Abstract
The aim of the study was to evaluate the levels of lipid and protein peroxidation markers, in the follicular fluids (FF) of 82 patients undergoing in vitro fertilization (IVF). This included, thiobarbituric acid-reactive substances (TBARS), protein carbonyl, and thiol groups. The oxidative stress markers were compared between the pregnancy positive and pregnancy negative patient groups. The two patient groups were compared in terms of their age, body mass index (BMI), cause of infertility, and the plasma hormone levels (AMH, E2, peak E2). Protein carbonyl and thiol groups were estimated using an ELISA assay and with Ellman's reagent (5, 5'-dithiobis-2-nitrobenzoic acid, DTNB), respectively. The mean FF TBARS level of 29 pregnant women was 0.954 ± 0.420 µmol/l, whereas it was twice as high (1.961 ± 0.796 µmol/l) in a group of 53 non-pregnant patients (p < 0.0001). In non-pregnant patients, we observed 2-fold elevated levels of protein carbonyl groups when compared to pregnant women (2.969 ± 0.723 vs. 1.523 ± 0.254; p < 0.0001). The mean age of women and BMI were significantly higher in the pregnancy negative vs. pregnancy positive group. There were no significant differences in protein thiols and in the levels of the hormones tested between patient groups. Our results demonstrate that elevated FF lipid and protein peroxidation level may have a negative impact on IVF outcome. The findings support the idea that increased level of oxidative stress markers in follicular fluid may play an important role in fertility.
Introduction
Reactive oxygen species (ROS) are produced as a part of cellular metabolism and are involved both in health and disease during our entire lifespan. The major types of ROS include superoxide anion (O2•-), hydrogen peroxide (H2O2), and hydroxyl radical (OH•). Antioxidant enzymes and small molecules e.g., glutathione, vitamins E, C, and A and thioredoxin 2 maintain the redox status of cells. In a healthy tissue, ROS (pro-oxidants) and antioxidants that serve to scavenge ROS remain in balance. Oxidative stress (OS) refers to the disruption of this balance and the overproduction of ROS. In vitro fertilization (IVF) is one of the most common assisted reproduction techniques. This method is a widely accepted infertility treatment and often remains the only chance of having a baby for infertile couples. Unfortunately, the success of this technique, measured as an average pregnancy rate per cycle, is only 30-40% [Das et al. Citation2006; Gerris et al. Citation1999]. Among the many reasons for the IVF failure, oxidative stress seems to be an important factor [Sikka Citation2004]. However, there is limited data on the possible effects of ROS on the female reproductive system [Agarwal et al. Citation2003; Oyawoye et al. Citation2003; Pasqualotto et al. Citation2004].
Free radicals and other oxidants may cause oxidation of lipids, proteins, and DNA, thereby increasing the likelihood of cardiovascular, neurodegenerative, inflammatory, and many other diseases [Łuszczewski et al. Citation2007]. Toxic products of radical reactions exert cytotoxic effects, cause cell membrane damage, and activate pathways of cell death. Therefore oxygen metabolites can alter cell function and even affect cell survival [Oral et al. Citation2006].
ROS serve as key signal molecules in a variety of physiological processes in a woman's body, from oocyte maturation to fertilization, pregnancy, and embryo development [Agarwal et al. Citation2005]. ROS play a role in the physiology of ovarian function. The expression of various oxidative stress biomarkers has been detected in normally functioning human ovaries [Suzuki et al. Citation1999]. There is evidence that ROS are involved in follicle maturation, folliculogenesis, corpus luteus function, and ovulation [Jozwik et al. Citation1999; Sabatini et al. Citation1999]. Moreover, stronger oxidative enzyme activity has been shown in cells involved in steroidogenesis such as theca cells, granulosa lutein cells, and hilus cells [Scully and Cohen Citation1964].
Numerous human studies have shown that increased ROS generation may be involved in birth defects and other situations such as abortions [Agarwal et al. Citation2005; Moreira da Silva et al. Citation2010]. Moreover, there is also some evidence for the role of ROS in the pathophysiology of infertility and assisted fertility [Agarwal and Allamaneni Citation2004; Gupta et al. Citation2009] but the existing data are conflicting and the effect of OS on IVF is not clear [Jozwik et al. Citation1999; Fujimoto et al. Citation2011].
