Endometrial BCL6 Overexpression in Eutopic Endometrium of Women With Endometriosis

Abstract

The objective of this study was to examine B-cell CLL/lymphoma 6 (BCL6) expression in human eutopic endometrium across the menstrual cycle in women with and without endometriosis and to establish a cutoff for future studies. This design was a series of case-control studies in tertiary University teaching hospitals. We examined BCL6 expression by messenger RNA and immunohistochemically in prospectively collected samples in both the proliferative (P) and the secretory phases. BCL6 is minimally increased in the mid-secretory phase of the menstrual cycle compared to the P phase in normal patients. BCL6 protein expression was significantly higher in the secretory phase of patients with endometriosis (n = 29) versus fertile controls without endometriosis at laparoscopy (n = 20; P < .0001). Normal fertile controls (n = 28) recruited for endometrial biopsy also had low levels of secretory phase BCL6 expression compared to women with unexplained infertility (UI; n = 119). A receiving-operator characteristic analysis of these data revealed an area under the curve of 94% (95% confidence interval 85%-100%; P < .0001) with an HSCORE cutoff of 1.4 to differentiate cases with and without endometriosis. Using this cutoff value, BCL6 was positive in 88% of cases with UI. Laparoscopic examination of a subset of 65 patients confirmed abnormalities in 98% of cases; 61 (93.8%) were found to have endometriosis, 3 (4.6%) with hydrosalpinx, and 1 (1.5%) with a normal pelvis. These data suggest that BCL6 is a promising candidate as a single diagnostic biomarker for detection of endometriosis in women with otherwise UI and may be associated with endometrial dysfunction, including progesterone resistance.

Keywords: BCL6; biomarker; endometriosis; endometrium; infertility.

Figure 1.

B-cell CLL/lymphoma 6 (BCL6) expression at different states. A, Quantitative RT polymerase chain reaction (RT-PCR) levels of BCL6 messenger RNA (mRNA) in whole normal endometrium evaluated each cycle phase, determined by cycle day and urine luteinizing hormone (LH) detection. Mid-secretory levels of BCL6 were significantly higher compared to proliferative phase (P = .02, Kruskal-Wallis with Dunn post hoc test). B, HSCORE values based on immunohistochemical expression of BCL6 across the menstrual cycle in normal women. No statistical difference was found among groups (Kruskal-Wallis). C, Quantitative RT-PCR levels of BCL6 mRNA in separated stromal and epithelium cells from whole endometrial tissue. A higher level of expression was found in the epithelial compartment (P = .0005, Mann-Whitney U test). D, Quantitative RT-PCR levels of BCL6 mRNA obtained from proliferative and secretory phase in women with and without endometriosis. Significant difference was found between secretory phases of both groups and between proliferative and secretory groups of endometriosis (P = .001 and 001, respectively—Kruskal-Wallis with Dunn post hoc test). E, Receiving–operator characteristic (ROC) analysis comparing laparoscopically proven endometriosis to fertile women without endometriosis at the time of laparoscopy. The dotted line represents the ROC-determined HSCORE cutoff of 1.4. F, Immunohistochemical expression (HSCORE) of BCL6 between fertile controls (Fertile) and fertile women without endometriosis at laparoscopy (L/S Controls) compared to those with endometriosis at laparoscopy (L/S E’osis; P < .0001, Mann-Whitney). A further comparison was made between immunohistochemical expression (HSCORE) of BCL6 between women with unexplained infertility (UI) and both fertile control groups (P < .0001, Mann-Whitney U test). All boxes in each graph represent the median levels of mRNA of expression or HSCORE.

Figure 2.

Representative B-Cell CLL/Lymphoma 6 (BCL6) protein immunostaining expression in endometrial paraffin sections over the menstrual cycle. (A) normal proliferative, (B) normal early secretory, (C) normal mid-secretory, (D) normal late secretory, and (E) endometriosis—mid-secretory. Positive (F) and negative controls (G).