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Systematic Review
Revised

Oral hormone pregnancy tests and the risks of congenital malformations: a systematic review and meta-analysis

[version 2; peer review: 3 approved]
PUBLISHED 29 Jan 2019
Author details Author details
OPEN PEER REVIEW
REVIEWER STATUS

Abstract

Background: Oral hormone pregnancy tests (HPTs), such as Primodos, containing ethinylestradiol and high doses of norethisterone, were given to over a million women from 1958 to 1978, when Primodos was withdrawn from the market because of concerns about possible teratogenicity. We aimed to study the association between maternal exposure to oral HPTs and congenital malformations.
Methods: We have performed a systematic review and meta-analysis of case-control and cohort studies that included data from pregnant women and were exposed to oral HPTs within the estimated first three months of pregnancy, if compared with a relevant control group. We used random-effects meta-analysis and assessed the quality of each study using the Newcastle–Ottawa Scale for non-randomized studies.
Results: We found 16 case control studies and 10 prospective cohort studies, together including 71 330 women, of whom 4,209 were exposed to HPTs. Exposure to oral HPTs was associated with a 40% increased risk of all congenital malformations: pooled odds ratio (OR) = 1.40 (95% CI 1.18 to 1.66; P<0.0001; I2 = 0%). Exposure to HPTs was associated with an increased risk of congenital heart malformations: pooled OR = 1.89 (95% CI 1.32 to 2.72; P = 0.0006; I2=0%); nervous system malformations  OR = 2.98 (95% CI 1.32 to 6.76; P = 0.0109 I2 = 78%); gastrointestinal malformations, OR = 4.50 (95% CI 0.63 to 32.20; P = 0.13; I2 = 54%); musculoskeletal malformations, OR = 2.24 (95% CI 1.23 to 4.08; P= 0.009; I2 = 0%); the VACTERL syndrome (Vertebral defects, Anal atresia, Cardiovascular anomalies, Tracheoesophageal fistula, Esophageal atresia, Renal anomalies, and Limb defects), OR = 7.47 (95% CI 2.92 to 19.07; P < 0.0001; I2 = 0%).
Conclusions: This systematic review and meta-analysis shows that use of oral HPTs in pregnancy is associated with increased risks of congenital malformations.

Keywords

pregnancy; congenital malformations, hormones

Revised Amendments from Version 1

We thank the reviewers for their positive comments about our manuscript and we have responded to the points made and revised version 1 of the paper in light of their comments. The major changes affect the introduction which has been revised in line with the peer review comments, We have also made changes to the references due to citation errors; uploaded revised excel data sheets as there was an error in the data in the first version, and made one correction in the text of the results to the ‘Exposure to oral HPTs was associated with a 40% increased risk of all congenital malformations: pooled odds ratio (OR) = 1.40 (95% CI 1.18 to 1.66; P < 0.0001; I2 = 0%).', which incorrectly stated the increased risk as 37%. We have also revised the forest plots (Figures 2-8) as the effect estimates were incorrectly labelled.

See the authors' detailed response to the review by Jesse Olszynko-Gryn and Cyrille Jean
See the authors' detailed response to the review by Olalekan Uthman
See the authors' detailed response to the review by David Healy

Introduction

Oral hormone pregnancy tests (HPTs), such as Primodos (known as Duogynon in Germany), were available as injections from 1950 and in tablet form in the UK from 1956 onwards, before the modern forms of urine pregnancy tests became available1. Oral HPTs contained ethinylestradiol and large doses of norethisterone (synthetic forms of estrogen and progesterone respectively), the latter in much larger amounts than those included in current combined oral contraceptives (see Table 1). The test principle was that they would induce bleeding similar to menstruation in those who were not pregnant.

Table 1. Doses of ethinylestradiol and norethisterone in various formulations of contraceptive steroids, ordered by increasing dose of norethisterone.

Indication (oral formulation)Ethinylestradiol doseNorethisterone acetate dose
Progestogen-only contraception*-350 micrograms
Combined oral contraceptive (Loestrin-20)20 micrograms1000 micrograms
Combined oral contraceptive (Norimin)35 micrograms1000 micrograms
Biphasic combined oral contraceptive (BiNovum)35 micrograms500/1000 micrograms
Triphasic combined oral contraceptive (Synphase)35 micrograms500/1000/500 micrograms
Combined oral contraceptive (Loestrin-30)30 micrograms1500 micrograms
Oral hormone pregnancy test (Primodos)20 micrograms10 milligrams
In endometriosis, dysmenorrhoea, dysfunctional
uterine bleeding, and menorrhagia, or to delay
menstruation*
-10–15 milligrams/day
Breast cancer*-40 milligrams/day

*Unbranded

In the UK more than a million women took HPTs2. However, evidence that they should not be used in pregnant women because of a risk of fetal malformations3 led the then Committee on Safety of Medicines in 1975 to conclude that a warning should be added to the Data Sheets, stating that HPTs should not be taken during pregnancy. (Supplementary File 1) Warnings about HPTs in pregnancy first emerged in 1956:4 accumulating concerns over an increased risk of malformations led to their withdrawal in a number of countries at different times. Norway cancelled the indication in pregnancy for HPTs in 1970; when the UK did so in 1978, the manufacturers of Primodos, Schering AG (taken over by Bayer AG in 2008), voluntarily stopped marketing the product; in Germany, Duogynon was taken off the market in 19811.

Since Primodos was withdrawn, the discovery of previously confidential documents has led to renewed concerns about its potential to cause harm5. In 2014, therefore, the Medicines and Healthcare products Regulatory Agency (MHRA) initiated a review, which was published in 2017 and reported that the evidence was insufficient, mixed, and too heterogeneous to support an association between oral HPTs and congenital malformations3.

