Document Type : Original Article
Authors
1 Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz, Iran
2 Women’s Reproductive Health Research Center, Alzahra Hospital, Tabriz University of Medical Sciences, Tabriz, Iran
3 Department of Biochemistry and Clinical Laboratories, School of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
4 Students Research Committee, Infertility and Reproductive Health Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
5 5Liver and Gastrointestinal Disease Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
6 6Department of Biology, Payame Noor University, Tehran, Iran
7 7Umbilical Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
Abstract
Keywords
Free radicals cause oxidative damages to the
cell membrane lipid content (
PON3 is synthesized in the liver and carried in the
blood in association with HDL. PON3 is also able
to prevent the oxidation of low density lipoproteins
(LDL) (
In this cross-sectional study, we gained the agreement of the Ethical Committee of Tabriz University of Medical Science, and all patients gave written informed consent. Fifty infertile couples referred to Tabriz Alzahra Women’s Hospital, Tabriz, Iran, for infertility treatment using assist- ed reproductive technique (ART) were selected within a three-month period. Out of 50 couples, 30 women with MFI were used as the control group (MFI group), and the remaining 20 women with FFI were used as the test group (FFI group).
Among selected patients, 60% of infertile
partner were male and 40% were female. Lack
of infections and husband with no smoking habit were defined as including criteria. The long
protocol, gonadotropin-releasing hormone agonist (GnRHa) and human menopausal gonado-
tropin (HMG) were used for all subjects as the
treatment protocol for the stimulation of ovulation (
PON3 has a unique ability to metabolize the lipophilic agents such as lovastatin and simvastatin (
PON3 activity was determined through monitoring of the conversion of simvastatin (SV) to β,δ-
dihydroxyacid simvastatin (SVA) using reverse
phase-high performance liquid chromatography
(RP-HPLC) method as described before (
MDA levels in FF were determined by the
thiobarbituric acid (TBA) method and expressed
as nmol MDA formed/mL FF (
Total antioxidant status (TAS) was measured in
FF using a commercial kit (Randox Laboratories,
France). The assay was performed by incubation of 2,2'-azino-di-(
HDL-C level was measured after extraction of the particles from FF by means of phosphotungstic solution and centrifugation. The amount of cholesterol in the supernatant was determined spectrophotometrically by cholesterol oxidase (Pars Azmon, Iran).
All data were expressed as mean ± SD. The normality test showed that all data were normal or nearly normally distributed. Statistical comparisons were performed using t test. ANOVA was used to evaluate the differences between the means of more than two groups. A value of p<0.05 was considered statistically significant. Statistical analysis was carried out by Statistical Package for the Social Sciences (SPSS; version 16, SPSS Inc., Chicago, USA).
The general information of the population study is shown in table 1. In the test group, 68% of the patients had normal ovaries, 18% had polycystic ovaries and 14% had abnormal ovaries (small ovaries, ovaries which were operated upon, and no eggs were produced), 4% of the test subjects had abnormal uteruses (small uterus and endometriosis) and 30% suffered from abnormal menstrual cycles.
General information of the studied women
Factors | Number (% ) |
---|---|
30 (60%) | |
20 (40%) | |
Ovary | |
Normal | 34 (68%) |
PCOS | 9 (18%) |
Abnormal | 7 (14%) |
Uterus | |
Normal | 48 (96%) |
Abnormal | 2 (4%) |
Menstrual cycle | |
Normal | 35 (70%) |
Abnormal | 15 (30%) |
PCOS; Polycystic ovary syndrome.
PON3 activity in the FF of the MFI group
was found to be significantly (p<0.001) higher than that in the women with FFI (4.7 ± 0.8
vs. 3.8 ± 0.7 µmol/min/ml). In contrast, the
concentration of MDA, a lipid peroxidation
indicator, in the FF of MFI group was 3.1 ±
1.4 nmol/ml compared to the value of 4.2 ±
1.7 nmol/ml measured in FF of women with
FFI (p=0.024). Therefore, the ratio of antioxidant to peroxidation, which was evaluated as
PON3/MDA value in the control group, was
also higher than the corresponding value in
the women with FFI. No statistically significant difference was found in the HDL-C level
in the FF of both groups (
Biochemical factors measured in the FF based on the number of oocytes are displayed in table 3. No significant difference was observed in the studied factors with respect to the number of oocytes.
Follicular fluid biochemical parameters in the studied population
Chromosomal polymorphic variations | MFI(Mean ± SD) | FFI(Mean ± SD) | P value |
---|---|---|---|
4.7 ± 0.8* | 3.8 ± 0.7 | <0.001 | |
21.6 ± 11.4 | 20.0 ± 6.1 | 0.556 | |
1.6 ± 0.3 | 1.6 ± 0.2 | 0.465 | |
3.1 ± 1.4 | 4.2 ± 1.7 | 0.024 | |
1.8 ± 0.9 | 1.0 ± 0.6 | 0.002 | |
0.4 ± 0.5 | 0.2 ± 0.1 | 0.097 | |
7.1 ± 4.0 | 6.5 ± 4.7 | 0.633 | |
3.0 ± 0.7 | 2.5 ± 0.5 | 0.013 | |
MFI; Male factor infertility, FFI; Female factor infertility and TAS; Total antioxidant status.
