Literature Review - Systematic Research 4 Pages

Spontaneous Abortion and a Diet Drug Containing Caffeine and Ephedrine: A Study within the Danish National Birth Cohort Penelope P. Howards1*, Irva Hertz-Picciotto2, Bodil H. Bech3, Ellen A. Nohr3, Anne-Marie

Nybo Andersen4, Charles Poole5, Jørn Olsen3

1 Department of Epidemiology, Rollins School of Public Health, Emory University, Atlanta, Georgia, United States of America, 2 Division of Environmental and

Occupational Health, Department of Public Health Sciences, University of California, Davis, California, United States of America, 3 Section for Epidemiology, Department of

Public Health, Aarhus University, Aarhus, Denmark, 4 Section of Epidemiology, Department of Public Health, University of Copenhagen, Copenhagen, Denmark,

5 Department of Epidemiology, Gillings School of Public Health, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America

Abstract

Background: Medications may be consumed periconceptionally before a woman knows she is pregnant. In this study, the authors evaluate the association of a prescription diet drug (Letigen) containing ephedrine (20 mg) and caffeine (200 mg) with spontaneous abortion (SAB) in the Danish National Birth Cohort.

Methods: Women were recruited during their first prenatal visit from 1996–2002. Pre-conception and early pregnancy medication use was reported on the enrollment form, and pregnancy outcome was determined by linking the mother’s Civil Registration Number to the Medical Birth Registry and the National Hospital Discharge Register. Of 97,903 eligible pregnancies, 4,443 ended in SAB between 5 and 20 completed gestational weeks, inclusive. Letigen use was reported for 565 pregnancies. Cox regression models accounting for left truncation were fit to estimate the effect of pre-conception and early pregnancy Letigen use on SAB.

Principal Findings: The estimated maternal age-adjusted hazard ratio for SAB was 1.1 (95% confidence interval 0.8–1.6) for any periconceptional Letigen use compared to no periconceptional use.

Conclusions: Although Letigen has high levels of caffeine (the recommended 3 pills/day are approximately equivalent to caffeine from 6 cups of coffee), periconceptional use does not appear to be associated with an appreciably increased hazard of clinically recognized SAB.

Citation: Howards PP, Hertz-Picciotto I, Bech BH, Nohr EA, Andersen A-MN, et al. (2012) Spontaneous Abortion and a Diet Drug Containing Caffeine and Ephedrine: A Study within the Danish National Birth Cohort. PLoS ONE 7(11): e50372. doi:10.1371/journal.pone.0050372

Editor: Hamid Reza Baradaran, Tehran University of Medical Sciences, Iran (Republic of Islamic)

Received July 16, 2012; Accepted October 19, 2012; Published November 16, 2012

Copyright: � 2012 Howards et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: The Danish National Research Foundation has established the Danish Epidemiology Science Centre that initiated and created the Danish National Birth Cohort. The cohort is furthermore a result of a major grant from this foundation. Additional support for the Danish National Birth Cohort was obtained from the Pharmacy Foundation, the Egmont Foundation, the March of Dimes Birth Defects Foundation, and the Augustinus Foundation. This particular study was supported by a grant from the Danish Epidemiology Science Centre and a dissertation completion fellowship from the Graduate School at the University of North Carolina, Chapel Hill. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: [email protected]

Introduction

Early gestation is a critical time in fetal development when

women may be unaware they are pregnant and therefore may use

medications, such as diet drugs, that they would not use knowing

they were pregnant. Recently, the obesity epidemic in the United

States has led the Food and Drug Administration (FDA) to weigh

the potential benefits of approving new weight loss drugs against

the potential risks, including the risk that reproductive age women

may use the drugs before becoming aware of pregnancy. In 2012,

the scientific advisory committee to the FDA reversed their 2010

position for one such drug despite concerns that the drug might be

associated with an increased risk of oral clefts [1]. In other

countries, including Denmark, the prevalence of obesity has also

been rising [2], and consequently, reproductive age women may

increasingly turn to prescription or over-the-counter weight loss

products where available. Letigen (NYCOMED, Denmark) was a

prescription diet drug that contained ephedrine and caffeine and

was available in Denmark prior to 2002.