To date, there has been no systematic review and meta-analysis of oral HPTs, using all the available data, to assess the likelihood of an association. We have therefore performed a systematic review to obtain all relevant data on hormone pregnancy tests and congenital malformations, used meta-analytical tools to obtain summary estimates of the likelihood of an association, and assessed the potential biases in these estimates.

Methods

Data sources

Full details of our search strategy are provided in Supplementary File 2. We searched Medline, Embase, and Web of Science (which yielded German papers and conference abstracts) and searched for regulatory documents online, including the UK Government’s “Report of the Commission on Human Medicines’ Expert Working Group on Hormone Pregnancy Tests”, which includes the original Landesarchiv Berlin Files3, and reference lists of retrieved studies from the start of the databases in 1946 to 20 February 2018.

We used the following search terms without date limits or language restrictions: (Primodos OR Duogynon OR "hormone pregnancy test" OR "sex hormones" OR "hormone administration" OR “norethisterone” OR “ethinylestradiol”) AND pregnancy AND (congenital OR malformations OR anomalies). Several comparable high-dose HPTs were available at the same time as Primodos; we performed additional searches for evidence relating to these (See Supplementary File 3 for List of HPTs included in evidence search).

Study selection

We included observational studies of women who were or became pregnant during the study and were exposed to oral HPTs within the estimated first three months of pregnancy and compared them with a relevant control group. When a study was described in more than one publication, we chose the publication that contained the most comprehensive data as the primary publication. We excluded studies where the intervention was oral hormones taken for other reasons (e.g., oral contraception) and it was not possible to extract data on hormone pregnancy tests. We did not restrict the language of publication. We checked additional relevant data and extracted them from the secondary publications when necessary.

Data extraction and risk of bias assessment

Two reviewers (CH and ES) applied inclusion and quality assessment criteria, compared results, and resolved discrepancies through discussion with the other authors. We used a review template to extract data on study type, numbers of pregnancies exposed and not exposed to oral HPTs, and types and numbers of outcomes. Where available, we extracted data about the women studied, including ascertainment of cases, age, parity, setting, exposure to other medications, and confounding variables. In case-control studies, if data were reported on more than one control group, we extracted data where possible for non-disease/non-abnormality controls, and combined control groups if necessary.

The primary outcome of interest was all major congenital malformations. We also categorized outcomes for the congenital anomaly in the offspring at any time into congenital cardiac, gastrointestinal, musculoskeletal, nervous system, and urogenital defects, and the VACTERL syndrome (Vertebral defects, Anal atresia, Cardiovascular anomalies, Tracheoesophageal fistula, Esophageal atresia, Renal anomalies, and Limb defects).

We assessed quality using the Newcastle–Ottawa Scale (NOS) for non-randomized studies included in systematic reviews6. The scale assesses the selection of study groups (cases and controls), comparability of study groups, including cases and controls, and ascertainment of the outcome/exposure. Each positive criterion scores 1 point, except comparability, which scores up to 2 points. The maximum NOS score is 9, and we interpreted a score of 1 to 3 points as indicating a high risk of bias7. To determine whether the study had controlled for the most important factors, we selected the items reported in the original paper and resolved disagreements through consensus, using a third author (IO). We examined whether there was a linear relation between methodological quality and study results, by plotting the odds ratios against the NOS scores, using Excel, and assessed the correlations of NOS scores with several confounding variables we collected8.

Data synthesis and statistical methods

We calculated study-specific odds ratios for outcomes and associated confidence intervals. We meta-analysed the data using a random-effects model. We assessed heterogeneity across studies using the I2 statistic and publication bias using funnel plots9. We performed a sensitivity analysis by removing single studies to judge the stability of the effect and to explore the effect on heterogeneity10, and we described any sources of variation. We also judged robustness by removing studies of low quality from the analysis. To examine whether the observed heterogeneity could be explained by differences in the NOS score, we also performed meta-regression using the NOS score as the covariate against the log OR as weights for traditional meta-regression using Stata version 14.

We planned subgroup analyses for the timing of administration of HPTs in relation to pregnancy and organogenesis and study design (case-control versus cohort) using Cochran’s Q test. We used RevMan v.5.3 for all analyses, except for meta-regression, for which we used Stata version 14. RevMan and Stata estimate the effects of trials with zero events in one arm by adding a correction factor of 0.5 to each arm (trials with zero events in both arms are omitted). We performed a sensitivity analysis by removing studies with zero events from the analyses.

We followed the reporting guidelines of the Meta-Analysis of Observational Studies in Epidemiology (MOOSE). A completed checklist is available as Supplementary File 411

Patient involvement

Members of the Association for Children Damaged by HPTs were involved in the original discussions of this review and provided input to the outcome choices, the search, the location of study articles, and translations. We plan to present the study findings to relevant patient groups and make available lay interpretations.

Results

Description of included studies

We retrieved 409 items for screening. After title and abstract screening and removal of duplicates (n = 18), we excluded 354 records as not being relevant to the aim of the review. We assessed the full texts of 37 articles and identified 24 articles for inclusion. Figure 1 shows the PRISMA flow diagram for the inclusion of studies.

be519e83-495e-47d1-bcac-bc52e12293b0_figure1.gif

Figure 1. Study flow diagram showing inclusion of relevant studies.

