An Unusual Case of a Monorchid Horse with an Abdominally Retained Testicle Open Access

Normal sex development in mammals is a highly complex three-step process under genetic control [1]. It includes determination of chromosomal sex, development of gonadal and phenotypic sex. A dysfunction at any step in these pathways and/or adverse environmental influences may lead to abnormal sex development [2]. Previously these disorders were described by terms like intersex, hermaphroditism and pseudo hermaphroditism in both human and veterinary medicine. A new classification system for such congenital conditions based on sex chromosome constitution, termed disorders of sex development (DSD), was first proposed for humans [3]. Soon after, a similar classification system emerged for horses [4] with four main classes proposed: 63,X (including mosaics and X anomalies); 64,XX (SRY-negative); 64,XY (SRY-positive and -negative); 64,XY (SRY unknown). It is well documented that chromosomal aberrations are responsible for numerous DSD and/or fertility problems in domestic animals [5]. Good fertility is a central element of any breeding enterprise and allows for higher selection intensity and selection potential, as well as for shorter generation intervals – resulting in faster genetic progress within a population [6]. Successful completion of the second step (gonadal sex) results in functional testes or ovaries.

Very few cases of true monorchidism have been reported in the horse, with a small number of single case studies or case series totalling about 30 horses found by the authors [7‒15]. Monorchidism is defined as the complete absence of one testicle and may be due to agenesis of the testicle and mesonephric duct or may be due to unilateral damage to the testicular vasculature [8].

In human medicine testicular degeneration is usually secondary to ischaemia often as the result of testicular torsion [8]. This may occur in neonates, adolescents and adults [8]. In man, there is good evidence to suggest that monorchidism results from testicular regression following in utero torsion [8, 16]. Testicular torsion in dogs usually occurs in mature cryptorchids with an abdominal testis [8, 17]. Though uncommon in the horse, testicular torsion has been reported in mature stallions with a descended testicle [18] as well as in adult cryptorchids [19]. One report described testicular torsion in a horse diagnosed upon routine cryptorchidectomy where the authors suggest had the castration been delayed, and the horse would have been diagnosed as monorchid upon surgical exploration [20]. According to Parks et al. [7], most cases of equine monorchidism can be attributed to testicular degeneration rather than agenesis.

Previous reports of equine monorchidism have described horses with a single descended testicle presented for cryptorchid castration then determined to have no further abdominal gonadal tissue as diagnosed by surgery, hormonal assays, or clinical behaviour. Monorchidism is not to be confused with cryptorchidism, although the final diagnosis may be challenging and only confirmed by surgery and histology in combination with hormonal assays [7]. Cytogenic and molecular testing have only previously been performed in one case of a monorchid horse – a phenotypic mare with 64, XY, SRY-positive karyotype [21].

Cryptorchidism is a frequently seen anomaly (2–8%) in new-born males and affects around 1% of boys at 1 year of age [22] and is also common in colts (8%) [23]. Cryptorchidism may occur as a congenital or acquired condition. Testicular descent in mammals depends on a functional hypothalamo-pituitary testicular axis and involves two major phases: the transabdominal and inguinoscrotal descent which result in the relocation of the testicles from a high abdominal location into the bottom of the scrotum [24, 25]. The transabdominal phase relies mainly on INSL3 and its receptor RXFP2, whereas the inguinoscrotal phase depends on androgens [26].

In humans and horses, unilateral cryptorchidism is much more frequent than bilateral cryptorchidism [26‒29]. Although different classes of cryptorchidism have been defined based on the anatomical location (scrotal and retractile testicles are considered normal, abdominal, inguinal, and ectopic testicles are abnormal [30]) of the retained testicle, phenotypic classification is not uniform and results in difficulties comparing scientific results such as prevalence estimates. Additionally, testicular regression (vanishing testes) may complicate a clear diagnosis [31]. This article serves to describe the first case in a horse of the diagnosis and laparoscopic removal of a retained, monorchid testicle in a standing sedated horse including detailed clinical, histological, and cytogenetic findings.

A four-year-old 412-kilogramme Irish Cob colt was presented to the veterinary teaching hospital for bilateral cryptorchid castration. The owners reported having known the horse since its purchase on auction at 6 months old. Since that time the colt had not changed ownership, no attempt had been made at castration. The referring veterinary surgeon was called to examine and routinely castrate the horse. On clinical examination, no palpable testes were present and the horse was referred for bilateral cryptorchidectomy.

