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Beta endorphin in serum and follicular fluid of PCOS- and non-PCOS women

Beta endorphin in serum and follicular fluid of PCOS- and non-PCOS women Purpose To compare the concentrations of beta endorphin in serum and follicular fluid (FF) of PCOS- and non-PCOS women. Secondarily, to investigate associations between beta endorphin and other parameters. Methods Fifty-nine women undergoing in vitro fertilization (IVF) were included in the study. Sixteen were stratified to the PCOS group using the Rotterdam criteria. The remaining 43 women served as controls. Follicular fluid was collected during oocyte retrieval and peripheral blood sampling was performed on the same day. Beta endorphin concentrations in serum and follicular fluid, serum levels of insulin, glucose, LH, estradiol and progesterone were measured. Additionally, testosterone was measured before starting the stimulation protocol. Results There was no difference in beta endorphin levels between PCOS- and non-PCOS women. The concentration of the peptide was higher in serum than in FF, likely due to collection of FF after ovulation induction and corresponding to the early luteal phase. We found a significant correlation between the number of mature Metaphase II (MII) oocytes retrieved and beta endorphin concentration in FF. In women with biochemical hyperandrogenemia, beta endorphin levels in FF cor- related with testosterone levels. Conclusion Beta Endorphin concentrations in serum and FF do not differ between PCOS- and non PCOS-women undergoing IVF. However, together with sex hormones, beta endorphin might play a key role in oocyte maturation. Keywords Opioids · Granulosa cells · PCOS · Beta endorphin Introduction All endogenous opioids share the same n-terminal ami- noacid pentasequence, the so-called “opioid motif” [6]. Pre- The polycystic ovary syndrome (PCOS) is one of the most opiomelanocortin (POMC), preprodynorphine and preproen- common endocrinopathies of women in reproductive age. kephaline are post-translationally processed and function as Many hypotheses regarding the pathogenesis of the disorder precursor molecules for the different opioid peptides [ 7–9]. have been postulated. Decades ago, neuroendocrine abnor- Three main opioid receptor classes have been described: µ, malities were thought to play a causative role [1]. 30 years κ and δ [10]. Although all opioid peptides can bind to each later, Gilling-Smith et al. reported about a primary dysfunc- receptor subclass, their affinity for the different receptors tion of ovarian steroidhormone synthesis in PCOS women vary [11]. While the role of endogenous opioids in the neu- [2]. roendocrine regulation of the menstrual cycle has been well The role of endogenous opioids, especially the POMC- established [12, 13], very little is known about the effects of derivate beta endorphin, in PCOS has been a research topic these molecules in the periphery. Beta endorphin has been for some time [3–5], but has not been extensively studied. shown to modulate pancreatic insulin and glucagon release [14]. Interestingly, the presence of sex steroids seems to be critical for beta endorphin action in the human pancreas [5]. In 1985, Petraglia et al. first reported that beta endorphin * Beata Seeber in human follicular fluid showed higher concentrations in the beata.seeber@i-med.ac.at follicular phase of the menstrual cycle and lower concentra- Department for Gynecological Endocrinology, Medical tions in the luteal phase [15]. In postmenopausal women, University of Innsbruck, Anichstrasse 35, 6020 Innsbruck, no beta endorphin was detectable in follicular fluid (FF). Austria Vol.:(0123456789) 1 3 218 Archives of Gynecology and Obstetrics (2018) 298:217–222 More recently, the expression of opioid receptors on human In both the sample types (FF and serum), beta endor- oocytes, as well as follicular granulosa cells has been con- phin was measured with a test kit (mdbioproducts, Egg near firmed [16, 17]. Together these data indicate a putative role Zurich, Switzerland) using enzyme-linked immunosorbent of opioids, especially beta endorphin, in reproductive func- (ELISA) technique with an assay sensitivity of 1 ng/ml and tion of women, both centrally and in the periphery (i.e. in no cross-reactivity between beta endorphin and other endog- the ovary). Specifically, altered opioid-induced regulation enous opioids. of granulosa cell VEGF expression has been suggested in PCOS- compared to non-PCOS women, leading to increased Statistical analysis VEGF secretion in the former [18]. Based on these prior studies, we hypothesized that (1) Baseline characteristics of the two study groups are concentrations of beta endorphin are higher in follicular expressed as mean ± SD or median with interquartile range. fluid than in serum, thus indicating a local production from Between-group comparisons of variables were performed granulosa cells and/or the oocyte and (2) concentrations of using unpaired t test and Mann–Whitney U test. Paired t beta endorphin in FF und serum differ between PCOS- and test was used to compare beta endorphin concentrations in non-PCOS