There has been little research on the effects of ephedrine on

pregnancy, but concern over the potential effect of caffeine on

spontaneous abortion (SAB) has resulted in a large but inconclu-

sive literature [3–26]. After considering this literature, the

American College of Obstetricians and Gynecologists published

a committee opinion in 2010 stating the current evidence did not

suggest that consuming less than 200 mg per day of caffeine

during pregnancy increased the risk of SAB, but that the evidence

for higher doses of caffeine was still unclear [27]. Similarly, the

United Kingdom’s Food Standards Agency recommended that

pregnant women limit their caffeine consumption to less than

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200 mg per day due to concerns about fetal growth restriction and

possibly SAB [28]. A number of methodological issues that are

difficult to resolve plague studies of caffeine consumption during

pregnancy and SAB making it difficult to draw conclusions about

the effect of exposure to high doses of caffeine [29–33]. Although

some of these methodological issues do not apply to pre-pregnancy

exposure to caffeine, high exposure to pre-pregnancy caffeine has

also been inconsistently associated with SAB with modestly

elevated effect estimates in some studies [9,12,18,19] but not

others [10,20,22]. Most studies of caffeine focused exclusively on

caffeine from beverages, including coffee, tea, and cola, that

contain other potentially fetotoxic components.

Letigen was a non-beverage source of high doses of caffeine

(200–600 mg/day) that some Danish women used pre-concep-

tionally and inadvertently early in pregnancy. It was banned in

Denmark in 2002 and similar products were banned in the United

States in 2004 due to reports of adverse cardiovascular events

[34,35]. We examine whether periconceptional Letigen use is

associated with SAB in the Danish National Birth Cohort.

Periconceptional Letigen use provides an alternative source of

information on exposure to caffeine and SAB with different

strengths and weaknesses from studies which focus on caffeine

exposure from coffee. Some strengths include that the dose of

caffeine is likely more consistent for women using Letigen

(600 mg/day if used as directed) compared with women who are

exposed to caffeine through coffee, which can be affected by the

type of coffee, how the coffee is prepared, as well as changes in

personal habits [36]. In addition, nausea is unlikely to lead to

mismeasurement of Letigen exposure, but it could affect

measurement of caffeine exposure from coffee [10,30,32,33].

Further, exposure to caffeine from Letigen is not confounded by

other potentially fetotoxic components of coffee although it could

be confounded by ephedrine if ephedrine affects SAB.

Materials and Methods

The Danish National Birth Cohort has been described in detail

elsewhere [37]. Briefly, between March 1, 1996 and November 1,

2002, pregnant women across Denmark were provided informa-

tion about the study during their first prenatal visit. According to a

pilot study, approximately 50% of all pregnant women received an

invitation to participate, and about 60% of those signed the

consent form [37]. The exclusion criteria were not having access to

a telephone, not speaking Danish, and not intending to carry the

pregnancy to term as of the first prenatal visit. A total of 101,051

pregnancies were enrolled.

The Danish National Birth Cohort was approved by the Danish

Scientific Ethics Committee, and this specific study was approved

Figure 1. Study timeline. Timelines based on gestational age showing the risk period for the outcome, the exposure period of interest, the timing of enrollment, and the exposure period covered by the enrollment forms used in a Danish National Birth Cohort study of spontaneous abortion and exposure to a diet drug composed of caffeine and ephedrine, 1996–2002. doi:10.1371/journal.pone.0050372.g001

SAB and a Diet Drug of Caffeine and Ephedrine

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Table 1. Summary of counts, fetus-time at risk, and rates per 10,000 for variables stratified on spontaneous abortion (SAB) vs. other pregnancy outcomes for the Danish National Birth Cohort (1996–2002).

Not SAB SAB

N Fetus-days* N Fetus-days* Rate of SAB/10,000 fetus- days

Letigen use{

None 92,930 6,547,875 4,408 108,617 6.6

Pre-conception only 219 17,680 13 274 7.2

Both pre-conception and early pregnancy 270 21,311 17 346 7.8

Early pregnancy only 37 2,725 5 71 17.9

Missing timing 4 327 0 0

Gestational age at entry (completed weeks)

5–8 29,102 2,766,449 2,878 76,695 10.1

9–12 40,260 2,886,894 1,435 30,006 4.9

13–16 18,680 839,147 118 2,465 1.4

17–20 5,418 97,428 12 142 1.2

Maternal age (years)

