1. Introduction
Preeclampsia is a complex disorder with a prevalence of 5%-8% in pregnant women [1].The disease is associated with hypertension, proteinuria, edema, clotting disorders in arteries, and dysfunction of endothelial cells [1]. These disorders mostly occur after the first week of pregnancy, although in the second half of pregnancy, during and after delivery it may also be observed. The etiology of this disease is unknown. Some studies have reported the role of genetic factors in the incidence of preeclampsia, especially the genes involved in oxidative stress, angiogenesis, connective tissue disorders and coagulation system [2, 3].
Studies have shown that matrix Metalloproteinases (MMPs) are involved in the development of some chronic connective tissue, cardiovascular, pulmonary, and autoimmune diseases [4, 5, 6]. These enzymes damage endothelial cells by destroying connective tissue components [7, 8]. MMP9 is a member of the MMP family, located on the long arm of chromosome number 20 (20q13.12), and is active in the reconstruction of placenta and uterine arteries. Functional –1562C>T polymorphism in the promoter region of MMP9 gene (rs3918242) is associated with high transcriptional activity and increased enzyme levels in tissue and biological fluid [9, 10]. In some studies, an association between rs3918242 polymorphism and increased incidence of preeclampsia have been reported, while in other studies, no association has been found between them [14, 15, 17]. Considering the contradictory results, this study aims to evaluate the frequency of rs3918242 polymorphism and its relationship with preeclampsia in pregnant women.

2. Materials and Methods
This case-control study was conducted on 100 pregnant women with preeclampsia (case group) and 100 healthy pregnant women (control group). Samples were recruited from the fertility center of Kowsar Hospital in Qazvin, Iran. The criteria for Preeclampsia was a systolic blood pressure >140 mmHg and/or a diastolic blood pressure >90 mmHg on two occasions >6 hours apart after 20 weeks of gestation but before the onset of labour, plus a proteinuria >2+ (on dipstick test) or >0.3 g/24. Exclusion criteria were: Multiple pregnancies, and a history of hypertension, cardiovascular disease, diabetes, kidney, liver and infectious diseases. Blood samples for molecular genetic assessments were collected in tubes containing EDTA as anticoagulant, and their DNAs were extracted from human leukocyte nuclei isolated from whole blood, and the target DNA was then amplified by Polymerase Chain Reaction (PCR) method. Restriction Fragment Length Polymorphism (RFLP) assays were designed for the identified polymorphisms. The PCR was performed by the forward primer (-5´-GCCTGGCACATAGTAGGCCC-3´) and reverse primer (-5´-CTTCCTAGCCAGCCGGCATC-3’). PCR product was digested with SphI restriction enzyme. Restriction fragments were visualized on 2% agarose gel stained with ethidium bromide. Data analysis was performed only on the data with no missing values (90 with preeclampsia and 99 healthy peers).

3. Results
The frequency of CC, CT and TT genotypes were 84.8%, 13.1% and 2% in the control group and 47.8%, 47.8% and 4.2% in the case group, respectively. The risk of developing preeclampsia in women with CT genotype was 6.2 times higher than in women with CC genotype (P<0.001). Although there was no statistically significant relationship between the frequency of TT and CC genotypes and the incidence of preeclampsia, the risk of developing preeclampsia in women with TT genotype was 4.3 times higher than in women with CC genotype.

4. Discussion and Conclusion
In this study, was investigated the association of rs3918242 polymorphism in the MMP9 gene and the risk of preeclampsia. The results showed that CT genotype and T allele of the polymorphism had association with increased risk of preeclampsia in pregnant women. Single nucleotide polymorphism of the MMP-9 gene (rs3918242) regulates the expression of this protein as well as its enzymatic activity by modulating the binding of transcription factors in the promoter region of MMP9 gene [18]. There are conflicting results regarding the association between this polymorphism of the MMP-9 gene and preeclampsia. Some studies have reported the association of CC genotype or C allele of rs3918242 polymorphism with an increased risk of preeclampsia in pregnant women [21, 22], while no such relationship has been found in other studies [17, 21, 23]. Coleman et al. suggested that the frequency of T allele in women with preeclampsia is a factor associated with reduced the risk of preeclampsia [24], which is consistent with our findings. Pali et al. reported a significant relationship between T allele and blood pressure, but its association with preeclampsia in pregnant women was not found [5]. In the study by Leonardo et al., no significant relationship between this polymorphism and preeclampsia was observed. 
The frequency of CC, CT and TT genotypes in patients in our study were 78%, 14% and 1%, respectively. Compared to the study by Leonardo et al. [25], the frequency of CC and CT genotype in our patients was lower while the frequency of TT genotype was higher. Meng et al. showed a significant association of rs3918242 polymorphism with the incidence of preeclampsia [26]. Consistent with our results, Rahimi et al. showed that the presence of TT and CT alleles of rs3918242 polymorphism is a biomarker of susceptibility to preeclampsia in pregnant women [17]. However, the frequency of TT and CT genotypes in patients was significantly different from those in our study. The discrepancy may be because of difference in ethnicity, race, lifestyle, diet and eating habits. Therefore, in order to investigate the effect of different genes on the incidence of preeclampsia, it is recommended that other factors and the severity of preeclampsia be examined in future studies

Ethical Considerations
Compliance with ethical guidelines
The present study obtained its ethical approval from the Research Ethics Committee of Qazvin University of Medical Sciences (Code: QUMS.REC.1396.244).

Funding
This study was extracted from MA. thesis of second author approved by Department of Clinical Biochemistry, School of Medicine, Qazvin University of Medical Sciences.

Authors' contributions
Writing – original draft, and writing – review & editing: Mehdi Sahmani and Amir Javadi; resources and validation: Nilofar Ahmadi, Somayeh Asadian and Amir Javadi; methodology and data analysis: Amir Javadi; editing & review and project administration: Mehdi Sahmani , Talaat Dabaghi Ghaleh

Conflict of interest
The authors declare no conflict of interest.

Acknowledgments
The authors would like to thank the efforts of Ms. Zahra Rashvand, a laboratory expert, during the study.

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