Ovarian cysts (OCs) of clinical relevance are commonly found in domestic animals [16] in which they cause gynecopathies [21828] and hyperestrogenism [15]. OCs can originate from different ovarian structures and their development, frequency, and size varies across species [16]. Histology is used to distinguish different types of OCs [1015]. The most common types are follicular cysts (FCs), cysts of subsurface epithelial structure (SES), cystic rete ovarii (CRO), lutein cysts (LCs), and cystic corpora lutea (CCL) [10151629]. While OCs and related gynecopathies are well characterized in sows [28] and cattle [2], the association of OC morphology based on histology and immunohistochemistry in relation to the clinical manifestation of the patient has rarely been investigated in dogs [46]. In particular, it should be noted, that histologically evaluated LCs in dogs have not been fully documented in recent reports [4].

The present study investigated advanced histology features of canine OCs in relationship to corresponding clinical manifestations that were observed when the bitch was presented in our clinic. It was hypothesized that multiple cyst types occur simultaneously on a single ovary in bitches having clinically manifest gynecopathies, and that it is possible to determine LCs histologically in bitches with gynecopathies.

Frequently, more than one type of cyst is present on a single ovary [126]. The multiple cellular differentiation processes that are involved in follicular growth and maturation may explain the differences in the prevalence of the different types of cysts. This is consistent with our findings in bitches with clinical signs of gynecopathies. We divided our cases into two groups: Group 1, only endocrine inactive OCs and Group 2, endocrine active and inactive OCs. In Group 1, we confirmed only two cases. In that group, only CROs were present (case No. 17 and 19) and were associated with clinical signs of gynecopathies. However, CROs do usually not produce hormones [26], although they may replace the surrounding physiological ovarian tissue [23], which could result in a functional loss. However, in the preliminary stages of our study, hyperestrogenism could be present due to delayed ovulation. Theoretically, the endocrine active OCs could have disappeared due to atresia or spontaneous ovulation, as has been described in cattle [17]. Other clinical observations in these two CRO cases, like edema of the vulva, the estrus cycle stage based on vaginal cytology, pyometra, and GLCHE would also relate to the prolonged presence of estrogen or a hormonal disturbance. Overall, CRO was the third most common type of OC detected in this study, although cells of the rete ovarii were present in the follicular stroma in a higher number than epithelial cells and granulosa cells. A possible explanation for this is, that rete ovarii tissue is physiologically uninvolved into sex-hormone-regulated differentiation and maturation processes [32]. Group 2 included the other 19 of 21 cases and was dominated by FCs (present in n = 17 bitches, 81.0%); FCs were the second most common cystic structures in this study. The transition of a mature physiologic follicle into a pathological FC is fluent and in general is based on an imbalance of the underlying endocrine regulatory mechanisms during estrus, on the local and superior levels [7]. In terms of clinical importance, hormone-producing FCs are the most often described cystic structure in relation to clinically persistent estrus or anestrus [525] as well as to infertility [1231]. In four cases of Group 2, FCs were present together with LCs. Clinical symptoms of FCs and LCs overlap and the bitches in this study had an estrogen-characterized clinical picture. LCs occur more frequently in sows and cows than in bitches [1629]. Hormone-producing canine LCs were described histologically by Dow [10] in 10% of 90 animals with cystic structures of ovaries that were submitted to routine post-mortem examination. In that study, clinical data of the bitches and histological pictures of LCs were not included, and evidence of oocyte residues in LCs was not a criterion in the histological evaluation. However, there are a few histological descriptions, but no histological figures, of canine LCs [1629]. In our study, we were able to detect more than twice the percentage of LCs reported by Dow [10] in dogs (n = 5/21, 23.8%) with medical histories of gynecopathies. The underlying pathological mechanism of LC, an insufficient LH-secretion of the adenohypophysis [23], is similar to the one described for FCs. A possible explanation for the difference in prevalences of LCs and FCs may be related to the sequence of cellular processes involved in follicular growth and maturation. The development of an LC ultimately requires luteinization of granulosa cells and therefore involves an additional cell differentiation step. Residues of oocytes can be found in FCs [3] and, as we have shown in our study, in LCs. We, therefore, propose to include this characteristic in the histological evaluation criteria for canine OCs as a contribution to clearly defining LCs in bitches (Table 1; “central cavity may contain degenerated oocytes” of LC). Moreover, this characteristic could help to distinguish histologically LC from CCL. The latter is characterized by a fossa ovarii [29] as a sign of ovulation. However, the possible misinterpretation of luteinized anovulatory follicles as LCs should be considered. Since only bitches which fulfill the inclusion criteria for OCs were included in this study, we are confident about our results. With regard to clinical presentation, we note that in cases of conservative therapy measures of cycle aberrations, such as the application of human chorion gonadotropin (hCG, e.g., Ovogest) or gonadotropin-releasing hormone analog (GnRHa, e.g., Receptal) [19], the simultaneous occurrence of LCs and FCs has to be considered, which could explain failures of conservative treatment.

In addition to OCs, corpora lutea (CL) occurred in 66.7% (n = 14/21) of our cases. This is consistent with previous findings [6]. Conservative treatment of pyometra with an antigestagen (Aglepristone) is often not effective [35] since hormonally active CL and OCs can have an enduring effect of the endometrium [35]. Regardless, hormonally active LCs may also contribute to conservative treatment failure.

