Changes of serum anti‐Müllerian hormone in a mare with granulosa cell tumour following surgery and reinitiation of follicular activity

Anti‐Müllerian hormone (AMH) has been reported to be elevated in mares with granulosa cell tumour (GCT). An 8‐year‐old Thoroughbred mare was presented for not being observed in oestrus after the beginning of the breeding season. Rectal palpation and ultrasonography revealed enlargement and cystic appearance of the left ovary while the right ovary was small with an anoestrous‐like appearance. The AMH concentration was 694.9 ng/ml. Presumptively diagnosed with GCT, the mare was subjected to tumour removal. Histopathology confirmed GCT. To evaluate changes of AMH concentration following surgery, blood samples were collected immediately prior to surgery, and on Days 1, 2, 4, 8, 16 and 32 after surgery. Thereafter, blood samples were collected monthly and also at the time the mare was observed in oestrus (148 days after tumour removal). The AMH concentrations decreased over the first 2 months after surgery (from 721.2 ng/ml to 0.1 ng/ml). Subsequently, AMH concentration increased and peaked at the time of oestrus expression (0.7 ng/ml). The mare then became anoestrous, and AMH concentration decreased and reached 0.2 ng/ml, which was not significantly different from the mean concentration of AMH in normal anoestrous mares (n = 5; 0.26 ± 0.07 ng/ml). In conclusion, the present report implies the potential use of AMH for determination of the time of follicular resumption in mares after GCT removal.     

Bilateral occurrence of granulosa-theca cell tumors in an Arabian mare

An Arabian mare was referred for right granulosa-theca cell tumor (GTCT) evaluation. The mare was presented 4.5 years later for a left GTCT, after successfully conceiving and delivering a normal foal in the interim. The concurrent or nonconcurrent occurrence of bilateral GTCT in mares appears to be rare.     

A Retrospective Analysis of 2,253 Cases Submitted for Endocrine Diagnosis of Possible Granulosa Cell Tumors in Mares

Endocrine diagnoses of granulosa cell tumors (GCTs) in the mare are frequently based upon determination of serum concentrations of inhibin, testosterone, and progesterone (GCT panel). In the present study, we examined results from 2,253 samples submitted for determination of GCT panel. In an additional subset of samples (n = 75), diagnosis of GCT was confirmed based upon surgery or necropsy. The objective of the current study was to examine the agreement between serum inhibin and testosterone concentrations in mares with serum progesterone concentrations less than or ≥1 ng/mL. Across all samples, elevations in serum inhibin or testosterone were noted in 15.2% or 17.3% of samples, respectively. For samples with progesterone <1 ng/mL, more (P = .001) samples had elevated inhibin than elevated testosterone, whereas for samples with progesterone ≥1 ng/mL, more (P < .0001) samples had elevated testosterone than elevated inhibin. For samples with progesterone <1 ng/mL, the agreement between elevated inhibin and testosterone was greater than that for samples with progesterone ≥1 ng/mL. In a subset of 53 samples with progesterone ≥1 ng/mL, 28 samples had endocrine evidence of a granulosa cell tumors based upon elevations of serum anti-Müllerian hormone, and 4 of these samples had endocrine evidence of pregnancy based upon estrone sulfate concentrations. For samples from mares with confirmed GCTs, 85% and 55% had elevated inhibin or testosterone, respectively. Based upon endocrine diagnosis, GCTs occur in mares with elevated progesterone and/or estrone sulfate, albeit at a low rate.     

Low Concentration of Anti-Müllerian Hormone in Mares with Delayed Uterine Clearance

Luteal dysfunction has been observed in mares with defective uterine clearance. Association of low ovarian reserve with luteal dysfunction and abnormal endometrial thickness has been reported in bovine. Anti-Müllerian hormone (AMH) has been indicated as a marker for ovarian reserve in bovine and originates primarily from the ovary in equine. The present study evaluated serum AMH concentration in mares with delayed uterine clearance versus that in mares without delayed uterine clearance. Of 49 mares assigned to the study, 12 and 37 mares were diagnosed with and without delayed uterine clearance, respectively. Delayed uterine clearance was determined based on history and the observation of intrauterine fluid in ultrasonographic examination 24 hours after natural breeding. Serum AMH concentration was measured during estrus. Concentration of AMH was lower in mares with delayed uterine clearance (0.4 ± 0.1 ng/mL) than in those without delayed uterine clearance (1.1 ± 0.1 ng/mL). In conclusion, the present study indicated possible associations between ovary-lined mechanisms and uterine clearance failure in mares.     

