Introduction
Infertility is a multifactorial disorder and life style has an important role in its occurrence. In WHO conducted Demographic Health Surveys from 1990 to 2010, responses from women were evaluated and it revealed one in every four couples in developing countries were affected by infertility. It also reported that the overall burden of infertility, in women from 190 countries was similar and that increasing trend was seen in that decade.1 This clearly shows that the burden remains high. According to Crosignani and Esteves, the evaluation of unexplained infertility in 30-50% of Couples was based on the following simple criteria:
Normal ovulatory function
Normal semen analysis for men
At least one patent fallopian tube2
Studies have shown that Assisted Reproductive techniques such as intrauterine insemination (IUI), In-vitro fertilization (IVF) are not always successful. It is worthwhile to investigate the factors which affect the success of this procedure.3 Ovary is responsible for production of reproductive hormones and oocytes. The oxidative stress in reproductive organs may play a vital role in preventing conception. K. H. Al-Gubory et al. reported that levels of reactive oxygen species and antioxidants influences the female reproductive function in different phases of menstrual cycle and the physiological process of pregnancy.4 Wang et al. (1997) and Polak et al. (2001) reported that higher levels of reactive oxygen species were found in peritoneal fluid of women with idiopathic infertility than fertile control. It was suggested that peritoneal fluid which enters the Fallopian tubes may cause damage to sperm, and creates oxidative stress (Storey, 1997). Fridovich in his study has shown antioxidant enzymes play critical roles in clearing the toxic products produced by superoxide dismutase.5
Role of vitamin E in fertility
Vitamin E the fat-soluble vitamin is found in the ovary especially in follicular fluid. Many studies have shown its role as an antioxidant within the body.6 In cells and organelles vitamin E is the first line of defence against lipid peroxidation. Rigotti, A has shown role of vitamin E in RBC flexibility and longevity in immune function, and its positive effects on fertility.7 Savita, et al. have shown that increased OS is associated with decrease in antioxidants and fertility.8 Vitamin E directly neutralizes superoxide anion, hydrogen peroxide, and hydroxyl radical. It is called a chain breaking antioxidant because of its ability to terminate a free radical chain reaction. Ruder, E.H. et al have shown that Vitamin E increases the number of embryos developing into the expanded blastocysts and increases the viability of embryos exposed to heat shock.9
Bayer, R. (1960) suggested that in a human trial, infertile couples given vitamin E have shown a significant increase in fertility.10 Plasma vitamin E levels were found to be higher in fertile women than in infertile women.11 Vitamin E supplementation in older mice partially prevented reduction in ovulation12 Vitamin E has a significant role in reproduction and sufficient levels give a better effectiveness in the treatment of unexplained infertile women.
Antioxidant status in females with unexplained infertility
Oxidative stress is due to the imbalance between pro-oxidant molecules and protective antioxidants. OS affects the entire reproductive capacity of a woman in many ways (i.e., oocyte maturation, ovulation, implantation, formation of blastocyst, luteolysis and luteal maintenance in pregnancy).13 Reactive oxygen species act, both as key signalling molecules in pregnancy and at higher levels cause adverse effect on female reproductive tract. The antioxidants scavenge free radicals, thus protecting the cell structures.14 Excess ROS in the follicular fluid overpowers the antioxidant defence capacity and damages the oocytes. This will affect the fertilization process. Peritoneal cavity microenvironment when flooded with ROS may allow fertilization but OS-induced apoptosis can cause implantation failure.15 Elevated ROS levels hinder the endometrial function, which is needed for the growth of the embryo16 and interferes in luteal regression and hormonal support which is responsible for the continuation of a pregnancy.17
MDA
There are studies showing increased marker of lipid peroxidation (MDA) in women with unexplained infertility when compared to primary infertile woman and male infertility. Chao HT et al and Tsuboi H etal showed that the oxidative damage could be due to factors like repeated drug induced ovarian stimulation or psychological stress.18 This necessitates further evaluation of factors responsible for oxidative stress in infertility workup protocol. Majid KH etal clearly correlate primary female infertility with significantly high levels of MDA and decreased levels of antioxidants.19
Antioxidant enzymes
Gonadotropin accelerate the up regulation of antioxidants such as catalase in the follicles and protects oocytes from ROS during steroidogenesis.20 Strong positive correlation was observed between SOD activity and intrafollicular oestradiol levels, which affects oocyte quality. Studies concluded that the ROS scavenging ability of antioxidant enzymes is related to fertilization outcomes21 GST is involved in the follicle maturation process by detoxifying harmful substances and enhancing the normal development of the oocyte.22 This study aims to establish the association of Oxidative stress, MDA and antioxidant defence in unexplained infertility.