The 24 included articles reported on 26 studies (16 case-control studies and ten prospective cohort studies); one article [Nora 78] included two case-control studies and one prospective study. We found no randomized controlled trials. Of these articles, two were unpublished reports (see Supplementary File 5 for full references). The studies included 71,330 women. The case-control studies included 28,761 mothers, 594 of whom were exposed to HPTs; the cohort studies included 42,569 mothers and 3,615 exposures to HPTs. The studies were published between 1972 and 2014, and all were performed either in Europe or the USA. They mostly recruited women and their infants at maternity centres or hospital paediatrics wards.

The choices of controls in the case-control studies varied; they included, at one extreme, healthy infants born on a date close to the case infants and, at the other extreme, infants with malformations other than those under investigation. Among the prospective cohort studies, the populations tended to be women recruited at antenatal clinics or birth centres (See Table 2. Characteristics of included studies).

Table 2. Characteristics of included studies.

Study IDStudy populationSettingConfounding variables collectedInformation on controls including
matching criteria in case-control
studies
Outcomes reported
Case-control studies
Ferencz 1980Mothers of 110 infants
with conotruncal
abnormalities of the
heart, born 1972–75.
Hospitals served by the
Maryland State Intensive
Care Neonatal Program,
USA.
Maternal health (hospitalisations,
illnesses, treatments); past reproductive
history; index pregnancy factors
including contraception used previously,
fertility treatments, symptoms, illnesses
and medications during pregnancy
including hormones; smoking; alcohol
intake; occupational history of mother
and father; exposure of mother to
fumes, paints and insecticides; family
history including history of congenital
abnormalities in previous children or in
close relatives.
For each case, three unaffected
controls were chosen from the
birth population: two matched on
eight characteristics related to
the likelihood of hormone-taking
(race, maternal age, parity, foetal
losses, gestational age, delivery
mode, time of prenatal registration,
private service), and one also on
the infant’s sex and birthweight; the
third control was chosen at random.
Congenital heart
disease (Conotruncal
malformations of the
heart)
Gal 1972100 mothers of infants
with spina bifida, and
controls
Hospital in London, UK,
for cases; unclear where
controls were recruited
from
Age, parity, reproductive history,
illnesses, illegitimacy, bleeding
Controls matched for week of
baby's birth; age of mother (5-year
bands), reproductive history, course
of pregnancy, sex of baby.
Spina bifida
Greenberg 1977Cases identified via
OPCS and matched
controls identified from
general practices of the
cases.
General practices in
the UK
Antenatal, personal, and family history
and drugs prescribed during the first
trimester.
Controls: babies born within 3
months of and based at the same
general practice as matched cases.
Antenatal, personal, and family
history and drugs prescribed during
the first trimester.
Neural tube defects, oral
clefts, limb malformations
and other non-minor
abnormalities
Janerich 1974 108 cases of congenital
limb defects and 108
unaffected controls
New York State, USAAge, parity, raceControls matched on birth date,
mother's race and age +/- 2 years;
and by default, due to adjacent
records for cases and controls
these matched well on county of
residence of the mothers.
Congenital limb defects
Janerich 1977104 cases with birth
certificate mentioning
CHD, 104 matched
controls
New York State, USAAge, country of residence, date of birth,
race, medications, infections
From adjacent birth record
matched by mother’s age, county of
residence, date of birth, race
Congenital heart disease
Lammer 1986 1,091 mothers of infants
with abnormalities born 1
July 190 to 20 June 1979,
(21% not completed data
collection)
Population registerRace, maternal education, family history,
socio-economic status, parity, previous
foetal loss
Control group was composed of
infants with malformations other
than the one under investigation.
e.g. for spina bifida, controls
were those with non-spina bifida
abnormalities.
Major malformations,
including anencephaly,
spina bifida, cleft lip, cleft
palate, Down syndrome,
oesophageal atresia,
small bowel atresia,
rectal anal atresia,
anterior abdominal wall
defects, diaphragmatic
hernia, limb reduction.
Laurence 19711968-1970, UK3 hospital birth centres
in the UK
Non-reported In London the controls were the
next baby with no abnormality born
in the same hospital; in Exeter,
control mothers were matched for
area of birth, parity and month of
conception; in Wales the control
mothers were those who had had
one baby with spina bifida or
anencephaly and had a subsequent
unaffected birth during the study
period; these last were not matched
individually.
Spina bifida and
anencephaly
Levy 197376 cases, 76 controlsHospital, Montreal,
Canada
Non-reported Controls were infants with
Mendelian disorders, matched for
date of birth.
Congenital heart defects
(transposition of the great
vessels)
Nora 197515 patients with multiple
congenital anomalies.
30 controls (15 with
chromosomal anomalies,
15 with functional heart
murmurs)
University of Colorado
Medical Center, Denver,
and affiliated hospitals,
USA
Age, race, socioeconomic status, area of
residence
Matched for age. 15 controls had
chromosomal abnormalities, 15 had
functional heart murmurs
VACTERL
Nora 1978 case
control 1
32 patients with
VACTERL, 60 controls
Hospital Age, date of birth, sex, gestational age,
race, socioeconomic levels, areas of
residences, parity
Matched as closely as possible for
age, date of birth, sex, gestational
age, race, socioeconomic levels,
areas of residences, parity
VACTERL
Nora 1978 case
control 2 and 3
236 patients with full
variety of cardiac lesions,
412 controls with known
single mutant gene and
chromosomal disorders
Hospital Sex, race, approximate date of birth,
area of residence
Matching was for sex, race,
approximate date of birth, area of
residence
Congenital heart disease
(congenital heart lesions)
Polednak 198399 singleton male births
with hypospadias and 99
matched controls
New York State, USAParity, maternal age, race, area of
residence
Most adjacent birth date, matched
for maternal age, race, area of
residence
Hypospadias
Rothman 1979390 cases, 1,254
controls. HPTS: 14/388
cases vs 35/1246
controls
State care service for
congenital heart disease
Parity, mother's education level, insulin
use, alcohol, tobacco
Controls: births within same 3
years of the study period; 1,254
respondents from contacts to births
selected randomly from the birth
register.
Congenital heart disease
Sainz 1987244 cases identified via
the national collaboration
of 42 hospitals registering
congenital abnormalities
between April 1976 to
Sept 1984
Spanish register of
congenital abnormalities
within 42 participating
hospitals
Sex, data and place of birthControls: unaffected births at same
hospital, matched on sex, date of
birth.
Spina bifida and
anencephaly
Tummler 2014296 cases, 3,676 infants
with abnormalities
Data from the
Malformation Monitoring
Centre Saxony-Anhalt,
Germany.
Non-reported No information on matchingCongenital bladder
exstrophy
Cohort studies
Fleming 1978RCGP Outcomes of
Pregnancy study 1975:
9,000 women; from
this was selected a
random sample of 500
pregnancies proceeding
to normal outcomes
General practices, UK Non-reported Any malformation
Goujard 1979 3,379 women pregnant
and attending
gynaecology clinics
between 1975 to 1977
Obstetrics and
gynaecology centres,
Paris and Lille, France
Information on current pregnancies
including symptoms and medications
taken; previous pregnancies and general
health backgrounds.
Congenital
malformations, also
congenital heart defects,
skeletal anomalies,
microencephaly.
Hadjigeorgiou
1982
Retrospective cohort,
Alexandra Maternity
Hospital Greece, births
1975-77. 15,535 live
births, 559 exposed
to sex hormones of
which 112 (20%)
exposed to HPTs,
14,976 no hormones,
congenital heart disease
studied confirmed
by cardiologist & lab
tests. Diseases and
medication reported at
admission prior to birth.
Hospital birth centreCytomegalovirus, infection,
toxoplasmosis, hepatitis, syphilis, rubella,
teratogenic drugs
Congenital heart disease
Haller 19743588 pregnant women,
recruited Oct 1969 to
April 1972, University
Hospital Göttingen; 617
(17.2 %) with hormonal
pregnancy test
Hospital birth centreNon-reported Congenital malformations
Kullander 19766,376 pregnancies,
Malmo, 1963-5,
resulting in 5,753 live
births, 5,002/5,753 no
abnormality, 751/5,753
with abnormality. 156
women took Primodos.
SwedenMajor and minor disease; the woman's
age, parity, marital status, and social
class. Birth weight, placental weight.
Major and minor
malformations
Meire 1978500 mothers consecutive
births in 3 hospitals in
Bruges, Belgium, 20 had
taken HPTs.
Hospital birth centresNon-reported Oesophageal atresia
Michaelis 198313,643 pregnanciesAntenatal clinics,
Germany
Detailed general and gynaecological
history, drug intake, exposure to
chemical agents, daily workload,
intercurrent diseases, accidents, surgical
operations and other factors.
Major malformations
Roussel 1968 Pregnancies 1966 to
1967
General practices, UKNRCentral nervous
system malformations
including anencephaly,
hydrocephaly,
microcephaly,
meningomyelocele,
myelocele, spina bifida.
Rumeau-
Rouquette 1978
1963-69, recruitment in
12 gynaecology clinics in
Paris; 12,764 women gave
birth to 12,895 children
in hospitals participating
in study; controls were
mothers of unaffected
infants selected at random
among women questioned
in same hospital
Hospital birth centres,
France
Medical history, course of pregnancy,
infectious diseases, inoculations,
reproductive history, social and
occupational category, use of alcohol,
tobacco
Congenital malformations
Torfs 198119,906 full term
pregnancies, 227 of
which exposed to HPTs.
Hospital birth centreAge, medical and reproductive history,
socio- economic information, ethnicity
Severe congenital
anomalies including
congenital heart
defects, neuroblastoma,
cleft lip and limb
reduction; non-severe
congenital anomalies
e.g. hypospadias of the
first degree, congenital
dis-location of the hip,
polydactyly.