Equine nonneoplastic abnormal ovary in a draft mare with high serum anti-Müllerian hormone: a case study

We performed a standing hand-assisted laparoscopic ovariectomy in a draft mare that presented with high serum anti-Müllerian hormone (AMH) level and had an enlarged single cystic ovary. Histopathological examination revealed no tumor cell proliferation in the ovary, but the presence of a large ovarian cyst was confirmed. In the diagnosis of abnormal ovaries in mares, a comprehensive assessment should be performed, including the monitoring of ovarian morphology and biomarkers over time, to determine the disease prognosis and treatment plan. The case of this mare with a nonneoplastic abnormal ovary and increased serum AMH level was rare. We suggest that standing hand-assisted laparoscopic ovariectomy is useful for the removal of large ovaries in draft mares.     

Determination of serum anti‐Müllerian hormone concentrations for the diagnosis of granulosa‐cell tumours in mares

Reasons for performing study: Endocrinological assays are important for evaluation of mares with granulosa‐cell tumours (GCTs), and our research in mares indicates that anti‐Müllerian hormone (AMH) may be a good biomarker for this type of ovarian tumour. Objectives: To evaluate the use of serum AMH concentrations for endocrine diagnosis of GCTs in mares. Methods: Archived serum samples (n = 403) previously assayed for determination of serum inhibin, testosterone and progesterone concentrations (GCT panel) were assayed for serum AMH concentrations using a heterologous enzyme‐linked immunosorbent assay previously validated by our laboratory. For a subset (n = 44) of these samples, a clinical diagnosis of GCT was confirmed by histopathology. Results: Overall, the sensitivity of AMH (98%) for detection of histologically confirmed GCTs was significantly (P<0.05) greater than that of either inhibin (80%) or testosterone (48%) or the combination of inhibin and testosterone (84%). Conclusions: Determination of serum AMH concentrations is a useful biomarker for detection of GCTs in the mare. Potential relevance: Measurement of serum AMH concentrations can be used for diagnosis of GCTs in the mare. As serum AMH concentrations do not vary significantly during the oestrous cycle or pregnancy, interpretation of these results is not confounded by these physiological states.     

Luteoma in two mares treated by ovariectomy

A 15-year-old Paint mare (Case 1) and a 15-year-old Arabian mare (Case 2) were presented for aggressive and undesired behaviour. Reproductive evaluation revealed, initially, an enlarged ovary with a smaller/normal sized contralateral ovary in each case. Granulosa cell tumour (GCT) panel testing revealed elevated inhibin B in both cases and elevated anti-Mullerian hormone (AMH) levels only in Case 1 determined from currently established reference ranges. Based on a presumptive diagnosis of GCT, bilateral standing ovariectomy was elected for both mares. In each case neoplastic tissue consistent with luteoma was detected only on histological examination in the smaller/normal sized left ovary; the right ovary appearing normal. Long-term follow-up was performed 1 year and 5 years post-operatively by telephone. The owners were satisfied with the outcome and each patient demonstrated resolution of aggressive stallion-like behaviour. Each case returned to the owners' intended use. In conclusion, luteoma should be considered as a differential diagnosis for ovarian neoplasms in horses related to behavioural abnormalities, even in normal-sized ovaries.     

Granulosa cell tumor in a mare with a functional contralateral ovary

A functional corpus luteum was found in the ovary contralateral to the ovary with a granulosa cell tumor in a 24-year-old Standardbred mare. The mare was ovariectomized because she was to be used as a jump mare for collection of semen from stallions. The blood concentration of progesterone was 2.2 ng/ml, and the luteal tissue progesterone concentration was 6.3 micrograms/mg. Atrophy of the contralateral ovary is one of the major signs used in diagnosis of granulosa cell tumor; however, our findings indicate that the ovary contralateral to a granulosa cell tumor is not invariably nonfunctional.     

