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Department of Physiology, Medicine and Nursing School, University of the Basque Country UPV/EHU, 48940 Leioa, SpainBioCruces Health Research Institute, Plaza de Cruces s/n, 48903 Barakaldo, Spain
Department of Physiology, Medicine and Nursing School, University of the Basque Country UPV/EHU, 48940 Leioa, SpainBioCruces Health Research Institute, Plaza de Cruces s/n, 48903 Barakaldo, Spain
Department of Physiology, Medicine and Nursing School, University of the Basque Country UPV/EHU, 48940 Leioa, SpainBioCruces Health Research Institute, Plaza de Cruces s/n, 48903 Barakaldo, Spain
Department of Physiology, Medicine and Nursing School, University of the Basque Country UPV/EHU, 48940 Leioa, SpainBioCruces Health Research Institute, Plaza de Cruces s/n, 48903 Barakaldo, Spain
The paraoxonases (PONs) are antioxidant enzymes associated with beneficial effects against several diseases and some exposures. Little is known, however, about the role of PONs in human reproduction. This work was conducted to investigate whether any association existed between the activities of the PON enzymes (1, 2, and 3) with the follicular size and fertility parameters in assisted reproduction. The study included 100 subfertile women (patients) and 55 proven fertile women (oocyte donors), all undergoing an ovarian stimulation cycle. Follicular fluid from small (diameter <12 mm) and large (diameter ≥18 mm) follicles was collected from each woman. The PONs were quantified in follicular fluid by immunoblotting. PON1 arylesterase and paraoxonase, PON2 methyl paraoxonase and PON3 simvastatinase activities from both donors and patients were significantly higher (P < 0.001) in follicular fluid from large follicles compared with small ones. In large follicles, PON3 activity was significantly higher (P < 0.01) in donors compared with patients. Follicular fluid PON1 arylesterase and paraoxonase activity was positively correlated with the number of retrieved oocytes in donors. This study shows an increase in the activities of PONs with follicle size, thus providing indirect evidence for the role of PONs in follicle maturation.
Reactive oxygen species (ROS) and antioxidants are involved in the regulation of reproductive processes, such as the corpus luteum cycle, as well as changes in the endometrium, the follicular development, ovulation, fertilization, embryogenesis, embryo implantation and placental differentiation and growth (
). In a previous study, we described that, in subfertile women undergoing an IVF cycle, treatment leading to ovarian stimulation was associated with an increased production of ROS (
). Therefore, serum resistance to in-vitro oxidation, in terms of the lag time (time to attain the maximal oxidation rate), total antioxidant capacity, and levels of α- and γ-tocopherol, were significantly reduced at the end of the IVF cycle (at the ovarian stimulation stage) compared with the basal values at the beginning of the cycle. Moreover, variation of the serum oxidation rate in the lag phase emerged as a predictor of pregnancy. In contrast, excessive ROS production has a negative effect on reproductive processes, such as spontaneous abortions, embryopathies, preeclampsia or fetal growth restriction (
Differences in biochemical parameters occurring in reproductive tissues and fluids may be reflected in the systemic circulation. Changes of these markers that take place in other tissues, however, may mask the actual changes occurring in the gonads. Therefore, ovarian follicular fluid represents a better choice than the blood as a source for detecting these differences. The follicular fluid is in part an exudate of serum, as small proteins and lipoproteins can diffuse freely through the follicular wall, but the fluid also contains substances produced locally by follicular cells. High-density lipoproteins (HDL) are the sole lipoprotein particles in human follicular fluid (
Regulation of high density lipoprotein receptor messenger ribonucleic acid expression and cholesterol transport in theca-interstitial cells by insulin and human chorionic gonadotropin.
The paraoxonase (PON) gene family is composed of three members (PON1, PON2, and PON3) that share considerable structural homology, are located in tandem on chromosome 7 in humans and have antioxidative properties (
). PON1 is found in serum and circulates in blood bound to HDL. This enzyme hydrolyzes the oxon forms of several organophosphorus compounds used as insecticides (paraoxon, chlorpyrifos oxon, diazoxon), as well as nerve agents; however, the protection is significant only for diazoxon and chloropyrifox oxon. Two polymorphisms in the coding region of the PON1 gene have been thoroughly studied. Human PON1 displays a polymorphism at position 192 (Q192R) toward paraoxon, rendering enzyme alloforms with very different activities. The three phenotypic groups: QQ, QR, and RR, represent low, intermediate, and high PON1 paraoxonase activity, respectively (
were the first to show that purified human PON1 could inhibit LDL oxidation in vitro. Other studies have reported that PON1 both prevents the formation of oxidized LDL and inactivates LDL-derived oxidized phospholipids once they are formed. PON1 also protects phospholipids in HDL and cells from lipid oxidative modifications (
). These data suggest that one physiological function of PON1 seems to be the metabolism of toxic oxidized lipids. PONs can also hydrolyze a number of lactone-containing pharmaceutical compounds (
). PON2 and PON3 lack paraoxonase or arylesterase activities but are similar to PON1 in that both hydrolyze aromatic and long-chain aliphatic lactones. PON3 in particular hydrolyzes widely used drugs such as the statin lactones lovastatin and simvastatin (
). Both PON2 and PON3 have antioxidant properties; PON3, similar to PON1, is predominately expressed in the liver and is associated with HDL. PON2 is more widely distributed and provides antioxidant protection in the mitochondria and endoplasmic reticulum and outer membrane (
Paraoxonase-2 is a ubiquitously expressed protein with antioxidant properties and is capable of preventing cell-mediated oxidative modification of low density lipoprotein.