Quality assessment of included studies

Of the 26 included studies, three were assigned a NOS score of 3 or below and were therefore judged as being at high risk of bias. One was a case-control study (Laurence 1971, a published abstract as a letter) and two were cohort studies (Fleming 1978 and Haller 1974, both unpublished). The NOS scores ranged from 2 to 9 (median 5). Twelve of the 26 included studies scored 7 to 9 and were judged to be at low risk of bias (see Table 3 of NOS scores in the data files). Item 5 of the NOS score addresses comparability of cases and controls based on design or analysis. Of the 16 case control studies, 12 controlled for the most important factor (item 5a) and nine controlled for important additional factors (item 5b). Of the ten cohort studies, six controlled for the most important factor (item 5a) and four controlled for important additional factors (item 5b). The mean Newcastle–Ottawa scale score was 6.1, indicating an overall moderate risk of bias. Table 2 also shows that seven studies did not report the confounding variables collected (Laurence 1971; Levy 1973; Tummler 2014; Fleming 1978; Haller 1974; Moire 1978; Rousel 1968). NOS scores correlated with the increasing number of confounding variables collected (r = 0.83). Supplementary File 6 shows the funnel plots for all congenital malformations and congenital heart disease; because of inadequate numbers of included studies, we did not use more advanced statistical methods to assess publication bias.

Table 3. Newcastle-Ottawa scale scores for included studies.

