Pathohistological Findings after Bilateral Ovariectomy in Mares with Behavioral Problems

Abstract

Behavioral problems in reproductively healthy mares are a challenging issue that is successfully treated with bilateral ovariectomy (BO). This laparoscopic procedure represents an alternative to conservative treatment for mares not intended for breeding and results in high owner satisfaction regarding behavioral improvement. However, a pathohistological explanation to justify surgical ovarian removal regarding animal welfare is lacking. Therefore, the objective of this study was to pathohistologically evaluate bilaterally removed, clinically unremarkable ovaries of mares with behavioral problems (bOE, n = 20) and to compare them with pathohistologically confirmed granulosa cell tumors of mares with neoplastic ovaries (GCT-uOE, n = 10). A complete data set including preliminary presentation, clinical examination, and serum anti-Müllerian hormone (AMH) and testosterone was further analyzed in both groups. Both hormones were significantly higher in GCT-uOE compared with bOE. Immunohistochemical expression of Ki-67, AMH, aromatase, epidermal growth factor receptor, calretinin, and epithelial cadherin in granulosa cells of large follicular structures in bOE did not differ from neoplastic granulosa cells in GCT-uOE. Ultrasonographically nondetectable early neoplastic changes were pathohistologically evaluated in 15% of mares and anovulatory-like follicles in 30% of mares in bOE and might be one explanation for the high success rate of BO in 85% of bOE in this study.

Keywords: anti-Müllerian hormone; behavior; equine ovary; granulosa cell tumor; immunohistochemical marker.

Figure 1

Comparison of serum anti-Müllerian hormone (AMH) concentrations between bilaterally ovariectomized mares (bOE, n = 20) and mares with granulosa cell tumors (GCT-uOE, n = 10). Mann–Whitney U (Wilcoxon rank-sum) nonparametric test was used due to not-normally distributed data (Shapiro–Wilk normality test). p-value < 0.001 shows very strong evidence for the (rank-sum) difference in serum AMH concentrations between bOE and GCT-uOE. Rank biserial correlation coefficient of −1.00 with 95% confidence interval indicates a very large effect size of serum AMH concentrations between the two groups. The red bullets show the median AMH concentrations; the green bullets show AMH concentrations of single cases. n_obs = total number of tested mares; x-axis: analyzed different groups, bOE = mares with bilaterally removed, clinically unremarkable ovaries, n = 20; GCT-uOE = mares with unilaterally removed granulosa cell tumors, n = 10; y-axis: measured serum AMH concentrations in pmol/L of each case with a range of 0.4–150 pmol/L.

Figure 2

Ovarian gross sections of bilaterally ovariectomized mares (bOE, (A)) and mares with granulosa cell tumors (GCT-uOE, (B)). (A1,A2) Ovary after fixation with preantral and tertiary follicles. (A3) Ovary with a corpus luteum (CL). (B1) Fragmented 5 kg granulosa cell tumor (GCT) immediately after removal via laparotomy. (B2,B3) GCT with typical multicystic appearance.

Figure 3

Early neoplastic changes (ENCs) in different ovaries of bilaterally ovariectomized mares (bOE, Case 29 and Case 10): (A) Gross section of a clinically unremarkable ovary with a pale area (red circle) near the ovulation fossa suspicious for ENCs (Case 29). (B) Immunohistochemical evaluation of this area in aromatase (AR) staining in different magnifications (B1,B2); note the granulosa cell (GC) nests with AR-positive cells resembling Leydig-like cells (LLCs) in between (asterisk), defined as ENC; Bars 200 µm, 100 µm. (C) Pathohistological findings in a clinically unremarkable ovary with detected ENCs (Case 10): spindle-shaped, neoplastic GCs with polyhedral, foamy cells in the theca interna cell layer resembling LLC. Figures are presented in hematoxylin and eosin (HE, (C1)) and different immunohistochemical staining with Ki-67 (Ki67, (C2)), anti-Müllerian hormone (AMH, (C3)), AR (C4), epidermal growth factor receptor (EGFR, (C5)), and calretinin (CAL, (C6)); bars 100 µm; GCs = granulosa cells, T = theca cell layer, Ti = theca interna cells.

Figure 4

Antral follicles in ovaries of bilaterally ovariectomized mares (bOE) in different immunohistochemical staining with hematoxylin (HE (A1,B1)), Ki-67 (Ki67 (A2,B2)), anti-Müllerian hormone (AMH (A3,B3)), aromatase (AR (A4,B4)), epidermal growth factor receptor (EGFR (B5)), calretinin (CAL (A5,B6)), and epithelial cadherin (E-Cad (A6,B7)): (A) Tertiary follicle with oocyte, granulosa cell (GC) layer, and theca cell layer (T); note the positive E-Cad staining of the zona pellucida (A6, arrow). (B) Preovulatory follicle with GC, theca interna (Ti), and theca externa cell layer (Te) and stroma (S); note the polyhedral cells with foamy cytoplasm in the theca interna cell layer (asterisks); bars 100 µm; GCs = granulosa cells, O = oocyte, S = stroma.

Figure 5

Large follicular structures in ovaries of bilaterally ovariectomized mares (bOE; (A,D)) compared with cyst-like structures in two different granulosa cell tumors (GCTs) of unilaterally ovariectomized mares (GCT-uOE; (B,C)) in hematoxylin and eosin (HE) and different immunohistochemical staining with Ki-67 (Ki67), anti-Müllerian hormone (AMH), aromatase (AR), epidermal growth factor receptor (EGFR), and calretinin (CAL). (A) Preovulatory follicle with granulosa cell (GC) layer, theca interna, and theca externa cell layer. (B) GCT with a multiple GC layer and numerous Leydig-like cells in the theca interna cell layer, some show an invasive growing character (asterisk). (C) GCT with microfollicular pattern of the GC layer. (D) Anovulatory-like follicle with a multiple GC layer, poorly developed theca interna, and theca externa cell layer; bars 100 µm; GCs = granulosa cells, Ti = theca interna cell layer, Te = theca externa cell layer, LLCs = Leydig-like cells.