On presentation, the horse was bright and alert and all his clinical parameters were within normal limits. The colt displayed marked stallion-like behaviour including aggression to other males and sexual interest and vocalisation towards females. On palpation, no epididymal or testicular tissue was palpable in the inguinal region. Examination of the skin revealed no scars or signs of attempt at castration. Laparoscopic surgery was advised and agreed to by the owners. The colt was starved for 36 h prior to surgery, but free access to water was allowed.

Pre-anaesthetic clinical examination and blood work were all within normal limits. The horse was pre-medicated with acepromazine (Calmivet) (0.03 mg/kg bwt i.m.), romifidine (Sedivet) (0.05 mg/kg bwt i.v.) morphine sulphate (0.2 mg/kg bwt i.v.). Procaine penicillin (Depocillin) (22 mg/kg i.m. q. 12 h) and flunixin meglumine (Meflosil) (1.1 mg/kg bwt i.v. q. 12 h) were given perioperatively. Standing sedation was then maintained with a detomidine (Detonervin) (5 μg/kg bwt/min) constant rate infusion. The colt was placed in stocks and the lateral abdominal walls were clipped and routinely aseptically prepared. Regional anaesthesia was performed with mepivicaine 2% (Mepidor) subcutaneously and intramuscularly at the intended portal sites. The first camera portal was made in the left paralumbar fossa, a stab incision was made and a 12-mm cannula with blunt trocar was inserted into the abdominal cavity through the incision. The abdomen was insufflated with carbon dioxide at approximately 10 mm Hg. A 30° 60-cm oblique laparoscope was inserted through the cannula and the abdomen was visualised. On examination of the abdomen, the left inguinal canal was located. Here the gubernaculum was visualised in the canal and followed cranially where the epididymis and vas deferens were seen coursing to the lateral ligament of the bladder. These were followed cranially and dorsally where a single large testicle was present within the mesentery of the descending colon. Two instrument portals were made under laparoscopic guidance on the left flank. The mesentery was incised with laparoscopic scissors allowing visualisation of the right portion of the testicle and associated gonadal structures coursing towards the right inguinal ring. A traumatic forcep was used to grasp the testicle and the testicle epididymis and vas deferens were then resected with the use of a Ligasure vessel-sealing device (Covidien). The testicle was held against the body wall and the middle portal was enlarged to allow removal. The tear in the mesentery allowed the laparoscope to be directed to the right side of the abdomen. Here the right inguinal canal was located. This again revealed an epididymis and vas deferens associated with the previously visualised testicular structure, coursing to the lateral ligament of the bladder and attached to the right inguinal canal. The contralateral epididymis and vas deferens were also removed with the Ligasure under tension from a traumatic forcep. The enlarged incision facilitated placement of a larger cannula through the left flank and the introduction of a unidirectional barbed suture (Covidien) which was used to close the hole in the mesentery in a continuous fashion. Routine three-layer closure was performed in the portals using continuous sutures of 3.5 m braided lactomer (Vicryl) in the muscle and subcutaneous tissue and cruciate sutures of 3.5 m Polypropylene (Surgipro) in the skin. Portals were covered with an island dressing (Primapore) and covered with adhesive foam (Polster plast). The horse recovered uneventfully from surgery with no complications. He was maintained on a course of broad-spectrum oral antimicrobials for 5 days and on a tapering dose of non-steroidal anti-inflammatories for 6 days. The horse was discharged from hospital 2 days later. Telephonic follow-up with the owner was performed at 16 weeks postoperatively. The owner reported behaviour consistent with a gelding and was pleased with the noticeable change in demeanour. The previously reported coltish behaviour had completely stopped and the horse was now able to be turned out with both mares and geldings. No post-surgical complications were reported by the owner.

Macroscopically the removed tissue consisted of an oval in shape, approximately 13 × 5.5 cm testicle with two ductal structures originating from both poles with circumferential band of mesenteric attachment at the midpoint. On the cut surface, the testicle showed a diffuse homogeneous pale tan appearance, and no septal structures dividing the parenchyma were observed.

