Association of M55L and Q192R polymorphisms of paraoxonase 1 gene (PON1) with recurrent pregnancy loss risk: A case–control study

Abstract Background Recurrent pregnancy loss (RPL) refers to the incidence of two or more abortions before the first half of pregnancy. Oxidative stress has been hypothesized to play a central role in RPL. Objective To investigate the relationship between Q192R and L55M polymorphisms of PON1 as antioxidant enzyme and the risk of RPL. Materials and Methods In this case–control study, 110 women with RPL (case) and 110 healthy fertile women (control) referred to the Research and Clinical Center for Infertility, Shiraz, Iran were enrolled. Genomic DNA was extracted from the peripheral blood in all participants. Polymorphisms were genotyped by polymerase chain reaction-restriction fragment length polymorphism method. Results Statistical analysis of Q192R polymorphism showed a significant difference for the RR genotype between the case and control group (OR = 11, CI = 1.39–86.87, p = 0.005) but none for the QR and QQ genotypes. No significant association was observed between the R and Q allelic frequency in the RPL participants compared to the control group (p = 0.53). Also, statistical analysis of the L55M polymorphism for MM genotype in the case group compared with the control group showed a significant difference (OR = 3.59, CI = 0.97–13.30, p = 0.042), but none for the LM and LL genotypes. Conclusion The findings showed a significant correlation between the Q192R polymorphisms and the L55M PON1 enzyme and RPL in this study population.


Introduction
Human reproduction is low enough so that 30% of fertilizers do not cause fetal development and abortion (1,2). Recurrent pregnancy loss (RPL) refers to the loss of the fetus before the 20 th day of pregnancy, which might be repeated twice or more, and are divided based on the time of abortion, abnormal preclinical abortions, and clinical abortions. In general, about 5% of couples struggle with this problem (2). It can hardly be described as a failure factor in reproduction because these problems may range from genetic causes to physiological events that occur either in the parent or in the fetus, and each of them has a cellular or particular molecular mechanisms. Therefore, genetic, environmental, immunological, hormone, infectious, and anatomical coagulation disorders are effective in its occurrence (3).
Oxidative stress and reduction of body antioxidant capacity are among the studies that might play an important role in pregnancyrelated disorders such as RPL (4)(5)(6). Biomarkers of oxidative stress that cause membrane degradation, such as lipid peroxidation products, increase before an abortion, which might lead to fatal injury and degeneration of syncytiotrophoblast in early pregnancy (7,8). Many studies have shown a significant reduction in the activity of antioxidants such as catalase (CAT) and glutathione peroxidase (GPX), and the level of selenium, as well as increased levels of malondialdehyde and lipid peroxidase in serum and embryonic tissue of RPL participants (8)(9)(10). The researchers found that the levels of antioxidants in healthy pregnant women are lower than nonpregnant women, while they are much less in women with RPL than nonpregnant women. Also, reactive oxygen species was higher in pregnant and RPL women than nonpregnant women (11).
The human body has a large number of free radical inhibition systems, including PON1, GPX, CAT and Superoxide dismutase. PON1 is linked to high-density lipoprotein and is responsible for detoxification of organophosphoric compounds and neutralization of free radicals soluble in lipid peroxidation lipids (12). The gene family of paraoxonase in mammals contains three genes of PON1, PON2, and PON3 (13). Antioxidant activity has been reported in the product of all three genes, but the most significant antioxidant role is related to PON1 (14). The two major polymorphisms in the PON1 gene-encoding region are Q192R (rs662) (replacing glutamine instead of arginine) and L55M (rs854560) (replacing leucine instead of methionine) (15).
PON1 is a protein with a molecular weight of 45-43 kDa and contains 345 amino acids (14). The main and important action of PON1 enzyme is the neutralization of free radicals and the suppression of oxidative stresses (16,17). This enzyme together with other enzymes and antioxidants in the body creates a strong protective barrier against oxidizing substances.
According to previous studies, infertility disorders such as RPL are affected by oxidative stress, and since the PON1 is known as an antioxidant, it could play a central role in inhibiting RPL (4)(5)(6).
For the first time, this study examines the relationship between Q192R and M55L polymorphisms and the risk of women's RPL.

