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Modifiable risk factors for ectopic pregnancy: a Mendelian randomization study

  • Tormod Rogne
    Affiliations
    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
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  • Zeyan Liew
    Affiliations
    Department of Chronic Disease Epidemiology, Yale Center for Perinatal, Pediatric, and Environmental Epidemiology, Yale School of Public Health, New Haven, CT
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  • Álvaro Hernáez
    Affiliations
    Centre for Fertility and Health, Norwegian Institute of Public Health, Oslo, Norway
    Consorcio CIBER, M.P. Fisiopatología de la Obesidad y Nutrición, Instituto de Salud Carlos III, Madrid, Spain
    Blanquerna-Ramon Llull University School of Health Science, Barcelona, Spain
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  • Ben Michael Brumpton
    Affiliations
    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
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  • Maria Christine Magnus
    Affiliations
    Centre for Fertility and Health, Norwegian Institute of Public Health, Oslo, Norway
    Medical Research Council Integrative Epidemiology Unit, University of Bristol, Bristol, England, United Kingdom
    Population Health Sciences, Bristol Medical School, University of Bristol, Bristol, England, United Kingdom
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Published:April 03, 2022DOI:https://doi.org/10.1016/j.ajog.2022.03.063

      Objective

      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.
      • Stulberg D.B.
      • Cain L.R.
      • Dahlquist I.
      • Lauderdale D.S.
      Ectopic pregnancy rates and racial disparities in the Medicaid population, 2004-2008.
      Several modifiable risk factors are associated with ectopic pregnancy, in particular tobacco smoking, although the role of residual confounding remains unclear.
      • Gaskins A.J.
      • Missmer S.A.
      • Rich-Edwards J.W.
      • Williams P.L.
      • Souter I.
      • Chavarro J.E.
      Demographic, lifestyle, and reproductive risk factors for ectopic pregnancy.
      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).
      • Davies N.M.
      • Holmes M.V.
      • Davey Smith G.
      Reading Mendelian randomisation studies: a guide, glossary, and checklist for clinicians.
      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).
      • Davies N.M.
      • Holmes M.V.
      • Davey Smith G.
      Reading Mendelian randomisation studies: a guide, glossary, and checklist for clinicians.
      Moreover, estimates can be biased by weak genetic instruments.
      • Hemani G.
      • Bowden J.
      • Davey Smith G.
      Evaluating the potential role of pleiotropy in Mendelian randomization studies.
      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),
      • Gaskins A.J.
      • Missmer S.A.
      • Rich-Edwards J.W.
      • Williams P.L.
      • Souter I.
      • Chavarro J.E.
      Demographic, lifestyle, and reproductive risk factors for ectopic pregnancy.
      and an instrument with an F statistic of <10 was considered weak.
      • Davies N.M.
      • Holmes M.V.
      • Davey Smith G.
      Reading Mendelian randomisation studies: a guide, glossary, and checklist for clinicians.
      Finally, as sample overlap between the exposure and outcome analyses may bias toward the confounded estimate,
      • Davies N.M.
      • Holmes M.V.
      • Davey Smith G.
      Reading Mendelian randomisation studies: a guide, glossary, and checklist for clinicians.
      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).
      Figure thumbnail gr1
      FigureMR analyses of modifiable risk factors for ectopic pregnancy
      Units are in 1 standard deviation increase of the exposure.
      BMI, body mass index; CI, confidence interval; LDL, low-density lipoprotein; MR, Mendelian randomization; OR, odds ratio.
      Rogne. Mendelian randomization study of risk factors for ectopic pregnancy. Am J Obstet Gynecol 2022.

      Conclusion

      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.
      • Gaskins A.J.
      • Missmer S.A.
      • Rich-Edwards J.W.
      • Williams P.L.
      • Souter I.
      • Chavarro J.E.
      Demographic, lifestyle, and reproductive risk factors for ectopic pregnancy.

      Supplementary Methods—Modifiable Risk Factors for Ectopic Pregnancy: a Mendelian Randomization Study

      Figure thumbnail fx1
      Supplemental Figure 1Schematic representation of MR analysis
      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.
      Figure thumbnail fx2
      Supplemental Figure 2MR analyses of modifiable risk factors for ectopic pregnancy in the FinnGen study
      Units are in 1 standard deviation increase of the exposure.
      BMI, body mass index; CI, confidence interval; LDL, low-density lipoprotein; MR, Mendelian randomization; OR, odds ratio.
      Rogne. Mendelian randomization study of risk factors for ectopic pregnancy. Am J Obstet Gynecol 2022.
      Supplemental Table 1Source of exposure genome-wide association study summary data
      TraitStudyAncestryNumber of participantsNumber of SNPs usedVarianceexplained (%)Median F statistic (range)
      Smoking initiationLiu et al,
      • Liu M.
      • Jiang Y.
      • Wedow R.
      • et al.
      Association studies of up to 1.2 million individuals yield new insights into the genetic etiology of tobacco and alcohol use.
      2019
      European1,232,0913412.041 (30–261)
      Alcoholic drinks per wkLiu et al,
      • Liu M.
      • Jiang Y.
      • Wedow R.
      • et al.
      Association studies of up to 1.2 million individuals yield new insights into the genetic etiology of tobacco and alcohol use.
      2019
      European941,280880.639 (30–1713)
      Body mass indexYengo et al,
      • Yengo L.
      • Sidorenko J.
      • Kemper K.E.
      • et al.
      Meta-analysis of genome-wide association studies for height and body mass index in ∼700000 individuals of European ancestry.
      2018
      European690,4955064.851 (29–1426)
      LDL cholesterolGraham et al,
      • Graham S.E.
      • Clarke S.L.
      • Wu K.H.
      • et al.
      The power of genetic diversity in genome-wide association studies of lipids.
      2021
      European1,231,2843993.970 (30–2578)
      Systolic blood pressureEvangelou et al,
      • Evangelou E.
      • Warren H.R.
      • Mosen-Ansorena D.
      • et al.
      Genetic analysis of over 1 million people identifies 535 new loci associated with blood pressure traits.
      2018
      European757,6014383.550 (30–628)
      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.
      • Hemani G.
      • Zheng J.
      • Elsworth B.
      • et al.
      The MR-base platform supports systematic causal inference across the human phenome.
      LDL, low-density lipoprotein; SNP, single-nucleotide polymorphism.
      Rogne. Mendelian randomization study of risk factors for ectopic pregnancy. Am J Obstet Gynecol 2022.
      Supplemental Table 2Genome-wide association analyses of ectopic pregnancy
      VariableVersionAncestryCase definitionControl definitionNumber of casesNumber of controlsh2λAnalysisLink
      FinnGenFreeze 5EuropeanICD-10: O00

