Ovarian brain-derived neurotrophic factor is present in follicular fluid from normally cycling women

Abstract

The process of oocyte maturation and follicle development is a complex and carefully orchestrated phenomenon, involving gonadotropin hormones and a rapidly expanding list of other intraovarian regulators (1Giudice L.C. Cataldo N.A. Thierry van Dessel H.J.H.M. Yap O.W.S. Chandrasekher Y.A. Growth factors in normal ovarian follicle development.Semin Reprod Endocrinol. 1996; 14: 179-196Crossref PubMed Scopus (21) Google Scholar). These regulators include growth factors belonging to the insulin-like growth factor family and the transforming growth factor-beta family. Examples of other known intraovarian regulators include cytokines and factors influencing angiogenesis (vascular endothelial growth factor). Additional intraovarian regulators are likely to be discovered in the coming years. One newly described growth factor present in the human follicle is brain-derived neurotrophic factor (BDNF) (2Seifer D.B. Feng B. Shelden R.M. Chen S. Dreyfus C.F. Brain-derived neurotrophic factor (BDNF) a novel human ovarian follicular protein.J Clin Endocrinol Metab. 2002; 87: 655-659Crossref PubMed Scopus (0) Google Scholar).Brain-derived neurotrophic factor is a neurotrophin widely recognized for its role within the mammalian nervous system, where it has been appreciated for its trophic action for survival and differentiation of neurons in the central and peripheral nervous system. Although originally described in the nervous system, BDNF has been shown to be expressed in a variety of non-neuronal tissues (3Yamamoto M. Sobue G. Yamamoto K. Terao S. Mitsuma T. Expression of mRNAs for neurotrophic factors (NGF, BDNF, NT-3 and GDNF) and their receptors (p75 NGFR, Trk A, Trk B, and Trk C) in the adult human peripheral nervous system and nonneural tissues.Neurochem Res. 1996; 21: 929-938Crossref PubMed Scopus (224) Google Scholar). Most recently, we demonstrated the presence of BDNF in the follicular fluid of preovulatory follicles from women undergoing ovulation induction in preparation for IVF (2Seifer D.B. Feng B. Shelden R.M. Chen S. Dreyfus C.F. Brain-derived neurotrophic factor (BDNF) a novel human ovarian follicular protein.J Clin Endocrinol Metab. 2002; 87: 655-659Crossref PubMed Scopus (0) Google Scholar). We further noted that the secretion of BDNF from cumulus granulosa is enhanced by cAMP, the second messenger of gonadotrophin stimulation, in a dose-dependent manner (2Seifer D.B. Feng B. Shelden R.M. Chen S. Dreyfus C.F. Brain-derived neurotrophic factor (BDNF) a novel human ovarian follicular protein.J Clin Endocrinol Metab. 2002; 87: 655-659Crossref PubMed Scopus (0) Google Scholar).In the present study, we examined whether BDNF is present in the follicular fluid of preovulatory follicles from normally cycling women. We addressed the question of whether BDNF is secreted within the context of normal ovarian physiology or exclusively within the context of supraphysiologic pharmacologic doses of exogenous gonadotrophins.Ten normal cycling women with a history of regular menstrual cycles of 26 to 32 days comprised the study population. One follicular fluid sample from a single dominant follicle from each of these 10 women had been frozen at −80°C; these were analyzed for inhibin/activin and follistatin, as reported as part of a larger cohort in a previous study (4Schneyer A.L. Fujiwara T. Fox J. Welt C.K. Adams J. Messerlian G.M. Taylor A.E. Dynamic changes in the intrafollicular inhibin/activin/follistatin axis during human follicular development relationship to circulating hormone concentrations.J Clin Endocrinol Metab. 