women. serum and FF. Spearman correlation analysis was used for investigating associations between different variables. To determine which variables were independent predic- tive factors for number of retrieved mature MII (Metaphase Materials and methods II) oocytes and beta endorphin levels in FF, respectively, we performed linear regression analysis. The study was approved by the ethical committee of the All statistical analyses were performed using SPSS Ver- Medical University of Innsbruck, Innsbruck, Austria. sion 22 (SPSS Inc., Chicago, IL). Fifty-nine reproductive age women (21–43 years) who P values < 0.05 were considered significant. presented for in vitro fertilization (IVF) between 2014 and 2016 were included in the study after obtaining informed consent. We used the Rotterdam Criteria [19] to differen- Results tiate between PCOS- and non-PCOS women. Specifically, biochemical hyperandrogenemia was defined as total testos- Of the 59 women included in our study, 16 were stratified terone > 0.4 µg/l measured by liquid chromatography-mass to the PCOS group according to the Rotterdam criteria. The spectrometry (LC/MS) as established in our center. Oli- remaining 43 women were assigned to the control group. gomenorrhea was defined as a menstrual cycle > 35 days, and Patient characteristics are shown in Table 1. Women in amenorrhea as the absence of menstruation for > 3 months. the PCOS group were significantly younger than controls, The only exclusion criteria were the presence of endome- triosis and age < 18. The subjects underwent ovarian stimulation in agonist- or Table 1 Patient characteritics antagonist-protocols, as routinely performed in our clinic. In Control PCOS p value women with oligomenorrhea, stimulation was begun after Age 34.7 ± 5.0 29.3 ± 4.0 < 0.000 inducing a withdrawal bleed with progesterone and with- BMI 23.7 ± 5.1 25.2 ± 6.4 0.374 out the routine use of oral contraceptives. 36 h after ovula- Insulin (U/l) 7.4 (6.8) 12.0 (12.3) 0.171 tion induction using hCG or GnRH agonist, follicles were Glucose (mg/dl) 84 (14) 89 (22) 0.756 punctured, oocytes retrieved and FF was collected. The FF LH (U/l) 0.95 (1.9) 0.8 (3.0) 0.519 from the first follicle punctured from each side was collected Progesterone (µg/l) 5.75 (5.5) 10.65 (12.0) 0.011 separately without flushing. With this method, we aimed to Estradiol (ng/l) 1161 (815) 1606 (1244) 0.018 minimize dilution effects. Subsequently, the follicular fluids Testosterone (µg/l) 0.21 (0.13) 0.35 (0.31) 0.003 of these two follicles were pooled, immediately centrifuged Number of oocytes 8 (8) 21 (15) < 0.000 and stored at – 80 °C until the day of the assay. Number of MII 7 (8) 18 (13) < 0.000 Serum samples for beta endorphin were collected on BetaEnd_S (ng/ml) 0.64 (0.31) 0.53 (0.36) 0.186 retrieval day (corresponding to the early luteal phase) and BetaEnd_FF (ng/ml) 0.37 (0.28) 0.46 (0.23) 0.117 were stored at – 80 °C until analysis. In addition, fasting insulin, glucose, LH, estradiol and progesterone were meas- Data presented as mean ± SD and median with interquartile range, ured per routine by the central laboratory of the Medical respectively University of Innsbruck. Testosterone was measured before MII MetaphaseII oocytes, BetaEnd_S beta endorphin in serum, starting the stimulation protocol. BetaEnd_FF beta endorphin in follicular fluid 1 3 Archives of Gynecology and Obstetrics (2018) 298:217–222 219 but no differences in BMI were noted. As expected, women in the PCOS group had higher baseline testosterone values and had higher estradiol and progesterone concentrations on retrieval day than women in the control group, the latter two measurements being proportional to the higher number of follicles and oocytes produced. The two groups did not differ in fasting insulin and glucose measurements. Furthermore, significantly more oocytes and mature oocytes (metaphase II oocytes) were retrieved in the PCOS group. 12 women (75%) in the PCOS group were treated using an antagonist protocol and the remaining 4 (25%) with an agonist protocol. In the control group, 17 (40%) were treated with an agonist-, 26 (60%) with an antagonist-protocol, respectively. There was no difference in distribution among the two protocols between the PCOS-group and the control- group (p = 0.365). There was no difference in the concentration of beta endorphin in serum, nor in FF between the two groups, 0.64 versus 0.53 ng/ml (p = 0.186) and 0.37 versus 0.46 ng/ml Fig. 1 Regression analysis: r  = 0.228, p = 0.003 (p = 0.117). Unexpectedly, for all women, we found signifi- cantly lower beta endorphin concentrations in FF than in serum (0.45 vs. 0.65 ng/ml, p = 0.001). estradiol, testosterone) predicted beta endorphin levels in FF. None of the variables measured correlated with beta We further elucidated which potential factors predicted the number of retrieved MII oocytes using linear regression endorphin concentration in FF or serum. Based on previously published data that androgens regu- analysis (least square fit) with number of MII oocytes as the dependent variable. Estradiol, progesterone, testoster- late granulosa cell POMC expression [20], we