In contrast to previous findings, in which FCs were the most cystic ovarian structures in bitches [1020], the number of cysts of SES dominated all other types of OCs in this study. The development of cysts of SES may be induced by accidental scattering of ovarian serosa that was introduced to the ovarian tissue during ovulation [37]. This process can potentially occur during any physiological heat of the bitch. Gulliver and Hurst [14] reported that estradiol exposure can result in ovarian surface epithelium hypertrophy and hyperplasia in mouse ovaries. On that basis, Chuffa et al. [9] analyzed the expression of sex steroid receptors in polycystic ovaries of dogs and noted moderate expression levels in unspecified OCs. It is likely that canine cysts of SES also express estrogen receptors, which could explain their frequent occurrence in bitches with high estrogen levels (case No. 1, 2, 4, 7, 10, 14, 16, 21). Overall, it should be critically noted, that only OCs ≤ 1 cm in diameter were sampled for histological examination, and OCs > 1 cm in diameter (which were present in 12 of the 21 bitches in this study; Table 2), were sampled for the presence of corresponding cyst fluid. Results of the estradiol-17ß and progesterone concentrations for these bitches have been published elsewhere [18]. Case No. 1 had to be excluded from Group 1 and transferred into Group 2 due to additional 15 OCs with a diameter > 1 cm that were not histologically evaluated, but contained increased estradiol-17ß and progesterone concentrations in cyst fluid published elsewhere [18]. Moreover, for case No. 21 we considered that at least one more hormonally active OC was present in addition to the histologically evaluated LC. Nevertheless, the clinical symptoms did not differ, as shown in Table 2.

Based on previous reports, the age of female dogs with OCs varies between 1 and 18 years [10112231]. This was confirmed by our results in which bitches had an average lifetime of 9.5 ± 3 years. However, we were unable to confirm that the prevalence of OCs is in direct proportion to dog age, as reported by Marino et al. [22]. Since life expectancy of different dog breeds varies, there is a tendency of increasing OC prevalence in dogs over the age of six. A relationship between obesity and OC incidence has been reported in human [21], mouse [27], and rat [36], but investigations into that association in dogs are lacking. Our study population included 38.9% (n = 7/18) overweight bitches, whereas 61.1% (n = 11/18) were normal weighted. A nutritional assessment was carried out during the dogs' first clinical examination and followed generally accepted protocols. However, based on our results we can only speculate that, with an increase in adipose tissue, the risk of developing OCs also increases in dogs; further investigations are needed. Most female dogs with OCs were nulliparous (n = 12/15, 80.0%), which supports findings reported elsewhere [610].

In Germany and other prosperous societies, most dogs are well integrated into households and are not allowed to breed. Under the assumption that most female dogs in Germany are nulliparous, it is not surprising that a similar situation was reflected in our OC study population. Studies that include free-ranging stray bitches may derive different results. Referring to the medical and reproductive histories of our bitches, there was no correlation between previous treatment of bitches with hormones to suppress their physiological estrus cycle or induce abortion and the prevalence of OCs. But, under the assumption that treatment with hormones can promote OC formation [5811], we recommend further investigations, including studies with standardized control and patient groups. Prolonged or shortened inter-estrus intervals of the past heats seem to be relatively low-risk factors for the development of OCs. Pet owners reported irregular past heats in 25% of the study animals, which did not reveal any gynecopathies. This could be due to the spontaneous healing of endocrine active OCs, as has been described in cattle [17].

The leading symptom in the bitches with OCs in our study was prolonged vaginal discharge (16 of 20 examined cases, 80.0%), which was consistent with a previous report on bitches with OC syndrome [6]. The most common OC-accompanying diagnosis was pyometra in 16 of 21 cases (76.2%). In this study population, the endometrium of 11 bitches (68.8%) also showed GLCHE. Another dog had a GLCHE accompanied by a mucometra. Overall, 13 bitches with OCs had GLCHE, which is indicative of longer-term exposure of the endometrium to estrogen and progesterone. Due to the association of OCs with pyometra in the majority of animals in this study (n = 16/21, 76.2%), we qualified the statement of an estrogen-induced leukocytosis with neutrophilia. Blood parameters of bitches with OCs should be separately evaluated from those in bitches with OCs and pyometra due to in utero inflammatory processes obscuring the changes. Our results indicated leukocytosis without accompanying pyometra in one of four dogs with OC. OCs may trigger hyperestrogenism through the non-regulated release of steroid sex hormones. However, dogs are estrogen sensitive [29]. An estrogen-induced myelotoxic effect on the hematopoietic system can lead to anemia and a loss of condition [253033]. In our study, we found one bitch with a thrombocyte level clearly below the physiological range (28 G/L), and that level in five further animals was in the lower part of the normal range (280–200 G/L). Only in one case (No. 19), was this linked with an erythropenia (3.9 T/L) and a loss of condition. Despite the low number of animals in this study, we suggest, that hemograms are not useful as a proxy for the presence of hyperestrogenism. It should, however, be emphasized that a thorough clinical examination must include a hemogram in order to exclude life-threatening conditions in bitches with gynecopathies and to determine the most appropriate treatment approach (conservative or surgical).

Supporting our hypothesis that multiple cyst types occur simultaneously on a single ovary in bitches having clinically manifest gynecopathies, we observed more than one type of OC on single ovaries isolated from bitches with clinical signs of gynecopathies. Despite the relatively small study population (n = 21) we identified four of five OC types with the occurrence of LCs being higher than that reported previously. Because we surprisingly could not detect a clinical difference between dogs with endocrine active and endocrine inactive OCs at the time of histological classification, our results indicate that every OC-affected bitch should be considered as an individual case. Therefore, further investigations are needed to identify the primary factors that trigger ovarian dysfunction and to better understand the complex pathophysiology of OC development.