2000; 85: 3319-3330Crossref PubMed Scopus (101) Google Scholar). The women had taken no medications for 3 months prior to aspiration of their dominant follicle. Pelvic ultrasonography was performed in the follicular phase of their cycle to document dominant follicle growth. Aspiration of the dominant follicle (mean diameter: ≥15 mm, range diameter: 15–23 mm) was accomplished after the largest follicle from among the cohort of antral follicles was determined. Approval for analyses of these samples was obtained from the institutional review boards at the Massachusetts General Hospital and Brigham and Women’s Hospital.Follicular fluid from 20 age-matched women undergoing ovulation induction for IVF for male factor infertility or who were oocyte donors were chosen as a comparison group. Stimulation protocols were similar to those previously reported, with aspiration of follicles (mean diameter: ≥17 mm, range diameter: 17–24 mm) 36 hours after hCG injection (2Seifer D.B. Feng B. Shelden R.M. Chen S. Dreyfus C.F. Brain-derived neurotrophic factor (BDNF) a novel human ovarian follicular protein.J Clin Endocrinol Metab. 2002; 87: 655-659Crossref PubMed Scopus (0) Google Scholar). Follicular fluid samples had been frozen at −80°C. Approval for analyses of these samples was obtained from the institutional review board at UMDNJ–Robert Wood Johnson Medical School.Brain-derived neurotrophic factor levels were determined using the commercially available BDNF Emax immunoassay system (Promega Corp., Madison, WI). The ELISAs were performed according to the manufacturer’s protocol and as previously described (2Seifer D.B. Feng B. Shelden R.M. Chen S. Dreyfus C.F. Brain-derived neurotrophic factor (BDNF) a novel human ovarian follicular protein.J Clin Endocrinol Metab. 2002; 87: 655-659Crossref PubMed Scopus (0) Google Scholar). To validate the BDNF assay for follicular fluid, serial dilutions of three follicular fluid samples were checked for linearity, and recovery of two doses of exogenous BDNF was assessed in IVF follicular fluid. Statistical analysis of data was prepared using a nonparametric Mann-Whitney U-test for group comparisons.Patient groups were matched for age, with a mean age of 32.5 ± 6.6 years in the normal cycle patients and 33.0 ± 5.2 years in the IVF patient group. We noted the presence of BDNF in all follicular fluid samples, except for two normal follicles in which BDNF levels were at the limit of sensitivity for the assay. A 27-fold difference in BDNF follicular fluid concentrations was noted between normally cycling women and women undergoing ovulation induction in preparation for IVF (Fig. 1). Median (interquartile range) BDNF levels in follicular fluid were 28.8 pg/mL (61.9 pg/mL) in normally cycling women and 780.0 pg/mL (314.9 pg/mL) in women undergoing ovulation induction in preparation for IVF (P<.0001). Follicular fluid BDNF concentrations did not vary as a function of follicle size in normal follicles (data not shown). The BDNF assay was valid for human follicular fluid measurement as demonstrated by the linear dilution of three IVF fluid samples. In addition, mean recovery of BDNF in follicular fluid was 89% at 125 pg/mL and 94% at 62.5 pg/mL.This is the first evidence that BDNF is present in the follicular fluid of normally cycling women, raising the possibility that BDNF is a physiologic regulator of normal follicle maturation. The BDNF levels in follicular fluid appear to be up-regulated by gonadotropin stimulation, in that BDNF levels were, on average, 27 times higher in follicular fluid from women undergoing ovulation induction as compared to normally cycling women. These findings are consistent with previous work (2Seifer D.B. Feng B. Shelden R.M. Chen S. Dreyfus C.F. Brain-derived neurotrophic factor (BDNF) a novel human ovarian follicular protein.J Clin Endocrinol Metab. 2002; 87: 655-659Crossref PubMed Scopus (0) Google Scholar) that demonstrated exogenous gonadotrophins increase cAMP, which in turn leads to greater cumulus cell secretion of BDNF into the follicular fluid.Prior to this study, the assay for BDNF had been validated only for urine samples (5BDNF Emax ImmunoAssay System. Promega Technical Bulletin #257. Madison, WI, revised 1/98Google Scholar). The data presented in this report validate the BDNF immunoassay for application to human follicular fluid samples. The process of oocyte maturation and follicle development is a complex and carefully orchestrated phenomenon, involving gonadotropin hormones and a rapidly expanding list of other intraovarian regulators (1Giudice L.C. Cataldo N.A. Thierry van Dessel H.J.H.M. Yap O.W.S. Chandrasekher Y.A. Growth factors in normal ovarian follicle development.Semin Reprod Endocrinol. 