1 Department of Obstetrics and Gynaecology, and 4 Department of Primary Care and Population Sciences, Royal Free and University College Medical School, Royal Free Campus, London, 2 London Female and Male Fertility Centre, London and 3 Department of Biological Sciences, Brunel University, Uxbridge, UK.
5 To whom correspondence should be addressed at Department of Obstetrics and Gynaecology, Royal Free and University College Medical School, Royal Free Campus, Rowland Hill Street, London NW3 2PF, UK. e-mail: hardiman{at}rfc.ucl.ac.uk
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Abstract |
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Key words: antioxidants/follicular fluid/free radicals/reproduction
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Introduction |
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In men, subfertility has been ascribed to excess production of ROS which overwhelms the available tissue antioxidants. On this basis, vitamins C, E and selenium are often used to treat male factor subfertility, but non-enzymatic antioxidant levels do not appear to be reduced in subfertile men, and the effectiveness of these treatments remains controversial (Tarin et al., 1994; Kessopoulou et al., 1995
; Suleiman et al., 1996
). At the same time, there are reports of some beneficial roles of ROS on sperm function. Aitken et al. (1989
) reported increased spermzona interaction following induction of limited peroxidation in human sperm, while Bize et al. (1991
) found that increased generation of hydrogen peroxide by the addition of glucose oxidase to a hamster sperm preparation accelerated the onset of the acrosome reaction and capacitation.
The role of ROS and antioxidants in relation to female reproductive function has, in contrast, received relatively little attention, although there is some evidence of both physiological and pathological effects (Guerin et al., 2001). Ho et al. (1998
) and Matzuk et al. (1998
) reported decreased litter size and number of litters per month in female homozygous mutant mice lacking superoxide dismutase (SOD). In the human, Paszkowski found decreased levels of SeGPx in follicular fluid of women with unexplained infertility (Paszkowski et al. (1995; Paszowski and Clarke, 1996
). In the same study, SeGPx levels were higher in follicles yielding oocytes that subsequently were successfully fertilized compared with those from follicles whose oocytes failed to fertilize. Yang et al. (1998
) found higher levels of hydrogen peroxide in fragmented compared with non-fragmented embryos and unfertilized oocytes, whilst Paszkowski and Clarke (1996
) reported increased antioxidant consumption (suggesting increased ROS activity) during incubation of poor quality embryos. On the other hand, Attaran et al. (2000
) reported a beneficial role for ROS, with higher levels in follicular fluid in IVF conception cycles compared with non-conception cycles. The authors did not, however, find any association between total antioxidant capacity (TAC) and conception. Not surprisingly, in view of this uncertainty, the therapeutic role of exogenous antioxidants in the treatment of male and female subfertility remains controversial (Kessopoulou et al., 1995
). The present study was designed to assess the stability and role of non-enzymatic antioxidants in human follicular fluid in relation to female reproduction.
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Materials and methods |
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Only neat follicular fluid with insignificant contamination with blood or culture medium was used in the study. Immediately after removal of the oocyte, the fluid was centrifuged at 600 g for 5 min to remove cellular components and the supernatant kept at 80°C for no longer than 1 week until analysed.
Approval for this study was obtained from the local research ethics committee of Enfield and Haringey Health Authority.
Methods
The total (peroxyl) radical-trapping antioxidant parameter (TRAP; Wayner et al., 1985) is widely used to study antioxidants in biological fluids. This method is based on the length of time that the biological fluid in question is able to resist artificially induced peroxidation using an oxygen electrode to detect the end of the lag period. We used a derivative of this method, the trolox equivalent antioxidant capacity (TEAC) assay (Rice-Evans and Miller, 1994
) that uses the more readily available spectrophotometric detection. The ferric reducing/antioxidant power (FRAP; Benzie and Strain, 1996
, 1999) assay has also been described for measuring antioxidants but has never been used for follicular fluid. We chose to use the FRAP assay for our study because it is sensitive, rapid and easier to perform.
The TEAC assay
A 50 µl aliquot of follicular fluid samples was added to a pre-mix containing 750 µl of phosphate-buffered saline (PBS), 100 µl of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS; Aldrich) (5 mmol/l) and 100 µl of methaemoglobin (Sigma) (50 µmol/l) in a semi-micro plastic cuvette. To this, 50 µl of 3 mmol/l hydrogen peroxide was added and the absorbance at 723 nm (A723) was followed at 25°C in a recording spectrophotometer (Perkin Elmer 5, UK). The length of time of the lag phase (before the oxidation of ABTS began) was taken as a measure of the TEAC. The reaction was calibrated using aliquots of trolox (Aldrich: prepared directly as 2.5 mmol/l in PBS).
