Decreased Level of Neurotrophic Factor Neuritin 1 in Women with Ovarian Endometriosis after Receiving Gonadotropin-Releasing Hormone Agonist Treatment

Abstract

This study aimed to investigate the effect of gonadotropin-releasing hormone agonist (GnRHa) treatment on the expression of neuritin 1 (NRN1) in women with ovarian endometriosis. We collected tissues and serum from women with endometriosis treated with (n = 45) or without (n = 37) GnRHa. NRN1 mRNA and protein levels were measured using qPCR and Western blot. Immunolocalization of NRN1 in endometriotic tissues was examined using immunohistochemistry. In addition, a follow-up study was carried out to monitor the serum level of NRN1 in patients before and after GnRHa treatment. Both mRNA (p = 0.046) and protein (p = 0.0155) levels of NRN1 were significantly lower in endometriotic tissues from patients receiving GnRHa treatment compared to the untreated group. Both epithelial and stromal cells of endometriotic tissues from untreated women with endometriosis exhibited stronger staining of NRN1 but not in those who were treated with GnRHa. The follow-up study showed that the serum level of the NRN1 concentration decreased significantly from 1149 ± 192.3 to 379.2 ± 80.16 pg/mL after GnRHa treatment (p = 0.0098). The expression of NRN1 was significantly lower in women with ovarian endometriosis treated with GnRHa. These results suggest that NRN1 may be a biomarker response to the effect of GnRHa treatment for patients with ovarian endometriosis.

Keywords: GnRHa; NRN1; gonadotropin-releasing hormone agonist; neuritin 1; ovarian endometriosis.

Figure 1

mRNA expression of Neuritin 1 (NRN1) in endometriotic tissues. (A) Expression of the NRN1 mRNA in patients treated with a gonadotrophin-releasing hormone agonist (GnRHa (+), n = 12) and in those not treated with a GnRHa (GnRHa (−), n = 14); * p < 0.05. (B) NRN1 mRNA levels in the GnRHa (−) group based on the menstrual cycle: the proliferative (n = 7) and secretory (n = 7) phases. (C) NRN1 mRNA expression in the GnRHa (+) group in patients who received treatment for 1 month (n = 3) and in those who received treatment for ≥2 months (n = 9). The TATA-box-binding protein gene was used as an internal control for qPCR. The data are expressed as the mean ± standard error of the mean. n, number of patients. The non-parametric Mann–Whitney U test was used for statistical analysis. Significance was set at p < 0.05.

Figure 2

Expression of the NRN1 protein in endometriotic tissues. (A) Western blot images of NRN1 protein expression in patients treated with a gonadotrophin-releasing hormone agonist (GnRHa (+), n = 10) and in those not treated with a GnRHa (GnRHa (−), n = 8). P, proliferative; S, secretory; 1, patients treated for 1 month; 2, patients treated for 2 months. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was the loading control. (B) The intensity of the NRN1 and GAPDH bands was quantified and is shown as a bar plot. * p = 0.0155. (C) NRN1 protein level in the GnRHa (−) group in the proliferative (n = 3) and secretory (n = 5) phases of the menstrual cycle. (D) The NRN1 protein level in the GnRHa (+) group in patients who were treated for 1 month (n = 7) and in those who were treated for ≥2 months (n = 3). The data are expressed as the mean ± standard error of the mean. n, number of patients. The non-parametric Mann–Whitney U test was used for statistical analysis. Significance was set at p < 0.05.

Figure 3

NRN1 protein expression and localization in endometrial tissues. Immunohistochemistry was used to localize the NRN1 protein in the normal endometrium (A), in the eutopic endometrium of women with endometriosis (B), and in endometriotic cysts with (D) and without (C) GnRHa treatment. Positive NRN1 staining is seen as a dark-brown precipitate on the surfaces of the cells. Hematoxylin and eosin staining were performed in each specimen to provide a tissue overview. Magnification, 400×; scale bar, 50 μm.

Figure 4

Serum level of NRN1, endometrial thickness, uterine volume and ovarian endometrioma diameter in women with endometriosis following GnRHa treatment. (A) NRN1 concentration was measured in the serum from 10 patients with endometriosis before and after GnRHa treatment by ELISA. The concentration of NRN1 in the serum decreased in nine out of the 10 patients. ** p < 0.01. (B) Endometrial thickness in women with endometriosis following GnRHa treatment; n = 10; * p < 0.05. (C) Uterine volume in women with endometriosis following GnRHa treatment, n = 10; * p < 0.05. (D) Ovarian endometrioma diameter in women with endometriosis following GnRHa treatment, n = 10; p = 0.875. The Wilcoxon signed-rank test was used for the analysis in this follow-up study. n, number of patients; tx, treatment. Significance was set at p < 0.05.

Figure 5

Level of NRN1 in serum of women with endometriosis was measured by ELISA. (A) Serum level of NRN1 in patients who were treated with gonadotrophin-releasing hormone agonist (GnRHa (+), n = 38) and in those who were not treated with the GnRHa (GnRHa (−), n = 28). (B) The level of NRN1 in the serum from the GnRHa (−) group in the proliferative (n = 14) and secretory (n = 14) phases of the menstrual cycle. (C) The level of NRN1 in the serum in the GnRHa (+) group in patients who were treated for 1 month (n = 24) and those who were treated for ≥2 months (n = 14). The non-parametric Mann–Whitney U test was used for statistical analysis. The data are expressed as the mean ± the standard error. Significance was set at p < 0.05. n, number of patients; tx, treatment.