15–19 987 64,538 40 1,120 6.1

20–24 11,399 818,878 464 11,806 5.6

25–29 38,814 2,797,147 1,609 40,299 5.7

30–34 31,581 2,183,600 1,506 37,775 6.8

35–39 9,756 666,161 683 15,781 10.0

40–46 923 59,594 141 2,527 22.7

Gravidity

No prior pregnancy 30,573 2,215,384 966 25,344 4.3

$1 prior pregnancy 57,061 3,988,842 2,171 55,864 5.4

Missing 5,826 385,692 1,306 28,100

Planned pregnancy

Planned 66,705 4,785,563 2,440 63,180 5.0

Partially planned 11,080 765,877 371 9,349 4.8

Not planned 9,859 654,187 328 8,757 4.9

Missing 5,816 384,291 1,304 28,022

Infertility treatment

Yes 5,700 386,549 167 4,581 4.3

No 81,923 5,817,343 2,972 76,705 5.0

Missing 5,837 386,026 1,304 28,022

Prepregnancy body mass index

Underweight (,18.5) 3,870 272,001 163 4,507 5.9

Normal (18.5–24.9) 58,493 4,138,880 2,076 52,949 5.0

Overweight (25.0–29.9) 16,707 1,182,334 613 15,805 5.1

Obese (. = 30.0) 7,160 510,755 255 7,114 4.9

Missing 7,230 485,948 1,336 28,933

Exercise (hours/week)

None 55,769 3,920,465 1,589 43,290 4.0

.0 to ,2 17,494 1,241,710 626 16,024 5.0

. = 2 to ,4 9,599 697,141 543 13,291 7.6

. = 4 to ,7 3,504 254,635 256 5,895 9.8

. = 7 1,271 91,193 124 2,774 13.2

Missing 5,823 384,774 1,305 28,034

Smoked during pregnancy (cigarette equivalents/day1)

None 67,521 4,781,746 2,342 60,576 4.8

.0 to 10 14,741 1,046,138 569 14,975 5.4

SAB and a Diet Drug of Caffeine and Ephedrine

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by the Danish Data Protection Board. All women participating in

the cohort provided written informed consent.

Pregnancy Outcome Pregnancy outcome was assessed from Danish national regis-

tries, which can be linked using the unique identifier, the Civil

Registration Number. The Medical Birth Registry and the Civil

Registration System were used to obtain data on live and still

births. Other pregnancy outcomes were identified in the National

Hospital Discharge Register, and emigration prior to the end of

pregnancy was determined from the Civil Registration System.

Less than one percent of the study pregnancies could not be linked

to registry data; in these cases, outcome information from the

interviews was used instead [4]. We excluded 34 pregnancies with

unknown outcomes and 142 ectopic and molar pregnancies.

We defined SAB as an involuntary intrauterine loss between

gestational days 35–146, inclusive (i.e. 5–20 completed weeks)

(Figure 1). Primarily, we based gestational age on the National

Hospital Discharge Register, which contained an estimate of

gestational age that was usually based on a sonogram. However,

for 391 pregnancies, we used the last menstrual period (LMP) date

reported on the enrollment form because it seemed reasonable and

the date from the National Hospital Discharge Register was

missing or seemed incorrect. We excluded 124 pregnancies with

missing or erroneous gestational ages, 586 pregnancies enrolled

after the outcome, 172 pregnancies allegedly enrolled prior to

28 days post-LMP, and 2,090 pregnancies enrolled after 146 days

gestation (i.e. no observed time at risk for SAB), yielding a total of

97,903 pregnancies. Eligible multiple births (n = 2,045) were

treated as a single event; we treated the one eligible pregnancy

with discrepant outcomes as a SAB.

Exposure Assessment The exposure of interest was periconceptional use of Letigen.

Each pill contained 20 mg of ephedrine and 200 mg of caffeine;

the recommended dose was three pills per day [38]. Thus, women

using Letigen as directed ingested caffeine approximately equiv-

alent to drinking five to six cups of coffee a day [39], as well as

ephedrine, and possibly caffeine from other sources. Letigen use

was reported on the enrollment form prior to the pregnancy

outcome. A pregnancy was defined as exposed if any Letigen use

was reported on the enrollment form during the four weeks prior

to the woman’s LMP through 13 completed weeks post-LMP

(n = 565). Twenty-two pregnancies were classified as unexposed

Table 1. Cont.