1996; 14: 179-196Crossref PubMed Scopus (21) Google Scholar). These regulators include growth factors belonging to the insulin-like growth factor family and the transforming growth factor-beta family. Examples of other known intraovarian regulators include cytokines and factors influencing angiogenesis (vascular endothelial growth factor). Additional intraovarian regulators are likely to be discovered in the coming years. One newly described growth factor present in the human follicle is brain-derived neurotrophic factor (BDNF) (2Seifer D.B. Feng B. Shelden R.M. Chen S. Dreyfus C.F. Brain-derived neurotrophic factor (BDNF) a novel human ovarian follicular protein.J Clin Endocrinol Metab. 2002; 87: 655-659Crossref PubMed Scopus (0) Google Scholar). Brain-derived neurotrophic factor is a neurotrophin widely recognized for its role within the mammalian nervous system, where it has been appreciated for its trophic action for survival and differentiation of neurons in the central and peripheral nervous system. Although originally described in the nervous system, BDNF has been shown to be expressed in a variety of non-neuronal tissues (3Yamamoto M. Sobue G. Yamamoto K. Terao S. Mitsuma T. Expression of mRNAs for neurotrophic factors (NGF, BDNF, NT-3 and GDNF) and their receptors (p75 NGFR, Trk A, Trk B, and Trk C) in the adult human peripheral nervous system and nonneural tissues.Neurochem Res. 1996; 21: 929-938Crossref PubMed Scopus (224) Google Scholar). Most recently, we demonstrated the presence of BDNF in the follicular fluid of preovulatory follicles from women undergoing ovulation induction in preparation for IVF (2Seifer D.B. Feng B. Shelden R.M. Chen S. Dreyfus C.F. Brain-derived neurotrophic factor (BDNF) a novel human ovarian follicular protein.J Clin Endocrinol Metab. 2002; 87: 655-659Crossref PubMed Scopus (0) Google Scholar). We further noted that the secretion of BDNF from cumulus granulosa is enhanced by cAMP, the second messenger of gonadotrophin stimulation, in a dose-dependent manner (2Seifer D.B. Feng B. Shelden R.M. Chen S. Dreyfus C.F. Brain-derived neurotrophic factor (BDNF) a novel human ovarian follicular protein.J Clin Endocrinol Metab. 2002; 87: 655-659Crossref PubMed Scopus (0) Google Scholar). In the present study, we examined whether BDNF is present in the follicular fluid of preovulatory follicles from normally cycling women. We addressed the question of whether BDNF is secreted within the context of normal ovarian physiology or exclusively within the context of supraphysiologic pharmacologic doses of exogenous gonadotrophins. Ten normal cycling women with a history of regular menstrual cycles of 26 to 32 days comprised the study population. One follicular fluid sample from a single dominant follicle from each of these 10 women had been frozen at −80°C; these were analyzed for inhibin/activin and follistatin, as reported as part of a larger cohort in a previous study (4Schneyer A.L. Fujiwara T. Fox J. Welt C.K. Adams J. Messerlian G.M. Taylor A.E. Dynamic changes in the intrafollicular inhibin/activin/follistatin axis during human follicular development relationship to circulating hormone concentrations.J Clin Endocrinol Metab. 