      ICD-9: 633

      ICD-8: 631
      Females not classified as case311189,4300.041.02Analyses conducted using SAIGE,
      • Zhou W.
      • Nielsen J.B.
      • Fritsche L.G.
      • et al.
      Efficiently controlling for case-control imbalance and sample relatedness in large-scale genetic association studies.
      adjusting for genetic relatedness, birth year, and first 4 principal components
      https://r5.finngen.fi/pheno/O15_PREG_ECTOP
      UK BiobankUKB-TOPMedEuropeanICD-10: O00Females not classified as case330212,2490.001.00Analyses conducted using SAIGE,
      • Zhou W.
      • Nielsen J.B.
      • Fritsche L.G.
      • et al.
      Efficiently controlling for case-control imbalance and sample relatedness in large-scale genetic association studies.
      adjusting for genetic relatedness, birth year, and first 4 principal components
      https://pheweb.org/UKB-TOPMed/pheno/634.3
      Michigan Genomics InitiativeFreeze 3EuropeanICD-10: O00Females not classified as case11425,2840.741.00Analyses conducted using SAIGE,
      • Zhou W.
      • Nielsen J.B.
      • Fritsche L.G.
      • et al.
      Efficiently controlling for case-control imbalance and sample relatedness in large-scale genetic association studies.
      adjusting for genetic relatedness, birth year, and first 4 principal components
      https://pheweb.org/MGI-freeze3/pheno/X634.3
      Meta-analysisEuropeanAs aboveFemales not classified as case3556327,7330.041.00Fixed-effect meta-analysis in METAL
      • Willer C.J.
      • Li Y.
      • Abecasis G.R.
      METAL: fast and efficient meta-analysis of genomewide association scans.
      (version 2011-03-25) with standard errors as weights
      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.
      • Bulik-Sullivan B.K.
      • Loh P.R.
      • Finucane H.K.
      • et al.
      LD score regression distinguishes confounding from polygenicity in genome-wide association studies.
      The population incidence of ectopic pregnancy was assumed to be 0.52% for women aged 15 to 44 years
      • Stulberg D.B.
      • Cain L.R.
      • Dahlquist I.
      • Lauderdale D.S.
      Ectopic pregnancy rates and racial disparities in the Medicaid population, 2004-2008.
      ; 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.

      References

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        Ectopic pregnancy rates and racial disparities in the Medicaid population, 2004-2008.
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        • Missmer S.A.
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        Demographic, lifestyle, and reproductive risk factors for ectopic pregnancy.
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      Supplemental References

        • Liu M.
        • Jiang Y.
        • Wedow R.
        • et al.
        Association studies of up to 1.2 million individuals yield new insights into the genetic etiology of tobacco and alcohol use.
        Nat Genet. 2019; 51: 237-244
        • Yengo L.
        • Sidorenko J.
        • Kemper K.E.
        • et al.
        Meta-analysis of genome-wide association studies for height and body mass index in ∼700000 individuals of European ancestry.
        Hum Mol Genet. 2018; 27: 3641-3649
        • Graham S.E.
        • Clarke S.L.
        • Wu K.H.
        • et al.
        The power of genetic diversity in genome-wide association studies of lipids.
        Nature. 2021; 600: 675-679
        • Evangelou E.
        • Warren H.R.
        • Mosen-Ansorena D.
        • et al.
        Genetic analysis of over 1 million people identifies 535 new loci associated with blood pressure traits.
        Nat Genet. 2018; 50: 1412-1425
        • Hemani G.
        • Zheng J.
        • Elsworth B.
        • et al.
        The MR-base platform supports systematic causal inference across the human phenome.
        eLife. 2018; 7e34408
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        • Nielsen J.B.
        • Fritsche L.G.
        • et al.
        Efficiently controlling for case-control imbalance and sample relatedness in large-scale genetic association studies.
        Nat Genet. 2018; 50: 1335-1341
        • Willer C.J.
        • Li Y.
        • Abecasis G.R.
        METAL: fast and efficient meta-analysis of genomewide association scans.
        Bioinformatics. 2010; 26: 2190-2191
        • Bulik-Sullivan B.K.
        • Loh P.R.
        • Finucane H.K.
        • et al.
        LD score regression distinguishes confounding from polygenicity in genome-wide association studies.
        Nat Genet. 2015; 47: 291-295
        • Stulberg D.B.
        • Cain L.R.
        • Dahlquist I.
        • Lauderdale D.S.
        Ectopic pregnancy rates and racial disparities in the Medicaid population, 2004-2008.
        Fertil Steril. 2014; 102: 1671-1676