Not SAB SAB

N Fetus-days* N Fetus-days* Rate of SAB/10,000 fetus- days

.10 5,373 376,952 224 5,656 5.9

Missing 5,825 385,082 1,308 28,101

Alcohol during pregnancy (drinks/week)

No drinks 48,727 3,507,924 1,560 41,792 4.4

.0 to ,4 37,112 2,578,283 1,427 36,548 5.5

. = 4 1,823 120,506 152 2,946 12.3

Missing 5,798 383,205 1,304 28,022

Coffee (cups/day)

None 48,693 3,507,238 1,477 40,674 4.2

.0 to ,4 27,519 1,922,961 1,016 25,636 5.2

4 to ,8 8,493 575,907 440 10,171 7.5

. = 8 2,932 198,957 203 4,734 10.0

Missing 5,823 384,855 1,307 28,093

Tea (cups/day)

None 32,583 2,311,375 1,200 31,184 5.1

.0 to ,4 39,427 2,793,264 1,334 34,381 4.7

4 to ,8 11,928 842,605 428 11,156 5.0

. = 8 3,699 257,799 176 4,534 6.7

Missing 5,823 384,875 1,305 28,053

Cola (liters/week)

None 29,967 2,125,366 1,136 30,021 5.3

.0 to ,1 42,896 3,026,527 1,310 33,935 4.3

. = 1 14,758 1,051,921 689 17,210 6.4

Missing 5,839 386,104 1,308 28,142

*Observed fetus days at risk (i.e. time from entry to outcome, censoring, or end of risk period). { Pre-conception includes the 4 weeks prior to last menstrual period through gestational age 2 completed weeks, early pregnancy includes gestational age 3–13

completed weeks, missing time includes Letigen use during an undefined time. 1 1 cigarette = 1 cigarette equivalent, 1 cherrot = 2 cigarettes equivalents, 1 cigar = 2 cigarette equivalents, 1 pipe = 1.5 cigarette equivalents.

doi:10.1371/journal.pone.0050372.t001

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despite reported Letigen use because the use predated the

periconceptional period. Four women did not report when they

used Letigen; we assumed they were exposed. We also defined pre-

conception Letigen use as occurring four weeks prior to LMP

through two completed gestational weeks, and early pregnancy use

as occurring from three to 13 completed gestational weeks

(Figure 1).

Classification of exposure status was complicated by the fact

that there were two versions of the enrollment form. On the

original form, used for 65% of the pregnancies, medication type

and timing of use were reported in text fields for the three months

prior to filling out the form (Figure 1). On the revised form, the

participant listed medications consumed and marked boxes to

indicate weeks when the medication was used. The boxes covered

the four weeks prior to the participant’s LMP through enrollment

or 13 completed weeks post-LMP, whichever came first (Figure 1).

Four factors affected Letigen exposure assignment. First, women

enrolled at different times in their pregnancies and therefore

reported Letigen use through different gestational ages (Figure 1).

We assumed that no one took Letigen after they knew they were

pregnant. Given that all women knew they were pregnant at

enrollment, this translated into assuming that no one took Letigen

after enrollment. This assumption seems reasonable because

almost all women specifically stated that they stopped taking

Letigen when they became aware of their pregnancy or clearly

reported cessation of use prior to enrollment. This is also

consistent with a recent United States study of birth defects and

retrospectively-reported weight loss products (including over-the-

counter products) that reported exposure dropping from over 1%

prior to pregnancy to 0.2% by the third month of pregnancy [40].

Table 2. Descriptive statistics by Letigen* exposure status for the Danish National Birth Cohort (1996–2002).

Non-Letigen* Letigen*

N (%) N (%)

Gestational age at entry (completed weeks)

5–8 31,703 32.6 277 49.0

9–12 41,481 42.6 214 37.9

13–16 18,738 19.3 60 10.6

17–20 5,416 5.6 14 2.5

Maternal age (years)