Material and Methods
Control group
Pregnant women with normal ovulation who attend the antenatal clinic in Obstetrics unit. Samples were collected from them at first-trimester of their pregnancy. 70 of these participants who had an uneventful pregnancy were included as control group.
Inclusion criteria
Women with normal ovulation, aged between 28 and 38 years who have conceived within 12 months of contraceptive free intercourse, and who have an uneventful pregnancy.
Cases
70 married Women with unexplained infertility were diagnosed in the Reproductive Medicine Unit at CMC, Vellore when they came for infertility treatment procedure.
Inclusion criteria
Sample size
In order to detect this difference as statistically significant with an alpha error of 5% and the power of 90%, we included 70 unexplained infertile women and 70 controls who were normal.
Study approved by IRB and consent was obtained from all participants.
Statistical analysis
Data was analysed using SPSS Software. Continuous parameters were analysed using Mann Whitney U test. P value of < 0.05 was considered significant.
Sample collection and preservation
Blood samples (5 ml) were collected in serum tubes (red clotted tube) and centrifuged at 3000 rpm for 10 min. Serum were separated and stored in 0.5ml micro tubes at -20∘C until analysis.
Calibration
Vitamin E Standard stock solution was prepared by dissolving 10mg of alpha-tocopherol in 1ml ethanol working standards 12.5, 25, 50 and100 ug/ml were prepared from stock 10mg/ml tocopherol acetate internal standard stock solution was prepared in ethanol.
Steps in sample preparation
100 µl of serum with 100 µl internal standard were mixed for 15 sec in a vortex mixer.
Tube kept in ice for 5 min. 1 ml of hexane added and vortexed for exactly 1 min.
Centrifuged at 25°C for 5 min at 1,500 rpm.
Upper clear hexane layer was separated and evaporated under nitrogen gas.
100μl of methanol was added and vortexed for 15 seconds.
The aliquot was transferred to a HPLC glass vial and loaded for further analysis.
The Figure 1, Figure 2, Figure 3, Figure 4 represents the peak of different concentration of vitamin E standards.
Antioxidant enzymes
Catalase – measured by Mahmoud H. Hadwan principle: 2H2O2 Catalase → 2H2O + O2Catalase activity was measured by incubating the sample in 1.0 ml substrate at 37°C for 3 minutes. Ammonium molybdate was added to stop the reaction. Absorbance of the yellow complex of molybdate and hydrogen peroxide is measured at 374 nm against the blank.
GST – measured by Boyland, E. and Chasseaud using CDNB as a substrate principleGlutathione –SH + → Glutathione –S-CDNBThe reaction is assessed by monitoring the conjugation of 1-chloro, 2, 4-dinitrobenzene (CDNB) with reduced glutathione (GSH). This is done by an increase in absorbance at 340nm.
SOD – measured by Markland’s method measured by Markland’s methodPrinciple: Superoxide dismutase reacts with pyrogallol and inhibits its autooxidation.The rate of autooxidation of pyrogallol calculated from the absorbance of orange colour compound at 420nm.