evaluated women with PCOS and biochemical hyperandrogenemia one, insulin, beta endorphin concentration in FF and age were used as independent variables. The results are shown (n = 5) and repeated the above described regression analysis, showing that T was the only highly predictive variable for in Table 2. In addition to age, beta endorphin and testosterone were beta endorphin levels in FF (r = 0.899, p = 0.014) (Fig. 2). each found to be independent predictors with the highest regression coefficients of 12.26 and 25.05, respectively. Discussion This indicates that for each increase of testosterone and beta endorphin by one unit (1 µg/l or 1 ng/ml), the num- While the neuroendocrine regulation of follicle maturation ber of retrieved MII oocytes increases by the above noted coefficient. has been well known for decades, elucidating the complex para- and autocrine mechanisms in the ovary has proven to With an increasing number of total oocytes isolated, the probability for a higher number of retrieved MII oocytes also be difficult. Over 30 years ago, Petraglia et al. [ 15] reported higher beta endorphin concentrations in FF than in serum increases. Thus, we calculated a ratio of MII oocytes:number of oocytes. This ratio also correlated significantly with beta and declining concentrations of the peptide in follicular fluid in the luteal phase. We have detected the beta endorphin endorphin content in FF (r  = 0.228, p = 0.003) (Fig. 1). We found no correlation between beta endorphin concen- precursor POMC in primary granulosa cells using immu- nofluorescence technique (unpublished data, Lunger F.), tration in serum and FF (p = 0.353). Similarly, we performed regression analysis to evaluate thus verifying a local production of endogenous opioids in the ovary. Furthermore, a basal POMC expression has which independent variables (age, insulin, LH, progesterone, Table 2 Regression analysis E2 P T Insulin Beta endorphin FF Age with number of MII oocytes as 2 2 2 2 2 2 the dependent variable MII oocytes r = 0.097 r = 0.331 r = 0.196 r = 0.079 r = 0.115 r = 0.395 B = 0.002 B = 0.622 B = 25.05 B = 0.312 B = 12.26 B = − 0.923 p = 0.016 p ≤ 0.000 p ≤ 0.000 p = 0.034 p = 0.044 p ≤ 0.000 1 3 220 Archives of Gynecology and Obstetrics (2018) 298:217–222 endorphin was a strong independent predictor of retrieved MII oocytes even when controlling for age. Agirregoitia et  al. have shown that G-Protein coupled (GPC) opioid receptors are expressed on human oocytes and that the localization of the µ-opioid receptor changes during oocyte maturation. While in germinal vesicle (GV) phase the recep- tor is mainly found peripherally (plasma membrane), a more homogenous distribution of the receptor in MII has been described, thus indicating an internalization during oocyte maturation [17]. After ligand binding (activation), GPC opi- oid receptors are desensitized and internalized [24]. As the µ-opioid receptor has been found to be mainly internalized in MII and the beta endorphin content of FF correlates with the number of retrieved MII oocytes, one could speculate that the activation (and sequential internalization) of µ-opioid receptors through beta endorphin binding promotes meiosis progression and therefore influences oocyte maturation. Fig. 2 Regression analysis in women with biochemical hyperandro- The presence of sex steroids seems to be critical for genemia. Dependent variable: beta endorphin in FF, independent beta endorphin’s influence on oocyte maturation. In bovine variable: testosterone oocyte maturation, an inhibitory effect of beta endorphin has been demonstrated, but only in the absence of sex hormones been described in porcine granulosa cells [21]. Therefore, [25]. This is consistent with the known effects of beta endor - we expected higher concentrations of beta endorphin in FF phin in the human pancreas and hypothalamus, where the than in serum in line with a local production. In fact, we presence of sex hormones is pivotal for stimulating insulin/ even found lower beta endorphin concentrations in FF com- glucagon release and inhibition of GnRH pulsatility, respec- pared to serum. A possible explanation for this observation tively [5, 26]. is the cycle phase in which the FF was collected, namely Our finding that testosterone levels positively correlated post-ovulation trigger on the day of oocyte retrieval, cor- with the number of MII oocytes is consistent with the studies responding to the early luteal phase. In mouse models, beta that have reported beneficial effects of androgens on follicu- endorphin concentration in FF significantly falls 10 h after lar maturation and oocyte yield [27, 28]. hCG administration [22]. Hence, the timeframe in our study In our experiment, the correlation between beta endor- between ovulation induction and FF collection is sufficient phin content in FF and testosterone was only significant in to explain the lower concentrations in FF. Also, Petraglia women with biochemical hyperandrogenemia, suggesting et al. found higher beta endorphin concentrations in FF when that 90% of variation of beta endorphin concentrations in FF collected in follicular phase. We did