). In this study, we determined the activities of the paraoxonases in follicular fluid from large and small follicles to asses any possible role of PON activities in follicle growth. We also compared the activities from large follicles in patients with those of a control group (oocyte donors with proven fertility) to examine any relationship with fertility parameters in IVF and intracytoplasmic sperm injection (IVF–ICSI).
Materials and methods
Study design and study population
This is a prospective case-control study including 155 women that were recruited at the Valencian Institute of Infertility in Bilbao (www.ivi.es, Vizcaya, Spain). The control group (donor) comprised fertile women (with at least one child alive), who entered to the Egg Donor Program of the clinic (n = 55), and the study group (patient), which included women with fertility problems, who attended the clinic to undergo IVF–ICSI treatment (n = 100). Criteria for participation in the study were as follows: no vitamin supplementation; no cardiovascular medical history; no hypertensive disorder; no metabolic disease; no polycystic ovary syndrome; and no endometriosis.
Egg donors were women who fulfilled the following inclusion criteria: age 18–35 years; body masa index (BMI) 18–29 kg/m2; and regular menstrual cycle of 26–35 days. The main exclusion criteria were abnormal karyotype and recurrent miscarriage.
An additional cohort of 18 women (patients) participated in the study to determine the relationship between serum and follicular fluid for PON1 and PON3 concentrations and activities. The same inclusion and exclusion criteria as for the patients described above were considered.
All women underwent gonadotrophin stimulation according to a clinical protocol described previously (
). Transvaginal ultrasonography and serum oestradiol levels were carried out routinely during ovarian stimulation to assess ovarian follicle maturation. Follicular puncture was carried out by transvaginal aspiration 36 h after HCG administration.
Antral follicle count (AFC) was the total number of follicles with a diameter between 2 and 10 mm in both ovaries on day 15 before the start of stimulation, as measured by transvaginal ultrasound. Oocytes were harvested and the total and metaphase II (MII)-stage oocytes were counted. Most of matured oocytes were subjected to ICSI (59%) or mixed ICSI–IVF (15%), whereas the remainder (26%) were cryopreserved by vitrification (
). Clinical pregnancy was confirmed by measuring beta-HCG concentrations and sonographic evidence of an intrauterine gestational sac after embryo transfer. Live birth was defined as a delivery resulting in the birth of at least one live born infant. In this study, a multiple pregnancy was regarded as one pregnancy.
Sample collection
In each patient, follicular fluid from large (diameter ≥18 mm), and small follicles (diameter <12 mm) were obtained. The follicular fluids from two contralateral follicles of the same size were individually aspirated. Follicle flushing was not carried out. Fluids were visually tested for blood contamination and samples contaminated with blood were discarded. Immediately after removing the oocytes, the two samples of the same size were pooled and centrifuged at 3000 g for 10 min to remove debris and granulose cells. The follicular fluid supernatant was then transferred to sterile polypropylene tubes and stored in liquid nitrogen. The tubes were carried to the university maintaining the cold-chain, and kept at −80 °C until analysis. The Free Radicals and Oxidative Stress (FROS) research group of the university (www.ehu.eus/radicaleslibres/) was responsible for the biochemical analyses, and was blinded to the clinical outcomes during the assay procedures. At the moment of oocyte retrieval, serum and follicular fluid were obtained from the same woman. For protein quantification assays follicular fluid from large follicles was used.
Ethical approval
The Ethics Committee of the University UPV/EHU (Ethics Committee for Research involving Human Subjects, CEISH) approved the human subject protocols (CEISH/96/2011/RUIZLARREA and M30_2015_187_RUIZ LARREA) on 27 February 2012 and 28 October 2015, and the study was carried out according to the UPV/EHU and IVI-Bilbao agreements, references 2012/01 and IVI_02_2015 RUIZ LARREA. The project complies with the Spanish Law of Assisted Reproductive Technologies (14/2006). Written informed consent was obtained from all trial participants.