Diagnosis and surgical removal of a granulosa-theca cell tumor in a mare

An 8-year-old mare showed stallion-like behavior and aggressiveness. A granulosa-theca cell tumor was tentatively diagnosed based on history, transrectal palpation, and ultrasonography, and surgically removed. Serological levels of testosterone and inhibin were elevated. Histopathological examination confirmed the diagnosis. The mare recovered and by day 45 could be turned out with other horses.     

Dimeric inhibin concentrations in mares with granulosa-theca cell tumors

OBJECTIVE: To determine whether concentrations of dimeric inhibin (CaCA) are greater in plasma and tumor fluid from mares with granulosa-theca cell tumors (GTCT), compared with concentrations in plasma and equine follicular fluid (eFF) from control mares. ANIMALS: 6 mares with GTCT and 12 clinically normal mares. PROCEDURE: The alphabetaA immunoradiometric assay used 2 antibodies, one against each subunit of inhibin (alpha and betaA subunits). Tumor tissue, tumor fluid, and a single blood sample were collected at the time of surgical removal of the GTCT. A single blood sample was collected from 7 control mares during various stages of the estrous cycle. Five other control mares were ovariectomized when their ovaries contained growing follicles of 25 to 35 mm in diameter. A blood sample and eFF from the largest follicle were collected at the time of ovariectomy. RESULTS: Mares with GTCT had significantly greater plasma concentrations of betabetaA (mean +/- SEM, 0.86 +/- 0.53 ng of recombinant human-alphabetaA/ml), compared with control mares (0.14+/-0.02 ng/ml). Concentrations of alphabetaA in tumor fluid and eFF were similar. Concentrations of alphabetaA were significantly lower after ovariectomy. CONCLUSIONS AND CLINICAL RELEVANCE: Dimeric inhibin concentration was higher in plasma from mares with GTCT than in plasma from control mares. Increased granulosa cell mass and loss of mechanisms regulating alphabetaA release in mares with GTCT likely accounted for the increase in plasma concentrations. Measurement of alphabetaA concentrations may be useful for identifying mares with GTCT.     

Anti‐Muellerian hormone concentration in bitches with histopathologically diagnosed ovarian tumours and cysts

Increased concentrations of Anti‐Muellerian hormone (AMH) can indicate a granulosa cell tumour as shown in women, mares and cows. To investigate AMH to differentiate canine granulosa cell tumour from other ovarian pathologies, we evaluated the ovaries of 63 bitches. Blood serum samples were collected before surgery for AMH analysis. Ovaries were submitted for histopathological examination. Fourteen bitches showed normal ovaries. These bitches had AMH values between 0.12 and 0.99 ng/ml. In 20 bitches ovarian cysts i.e., follicular cysts (n = 8), corpora lutea cysts (n = 7), subsurface cysts (n = 5) were diagnosed. These dogs had AMH values of 0.11–2.09 ng/ml. Bitches with small luteinized follicular cysts had slightly higher AMH values than those without ovarian alteration. In 29 cases ovarian neoplasms i.e., granulosa cell tumour (n = 9), epithelial tumours (n = 16), dysgerminomas (n = 3) and one sarcoma were identified. Anti‐Muellerian hormone values of bitches with an ovarian neoplasm except granulosa cell tumour ranged from 0.18 to 1.18 ng/ml. The AMH values of bitches with granulosa cell tumour ranged from 1.12 to ≤23 ng/ml and were significantly higher (p < .05) than in all of the other bitches. The cut‐off of 0.99 ng/ml gave a sensitivity of 100% and a specificity of 94.44% to diagnose granulosa cell tumour. In conclusion, markedly elevated AMH concentrations in bitches are indicative for a granulosa cell tumour. However, negative testing does not rule out the existence of small one. Differentiation of GCT from luteinized follicular cysts may especially be difficult.     