not find a correlation was explained by testosterone levels. This may be analogous between FF and serum concentration of beta endorphin, thus to the known physiology of androgens in both animal models supporting the concept of a local follicular regulation of beta as well as in the human prostate, where a critical androgen endorphin expression, secretion and clearance. threshold needs to be reached before androgens can exert Our findings are not consistent with those of Kialka et al., their regular actions in vitro [29]. who reported higher beta endorphin concentrations in the Our study has several limitations. The sample size of serum of PCOS women compared to controls, independent PCOS women is relatively small. We pooled the FF of the of the BMI [23]. However, the authors used the NIH crite- first two follicles from each side to avoid dilution due to ria for PCOS and included only women with biochemical subsequent flushing. We purposely did not pool all the FF hyperandrogenemia in the PCOS group, while we used the from larger and smaller follicles. Thus, we cannot make Rotterdam criteria. We did not have sufficient women in the any statement regarding relationship between follicle size, PCOS group to perform extensive subgroup analysis based oocyte maturity and beta endorphin content per follicle. on PCOS phenotype. Moreover, we collected serum samples We acknowledge that the follicular fluid content of beta in the context of IVF and we do not know how the stimula- endorphin may differ per follicle. Furthermore, we did not tion protocols may impact beta endorphin concentrations measure testosterone on retrieval day, but before starting in serum. the stimulation protocol. One study [30] has found a small The most intriguing finding of our study is the positive increase of serum testosterone mid-cycle which may serve correlation between the proportion of mature metaphase as an explanation for the high pre-ovulatory beta endorphin II oocytes (MII) and beta endorphin content in FF. Beta concentrations in FF as described by Petraglia et al. 1 3 Archives of Gynecology and Obstetrics (2018) 298:217–222 221 7. Kakidani H, Furutani Y, Takahashi H, Noda M, Morimoto Y, In summary, to the best of our knowledge, we are the first Hirose T et al (1982) Cloning and sequence analysis of cDNA to report a potential role of beta endorphin in oocyte matu- for porcine beta-neo-endorphin/dynorphin precursor. Nature ration in humans. The lack of correlation between serum 298(5871):245–249 and follicular fluid levels supports the hypothesis of a local 8. Noda M, Furutani Y, Takahashi H, Toyosato M, Hirose T, Inay- ama S et al (1982) Cloning and sequence analysis of cDNA for expression, secretion and clearance of the peptide. When tes- bovine adrenal preproenkephalin. Nature 295(5846):202–206 tosterone levels are high, beta endorphin content in follicular 9. Nakanishi S, Inoue A, Kita T, Nakamura M, Chang AC, Cohen SN fluid might be almost exclusively regulated by androgens. et al (1979) Nucleotide sequence of cloned cDNA for bovine cor- We do not know what other molecules are involved in regu- ticotropin-beta-lipotropin precursor. Nature 278(5703):423–427 10. Paterson SJ, Corbett AD, Gillan MG, Kosterlitz HW, McKnight lating granulosa cell POMC expression and if these factors AT, Robson LE (1984) Radioligands for probing opioid recep- are secreted systemically, locally, or both. Future studies tors. J Recept Res 4(1–6):143–154 elucidating these regulating factors will improve our under- 11. Mansour A, Taylor LP, Fine JL, Thompson RC, Hoversten MT, standing of opioid physiology in the ovary. Mosberg HI et al (1997) Key residues defining the mu-opioid receptor binding pocket: a site-directed mutagenesis study. J Neurochem 68(1):344–353 Acknowledgements Open access funding provided by University of 12. Wardlaw SL, Wehrenberg WB, Ferin M, Antunes JL, Frantz AG Innsbruck and Medical University of Innsbruck. We would like to thank (1982) Effect of sex steroids on beta-endorphin in hypophyseal Dr. W. Biasio and D. Rosenfellner for their cooperation in the IVF portal blood. J Clin Endocrinol Metab 55(5):877–881 laboratory. 13. Wehrenberg WB, Wardlaw SL, Frantz AG, Ferin M (1982) beta- Endorphin in hypophyseal portal blood: variations throughout Author contributions JNP: data collection and analysis, manuscript the menstrual cycle. Endocrinology 111(3):879–881 writing. LF: data collection and analysis. WL: project development, 14. Reid RL, Yen SS (1981) Beta-endorphin stimulates the secretion manuscript editing. SB: project development, manuscript writing and of insulin and glucagon in humans. J Clin Endocrinol Metab editing. 52(3):592–594 15. Petraglia F, Segre A, Facchinetti F, Campanini D, Ruspa M, Compliance with ethical standards Genazzani AR (1985) Beta-endorphin and met-enkephalin in peritoneal and ovarian follicular fluids of fertile and postmeno- pausal women. Fertil Steril 44(5):615–621 Conflict of interest The authors declare that they have no conflict of 16. Lunger F, Vehmas AP, Fürnrohr BG, Sopper S, Wildt L, Seeber interest. B (2016) Opiate receptor blockade on human granulosa cells inhibits VEGF release. Reprod Biomed Online 32(3):316–322 Open Access This article is distributed under the terms of the Crea- 17. 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Beta endorphin in serum and follicular fluid of PCOS- and non-PCOS women