Lipid peroxidation marker (MDA)- measured by Thiobarbituric acid method
Results
A total of 140 women, (70 normal and 70 women with unexplained infertility) in the reproductive age group of 28-38 were included in this study. The mean ages of the participants were 26.70 ± 4.40 and 30.39 ± 4.36 respectively for control and cases. (Table 1)
The median of MDA level was found to be significantly higher in cases than in control (p < 0.005) (Table 3).
This is clearly represented in boxplot (Figure 7)
The antioxidant enzyme activities were significantly lower in case group compared to the control subjects (Table 3).
SOD showed significant decrease in median value in study group compared to control group. p=<0.001.
Catalase activity in study group decreased compared to control group p= <0.001.
GST in serum levels showed significant decrease in median compared to control group p =< 0.001.
Unexplained infertile group showed lower levels of vitamin E in serum compared to control group.
This difference were statistically significant. This is clearly represented in boxplot (Figure 7).
Serum vitamin E analysis by HPLC method showed good recovery.(Table 2)
Table 1
Baseline characteristics of study subjects
Table 2
Descriptive statistics for antioxidant enzyme, MDA and Vitamin E
Table 3
Descriptive statistics for antioxidant enzyme, MDA and Vitamin E
Discussion
Unexplained infertility is a major problem with a significant public health concern. ROS and reactive nitrogen species (RNS) act as signal molecules in physiological and pathological process in female reproductive tract.23 In this study the mean serum levels of MDA were significantly higher in the unexplained infertile group (Table 3), a finding similar to the study by Majid KH et al.24 The higher MDA level in unexplained infertile group shows that there is considerable oxidative damage in this group of women decreasing the possibility of conception. This finding also implies the high levels of ROS has used up the antioxidant enzymes in scavenging them. Studies have shown ROS over production in ovulatory follicles may affect the oocytes. Higher MDA concentration has been noted in human endometrium with negative impact on menstruation process.25 Pregnant women were more susceptible to oxidative stress compared to non-pregnant women with increased ROS and decreased antioxidants.26 So, evaluating the oxidative marker in the unexplained group would be helpful for their treatment.
Adequate levels of antioxidants are important for oocyte quality, maturation, fertilization, and implantation. They have a vital role in reducing oxidative stress, a process known to impair conception and its sustenance.27 Various studies on Antioxidant enzymes in serum levels confirmed decreased levels of catalase, SOD and GST in association with infertility.28 The results of this study clearly support this finding. Infertile couples were suggested antioxidants in their diet and also adopt a healthy life style.29 Very few studies on oxidative stress markers like catalase, SOD, GST and MDA were studied collectively in Indian women with unexplained infertile woman. Findings from this study emphasizes the non-invasive protocol including oxidative stress markers.
Vitamin E (α-tocopherol) the lipid-soluble antioxidant is considered a “fertility factor”. It is said to be a direct free radical scavenger by enhancing the antioxidant enzymes and protects the cell membranes from lipid peroxidation. In this present study, unexplained infertility group have lower concentration of serum vitamin E levels as compared with control group (p= <0.001). This study points out the importance of vitamin E and its effect on female fertility. Many studies showed low Vitamin E levels in infertile women with reduced total antioxidant status. According to Naseer et al Vitamin E levels in sera and cervical secretions of infertile women with unexplained infertility show a significant decrease when compared with fertile controls.30
Conclusion
The study clearly shows an imbalance in the antioxidant status with increased MDA level, decreased vitamin E and antioxidant enzyme level in the study group as compared to control group. Vitamin E levels can be measured using this HPLC method and can be titrated to optimal levels. The antioxidant enzymes if included in the fertility workup and found deficient, can be given orally in the treatment protocol.
Limitation
Limitations of our study is the small number of subjects taken. A larger group might have been helpful in drawing better correlation between the various parameters. Other OS markers could have been included.
Interventional study with antioxidant supplementation and evaluation of OS markers before and after the treatment can be the future study. Measurement of these markers in follicular fluid can also be undertaken.
