{"paper_id":"29446dce-62ff-4175-a460-e8fe90d287e3","body_text":"The follicular fluid (FF) is produced from blood plasma components and secretions\nfrom the granulosa and thecal cells (1). FF provides the microenvironment for oocyte\nmaturation (2). Indeed, the follicular microenvironment will determine whether a\nfollicle is selected or not for ovulation (3). Adequate folliculogenesis requires\nmetabolic stability, and alterations in follicular development may induce a\npremature loss of reproductive capacity. Therefore, FF biochemistry may determine\noocyte quality and the potential to achieve successful fertilization and embryonic\ndevelopment (4-6). To test this hypothesis, we developed a preliminary study to\nexamine whether the biochemical composition of FF remains stable or presents\nalterations that could influence oocyte quality. In fact, in the last 50 yr, several\nstudies have assessed the potential relationship between the composition of FF and\nreproductive outcomes (7-9). In recent years, several peptidergic systems have been\nassociated with the regulatory processes of male and female fertility (10, 11).\nThese systems include local endogenous opioidergic systems (12, 13)\n(gamma-endorphin, beta-endorphin, met-enkephalin, immunoreactive beta-lipotropin),\nand components of the renin-angiotensin system (14, 15) (angiotensin 1-8,\nangiotensin 1-7 or angiotensin II). Their distinct cellular localization pattern in\nhuman ovarian tissue during folliculogenesis and in luteal tissue suggests a role in\nthe growth and differentiation of luteal, granulosa and theca cells (5).\nFF is easily accessible during ovum pick-up and can be aspirated along with the\noocyte. To explore the correlation between FF biochemistry and oocyte quality, each\nfollicle must be aspirated separately (6). The reason is that metabolic differences\nmay be observed in FF samples of the same individual obtained from separate\naspirations.\nIn this study, we analyzed peptide activity in the FF of women undergoing in vitro\nfertilization. To such purpose, 3 samples of FF were analyzed per individual. The\nstudy variables included the presence/absence of oocyte in the FF, the age of the\nwomen, and conditions related/unrelated to ovarian diseases, i.e., endometriosis and\nsterility of unknown etiology, known as unexplained infertility (16, 17). To assess\npeptidergic metabolic stability, we assessed local encephalinergics:\naminopeptidase-N (neutral aminopeptidase, APN), puromycin sensitive aminopeptidase\n(PSA) and neutral endopeptidase (NEP); and components of the renin-angiotensin\nsystem of the reproductive tract: prolyl endopeptidase (PEP), which converts\nangiotensin I (AI) and AII to A1-7; APN, which converts AIII to AIV;\naspartate-aminopeptidase (Asp-AP), which transforms AII to AIII; and basic\naminopeptidase (aminopeptidase B or APB), with converts AIII to AIV. The aim was\ntwofold: i) to assess whether the biochemical composition of FF remains stable or\notherwise presents alterations, and ii) to better understand the role of these\nenzymes and their substrates in follicular physiology (or pathophysiology, in the\ncase of infertility). Finally, we aimed to explore potential biomarkers that are\nuseful in predicting and assessing female infertility and help improve reproductive\noutcomes.\n\nThis was an experimental study. FF samples were obtained from women aged 19-42 yr\nundergoing a fertility treatment in the In Vitro Fertilization Unit of the\nGynecological Clinic of Dr. Juana Hernández (Logroño, Spain). Different\ninclusion criteria were applied to each group. The control group included oocyte\ndonors between 19 and 31 yr of age without known diseases. The endometriosis\ngroup (END group) included women aged 31-39 with an established diagnosis of\nendometriosis. The group of unexplained infertility (UI group) was composed of\nwomen aged 33-38 yr with infertility of unknown etiology diagnosed after an\ninfertility work-up. The  \n > \n  39-yr group consisted of women aged 39 yr or more without\nknown diseases. The exclusion criteria included age  \n < \n  18 or  \n > \n  43 yr, or declining to participate in the study.\nA total of 30 women participated in this study. 3 samples of FF were obtained per\nindividual (90 samples). 8 samples were excluded from the analysis because they\nwere hemolyzed, so the total number of samples in our study was 82. The samples\nof FF were categorized into 4 groups: controls (n = 36, donors without fertility\nproblems); END (n = 15);  \n > \n  39 (n = 12); and UI (n = 19). The sample volume obtained\n(minimum 3 ml) was always much higher than the necessary amount to make\nbiochemical determinations.