open access protocol long-term follow-up of children

1Simons NE, et al. BMJ Open 2021;11:e053066. doi:10.1136/bmjopen-2021-053066

Open access

Long- term follow- up of children exposed in- utero to progesterone treatment for prevention of preterm birth: study protocol of the AMPHIA follow- up

Noor E Simons ,1 Emilie V J van Limburg Stirum,1 Aleid G van Wassenaer- Leemhuis,2 Martijn J J Finken ,3 Cornelieke S H Aarnoudse- Moens,2 Jaap Oosterlaan,4 Anneloes van Baar,5 Tessa J Roseboom,1,6 Arianne C Lim,7 Madelon van Wely,1 Marjon A de Boer,8 Rebecca C Painter,1 Eva Pajkrt,1 Martijn A Oudijk ,1 Janneke van t Hooft,1 AMPHIA study group

To cite: Simons NE, van Limburg Stirum EVJ, van Wassenaer- Leemhuis AG, et al. Long- term follow- up of children exposed in- utero to progesterone treatment for prevention of preterm birth: study protocol of the AMPHIA follow- up. BMJ Open 2021;11:e053066. doi:10.1136/bmjopen-2021-053066

► Prepublication history and additional supplemental material for this paper are available online. To view these files, please visit the journal online (http:// dx. doi. org/ 10. 1136/ bmjopen- 2021- 053066).

Received 03 May 2021Accepted 27 August 2021

For numbered affiliations see end of article.

Correspondence toDr Noor E Simons; n. e. simons@ amsterdamumc. nl

Protocol

© Author(s) (or their employer(s)) 2021. Re- use permitted under CC BY- NC. No commercial re- use. See rights and permissions. Published by BMJ.

ABSTRACTIntroduction Preterm birth is one of the main problems in obstetrics, and the most important cause of neonatal mortality, morbidity and neurodevelopmental impairment. Multiple gestation is an important risk factor for preterm birth, with up to 50% delivering before 37 weeks. Progesterone has a role in maintaining pregnancy and is frequently prescribed to prevent (recurrent) preterm birth and improve pregnancy outcomes in high- risk patients. However, little is known about its long- term effects in multiple gestations. The objective of this follow- up study is to assess long- term benefits and harms of prenatal exposure to progesterone treatment in multiple gestations on child development.Methods and analysis This is a follow- up study of a multicentre, double- blind, placebo- controlled randomised trial (AMPHIA trial, ISRCTN40512715). Between 2006 and 2009 women with a multiple gestation were randomised at 16–20 weeks of gestation to weekly injections 250 mg 17α-hydroxyprogesterone caproate or placebo, until 36 weeks of gestation or delivery. The current long- term follow- up will assess all children (n=1355) born to mothers who participated in the AMPHIA trial, at 11–14 years of age, with internationally validated questionnaires, completed by themselves, their parents and their teachers.Main outcomes are child cognition and behaviour Additional outcomes are death (perinatal and up to age 14), gender identity, educational performance and health- related problems. We will use intention- to- treat analyses comparing experimental and placebo group. To adjust for the correlation between twins, general linear mixed- effects models will be used.Ethics and dissemination Amsterdam UMC MEC provided a waiver for the Medical Research Involving Human Subjects Act (W20_234#20.268). Results will be disseminated through peer- reviewed journals and summaries shared with stakeholders, patients and participants. This protocol is published before analysis of the results.

Trial registration number NL8933.

INTRODUCTIONBackground and rationalePreterm birth, defined as birth before 37 weeks of gestation, is one of the main problems in obstetrics and is the most important cause of neonatal morbidity and mortality.1 It compli-cates 5%–13% of all pregnancies worldwide. Most importantly, children born preterm more often have neurodevelopmental deficits including impairments in cognitive, motor, behavioural and emotional functioning in

Strengths and limitations of this study

► This follow- up study is the first to evaluate outcomes in early adolescence (11–14 years) after maternal progesterone administration in pregnancy.

► Children will be evaluated using internationally vali-dated questionnaires, filled out by themselves, their parents and their teachers, using local normative data.

► We will collect data on school performance at the end of primary school, using a well- validated nation-wide registration system.