Review of seven cases of granulosa cell tumour of the equine ovary

This paper reviews the equine granulosa cell tumour (GCT) and describes the clinicopathological features, treatment and outcome in seven cases of GCT in mares. Mares were presented with unilateral ovarian enlargement during the 2007 to 2010 breeding seasons. The mean (sd) age of the mares was 11.7 (5.96) years. Three mares were multiparous barren, three were nulliparous and one was primigravida. Behaviour at presentation was 57 per cent anoestrus, 28 per cent with stallion-like behaviour and 14 per cent with persistent oestrus. All mares had unilateral ovarian enlargement. Six non-pregnant mares had a small and inactive contralateral ovary; the pregnant mare had a single small corpus luteum on the contralateral ovary and was at three-and-a-half months' gestation. Enlarged ovaries measured 7 cm to an estimated 30 cm in diameter. 28 per cent had a multicystic ultrasound appearance, 57 per cent were dense structures and 14 per cent were of mixed appearance. Mean concentrations of progesterone were <1 ng/ml, oestrone sulphate 3.06 (2.32) ng/ml and testosterone 0.58 (0.64) nmol/l in non-pregnant mares. Inhibin was elevated in all non-pregnant cases at 7.6 (12.45) ng/ml. The pregnant mare had concentrations of progesterone 2.5 ng/ml, oestrone sulphate 81.0 ng/ml, testosterone 1.9 nmol/l and inhibin 1.31 ng/ml. Mares demonstrating stallion-like behaviour had a significantly higher (P<0.001) testosterone concentration (1.85 [0.07] nmol/l) than those that did not (0.34 [0.26] nmol/l). Three mares underwent unilateral ovariectomy and resumed cyclic ovarian activity within nine months of surgery.     

Hyperleptinemia in Mares: Prevalence in Lactating Mares and Effect on Rebreeding Success

Previous research from our laboratory showed that approximately one third of obese, nonfoaling mares displayed a condition of hyperleptinemia coupled with hyperinsulinemia that resembled type 2 diabetes in humans. The current study was performed to evaluate the prevalence of the hyperleptinemic syndrome in lactating mares and its possible impact on their rebreeding success. Additionally, we investigated possible relationships between leptin levels in lactating versus nonlactating mares. In experiment 1, jugular blood samples were collected from 198 lactating mares on two occasions approximately 2 weeks apart. The mares resided on eight farms in Louisiana; breeds included Thoroughbred (n = 86), Quarter Horse (n = 71), Warmblood (n = 24), and draft-type (n = 17). Body condition scores (BCS) were measured at the time of blood sampling; plasma samples were assessed for leptin and progesterone concentrations. Reproductive and medical histories, as well as feeding regimens, were compiled on each mare. Based on our previous reports and examination of the current data, a mare was considered hyperleptinemic if her plasma samples contained greater than10 ng/mL leptin; normal was considered 6.0 ng/mL or less; mares with levels above 6.0 and 10 ng/mL or greater were classified as intermediate. Overall mean leptin concentration was 4.7 ng/mL, and average BCS was 5.5. After analysis, 24 mares were classified as hyperleptinemic (12%), 138 were classified as normal (70%), and 36 were classified as intermediate (18%). Leptin concentrations were affected by BCS (P = .08), with higher concentrations in mares with higher body condition; however, there were hyperleptinemic mares with BCS of 4 to 5.5. Feeding regimen affected leptin concentrations (P < .01), with mares on pasture full-time having the highest concentrations. There was no effect of breed, mare age, number of years the mare had been bred, number of live foals, progesterone concentrations, or last foaling date on leptin concentrations. Rebreeding success averaged 81% overall and was not affected by leptin classification. In experiment 2, nonfoaling mares kept on pasture had mean leptin concentrations of 7.0 ng/mL; 8 of 31 mares (26%) displayed hyperleptinemia. Mean leptin concentration was correlated with BCS (R² = 0.65; P < .02) but was not affected by age of the mare. It was concluded that the hyperleptinemic condition occurs in lactating broodmares, even at BCS as low as 4. The overall incidence appears to be lower in broodmares than in nonfoaling mares, likely because of their lower BCS in general and the energy demands of lactation. Hyperleptinemia did not affect rebreeding success at the end of the breeding season.     

