Proteomic identification of neurotrophins in the eutopic endometrium of women with endometriosis

Abstract

Objective: To evaluate neurotrophin (NT) expression in the endometrium of women with and without endometriosis.

Design: Prospective, cross-sectional, translational study.

Setting: Academic hospital.

Patient(s): Thirty-three reproductive-age women undergoing laparoscopy for infertility, pelvic pain, intramural fibroids, or tubal ligation.

Intervention(s): Endometrial biopsies, protein microarrays, reverse transcriptase-polymerase chain reaction, ELISAs, and Western blotting.

Main outcome measure(s): Neurotrophin proteins and mRNAs in eutopic endometrial biopsies.

Result(s): Among seven neurotrophic proteins detected on the antibody microarrays, reverse transcriptase-polymerase chain reaction analysis confirmed nerve growth factor, NT-4/5, and brain-derived neurotrophic factor mRNAs in endometrial tissue. Quantitative ELISAs revealed that NT-4/5 (806 ± 701 vs. 256 ± 190 pg/100 mg protein) and brain-derived neurotrophic factor (121 ± 97 vs. 14 ± 11 ng/100 mg protein) concentrations were significantly higher in women with endometriosis. Nerve growth factor (100 ± 74 vs. 93 ± 83 pg/100 mg protein) levels did not differ between cases and controls.

Conclusion(s): Neurotrophins are synthesized in situ within the endometrium. NT-4/5 and brain-derived neurotrophic factor proteins were more concentrated in biopsies from endometriosis cases than controls, whereas nerve growth factor levels were similar. We hypothesize that the local production of NTs induces sensory innervation of endometrium of women with endometriosis. These NTs represent novel targets for the diagnosis and treatment of endometriosis.

Figure 1

Figure 1A. RT-PCR confirmed the expression of NGF mRNA (predicted amplicon=340 bp) and GAPDH mRNA (predicted amplicon=185 bp) in eutopic endometrial biopsies from two independent and representative controls (C) and two independent endometriosis (E) subjects. Amplicon lengths are indicated as base pairs (bp) and correspond with the molecular weight (MW) ladder in the left lane. Figure 1B. RT-PCR confirmed the expression of BDNF mRNA (predicted amplicon=120 bp), NT-4/5 mRNA (predicted amplicon=96 bp) and GAPDH mRNA (predicted amplicon=185 bp) in eutopic endometrial biopsies from a representative control (C) and two independent endometriosis (E) subjects. GAPDH amplification was performed in duplicate. Molecular weight (MW) ladder is shown at left.

Figure 1

Figure 1A. RT-PCR confirmed the expression of NGF mRNA (predicted amplicon=340 bp) and GAPDH mRNA (predicted amplicon=185 bp) in eutopic endometrial biopsies from two independent and representative controls (C) and two independent endometriosis (E) subjects. Amplicon lengths are indicated as base pairs (bp) and correspond with the molecular weight (MW) ladder in the left lane. Figure 1B. RT-PCR confirmed the expression of BDNF mRNA (predicted amplicon=120 bp), NT-4/5 mRNA (predicted amplicon=96 bp) and GAPDH mRNA (predicted amplicon=185 bp) in eutopic endometrial biopsies from a representative control (C) and two independent endometriosis (E) subjects. GAPDH amplification was performed in duplicate. Molecular weight (MW) ladder is shown at left.

Figure 2

Figure 2A. Quantitative ELISAs of NGF and NT-4/5 proteins in lysates of endometrial biopsies from endometriosis (n=18) and control (n=15) subjects. Data are shown as mean ± SD (* P=0.04, Mann-Whitney U test). Figure 2B. Quantitative ELISA of BDNF protein in lysates of endometrial biopsies from endometriosis (n=18) and control (n=15) subjects. Data are shown as mean ± SD (** P<0.01, Mann-Whitney U test).

Figure 3

Figure 3A. Western blot of NGF in lysates of endometrial biopsies. Two representative controls (C) and endometriosis (E) cases are shown. Molecular weights are shown in kilodaltons (kD) as determined from the standard ladder shown at the right (MW). The faint band at 13 kD represents mature NGF processed from the 27 kD precursor. β-actin, a housekeeping control protein, was immunoblotted to verify equal loading. Figure 3B. Western blots of NT-4/5 and BDNF in lysates of endometrial biopsies. Two representative controls (C) and endometriosis (E) cases are shown. Molecular weights are shown in kilodaltons (kD) at the right. The predominant bands of 22 kD and 28 kD represent proNT-4/5 and proBDNF, respectively. The faint bands at 14 kD represent mature, processed NT-4/5 and BDNF. β-actin, a housekeeping control protein, was immunoblotted to verify equal loading.

Figure 3

Figure 3A. Western blot of NGF in lysates of endometrial biopsies. Two representative controls (C) and endometriosis (E) cases are shown. Molecular weights are shown in kilodaltons (kD) as determined from the standard ladder shown at the right (MW). The faint band at 13 kD represents mature NGF processed from the 27 kD precursor. β-actin, a housekeeping control protein, was immunoblotted to verify equal loading. Figure 3B. Western blots of NT-4/5 and BDNF in lysates of endometrial biopsies. Two representative controls (C) and endometriosis (E) cases are shown. Molecular weights are shown in kilodaltons (kD) at the right. The predominant bands of 22 kD and 28 kD represent proNT-4/5 and proBDNF, respectively. The faint bands at 14 kD represent mature, processed NT-4/5 and BDNF. β-actin, a housekeeping control protein, was immunoblotted to verify equal loading.