15–19 1,023 1.1 4 0.7

20–24 11,764 12.1 99 17.5

25–29 40,183 41.3 240 42.5

30–34 32,911 33.8 176 31.2

35–39 10,399 10.7 40 7.1

40–46 1,058 1.1 6 1.1

Gravidity

No prior pregnancy 31,374 32.2 165 29.2

$1 prior pregnancy 58,874 60.5 358 63.4

Missing 7,090 7.3 42 7.4

Planned Pregnancy

Planned 68,859 70.7 286 50.6

Partially planned 11,336 11.6 115 20.4

Not planned 10,065 10.3 122 21.6

Missing 7,078 7.3 42 7.4

Infertility treatment

Yes 5,852 6.0 15 2.7

No 84,388 86.7 507 89.7

Missing 7,098 7.3 43 7.6

Prepregnancy body mass index

Underweight (,18.5) 4,032 4.1 1 0.2

Normal (18.5–24.9) 60,408 62.1 161 28.5

Overweight (25.0–29.9) 17,109 17.6 211 37.3

Obese (. = 30.0) 7,272 7.5 143 25.3

Missing 8,517 8.7 49 8.7

Exercise (hours/week)

None 57,010 58.6 348 61.6

.0 to ,2 18,035 18.5 85 15.0

. = 2 to ,4 10,086 10.4 56 9.9

. = 4 to ,7 3,739 3.8 21 3.7

. = 7 1,382 1.4 13 2.3

Missing 7,086 7.3 42 7.4

Smoked during pregnancy (cigarette equivalents/day{)

None 69,511 71.4 352 62.3

.0 to 10 15,190 15.6 120 21.2

.10 5,546 5.7 51 9.0

Missing 7,091 7.3 42 7.4

Alcohol during pregnancy (drinks/week)

No drinks 49,980 51.3 307 54.3

.0 to ,4 38,336 39.4 203 35.9

. = 4 1,962 2.0 13 2.3

Table 2. Cont.

Non-Letigen* Letigen*

N (%) N (%)

Missing 7,060 7.3 42 7.4

Coffee (cups/day)

None 49,842 51.2 328 58.1

.0 to ,4 28,410 29.2 125 22.1

4 to ,8 8,883 9.1 50 8.8

. = 8 3,115 3.2 20 3.5

Missing 7,088 7.3 42 7.4

Tea (cups/day)

None 33,567 34.5 216 38.2

.0 to ,4 40,532 41.6 229 40.5

4 to ,8 12,300 12.6 56 9.9

. = 8 3,853 4.0 22 3.9

Missing 7,086 7.3 42 7.4

Cola (liters/week)

None 30,976 31.8 127 22.5

.0 to ,1 43,981 45.2 225 39.8

. = 1 15,276 15.7 171 30.3

Missing 7,105 7.3 42 7.4

*Letigen is composed of 20 mg ephedrine and 200 mg caffeine. { 1 cigarette = 1 cigarette equivalent, 1 cherrot = 2 cigarettes equivalents, 1

cigar = 2 cigarette equivalents, 1 pipe = 1.5 cigarette equivalents. doi:10.1371/journal.pone.0050372.t002

SAB and a Diet Drug of Caffeine and Ephedrine

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For each version of the enrollment form, the mean gestational

age at entry was ten (standard deviation (SD) three) weeks.

However, the two forms referred to different timeframes (Figure 1).

The exposure period of interest was covered by the checkbox

enrollment form and the text-based form for women enrolling by

eight weeks. However, women who used the text-based form and

enrolled after eight completed weeks were asked only about part of

this timeframe. In practice, Letigen users who completed the text-

based form and enrolled late tended to report medication usage

covering the entire timeframe of interest (i.e. usage more than

three months prior to enrollment). However, women who used the

text-based enrollment form and did not report Letigen use might

be mistakenly classified as unexposed if they used Letigen pre-

conceptionally and enrolled late. We examined this issue

analytically by performing sub-analyses where we excluded late

enrollers or stratified on the version of the form used.

The third issue was that the timing of Letigen use was often

reported vaguely on the text-based form. We reviewed each text

field individually and developed rules regarding the timing of

exposure for commonly used phrases (e.g., six days in early

September was assumed to be the middle six days in the first ten

days of the month). Because we assessed broad exposure windows,

most women were clearly exposed or unexposed periconception-

ally, despite nonspecific responses.

The final challenge was that women reported Letigen use

relative to self-reported LMP, which was not always consistent

with the estimated LMP based on the National Hospital Discharge

Register. For 87% of the women, the dates were within a week of

each other (30% were identical) so reports pertained to the

relevant timeframe despite minor date discrepancies. Of the 1,452

pregnancies where LMP dates differed by more than four

completed weeks and the National Hospital Discharge Register

date was determined to be the best estimate, 11 women reported

using Letigen. Their exposure status did not change regardless of

the gestational age definition used. Some of the 1,441 women who

were classified as unexposed might have been exposed if they had

reported on the relevant timeframe only, but the number is likely

to be small. We addressed this uncertainty through a sensitivity

analysis excluding pregnancies for which gestational age was based

on the National Hospital Discharge Register and for which the

self-reported and Register-based LMPs differed by more than four

completed weeks.