Newcastle–Ottawa scale case-control studies
Comparability of cases and controls
on the basis of the design or analysis
Study IDIs the case
definition
adequate?
Are the cases
representative?
Selection
of controls
adequate
Definition
of controls
adequate
a) Study
controls for the
most important
factor
b) Study controls
for important
additional factors
Ascertainment
of exposure
adequate
Same method of
ascertainment
for cases and
controls
Non-response
rate adequate
Total
score /9
Ferencz 1980yesyesyesyesyesyesyesyesyes9
Gal 1972unclearunclearyesyesyesyesyesyesunclear6
Greenberg 1977yesyesyesyesyesyesunclearyesyes8
Janerich 1974 noyesyesnoyesnoyesyesunclear 5
Janerich 1977yesyesunclearyesyesyesyesunclearyes7
Hellstrom 1976yesunclearnoyesyesnounclearyesunclear4
Lammer 1986 yesyesunclearyesyesyesyesyesno7
Laurence 1971yesunclearunclearyesnounclearunclearunclearunclear2
Levy 1973yesyesnoyesyesnounclearunclearunclear4
Nora 1975yesyesyesyesyesnoyesyesyes8
Nora 1978
case control 1
yesyesyesnoyesyesyesyesyes8
Nora 1978
case control 2
and 3
yesyesyesnoyesyesyesyesunclear7
Polednak 1983yesyesyesyesyesnounclearyesunclear6
Rothman 1979yesyesnononoyesyesyesunclear5
Sainz 1987unclearyesyesunclearyesyesunclearyesunclear5
Tummler 2014yesnonononononoyesyes3
Newcastle–Ottawa scale cohort studies
Comparability of cases
and controls on the
basis of the design or
analysis
Are the participants
representative
of the exposed
cohort?
Is the non-
exposed cohort
similar to the
exposed?
Is the
ascertainment
of exposure
accurate?
Is there
evidence that
the outcome
of interest was
not present at
start of study?
a) Study
controls
for the
most
important
factor
b) Study
controls for
important
additional
factors
Was the
assessment
of outcome
adequate?
Was follow-up
long enough
for outcomes
to occur?
Was there
adequacy
of follow
up of
cohorts?
Total
score /9
Fleming 1978yesunclearunclearyesunclearunclearunclearyesunclear3
Goujard 1979 yesyesunclearyesyesnounclearyesyes6
Hadjigeorgiou
1982
yesyesyesyesnoyesyesyesunclear7
Haller 1974unclearunclearunclearyesnonounclearyesunclear2
Kullander
1976
yesyesyesyesyesunclearyesyesyes8
Meire 1978yesnoyesyesyesnounclearyesyes6
Michaelis 1983yesyesyesyesyesyesyesyesyes9
Roussel 1968 yesyesyesyesnonounclearyesyes6
Rumeau-
Rouquette
1978
yesunclearyesyesyesyesyesyesno7
Torfs 1981yesyesyesyesyesyesyesyesyes9

Association of exposure to HPT with the risks of malformations

Nine studies, including 61,642 mothers of infants and 3,274 exposed to HPTs, examined the association in pregnancy with all congenital malformations. Two were case-control studies (Greenberg 1977; Sainz 1987) and seven were cohort studies (Fleming 1987; Goujard 1979; Haller 1974; Kullander 1976; Michaelis 1983; Rumeau-Rouquette 1978; Torfs 1981) (Figure 2). Exposure to oral HPTs was associated with a 40% increased risk of all congenital malformations: pooled odds ratio (OR) = 1.40 (95% CI 1.18 to 1.66; P < 0.0001; I2 = 0%). For the two case-control studies only, pooled OR = 1.70 (95% CI 1.01 to 2.86; P = 0.04; I2 = 63%) and for the seven cohort studies, pooled OR = 1.28 (95% CI 1.05 to 1.56; P = 0.02; I2 = 0%). The test for subgroup differences was not significant (P = 0.32). In a post-hoc sensitivity analysis, removing the studies that collected no confounding variables (Haller 74 and Fleming 78, both of low quality) did not affect the significance of the result (OR 1.44; 95% CI 1.18 to 1.75; P = 0.0004, I2 = 11%). The meta-regression showed no association between total NOS score and increased risk (P = 0.51).

be519e83-495e-47d1-bcac-bc52e12293b0_figure2.gif

Figure 2. Association of exposure to oral HPTs in pregnancy with all malformations in the offspring.

Seven studies, including 19,267 mothers of infants and 218 exposed to oral HPTs, analysed congenital heart malformations. Five were case-control studies (Ferencz 1980; Janerich 1977; Levy 1973; Nora 1978-2/3) and two were cohort studies (Hadjigeorgiou 1982; Torfs 1981) (Figure 3). The pooled relative OR = 1.89 (95% CI 1.32 to 2.72; P = 0.0006; I2 = 0%).

be519e83-495e-47d1-bcac-bc52e12293b0_figure3.gif

Figure 3. Association of exposure to oral hormone pregnancy tests (HPTs) in pregnancy with congenital heart disease in the offspring.

In a post-hoc sensitivity analysis, removing one study that collected no confounding variables (Levy 73, a low-quality study) did not affect the significance of the result (OR = 1.88; 95% CI 1.25 to 2.85; P = 0.003, I2 = 12%) For the five case-control studies only, the pooled OR = 1.87 (95% CI 1.23 to 2.85; P = 0.004; I2 = 9%); for the two cohort studies the pooled OR = 1.95 (95% CI 0.44 to 8.69; P = 0.38; I2 = 32%). The meta-regression was not significant (P = 0.94).