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Springer Journals
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Copyright © 2018 by The Author(s)
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Medicine & Public Health; Gynecology; Obstetrics/Perinatology/Midwifery; Endocrinology; Human Genetics
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0932-0067
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10.1007/s00404-018-4793-6
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Abstract

Purpose To compare the concentrations of beta endorphin in serum and follicular fluid (FF) of PCOS- and non-PCOS women. Secondarily, to investigate associations between beta endorphin and other parameters. Methods Fifty-nine women undergoing in vitro fertilization (IVF) were included in the study. Sixteen were stratified to the PCOS group using the Rotterdam criteria. The remaining 43 women served as controls. Follicular fluid was collected during oocyte retrieval and peripheral blood sampling was performed on the same day. Beta endorphin concentrations in serum and follicular fluid, serum levels of insulin, glucose, LH, estradiol and progesterone were measured. Additionally, testosterone was measured before starting the stimulation protocol. Results There was no difference in beta endorphin levels between PCOS- and non-PCOS women. The concentration of the peptide was higher in serum than in FF, likely due to collection of FF after ovulation induction and corresponding to the early luteal phase. We found a significant correlation between the number of mature Metaphase II (MII) oocytes retrieved and beta endorphin concentration in FF. In women with biochemical hyperandrogenemia, beta endorphin levels in FF cor- related with testosterone levels. Conclusion Beta Endorphin concentrations in serum and FF do not differ between PCOS- and non PCOS-women undergoing IVF. However, together with sex hormones, beta endorphin might play a key role in oocyte maturation. Keywords Opioids · Granulosa cells · PCOS · Beta endorphin Introduction All endogenous opioids share the same n-terminal ami- noacid pentasequence, the so-called “opioid motif” [6]. Pre- The polycystic ovary syndrome (PCOS) is one of the most opiomelanocortin (POMC), preprodynorphine and preproen- common endocrinopathies of women in reproductive age. kephaline are post-translationally processed and function as Many hypotheses regarding the pathogenesis of the disorder precursor molecules for the different opioid peptides [ 7–9]. have been postulated. Decades ago, neuroendocrine abnor- Three main opioid receptor classes have been described: µ, malities were thought to play a causative role [1]. 30 years κ and δ [10]. Although all opioid peptides can bind to each later, Gilling-Smith et al. reported about a primary dysfunc- receptor subclass, their affinity for the different receptors tion of ovarian steroidhormone synthesis in PCOS women vary [11]. While the role of endogenous opioids in the neu- [2]. roendocrine regulation of the menstrual cycle has been well The role of endogenous opioids, especially the POMC- established [12, 13], very little is known about the effects of derivate beta endorphin, in PCOS has been a research topic these molecules in the periphery. Beta endorphin has been for some time [3–5], but has not been extensively studied. shown to modulate pancreatic insulin and glucagon release [14]. Interestingly, the presence of sex steroids seems to be critical for beta endorphin action in the human pancreas [5]. In 1985, Petraglia et al. first reported that beta endorphin * Beata Seeber in human follicular fluid showed higher concentrations in the beata.seeber@i-med.ac.at follicular phase of the menstrual cycle and lower concentra- Department for Gynecological Endocrinology, Medical tions in the luteal phase [15]. In postmenopausal women, University of Innsbruck, Anichstrasse 35, 6020 Innsbruck, no beta endorphin was detectable in follicular fluid (FF). Austria Vol.:(0123456789) 1 3 218 Archives of Gynecology and Obstetrics (2018) 298:217–222 More recently, the expression of opioid receptors on human In both the sample types (FF and serum), beta endor- oocytes, as well as follicular granulosa cells has been con- phin was measured with a test kit (mdbioproducts, Egg near firmed [16, 17]. Together these data indicate a putative role Zurich, Switzerland) using enzyme-linked immunosorbent of opioids, especially beta endorphin, in reproductive func- (ELISA) technique with an assay sensitivity of 1 ng/ml and tion of women, both centrally and in the periphery (i.e. in no cross-reactivity between beta endorphin and other endog- the ovary). Specifically, altered opioid-induced regulation enous opioids. of granulosa cell VEGF expression has been suggested in PCOS- compared to non-PCOS women, leading to increased Statistical analysis VEGF secretion in the former [18]. Based on these