\nFF samples were obtained by ovum pick-up. During the procedure, 3 random\nfollicles were sequentially aspirated and marked. Then, aspirates were sent to\nthe laboratory of embryology to assess the presence of oocytes in the FF\nsamples. Immediately after oocyte retrieval, the FF was stored at -80°C for\nlater analysis of enzymatic activity. Cautionary measures were adopted to avoid\ncontamination of the fluid samples with blood. The FF samples were thawed to\neliminate impurities and centrifuged at 3000 g at 4°C for 5 min. Later, the\nsupernatant was removed, and specific enzymatic activity was assessed. The study\nvariables included the presence or absence of oocytes in the FF, the age of the\nwomen, and diseases related/unrelated to ovarian disorders (endometriosis and\nsterility of unknown etiology).\nWe assessed the local metabolism of enkephalins, including APN, PSA, and NEP. The\nlocal angiotensin renin system was also analyzed, including PEP, which converts\nAI and AII to A1-7; APN, which converts AIII to AIV; Asp-AP, which hydrolysis\nAII to AIII; and APB, which converts AIII to AIV. By this method, we analyzed\nthe complete metabolic degradation of peptidases in the 2 systems.\nAminopeptidase activity was measured by fluorometry according to previously\ndescribed methods (18). The assay is based on the fluorescence of products\ngenerated from the hydrolysis of a specific substrate by each enzyme present in\nthe sample. Several aminoacyl-2-naphthylamide derivatives (Sigma Aldrich, St.\nLouis, MO, USA) were used as enzymatic substrates. Substrate solutions were\nprepared in a 50 mM phosphate buffer (pH 7.4) containing 0.25 mg/ml of bovine\nserum albumin for APN (0.5 mM), APB (0.5 mM; pH 6.5), APA (0.125 mM) and PEP\n(0.125 mM), and in a 50 mM Tris-HCl buffer (pH 7.4) containing 0.25 mg/ml of\nbovine serum albumin for NEP.\nReactions were triggered by adding 10 μl of the FF sample to 1 ml of the\nincubation mixture with fluorogenic-derived substrates (0.125 mM\naminoacyl-β-naphthylamide). Alanine aminopeptidase activities (APN and PSA) were\nmeasured using Ala-β-naphthylamide as substrate.\nIncubations with the specific PSA inhibitor puromycin (40 μM) (Sigma Aldrich, St.\nLouis, MO, USA) were performed in parallel to discriminate APN and PSA activity\nfrom total alanine aminopeptidase activity. APB (basic aminopeptidase) and\nAsp-aminopeptidase activity were quantified based on Arg-β-naphthylamide and\nAsp-β-naphthylamide levels. PEP activity was assayed using\nZ-Gly-Pro-β-naphthylamide (Bachem, CA, USA) (18, 19).\nN-Dansyl-D-Ala-Gly-p-Nitro-Phe-Gly (DAGNPG) (Sigma Aldrich, St Louis, MO, USA)\nwas used as a fluorogenic substrate to measure NEP activity (19, 20). The\nsubstrate solutions for PSA, APN, and PEP (pH 7.4) and for APB (pH 6.5) were\nprepared in a 50 mM phosphate buffer solution. Asp-AP activity was assayed in\nTris-HCl buffer (50 mM; pH 5.9). After incubation (30 min, 37°C), the enzymatic\nreaction was stopped by adding 1 ml of 0.1 M sodium acetate buffer (pH 4.2). The\nreleased β-naphthylamine was determined by measuring the fluorescence intensity\nin the reaction mixture at 412 nm (with an excitation wavelength of 345 nm)\nusing a spectrofluorometer RF540, Shimadzu, Japan. To determine the released\nDAGNPG in the NEP activity assay, fluorescence was measured at 410 nm and 342 nm\nexcitation. Fluorescence was converted to pmol of product using a standard curve\nmade with increasing concentrations of β-naphthylamine or decreasing\nconcentrations of DAGNPG (Sigma Aldrich, St. Louis, MO, USA).\nAll assays were performed in triplicate. Results were expressed as units of\npeptidase activity per liter of sample: UP/L. A UP is the enzyme that hydrolyzes\n1 pmol of fluorogenic substrate per min.\nThis study was carried out in accordance with the international standards on\nclinical trials. In the preliminary interview, women were made aware that\nparticipation was entirely voluntary, were informed about the anonymity and\nconfidentiality of the data, and read and signed an informed consent form. The\nstudy protocol was reviewed and approved by the Ethics Committee of the Quirón\nClinic hospitals, Spain (Code: GHQZ 01/06/08).\nDifferences between the quantitative variables were assessed using student's\n t  test for independent groups. When a statistically\nsignificant difference was obtained through the Shapiro-Wilk test, the\nMann-Whitney U test was performed. Repeated measures ANOVA with Sidak adjustment\nfor multiple comparisons was used to compare the means across 1 or more\nvariables; the purpose was to assess differences in peptide activity across the\n3 FF samples of the same individual. All statistical tests were bilateral,\nconsidering 95% confidence intervals. All statistical analyses were performed\nusing the Statistical Package for the Social Sciences (SPSS), version 21.0 (SPSS\nInc., Chicago, IL, USA) and the R Commander 3.3.3 software package. Significant\ndifference is considered from p  \n < \n  0.05.