Other Variables Maternal age at LMP was calculated using birth date from the

Civil Registration System. Information on all other covariates was

collected through telephone interviews. Of the 97,903 pregnancies

in this study, 7,094 were never interviewed. Of the women

Table 3. Summary statistics for the number of weeks that Letigen* was used at least once among women in the Danish National Birth Cohort (1996–2002).

Weeks

Timeframe of use{ N Mean SD Minimum Maximum

Total eligible time 561 6.3 3.3 1 16

Pre-conception only 232 4.1 1.9 1 7

Both pre-conception and early pregnancy 287 8.7 2.5 2 16

Pre-conception time 6.0 1.9 1 7

Early pregnancy time 2.7 1.5 1 9

Early pregnancy only 42 2.3 1.5 1 8

*Letigen is composed of 20 mg ephedrine and 200 mg caffeine. { Total eligible time includes the 4 weeks prior to last menstrual period through gestational age 13 completed weeks, pre-conception includes the 4 weeks prior to last

menstrual period through gestational age 2 completed weeks, early pregnancy includes gestational age 3–13 completed weeks. Abbreviations: SD, standard deviation. doi:10.1371/journal.pone.0050372.t003

Table 4. Unadjusted and age-adjusted hazard ratios and 95% confidence intervals for the association between Letigen* use and spontaneous abortion (SAB) in the Danish National Birth Cohort (1996–2002).

Unadjusted Age adjusted{

Timeframe of exposure1 SAB Fetus-days$ HR (95% CI) HR (95% CI)

None 4,408 6,656,492 1.0 ref 1.0 ref

Pre-conception only 13 17,954 1.0 0.6; 1.6 1.0 0.6; 1.7

Both pre-conception and early pregnancy 17 21,657 1.0 0.6; 1.7 1.1 0.7; 1.7

Early pregnancy only 5 2,796 2.6 1.1; 6.5 2.7 1.1; 6.6

*Letigen is composed of 20 mg ephedrine and 200 mg caffeine. { Adjusted for maternal age using the following categories: 15–19, 20–24, 30–34, 35–39, 40–46 with 25–29 as the referent.

1 Pre-conception includes the 4 weeks prior to last menstrual period through gestational age 2 completed weeks; early pregnancy includes gestational age 3–13

completed weeks. $Observed fetus days at risk (i.e. time from entry to outcome, censoring, or end of risk period). Abbreviations: HR, hazard ratio; CI, confidence interval. doi:10.1371/journal.pone.0050372.t004

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contacted for the first pregnancy interview, 3,144 were no longer

pregnant and therefore were not interviewed at that time. The

mean gestational age at interview for women who were still

pregnant was 16 completed weeks (range 5–37 weeks). Approx-

imately 80% of the women who were no longer pregnant agreed to

participate in an alternative interview, which for our purposes, was

comparable to the pregnancy interview.

Statistical Analyses We fit Cox regression models accounting for left truncation to

estimate the effect of Letigen on SAB. We used gestational age in

days with entry into observation defined as enrollment in the

study. We employed the robust Lin and Wei confidence interval

option in SAS 9.2 (Cary, North Carolina) to account for the fact

that some women (n = 8,099) contributed more than one

pregnancy to the study. Pregnancies that ended in an induced

abortion (n = 425) and where the mother died (n = 3) or emigrated

(n = 48) prior to 20 completed weeks gestation were censored.

We considered the following potential confounders: maternal

age, pre-pregnancy body mass index (BMI), exercise, smoking,

alcohol consumption, and consumption of caffeine from beverag-

es. Although these factors could conceivably be confounders or

proxies for confounders, none substantially changed the effect-

measure estimates for Letigen separately or as a group (,3%

change in estimate). Given that a higher proportion of women

with SABs did not have interviews compared with women with

other pregnancy outcomes (29% vs. 6%) and the fact that none of

the other covariates appeared to confound the effect-measure

estimate considerably, we opted to report the results for the model

adjusted for maternal age only. This allowed us to include all

eligible pregnancies.