The removed testicular tissue was fixed in 10% neutral buffered formalin and submitted for histological examination. Sections of the main body of the testicle and of both sets of ductal structures were embedded in paraffin wax and routinely stained with haematoxylin and eosin stain (H&E). The main body of the testicle was composed of seminiferous tubules lined by a single layer of Sertoli cells, often showing mild vacuolation of the cytoplasm, and no germinal cells were identified (“Sertoli cell-only pattern or Sertoli cells only syndrome”). The interstitium was expanded by small increased amount of fibrous connective tissue (mild fibrosis) and multifocal groups of Leydig cells were observed between seminiferous tubules sometimes showing small amount of intracytoplasmic brown pigmentation. No evidence of malignant neoplasia was observed. Close to the poles of the testicle, near the ductal structures, the testicle exhibited numerous tubules lined by a single layer of flat epithelium which was consistent with rete testis. Both ductal tissues were composed of epididymal tubular structures. In proximal sections (close to the testicle) the tubules were lined by a ciliated pseudostratified columnar epithelium continued by an outer thin layer of smooth muscle cells. In distal sections, the tubules were lined by a simple columnar epithelium which formed papillary folds extending to the lumen, the outer smooth muscle layer was markedly thicker compared to proximal sections. In all regions the interstitium was expanded by fibrous connective tissue (fibrosis).

Serum blood tests for testosterone and anti-Mullerian hormone (AMH) were performed 4 weeks postoperatively. At this time point Testosterone was 0.17 mmol/L (range-entire male: 5.0–30; cryptorchid 0.3–4.3; gelding <0.15). Anti-Mullerian hormone, which has been shown to be a sensitive biomarker for the presence of testicular tissue [32] was 0.07 ng/mL (range-entire male: 14.7 ± 2.4; cryptorchid 32.4 ± 5.0; gelding 0.07 ± 0.01). These results confirmed that AMH was not significantly increased; suggesting that retained testicular tissue was very unlikely to be present.

EDTA- and heparin-treated blood samples were used for molecular and cytogenetic analysis, respectively. Genomic DNA was isolated using the QIAGEN DNeasy^® Blood & Tissue Kit (QIAGEN GmbH, Germany). PCR amplification of the SRY gene from genomic DNA was performed using a primer pair previously described [33], resulting in a 714-bp amplification product typical for stallions and geldings. Metaphase chromosomes of lymphocytes were prepared following a modified protocol of Arakaki and Sparkes [34]. Modifications consisted of longer colcemide treatment (2 h) and keeping the freshly prepared fixative at −30°C prior to use. After drop-spreading the slides, chromosomes were DAPI-banded (Thermo Fisher Scientific, D1306, Switzerland) by pipetting 25 µL Vectashield H-1200 (1.5 µg/mL DAPI in anti-fade mounting medium; Vector Laboratories, USA) onto the slide. A total of 190 metaphases were analysed. Fluorescence in situ hybridisation (FISH) analyses of the Y and X chromosome were carried out with in-house developed equine whole Y and X chromosome painting probes according to Pieńkowska-Schelling and co-workers [35]. The Y chromosome painting probe hybridises along the entire acrocentric Y chromosome and to the heterochromatic block of the X chromosome (Xq17q21). However, it does not detect the pseudoautosomal region on Xp. The X chromosome painting probe hybridises to the entire length of the X chromosome and does not detect the pseudoautosomal region on the Y chromosome. The two painting probes were hybridised separately or together (double FISH) onto metaphase chromosomes and interphase nuclei. The results of cytogenetic and FISH experiments were analysed with a Zeiss Axio Imager Z1 microscope (Zeiss, Switzerland). Pictures were taken with a CoolCube CCD camera (Princeton Instruments) using Ikaros/Isis-software software from MetaSystems GmbH (Germany). Almost 1,000 interphase nuclei were examined, and micronuclei were counted.

After investigating peripheral blood lymphocytes, the horse showed a mosaic karyotype (Fig. 1) consisting of a normal (64,XY) and an aneuploid cell line (63,X) in which the second sex chromosome was absent. Gross structural aberrations of the autosomes were not observed. PCR confirmed the presence of a SRY gene sequence (not shown). The aneuploid cells (Fig. 1b) were present in only 2% of the cells analysed and pointed initially at a case of low-level chromosomal mosaicism. After examining almost 1,000 interphase nuclei, a high micronuclei rate (6.5.%) was observed. Double FISH using chromosome painting probes for the sex chromosomes showed that some Y chromosomes were embedded in a so-called micronucleus. Some micronuclei contained whole Y chromosomes, while others contained only a fragment of the male sex chromosome. Very rarely micronuclei without sex chromosomes were observed (Fig. 2a–c). An incidental finding was the presence of two X and two Y chromosomes in one interphase nucleus (Fig 2a).