\n\nWe assessed the enzymatic activity of 6 peptidases in the FF of 4 groups of women:\ndonors (control group), women with endometriosis (END group), women with unexplained\ninfertility (UI group), and women aged  \n > \n  39 yr ( \n > \n  39 group). The enzymes analyzed included APN, PSA, PEP, Asp-AP,\nAPB, and NEP (Figure 1). The peptidases with the highest activity were NEP and APN,\nwhereas PEP showed the lowest activity. Figure 1 also shows the enzymatic activity\nof the 6 peptidases according to the presence or absence of an oocyte. As observed,\nthe 3 samples of FF of each individual showed similar peptidase activity, regardless\nof whether they contained an oocyte, without statistically significant differences.\nThe peptidase with the highest enzymatic activity was NEP, followed by APN, APB, and\nPSA, whereas PEP showed the lowest activity. Within-subject variations were not\nobserved, and no visible correlations were found. In summary, there were no\nstatistically significant differences in the mean enzymatic activity in the FF\nsamples according to the presence or absence of an oocyte.\nTable I shows the results obtained for the comparisons between the groups in the\nstatistical analysis. Figure 2 summarizes the results for the peptidases that showed\nstatistically significant differences across the groups. PSA activity was\nsignificantly increased in the END and age  \n > \n  39 groups (Figure 2a). PSA activity in the  \n > \n  39 group was 1.5 times higher than in the END group, which was a\nstatistically significant difference (p = 0.018). Consistently, PSA activity was 1.3\ntimes lower in the control group (donors), as compared to in the  \n > \n  39 group, which was a statistically significant difference (p =\n0.033).\nFigure 2b shows the APB activity in the different study groups. APB activity was\nsignificantly reduced in END with statistically significant differences with all\ngroups. As compared to the UI and age  \n > \n  39 groups, APB activity was 2 times lower in the END group (END\nvs. UI, p = 0.007; END vs.  \n > \n  39 group, p = 0.013). The differences with respect to the donors\nwere slightly lower, with APB activity being 1.7 times higher in the donors than in\nthe END group (p = 0.031). All differences were statistically significant.\nFigure 2c shows Asp-AP activity by study group. The lowest Asp-AP activity was\nobserved in the END group, whereas the group with the highest Asp-AP activity was\nUI, followed by the  \n > \n  39 group. Asp-AP activity was 1.5 times higher in the UI group\nthan in the END group, and this difference was statistically significant (p =\n0.049).\nThe group with the most elevated NEP activity was the END group, whereas the lowest\nNEP activity was observed in the  \n > \n  39 group (Figure 2d). NEP activity was 2 times lower in the  \n > \n  39 group than in the END group, and this difference was\nstatistically significant (p = 0.003). Similarly, NEP activity was 1.6 times lower\nin the UI group than in the END group, which was a statistically significant\ndifference (p = 0.019).\nEnzymatic activity of 6 peptidases in FF by study group, including the\npresence or absence of an oocyte. The statistical mean for each enzyme is\npresented. Enzymatic activity is expressed as UP/L (pmol of hydrolyzed\nfluorogenic substrate/min/liter of the sample). PSA: Puromycin sensitive\naminopeptidase, APN: Aminopeptidase-N, PEP: Prolyl-endopeptidase, APB:\nBasic-aminopeptidase, ASP: Aspartate-aminopeptidase, NEP: Neutral\nendopeptidase.\nResults of the peptidases in which statistically significant differences were\nobserved across groups: 2a: Puromycin sensitive aminopeptidase (PSA), 2b:\nBasic-aminopeptidase (APB), 2c: Asp-aminopeptidase (ASP), 2d: Neutral\nendopeptidase (NEP). *P  \n < \n  0.05, **P  \n < \n  0.01.\nMean comparisons between the groups\n\nThe present study sought to describe the activity of enkephalin-degrading enzymes\n(PSA, APN, and NEP) and renin-angiotensin system enzymes (PEP: AI and AII to A1-7;\nAPN: AIII to AIV; Asp-AP: AII to AIII; APB: AIII to AIV) in human FF. The purpose\nwas to better understand the role of these enzymes and their substrates in\nfollicular physiology (or pathophysiology in the case of infertility). Secondly, we\naimed to explore potential biomarkers that help predict or assess female infertility\nand can contribute to improved reproductive outcomes. FF components emerge as\npotential predictors of oocyte quality. We described and compared enzymatic activity\nin FF based on the presence or absence of an oocyte. We also analyzed these\nmetabolisms in relation to the presence/absence of conditions such as endometriosis\nand unexplained infertility.\nThe reason why some FF samples contain an oocyte while others do not is unclear. Some\nauthors have posited that the oocyte could remain in residual FF when FF is\npartially aspirated (21). Another reason could be that oocytes with complex\naneuploidies remain strongly adhered to the follicular wall, thus resisting\naspiration (22). Borderline empty follicle syndrome could explain why, in some\ncycles, the number of oocytes retrieved does not fit the number of follicles counted\nby ultrasound. Empty follicle syndrome has been described, and several hypotheses\nhave been posited, including error in drug administration, advanced age,\nlong-standing infertility, low estrogen levels, and endometriosis, to name a few\n(23-25). Some researchers have suggested an intrinsic ovarian alteration or a\ngenetic etiology. In any case, assessing whether the presence or absence of an\noocyte in FF is associated with biochemical alterations in the FF would shed some\nlight. Since 3 FF samples were obtained from each woman in this study, a twofold\nmethod was employed for comparison of the results.