PON1 activities
Because of the wide range of substrates PON1 is able to hydrolyze, different methods for determining PON1 activity have been described. In the present study, both paraoxonase activity (using diethyl p-nitrophenyl phosphate–paraoxon as substrate) and arylesterase activity (using phenylacetate as substrate) were analysed. The enzyme activities were measured spectrophotometrically based on the method described by
adapted to 96-well plates. All reactions were carried out in triplicate.
PON1 paraoxonase activity
A total of 40 µl of follicular fluid 1:20 diluted in buffer was added per well to 60 µl of working buffer (225 mM Tris-HCl, 2.25 mM CaCl2, and 2.14 M NaCl, pH 8.5). The reaction was started by the addition of 100 µl of 6 mM paraoxon (200 µl final volume). The reaction was followed at 37°C by monitoring the change in absorbance at 405 nm every 52 s for 12 min. Blanks were included to correct for the spontaneous hydrolysis of paraoxon. Reaction rates were derived from the corresponding slopes. Results were expressed as nmol per min per ml by applying the experimental molar extinction coefficient of 10.104 mM−1 for p-nitrophenol.
PON1 arylesterase activity
A total of 40 µl of follicular fluid diluted 1:500 in buffer was added per well to 60 µl of the reaction buffer (9 mM Tris-HCl, 1 mM CaCl2, pH 8). The reaction was started by the addition of 100 µl of 24 mM phenyl acetate in the reaction buffer (200 µl total volume). The reaction was followed at 37°C by monitoring the change of absorbance at 270 nm every 50 s for 10 min. Blanks were included to correct for the spontaneous hydrolysis of phenyl acetate. Reactions rates were derived from the corresponding slopes and the results were expressed as µmol/min/ml by applying the experimental molar extinction coefficient of 0.8197 mM−1 for phenol.
Paraoxonase to arylesterase ratio
PON1 phenotype can be determined by kinetic enzyme assays. The paraoxonase/arylesterase ratio was calculated for each woman, and this ratio was used to classify PON1 Q192R phenotypes (
PON2 activity was determined using methyl paraoxon as the substrate. PON2 methyl paraoxonase activity was measured spectrophotometrically by a method adapted to 96-well plates. The reaction buffer consisted of 225 mM Tris-HCl, 2.25 mM CaCl2, and 2.14 mM NaCl, pH 8.5. A total of 40 µl of follicular fluid 1:20 diluted in buffer was added to 60 µl of reaction buffer per well in triplicate. Blanks were included to correct for the spontaneous hydrolysis of methyl paraoxon. The reaction started by the addition of 100 µl of 6 mM methyl paraoxon (200 µl total volume). The reaction was followed at 37°C by monitoring the change of absorbance at 405 nm every 52 s for 12 min. Reactions rates were derived from the corresponding slopes. Results were expressed as nmol per min per ml by applying the experimental molar extinction coefficient of 10.104 mM−1 for p-nitrophenol.
PON3 activity
The determination of PON3 activity was carried out according to
, with modifications to adapt the method to follicular fluid samples. The PON3 protein does not show the capacity to hydrolyze paraoxon and has a very low arylesterase activity; however, the enzyme exhibits lactonase activity on lovastatin and simvastatin compounds. The method measures the lactonase activity of PON3 using simvastatin lactone (SVL) as substrate. PON3 converts simvastatin lactone to its acid form (ß,δ-dihydroxyacid simvastatin, SVA). Both the substrate (SVL) and product (SVA) of the reaction were separated by reverse-phase HPLC and the reaction rate was estimated by the product UV-quantitation (see Supplementary material).
The intra- and inter-assay coefficients of variation for the enzyme activities were as follows: 2.9% and 5.4% for PON1 paraoxonase, 2.3% and 5.1% for PON1 arylesterase, 3.0% and 5.2% for PON2 methyl paraoxonase, and 6.4% and 8.6% for PON3 simvastatinase. The minimal levels of quantitative detection were 500 nmol/min/ml for PON1 arylesterase, 60 nmol/min/ml for PON1 paraoxonase, 10 nmol/min/ml for PON2, and 0.2 nmol/min/ml for PON3 activity.