2000; 85: 3319-3330Crossref PubMed Scopus (101) Google Scholar). The women had taken no medications for 3 months prior to aspiration of their dominant follicle. Pelvic ultrasonography was performed in the follicular phase of their cycle to document dominant follicle growth. Aspiration of the dominant follicle (mean diameter: ≥15 mm, range diameter: 15–23 mm) was accomplished after the largest follicle from among the cohort of antral follicles was determined. Approval for analyses of these samples was obtained from the institutional review boards at the Massachusetts General Hospital and Brigham and Women’s Hospital. Follicular fluid from 20 age-matched women undergoing ovulation induction for IVF for male factor infertility or who were oocyte donors were chosen as a comparison group. Stimulation protocols were similar to those previously reported, with aspiration of follicles (mean diameter: ≥17 mm, range diameter: 17–24 mm) 36 hours after hCG injection (2Seifer D.B. Feng B. Shelden R.M. Chen S. Dreyfus C.F. Brain-derived neurotrophic factor (BDNF) a novel human ovarian follicular protein.J Clin Endocrinol Metab. 2002; 87: 655-659Crossref PubMed Scopus (0) Google Scholar). Follicular fluid samples had been frozen at −80°C. Approval for analyses of these samples was obtained from the institutional review board at UMDNJ–Robert Wood Johnson Medical School. Brain-derived neurotrophic factor levels were determined using the commercially available BDNF Emax immunoassay system (Promega Corp., Madison, WI). The ELISAs were performed according to the manufacturer’s protocol and as previously described (2Seifer D.B. Feng B. Shelden R.M. Chen S. Dreyfus C.F. Brain-derived neurotrophic factor (BDNF) a novel human ovarian follicular protein.J Clin Endocrinol Metab. 2002; 87: 655-659Crossref PubMed Scopus (0) Google Scholar). To validate the BDNF assay for follicular fluid, serial dilutions of three follicular fluid samples were checked for linearity, and recovery of two doses of exogenous BDNF was assessed in IVF follicular fluid. Statistical analysis of data was prepared using a nonparametric Mann-Whitney U-test for group comparisons. Patient groups were matched for age, with a mean age of 32.5 ± 6.6 years in the normal cycle patients and 33.0 ± 5.2 years in the IVF patient group. We noted the presence of BDNF in all follicular fluid samples, except for two normal follicles in which BDNF levels were at the limit of sensitivity for the assay. A 27-fold difference in BDNF follicular fluid concentrations was noted between normally cycling women and women undergoing ovulation induction in preparation for IVF (Fig. 1). Median (interquartile range) BDNF levels in follicular fluid were 28.8 pg/mL (61.9 pg/mL) in normally cycling women and 780.0 pg/mL (314.9 pg/mL) in women undergoing ovulation induction in preparation for IVF (P<.0001). Follicular fluid BDNF concentrations did not vary as a function of follicle size in normal follicles (data not shown). The BDNF assay was valid for human follicular fluid measurement as demonstrated by the linear dilution of three IVF fluid samples. In addition, mean recovery of BDNF in follicular fluid was 89% at 125 pg/mL and 94% at 62.5 pg/mL. This is the first evidence that BDNF is present in the follicular fluid of normally cycling women, raising the possibility that BDNF is a physiologic regulator of normal follicle maturation. The BDNF levels in follicular fluid appear to be up-regulated by gonadotropin stimulation, in that BDNF levels were, on average, 27 times higher in follicular fluid from women undergoing ovulation induction as compared to normally cycling women. These findings are consistent with previous work (2Seifer D.B. Feng B. Shelden R.M. Chen S. Dreyfus C.F. Brain-derived neurotrophic factor (BDNF) a novel human ovarian follicular protein.J Clin Endocrinol Metab. 2002; 87: 655-659Crossref PubMed Scopus (0) Google Scholar) that demonstrated exogenous gonadotrophins increase cAMP, which in turn leads to greater cumulus cell secretion of BDNF into the follicular fluid. Prior to this study, the assay for BDNF had been validated only for urine samples (5BDNF Emax ImmunoAssay System. Promega Technical Bulletin #257. Madison, WI, revised 1/98Google Scholar). The data presented in this report validate the BDNF immunoassay for application to human follicular fluid samples.