prior studies, we hypothesized that (1) Baseline characteristics of the two study groups are concentrations of beta endorphin are higher in follicular expressed as mean ± SD or median with interquartile range. fluid than in serum, thus indicating a local production from Between-group comparisons of variables were performed granulosa cells and/or the oocyte and (2) concentrations of using unpaired t test and Mann–Whitney U test. Paired t beta endorphin in FF und serum differ between PCOS- and test was used to compare beta endorphin concentrations in non-PCOS women. serum and FF. Spearman correlation analysis was used for investigating associations between different variables. To determine which variables were independent predic- tive factors for number of retrieved mature MII (Metaphase Materials and methods II) oocytes and beta endorphin levels in FF, respectively, we performed linear regression analysis. The study was approved by the ethical committee of the All statistical analyses were performed using SPSS Ver- Medical University of Innsbruck, Innsbruck, Austria. sion 22 (SPSS Inc., Chicago, IL). Fifty-nine reproductive age women (21–43 years) who P values < 0.05 were considered significant. presented for in vitro fertilization (IVF) between 2014 and 2016 were included in the study after obtaining informed consent. We used the Rotterdam Criteria [19] to differen- Results tiate between PCOS- and non-PCOS women. Specifically, biochemical hyperandrogenemia was defined as total testos- Of the 59 women included in our study, 16 were stratified terone > 0.4 µg/l measured by liquid chromatography-mass to the PCOS group according to the Rotterdam criteria. The spectrometry (LC/MS) as established in our center. Oli- remaining 43 women were assigned to the control group. gomenorrhea was defined as a menstrual cycle > 35 days, and Patient characteristics are shown in Table 1. Women in amenorrhea as the absence of menstruation for > 3 months. the PCOS group were significantly younger than controls, The only exclusion criteria were the presence of endome- triosis and age < 18. The subjects underwent ovarian stimulation in agonist- or Table 1 Patient characteritics antagonist-protocols, as routinely performed in our clinic. In Control PCOS p value women with oligomenorrhea, stimulation was begun after Age 34.7 ± 5.0 29.3 ± 4.0 < 0.000 inducing a withdrawal bleed with progesterone and with- BMI 23.7 ± 5.1 25.2 ± 6.4 0.374 out the routine use of oral contraceptives. 36 h after ovula- Insulin (U/l) 7.4 (6.8) 12.0 (12.3) 0.171 tion induction using hCG or GnRH agonist, follicles were Glucose (mg/dl) 84 (14) 89 (22) 0.756 punctured, oocytes retrieved and FF was collected. The FF LH (U/l) 0.95 (1.9) 0.8 (3.0) 0.519 from the first follicle punctured from each side was collected Progesterone (µg/l) 5.75 (5.5) 10.65 (12.0) 0.011 separately without flushing. With this method, we aimed to Estradiol (ng/l) 1161 (815) 1606 (1244) 0.018 minimize dilution effects. Subsequently, the follicular fluids Testosterone (µg/l) 0.21 (0.13) 0.35 (0.31) 0.003 of these two follicles were pooled, immediately centrifuged Number of oocytes 8 (8) 21 (15) < 0.000 and stored at – 80 °C until the day of the assay. Number of MII 7 (8) 18 (13) < 0.000 Serum samples for beta endorphin were collected on BetaEnd_S (ng/ml) 0.64 (0.31) 0.53 (0.36) 0.186 retrieval day (corresponding to the early luteal phase) and BetaEnd_FF (ng/ml) 0.37 (0.28) 0.46 (0.23) 0.117 were stored at – 80 °C until analysis. In addition, fasting insulin, glucose, LH, estradiol and progesterone were meas- Data presented as mean ± SD and median with interquartile range, ured per routine by the central laboratory of the Medical respectively University of Innsbruck. Testosterone was measured before MII MetaphaseII oocytes, BetaEnd_S beta endorphin in serum, starting the stimulation protocol. BetaEnd_FF beta endorphin in follicular fluid 1 3 Archives of Gynecology and Obstetrics (2018) 298:217–222 219 but no differences in BMI were noted. As expected, women in the PCOS group had higher baseline testosterone values and had higher estradiol and progesterone concentrations on retrieval day than women in the control group, the latter two measurements being proportional to the higher number of follicles and oocytes produced. The two groups did not differ in fasting insulin and glucose measurements. Furthermore, significantly more oocytes and mature oocytes (metaphase II oocytes) were retrieved in the PCOS group. 