Results

Table 1 provides counts and observed fetus-time at risk for the

study population by outcome. As expected, older women were

more likely to have a pregnancy loss, and as previously published,

SAB was associated with higher coffee consumption during

pregnancy [4]. Letigen use was strongly associated with higher

prepregnancy BMI (Table 2). In addition, Letigen users tended to

drink more cola than non-users, but the two groups were similar

on all other factors including reported coffee consumption and tea

consumption. Among Letigen users, the average number of

gestational weeks with any Letigen use was 6 (SD 3) during the

exposure period interest (Table 3). Most Letigen use was reported

during the pre-conception period rather than early pregnancy.

The maternal age-adjusted hazard ratio (HR) for any Letigen

use compared to no use was 1.1 (0.8–1.6). Table 4 shows

unadjusted and age-adjusted HRs from models examining Letigen

use during different timeframes. Only women who used Letigen

during early pregnancy exclusively had an elevated hazard

(adjusted HR 2.7; 1.6–6.6), but there were only five SABs exposed

to Letigen during this timeframe.

We conducted additional analyses to examine the sensitivity of

our results to possible misclassification of Letigen use. First, we

excluded women who used the text-based form and enrolled after

eight weeks gestation; the age-adjusted HR did not change

substantially from that of the complete study population (1.2; 0.8–

1.7). Excluding all women who enrolled after eight weeks

gestation, regardless of which enrollment form was used, also did

not change the estimate (1.1; 0.8–1.6). Limiting to pregnancies

dated by self-reported LMP (i.e. LMP from the enrollment form

where medication use was reported) or where the Register

estimated LMP differed from the self-reported LMP by fewer

than five weeks yielded an age-adjusted HR of 1.2 (0.9–1.7).

Finally, restricting our analysis to nulligravida women yielded in

an age-adjusted HR of 1.1 (0.5–2.4).

Discussion

Periconceptional Letigen use is not appreciably associated with

SAB in this study. Although pre-pregnancy Letigen use showed

little or no association with SAB, early pregnancy use was

associated with an elevated HR. However, the elevated HR was

based on only five exposed SABs. If Letigen were causally related

to SAB when taken in early pregnancy, it would be expected to be

harmful for any exposed pregnancy, whether or not the woman

also took it prior to conception. However, women exposed during

both periods showed no elevated risk. Further, the effect measure

estimate for Letigen use during early pregnancy only may be

confounded. Although adjusting for measured potential confound-

ers did not change the estimate meaningfully, it is possible that

women who began using Letigen after conception may have been

more likely to participate in unmeasured behaviors that increase

the hazard of SAB.

Some assumptions were made to determine exposure status

during the timeframe of interest; to the degree that we could test

these assumptions, the overall effect-measure estimates appeared

robust. We accounted for left truncation analytically. However,

this approach assumes that the pregnancies under observation at a

given gestational age are representative of all pregnancies at that

gestational age. Bias could be introduced if this was not the case,

especially if entry was associated with the exposure [41]. Further,

gestational age may be less accurate for SABs because live births

are more likely to have a sonogram. However, previous work

suggests bias due to differential accuracy in gestational age by

outcome is likely to be small given realistic assumptions about the

magnitude of these differences [42].

Letigen was composed of ephedrine and caffeine. To our

knowledge, no prior studies looked at weight loss products in

relation to SAB. A National Birth Defects Prevention Study

reported that periconceptional use of weight loss products

containing ephedra (a botanical source of the alkaloid ephedrine)

was associated with increased risk for some birth defects although

the effect-measure estimates were imprecise [40]. While the

relation between diet drugs and SAB has not been studied, an

extensive literature addresses caffeine and SAB [3–26]. Opinions

published in both the United States and the United Kingdom

could not reach consensus regarding the relation between

consumption of high doses of caffeine during pregnancy and

SAB, but both recommended women limit their consumption to

less than 200 mg per day [27,28].

High coffee consumption (greater than 4 cups per day) during

pregnancy is associated with late SAB in the Danish National Birth

Cohort [4], which contrasts with the results for periconceptional

Letigen consumption. The results may differ for several reasons.