Low-abundance protein enrichment from follicular fluid
The presence of major proteins (for example, albumin) in follicular fluid makes the detection of minority proteins (PON2) extremely challenging. Before the analysis of PON2 by Western blot, follicular fluid samples were processed with the ProteoMiner Protein Enrichment Large-Capacity commercial kit (Bio-Rad, Madrid, Spain). The columns contained in the kit remove a large proportion of the major proteins in a complex mixture, which facilitates the detection of less abundant proteins. The sample is mixed with the column-containing mixture of hexapeptides attached to chromatographic beads. Major proteins rapidly saturate their hexapeptide ligands, and excess proteins can be washed away, while minor proteins remain bound to their ligands by concentrating on the chromatographic support. The procedure was carried out as indicated by the manufacturer. The storage solution of the column was removed and the column was washed twice with phosphate-buffered saline (150 mM NaCl, 10 mM NaH2PO4, pH 7.4, washing buffer). Follicular fluid (1.8 ml) was applied to the column, and remained rotating for 2 h at room temperature. Afterwards, the column was washed twice with washing buffer to remove the excess of unbound proteins. Finally, the proteins bound to the ligands were eluted by the addition of 200 µl of 4 M urea, 1% CHAPS, and 5% acetic acid. Preliminary experiments indicated that this method was useful for enriching PON2; however, it was not efficient in enriching PON1 and PON3 (data not shown); therefore, it was only used for PON2 analysis.
PON1, PON2 and PON3 quantification by Western blot
Enriched (for PON2) and non-enriched follicular fluid samples (for PON1 and PON3), serum and human recombinant PON1, PON2 (ProsPec-Tany TechnoGene Ltd., Ness Ziona, Israel), and PON3 proteins (ThermoFisher Scientific, Waltham, MA, USA) were denatured in Laemmli buffer 5x (300 mM Tris-HCl, 50% glycerol, 10% sodium dodecylsulphate, 250 mM dithiothreitol, and 0.01% bromophenol blue, pH 6.8) (
). Samples of follicular fluid (0.6 µl, 30 µl and 3.2 µl for PON1, PON2, and PON3 analyses, respectively) or serum (0.4 µl for PON1 and PON2 analyses) were loaded onto the gel. Proteins were separated by vertical slab gel electrophoresis with a resolving gel of 12% polyacrylamide and a stacking gel of 4% polyacrylamide under denaturing conditions in 25 mM Tris-HCl, pH 8.3, buffer containing 192 mM glycine and 0.1% SDS. Electrophoresis was developed for 90 min at constant voltage of 175 V for PON1, and 240 min at a constant voltage of 100 V for PON2 and PON3. In order to analyse PON1, after electrophoresis, the separated proteins were transferred onto PVDF membranes (Immobilon-P, Millipore, Spain) by electroblotting in a semi-dry transfer device (Bio-Rad, Madrid, Spain) with constant amperage (1 mA/cm2) for 1 h. In the case of PON2 and PON3, a wet transfer was carried out; proteins were transferred onto polyvinylidene difluoride membranes in a mini trans-blot module (Bio-Rad, Madrid, Spain) with constant voltage (26 V) for 16 h at 4°C. After blocking for 1 h at room temperature in TTBS buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl and 0.05% Tween20) and 5% skimmed, membranes were incubated overnight at 4°C with the corresponding primary monoclonal antibody (R&D System, Abingdon, UK) at dilutions of 7:7000, 6:7000 and 6:7000 for the detection of PON1, PON2 and PON3, respectively. After extensive washing in TTBS to remove the residual primary antibody, membranes were probed with the secondary antibody conjugated to horseradish peroxidase (R&D System, Abingdon, UK) for 1 h at room temperature. The immunoreactive proteins were detected by incubation with the Clarity Western ECL substarte (Bio-Rad, Madrid, Spain), and the blots were imaged by scanning with the C-DiGit LI-COR blot scanner (Bonsai Advanced Technologies, Madrid, Spain). Quantitative data were derived from the corresponding standard curve on each electroblotting. No cross-reactivity was observed for the PON antibodies (Figure 1).
Figure 1Specificity of anti-PON1, anti-PON2 and anti-PON3 antibodies for PON proteins. Thirty nanograms of each human recombinant PON1, PON2, and PON3 protein were loaded onto the gel for Western blotting in a semi-dry transfer device.
Sample size was calculated for a significance level of 5% and a power higher than 80% to detect a difference between means higher than 15%. StatMate for Windows (GraphPad Software, USA) was used for sample size calculations. Univariate descriptive statistics (mean, standard deviation, and frequency) and bivariate (Student's t-test, Mann–Whitney and Wilcoxon tests) were conducted. Statistical comparisons for categorical variables were carried out using the chi-square test. The Kolmogorov–Smirnov test was used to assess if the variables followed a normal distribution. The Student's t-test for independent samples and its non-parametric equivalent (Mann–Whitney test) were applied to compare the Donor and Patient groups in terms of anthropometric and biochemical variables. The Student's t-test for paired samples and the Wilcoxon test (non-parametric equivalent) were applied to compare large and small follicles within each group (Donor and Patient), and between serum and follicular fluid. Associations among quantitative variables were analysed using the Pearson's correlation coefficient. All of the tests were bilateral, with a significance level of P < 0.05. Stepwise forward multiple linear regression analysis was used for the evaluation of group, age, body mass index, number of retrieved oocytes, and fertilization rate as predictors of the intrafollicular PON3 activity.