12 women (75%) in the PCOS group were treated using an antagonist protocol and the remaining 4 (25%) with an agonist protocol. In the control group, 17 (40%) were treated with an agonist-, 26 (60%) with an antagonist-protocol, respectively. There was no difference in distribution among the two protocols between the PCOS-group and the control- group (p = 0.365). There was no difference in the concentration of beta endorphin in serum, nor in FF between the two groups, 0.64 versus 0.53 ng/ml (p = 0.186) and 0.37 versus 0.46 ng/ml Fig. 1 Regression analysis: r  = 0.228, p = 0.003 (p = 0.117). Unexpectedly, for all women, we found signifi- cantly lower beta endorphin concentrations in FF than in serum (0.45 vs. 0.65 ng/ml, p = 0.001). estradiol, testosterone) predicted beta endorphin levels in FF. None of the variables measured correlated with beta We further elucidated which potential factors predicted the number of retrieved MII oocytes using linear regression endorphin concentration in FF or serum. Based on previously published data that androgens regu- analysis (least square fit) with number of MII oocytes as the dependent variable. Estradiol, progesterone, testoster- late granulosa cell POMC expression [20], we evaluated women with PCOS and biochemical hyperandrogenemia one, insulin, beta endorphin concentration in FF and age were used as independent variables. The results are shown (n = 5) and repeated the above described regression analysis, showing that T was the only highly predictive variable for in Table 2. In addition to age, beta endorphin and testosterone were beta endorphin levels in FF (r = 0.899, p = 0.014) (Fig. 2). each found to be independent predictors with the highest regression coefficients of 12.26 and 25.05, respectively. Discussion This indicates that for each increase of testosterone and beta endorphin by one unit (1 µg/l or 1 ng/ml), the num- While the neuroendocrine regulation of follicle maturation ber of retrieved MII oocytes increases by the above noted coefficient. has been well known for decades, elucidating the complex para- and autocrine mechanisms in the ovary has proven to With an increasing number of total oocytes isolated, the probability for a higher number of retrieved MII oocytes also be difficult. Over 30 years ago, Petraglia et al. [ 15] reported higher beta endorphin concentrations in FF than in serum increases. Thus, we calculated a ratio of MII oocytes:number of oocytes. This ratio also correlated significantly with beta and declining concentrations of the peptide in follicular fluid in the luteal phase. We have detected the beta endorphin endorphin content in FF (r  = 0.228, p = 0.003) (Fig. 1). We found no correlation between beta endorphin concen- precursor POMC in primary granulosa cells using immu- nofluorescence technique (unpublished data, Lunger F.), tration in serum and FF (p = 0.353). Similarly, we performed regression analysis to evaluate thus verifying a local production of endogenous opioids in the ovary. Furthermore, a basal POMC expression has which independent variables (age, insulin, LH, progesterone, Table 2 Regression analysis E2 P T Insulin Beta endorphin FF Age with number of MII oocytes as 2 2 2 2 2 2 the dependent variable MII oocytes r = 0.097 r = 0.331 r = 0.196 r = 0.079 r = 0.115 r = 0.395 B = 0.002 B = 0.622 B = 25.05 B = 0.312 B = 12.26 B = − 0.923 p = 0.016 p ≤ 0.000 p ≤ 0.000 p = 0.034 p = 0.044 p ≤ 0.000 1 3 220 Archives of Gynecology and Obstetrics (2018) 298:217–222 endorphin was a strong independent predictor of retrieved MII oocytes even when controlling for age. Agirregoitia et  al. have shown that G-Protein coupled (GPC) opioid receptors are expressed on human oocytes and that the localization of the µ-opioid receptor changes during oocyte maturation. While in germinal vesicle (GV) phase the recep- tor is mainly found peripherally (plasma membrane), a more homogenous distribution of the receptor in MII has been described, thus indicating an internalization during oocyte maturation [17]. After ligand binding (activation), GPC opi- oid receptors are desensitized and internalized [24]. As the µ-opioid receptor has been found to be mainly internalized in MII and the beta endorphin content of FF correlates with the number of retrieved MII oocytes, one could speculate that the activation (and sequential internalization) of µ-opioid receptors through beta endorphin binding promotes meiosis progression and therefore influences oocyte maturation. Fig. 2 Regression analysis in women with biochemical hyperandro- The presence of sex steroids seems to be critical for genemia. Dependent variable: beta endorphin in FF, independent beta endorphin’s influence on oocyte maturation. In bovine variable: testosterone oocyte maturation, an inhibitory effect of beta endorphin has been demonstrated, but only in the absence of sex hormones been described in porcine granulosa cells [21]. Therefore, [25]. This is consistent with the known effects of beta endor - we expected higher concentrations of beta endorphin in FF phin in the human pancreas and hypothalamus, where the than in serum in line with a local production. In fact, we presence of sex hormones is pivotal for stimulating insulin/ even found lower beta endorphin concentrations in FF com- glucagon release and inhibition of GnRH pulsatility, respec- pared to serum. A possible explanation for this observation tively [5, 26]. is the cycle phase in which the FF was collected, namely Our finding that testosterone levels positively correlated post-ovulation trigger on the day of oocyte retrieval, cor- with the number of MII oocytes is consistent with the studies responding to the early luteal phase. In mouse models, beta that have reported beneficial effects of androgens on follicu- endorphin concentration in FF significantly