Follicular fluid (FF) creates the microenvironment for the developing oocyte and has a direct impact on oocyte quality, implantation, and early embryo development. An imbalance in ROS production in ovarian follicular fluid may have an adverse effect on the above processes. Increased ROS activity in FF may be toxic to embryo formation whereas a physiologic ROS level may be indicative of healthy developing oocytes [Jana et al. Citation2010]. Therefore, the aim of this study was to measure the levels of lipid and protein peroxidation in the follicular fluid of women after IVF and to assess their impact on outcome of pregnancy success. The concentrations of thiobarbituric acid-reactive substances (TBARS) as well as carbonyl (CO) and thiol groups were evaluated as the most general and well-used biomarkers of severe oxidative lipid and protein damage in a variety of human diseases [Shacter Citation2000; Levine Citation2002; Dalle-Donnel et al. 2003; Cherubinia et al. Citation2005; Lykkesfeldt Citation2007]. Measurement of these parameters could have clinical relevance. Assessment of the oxidative stress rate may be helpful in evaluating in vitro fertilization.
Results
Twenty-nine pregnancies were recorded among the 82 women enrolled in the study. The two groups of patients, pregnancy positive (group I) and pregnancy negative (group II), were compared in terms of the levels of three oxidative stress markers measured in FF: TBARS, protein carbonyl, and thiol groups. The results are shown in .
Table 1. Oxidative stress markers in follicular fluid from women after IVF.
The mean TBARS concentration in the FF of 29 pregnant women was 0.954 ± 0.420 µmol/l. It was twice as high (1.961 ± 0.796 µmol/l) in non-pregnant patients (p < 0.0001). Moreover, in the FF of pregnancy negative patients a two-fold elevated concentration of protein CO groups relative to group I (2.969 ± 0.723 vs. 1.523 ± 0.254; p < 0.0001) was observed. There were no statistically significant differences between patient groups in terms of their protein thiol concentrations (0.317 ± 0.118 in group II vs. 0.343 ± 0.096 in group I; p = 0.1152). Furthermore, the two patient groups were compared in terms of their age, body mass index (BMI), and concentration of plasma hormones including anti-Müllerian hormone (AMH), estradiol (E2) as well as peak estradiol levels (peak E2). The mean age of women and BMI were significantly higher in the pregnancy negative compared to the pregnancy positive group (34.0 ± 4.35 vs. 27.2 ± 4.18 y; p < 0.05 and 27.3 ± 2.26 vs. 22.7 ± 3.52; p < 0.05, respectively; .). There were no significant differences in terms of the levels of all tested hormones between patient groups. The levels of hormones measured in the laboratory of Fertility Center Gameta in Rzgow are shown in .
Table 2. Values of BMI and mean age of women undergoing IVF.
Table 3. Level of hormones estimated in plasma of women undergoing IVF.
Discussion
In the present study we analyzed the levels of lipid peroxidation and protein oxidation products (expressed as TBARS, protein carbonyl, and thiol group concentrations) in the FF of women after IVF. We aimed to evaluate the impact of these oxidative stress markers on the outcome of pregnancy success.
Follicular fluid constitutes a valuable study material for assessment of the developmental competence of female gametes. It is rich in low-molecular metabolites that are direct or indirect regulators of OS and antioxidant protection [Tamura et al. Citation2008]. Growing evidence indicates that increased ROS activity in FF may be toxic to embryo formation and may affect both embryo cleavage rate and the degree of embryo fragmentation [Yang et al. Citation1998; Attaran et al. Citation2000]. Thus, FF creates a metabolically active microenvironment in which the oocytes live and contains, among others, steroid hormones, growth factors, cytokines, granulosa cells, macrophages, and leukocytes [Agarwal and Allamaneni Citation2004].
The concentrations of TBARS and carbonyl groups were significantly higher in the FF of women who did not become pregnant after IVF relative to those who became pregnant, reflecting enhanced lipid and protein peroxidation. A non-significant tendency towards protein thiol oxidation has also been observed. In contrast, Jozwik et al. [1999] did not find any relationship between lipid peroxidation levels (as measured by the concentrations of conjugated dienes, lipid peroxides, and TBARS) and the pregnancy rate in women after IVF. This discrepancy may be explained by the fact that different methods were used to measure the concentration of TBARS and/or some unknown differences related to infertility that might have existed in the patient groups enrolled in the study. Also, conjugated dienes and lipid peroxides are unstable and their concentrations may change during longer storage. TBARS and carbonyl groups are more stable end products and more reliable markers of lipid and protein peroxidation. Moreover, no significant correlations between lipid peroxidation derivatives (13-hydroxy octadecatrienoic acid and 13-hydroperoxy octadecadienoic acid) or antioxidant enzyme activities (SOD, GPX, GR, GST) and embryo quality were observed in a recent study using a 1 follicle-1 oocyte/embryo approach [Fujimoto et al. Citation2011]. These results suggest that concentrations of most lipid peroxidation products and antioxidant enzyme activities within FF are not associated with the quality of embryos. The results of single FF with regard to the oocyte quality and subsequent fertilization and embryo developmental potential may be more important than total FF. Similarly, no correlation was seen among lipid peroxidation or total antioxidant capacity (TAC) levels and oocyte maturity, fertilization, cleavage, and embryo quality [Pasqualotto et al. Citation2004]. Pregnant patients were found to be significantly younger than those who did not become pregnant. After adjusting for age, a positive correlation was seen between pregnancy rate and lipid peroxidation and between pregnancy rate and TAC. These authors concluded that lipid peroxidation may be a good marker of metabolic activity within the follicle, and a certain threshold amount may be necessary to establish a pregnancy.
