Association between the TOX3 rs3803662 C>T polymorphism and recurrent miscarriage in a southern Chinese population

Abstract Background Studies have shown that some genetic polymorphisms associated with breast cancer susceptibility may also be associated with abortion. The TOX3 gene plays a key role during the onset of breast cancer, and reproductive factors such as abortion are risk factors for breast cancer. However, there is currently no study describing the relationship between the TOX3 rs3803662 C>T polymorphism and the risk of recurrent miscarriage. Therefore, we investigated whether the TOX3 rs3803662 C>T polymorphism is associated with recurrent miscarriage susceptibility in this case‐control study. Methods We recruited 248 recurrent miscarriage patients and 392 healthy controls from the southern Chinese population and performed genotyping using the TaqMan method. Results The results showed no evidence that TOX3 rs3803662 C>T is associated with recurrent miscarriage (CT and CC: corrected OR = 1.038, 95% CI = 0.737‐1.461, P = .8321; TT and CC: adjusted OR = 0.989, 95% CI = 0.591‐1.656, P = .9659; dominant model: adjusted OR = 1.027, 95% CI = 0.742‐1.423, P = .8712; recessive model: adjusted OR = 0.969, 95% CI = 0.600‐1.566, P = .8975). Conclusion According to this study, the TOX3 rs3803662 C>T polymorphism may not be associated with recurrent miscarriage in the southern Chinese population. A larger multicenter study is needed to confirm the results.


| INTRODUC TI ON
Recurrent miscarriage (RM) is defined as two or more consecutive spontaneous abortions before the 20th week of gestation in women with the same sexual partner. 1,2 Although the cause of RM is still unclear, an increasing number of studies have shown that genetic susceptibility plays an important role. [3][4][5] Therefore, exploration of the causes of recurrent abortion has important implications for the prevention and treatment of recurrent abortion. Recent studies have shown a link between abortion and breast cancer risk, and induced abortion may be a risk factor for breast cancer. 6,7 Moreover, there are studies confirming that breast cancer may be associated with reproductive risk factors. 8 In addition, other studies have shown that certain genetic polymorphisms are associated with breast cancer and may also be related to abortion, for example, the MDM2 Del1518 polymorphism and miR-196a (rs11614913). [9][10][11] Therefore, it is helpful to understand the causes of RM by studying whether some genes involved in breast cancer pathogenesis are associated with RM.
Located on 16q12.1, the TOX3 gene is 143 kb long and consists of seven exons. 12 Studies have shown that the genetic variants in the TOX3 gene are associated with multiple diseases, including lung cancer, familial ovarian cancer, polycystic ovary syndrome (PCOS), and breast cancer, [13][14][15][16][17][18][19] and Wang Q et al found that the rs3803662 C>T polymorphism is strongly related to an increased risk of breast cancer in both Asian and Caucasian populations. 20 Moreover, studies have confirmed that TOX3 methylation abnormalities may be closely related to the occurrence of PCOS and may play a role in the development of PCOS pathology by regulating changes in TOX3 protein expression. 21 In recent years, studies have confirmed that rs3803662 C>T is the most important genetic variation in the TOX3 locus, which is associated with an increased risk of breast cancer in women. 20,22 The SNP rs3803662 C>T also shows significant associations with estrogen receptor status, 23 and patients with PCOS have a higher risk of spontaneous abortion. 24 Additionally, reproductive factors such as abortion are known risk factors for breast cancer. 25,26 A recent study of ours found that some lncRNA gene polymorphisms associated with breast cancer susceptibility are also associated with RM, such as lncRNA CCAT2 and lncRNA MALAT1. 27,28 However, it remains unclear whether the TOX3 gene is also associated with recurrent abortion susceptibility, similar to lncRNA CCAT2. These previous studies suggest that genetic variation in the TOX3 locus may be associated with susceptibility to RM. However, there is currently no study on the relationship between the TOX3 rs3803662 C>T polymorphism and the risk of RM. These findings prompted us to evaluate whether the rs3803622 variant of the TOX3 gene is associated with RM susceptibility and whether rs3803622 C>T can be used as a biomedical indicator or a potential risk factor. Women with a history of autoimmune disease; liver and kidney dysfunction; metabolic disorders; endocrine, uterine anomalies, arterial, or venous thrombosis; and other diseases were excluded from the healthy control and RM groups.

| Statistical analysis
Hardy-Weinberg equilibrium (HWE) in the control group was confirmed by the goodness-of-fit chi-square test. Unconditional univariate logistic regression analysis was applied to analyze genotypic and demographic differences between the RM patients and healthy individuals. Furthermore, the association between rs3803622 C>T polymorphism and susceptibility to RM was assessed by the 95% confidence interval (CI) and odds ratio (OR).

| Ethics statement
The study was approved by the Medical Ethics Committee of Guangzhou Women and Children's Medical Center. Written informed consent was obtained from each RM patient and control subject before participation in the study.

| Demographic characteristics
The demographic characteristics of the RM patients (approximately 68.15% of RM patients experienced two or three spontaneous abortions, and more than 31.85% experienced four or more) and healthy controls are listed in Table 1. Among the 248 RM patients and 392 healthy controls included, no significant differences in age were found (31.00 ± 4.83 vs 31.44 ± 4.39 years, P = .7225).

| Association between TOX3 polymorphisms and RM susceptibility
The genotype distribution of the TOX3 rs3803662 C>T polymorphism in RM patients and controls is shown in Table 2. The control group showed HWE for the TOX3 rs3803662 C>T genotypes (HWE = 0.636).
However, there was no significant association between the TOX3

| Stratification analysis
Stratified analysis in which the subjects were stratified by age and the number of abortions was conducted to further evaluate the effects of the TOX3 rs3803662 C>T polymorphism in RM patients and controls ( Table 3). The results showed that the TOX3 rs3803662 C>T polymorphism was not significantly associated with RM risk in different age groups or based on the number of abortions.

| D ISCUSS I ON
Genetic susceptibility may be involved in the onset of recurrent miscarriage and play an important role in the occurrence and development of RM 30 ; such genes include FOXP3, IL-10, and TNF-α. [31][32][33][34] However, few studies have explored the association of long  The study found that as the maternal age of pregnant women increases, the risk of miscarriage increases, and maternal age is an important risk factor for spontaneous abortion. 39