Endocrinological characterization of an ovarian sex cord–stromal tumor with a Sertoli cell pattern in a Japanese Black cow

A Japanese Black cow was evaluated for prolonged post‐partum anestrus and enlargement of the right ovary. Transrectal ultrasonography revealed that the right ovary was markedly enlarged and had a solid appearance, while the left ovary was small and inactive. The presumptive diagnosis was directed towards granulosa‐theca cell tumour (GTCT) which was supported by markedly elevated plasma anti‐Müllerian hormone (AMH; 332.0 ng/ml), oestradiol (E₂; 103.3 pg/ml) and immunoreactive inhibin (ir‐INH; 2.1 ng/ml) in comparison with the diagnostic cut‐off points for bovine GTCTs. Since the cow had been infertile and had swelling of the udder, slaughter was chosen. Histopathological examination revealed that the tumour was an ovarian sex cord–stromal tumour (SCST) with a Sertoli cell pattern. These findings suggest that plasma AMH, ir‐INH and E₂ could be possible biomarkers for bovine ovarian SCST with a Sertoli cell pattern, whereas this case could not be distinguished from GTCTs based on endocrinological profile.     

Changes in Serum Concentrations of Prolactin,Progestagens, and Estradiol-17β and Biochemical Parameters During Peripartum in an Agalactic Mare

The purpose of this study was to investigate the changes in serum concentrations of prolactin (PRL), progestagens, and estradiol-17β and biochemical parameters during peripartum in an agalactic mare, as well as to study the periparturient indices of the foal. A 4-year-old Thoroughbred, primiparous, pregnant mare was diagnosed as agalactic from clinical conditions such as absence of observable udder development and weight loss of her foal after parturition. Serum PRL concentrations of the agalactic mare during prepartum tended to be lower than those of the control mares (19.5 ng/mL and 67.0 ± 15.0 ng/mL on the day of parturition, respectively). The progestagens and estradiol-17β concentrations were not markedly different between the agalactic mare and the control mares. Concentrations of γ-glutamyl transferase of the agalactic mare were higher than those of the control mares (P < .05). Although the serum immunoglobulin G concentrations of the foal of the agalactic mare after 24 hours of age were lower than those of the foals of the control mares (P < .05), all periparturient indices regarding the agalactic mare were considered to be within the normal range. In contrast, insufficient udder development and milk production was observed in the agalactic mare through the pre- and postpartum periods. We have ruled out the known causes for agalactia in mares based on other parameters, clinical symptoms, and nutritional causes. Clinical symptoms and PRL parameters in this case report are consistent with the category of agalactia in mares of unexplained causes, and we have determined additional blood parameters associated with agalactia in mares that are consistent with this category.     

Collection Rates and Morphology of Equine Oocytes Obtained from Immature Follicles after Treatment with Equine Pituitary Extract (EPE) and Human Chorionic Gonadotropin

This study examined the effect of treating mares with equine pituitary extract (EPE) in combination with human chorionic gonadotropin (hCG; EPE/hCG) on the recovery rate of immature oocytes by ovum pick-up (OPU) and on oocyte morphology. Ten mares were subjected to each of two treatments in a random sequence: superstimulated with EPE (25 mg, twice daily) and treated with hCG (2,500 IU) or control (no exogenous treatment). The cytoplasmic morphology of oocytes recovered was evaluated through transmission electron microscopy. Follicular fluid was collected at aspiration for progesterone analysis, which was performed by radioimmunoassay. The EPE/hCG did not increase the oocyte recovery rate from immature follicles when compared with the controls (15.5% and 16.7%, respectively). A significantly higher oocyte recovery rate per mare was observed (70% versus 50%). However, precocious granulosa cell expansion was observed with EPE/hCG treatment in contrast to the control (64.4% and 33% of follicles with expanded cumulus, respectively), and increased intrafollicular progesterone concentration was also seen (158.80 ng/mL versus 82.05 ng/mL). The ultrastructural analysis of oocytes from both groups showed morphologic features related to immaturity. Numerous vesicles containing cortical granules were found, distributed in clusters into the cytoplasm, and junctional complexes were still seen between oocyte and granulosa cells. In conclusion, EPE/hCG treatment induced some follicular modifications, but the recovery rate was not increased. All oocytes examined presented signs of immaturity.     