In comparison, Oyawaye et al. [2003] investigated ROS in FF in IVF cases and reported significantly lower levels of OS markers in successfully fertilized and transferred oocytes. Similarly, in the study reported in this communication we established a reverse relationship between levels of OS markers in FF and pregnancy success, i.e., the concentration of ROS in FF may be important both for fertilization and implantation of eggs. Therefore, our results are in agreement with previous findings of other research groups [Oyawoye et al. Citation2003; Gupta et al. Citation2009] that ROS plays an important role in the female reproductive system. Saikat et al. [2010] suggested that significantly increased ROS production, high lipid peroxidation, and decreased total antioxidant capacity correlate with poor oocyte and embryo quality. It has been reported that active metabolism and steroid production are the major causes of high ROS generation in female reproductive tissues [Gupta et al. Citation2009]. Oxidative stress seems to be an important causative factor in the etiology of many diseases including polycystic ovary syndrome, endometriosis, and tubal, peritoneal, or unexplained infertility. It is therefore necessary to conduct further research to determine the optimal levels of ROS in FF that allows successful IVF to be carried out.
Analysis of data obtained from the Fertility Center Gameta showed that both mean age and BMI of women who failed to become pregnant following IVF were higher compared to the pregnancy positive group. This may confirm that increased OS with age and obesity are important factors in the failure to become pregnant after IVF.
In summary, our results demonstrate that elevated levels of FF lipid and protein peroxidation may have a negative impact on IVF outcome. The findings support the idea that the increased level of oxidative stress markers in FF may play an important role in fertilization. The effects of ROS on female reproduction are still not clear and need further investigation. Our results are in agreement with the findings of several research groups who have observed that increased ROS generation is associated with poor oocyte quality and a low fertilization rate. We believe that the results of this study might help to identify potential causes of poor pregnancy rate associated with IVF. One possible approach to consider would be to increase levels of antioxidants in FF to control ROS generation. Such a reduction in ROS generation by exogenous antioxidants might result in the formation of good quality embryos and improvement of fertilization rate during IVF.
Materials and Methods
Study participants and ovarian stimulation protocols
A total of 82 women, aged 24–39 (mean age 31.5 y) attending an IVF treatment session in the Fertility Center Gameta in Rzgow, Poland, were studied. This study was approved by the local Ethics Committee (no RNN/57/06/KE) and all women gave their informed consent prior to being included in the study. Patients were given a short questionnaire to obtain information about race, age, smoking, alcohol consumption, physical exercise, and diagnosed diseases. None of the women had a history of thrombosis, hypertension, diabetes, or any metabolic disorder. They did not take any cholesterol-lowering drugs, antioxidant-vitamin supplements, or any medication that might affect the results of the present study. These patients were divided into 2 groups: group I (pregnancy positive) which included 29 women (mean age 27.2 y) who became pregnant after embryo transfer, and group II (pregnancy negative) that included 53 patients (mean age 34.4 y) who failed to become pregnant after the transfer. All patients were new to the IVF procedure. Blood levels of AMH, E2, and E2 peak were measured. AMH testing is one of the markers for the ovarian reserve. The level of AMH decreases with age. Peak E2 determines a concentration of estradiol after hormonal stimulation and expresses maturity of oocytes. The level of E2 indicates whether a patient should be still stimulated during the IVF procedure. In the treatment groups the following causes of infertility were identified: endometriosis (31%), PCOS (12%), male factor (10%), tubal factor (9%), iatrogenic causes (6%), and idiopathic infertility (32%).
Patients underwent IVF and embryo transfer with a standardized ovarian-stimulation protocol as reported previously [Taketani et al. Citation2007; Revelli et al. Citation2009]. A short protocol of stimulation was applied. Injections of the gonadotrophin-releasing hormone agonist (GnRHa) triptorelin acetate (Decapeptyl, Ferring, Germany) at a dose of 0.1 mg starting on Day 1 was followed by the administration of gonadotrophins, follicle stimulating hormone (FSH, Gonal F; Serono, Italy) in individual doses for every patient (equivalent to 150-300 IU FSH) starting on Day 3 of a cycle. Ovarian stimulation was monitored using the serum estradiol assay together with ultrasound measurements of follicle numbers and diameters. The induction of ovulation with 5000 IU of human chorionic gonadotrophin (HCG, Pregnyl; Organon, Holland) was performed when the leading follicles reached 18–20 mm in diameter and serum estradiol concentration per follicle was 150–200 ng/l.