For the association between exposure to oral HPTs and nervous system malformations in the offspring, five studies provided data: three case-control studies (Gal 1972; Laurence 1971; Sainz 1987) and two cohort studies (Roussel 1968; Torfs 1981), including 12 486 mothers of infants and 127 exposed (Figure 4). The pooled OR = 2.98 (95% CI 1.32 to 6.76; P = 0.009; I2 = 78%). In a post-hoc sensitivity analysis, removing the two studies that collected no confounding variables (Laurence 71; Roussel 68) did not affect the significance of the result and removed the heterogeneity (OR 6.04; 95% CI 3.33 to 10.78; P < 0.00001, I2 = 0%).

be519e83-495e-47d1-bcac-bc52e12293b0_figure4.gif

Figure 4. Association of exposure to oral hormone pregnancy tests (HPTs) in pregnancy and nervous system malformations in the offspring.

Gastrointestinal malformations and exposure to oral HPTs were reported in three studies: a case-control study (Lammer 1986) and two cohort studies (Meire 1978 and Torfs 1981), providing data on 2,722 mothers of infants, including 79 exposed to HPTs (Figure 5). The pooled OR = 4.50 (95% CI 0.63 to 32.20; P = 0.13; I2 = 54%). One case-control study (Polednak 1983) and one cohort study (Torfs 1981) examined the relationship between exposure to oral HPTs in pregnancy and urogenital malformations: pooled OR = 2.63 (95% CI 0.84 to 8.28; P = 0.10; I2 = 0%) (Figure 6).

be519e83-495e-47d1-bcac-bc52e12293b0_figure5.gif

Figure 5. Association of exposure to oral hormone pregnancy tests (HPTs) in pregnancy and gastrointestinal malformations in the offspring.

be519e83-495e-47d1-bcac-bc52e12293b0_figure6.gif

Figure 6. Association of exposure to oral hormone pregnancy tests (HPTs) in pregnancy and urogenital malformations in the offspring.

A relation between the exposure to oral HPTs and musculoskeletal malformations was reported in three studies: three case-control studies (Hellstrom 1976; Janerich 1977; Lammer 1986) and one cohort study (Torfs 1981) (Figure 7), based on 2,464 women, with 79 exposed to HPTs. The pooled OR = 2.24 (95% CI 1.23 to 4.08; P = 0.009; I2 = 0%). Removal of the zero study events (Torfs 1981) did not affect this result. The association of VACTERL with HPT exposure was reported in two case-control studies (Nora 1978-1 and Nora 1975), based on 135 women and infants and 27 exposed to HPTs; the OR was 7.57 (95% CI 2.92 to 19.07; P < 0.0001; I2 = 0%) (Figure 8).

be519e83-495e-47d1-bcac-bc52e12293b0_figure7.gif

Figure 7. Association of exposure to oral hormone pregnancy tests (HPTs) in pregnancy and musculoskeletal malformations in the offspring.

be519e83-495e-47d1-bcac-bc52e12293b0_figure8.gif

Figure 8. Association of exposure to oral hormone pregnancy tests (HPTs) in pregnancy with Vertebral defects, Anal atresia, Cardiovascular anomalies, Tracheoesophageal fistula, Esophageal atresia, Renal anomalies, and Limb defects (VACTERL) syndrome in the offspring.

Dataset 1.Study extraction sheet.

Discussion

We found 24 articles containing 26 studies that reported the association between exposure to oral hormone pregnancy tests in mothers and malformations in their infants: 16 were case-control studies and ten were prospective cohort studies. The overall quality of the evidence, assessed by the Newcastle–Ottawa Scale, was moderate.

We found significant associations for all congenital malformations pooled and separately for congenital heart malformations, nervous system malformations, musculoskeletal malformations, and the VACTERL syndrome. Many of these pooled analyses had zero heterogeneity, and the direction of effect favoured the controls in 30 of the 32 analyses undertaken (Torfs 81 provided the only effect estimate favouring HPT exposure). The analyses were also robust to sensitivity analyses, and there was no relation between NOS score and increasing risk.

Based on the assumptions that a teratogenic effect of HPTs would be mediated by actions on estrogen and progestogen receptors, and that concentrations of ethinylestradiol and norethisterone in the fetus would be too low to have a significant effect on those receptors, it has been suggested that there is no mechanistic argument for teratogenicity3. However, other unknown mechanisms might be at play. For example, Isabel Gal first reported concerns of malformations in the children of mothers exposed to HPTs in 196712, pointing out that bleeding often occurred in pregnant women soon after exposure and suggesting that that would affect the “equilibrium” of the uterus. Between 5 and 11% of exposed women had bleeding, and the RCGP survey reported induced abortions in about 10% of women13.

The drugs in Primodos were not tested for animal toxicity and teratogenicity at the time, which, although not unusual, meant that there was a gap in mechanistic understanding. A 2018 study showed that the components in Primodos are associated with dose-dependent and time-related damage in zebrafish embryos, and affect nerve outgrowth and blood vessel patterning in zebrafish12,14. Although it is difficult to compare drug actions between species, and evidence from animal studies is limited, the drugs accumulated in the zebrafish embryos, persisted for some time, and led to rapid embryonic damage12,14. In contrast, other animal studies have shown minimal effects on embryo development15. There is also evidence that estradiol and progestogens increase the expression of mRNA for isoforms of vascular endothelial growth factor (VEGF) in Ishikawa cells from human endometrial adenocarcinoma16.

Strengths and weaknesses

Establishing causal associations in the absence of randomization can be difficult. However, the lack of randomized trials in our analysis should not be seen as a barrier to interpreting our findings. It would have been unethical to randomize individuals to drugs with known concerns, and randomization, like systematic reviews, was not the norm at the time. Furthermore, for questions about harms, the Oxford CEBM levels of evidence puts systematic reviews of case-control studies on a par with systematic reviews of randomized trials17.

