Combined Oral Contraceptives Use, Genetic Variants And The Risk Of Stroke: A Case-control Study In China

Use of oral contraceptives is considered as an innovative method for contraceptionin human beings. Combined oral contraceptives (COC) are combined estrogen andprogesterone. Clinical practice has demonstrated that COC use is one of the mosteffective and safest short-effect contraception methods for reducing unexpectedpregnancy. However, increased risk of cardio-cerebrovascular diseases such as stroke,myocardial infarction, and venous thromboembolism was reported to be associatedwith COC use since it was on the market in the developed countries in1960s.Although the development of low estrogen doses and altered progestin levels led tofewer cardio-cerebrovascular complications, the increased risk was still documentedin subsequent studies.More attention has been paid to stroke among all COC related health risks. Theincidence of stroke is much higher in Chinese population. Moreover, with theacceleration of an aging population and changed lifestyle, health and social problemsarising from stroke will be more prominent. Compared with Caucasians, Chinesediffer in incidence rate, phenotypes, clinical characteristics, and risk factors of stroke.Therefore, to explore the associations between COC use and stroke risk will benefitfor stroke prevention in Chinese women.Stroke is believed to be a kind of complex diseases arising from multiple environmental and genetic factors. Nevertheless, environmental factors could merelyexplain a small part of stroke, for instance, only a few of COC users suffer fromstroke. Therefore, there are differences in genetic susceptibilities in individuals whenthey are exposed to similar environmental factors. Recently, genome-wide associationstudies (GWAS) which analyze the genetic variants covering whole genome havebecome a promising method for the studies of genetic susceptibilities. With theadvances of GWAS, many new single nucleotide polymorphisms (SNPs) werereported to be associated with stroke risk. However, most of these GWAS of strokewere conducted in Caucasians. It is known there is difference of allelic frequencybetween Caucasians and Chinese. Therefore, it is an interesting topic to check theassociations between GWAS SNPs and the risk of stroke in Chinese population.The interactions between risk factors may have an impact on the development of acomplex disease. Studies showed that in migrainous women, coexistent use of oralcontraceptives or a history of high blood pressure or smoking had greater thanmultiplicative effects on the odds ratios for ischemic stroke associated with migrainealone. In addition, the risk of stroke was significantly elevated in combination ofCOC use with gene polymorphisms of coagulation system and rennin-angiotensinsystem. To explore the possible gene-gene and gene-environment interactions willhelp in elucidating the mechanisms for stroke.This study was a case-control study based on the prospective cohort which wasestablished with the support of National Key project ‘Study on the long term healtheffects of contraceptives’. Traditional epidemiological and molecular epidemiologicalmethods were applied integratedly in this study. The risk factors for stroke in Chinesewomen, including environmental factors such as COC use, and genetic variantssuggested by GWAS were investigated. Furthermore, the gene-gene andgene-environment interactions were discussed. The results from this study will behelpful in instructing contraceptive use, preventing stroke, and promoting women’shealth. Part ⅠCOC Use and the Risk of Stroke in Chinese WomenCOC is a widely used and effective method for contraception. About8.8%women of childbearing age take COC for contraception. Although there are a relativefew women taking COC in China, compared with those in developed countries, thetotal COC users are with a large number due to many women at reproductive age.Moreover, the accumulated number of women who have ever taken domestic-madeCOC since1970s is more than tens of millions. Many studies have been conducted toreduce estrogen doses and change the formulation of COC so as to protect women’shealth, however, there is few study on the long-term health effects related todomestic-made low-dose COC.[Objective] To illuminate the associations between stroke and environmental factors(COC use) in Chinese women, and explore the possible interactions between theseenvironmental factors.[Methods] On the basis of a prospective female cohort of contraceptive use, thefirst-ever stroke cases, as well as, two age-and region-matched controls (includingneighbourhoods and hospitalized patients) were recruited. An epidemiologicalinvestigation was conducted to obtain relevant information on possible risk factors,for instance, COC use. Blood pressure, height, and body weight were measured.Peripheral venous blood was taken for lipid tests after an overnight fast.