\nFirstly, we explored the potential correlation between the presence/absence of an\noocyte and peptidase activity in the 3 FF samples of each woman. Individual data do\nnot show appreciable differences between them thus, significant differences were not\nobserved in the mean enzymatic activity between oocyte-containing FF and empty FF.\nThere were no clear within-subject differences in peptide metabolism across the 3\nindividual FF samples, regardless of them containing a gamete or not. This means\nthat antral fluid containing oocytes remained stable and gamete-containing FF was\nnot affected. Therefore, none of the enzymes assayed emerged as a potential\nbiomarker.\nFemale fertility peaks at 20-24 yr and progressively decreases over time (7, 26, 27).\nThe pregnancy rate per in vitro fertilization cycle reaches near 40% at 34 yr and\ndrops dramatically in the following 5 yr, a tendency that remains stable throughout\nreproductive life. The pregnancy rate at age 45 is virtually 0, since it is near\nmenopausal age. We compared enzyme levels in 2 age groups: women older and younger\nthan 39 yr. Differences in activity levels were also assessed across the 3 samples\nobtained from each woman. Like in the case of the presence or absence of a gamete,\nindividual data did not show any correlation. There were no clear differences in the\nenzymatic activity across the 3 FF samples of each woman. The behavior of several\npeptidases in the FF of fertile women categorized into 5 age ranges has been\npreviously described (11). The study did not reveal significant age-based\ndifferences in the activity of APN, dipeptidyl peptidase IV, glu-aminopeptidase, PEP\nor pGlutamyl-aminopeptidase I. In contrast, statistically significant differences\nwere found in PSA, APB, Cys-aminopeptidase, and Asp-AP from 40 yr of age. In the\nsame line, the mean levels of enzymatic activity in the FF samples of women  \n < \n  39 vs.  \n > \n  39 (2 groups) revealed statistically significant differences in\nPSA (the highest difference) and APB between the groups. To the best of our\nknowledge, this is the first study to uncover a significant decrease of NEP in women  \n > \n  40. This finding confirms PSA as a potential biomarker of\nfollicular quality and, consequently, of oocyte quality. This is consistent with\nseveral other studies which have documented that the activity of important proteases\ninvolved in peptide metabolism increases as the age of the tissue increases\n(27).\nTo analyze the potential effect of diverse conditions on FF composition, we\nconsidered those most strongly related to ovarian alterations. In this case, we\nassessed metabolic activity in women with endometriosis as compared to fertile\ndonors or women with unexplained infertility, which affects couples without any\napparent fertility problems. Endometriosis is defined as endometrial tissue out of\nthe uterine cavity. Endometriosis has been posited to be associated with poorer\noocyte quality owing to an adverse follicular environment, thereby resulting in\nlower fertilization, embryonic development, and implantation rates (16). In our\nstudy, comparison of enzymatic levels per individual did not show any correlation.\nWe found, however, statistically significant differences in the total mean values\nfor APB between the control group and the END group. There were statistically\nsignificant differences between the UI and the END groups in terms of APB, Asp-AP,\nand NEP activity. We found statistically significant differences between the\ncontrols and the UI group as well. These results confirm the null hypothesis: there\nare no differences in the metabolism of follicular peptides between donors (fertile\nwomen without conditions) and women with unexplained infertility, since the gamete\nmicroenvironment remains stable. This contrasts with the behavior of FF in women\nwith endometriosis, who show alterations in virtually all the peptides studied (as\nthe local opioidergic system is especially affected).\n\nThe results obtained in this study suggest that an adverse follicular environment\nshows alterations in the metabolism of enkephalins and angiotensins. Therefore, some\nof these peptides can be used as diagnostic biomarkers. However, due to the\nlimitations of the study (i.e., the limited number of samples), further studies are\nneeded to verify the role of these peptides in FF stability and to validate the\nvalue of FF components as predictive biomarkers of reproductive success.\n\nThe authors declare that there is no conflict of interest.","source_license":"CC0","license_restricted":false}