► A focus group meeting of women who delivered preterm identified the most essential long- term outcomes after obstetrical interventions, which are used in this study and, therefore, making our results of utmost importance for daily clinical practice.

► The main limitation is that even though question-naires are highly feasible and relatively inexpensive assessment tools, face- to- face assessment to eval-uate development in children could be more useful to detect mild problems.

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2 Simons NE, et al. BMJ Open 2021;11:e053066. doi:10.1136/bmjopen-2021-053066

Open access

early adolescence.2–5 Progesterone has a role in main-taining pregnancy. Antenatal progesterone treatment is effective in preventing preterm birth, and consequently improving neonatal outcomes among women considered at high risk of preterm delivery, for example, due to a history of preterm birth or short cervix in mid trimester.6 7 Multiple gestation is an important risk factor of preterm birth, with 53% of multiple gestations in the Nether-lands born before 37 weeks of gestation.8 Consequently, we evaluated the effect of the synthetic form of proges-terone, 17α hydroxyprogesterone caproate (17- OHPC), on preterm birth prevention in multiple gestations in the AMPHIA trial. We found that 17- OHPC did not prevent neonatal morbidity or preterm birth in multiple gesta-tions. The findings of our study were confirmed by other trials, and showed that progesterone did not reduce the risk of preterm birth in multiple gestations.9

Animal models suggest that natural progestogens have neuroprotective properties.10 11 One of the neuroactive metabolites derived from natural progesterone is allo-pregnanolone. Pharmacological suppression of allopreg-nanolone might increase cell death in the fetal brain.11 12 Furthermore, rats treated with progesterone following a traumatic brain injury, stroke or for Alzheimer’s disease showed improved memory, learning and sensorimotor outcomes compared with untreated rats.13 Animal studies also suggested that perinatal progesterone exposure may alter behaviour. Male rat pups treated neonatally with progesterone showed demasculinisation of behaviour patterns, whereas in female rat pups progesterone was suggested to protect against the virilising effects of sex hormones.14 15 17- OHPC is relatively resilient to metab-olism by classical pathways. Treatment with 17- OHPC, as compared with natural progesterone, is therefore accom-panied by lower quantities of progesterone metabolites, including allopregnanolone, leading to a distinct acti-vation pattern of progesterone receptors.16 The unin-tended effects of synthetic 17- OHPC on the developing brain might therefore be different from those observed after natural progesterone.

Previous studies found no evidence for long- term developmental effects of prenatal progesterone exposure in children aged up to 8 years.17–23 The included studies had used different tests and ages of follow- up, precluding aggregation of evidence. Of these studies, three reported on the follow- up of children following progesterone or placebo use in twin pregnancies.19 22 23 Though the majority of children appear to have a normal develop-ment, these studies suggest progesterone use might lead to lower neurodevelopmental concerns. As expected, the attrition rates at the age of six were high in the studies, limiting the validity of the results and underscoring the necessity of further follow- up studies in children. More-over, no studies evaluated the long- term effect of proges-terone use in multiple gestations in late childhood or early adolescence, when more complex and differenti-ated cognitive functions and behavioural and emotional behaviour become apparent.

To contribute to this knowledge gap, we will study the cognitive, behaviour, mortality, gender identity, educa-tional performance and health- related problems in chil-dren born from mothers that participated in the AMPHIA trial, in children between 11 and 14 years old.

ObjectivesThe aim of this follow- up study is to assess the long- term effect of prenatal exposure of progesterone treatment versus placebo in multiple gestations on child develop-ment (ie, cognition, behaviour, mortality, gender identity, educational performance and health- related problems) in early adolescence.

METHODS AND ANALYSISStudy settingDescription of original AMPHIA trialThis is a follow- up study of a multicentre, double- blind, placebo- controlled randomised controlled trial (AMPHIA trial, ISRCTN40512715, MEC 05/102).24 Between 2006 and 2009 women with a multiple gestation were randomised to receive weekly injections of either 250 mg 17- OHPC or placebo, starting between 16 and 20 weeks of gestation and continued until 36 weeks of gesta-tion or delivery, whichever came first. In the AMPHIA trial participants were followed until discharge from the hospital. Participants, caregivers and investigators of the original trial were blinded for treatment allocation. The number of women that requested to unblind their treatment allocation after the trial is unknown. The primary outcome of the AMPHIA trial was a composite measure of adverse neonatal outcome (ie, severe respi-ratory distress syndrome, bronchopulmonary dysplasia, intraventricular haemorrhage, necrotising enteroco-litis, sepsis and death). This was present in 110 children (16%) born to mothers in the progesterone group, and in 80 children (12%) born to mothers in the placebo group (relative risk 1.34, 95% CI 0.95 to 1.89). Mean gestational age at delivery was 35.4 weeks for the proges-terone group and 35.7 weeks for the placebo group (p value 0.32).24