Results
The characteristics of the study population are shown in Table 1. The mean age of the patients was significantly higher than that of the donors (P < 0.001). The body mass index was similar in both groups. No differences were found in the percentage of smokers between donors and patients. Among the patients, the cause of infertity included 9.8% recurrent pregnancy loss, 4.9% tubal factor and 6.9% genetic factor. Unexplained infertility was 78.4%. Some type of male infertility in combination with other causes was found in 58% of the cases.
After an ovarian stimulation cycle, large and small follicles were obtained from each woman, and the follicular fluid was separated from oocytes. The different activities of PON1, PON2, and PON3 were measured in follicular fluid from follicles of both sizes (Figure 2). No statistically significant differences were found in PON activities between smokers and non-smokers (data not shown), so this criterion was not considered for subsequent comparisons. Arylesterase showed the highest activity, three orders of magnitude higher than paraoxonase, methyl-paraoxonase, and simvastatinase activities. Highly significant differences depending on the follicle size were found for all studied PON activities. In all cases the activities were markedly higher in large follicles, compared with small ones (P < 0.001), suggesting a role for PON in ovarian follicle maturation. In the case of PON3, significant differences were found between donors and patients for large follicles (P < 0.01), with PON3 activity being 20.7% higher in donors than in patients (16.3 ± 1.0 versus 13.5 ± 0.5 nmol/min/ml). Values remained significantly different after adjusting for age, body mass index, number of retrieved oocytes, and fertilization rate (P = 0.020) (Table 2).
Figure 2Intrafollicular PON1 arylesterase, PON1 paraoxonase, PON2, and PON3 activities of large and small follicles from donors and patients. Box-and-whisker plot shows median, 25th and 75th percentiles, minimum and maximum values, and the outliers of the distribution. ***P < 0.001; ##P < 0.01.
The phenotype distribution of the Q192R polymorphism of PON1 was analysed. The cumulative distribution of women with respect to the ratio of paraoxonase/arylesterase activity revealed a trimodal distribution, in which the three QQ (ratio <2.0), QR (ratio ≥2.0 and <6.0), and RR (ratio ≥6.0) genotypes could be assigned (Figure 3). The distribution of the phenotypes was not significantly different between donors (43.6% QQ, 47.3% QR, and 9.1% RR) and patients (45.4% QQ, 45.4% QR, and 9.2% RR). The polymorphism clearly showed an influence on the paraoxonase activity, with an increasing activity in the order QQ < QR<RR (Figure 4). When the effect of the polymorphism was taken into account, however, no significant differences were found between donors and patients (Figure 5).
Figure 3Cumulative distribution of women with respect to the ratio of paraoxonase/arylesterase activity. PON1 activities were measured in follicular fluid from large follicles. Arrows at ratios of 2.0 and 6.0 are where we divide the phenotypes. PON1 Q192R phenotypes: homozygous QQ, heterogygous QR, and homozygous RR.
Figure 4Intrafollicular PON1 paraoxonase activity across the PON1 Q192R polymorphism. PON1 activity was determined in follicular fluid of large follicles from the overall cohort. Box-and-whisker plot shows median, 25th and 75th percentiles, minimum and maximum values, and the outliers of the distribution. ***P < 0.001.
Figure 5Intrafollicular PON1 paraoxonase activity of large follicles in donors and patients according to PON1 Q192R polymorphism. Box-and-whisker plot shows median, 25th and 75th percentiles, minimum and maximum values, and the outliers of the distribution.
We identified and quantified PON proteins in follicular fluid by Western blot. As described in the ‘Materials and methods section’, PON2 was quantified after depletion of high abundant proteins in follicular fluid samples. On each gel a standard curve was derived from increasing concentrations of human recombinant proteins expressed in Escheria coli, all of them with an additional His-tag. The apparent molecular weights for PON proteins in follicular fluid calculated from the blots using molecular weight markers were 45 kD (PON1), 42 kD (PON2), and 39 kD (PON3). Theoretical values for these proteins vary from 38 to 39.7 kDa. The higher molecular weight for PON1 in follicular fluid corresponds to the extensive post-translational modifications (
). Results revealed that PON1 was the most abundant protein, about 15 times more concentrated than PON3, and 1,600-fold than PON2 (Figure 6).