However, observational methods have limitations18. First, interpretation can be affected by confounding factors. Although most of the studies in this review used matched controls, our analysis was based on raw data from the publications and did not adjust for confounders. Secondly, susceptibility bias can occur, as women with threatened abortions might be more likely to present and take the medication. Both of these problems can be mitigated by careful matching; 13 of the 16 studies controlled for the most important factor, item 5a on the NOS scale. Thirdly, the severity of malformations studied will have led to differing risk estimates across studies. Fourthly, inappropriate methods of ascertainment of the malformations and exposures could have introduced bias. Finally, incomplete and uneven reporting, along with publication bias (since it is likely that unreported studies exist) could introduce bias and alter the effect estimates.

The use of scoring systems to assess quality has been criticized. However, the NOS scale has been used widely in assessing the quality of non-randomized studies1924. A NOS score between 0 and 9 has previously been used as a potential moderator in meta-regression25, and has been recommended by the Cochrane Collaboration26. A weakness of the NOS scale is the possible low agreement between assessors27. This was particularly the case when authors had limited experience in doing systematic reviews, but training, even of novices, improves agreement19.

The effects were also stable to sensitivity analyses, and changes in NOS score did not affect the risk estimates. The absence of subgroup differences between study designs for the risk estimates supports the robustness of the findings. We also tried to overcome publication bias by translation and assessment of unpublished data. The sample sizes in the studies for all congenital malformations, congenital heart disease, and nervous system malformations were sufficiently large to suggest that small unpublished studies would have little effect on the estimates unless they were highly heterogeneous. The analyses of gastrointestinal, urogenital, musculoskeletal, and VACTERL malformations were limited by their small sample sizes and low number of events: the interpretation of these effects should, therefore, be treated more cautiously. The significant effect observed for VACTERL should also be treated cautiously, as the confidence intervals for this effect were wide.

Our study has several strengths. We used standard systematic review methods, and by asking a focused question solely on exposure to HPTs, and excluding exposure to other hormones, we have been able to assess the heterogeneity of the effect estimates. However, as with any observational studies, there is always the possibility that an unknown confounder could be the cause of the observed difference. While such a possibility cannot be ruled out, the lack of heterogeneity means that such a confounder would potentially have to act in the same direction, despite many different confounders being collected and controlled for. Confounding factors with variable effects on the effect estimates would have probably led to a high degree of heterogeneity, which would have prevented pooling; this was not the case.

Conclusion

Regulators were first made aware of the link between exposure to HPTs and congenital malformations in 1967. After 1975, the Primodos label was changed to state that the medication should not be used in pregnancy because of a risk of malformations (see Figure 9). The evidence of an association has previously been deemed weak, and previous litigation and reviews have been inconclusive. However, we believe that this systematic review shows an association of oral HPTs with congenital malformations.

be519e83-495e-47d1-bcac-bc52e12293b0_figure9.gif

Figure 9. Primodos label 1975 and 1978.

Our results show the benefit of undertaking systematic reviews, a study type not in routine use when most of these studies were done. For example, only one study (Greenberg 1997) out of nine reported a significant effect for all congenital malformations; the pooled estimate was significant. Much of the discussion over the associations of HPTs with congenital malformations at the time these studies were published focused on the lack of significance of individual studies28, although it was also recognized that the numbers involved were insufficient to reject the hypotheses29.

Declarations

Data availability

F1000Research: Dataset 1. Study extraction sheet, https://dx.doi.org/10.5256/f1000research.16758.d23349430

Comments on this article Comments (4)

Version 2
VERSION 2 PUBLISHED 29 Jan 2019
Revised
Version 1
VERSION 1 PUBLISHED 31 Oct 2018
Discussion is closed on this version, please comment on the latest version above.
  • Reader Comment 27 Nov 2018
    Nick Brain, Association for Children Damaged by Hormone Pregnancy Tests, UK
    27 Nov 2018
    Reader Comment
    As Prof. Heneghan and colleagues note, previous studies of the association of Hormone Pregnancy Tests (HPTs) with congenital malformations in children have been acknowledged as being too small to definitively ... Continue reading
  • Reader Comment 08 Nov 2018
    Sarah-Jane Richards, Fortitude Law, UK
    08 Nov 2018
    Reader Comment
    Heneghan et al. 2018 report on a meta-analysis of all available studies on the historical risks of congenital malformations attributable to the oral hormone pregnancy tests (HPTs) – principally Primodos ... Continue reading
  • Reader Comment 06 Nov 2018
    Leonard Lofts, The Northam Care Trust, UK
    06 Nov 2018
    Reader Comment
    Excellent research. Looking forward to observing peer review. The research is so important for so many families.