Participants
This investigation was carried out as a casecontrol study on two different groups of women When enrolling in the experiment, tests were taken using 5-ml peripheral blood, after which the storage of the samples was performed in a tube covered with anticoagulant EDTA.

Genetic analysis
For genotype analysis, genomic DNA was extracted from the whole blood sample using a salting-out method described by Miller (18).
The Q192R polymorphism was amplified using: Electrophoresis in 3% Agarose gels was used to separate the fragments after digestion, and then staining with green viewer was applied for detection.
As shown in Figure 1, three different electrophoretic patterns were obtained RFLP analysis. Sanger method was followed to purify and sequence-relative PCR fragments for every pattern using forward and reverse primers with the use of an automated sequencer (ABI3500; Applied Biosystems). A gel visualization system (Syngene GBOX Gel Documentation 680X) was used to observe the fragments.  The study points were explained to the participants prior to the study, and informed consent was obtained from all.

Statistical analysis
SPSS16 (Statistical Package for the Social Sciences, SPSS, Inc., Chicago, IL, USA) was used to analyze data statistically. Chi-square test was applied to calculate goodness of fit for the genotypes determined by observations and estimations as well as allele frequencies.
The calculation of odds ratio was performed at confidence intervals of 95%. P-values of < 0.05 indicated the significance of the differences. Evaluation of SNPs was carried out regarding deviations from Hardy-Weinberg Equilibrium with the use of Chi-square tests.

Results
In the present study, we evaluated two types of polymorphisms in the PON1 genes in women with a history of at least two or three miscarriages compared with women with no history of spontaneous abortion (Table I). Table  II shows the genotype distribution and allelic frequency of PON1 gene polymorphisms in case and healthy women. The genotype distributions of all polymorphisms in cases and controls were in Hardy-Weinberg equilibrium.

Discussion
The phenomenon of RPL is a difficult and stressful problem for both doctors and couples. Generally, abortion is defined as the loss of pregnancy before the 20 th wk, and RPL as two or more abortions before the 20 th wk (4). In 50% of the RPL cases, no specific etiologic factor is found and as such they are categorized as idiopathic, while the remaining 50% include anatomical, immunological, genetic, endocrine, thrombophilic, oxidative stress, and environmental factors. According to previous studies, one of the predisposing factors in infertility disorders, in particular, the RPL is the presence of alleles and genes in the people which leads to clinical manifestations in the participants. One of these effects is a different including in RPLs (6,11,20). Therefore, to prevent this, several antioxidant defense barriers function in the body. One of these defensive factors is the PON1 enzyme, which is linked to highdensity lipoprotein and prevents LDL oxidation Mashayekhi and colleagues, in their studies of the effects of Q and R polymorphism on infertility in Iranian women, observed that there is a significant difference between Q and R genotypes between control and infertile women. They observed that the risk of infertility is potentially reduced in populations with QR genotype (30). Data from these studies showed that QR polymorphism is associated with a reduction in the risk of infertility. The results of this study confirmed our results. The only study related to our work, conducted by Ozturk and colleagues, found no significant association between the enzymatic polymorphisms of PON1 and RPL. It should be noted that this study was a preliminary study in which the sample size was small. The results of this study were not consistent with the results of our study (33).
The results obtained in this study might have been influenced by the race of the subjects studied and the geographical distribution and the difference in sample selection criteria that should be considered. The study of other PON1 polymorphisms is recommended in subsequent studies because it has been shown that paraoxonase gene polymorphisms weaken or exacerbate each other's effects (34).

International Journal of Reproductive BioMedicine
Alizadeh et al.

Conclusion
The findings of this study, which was conducted in Iran for the first time, showed that there is a clear correlation between the Q192R and L55M PON1 enzyme polymorphisms and RPL. In the near future, with the possibility of determining the genetic map and identifying the polymorphisms of each person, the frequency and susceptibility of each person to the diseases can be understood and treated for each person specifically. Hence the polymorphism studies help us for novel treatment concepts of RPL.
In general, statistical studies should be repeated in different populations. Therefore, the present study cannot conclusively prove the relationship between Q192R and L55M polymorphism with RPL until it is repeated and confirmed by others.
In the present study, PON1 polymorphisms were investigated, and it is recommended that the serum levels and activity be investigated in subsequent studies to be more conclusive.