Figure 6Protein levels of PON1 and PON3 in follicular fluid and PON2 in enriched follicular fluid. Representative Western blots for (A) PON1, (B) PON2, and (C) PON3. Each lane corresponds to increasing quantities (5–20 ng PON1, 1–10 ng PON2, and 0.75–7.5 ng PON3) of the corresponding human recombinant proteins (STD) and different follicular fluid (FF) samples from large follicles from patients. Arrows indicate the position and the calculated apparent molecular weight of the specific PON protein in follicular fluid. (D) Values correspond to the mean ± standard error of the quantified samples (47, 4, and 31 for PON1, PON2, and PON3, respectively).
As PON1 and PON3 are proteins secreted in the blood and follicular fluid is in part a plasma exudate, these proteins were quantified in both serum and follicular fluid from large follicles from the same woman, and the enzyme activities were analysed. Results indicated that there were no statistically significant differences in the concentration of PON1 between serum and follicular fluid (Figure 7). The amount of PON3 was markedly higher in serum than in follicular fluid. Regarding enzyme activities, PON1 arylesterase and paraoxonase were significantly increased in serum compared with follicular fluid (P < 0.001). In the case of PON3, despite its higher concentration in serum, no differences in simvastatinase activity were found between both fluids (Figure 8).
Figure 7Protein levels of PON1 and PON3 in serum and follicular fluid. Representative Western blots for (A) PON1 and (B) PON3 in serum (S) and follicular fluid (FF) from large follicles from patients. Different paired samples (serum and follicular fluid from the same woman) were analysed on each Western blot along with increasing quantities (5–20 ng PON1 and 0.75–7.5 ng PON3) of the corresponding recombinant proteins (STD). Arrows indicate the position of the intrafollicular PON proteins. (C) Bars represent the mean + standard error of the quantified proteins (n = 18 paired samples). ***P < 0.001.
Figure 8PON1 and PON3 activities in serum and follicular fluid. Samples of serum and follicular fluid from large follicles were obtained from the same woman (paired samples). Bars represent the mean + standard error; ***P < 0.001.
The fertility parameters are shown in Table 3. As expected, the mean antral follicle count (AFC) was higher in donors than in patients (P < 0.001). Correlation analysis showed that AFC was positively correlated with the number of retrieved oocytes (R = 0.445, P < 0.001) and MII-stage oocytes (R = 0.448, P < 0.001), and inversely with the woman's age (R = −0.409, P < 0.001). The mean number of retrieved oocytes and MII-stage oocytes were markedly higher (P < 0.001) in donors than in patients. The fertilization rate was similar in both populations (Table 3).
Table 3Fertility parameters after an ovulatory stimulation cycle.
Pearson's correlation analyses were carried out to study possible associations between the biochemical parameters and fertility outcomes. Only those correlations that were statistically significant in the donor and patient populations are shown in Figure 9. In donors, positive correlations were found between PON1 arylesterase (R2 = 0.078, P < 0.05) and paraoxonase activities (R2 = 0.073, P < 0.05) with the total number of retrieved oocytes. Values adjusted for age gave the same statistical significances. These correlations were not found in the patient population. In this group, none of the activities of the PON system differed depending on the pregnant condition (data not shown); around 50% of women receiving an embryo transfer became pregnant after only one IVF cycle.
Figure 9Correlation of total number of retrieved oocytes with (A) PON1 arylesterase activity; and (B) PON1 paraoxonase activity in follicular fluid from large follicles in the donor group.
The main aim of this study was to examine the role of the PONs in the follicular fluid in relation to follicle maturation, and compare these markers in subfertile women with those in a control group (oocyte donors with proven fertility) to establish any relationship with fertility parameters in IVF–ICSI. Follicular size and serum concentration of oestradiol are commonly used as indicators of oocyte maturity, and larger follicles yield oocytes with increased developmental capacity and fertilization rate than those yielded by smaller follicles in IVF (
). The fluid from large follicles is distinct in biochemical nature from that from small follicles, probably reflecting differences in the maturational stage of the follicles (
). In the present study, we report that PON1 paraoxonase and arylesterase activities, PON2 methyl paraoxonase, and PON3 simvastatinase were significantly higher in the follicular fluid of large follicles compared with small ones both from fertile and subfertile women. Moreover, PON3 activity was dramatically higher in follicular fluid of large follicles from fertile women than from subfertile ones. These data provide evidence for a possible role of the PONs in follicle development. To the best of our knowledge, this is the first study focusing on the measurement of PON activities in relation to this aspect of human reproduction. In a pilot study,
Distributions of high-density lipoprotein particle components in human follicular fluid and sera and their associations with embryo morphology parameters during IVF.
analysed the PON1 and PON3 activities in human follicular fluid in connection with HDL particles contained in this ovary fluid, and suggested a role for PON arylesterase activity in early embryo development. In the present study we report in the donor population a clear correlation between PON1 arylesterase and paraoxonase activities with the number of retrieved oocytes. Certain antioxidants (such as melatonin) in the growing follicle may be important factors in avoiding atresia, and, thus, allowing follicle growth and, therefore, oocyte growth (
). Perhaps the different antioxidant status, particularly PON3 antioxidant activity, in the follicles between the donor and patient groups could account for the different correlation findings on the studied populations. Oocyte growth involves an increase in the number of mitochondria, where oxidative phosphorylation takes place in order to produce the required energy for oocyte maturation from the germinal vesicle stage (
Age related changes in mitochondrial function and new approaches to study redox regulation in mammalian oocytes in response to age or maturation conditions.
