Oxidative stress is one of the possible causal mechanisms to increase the incidence of infertility, covering environmental influences, modern lifestyle, psychological factors, and potential diseases. So far, the role of oxidative stress in female reproduction is a little researched area. The level of mitochondria in the reproductive organs is very high in comparison with other organs in the human body, so it is understandable that oxidative stress is important in various physiological processes, including the maturation and fertilization of the egg. Oxidative stress disrupts the relationship between oxidants and antioxidants. The ratio may be altered due to increased free radical concentrations or decreased levels of antioxidant protection. Regardless of the origin, an excess of reactive free radicals in the body can cause a range of structural and functional damage in the cell itself and the development of various pathological conditions. Injuries can be also epigenetic or genetic. In the aging process, eggs in women become poorer quality and contain more damaged mitochondrial DNA. There are also several damaged cell membranes and acquired chromosomal aneuploidies. Similar changes may also occur in infertile women in association with oxidative stress. The this study we try to assess the status of oxidative stress and explain its role in relation to Antimüllerian hormone (AMH) and estradiol in follicular fluid, and also to assess the possible association of these markers with oocyte maturity and fertilization. We included 409 randomized patients who, for the first time between March 2013 and December 2014, entered the Biomedical Assisted Reproduction procedure and signed a consent form to participate in the study. According to the cause of infertility, they were divided into 4 groups: 36 patients with polycystic ovary syndrome (PCOS), 72 patients with endometriosis, 41 patients with tuboperitoneal infertility factor (TPF) and 48 healthy women with the cause of infertility in male partner, who were treated as control. A follicular fluid sample from the leading dominant follicle was used in each patient. Follicle concentrations of AMH, estradiol, and selected oxidative stress markers were measured: total antioxidant status (TAS), 8-isoprostane (8-IP), 8-hydroxy-2’-deoxyguanosine (8-OHdG), dROM, and PAT. AMH concentrations did not differ significantly between groups with different diagnoses. The median estradiol concentrations in follicular fluid were statistically significantly lower in the PCOS group (P = 0.0460) and also in the endometriosis group (P <0.0001) compared to the control group. No statistically significant differences in estradiol concentrations in follicular fluid were observed between the TPF and control group. No association was found between AMH concentrations and estradiol concentrations (Spearman's rho coefficient was 0.0499). In the case of oxidative stress markers, several differences were confirmed between the groups. The 8-IP concentration was statistically significantly higher in the healthy group compared to all three groups of patients (253.35 pg / mL compared to 85.97 pg / mL in PCOS, 91.07 pg / mL in endometriosis and 91.8 pg / mL at TPF; P≤0.0005). Such a result was not expected, as less damaged lipids would be expected in healthy patients. Measurements of 8-OHdG in the study did not differentiate between follicular fluids of patients with endometriosis and healthy ones. Significantly lower concentrations of 8-OHdG (6.82 ng / mL in PCOS and 14.81 ng / mL in healthy; P = 0.0001) were detected in the follicular fluid of patients with PCOS. TAS measurements in our study did not confirm elevated levels of antioxidant protection, as they were expected to be lower in follicular fluids in all patient groups compared to the control group. According to the reference limits of the dROM test, marked oxidative stress was detected in patients with endometriosis (422 UCarr) and PCOS (414 UCarr). Measurements of dROM in follicular fluid in patients with TPF showed moderate oxidative stress (375 UCarr), and the control group deviated significantly (302.6 UCarr; P <0.0001), with mostly negative values. In the PAT test, increased antioxidant activity was detected in all three groups of patients compared to the control group (2751.5 UCor in endometriosis, 2618 UCor in PCOS, 2954.7 UCor in TPF and 2365 UCor in controls; P≤0.0020; mean or median applied according to distribution). The ratio of dROM / PAT tests showed a significantly higher level of oxidative stress in patients with PCOS and endometriosis compared to the control group (P = 0.0076 in endometriosis and P = 0.0049 in PCOS). In the follicular fluid of patients with TPF, the difference in the ratio between the two tests was not statistically significant compared to the control group (P = 0.4933). The relationship between the concentration of a single marker in the follicular fluid in each group of patients and the maturity and fertilization of a cell from that follicle was verified by ROC analysis. However, the concentration of 8-OHdG is important stood out in the group of patients with PCOS (AUC = 0.886 for the prognosis of mature and AUC = 0.839 for the prognosis of egg fertilization). A lower concentration of 8-OHdG indicates a better outcome of the procedure. In the PCOS group, we also detected a stronger predictive value of the dROM / PAT ratio for both maturity and fertilization of oocytes. A lower value of the quotient indicates a higher probability that the cell will be mature or fertilized. The first hypothesis in which we predicted that the concentrations of biochemical markers of oxidative stress in the follicular fluids of patients with PCOS elevated were confirmed with the markers dROM, PAT and TAS and rejected with the markers 8-OHdG and 8-IP. AMH concentrations were not significantly higher in PCOS patients compared to the control group as predicted in the first hypothesis. In the first hypothesis, we also predicted a smaller number of mature and fertilized eggs in the group of women with PCOS. We did not confirm this, as we obtained the same number of mature and fertilized eggs as in the control group. The second hypothesis in which we predicted that the concentrations of biochemical markers of oxidative stress in the follicular fluids of patients with endometriosis elevated were confirmed with the markers dROM, PAT and TAS and rejected with the markers 8-OHdG and 8-IP. AMH concentrations were not significantly lower in patients with endometriosis compared with the control group as predicted in the second hypothesis. In the second hypothesis, we also predicted the same number of mature and fertilized eggs in the group of women with endometriosis and confirmed it. Our research was among the first to measure 8-IP, dROM, PAT in follicular fluid and in general the first to measure the concentrations of all markers used (in addition to 8-OHdG, AMH and estradiol) in a separate follicular fluid of the leading follicle and searched possible links with oocyte maturity and fertilization ability. We found an association between 8-OHdG concentration and dROM / PAT ratio in follicular fluid with oocyte maturity and their fertility ability in patients with PCOS. This means that the 8-OHdG marker and the dROM / PAT ratio in each follicular fluid could be used as a prognostic marker, indicating a greater chance that the egg will be successfully fertilized. A lower concentration of 8-OHdG, i.e. less DNA damage, indicates a better outcome of the procedure. A lower dROM / PAT ratio indicates a lower level of oxidative stress and thus a higher probability that the cell will mature or fertilize, as expected. The dROM / PAT ratio is a particularly interesting marker for such use, as it is determined on Point-of-care system. The analysis could be performed immediately after collection of the cells. The described results can be a good starting point for further research and development of markers for monitoring oxidative stress as predictors of fertilization in Biomedical Assisted Reproduction procedures.
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