Role of CYP1A1, CYP2D6, and NOS3 gene polymorphisms in idiopathic recurrent pregnancy loss in the Iranian Azeri population: A case-control study

Abstract Background It is estimated that 1-5% of couples suffer from recurrent pregnancy loss (RPL). Recent studies have shown the effects of gene polymorphisms in RPL. Objective The aim of this study was to evaluate 3 gene polymorphisms including rs1048943 of CYP1A1, rs28371725 of CYP2D6, and rs7830 of NOS3 in idiopathic RPL to identify their association with RPL. Materials and Methods Blood samples were collected from 136 women with at least 2 consecutive idiopathic miscarriages (case group) and 136 women with no history of miscarriage and at least one successful pregnancy (control group) from the Iranian Azeri population. This study was carried out between April 2018-April 2020. Amplification-refractory mutation system polymerase chain reaction was used for the rs7830, rs1048943 and rs28371725 polymorphisms in order to genotype each extracted genomic DNA sample. After that, Chi-square, Fisher's exact test and logistic regression were used to investigate whether each of these polymorphisms is associated with RPL. Results Among these polymorphisms, only rs1048943 of CYP1A1 showed a statistically significant association with RPL in the Iranian Azeri women studied. Conclusion Our results suggest that CYP1A1 gene polymorphisms might be associated with a reduced risk of RPL. Further studies in other populations and in the same population with a larger sample size, as well as functional genomics analyses such as gene expression analyses or epigenetic studies are required to validate our results.


Introduction
Pregnancy loss is defined as a loss of pregnancy before the end of the 20 th wk of gestation (1).
Several studies have examined various polymorphisms of candidate genes that encode different mediators which may affect susceptibility to idiopathic RPL (4,10,11). A group of these genes belongs to metabolic enzymes. Genetic polymorphisms of these genes may affect the balance of phase I / phase II detoxification enzymes (12). One of these enzymes is encoded by CYP1A1 which is located on 15q24.1 and includes 7 exons. This gene acts in a 2-step process of detoxifying toxins. In the first step, CYP1A1 is required for activation of toxic components. These components are required for the 2 nd detoxification step. The polymorphisms of this gene can be directly linked with functional disturbance diseases and conditions like cancers and idiopathic male infertility. A recent study showed that this gene can influence normal estrogen metabolism and placental function (13). It seems that its polymorphisms may also lead to RPL.
Another gene of this pathway is CYP2D6 which is located on 22q13.2 and includes 9 exons (14,15). This gene is important for pharmacogenetics; it is involved in the metabolism of over 150 drugs and has been studied in individuals with suicidal thoughts or depression (16). CYP2D6 has an important role in catalyzing the oxidation of testosterone to androstenediones (17), both of which increase during pregnancy. So, it seems that any change in this enzyme may lead to an increased risk of RPL.
There is no autonomic innervation in fetoplacental blood vessels and the regulation of vascular functions at the fetomaternal interface is mediated by endothelial nitric oxide synthase (NOS3), which is a vasoactive mediator (18). This gene is located on 17q36.1 and includes 26 exons. It encodes an enzyme that is important in producing vascular NO (19,20). "NO is a gaseous molecule, which serves different physiological regulatory functions in the regulation of reproduction, such as the formation of new blood vessels, enhancement of blood supply through the maternal arteries to the placenta, regulation of the placental vessel tones, and immune protection of the fetus. All these factors are required for a successful pregnancy outcome and any disturbance in these steps may increase the risk of miscarriage" (21). In the first trimester of pregnancy, trophoblast cells express a large amount of NOS3, so any polymorphism in coding or noncoding regions of the gene may change its activity or expression level, which may lead to RPL (19).
The aim of this study was to determine the relationship between 3 polymorphisms of these genes and RPL in the Iranian Azeri population.

Sampling
miscarriages and no successful pregnancies. It should also be considered that all the women in both groups were Iranians with Azeri origin.
All women with RPL due to infections, uterine conformational abnormalities, immune disorders, hormonal abnormalities (including thyroid and prolactin disorders) and chromosomal abnormalities in themselves or their spouses were excluded from the study.  instructions. The quality of each sample was then estimated using a nanodrop for assessment of nucleic acid purity.

Genotyping
To confirm each polymorphism in its genetic

Reverse inner ACTCCCTTCAGGCAGTCCTTTAGCCA
T-ARMS-PCR: Tetra amplification refractory mutation system polymerase chain reaction

Ethical considerations
The

Results
The mean age of the women in the control group was 31.9 ± 6.3 yr (range 19-45) and 28.9 ± 6.3 (range  in the case group.