). Mitochondria are the main organelles responsible of ROS production in the cells, so that ROS generation must be tightly controlled by antioxidant systems. Remarkably, both PON2 and PON3 proteins have been detected in mitochondria (
). The present data show associations that do not necessarily imply causal effect; thus, further studies with humans and mammals are needed to establish causality.
To what extent the differences found in PON activities between small and large follicles are not due to an increased vascularization during follicle maturation are not actually known. In this study, we have characterized the activities and mass expression of the extracellular PON1 and PON3 proteins in serum and follicular fluid. PON1 activities (paraoxonase and arylesterase) were higher in serum than in follicular fluid; however, no difference was observed for the total amount of protein between both fluids. In contrast, similar PON3 activities were found in serum and follicular fluid; however, a significantly lower amount of protein was found in follicular fluid than in serum. These results on PONs expression suggest that the activities determined in the present report do not simply reflect an increased HDL infiltration rate, but indicate that PON1 and PON3 are subjected to strict control in the follicular fluid. PON2 was also detected in follicular fluid. Using a proteomic approach
identified PON1 and other proteins with antioxidant activities in relatively high amounts in follicular fluid of normovulatory women. The authors suggest that these enzymes probably assist in the protection of the follicle from oxidative stress during ovulation. Recently, it has been reported the expression of the three PON isoenzymes in granulosa cells of dairy cows (
), although so far there are no data concerning PONs expression in these ovary cells in women.
One limitation of the present study is the use of pooled follicular fluid, loosing the presumed variability of the analysed biochemical parameters between individual follicles. In all cases, however, the samples came from the same number of follicles, and the measured enzyme activities have an estimated variability between women higher than between follicles (
). The observed intrafollicular changes detected in the PON antioxidant system with follicular maturation would assess the normal oocyte development inside the follicle. One advantage of this work is that each sample from large and small follicles came from the same woman, i.e. the statistical analysis has been carried out for paired data, thus reducing the variability of mean values when independent groups with different subjects are used.
No significant differences in intrafollicular PON1 mean activities were found between donors and patients. This lack of differences was not due to a dissimilar frequency of the PON1 Q192R polymorphism between both populations. Regarding the statistically significant differences found for PON1 activities between small and large follicles, these changes are independent of the polymorphism, as follicle samples of different sizes came from the same woman.
Previous studies have shown the relationship between the paraoxonases and diseases, such as atherosclerosis, hypercholesterolaemia and obesity, attributed to their antioxidant activity against lipid oxidation (
Low levels of serum paraoxonase activities are characteristic of metabolic syndrome and may influence the metabolic-syndrome-related risk of coronary artery disease.
). Little is known, however, on the role of the antioxidant PON enzymes in reproduction and female infertility. The physiological roles of PONs may include the detoxification of lactones (exogenous or endogenous lactones derived from oxidized lipids) and organophosphates, although the physiological substrate(s) of PONs are unknown. It has been shown that purified recombinant human PONs hydrolyze oestrogen esters at position 3 of the steroid A-ring. Oestrogen esters are substrates for the PONs and are efficiently hydrolyzed, particularly by PON3 (
). Nevertheless, they require a free phenolic hydroxyl group to exhibit their antioxidant properties, the corresponding 3-esters having a dramatically reduced antioxidant activity (
). The ability of PONs to hydrolyze esters of phenolic compounds will result in their conversion to more potent antioxidant species and this may represent a potential mechanism by which PON1/3 could confer antioxidant properties to HDL, the lipoprotein particle present in follicular fluid. Moreover, the mean oestradiol ester concentration in follicular fluid in pregnancy is higher than 100 nmol/l, a much higher amount than that reported in plasma, where it varies from 40 pmol/l (first trimester) to 400 pmol/l (third trimester) (
The isolation and characterization of estradiol-fatty acid esters in human ovarian follicular fluid. Identification of an endogenous long-lived and potent family of estrogens.
