Introduction
Polycystic ovarian syndrome (PCOS) is a complicated endocrinopathy occurring in 6% to 8% of women [
]. Furthermore, it is also related with harmful alteration of other markers of risk and insulin resistance [
,
]. Women with PCOS are known to have an increased risk of preterm birth, gestational diabetes, and pre-eclampsia [
,
]. Increased oxidative stress is related with atherosclerosis, diabetes, metabolic syndrome, and obesity. The first report documenting increased oxidative status in women with PCOS was presented a decade ago [
]. Complementary to the increase of oxidative stress, women with PCOS also have reduced total antioxidant status [
].
In vitro fertilization and embryo transfer (IVF-ET) is a widely accepted assisted reproduction technique (ART), and this technology is a chance for infertile women with PCOS to get pregnant. However, the average successful pregnancy rate of IVF-ET is approximately 40% to 50% [
,
]. In the many possible reasons for IVF-ET failure, the antioxidant system was a significant factor [
]. In the female reproductive system, antioxidant activity and oxidative stress perform physiological roles during oocyte maturation and may also be associated with conditions that limit the success of ART [
].
Follicular fluid, a product of blood–plasma component that crosses the blood–follicular barrier, is altered by the theca cells and the granulosa cells (GCs). The constituent changes in the follicular fluid proved that the selective filtering capacity of the GCs could modify the serum metabolites. In the follicular fluid, many metabolites play an important role in oocyte quality, embryo development, embryo quality, and viability [
]. Several studies have focused on the micro-environment surrounding the oocyte and on radical scavenging capacity of the follicular fluid [
]. An imbalanced antioxidant activity in follicular fluid might have a hostile effect on the above processes [
]. Adequate antioxidant activity of follicular fluid during oocyte development is related with embryo quality and clinical pregnancy [
,
]. In addition, follicular fluid has its antioxidant system, including glutathione peroxidase, catalase, and superoxide dismutase [
,
] and vitamins [
,
]. The activity of free radicals in follicular fluid was also related with ovarian responsiveness and IVF success rate [
]. However, studies analyzing the relationship between the free radical scavenging capacity and embryo quality of infertile women with PCOS were lacking. In this study, we tested the free radical scavenging capacity of infertile women with PCOS, with the aim of determining the relationship between free radical scavenging window of women with PCOS and their embryo quality.
Materials and methods
Chemicals
a,a-Diphenyl-b-picrylhydrazyl (DPPH) (Sigma-Aldrich, USA) and 2,2-azinobis (3-ethylbenzthiazoline-6-sulfonic) acid (ABTS) (Fluka, USA) were used in this study.
Study protocol and sample collection
This is a non-interventional study of patients undergoing routine practice. Ethics approval of this study was obtained from the ethics committee of Tongji Hospital. All enrolled women who signed written informed consent underwent routine IVF-ET process at the Reproductive Medicine Center, Tongji Hospital. The reason for infertility of all women was PCOS, and the duration of infertility was more than 1 year. Patients underwent controlled ovarian stimulation with the use of a GnRH agonist long protocol [
,
]. A total of 402 follicular fluid samples from 41 infertile patients undergoing IVF were collected for the present study. The collection process of a follicular fluid sample was reported in published reference [
]. In addition, samples from follicles containing two or three or no oocytes were excluded from this study.
Embryo culture and grading
The process of semen preparation, insemination, embryo culture, and transfer was previously shown [
,
]. Briefly, sperm was collected in sterile containers after 3 to 6 days of sexual abstinence. Keeping it for 30 min and then subjecting it to liquefaction, the researchers analyzed the semen samples for motility, morphology, and concentration. During the IVF cycles, every oocyte was inseminated with 10 000 motile spermatozoa, 4 h after retrieval. Routinely, the fertilized oocytes were transferred into G1 culture medium (Vitrolife, Sweden) for 3 days. According to the morphological score, embryos were classified after 48 and 72 h based on the blastomeres, symmetry, multinucleation, and fragmentation. Embryos were evaluated on a range of I (high score) to IV (low score) [
].
Determination of DPPH radical scavenging capacity
The DPPH assay was reported previously [
24], with a minor difference in this study. Follicular fluid sample was mixed with 3.6 ml 0.2 mmol/L DPPH solution in methanol. Then, they were centrifuged at 1000×
g for 10 min. Finally, the absorbance of samples was measured at 517 nm. DPPH radical scavenging capacity was calculated as the following percentage: [(
ADPPH −
As)/
ADPPH] × 100 (
ADPPH = result of DPPH alone and
As = result of DPPH in the presence of different follicular fluid samples).
Determination of ABTS radical scavenging capacity
The ABTS radical scavenging capacity of follicular fluid was evaluated by reference [
]. The ABTS radical solution was produced by the overnight reaction of 7 mmol/L ABTS and 2.45 mmol/L potassium persulfate. Then, the ABTS radical solution was diluted with ethanol to acquire an absorbance of 0.700 at 734 nm. Follicular fluid samples (0.3 ml) were reacted with 2.7 ml ABTS solution, and the absorbance of this solution at 734 nm was determined. The level of radical scavenging was obtained using the equation described in DPPH assay.
Determination of superoxide radical scavenging capacity
A pyrogallol auto-oxidation method was used to test the capacity of follicular fluid to scavenge superoxide radicals [
]. Briefly, a 0.5 ml follicular fluid sample in 4.50 ml Tris–HCl buffer was mixed with pyrogallic acid. The absorbance of the mixed solution at 325 nm was determined. The
Kb value was processed from the
A325 nm and time.