First, the study of coffee and SAB only included late losses because

pregnancies interviewed after the outcome were excluded to avoid

recall bias. In contrast, earlier losses were included for pericon-

ceptional Letigen use, which was reported at enrollment,

precluding recall bias. Second, although both coffee and Letigen

contain caffeine, coffee also contains other substances that may

affect SAB. Additionally, caffeine levels from coffee were likely

based on coffee consumption at the time of the interview, which

probably represented average daily exposure after becoming

aware of pregnancy. In contrast, while the dose of caffeine may be

consistent for women taking Letigen (ranging from 200 to 600 mg

of caffeine depending on the number of pills consumed), the timing

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PLOS ONE | www.plosone.org 7 November 2012 | Volume 7 | Issue 11 | e50372

of use during early pregnancy may have been brief or sporadic.

Letigen use was reported for an average of less than three weeks

during the exposure period of interest. It is possible that regular

exposure to high doses of caffeine affects SAB but sporadic

exposure to high doses does not.

One concern in studies of caffeine consumption during

pregnancy is that pregnancy induced nausea can lead to food

and beverage aversions, while lack of nausea is strongly associated

with an increased risk of SAB. As a result, observed associations

between caffeine and SAB could be spurious [10,30,32,33]. An

advantage of assessing pre-pregnancy caffeine consumption is that

the exposure predates the onset of nausea so any observed

association is not a result of nausea’s effect on caffeine

consumption. However, pre-conception caffeine exposure cannot

be generalized to caffeine exposure during pregnancy because

effects may differ during different developmental periods. In

addition, pre-conception caffeine exposure may have an effect on

early pregnancy loss (,5 completed weeks), but not on clinically

recognized SAB.

Women tended to cease using Letigen once they became aware

of their pregnancy. Therefore, there was limited early pregnancy

exposure in this study, but Letigen users were exposed to caffeine

for longer periods prior to conception. Researchers examining pre-

pregnancy (beverage) caffeine consumption and SAB

[9,10,12,14,18–20,22] reported inconsistent results even for high

levels of caffeine. However, the effect-measure estimates for high

caffeine intake prior to pregnancy are smaller than estimates for

high caffeine intake during pregnancy in studies that looked at

both periods. We also found a stronger association between SAB

and Letigen use during early pregnancy compared to pre-

conceptional use although relatively few women consumed

Letigen in early pregnancy exclusively. The association between

SAB and Letigen use during both periods was essentially null.

We did not have adequate data to evaluate whether caffeine

from other medications might confound the association between

Letigen and SAB. Some painkillers and migraine medications that

contained caffeine were available during the study period. These

types of drugs were likely used sporadically and had much lower

caffeine content (50–100 mg/pill) than Letigen (200 mg/pill).

Therefore, it seems likely that few study participants were regularly

exposed to high doses of caffeine from other medications.

We evaluated whether caffeine from beverages could confound

this study. Although women were asked about coffee, tea, and cola

consumption, the questions were not anchored to a specific time

period. We considered whether reported caffeine intake from

beverages could approximate periconceptional caffeine consump-

tion as well as whether caffeine from beverages might be an

intermediate between Letigen and SAB. If the latter were true,

adjustment for beverage caffeine would be inappropriate. Only

cola consumption appeared to be associated with Letigen use in

our data. High prior cola consumption could contribute to obesity,

which could lead to Letigen use, or alternatively, obesity could

lead to both Letigen use and diet cola consumption. In both of

these scenarios cola could be a confounder if caffeine from cola

caused SAB. Adjustment for cola consumption did not change the

results appreciably (1.0; 0.7–1.5) although residual confounding

due to imprecise measurement of cola consumption is possible.

In the Danish National Birth Cohort, periconceptional use of a

diet pill containing ephedrine and caffeine does not appear to be

appreciably associated with clinically recognized SAB.

Acknowledgments

The authors thank Gitte Nielsen for help with the translations; Antonio

Sylvester Vethanayagam for technical support; and Dr. Ivanka Orozova-

Bekkevold, Inge Eisensee, and Lone Fredslund Møller for data extraction.

The authors thank the Helen Riaboff Whiteley Center for providing a

work environment conducive to writing this manuscript.

Author Contributions

Conceived and designed the experiments: PPH IHP BHB EAN AMNA CP

JO. Analyzed the data: PPH. Contributed reagents/materials/analysis

tools: JO. Wrote the paper: PPH IHP BHB EAN AMNA CP JO.

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