). It has been proposed that PONs may hydrolyze oestrogen 3-esters, which are likely to be sequestered by HDL owing to their hydrophobic nature, resulting in the unmasking of their antioxidant capacity (
The role of PON2 in physiology has been poorly documented, although some evidence relates its presence to the control of mitochondrial ROS production (
). As mentioned above, PON2 is an intracellular protein in various cell types. In our study, PON2 was detected at low concentrations in follicular fluid. Although it has been reported that methyl paraoxonase activity is more specific for PON2 than PON1 (
), the low intrafollicular PON2 concentration makes it possible that the detected methyl paraoxonase activity could be ascribed to PON1. Most data on PON3 are related to its role in the prevention of LDL oxidation, with minor attention to the role of PON3 in fertility. However, disruption of the Pon3 gene causes embryonic lethality in mice (
). The major cause of homocysteine (Hcy) toxicity is thought to be Hcy metabolites. Specifically Hcy-thiolactone, whose synthesis increases with an increase in Hcys levels, is a reactive metabolite that causes protein N-homocysteinylation, thus impairing protein function (
). The Hcy-thiolactonase activity of PON is a determinant of plasma N-Hcy-protein levels, leading to the conclusion that PON protects proteins against N-homocysteinylation in vivo (
). The reported differences in intrafollicular PON status in this study, and the possible role that these enzymes play in the follicular fluid protection from Hcy-triggered toxicity and the outcomes in reproduction, needs to be unravelled.
In the present study, significant differences in age were observed between donors and patients, fertile women being significantly younger than patients. The PON1 paraoxonase activity from plasma and serum has been reported to vary with age (
). In our study, no correlations were observed between follicular fluid PON1 activities (arylesterase or paraoxonase) and the age (range of the study, 19–46 years). In the study by
, the recruited population was aged between 22 and 89 years, allowing a wide range of differences in PON1 paraoxonase to be observed, which could be related to the development of oxidative stress conditions with ageing. When adjusted for age, we found very significant variations of follicular PON3 activity between donors and patients, with PON3 simvastatinase activity being lower in patients, ruling out the age as the explaining factor for PON3 activity differences.
In conclusion, to the best of our knowledte, this is the first study showing that the activities of PONs are increased in large follicles compared with small ones from either fertile or subfertile women undergoing a controlled ovarian stimulation cycle. The data indicate that the activity of PONs may increase during follicle maturation. The activity of PON3 was higher in follicles from fertile women than from patients, suggesting the involvement of the enzyme in fertility. PON1 activities were associated with the number of total oocytes, providing indirect evidence of the involvement of the redox balance in follicle growth.
Acknowledgement
This work was supported by the ‘PN de I + D + I’ of the Spanish Ministry of Science and Innovation, ‘ISCIII-Subdirección General de Evaluación y Fomento de la Investigación’ and FEDER (reference FIS/FEDER PI11/02559), the Basque Government (Dep. Education, Universities and Research, ref. IT687-13), and UPV/EHU (CLUMBER UFI11/20 and PES13/58). SM was awarded predoctoral fellowships from Fundación Jesús Gangoiti-Barrera. IP was awarded a predoctoral fellowship from Gobierno Vasco.
Appendix. Supplementary material
The following is the supplementary data to this article:
Distributions of high-density lipoprotein particle components in human follicular fluid and sera and their associations with embryo morphology parameters during IVF.
Age related changes in mitochondrial function and new approaches to study redox regulation in mammalian oocytes in response to age or maturation conditions.
The isolation and characterization of estradiol-fatty acid esters in human ovarian follicular fluid. Identification of an endogenous long-lived and potent family of estrogens.
Regulation of high density lipoprotein receptor messenger ribonucleic acid expression and cholesterol transport in theca-interstitial cells by insulin and human chorionic gonadotropin.
Low levels of serum paraoxonase activities are characteristic of metabolic syndrome and may influence the metabolic-syndrome-related risk of coronary artery disease.
Paraoxonase-2 is a ubiquitously expressed protein with antioxidant properties and is capable of preventing cell-mediated oxidative modification of low density lipoprotein.
Dr Ruiz-Larrea is Professor of the Department of Physiology in the Basque Country University. She works in the field of free radicals and oxidative stress in human pathophysiology, and leads a research group (FROS) in this area. One of her major interests involves free radicals in reproduction, particularly in infertility.
Key message
Intrafollicular paraoxonase (PON) activities are higher in large follicles compared with small ones in women undergoing ovarian stimulation, indicating that PONs activities may increase during follicle maturation. The activity of PON3 was higher in follicles from fertile women than from patients, thus suggesting the involvement of the enzyme in fertility.
Article info
Publication history
Published online: June 22, 2017
Accepted:
June 8,
2017
Received in revised form:
May 31,
2017
Received:
July 29,
2016
Declaration: The authors report no financial or commercial conflicts of interest.
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