Oviductal prolapse associated with a leiomyoma in a Hermann ’ s tortoise ( Testudo hermanni )

A twelve-year-old, female Hermann’s tortoise (Testudo hermanni) was presented with an oviductal prolapse after showing tenesmus for three days prior to presentation. Radiography revealed a soft tissue opacity in the caudal coelomic cavity that ultrasonographically presented as a congested, invaginated tubular structure and was deemed to comprise the left oviduct. After repositioning the oviductal prolapse, coelioscopy confirmed that the invaginated structure consisted of the anterior part of the left oviduct and incorporated a part of the ipsilateral mesovarium and mesosalpinx. A transplastron osteotomy was carried out to perform unilateral salpingectomy and bilateral ovariectomy. A definitive diagnosis of an oviductal leiomyoma was made based on histopathological and immunohistochemical examination of the invaginated part of the oviduct. In the present case, the authors highlight that neoplastic disorders should be considered as a cause of oviductal invagination and prolapse in chelonians. To the authors’ knowledge, this is the first report of the diagnosis and successful treatment of an oviductal leiomyoma in a Hermann’s tortoise.

BSTRACT A twelve-year-old, female Hermann's tortoise (Testudo hermanni) was presented with an oviductal prolapse after showing tenesmus for three days prior to presentation. Radiography revealed a soft tissue opacity in the caudal coelomic cavity that ultrasonographically presented as a congested, invaginated tubular structure and was deemed to comprise the left oviduct. After repositioning the oviductal prolapse, coelioscopy confirmed that the invaginated structure consisted of the anterior part of the left oviduct and incorporated a part of the ipsilateral mesovarium and mesosalpinx. A transplastron osteotomy was carried out to perform unilateral salpingectomy and bilateral ovariectomy. A definitive diagnosis of an oviductal leiomyoma was made based on histopathological and immunohistochemical examination of the invaginated part of the oviduct. In the present case, the authors highlight that neoplastic disorders should be considered as a cause of oviductal invagination and prolapse in chelonians. To the authors' knowledge, this is the first report of the diagnosis and successful treatment of an oviductal leiomyoma in a Hermann's tortoise.

INTRODUCTION
The cloaca of reptiles is a vestibule where the gastrointestinal, urinary and reproductive tract terminate (McArthur and Machin, 2019). Accordingly, a cloacal prolapse in reptiles may consist of everted cloacal tissue or may involve the colon, urinary bladder (chelonians and some lizards), oviduct, hemipenis (snakes and lizards) or phallus/clitoris (chelonians) (Hedley and Eatwell, 2014). Correct identification of the involved anatomical structure may be challenging but is an essential initial step in the diagnostic and therapeutic approach for a cloacal prolapse in reptiles (Music and Strunk, 2017). Prolapse of the phallus or hemipenes is observed most frequently, followed by a prolapse of cloacal tissue or colon, the oviduct and the urinary bladder (Hedley and Eatwell, 2014). Prolapse of the oviduct in reptiles is most frequently associated with dystocia but has also been associated with other reproductive tract disorders, such as salpingitis, oviductal rupture, follicular stasis and oviductal neoplasia (Stahl and DeNardo, 2019). In addition, hypocalcemia, dehydration, systemic infections, uro-and coprolithiasis, obstipation and other space-occupying intracoelomic processes might predispose to the development of oviductal prolapse in reptiles (Stahl and DeNardo, 2019). In the present report, the diagnosis and successful surgical treatment of an oviductal leiomyoma associated with an oviductal prolapse in a Hermann's tortoise (Testudo hermanni) are described.