Follicular fluid biochemical parameters according to the oocyte numbers in studied women
Factors | Number of the oocytes (number of subject) | P value | ||
---|---|---|---|---|
1-5 (n=12) | 5-10 (n=24) | >10 (n=14) | ||
4.1 ± 1.2 | 4.3 ± 0.9 | 4.5 ± 0.8 | 0.550 | |
18.4 ± 3.6 | 20.0 ± 10.2 | 23.5 ± 10.2 | 0.379 | |
1.5 ± 0.9 | 1.6 ± 0.2 | 1.7 ± 0.2 | 0.515 | |
3.1 ± 0.9 | 3.8 ± 1.9 | 3.5 ± 1.6 | 0.566 | |
1.4 ± 0.4 | 1.5 ± 1.0 | 1.6 ± 0.9 | 0.840 | |
0.2 ± 0.1 | 0.2 ± 0.1 | 0.4 ± 0.6 | 0.356 | |
7.2 ± 3.7 | 7.5 ± 5.2 | 6.4 ± 3.8 | 0.681 | |
2.6 ± 0.9 | 2.7 ± 0.6 | 2.8 ± 0.8 | 0.791 | |
TAS; Total antioxidant status. Values are expressed as mean ± SD.
Comparison of number of the oocytes (t=1.32, p=0.13), EFS (t=0.81, p=0.41), ECN (t=0.89, p=0.32) and FR (t=1.52, p=0.11) between women with MFI and FFI by t test showed no significant difference between IVF and ICSI techniques. The relations between FF biochemical parameters with EFS, ECN and FR were assessed using regression analysis and the results have been summarized in table 4. A significant negative correlation was found between PON3 activity (r=-0.65, p=0.02) and PON3/MDA (r=-0.63, p=0.001) with EFS, whereas there was a positive correlation between EFS and MDA (r=0.55, p=0.002) which indicates that an increase in antioxidant/peroxidation value is accompanied with an increase in the embryo quality. No significant correlation was found between ECN and FF biochemical parameters. A negative correlation between FR and MDA (r=- 0.42, p=0.02), while a positive relations between FR and PON3 activity (r=0.56, p=0.004), HDL- C (r=0.35, p=0.041) and PON3/MDA (r=0.59, p=0.001) could be indicative of the beneficial effects of antioxidant in the success of ART. The same pattern was also observed when correlations were examined separately for women with MFI and FFI.
Correlation of follicular fluid biochemical factors with embryo quality and fertilization rate
EFS | ECN | FR | ||
---|---|---|---|---|
-0.65 (0.02) | 0.24 (0.140) | 0.56 (0.004) | ||
-0.25 (0.310) | 0.07 (0.821) | 0.35 (0.041) | ||
-0.16 (0.425) | 0.18 (0.283) | 0.14 (0.404) | ||
0.55 (0.002) | -0.38 (0.029) | -0.42 (0.020) | ||
-0.63 (0.001) | 0.22 (0.215) | 0.59 (0.001) | ||
-0.17(0.406) | 0.15 (0.314) | 0.22 (0.214) | ||
-0.20 (0.374) | 0.09 (0.703) | 0.25 (0.246) | ||
EFS; Emberyo fragmentation score, ECN; Emberyo cell number, FR; Fertilization rate and TAS; Total antioxidant status. The numbers in brackets are p values.
It has been shown that the concentration of lipidic hydroperoxides and active substances of
thiobarbituric acid in the FF is lower than serum
in women who underwent IVF. This confirms the
presence of a suitable antioxidant in oocyte’s environment prior to ovulation (
PON3 is synthesized in the liver, attached to
HDL and carried by the fluids in the body (
In our study, no significant difference was observed in the concentration of TAS in the two
groups. In similar studies, no significant difference
was found in the TAS levels in the women with
FFI who suffered from endometriosis when compared with the TAS concentration of the MFI (
The results obtained in the present study show that the PON3/MDA ratio in the women with FFI is significantly lower than the corresponding value in the women with MFI (p=0.002). It could be stated that the ratio of antioxidant to peroxidation in the FF is a suitable factor for assessing the oxidative stress in the follicles.
PON3 is a strong antioxidant in FF. Closshey
et al. (
On the other hands, we were not able to find any
significant difference between PON3 activity and
the number of oocytes. Plachot et al. (
A significant negative relation between PON3 and PON3/MDA with EFS, and a positive relation between these parameters with FR may indicate that PON3 plays an important role in fertilization and the quality of embryo. A high level of PON3 in FF could be indicative of its specific role in development and maturation of good oocyte which, in turn, can lead to a healthy embryo.
The role of PON3 in fertility has not received
enough attention. Browne et al. (
Our findings confirm that PON3, as an antioxidant potential in follicular fluid, has a major role in regulating fertility and maintaining embryonic growth. Thus, PON3 could be a valuable therapeutic target to improve the success rate of ART.