Current follow-up studyIn this long- term follow- up study we will assess children born to mothers following their participation in the AMPHIA trial, and therefore exposed to 17- OHPC or placebo as fetuses. Data collected in this follow- up study will be linked to maternal, obstetrical and neonatal data collected during the AMPHIA trial. The study protocol is designed, constructed and reported according to the recommendations given in the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) (see online supplemental file 1, SPIRIT checklist for reporting randomised trials and online supplemental file 2, SPIRIT schematic diagram of enrolment).

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Participants/eligibility criteriaThe study population consists of children (n=1355) born to mothers (n=671) who participated in the AMPHIA trial between 11–14 years of age and attending secondary school.

Exclusion criteriaNot able to read and speak Dutch (ie, not able to give informed consent and fill out questionnaires for this follow- up study).

Study designThe follow- up study will be performed within the Dutch consortium for Healthcare Evaluation and Research in Obstetrics and Gynaecology (NVOG Consortium; https:// zorg eval uati ened erland. nl/ NVOG). Research nurses from participating hospitals will be asked to cross-check medical records for possible deaths (of women and offspring) before contacting mothers and their children for participating in this follow- up study. All mothers will be contacted by mail (see online supplemental file 3). Chil-dren have to sign their own informed consent in addition to the parental informed consent form if they are ≥12 years of age. After receiving informed consent of both parents and child(ren), a weblink to the online question-naires will be sent by email. Additionally, this informed consent will allow us to contact teachers or tutors of the children, asking them to participate in an online ques-tionnaire. First inclusion was on 29 September 2020. The expected end date will be in December 2023.

BlindingIn the follow- up study, investigators involved in data collection will be blinded for treatment allocation. We expect that most participants are still blinded for treat-ment allocation. Blinding status will be asked during follow- up. Women who request unblinding during this follow- up study, will be offered allocation information after completion of the follow- up measurements.

Patient involvementOur research team has involved patients in establishing several follow- up studies, all evaluating perinatal interven-tions to improve maternal and neonatal health. Most of these studies evaluated long- term neurodevelopmental outcomes after interventions to prevent preterm birth. Late neurodevelopmental morbidity is one of the core outcomes mentioned in the core outcome set for studies investigating prevention of preterm birth.25 The Dutch patient organisation for parents with a (very) preterm born child ( care4neo. nl) participated in an online survey emphasising the importance and willingness to participate in follow- up studies. Furthermore, our team organised a focus group meeting in 2019 with mothers of children born preterm, to explore the different aspects of long- term development of their children, and their opinion on crucial outcomes that should be assessed in future long- term follow- up studies. The results of this focus group meeting have driven the choices of outcomes

in this follow- up study (see online supplemental file 4, GRIPP2 short form.

OutcomesThe main outcomes of this follow- up study are cogni-tive as well as behavioural and emotional functioning. All outcomes will be evaluated using questionnaires completed by parents, children and teachers. We will report the outcomes of these questionnaires for each informant (eg, cognition reported by parents, children and teachers) and as a composite (eg, abnormal score on cognitive questionnaire by at least one informant). Finally, we will present a composite outcome consisting of abnormal score(s) on cognition and/or behavioural and emotional functioning. See table 1 for an overview of all questionnaires, outcomes and the analyses that will be used. We will report means of continuous scores (with SD) as well as dichotomised scores on basis of the cut- off for abnormal outcome. Abnormal outcomes are defined as ≤−2SD or ‘within clinical range’ following the scoring manual of the questionnaires used.