CASE REPORT
A twelve-year-old, female Hermann's tortoise of 1.630 kg was presented with a cloacal organ pro-lapse after showing decreased appetite and tenesmus for three days. The tortoise was kept individually in an outdoor pen. The applied management was considered adequate. Physical examination revealed the presence of a cloacal organ prolapse that presented as a congested, tubular structure with an approximately diameter of 1.8 cm and a length of 4.5 cm that protruded from the cloacal vent and showed focal dark discolorations, superficial lacerations and hemorrhage ( Figure 1). Although longitudinal striation indicative for oviductal tissue was not observed, the prolapsed tissue was considered to comprise the oviduct based on the thick muscular appearance and tubular presentation, luminal patency of the colon and voiding of urine and feces during physical examination (McArthur and Machin, 2019). The tortoise presented active and alert behavior and no other clinical abnormalities were noticed. Parasitological examination of a fecal sample yielded negative results. A blood sample was collected by jugular venipuncture for hematological and biochemical examination at the time of initial presentation and the test results were within physiological reference ranges (Andreani et al., 2014;Leineweber et al., 2019). Dorsoventral and laterolateral full body radiographs were obtained and revealed the presence of an ovoid soft tissue mass (length: 2.3 cm, height: 3.24 cm) with a dense border and slightly more radiolucent center ( Figure 2A). Ultrasonographic images were obtained before repositioning the prolapse to avoid interference with assessment of the coelomic cavity ( Figure 2B). On ultrasonography, the mass presented as a large, well-defined, luminal structure (2.72 cm x 4.45 cm), heterogeneously filled, with a prominent hypoechoic wall (0.76 -0.96 cm), which was strongly vascularized based on color flow Doppler sonography (data not shown). A presumptive diagnosis of invagination of the anterior left oviduct was made based on these findings.
Next, cloacoscopy and coelioscopy were performed following induction with 10 mg/kg alfaxalone (Alfaxan ® ; intravenous injection; Jurox Limited, Crawley, United Kingdom) via the right jugular vein and intubation with an uncuffed 2.5 mm inner diameter endotracheal tube. Anesthesia was maintained with 1.5 -2 % isoflurane (Isoflo ® , Abbott Logistics B.V., Breda, the Netherlands) in 1 L medical oxygen per minute with intermittent positive-pressure ventilation. After the tortoise was positioned in dorsal recumbency, the prolapsed tissue was repositioned in the cloaca and subsequently reduced through the left oviductal papilla under cloacoscopic guidance using a cotton tipped swab. Next, the tortoise was positioned in right lateral recumbency to perform a routine coelioscopy from left prefemoral fossa as previously described by Proença and Divers (2015) and confirmed the presence of an invagination of the anterior part of the left oviduct that incorporated a part of the ipsilateral mesovarium and mesosalpinx ( Figure  3). Endoscopy-assisted reduction of the invagination was attempted by performing gentle traction on the mesovarium using an atraumatic forceps but was unsuccessful. Accordingly, trapezoidal plastron osteotomy was performed using an oscillating cast saw as previously described by Di Girolamo and Mans (2016), in an endeavor to obtain the desired accessibility of the invaginated structure. The left oviduct was visualized after exteriorization of the left ovary ( Figure 4A). Manual reduction of the left oviduct could be achieved but required longitudinal incision of the part of the oviduct that was involved in the invagination over a length of approximately 1 cm ( Figure 4B). The anterior part of the oviduct that was previously incorporated in the invagination, showed a thickened wall and a white-yellow glistering mucosal surface. Considering the abnormal appearance of the left oviduct, salpingectomy was performed using bipolar electrocoagulation after applying a Miller's knot with poliglecaprone (Monocryl 4/0, Ethicon, Diegem, Belgium) at the most posterior part of the oviduct. The right oviduct was inspected, but no abnormalities were observed. Subsequently, routine bilateral ovariectomy was performed (Di Girolamo and Mans, 2016). Coelomic inspection revealed no other abnormalities. The coelomic membrane was routinely closed and the plastron osteotomy flap was repositioned and stabilized using a fast-curing two-component resin based on methyl-methacrylate (Technovit ® 6091, Kulzer GmbH, Hanau, Germany). Recovery from anesthesia was uneventful. Tramadol (Tramadol EG 50 mg Tablets; 8 mg/kg per orally q48h; Eurogenerics NV, Brussels, Belgium) was chosen as analgesic and given three times. Administration of 20 mg/kg ceftazidime (Glazidim; intramuscular injection q72h; GlaxoSmithKline, Wavre, Belgium) as a prophylactic antibiotic was repeated three times. Epicoelomic fluid therapy was provided for two days to restore perioperative losses. Assist-feeding was provided every three days until spontaneous food uptake was observed ten days postoperatively. No clinical or ultrasonographic abnormalities could be identified in the tortoise during a control visit that was planned twelve months postoperatively.
At cross section, the infundibulum and uterine tube of the surgically removed oviduct appeared swollen and well vascularized, showing a white-yellow glistering surface on the cut ( Figure 4C). Collected tissues were fixed in 10% neutral buffered formalin, processed routinely, embedded in paraffin and stained with hematoxylin and eosin for histopathologic examination. Evaluation of histological sections of the oviduct revealed focal proliferation of smooth muscle cells in a palisading and swirling pattern ( Figure 5A). The lamina propria and submucosal tissue of the oviductal wall showed mild but diffuse infiltration with