Granulosa-theca cell tumor associated with an ovulation fossa and normal ovarian stroma in a mare.

A granulosa-theca cell tumor was found in an ovary that had an ovulation fossa and normal ovarian tissue. The ovary was removed from a mare with a history of ovarian enlargement and behavioral changes. The affected ovary had a multicystic appearance on ultrasonographic examination performed before surgery, and an ovulation fossa was not palpable on examination per rectum. However, during surgery, the affected ovary was found to be within normal size limits, with an enlargement on 1 pole, and to contain an ovulation fossa. Atrophy of the infundibulum of the affected ovary helped to confirm the diagnosis of granulosa-theca cell tumor, and the ovary was removed. The mare's testosterone concentrations were normal. Granulosa-theca cell tumors are usually associated with a spherical ovary, attributable to ablation of the ovulation fossa, with no normal ovarian tissue present.     

Enlarged ovary in a mare: review of the literature and a case report

Mares regularly have an enlarged ovary. The main causes are haematomas, anovulatory follicles, abscesses, and neoplasia. The granulosa-theca-cell tumour is by far the most common neoplasia of the ovary (about 97%) and accounts for 2.5% of all equine tumours. In this article the differential diagnosis of an enlarged ovary and the background of granulosa-theca cell tumours are reviewed. A case is described of a mare with a very large granulosa-theca cell tumour in the left ovary, which was discovered 1 month after delivery of a healthy foal. This case is special not only because the tumour was enormous (diameter 60 cm) but also because the contralateral ovary was functional and there were no behavioural changes. The fact that a haematoma had been found 2 years earlier made the diagnosis even more difficult.     

Anti-Müllerian hormone profiles as a novel biomarker to diagnose granulosa-theca cell tumors in cattle

This study was carried out to evaluate the blood profile and tissue expression of Anti-Müllerian hormone (AMH) as a biomarker for granulosa-theca cell tumors (GTCTs) in cattle. Five cases with unilateral ovarian GTCTs (GTCT group) were investigated in comparison to other groups of Japanese Black cows, which had either cystic ovarian disease (COD group, n=5), a functional corpus luteum on Days 9 to 11 of the estrous cycle (Day 0=estrus; CL group, n=13) or received superovulation treatment (SOT group, n=13). We used transrectal ultrasonography and measured plasma AMH, estradiol-17β (E(2)), progesterone (P(4)) and testosterone (T) levels. Moreover, GTCT tissues were collected and examined by immunohistochemical staining (IHC) for AMH. In the GTCT group, ultrasound images of GTCTs were variable and not definitive. However, the AMH level in the GTCT group (n=3, 58.1 ± 66.3 ng/ml) was significantly higher than in the COD, CL and SOT groups (0.1 ± 0.1 ng/ml for GTCT vs. COD, P<0.05; 0.2 ± 0.1 and 0.3 ± 0.2 ng/ml, respectively for GTCT vs. CL and SOT, P<0.01). The other hormonal levels in the GTCT group had no significant differences compared with the COD or SOT group. Neoplastic granulosa cells labeled with AMH antibody clearly demonstrated a variety of tissue patterns in all cases by IHC. To the best of our knowledge, this is the first study to investigate the blood profile and IHC of AMH in bovine GTCTs. Our findings indicate that AMH may be a novel biomarker to diagnose GTCTs in cattle.     

Equine granulosa cell tumors.

Unilateral ovariectomy was performed on 3 mares affected with granulosa cell tumors. Tumor fluid in each mare was found to contain estrogen, testosterone, and progesterone. In 2 mares, preoperative blood plasma concentrations of these hormones were comparable to those of a series of clinically normal mares. The other mare, which had a history of aggressive, masculine behavior, had higher testosterone content in the tumor fluid and in the preoperative blood sample. After surgical removal of the tumors, each mare developed follicles and ovulated with the remaining ovary. Each was eventually bred and 2 conceived. The probability of metastasis of these tumors in mares appears uncertain. Data from other species suggests a guarded long-term prognosis may be justified.