    Dr Len Lofts
    Competing Interests: No competing interests were disclosed.
  • Reader Comment 06 Nov 2018
    Marie Lyon, Assocation for Children Damaged by Hormone Pregnancy Tests, UK
    06 Nov 2018
    Reader Comment
    Prof. Henegan's systematic analyses of epidemiological studies, is a scientific review which members of the Association for children damaged by HPT's have waited over 45 years for. The findings are ... Continue reading
  • Discussion is closed on this version, please comment on the latest version above.
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Heneghan CJ, Aronson JK, Spencer E et al. Oral hormone pregnancy tests and the risks of congenital malformations: a systematic review and meta-analysis [version 2; peer review: 3 approved]. F1000Research 2019, 7:1725 (https://doi.org/10.12688/f1000research.16758.2)
NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.
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ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions
Version 1
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PUBLISHED 31 Oct 2018
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Reviewer Report 14 Dec 2018
Olalekan Uthman, Division of Health Sciences, Warwick Centre for Applied Health Research and Delivery, Warwick Medical School, University of Warwick, Coventry, UK 
Approved
VIEWS 43
The manuscript reads well. The manuscript was well written. It has the potential to add to the body of knowledge in the field. The method was described in detail to allow for replication of the study. I have no major ... Continue reading
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CITE
HOW TO CITE THIS REPORT
Uthman O. Reviewer Report For: Oral hormone pregnancy tests and the risks of congenital malformations: a systematic review and meta-analysis [version 2; peer review: 3 approved]. F1000Research 2019, 7:1725 (https://doi.org/10.5256/f1000research.18318.r40887)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Author Response 17 Jan 2019
    Carl Carl, University of Oxford, UK
    17 Jan 2019
    Author Response
    Many thanks for these positive comments. The methods and results are clear about the type of studies included, and we consider the conclusions do not require the addition of the ... Continue reading
COMMENTS ON THIS REPORT
  • Author Response 17 Jan 2019
    Carl Carl, University of Oxford, UK
    17 Jan 2019
    Author Response
    Many thanks for these positive comments. The methods and results are clear about the type of studies included, and we consider the conclusions do not require the addition of the ... Continue reading
Views
78
Cite
Reviewer Report 29 Nov 2018
Jesse Olszynko-Gryn, Department of History and Philosophy of Science, University of Cambridge, Cambridge, UK 
Cyrille Jean, Sciences Po, Paris, France 
Approved
VIEWS 78
This is a timely and much-needed paper that deserves to be widely read and cited. It provides the first systematic review and meta-analysis of old epidemiological data pointing towards a long-acknowledged association between HPTs and birth defects. Most of the ... Continue reading
CITE
CITE
HOW TO CITE THIS REPORT
Olszynko-Gryn J and Jean C. Reviewer Report For: Oral hormone pregnancy tests and the risks of congenital malformations: a systematic review and meta-analysis [version 2; peer review: 3 approved]. F1000Research 2019, 7:1725 (https://doi.org/10.5256/f1000research.18318.r40291)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Author Response 17 Jan 2019
    Carl Carl, University of Oxford, UK
    17 Jan 2019
    Author Response
    Many thanks for these positive comments. 

    We have amended the introduction with the following text: 'Oral hormone pregnancy tests (HPTs), such as Primodos (known as Duogynon in Germany), were available as ... Continue reading
COMMENTS ON THIS REPORT
  • Author Response 17 Jan 2019
    Carl Carl, University of Oxford, UK
    17 Jan 2019
    Author Response
    Many thanks for these positive comments. 

    We have amended the introduction with the following text: 'Oral hormone pregnancy tests (HPTs), such as Primodos (known as Duogynon in Germany), were available as ... Continue reading
Views
159
Cite
Reviewer Report 05 Nov 2018
David Healy, School of Medical Sciences, Bangor University, Bangor, UK 
Approved
VIEWS 159
I am fully supportive of this article on the effects of hormone pregnancy tests as it stands. I have no substantive criticism of the content or methods.

I am of course interested in why the regulator (MHRA) ... Continue reading
CITE
CITE
HOW TO CITE THIS REPORT
Healy D. Reviewer Report For: Oral hormone pregnancy tests and the risks of congenital malformations: a systematic review and meta-analysis [version 2; peer review: 3 approved]. F1000Research 2019, 7:1725 (https://doi.org/10.5256/f1000research.18318.r40053)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Author Response 17 Jan 2019
    Carl Carl, University of Oxford, UK
    17 Jan 2019
    Author Response
    Many thanks for these positive comments.

    Signature defects are only likely to occur if the timing of exposure is the same in all cases. However, when the timing of exposure varies, ... Continue reading
COMMENTS ON THIS REPORT
  • Author Response 17 Jan 2019
    Carl Carl, University of Oxford, UK
    17 Jan 2019
    Author Response
    Many thanks for these positive comments.

    Signature defects are only likely to occur if the timing of exposure is the same in all cases. However, when the timing of exposure varies, ... Continue reading

Comments on this article Comments (4)

Version 2
VERSION 2 PUBLISHED 29 Jan 2019
Revised
Version 1
VERSION 1 PUBLISHED 31 Oct 2018
Discussion is closed on this version, please comment on the latest version above.
  • Reader Comment 27 Nov 2018
    Nick Brain, Association for Children Damaged by Hormone Pregnancy Tests, UK
    27 Nov 2018
    Reader Comment
    As Prof. Heneghan and colleagues note, previous studies of the association of Hormone Pregnancy Tests (HPTs) with congenital malformations in children have been acknowledged as being too small to definitively ... Continue reading
  • Reader Comment 08 Nov 2018
    Sarah-Jane Richards, Fortitude Law, UK
    08 Nov 2018
    Reader Comment
    Heneghan et al. 2018 report on a meta-analysis of all available studies on the historical risks of congenital malformations attributable to the oral hormone pregnancy tests (HPTs) – principally Primodos ... Continue reading
  • Reader Comment 06 Nov 2018
    Leonard Lofts, The Northam Care Trust, UK
    06 Nov 2018
    Reader Comment
    Excellent research. Looking forward to observing peer review. The research is so important for so many families.

    Dr Len Lofts
    Competing Interests: No competing interests were disclosed.
  • Reader Comment 06 Nov 2018
    Marie Lyon, Assocation for Children Damaged by Hormone Pregnancy Tests, UK
    06 Nov 2018
    Reader Comment
    Prof. Henegan's systematic analyses of epidemiological studies, is a scientific review which members of the Association for children damaged by HPT's have waited over 45 years for. The findings are ... Continue reading
  • Discussion is closed on this version, please comment on the latest version above.
Alongside their report, reviewers assign a status to the article:
Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions
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