The present article presents an Irish cob with an unusual phenotype: monorchidism and abdominal cryptorchidism of the developed testicle and otherwise normally developed male genitalia. To the best knowledge of the authors no case with this phenotype has been reported for the horse. Laparoscopy has been described as vital to the diagnosis of cryptorchids as well as monorchids [8, 9, 15], but most previous reports have required general anaesthesia for either or both laparoscopic diagnosis and surgical removal with the horse in dorsal recumbency. Laparoscopic examination in the standing horse provides a minimally invasive means of clear bilateral visualisation from the internal inguinal ring to the level of the caudal pole of the kidney [8]. This is ideally suited for cases where a diagnostic examination is required to identify the position of the retained testicle [8]. It avoids the need for general anaesthesia thus mitigating the associated risks and cost and facilitates a rapid return to function with a low rate of morbidity [8].

The aetiology of monorchidism in the horse has previously thought to be a result of either agenesis or testicular degeneration as a result of vascular trauma and it is thought that the latter is the most common cause in the horse [8]. A definitive diagnosis of monorchidism, as previously explained, is a complex procedure and requires multiple components (surgery, hormonal assays, and histology) before being confirmed [36]. Circulating concentrations of oestrogen and testosterone have been used as the mainstay in testing hormonal markers for the cryptorchid diagnosis in the horse [32]. Recently, a number of studies have shown that AMH can be used as a biomarker for cryptorchidism because circulating concentrations will be significantly higher in stallions and cryptorchids than in geldings [32]. Some data suggest that AMH is more suitable to determine whether a horse has retained testicular tissue than testosterone levels or oestrogen sulphate although this still requires validation [32]. This specificity is thought to be due to the fact that Sertoli cells are the only tissue source of AMH in male horses, while testosterone can be either testicular or adrenal in origin. When using the hCG stimulation test or testosterone levels it has been reported that up to 8 weeks might be required for the test to be completely accurate [36]. The hormonal analyses after surgery support the diagnosis of true monorchidism in this horse.

Histopathology of the undescended testicle showed Sertoli cells only without any germ cells in the seminiferous tubules. Congenital Sertoli cell-only syndrome (SCOS) is a multifactorial condition which is found in less than 10% of infertile men [37]. Even though the aetiology is not fully clear, pathogenetic factors including chromosomal aberrations and environmental agents have been proposed to be involved in the development of SCOS [38]. Primordial germ cells are the first cells of the germ lineage and are specified by inductive signals from embryonic and extraembryonic sources [39]. By active and passive migration processes primordial germ cells are relocated from the embryonic epiblast via the hindgut epithelium to the genital ridges [40]. All these processes are under genetic control and genetic and epigenetic mutations may result in failure of colonisation of the genital ridges by primordial germ cells. In human, abnormal karyotypes (mostly Klinefelter), microdeletions of the Y chromosome, copy number variations, and cryptorchidism were associated with SCOS [41]. SCOS is also observed frequently in DSD horses with cryptorchidism. Without scientific proof, we speculate that similar factors lead to SCOS in this horse.

Cryptorchidism has been observed in horses with normal karyotypes [42], in sex reversed horses with constitutional karyotypes 64,XX [43] or 64,XY [44] and in horses with other DSD displaying usually mosaic karyotypes [45]. Cytogenetic and molecular analysis showed that the horse presented here had a 63,X/64,XY mosaic karyotype and was positive for the SRY gene, respectively. It can be assumed that SRY acted in a timely manner and exceeded the required threshold to trigger SOX9 expression and subsequent male development. This is also supported by the normal-sized penis and histologically normal epididymis and vas deferens which showed androgen exposure in utero, as stabilisation of the Wolffian ducts and masculinisation of the urogenital sinus, genital tubercle, and genital swellings during the critical embryonic period is dependent on secretion of testosterone and its conversion to dihydrotestosterone [46].

Cryptorchidism has a negative impact on fertility and increases the risk for testicular cancer in adulthood [26]. It is assumed that in horses the same cancer risks exist, but the frequency of testicular tumours is difficult to assess because most animals are castrated at an early age [47]. Like in other domestic species, breed-specific prevalence rates and heritability of 0.12–0.32 (SE 0.08–0.12) have been reported for cryptorchidism in the horse [23, 48, 49] allowing for genetic selection. Cryptorchidism occurs in connection with many genetic disorders and syndromes (syndromic cryptorchidism) or it may be observed as an isolated condition (non-syndromic cryptorchidism) [31].