Determination of reactive oxygen species
The reactive oxygen species (ROS) levels of follicular fluid were tested by the published method [
]. A 400 ml follicular fluid sample was added into the cuvette with 10 ml luminol. Each mixture was determined for 10 min. The counted photons per second represented the ROS levels.
Statistical analysis
All data analyses were processed using Statistical Package for Social Sciences version 13.0. All experimental results were reported as mean±SD. The control chart (3 sigma) was processed by a moving range. The nonparametric Mann–Whitney U test and c2 test were analyzed between groups. Significant difference was determined at P<0.05.
Results
The results of DPPH radical assay are shown in Fig. 1A. The follicular fluid from grades I and II embryos was significantly higher than follicular fluid from grades III and IV embryos (P<0.05). In the ABTS radical scavenging assay, results from follicular fluid (Fig. 1B) showed no statistical difference between grades I and II embryos and grades III and IV embryos (P>0.05). In addition, the results of ROS level are shown in Fig. 1C. The ROS level in follicular fluid of grades I and II embryos was significantly lower than that of grades III and IV embryos (P<0.05). The results of the correlation between superoxide radical scavenging capacity in follicular fluid of women with PCOS and their embryo quality are shown in Table 1. Similar results with ABTS scavenging capacity were found in the superoxide radical scavenging results. The Kb value of grades I and II embryos was similar to grades III and IV embryos (P>0.05).
Furthermore, the lower control limit of DPPH radical scavenging capacity and upper control limit of ROS level in follicular fluid of women with PCOS was determined by using a control chart. As shown in Fig. 2A and 2B, the lower control limit of DPPH radical scavenging capacity and upper control limit of ROS level were 13.2% and 109.0 cps, respectively. These findings mean that follicular fluid of women with PCOS may adversely affect embryo development when the DPPH radical scavenging ability was below 13.2% or when the ROS level was above 109.0 cps. These calculated lower and upper control limits were further confirmed in the follicular fluid of embryos of all grades (Table 2). In DPPH radical scavenging assay, the grades I and II embryo proportions were significantly higher (74.4%) in PCOS patients with DPPH radical scavenging capacity>13.2% compared with those of<13.2% (P<0.01). A similar result was shown in ROS test. In the PCOS patients with ROS level<109 cps, the percentage of grades I and II embryo formations was 74.6%, which was significantly higher than that of>109 cps (P<0.001).
Discussion
In this study, we evaluated the free radical scavenging capacity of infertile women with PCOS and the relationship between the free radical scavenging capacity and their embryo quality. Embryo grading refers to the established criteria [
]. Embryo Grade I: even, spherical, regular, and equal size blastomeres,<5% fragments. Embryo grade II: uneven or irregular, unequal size blastomeres,<10% fragments. Embryo grade III: defects of cytoplasm,<50% fragments. Embryo grade IV:>50% fragmentation with gross. We found that the free radical scavenging capacity of follicular fluid was statistically related to embryo quality in infertile women with PCOS.
Antioxidants in follicular fluid might have a valuable effect on the oocyte maturation, development, and quality. Furthermore, several studies focus on the antioxidant status of follicular fluid in recent years [
]. A recent result of insulin resistance and hyperglycemia showed that they have adverse effect on oxidative status by increasing the level of superoxide dismutase (SOD) [
,
]. Another research determined the effect of
N-acetyl-cysteine (NAC). The results showed that glutathione (GSH) has a positive effect on insulin resistance in infertile women with PCOS [
].
Furthermore, the imbalance between free radical and the antioxidants could lead to tissue damage in biological systems. These free radicals come from oxidative stress and could reduce GSH, SOD, catalase enzyme, and glutathione peroxidase. Many results showed that the oxidative markers mentioned above were cell damage indicators, which were determined over the normal range in women with PCOS [
,
]. To prove this finding, analysis of scavenging free radical was performed. In our study, we detected the free radical scavenging activity of follicular fluid from women with PCOS undergoing IVF. We found that DPPH radical scavenging capacity of follicular fluid was positively correlated with embryo quality. When the DPPH solution reacted with antioxidants, bleaching of the purple color in the solution occurred. The decreasing absorbance levels indicated increasing free radical scavenging capacity of follicular fluid [
]. Another research also showed that decreased antioxidant activity was related with low embryo quality [
]. The results of our ROS level determination studies showed that ROS level of follicular fluid was negatively correlated with embryo quality. A significant relationship between increased ROS levels and embryo fragments was reported. It means that increased ROS level in follicular fluid was harmful to embryo development [
]. In addition, our results showed that the follicular fluid represented a wide range in activity of scavenging DPPH radical (11.03% to 49.60%) and ROS level (21 cps to 120 cps). These results also found that stronger antioxidant capacity in follicular fluid correlated with better embryo grades, which was similar to previous reports. Similar results were also obtained in animal experiments. Some reports found that antioxidants may promote the
in vitrodevelopment of embryos from pigs and cows [
]. Then, we used the control chart to describe the free radical scavenging windows, beyond which embryo development is unfavorable.
In conclusion, the result indicated that going beyond the lower limit of activity of scavenging DPPH radical and the upper limit of ROS level may lead to a negative effect on embryo quality (Fig. 2). These cut-off values of free radical scavenging of follicular fluid from women with PCOS could assist embryologists in choosing the development of embryo from PCOS patients undergoing IVF. However, the antioxidant status of follicular fluid needs to be better researched in the future. In addition, more research is needed to better understand the mechanism of oxidative stress in PCOS patients and its effects on embryo development.
Higher Education Press and Springer-Verlag Berlin Heidelberg
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