[Results]1. The association between COC use and stroke risk in Chinese womenCOC users had a1.56-fold increased risk of stroke, compared with the non-users.Current COC use increased stroke risk by3.05-fold, while past use only slightlyincreased the risk. The stroke risk gradually elevated with the increasing cumulativetime of COC use. Moreover, COC use was significantly associated not with ischemicstroke but with hemorrhagic stroke subtype (OR2.23,95%CI1.43-3.48).2. Analyses of risk factors for stroke in Chinese women The risk factors for stroke included family history of stroke, COC use,hypertension, hyperlipidemia, and diabetes mellitus. Family history of stroke, COCuse, hypertension, and diabetes mellitus were risk factors for hemorrhagic stroke.While age, family history of stroke, hypertension, hyperlipidemia, and diabetesmellitus were risk factors for ischemic stroke. Most of the stroke could be attributableto hypertension.3. Interactions between risk factors on stroke riskCompared with normotensive women without family history of stroke,hypertensive women with family history of stroke had a22-fold increased risk ofstroke. In combination of hypertension and hyperlipidemia increased the risk ofischemic stroke by35-fold. COC use combined with hypertension elevated the risk ofhemorrhagic stroke by18-fold. Additive interactions between these risk factors weredetected.[Conclusion] COC use was the risk factor for stroke in Chinese women. Theinteractions between hypertension and other risk factors (COC use, hyperlipidemia,family history of stroke) significantly increased the risk of stroke.Part ⅡGWAS Suggested Genetic Variants and the Risk of Stroke in Chinese WomenOnly a part of stroke could be attributed to the traditional risk factors such ashypertension. Genetic susceptibility does differ in individuals. Recently growing areaof GWAS has suggested several new SNPs associated with stroke risk. Nevertheless,most of these GWAS were conducted in Caucasians. There are differences in strokesubtypes and genetic characteristics when compared Chinese with Caucasians. Thepercentage of hemorrhagic stroke among overall stroke is higher in Chinese,compared with that in Caucasians. Furthermore, there are differences in thedistributions of allelic frequency. It is unknown if these positive associations reportedby GWAS of stroke in Caucasians could be replicated in Chinese.[Objective] To demonstrate the associations between genetic variants suggested by GWAS and stroke risk in Chinese women, and explore the possible gene-gene andgene-environment (COC use) interactions.[Methods] Blood samples were taken from first-ever stroke cases and controls.Genomic DNA was extracted by proteinase K digestion and phenol-chloroformextraction. Genotyping of selected GWAS SNPs was performed by the polymerasechain reaction assay with TaqMan probes. The replicated positive associations wereconfirmed in another independent population.[Results]1. Associations between GWAS suggested genetic variants and stroke risk in ChinesewomenThe results showed that SNPs rs9943582, rs10958409, rs1333040, and rs10986769were associated with the risk of stroke. SNPs rs10958409and rs1333040wereassociated with increased risk of hemorrhagic stroke with adjusted ORs of1.83(95%CI1.12-3.01) and4.57(95%CI1.38-15.09), respectively, under dominant model.In another independent population, only the association between rs1333040andstroke was verified. Risk T allele carriers of rs1333040were demonstrated to beassociated with a2.41-fold increased risk of stroke when compared with thenon-carriers.The results from the pooled samples suggested the associations between two SNPs(rs10958409and rs1333040) and stroke (overall stroke and ischemic/hemorrhagicsubtypes) in Chinese women.2. Joint effects between individual SNP: None.3. Joint effects between COC use and genetic variants on stroke riskCompared with non-users without genetic variant, COC users carried geneticvariants of rs10958409or rs1333040significantly increased the risk of stroke,especially hemorrhagic stroke. COC use combined with genetic variants ofrs10958409or rs1333040increased the risk of hemorrhagic stroke by4.81-and 15.06-fold, respectively. The joint effects were noticeable in younger subjects (<50years).[Conclusion] SNPs rs10958409and rs1333040were confirmed to be associated withthe risk of stroke and its subtypes in Chinese women. COC use combined with thesegenetic variants significantly increased the risk of stroke, especially hemorrhagicsubtype.