Follicular fluid (FF) represents a very important microenvironment. It is a metabolically active system that plays a critical role in constituting a complex, regulated ovarian microenvironment. This environment, in addition to granulosa cells, cytokines and macrophages all produce reactive oxygen species (
Several studies have provided evidence that OS in antral follicle and in media culture cause deleterious effects in human oocytes and embryos
In the literature there are numerous studies that have considered life style to be a cause of OS. The prominent risk factors for increased lipid peroxidation are smoking (
In this prospective observational study enrolled women who underwent assisted reproduction treatment from July 2010 to April 2011 at Isfahan Fertility and Infertility Center. This study was approved by the Institutional Review Board and Ethics Committee of Tehran University of Medical Sciences.
Inclusion criteria were women 18-40 years of age who used their own oocytes (autologous) for fertility treatments. Male factors according to World Health Organization criteria (
On day three of a spontaneous menstrual cycle, we calculated BMI by dividing weight in kilograms by height in meters squared.
A validated semi-quantitative food frequency questionnaire (
PA was measured using the original International Physical Activity Questionnaire. This questionnaire assessed energy expenditure in total, by moderate and vigorous intensity (
The long protocol that involved a gonadotrophin-releasing hormone (GnRH) agonist and human menopausal gonadotrophin (hMG) administration was consistent. Follicular maturation was monitored by ultrasound examination. At oocyte retrieval, fluid from an average of one to 5 follicles that were 16-mm diameter or larger was pooled. Samples that appeared blood stained or without oocytes were discarded. The samples were immediately centrifuged at 300 rpm for 17 minutes, after which 2 ml of the supernatant was stored in microtubes at -70˚C for a maximum of 2 weeks until analyses for malondialdehyde (MDA) and total antioxidant capacity (TAC).
FF lipid peroxidation and antioxidant defense activity were measured as the levels of MDA and TAC. Follicular MDA was determined by the 2-thiobarbituric acid reactive substances (TBARS) method (
Statistical analysis was conducted using SPSS version 13.0 (SPSS, Chicago, IL, USA). The data were analyzed using multivariable liner regression analysis (adjusted for age, etiology of infertility, duration of infertility and number of used gonadotrophin and passive smoking). All variables were entered in the model and unstandardized regression coefficients (B) with 95% confidence interval (95% CI) were reported. The etiology of infertility and passive smoking were used as qualitative with a dummy code. Also, ANOVA was used for comparing age, BMI, PA, etiology of infertility, duration of infertility and number of used gonadotrophin between women whom FF biomarkers were assessed and those with FF were not available. P-values of <0.05 were considered significant.
Of the 240 eligible women (18-40 years of age) who participated, 4 individuals withdrew from the assisted reproduction program prior to study completion. FF biomarkers for 17 participants were not assessed because their follicles did not have oocytes or the FF sample volumes were insufficient. The age, BMI, PA, etiology of infertility, duration of infertility and number of used gonadotrophin did not differ between women whose FF biomarkers were assessed and those whose markers were unavailable (data not shown).
The baseline data and characteristics of 219 patients with the mean age of 31.54 years are presented in table 1.
Characteristics of subjects
|Variables||Mean (SD) or %|
SD; Standard deviation, PA; Physical activity, BMI;Body mass index, PCOS; Polycystic ovarian syndrome, MDA; Malondialdehyde and TAC; Total antioxidant capacity
Relation between energy expenditure related factors and oxidative stress (OS) in follicular fluid (FF)
|Model A: Outcome MDA R2=0.050|
|Unstandardized coefficient||Standardizedcoefficient||Sig||95% Confidence interval (95% CI)|
MDA; Malondialdehyde, TAC; Total antioxidant capacity, PCOS; Polycystic ovaries syndrome, PA; Physical activity, BMI; Body mass index, Sig; Significance, NS; Non-significant and SE; Standard error.
According to regression analysis when adjusted for age, etiology of infertility, duration of infertility and the number of used gonadotropin, there was no relation between MDA levels in FF and BMI (B=-0.06, CI=-0.01- 0.02), PA (B=0.01, CI=-0.18- 0.16) and total calorie intake (B=-0.36, CI=-1.49- 0.78). There was a positive relation between TAC level and BMI (B=73.30, CI=14.15- 47.31, p=0.001), but there was no relation to PA (B=1.31, CI=-212.46- 215.08) and total calorie intake (B=-126.05, CI=-20.54- 272.64).
Of the influential factors, we observed a positive relation between anovulation disorder (B=0.13, CI=0.02- 0.25, p=0.02) and MDA levels in FF. Other infertility etiologies (tubal factor, hypothalamic amenorrhea and unexplained factor) had a negative association (B=-0.14, CI=-0.25- 0.03, p=0.02) to MDA levels in FF.
The mean MDA level (F=1.20, p=0.31) and the TAC levels (F=0.29, p=0.84) did not differ in the four groups based on etiology of infertility. The MDA levels (B=-0.01, CI=-0.01- 0.02) and the TAC levels (B=-3.78, CI=-13.05- 5.48) in FF were not related to age.
This study presents the first clinical evidence based on human assisted reproduction for a relation between life style factors and OS in the follicular microenvironment, considering PA and calorie intake on assisted reproduction parameters as factors of energy expenditure.
Several lines of evidence suggest the importance of age in a decrease in the efficiency of the follicular antioxidant defence system (
Another finding of the present study revealed that independent to age, etiology of infertility, number of used gonadotrophin, PA and calorie intake, the TAC levels in FF was positively related to BMI. This was consistent with a previous finding. Previously it was explained that dead granulosa cells could theoretically contribute to the passive release of antioxidants into the FF and contribute to a negative outcome of
The increased TAC levels in FF in women with higher BMI might refer to antioxidant effect of estradiol (
Numerous studies have demonstrated that metabolic and hormonal changes, followed by obesity could induce systemic OS (
It was demonstrated that follicle-stimulating hormone stimulated from glutathione synthesis suppressed the production of reactive oxygen species and decreased the rate of apoptosis in cultured follicles (
In contrast to TAC, there was no relation between MDA level in FF and BMI. However, the increased TAC level in overweight women might protect the follicular environment from lipid peroxidation.
Another finding of the present study revealed that OS biomarkers were not related to total calorie intake and PA. Therefore the benefit effect of PA on reproduction (
The present study indicated that the MDA level in FF did not differ in women with different etiologies of infertility, but the MDA level in FF, independent of energy expenditure related factors, had a positive relation to anovulation.
This finding suggested that OS in follicular environment or OS-induced factors might contribute to anovulation and infertility. A negative relation between other etiologies with normal ovarian function (such as tubal factor and primary amenorrhea) and MDA level in FF might power this suggestion. Therefore, antioxidant supplements might benefit women with anovulation disorder.
We did not observe any relation between PCOS and endometriosis and OS in FF. Our observations were consistent with previous studies that reported OS in plasma and FF was not related to etiology of infertility (
In the present study women with systemic disorders such as hyperlipidemia and insulin resistance were excluded. The adverse effect of obesity on OS in FF might link with these disorders.
This study demonstrated that age and BMI independent of PA, calorie intake and etiology of infertility did not have important roles in inducing OS in the follicular environment. The duration of infertility was shown to have a stronger positive relation with decreased potent antioxidant defense in FF. Among etiologies of infertility, we have shown that anovulation was related to potent antioxidant defense in the follicular environment. Antioxidant supplements may be helpful for this group.