The FRAP assay
The FRAP assay uses antioxidants as reductants in a redox-linked colorimetric method with absorbance measured with a spectrophotometer (U-1500, Toshiba, UK). A 300 mmol/l acetate buffer of pH 3.6 (3.1 g of sodium acetate·3H2O + 16 ml of glacial acetic acid made up to 1 l with distilled H2O), 10 mmol/l 2,4,6-tri-(2-pyridyl)-1,3,5-triazine, 98% (Sigma-Aldrich) (3.1 mg/ml in 40 mmol/l HCl) and 20 mmol/l FeCl3·6H2O (5.4 mg/ml in distilled H2O) were mixed together in the ratio of 10:1:1, respectively, to give the working FRAP reagent. A 50 µl aliquot of follicular fluid was added to 1 ml of FRAP reagent in a semi-micro plastic cuvette. Absorbance measurement was taken at 593 nm (A593) exactly 10 min after mixing using 50 µl of water as the reference. To standardize, 50 µl of the standard (FeSO4·7H2O, 1 mmol/l) was added to 1 ml of FRAP reagent. All measurements were taken at room temperature with samples protected from direct sunlight.
Each sample of fluid was assayed twice. The second assay was done 72 h after the initial assay, keeping the sample in the dark at room temperature in between assays. This gives a measure of the relative stability of the antioxidants in the fluid samples. Both baseline mean TAC and the percentage loss of TAC after 72 h were examined as markers of oxygen radical activity and correlated with the presence or absence of an oocyte in the follicle, fertilization and the status of the embryo on the day of embryo transfer.
Bradford assay for follicular fluid protein
The protein content of fluid samples was routinely estimated by the Bradford method (Bradford, 1976). The Bradford reagent used contained Coomassie brilliant blue G250 [0.01% (w/v)], ethanol (0.8 mol/l) and phosphoric acid (1.7 mol/l) in H2O. Bovine serum albumin was used as a standard. The assay was performed by adding 50 µl of sample containing 0400 µg/ml of protein to 950 µl of the Bradford reagent and measuring the A595 after 10 min at room temperature. Follicular fluid samples were diluted x250 into PBS prior to assay.
Statistical analysis
Data were analysed using analysis of variance (ANOVA) and Student t-test as appropriate. All analyses were performed with SPSS (SPSS Inc., Chicago). Statistical significance was defined as P < 0.05.
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Results |
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Validation of the FRAP assay: sample variance. FRAP assays were carried out in triplicate on 10 randomly selected fluid samples. The mean coefficient of variation (CV) for replicates was 4.0% (sampling error). The CV for the 10 sample means was 10.5%. ANOVA showed that the differences between sample means are unlikely to derive from sampling errors (F = 14.88: P < 0.0001).
Conversion of FRAP to TAC. The FRAP assay was standardized using Fe2+; TAC was taken as being equal to the equivalent Fe2+ concentration. It should be noted that this would correspond to twice the concentration of either ascorbate or urate in the follicular fluid since both are able to reduce 2 mol of ferric iron per mol of antioxidant. A593 versus Fe2+ was linear over the absorbance range 00.8 with a slope of 19 500/mol/l/cm.
Protein assay: validation and comparison with the FRAP assay. Protein content of follicular fluid lies within a fairly narrow range of 3.8 ± 0.4 mg% (Shapira et al., 1996). For a random selection of fluid samples, the average total protein content from this study was 3.6 ± 0.35 g/100 ml.
Both FRAP and protein assays (Bradford method) were carried out on 10 randomly selected samples of fluid. Each sample was measured in triplicate for both assays. ANOVA on the protein assay (F = 1.01; P = 0.46) indicated that differences between samples could be due to random sampling errors (CV = 11.2%; larger than that for the FRAP replicates: 4.0%). Of more importance, there was no correlation between FRAP and protein estimates of the same samples (slope = 0.029; R2 = 0.0001).
Follicle analysis
A total of 303 follicular aspirates collected from 63 women during egg retrieval for IVF were examined for TAC using the FRAP assay. Fluid from different follicles was individually analysed and though the oocytes were pooled at the time of fertilization, each was marked so that resulting embryos could be clearly identified. A total of 218 (71.9%) of 303 follicles yielded oocytes, of which 169 (77.5%) fertilized; 134 (79.3%) of the embryos survived till the day of transfer.