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heterophils. Immunohistochemical staining using antibodies directed against alpha-smooth muscle actin (α-SMA) was positive ( Figure 5B). Based on the histologic examination, a definitive diagnosis of an oviductal leiomyoma was made.
Leiomyomas are mesenchymal tumors arising from smooth muscle cells (Devereaux and Schoolmeester, 2019) and represent the most commonly encountered tumors of the uterus in humans, dogs and cats (Miller et al., 2003;Sabry and Al-Hendy, 2012;Saba and Lawrence, 2013) and the (meso)salpinx in laying hens (Berry et al., 2006;Carrasco and González, 2017). In general, leiomyomas of the uterus or oviduct are considered benign, slow growing and non-invasive with a low metastatic potential (Parker, 2007;Saba and Lawrence, 2013;Carrasco and González, 2017) and often constitute incidental findings that are made during routine surgical procedures or necropsy. Although the exact pathogenesis of uterine and oviductal leiomyomas has not been revealed, in humans, guinea pigs and laying hens, estrogen and progesterone seem to play a predisposing role in the development of leiomyomas by eliciting an increased production of growth factors (e.g. transforming growth factor-b) and cytokines,

. The plastron osteotomy flap is reflected caudally and is covered with a moist sterile compress (star). The coelomic membrane is opened between both abdominal veins (arrowheads) allowing visualization of the invaginated anterior part of the left oviduct. Note the congested oviductal wall (arrow) and the ipsilateral mesovarium and mesosalpinx (asterix) that is incorporated in the invagination. B. The incorporated mesentery is released and the invaginated oviduct is retracted after a longitudinal incision was made in the congested oviductal wall. C. Longitudinal section view of the anterior oviduct following salpingectomy reveals a swollen, wellvascularized oviductal wall with a white grey to yellow glistering surface on the cut. The ostium of the infundibulum is kept intact (arrow). C A B
which in turn may result in an increase in cell proliferation, cell hypertrophy and/or extracellular matrix (Porter et al., 1995, Sozen andArici, 2002;Walker andStewart, 2005, Berry et al., 2006). Consequently, leiomyomas mostly develop during the reproductive years, while regression may be observed during menopause or pseudomenopause (Sabry and Al-Hendy, 2012). Although the presence of estrogen and progesterone receptors have been demonstrated at the level of the oviduct of various reptile species (Girling, 2002), the promoting effect of these hormones towards the development of oviductal leiomyomas has not been studied.
Clinical signs that have been attributed to uterine leiomyomas in humans and conventional pets include infertility, vaginal discharge, pyometra, abdominal pain and the secondary development of urinary tract disease. These signs may be directly associated with the neoplasm but may also result from the space-occupying effect in those cases where uterine leiomyomas reach a considerable size (Miller et al. 2003;Parker, 2007;Nelis et al., 2013;Saba and Lawrence, 2013). In laying hens, leiomyomas appear to have a limited effect on the oviductal function and egg production (Carrasco and González, 2017). In the present case, a cloacal prolapse was seen following vague symptoms consisting of decreased appetite and tenesmus. Although interspecies differences exist, histologically, the reptilian oviduct can be divided into five regions: the infundibulum (the most anterior part of the oviduct), the uterine tube, the isthmus, the uterus and the vagina (Girling, 2002;Stahl and DeNardo 2019). The exact regions that were affected by the leiomyoma in the tortoise could not be identified based on gross anatomical features or histological findings. Taking into account the gross anatomical location however, the infundibulum