Discussion
CYP1A1 plays an important role in the oxidation of polycyclic aromatic hydrocarbons like benzopyrene and polychlorinated biphenyls.
These substances are unusual environmental toxicants (13). The role of this enzyme in activating carcinogens in cancers has been shown in previous studies (23)(24)(25). A recent study also showed that placental CYP1A1 mRNA levels were higher in women with RPL (26). We also know that CYP1A1 can affect the metabolism of estrogen and normal functions of the placenta (13). In fact, this enzyme participates in the metabolism of estrogen by catalyzing the 2-hydroxylation of stradiol (27) and can convert endogenous estrogens into more hydrophilic compounds (28). This enzyme also has an important role in the metabolism of enzymatic xenobiotics that are specifically induced in women exposed to tobacco smoke. These xenobiotics may transfer through the placenta to the fetus and cause toxicity in the fetus or may affect the expression or production of placental hormones and change the function of proteins (29). Thus, it seems that it could be a good candidate gene in relation to RPL.
One of its polymorphisms is rs1048943 A>G which is located in 2455 nucleotides and causes Ile>Val in the amino acid chain. This amino acid change occurs near the hem group of the protein and causes the enzyme activity to double (30).
This polymorphism is a risk factor for pharyngeal, prostate, lung, oral, ovarian, bladder, colorectal, and cervical cancers (31). A protective association also was shown between this polymorphism and coronary artery disease in a study conducted with the Han population of China in 2017 (32). In our study, we found that rs1048943 was associated with a reduced risk of RPL in women of Iranian Azeri origin. However, another study done in 2014 in Russia did not show any association between this polymorphism and RPL (33). Given the varied results, studying this common polymorphism of CYP1A1 in larger populations with different genetic backgrounds is recommended.
CYP2D6 is active in catalyzing the oxidation of testosterone to androstenediones (17). Both of these hormones increase during pregnancy.
The normal fertility process requires an adequate supply of sex hormones and the normal functioning of CYP2D6 is needed to reach this threshold.
CYP2D6 is also an important modulator of the detoxification system, and can protect the environment of the utero-placental tissue from being affected by overwhelming oxidative stress (6). The activity of this enzyme begins to increase in the early stages of the 2 nd trimester of pregnancy and continues to increase as the pregnancy progresses. Nowadays it is also known that many commonly used drugs in pregnancy are metabolized by this enzyme. Considering the highly polymorphic nature of this gene (28), it seems that this could be a good candidate gene in RPL associated studies. This enzyme is also important in pharmacogenetics and is active in the biotransformation of more than 150 drugs including antipsychotics, antidepressants, analgesics, beta-blockers, and antiarrhythmics (16).  (34). To the best of our knowledge, ours is the first study to evaluate the effect of this SNP in RPL. Our research showed that the association between this SNP and RPL in the Iranian Azeri population was not significant.
Another study which was conducted in the South of India in 2004 investigated the association between rs3892097 of this gene and RPL but it also did not find any positive or negative association between rs3892097 and RPL (35).
"NOS3 encodes an enzyme that generates NO in endothelial cells and is involved in the regulation of vascular functions". The endometrial expression of NOS3 reaches its peak in humans during implantation (36). NO also plays an important role in blood pressure control, cardiovascular homeostasis, the metabolism of glucose, and insulin resistance during pregnancy. A reduction of NO production may cause pregnancy-related vascular problems like RPL. Additionally, alteration in NO synthesis may cause premature labor by affecting the inflammatory response and uterine contractility regulation (37). As a vasoactive agent, NO causes vasodilation and increases the rate of nutrient supply and oxygen perfusion in the umbilical cord. So, any decrease in its rate of production may cause intraurine hypoxia, restriction in fetal growth and increased fetoplacental vascular resistance (38). We also know that the inhibition of NO synthesis in pregnant rats causes hypertension, proteinuria, thrombocytopenia and fetal growth restriction that can all lead to miscarriage (39). Hence, this gene could also be a good candidate gene for RPL associated studies.
Rs7830 (G>T) is associated with 2 different genes: NOS3 and ATG9B, but because it is located within a silencer motif, TGGGGAC, where a G>T change can lead to a different transcript, it seems that it influences the NOS3 gene more. Although we did not find any association between this SNP and RPL, the association of this SNP with end-stage renal disease has been previously demonstrated (40). The association between NOS polymorphisms and renal dysfunction (41), atherosclerotic vascular diseases (42), and advanced diabetic nephropathy

Conclusion
Our results indicated that while rs1048943 of