Baseline TAC (Table I). The mean TAC of fluid from follicles containing oocytes was not different from that of empty follicles. The mean TAC in fluid from follicles that yielded oocytes that were successfully fertilized was significantly greater than the mean TAC from follicular fluid associated with oocytes that were not. Conversely, the mean TAC of fluid from follicles whose oocyte gave rise to an embryo that survived till time of transfer was significantly lower than the mean TAC in follicular fluid associated with oocytes that gave rise to non-viable embryos. The mean CV for the intraindividual interfollicular TAC levels was 17.5%.
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Percentage TAC loss (two-point assay) (Table II). The mean TAC loss in the samples after 72 h did not differ significantly between follicular fluid from follicles that contained an oocyte and from those that did not. Also, there was no significant difference in the mean TAC loss in the follicular fluid irrespective of whether the oocyte was successfully fertilized or not and whether the resulting embryo remained viable or not.
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Discussion |
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Our studies demonstrated an excellent correlation between FRAP and TEAC when applied to human follicular fluid to determine TAC. That the FRAP assay is as sensitive and reproducible as TEAC is important because the former is much easier and faster to perform. Moreover, the absence of correlation between the results of the FRAP and Bradford assays precludes the possibility that the observed variance in FRAP activity resulted from decreased low molecular weight antioxidant levels as a consequence of accidental dilution of follicular fluid with flush buffer at the time of oocyte retrieval. Otherwise both protein and FRAP values would be similarly decreased.
We found no evidence of any relationship between follicular antioxidant activity and the presence of oocytes in follicular fluid, both with baseline TAC and antioxidant consumption (two-point assay). The factors responsible for non-recovery of oocytes from follicles are not clear but there is some evidence that this may be related to oocyte immaturity, possibly secondary to low HCG levels, an underlying ovulatory disorder or premature oocyte atresia (Awonuga et al., 1998; Esposito and Patrizio, 2000
). It is therefore of interest that levels of glutathione, which is thought to be the main non-enzymatic defence system against the effects of oxygen radicals on oocytes and embryos (Takahashi et al., 1993
; Gardiner et al., 1998
), are higher in mature compared with immature hamster oocytes (Gwatkin and Haidri, 1974
; Perreault et al., 1988
).
We did find that lower levels of TAC predict decreased fertilization potential. As discussed previously, this is in accordance with the finding by Paszkowski et al. (1995) of lower levels of SeGPx in association with failed fertilization in the human. This, however, conflicts with the results of a study by Sabatini et al. (1999
) in which higher levels of SOD activity were present in fluid from follicles whose oocytes did not fertilize compared with follicular fluid whose oocytes did fertilize. This discrepancy may reflect methodological differences; we measured TAC, whilst Sabatini studied a single antioxidant enzyme. We also found that lower levels of TAC were associated with increased viability of the embryo up to the time of transfer. These results also conflict with the findings of Paszkowski and Clarke (1996
) and Yang et al, (1998
), who found that increased ROS activity was associated with impaired embryo development. The reasons for this discrepancy are also not clear. It appears that ROS may have different effects at different stages of embryo development.
Antioxidant consumption over 72 h, though not a true measure of free radical flux under physiological conditions, does provide an indicator of the potential of follicular fluid to counteract the effects of endogenously generated ROS. Differences between samples will, in the main part, be due to pro-oxidant potential of the fluid itself. The results of our study suggest that antioxidant consumption in follicular fluid had no predictive value with respect to successful fertilization and embryo viability up until embryo transfer.
Since all our subjects had at least two embryos transferred, it was not possible to ascribe a pregnancy to a particular follicle or embryo, hence our outcome measure was embryo viability rather than pregnancy. We are therefore unable to corroborate the results of a previous study in which ROS activity and TAC levels were measured using chemiluminescence in women undergoing IVF. The results demonstrated that women who became pregnant had higher levels of ROS than those who did not (Attaran et al., 2000), leading the authors to suggest that ROS, at low concentrations, may be a potential marker for predicting success of IVF patients. On the other hand, Ho et al. (1998
) and Matzuk et al. (1998
), in genetic manipulation studies in mice, found that mice that were made deficient in SOD had both decreased litter size and decreased number of litters per month.
In summary, the role of ROS in relation to human reproduction remains unclear. This confusion results from the differing materials (follicular fluid, embryos and culture medium), assay methods (TAC, antioxidants and antioxidant enzymes) and end points (presence of an oocyte, fertilization, embryo viability and pregnancy) used. It would seem that the role of ROS and oxygen radicals prior to ovulation is different from that in relation to fertilization or embryo viability. Further studies to clarify their physiological and pathological roles and their relationship to female reproduction should be undertaken, as they could lead to the development of novel strategies for fertility regulation in the human.
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Acknowledgements |
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Submitted on August 6, 2002; resubmitted on April 24, 2003; accepted on July 14, 2003.