In human and domestic animals, risk factors including exogenous substances (endocrine disruptors) are suspected to interfere with normal testicular descent [43, 50‒54]. Additionally, anatomic and mechanical factors as well as trauma may promote cryptorchidism [20, 24, 55]. Both testicular development and descent are under genetic control [39] and phenotypic variation in terms of location of the retained testicle reflects extensive genetic heterogeneity in syndromic and isolated cryptorchidism [24, 56, 57].

Monosomy X was observed only in 2% of analysed cells. Mosaicism is always a challenge for detection and interpretation of cytogenetic results. An artefact resulting from lymphocyte-culturing [58] could be excluded and the presence of true of low-level mosaicism was suspected [59]. The impact of low-level chromosomal mosaicism in which the aberrant cells are present at a rate <5–10% is not fully understood and its possible role for fertility is still under debate [60, 61]. However, using FISH methodology, Y chromosomes of the Irish cob were detected in micronuclei and this probably led to the initial impression that a sex chromosome was missing in few metaphases. Towards the end of mitosis damaged or lagging chromosomes tend to be enclosed in a fragile nuclear envelope and they end up in one daughter cell, separated from the main nucleus [62]. Such micronuclei may lead to chromothripsis. Micronuclei are established biomarkers of genotoxic events and genome instability [63] and they were observed with an abnormal high frequency of 6.6% in the Irish cob. Genome instability is defined as an increased tendency of the genome to acquire mutations [64] and acquired or inherited defects of DNA replication/repair, cell cycle control or chromosome segregation are suspected mechanisms, leading to genome instability [65].

Although data on micronuclei in domestic animals are scarce, a recent study in a cohort of healthy fertile mares of different ages reported micronuclei rates of around 1% [66], well below the proposed 5% baseline rate for micronuclei for human peripheral blood [67]. Another study found significant differences of micronuclei numbers in peripheral blood between mares diagnosed with early embryonic death and a control group [68]. Additionally, a recent study suggested a link between micronuclei rate and higher incidence of pregnancy failure after transfer of in vitro produced embryos [69]. The fate and consequences of micronuclei in cells are not yet fully clear, but it has been shown that they lead to aneuploid cells in subsequent cell divisions, drive cancer development or may cause congenital disorders [70, 71]. As shown in Figure 3, a Y chromosome of the Irish Cob was broken and might have caused segregation problems if it would have been reintegration in a nucleus. The existence of a structural defect of the Y chromosome was not recognised by conventional cytogenetic analysis indeed, according to international nomenclature of horse chromosomes from 1997, the Y is a small sub-metacentric. Though, more recently studies of isoY [72] and Y chromosome sequence organisation [73], show that the horse Y is an acrocentric chromosome. Structural aberrations including isochromosomes and deletions of the horse Y chromosome have been reported earlier [72, 74‒76].

A limitation of cytogenetic testing is the fact that the constitutional karyotype found in cultured lymphocytes of the adult animal might be not representative for cells at embryonic/foetal stages. For instance, it is well documented that postzygotic mutations are common and may influence health and disease [77].

In humans and domestic animals, the causes of isolated cryptorchidism and SCOS are not yet fully understood and for many of the cases no causative mutation can be identified. Nevertheless, mutations in RXFP2 have been shown to be responsible for bilateral familial cryptorchidism [78] and homozygous carrier of a deletion on equine chromosome 29 seems to be at risk for DSD and cryptorchidism in particular [79]. The exact causes of monorchidism and cryptorchidism in the present case remain unclear. However, the elevated rate of micronuclei is strong evidence for genome instability which might have been involved in the failure of normal testicular development and descent. Further research in domestic animals will be necessary to get more insight into the role of genome instability for developmental disorders in domestic animals.

The authors wish to thank the staff and students at the Leahurst Equine Hospital and the Veterinary Diagnostic Pathology services for their assistance with the care of the horse in this report.

This retrospective case report is covered by generic ethical approval granted by University of Liverpool Ethics Committee (VREC 1305) that allows anonymised patient data stored as part of horses’ clinical records to be used for the purposes of future clinical research where owner consent was obtained at hospital admission. This was a clinical case presented to the University of Liverpool’s Philip Leverhulme Equine Hospital and informed owner consent was obtained.

The authors have no conflicts of interest to declare.

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

M. Sinovich and P. Kelly were involved in the management of the case and the majority of the production of the manuscript. J. Monné Rodríguez provided pathology services and contributed to the production and editing of the manuscript. A. Pieńkowska-Schelling and C. Schelling performed molecular and cytogenetic testing and contributed to the production and editing of the manuscript.

All data generated or analysed during this study are included in this article. Further enquiries can be directed to the corresponding author.