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Department of Chronic Disease Epidemiology, Yale Center for Perinatal, Pediatric, and Environmental Epidemiology, Yale School of Public Health, One Church St., 6th Floor, New Haven, CT 06510
Department of Circulation and Medical Imaging, Gemini Center for Sepsis Research, Norwegian University of Science and Technology, Trondheim, Norway
Centre for Fertility and Health, Norwegian Institute of Public Health, Oslo, Norway
Department of Chronic Disease Epidemiology, Yale Center for Perinatal, Pediatric, and Environmental Epidemiology, Yale School of Public Health, New Haven, CT
Department of Public Health and Nursing, K.G. Jebsen Center for Genetic Epidemiology, Norwegian University of Science and Technology, Trondheim, Norway
Department of Public Health and Nursing, HUNT Research Centre, Norwegian University of Science and Technology, Levanger, Norway
Clinic of Medicine, St. Olavs Hospital, Trondheim University Hospital, Trondheim, Norway
Ectopic pregnancy is a condition where the fertilized ovum implants outside the main cavity of the uterus, and it is an important cause of pregnancy-related mortality.
Several modifiable risk factors are associated with ectopic pregnancy, in particular tobacco smoking, although the role of residual confounding remains unclear.
Because genetic variations are randomly assigned at conception, the presence or absence of risk-increasing alleles for a trait of interest is unaffected by disease status and lifestyle factors. Thus, one may use genetic variants as instruments in instrumental variable analyses—often called Mendelian randomization (MR) analyses—to greatly reduce the risk of reverse causation and confounding (Supplemental Figure 1).
This study aimed to evaluate the causal association between 5 modifiable risk factors—smoking initiation, alcohol consumption, low-density lipoprotein (LDL) cholesterol, systolic blood pressure, and body mass index (BMI)—and risk of ectopic pregnancy using MR.
Study Design
We conducted a 2-sample MR study. The instruments for the 5 exposures were collected from genome-wide association studies (GWASs) of subjects of European ancestry (Supplemental Table 1). For each exposure, we used as instruments single-nucleotide polymorphisms strongly associated with the exposure (P<.001) and independent (R2<0.001 in 10 MB windows, European sample in the 1000 Genomes Project) from each other.
As there was no published GWAS of ectopic pregnancy, we retrieved publicly available summary statistics from genome-wide association analyses from UK Biobank, FinnGen, and Michigan Genomics Initiative (Supplemental Table 2). Next, we performed a fixed-effect meta-analysis in METAL (version 2011-03-25; University of Michigan, Ann Arbor, MI) with standard errors as weights and used these results as the outcome in the MR analyses (3556 cases and 327,733 controls).
MR analyses were performed using the TwoSampleMR package (version 0.5.6) in R (version 3.6.2). We used the inverse-variance–weighted analysis as the main analysis. For a genetic instrument to be valid, its effect on the outcome needs to solely go through the exposure, and thus, estimates can be biased by horizontal pleiotropy (a genetic variant has direct effects on other pathways other than exposure).
Bias because of pleiotropy was explored through 3 sensitivity analyses: weighted mode, weighted median, and MR Egger. F statistics were calculated for all instruments using the formula F (beta/standard error),
we performed a sensitivity analysis using only the coefficient for the association with ectopic pregnancy from FinnGen. To account for testing 5 exposures, we set the threshold for statistical significance to P=.01.
Our study used publicly available data from sources with relevant ethical approvals.
Results
Smoking initiation (ever-smokers vs never-smokers) was significantly associated with the risk of ectopic pregnancy in the main analysis, with an odds ratio of 2.02 (95% confidence interval [CI], 1.22–3.36) per standard deviation increase in the prevalence of smoking initiation (Figure). Systolic blood pressure, LDL cholesterol, and BMI showed no clear effect. Alcohol intake showed some evidence of a positive association, but with wide CIs in part because of a smaller explained variance (0.6%) of the combined genetic instruments compared with the other exposures (2.0%–4.8%) (Supplemental Table 1). Our findings were robust in sensitivity analyses that accounted for pleiotropy (Figure) and sample overlap (Supplemental Figure 2). There was no weak instrument included in the analyses (Supplemental Table 1).
FigureMR analyses of modifiable risk factors for ectopic pregnancy
The concordance between our MR study and previous observational studies strongly suggested a causal relationship between tobacco smoking and risk of ectopic pregnancy. The underlying mechanism for this is unclear but may be because of an impairment of oocyte or embryo transportation because of smoking.
The figure illustrates the assumptions of an instrumental variable analysis with the use of genetic instruments (often called MR), using smoking initiation and risk of ectopic pregnancy as an example. The 3 key assumptions are as follows: the genetic instrument must be associated with the exposure (here, smoking initiation); the genetic instrument must not be associated with any confounders (eg, lifestyle factors); and there is no effect of the genetic instrument on the outcome (here, ectopic pregnancy) other than through the exposure.
MR, Mendelian randomization.
Rogne. Mendelian randomization study of risk factors for ectopic pregnancy. Am J Obstet Gynecol 2022.
Variance explained for each SNP was estimated using the get_r_from_bsen function in the TwoSampleMR package in R and summed across the independent SNPs.
(version 2011-03-25) with standard errors as weights
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Heritability (h2) and genomic inflation factor (λ) were estimated with LD Score Regression (version 1.0.1) using LD scores calculated from the 1000 Genomes European data as reference and filtered to 1,215,620 HapMap3 single-nucleotide polymorphisms.
; note that difference in h2 between cohorts in large part is explained by difference in capture of cases.
ICD-8, International Classification of Diseases, Eight Revision; ICD-9, International Classification of Diseases, Ninth Revision; ICD-10, International Classification of Diseases, Tenth Revision LD, linkage disequilibrium; LDSC, linkage disequilibrium score regression; SAIGE, Scalable and Accurate Implementation of GEneralized mixed model.
Rogne. Mendelian randomization study of risk factors for ectopic pregnancy. Am J Obstet Gynecol 2022.
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