Collection and management of oocytes
Oocytes were retrieved through vaginal access under ultrasound guidance, 34–36 h after HCG administration. Oocytes were separated and placed into media, whereas FF were collected. Only uncontaminated FF minimally stained with blood were retained for further determinations.
The oocytes were cultured in a modular incubator (Heraeus Intruments; Hanau, Germany) in an atmosphere of 5% CO2. The cells were examined for the presence of two pronuclei 19–20 h after insemination. Embryo transfer was carried out 3–5 d after insemination. Prior to the transfer, embryos were microscopically evaluated for morphology. IVF outcome was assessed by calculating the fertilization rate and by biochemical pregnancy test. Fertilization rate was calculated as the number of zygotes obtained divided by the number of oocytes retrieved. On the 12–14th day of the transfer the biochemical pregnancy test (blood β-HCG level) was performed to confirm pregnancy.
Follicular fluid collection and processing
Follicular fluid was carefully aspirated from the oocyte of each patient in 3.2% sodium citrate and centrifuged at 4,000 x g for 10 min to remove cellular components. Afterwards the supernatant was frozen at -32°C and kept for later analysis.
Lipid peroxidation measurement
Lipid peroxidation was assessed in FF by estimating the concentration of TBARS [Rice-Evans et al. Citation1991]. Briefly, equal volumes of the sample, 15% (m/v) trichloroacetic acid containing 0.25 M HCl and 0.375% (m/v) TBA (thiobarbituric acid) containing 0.25 M HCl were mixed, incubated at 95oC for 10 min and cooled. The sample was then centrifuged at 3500 × g for 20 min and absorbance readings were taken on the spectrophotometer at 535 nm. The concentration of TBARS was calculated using the molar extinction coefficient (ϵ = 156,000 M-1cm-1). All experiments were performed twice and results were expressed as μmol/l.
Protein carbonyl measurement
Carbonyl groups of proteins in FF were estimated by a sensitive ELISA method with anti-DNP antibody [Alamdari et al. Citation2005]. Protein samples diluted in PBS were adsorbed to the wells of an ELISA plate and then reacted with dinitrophenylhydrazine (DNPH). The protein-conjugated DNPH was probed by a commercial anti-DNP antibody, and then a second antibody conjugated with HRP was added for quantification. The method was calibrated using oxidized albumin and required only 1 µg protein per well.
Determination of thiol group levels
Protein thiol group concentrations in FF were measured using Ellman's reagent (5,5'-dithiobis-2-nitrobenzoic acid; DTNB) [Ellman Citation1959]. The samples were mixed with an equal volume of 10% SDS and DTNB solution. After incubation (1h, 37°C) absorbance was recorded at 412 nm. The concentration of –SH groups was calculated by using the molar extinction coefficient ϵ= 13,600 M-1 cm-1 and the results were expressed as μmol/l.
Statistical analysis
All data in this study were expressed as means ± SD. For checking normality of sample distribution the data were analyzed with a Shapiro-Wilk test using StatSoft Inc. Statistica v. 8.0 for Windows. When the distribution was not normal the non-parametric Mann-Whitney U test was used. Differences were considered to be statistically significant at p < 0.05.
Abbreviations
| FF: | = | follicular fluids |
| IVF: | = | in vitro fertilization |
| TBARS: | = | thiobarbituric acid-reactive substances |
| BMI: | = | body mass index |
| DTNB: | = | 5, 5'-dithiobis-2-nitrobenzoic acid |
| ROS: | = | reactive oxygen species |
| O2•-: | = | superoxide anion |
| H2O2: | = | hydrogen peroxide |
| OH•: | = | hydroxyl radical |
| OS: | = | oxidative stress |
| CO: | = | carbonyl |
| AMH: | = | anti-Müllerian hormone |
| E2: | = | estradiol (E2) |
| peak E2: | = | peak estradiol levels |
| TAC: | = | total antioxidant capacity |
| HCG: | = | human chorionic gonadotrophin |
| DNPH: | = | dinitrophenylhydrazine. |
Acknowledgments
The authors thank the Proper Medical Writing (infrared group) for the language assistance in the preparation of this paper.
Declaration of interest: The authors report no conflicts of interest.
Author contribution Conceived and designed the experiments: Marta Borowiecka, Ireneusz Polac, Halina M. Zbikowska Performed the experiments: Marta Borowiecka, Joanna Wojsiat Analyzed the data: Halina M. Zbikowska, Marta Borowicka , Ireneusz Polac, Joanna Wojsiat Contributed reagents/materials/analysis tools: Ireneusz Polac, Michal Radwan, Pawel Radwan Wrote the manuscript: Marta Borowiecka, Joanna Wojsiat, Halina M. Zbikowska.
Related Research Data
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