytology, respectively, does not exclude ectopic ovarian tissue as a possible cause for persistent menstruation or elevated ovarian hormones in ovariectomized female macaques.

The origin of both supernumerary and accessory ovaries is still unknown; however, possible theories discussed in the human literature include abnormal formation of the embryonic ovaries, postsurgical transplantation, and postinflammatory transplantation.^{6, 8,10,11} Some authors have proposed that during early embryonic development, supernumerary ovaries could be formed from arrested primordial germ cells, migrating from the yolk sac endoderm to the gonadal ridge (medial portion of the urogenital ridge) by the dorsal mesentery.⁸ These arrested germ cells would stimulate the mesothelium covering the dorsal mesentery to differentiate into ovarian stroma and form an ectopic ovary. Alternatively, other authors have proposed that a supernumerary ovary could be formed from a segment of the gonadal ridge transplanted to the dorsal mesentery after the colonization of the primordial germ cells.¹⁰ Accessory ovaries also may have an embryologic origin; however, it has been hypothesized that accessory and supernumerary ovaries arise from separate primordia.^8,11 Given their close proximity to eutopic ovaries, accessory ovaries may form secondary to division of the embryonic ovary after the primordial germ cells have fully descended and formed ovarian stroma.^8,11 In contrast, abnormally placed ovarian tissue perhaps results from detachment and transplantation of tissue during any surgery or severe inflammatory process involving the ovaries.⁶ This postoperative theory is based on the high percentage of human cases of ovarian ectopia with pelvic surgery.⁶ Although, the subject of this case report did have a bilateral ovariectomy, there was no evidence of inflammation, and both ovaries were confirmed intact grossly and histologically by a board-certified veterinary pathologist (JMC). In addition, the ectopic ovarian tissue in this case was surrounded by an epithelial capsule and had a defined cortical and medullary region similar to a normal ovary. Transplanted ovarian tissue from eutopic ovaries likely would lack organized tissue architecture and a capsule.

In light of the aforementioned observations, an embryologic etiology for the formation of the supernumerary ovary in the present case seems to be the most feasible explanation. Additional supportive evidence for embryologic derivation for any form of ectopic ovarian tissue is location and the presence of other congenital malformations involving the urogenital ridge. The supernumerary ovary in the current case was found in the greater omentum, which originates from the dorsal mesentery. Supernumerary ovaries in women have also been found in the mesentery and retroperitoneal regions, suggesting an embryologic background.¹¹ Although the current subject lacked other congenital malformations, abnormalities involving the urogenital tract have occurred in approximately one-third of women with ectopic ovaries.¹¹ Some of these malformations have included accessory fallopian tubes, agenesis or bifurcation of the fallopian tubes, accessory tubal ostium, agenesis of a kidney and ureter, bicornuate and unicornuate uteri, septate uterus, and bladder diverticulum.^9,11 Furthermore, ectopic ovaries in women have been observed to have a high prevalence of tumors that have an embryologic background, including teratomas, mucinous cystadenoma, and serous cystadenoma.⁶

Disagreement regarding the etiology of ectopic ovaries has lead to debate and confusion regarding terminology in the human literature. Wharton first defined ectopic ovaries as either supernumerary or accessory ovaries based on their distance from the 2 eutopic ovaries;¹¹ however, this terminology has been criticized because it assumes the presence of 2 normally placed ovaries and excludes the possibility of the aforesaid ectopic ovarian tissue resulting from postsurgical or postinflammatory implantation.⁶ Consequently, Lachman proposed that any inappropriately placed ovaries should be defined as ‘ectopic ovarian tissue’ with subcategorization based on etiology: 1) postsurgical implant; 2) postinflammatory implant; 3) truly embryologic.⁶ Although we agree that the term ‘ectopic ovarian tissue’ can be used for any misplaced ovarian stroma containing follicles and luteal tissue regardless of etiology, this terminology does not distinguish between different structural appearances of the ectopic ovarian tissue. Different structural features, such as the absence or presence of an epithelial capsule and cortical–medullary division, may suggest different etiology. In addition, this terminology does not account for location relative to the eutopic ovaries as with the terms ‘supernumerary’ and ‘accessory,’ which again may suggest origin.

As previously mentioned, other forms of ectopic ovarian tissue in cynomolgus macaques have been reported previously, including ectopic ovarian tissue located on the broad ligament, on the serosal surface of the uterus, and within the myometrium.^2,5 Unlike the current case, these clusters of uterine ovarian tissue did not resemble a normal ovary in structure. Most of these lesions were not visual grossly and often contained only a few scattered primordial, primary, small secondary, and atretic follicles. Given location and structural differences, the current supernumerary ovary and uterine ovarian tissue likely have different etiologies; however, the parametrial location of the uterine ovarian tissue (with no signs of trauma or inflammation) suggests an embryologic background. As such, we propose that terms be used in the nonhuman primate literature that reflects location, structure, and possible origin of these ectopic ovarian tissues (for example, ‘uterine ovarian tissue,’ ‘supernumerary ovary,’ or ‘ovarian remnant’ which results from incomplete removal of the ovary during surgery).