falls 10 h after lar maturation and oocyte yield [27, 28]. hCG administration [22]. Hence, the timeframe in our study In our experiment, the correlation between beta endor- between ovulation induction and FF collection is sufficient phin content in FF and testosterone was only significant in to explain the lower concentrations in FF. Also, Petraglia women with biochemical hyperandrogenemia, suggesting et al. found higher beta endorphin concentrations in FF when that 90% of variation of beta endorphin concentrations in FF collected in follicular phase. We did not find a correlation was explained by testosterone levels. This may be analogous between FF and serum concentration of beta endorphin, thus to the known physiology of androgens in both animal models supporting the concept of a local follicular regulation of beta as well as in the human prostate, where a critical androgen endorphin expression, secretion and clearance. threshold needs to be reached before androgens can exert Our findings are not consistent with those of Kialka et al., their regular actions in vitro [29]. who reported higher beta endorphin concentrations in the Our study has several limitations. The sample size of serum of PCOS women compared to controls, independent PCOS women is relatively small. We pooled the FF of the of the BMI [23]. However, the authors used the NIH crite- first two follicles from each side to avoid dilution due to ria for PCOS and included only women with biochemical subsequent flushing. We purposely did not pool all the FF hyperandrogenemia in the PCOS group, while we used the from larger and smaller follicles. Thus, we cannot make Rotterdam criteria. We did not have sufficient women in the any statement regarding relationship between follicle size, PCOS group to perform extensive subgroup analysis based oocyte maturity and beta endorphin content per follicle. on PCOS phenotype. Moreover, we collected serum samples We acknowledge that the follicular fluid content of beta in the context of IVF and we do not know how the stimula- endorphin may differ per follicle. Furthermore, we did not tion protocols may impact beta endorphin concentrations measure testosterone on retrieval day, but before starting in serum. the stimulation protocol. One study [30] has found a small The most intriguing finding of our study is the positive increase of serum testosterone mid-cycle which may serve correlation between the proportion of mature metaphase as an explanation for the high pre-ovulatory beta endorphin II oocytes (MII) and beta endorphin content in FF. Beta concentrations in FF as described by Petraglia et al. 1 3 Archives of Gynecology and Obstetrics (2018) 298:217–222 221 7. Kakidani H, Furutani Y, Takahashi H, Noda M, Morimoto Y, In summary, to the best of our knowledge, we are the first Hirose T et al (1982) Cloning and sequence analysis of cDNA to report a potential role of beta endorphin in oocyte matu- for porcine beta-neo-endorphin/dynorphin precursor. Nature ration in humans. The lack of correlation between serum 298(5871):245–249 and follicular fluid levels supports the hypothesis of a local 8. Noda M, Furutani Y, Takahashi H, Toyosato M, Hirose T, Inay- ama S et al (1982) Cloning and sequence analysis of cDNA for expression, secretion and clearance of the peptide. When tes- bovine adrenal preproenkephalin. Nature 295(5846):202–206 tosterone levels are high, beta endorphin content in follicular 9. Nakanishi S, Inoue A, Kita T, Nakamura M, Chang AC, Cohen SN fluid might be almost exclusively regulated by androgens. et al (1979) Nucleotide sequence of cloned cDNA for bovine cor- We do not know what other molecules are involved in regu- ticotropin-beta-lipotropin precursor. Nature 278(5703):423–427 10. Paterson SJ, Corbett AD, Gillan MG, Kosterlitz HW, McKnight lating granulosa cell POMC expression and if these factors AT, Robson LE (1984) Radioligands for probing opioid recep- are secreted systemically, locally, or both. Future studies tors. J Recept Res 4(1–6):143–154 elucidating these regulating factors will improve our under- 11. Mansour A, Taylor LP, Fine JL, Thompson RC, Hoversten MT, standing of opioid physiology in the ovary. Mosberg HI et al (1997) Key residues defining the mu-opioid receptor binding pocket: a site-directed mutagenesis study. J Neurochem 68(1):344–353 Acknowledgements Open access funding provided by University of 12. Wardlaw SL, Wehrenberg WB, Ferin M, Antunes JL, Frantz AG Innsbruck and Medical University of Innsbruck. We would like to thank (1982) Effect of sex steroids on beta-endorphin in hypophyseal Dr. W. Biasio and D. Rosenfellner for their cooperation in the IVF portal blood. J Clin Endocrinol Metab 55(5):877–881 laboratory. 13. Wehrenberg WB, Wardlaw SL, Frantz AG, Ferin M (1982) beta- Endorphin in hypophyseal portal blood: variations throughout Author contributions JNP: data collection and analysis, manuscript the menstrual cycle. Endocrinology 111(3):879–881 writing. LF: data collection and analysis. WL: project development, 14. 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Published: May 28, 2018

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