Cohort

Figure 1. Figure 1. Construction of the Two Study Cohorts and Propensity-Score Matching of Pregnant Women with Vaccine Exposure and Those without Vaccine Exposure, Yielding Five Outcome-Specific Cohorts. Values regarding exclusions do not sum to the total number because some pregnancies were excluded for more than one reason. Between the unmatched and matched analyses, all the excluded records were from unmatched pregnancies that did not have vaccine exposure. All the outcome-specific analyses were matched for maternal age, calendar year of pregnancy onset, and propensity score, and the analyses of spontaneous abortion and stillbirth were also matched for gestational age.

Using the Medical Birth Register13 and the National Patient Register,14 we identified all pregnancies in Denmark that ended in a singleton birth or an abortive outcome between October 1, 2006, and November 30, 2013 (Figure 1). We then estimated the date of pregnancy onset by subtracting the gestational age from the date of birth or abortion. Records of gestational age for live-born infants and stillbirths are based mainly on ultrasonography, whereas records of gestational age in abortive outcomes are based on ultrasonography or the first day of the last menstrual period.15 The records of gestational age for live births were validated to be accurate within 1 week for 87% of the records.16

We subsequently excluded pregnancies with missing or implausible data on gestational age, pregnancies with multiple overlapping records, and pregnancies in women who had not lived continuously in Denmark for the 2-year period before pregnancy onset. In addition, for analyses of spontaneous abortion, we excluded all cases of spontaneous abortion that occurred within the first 6 weeks of gestation, because many cases of spontaneous abortion during the first weeks of gestation are likely to be clinically unrecognized. Consequently, for analyses of spontaneous abortion we also excluded women who had been exposed to vaccination within the first 6 weeks of gestation to ensure that immortal time (a follow-up period during which the study outcome, by design, could not occur) was not introduced.

We then defined two cohorts: one cohort included all the pregnancies that ended in live birth and was used for the analyses of major birth defect, preterm birth, low birth weight, and small size for gestational age; and the second cohort included all the pregnancies and was used for the analyses of spontaneous abortion and stillbirth (Figure 1). The unique personal identification numbers that are given to every resident in Denmark enabled the individual-level linkage of our cohort with nationwide health and demographic registers containing information on vaccination, adverse outcomes, and potential confounders (see the Supplementary Appendix, available with the full text of this article at NEJM.org).17

The study was approved by the Danish Data Protection Agency. Informed consent is not required for registry-based research in Denmark. The funders and vaccine manufacturers had no role in the design and conduct of the study; the collection, management, analysis, and interpretation of the data; the preparation, review, or approval of the manuscript; or the decision to submit the manuscript for publication. The first and last authors take responsibility for the accuracy and completeness of the reported data and analyses.

Vaccination

During the study period, the quadrivalent HPV vaccine (Gardasil, Sanofi Pasteur MSD [manufactured in the United States by Merck]) was the sole HPV vaccine used in the Danish national vaccination program (see the Supplementary Appendix). The few women who were vaccinated with the bivalent HPV vaccine (Cervarix, GlaxoSmithKline Biologicals) before or during pregnancy were excluded from our study (Figure 1).

Information on quadrivalent HPV vaccinations that were given through the national vaccination program was obtained from the Danish Childhood Vaccination Database.18 The quadrivalent HPV vaccine was also available by prescription, so additional data on vaccination status were obtained from the Danish National Prescription Registry.19 We defined the date of exposure to the quadrivalent HPV vaccine as the date of the first vaccination or filled prescription. Exposure windows were categorized according to study outcome: the first trimester (pregnancy onset through week 12 of gestation) for the analysis of major birth defect, the start of week 7 through week 22 of gestation for the analysis of spontaneous abortion, the start of week 7 of gestation until birth for the analysis of stillbirth, before 37 completed weeks of gestation for the analysis of preterm birth, and at any time during pregnancy for the analyses of low birth weight and small size for gestational age. In each analysis, unexposed pregnancies were defined as pregnancies in women who were not vaccinated during the specified exposure window.