and uterine tube were considered as the most likely regions that were affected by the neoplasm and were involved in the prolapse. The mass effect of the intramural oviductal leiomyoma and the potential increased effects of steroid hormones on affected oviductal tissue may have resulted in increased oviductal peristalsis with the development of an invagination of the infundibulum and uterine tube (Nowak, 1999;Berry et al., 2006;Barton et al., 2020). Subsequently, the alterations to the oviduct and invagination may have elicited tenesmus and eventually the development of a prolapse of the posterior part of the oviduct. Further studies are warranted to evaluate if leiomyomas in reptiles also show an increase in steroid hormone receptors expression, and if this coincides with alterations of physiological secretions, ciliary activity and muscle contractions that may predispose to the development of oviductal invagination and prolapse.
Imaging, endoscopy and histopathological examination of collected tissue samples were essential to establish a definitive diagnosis of the primary neoplastic disorder of the oviduct in the present case. The use of advanced medical imaging modalities, such as computer tomography or magnetic resonance imaging, combined with cytological or histological evaluation of endoscopically collected tissue samples may have allowed a preoperative diagnosis of the oviductal tumor (Christman et al., 2017). Endoscopy has been proven to be an important tool in the diagnosis and treatment of chelonian oviductal disease (Mans and Sladky, 2012;Divers, 2019) and has been validated for elective and therapeutic ovariectomy in chelonians (Innis et al., 2007). In the present case however, the narrow prefemoral window did not allow exteriorization of the oviductal invagination, and endoscopy-assisted reduction of the invagination failed. Eventually, transplastron osteotomy was necessary to adequately approach and resect the invaginated oviduct.
Hemiovariosalpingectomy has been described to preserve reproductive potential in a loggerhead sea

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turtle (Caretta caretta) with a severely traumatized oviductal prolapse (Nutter et al., 2000). Since preservation of the reproductive potential was not of importance and no abnormalities were observed in the contralateral oviduct in the present case, bilateral ovariectomy and unilateral salpingectomy were performed. Leiomyomas in humans, cats, dogs and laying hens usually present as small white nodules to large, grey, well-vascularized masses and typically have a glistering surface on the cut (Miller et al., 2003;Nelis et al., 2013;Carrasco and González, 2017;Devereaux and Schoolmeester, 2019), similar to what has been observed in the present case. The histological findings for the leiomyoma in the present case largely complied to the main histological characteristics that are observed in uterine leiomyomas in humans and conventional pets as well as those that are seen in leiomyomas of the oviduct of birds. The main characteristics consist of a fascicular pattern of bundles of well-differentiated smooth muscle cells separated by wellvascularized connective tissue. Leiomyoma smooth muscle cells are elongated and show abundant eosinophilic cytoplasm, distinctive cell membranes and less than five mitotic figure per high power field. In general, leiomyomas show immunoreactivity against α-SMA, muscle specific actin, desmin, h-caldesmon and vimentin (Miller et al., 2003;Nelis et al., 2013;Devereaux and Schoolmeester, 2019;Parra-Herran, 2019).

CONCLUSION
Although oviductal leiomyoma seems to be a rare condition in tortoises and this neoplastic disorder may remain unnoticed during a prolonged period of time, the present case illustrates the need to apply a multidirectional approach towards the diagnosis and treatment of oviductal leiomyomas in Hermann's tortoises.