Outcomes

The outcome of major birth defects overall was defined as the first registered diagnosis of any major birth defect within the first year of life (see the Supplementary Appendix), as identified in the National Patient Register.14 A validation study of the National Patient Register estimated a predictive value of 88% for diagnoses of birth defects overall.20 Cases of spontaneous abortion (defined as fetal death occurring through 22 weeks of gestation) were identified in the National Patient Register. Validation has shown that the records were correct for 99% of the diagnoses of spontaneous abortion (details on abortive outcomes are provided in the Supplementary Appendix).21 Information on stillbirth (defined as fetal loss after 22 completed weeks of gestation), preterm birth (delivery before 37 completed weeks of gestation), low birth weight (<2500 g), and small size for gestational age (birth weight in the lowest 10th percentile of gestational age–specific birth weight in the cohort) was obtained from the Medical Birth Register. For the analyses of low birth weight and small size for gestational age, we excluded records of all the pregnancies resulting in live birth for which information on birth weight was missing.

Statistical Analysis

Because different exposure windows and exclusion criteria were applied to the analysis set of each distinct outcome, we created five unmatched outcome-specific cohorts (Figure 1). Selected baseline characteristics were then identified for each woman at pregnancy onset, and missing values were imputed with the use of mode imputation (Table S1 in the Supplementary Appendix). To account for potential confounders, we calculated propensity scores using logistic regression, which estimated the probability of quadrivalent HPV vaccination in outcome-specific exposure time windows, given all baseline characteristics (and all two-way interactions between demographic variables).

For each of the five outcome-specific cohorts, we then matched vaccinated women and unvaccinated women in a 1:4 ratio according to age (5-year categories), calendar year of pregnancy onset, and propensity score, thus creating a distinct matched analysis set for each of the five outcome-specific cohorts (Figure 1). We added gestational age as a matching criterion for the cohorts regarding spontaneous abortion and stillbirth, the risks of which are highly dependent on gestational age.

Matching was performed with the use of the nearest-neighbor matching algorithm (caliper width, 0.2 of the standard deviation of the logit score). We assessed the balance of covariates that was achieved from matching by evaluating standardized differences between vaccinated groups and unvaccinated groups. We considered covariates with a standardized difference of less than 10% to be well balanced.

We performed the analyses of spontaneous abortion and stillbirth using gestational age as the underlying time scale. Hazard ratios with 95% confidence intervals were estimated with the use of Cox proportional-hazards regression. The Wald test was used to assess the fulfillment of the proportional-hazards assumption, which was fulfilled for the analyses of stillbirth (P=0.83) and spontaneous abortion (P=0.46). In the analyses of major birth defect, preterm birth, low birth weight, and small size for gestational age, we used logistic regression to estimate prevalence odds ratios. The generalized estimating equation method was used for all analyses to account for the possible correlation between pregnancies within the same mother.

We performed a number of prespecified sensitivity analyses. In the analysis of major birth defect, we restricted the exposure window to weeks 4 to 10 of gestation, which corresponds to the period of maximal susceptibility to teratogenic agents.22 Basing the analysis of major birth defect only on live births could potentially introduce misclassification by the exclusion of stillbirths and induced abortions that were caused by major birth defects, thus potentially biasing results toward no association. We therefore conducted an analysis of major birth defect that included stillbirths and induced abortions (see the Supplementary Appendix). Furthermore, we performed a complete-case analysis for each subcohort, which excluded all the participants who had missing data. To investigate the potential for residual confounding we performed two additional analyses: one analysis incorporated 1:1 matching, because 1:1 matching increases the comparability between exposure groups,23 and a second analysis incorporated an increase in the granularity of the age variable that was used for matching, because vaccination and pregnancy outcomes are both heavily dependent on age.

Post hoc, we added two sensitivity analyses. First, we excluded pregnancies among women who were not exposed to vaccination during pregnancy but who had been vaccinated within 30 days before pregnancy onset. Second, since the date of filling a prescription may differ from the actual date of vaccination, we excluded women who were defined as having vaccine exposure when they filled a vaccine prescription during pregnancy as well as excluding their corresponding matches. SAS software, version 9.4 (SAS Institute), was used for all the analyses.