Cognition ► Executive function: The child’s executive functions

will be assessed with the Behaviour Rating Inven-tory of Executive Function (BRIEF) screener.26 This questionnaire evaluates the executive functions of children between 11 and 17 years of age in 14 items (self- report) or 18 items (parent report) on a 3- point Likert scale. Mean scores will be compared and scores will be transformed in a total score percentile rank, with a higher score indicating weaker executive func-tioning. Norm scores are age and gender dependent and a score of 1.5 SD above the mean of the Dutch reference group is considered abnormal.27

► Current school functioning: – Level of education will be asked in a questionnaire

filled out by parents. – Need for additional help/support inside or out-

side the classroom will be answered by the current teacher/mentor and parent of the child. Additional help is further specified in help with mathematics, reading, writing, physical education, social skills and/or speech therapy.

► Learning progress primary school: The child’s teacher in primary school will be asked to send results of the cito test scores of the final 6 years at primary school. The Dutch pupil monitoring system (cito) developed by the National Institute for Educational Measurement assesses academic performance thought primary school. Most primary schools in the Netherlands use these exams to evaluate the students learning progress. Scores of the cito exams will give us information on the learning progress of the child in primary school on the following subjects: mathematics, spelling, tech-nical reading and reading comprehension . Further-more, we will collect the eighth grade final cito test score (cito eindtoets) or another final test if applicable.

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► Grade repetition and special education: repeating a class/grade in primary and/or secondary school or following special education: This will be answered by the parents.

► Composite cognitive outcomes, at least one of the following applies: – Abnormal BRIEF screener (parent or self- report). – Attending special education.

Behaviour ► Behaviour: the Strength and Difficulties Question-

naire (SDQ) 4–17 years screens for behavioural prob-lems in children and consists of 25 items that inform on five subscales (emotional problems, conduct problems, hyperactivity, peer problems and prosocial behaviour).28 The first four subscales give a total diffi-culties score. A higher total difficulties score indicates more problems. The SDQ will be filled out by parents, children and teachers. A total difficulty score >90th

Table 1 Overview of questionnaires, outcomes and analyses of results

Questionnaire Participant Outcome(s) Outcome measurement

Cognition Behaviour Rating Inventory of Executive Function (BRIEF) Screener26

Parents Total score executive function Mean (SD)Cut- off according to age and gender

BRIEF Screener Self Report26 Child Total score executive function Mean (SD)Cut- off according to age and gender

Additional questions (part of general health questionnaire)

Parents Current level of educationRepeating a class/grade in primary/secondary school

Number (%) of children per educational level including special education.Number (%) of children who repeated a grade.

Additional questions Teacher secondary schoolParents

Additional help inside the classroom or outside the classroom

Number (%)

Citoscores* primary school Teacher primary school Learning progress primary school from third until eighth grade (ability scores)

Mean (SD) grade score from third until eighth grade.Longitudinal relation between learning progress and intervention/placebo.Final eighth grade score equal or below ‘level E’ (≤10 percentile)

Educational performance at the end of primary school

Central Bureau of Statistics Final eighth grade score Final eighth grade score equal or below ‘level E’ (≤10 percentile)

Behaviour Strength and Difficulties Questionnaire28

Parents Total difficulties score Mean (SD)Cut- off according to age and genderCut- off according to subscale

SDQ28 Children Total difficulties score Mean (SD)Cut- off according to age and gender

SDQ28 Teacher secondary school Total difficulties score Mean (SD)Cut- off according to age and gender

Strengths and Weaknesses of ADHD symptoms and Normal- behaviour rating scale41

Parents Attention deficithyperactivity/impulsivenesscombined

Mean (SD)Cut- off <5% (mean+1.65 sd, cut- off; ADHD- C 2.11, ADHD- I 2.48, ADHD- HI 2.00)

Mortality Medical records and the Dutch Personal Records Database

Child Number (%)

Gender identity Gender Identity Questionnaire31

Parents Mean (SD)Cut- off according to age

General health and sociodemographic characteristics

General Health Questionnaire† Parents Number (%)

*National pupil monitoring system scores of the Central Institute for Test Development.42

†A general health questionnaire, developed by our team and used in several other follow- up studies, includes questions on children’s current and previous health (including hospital visits, medication and mental health).ADHD- C, attention deficit hyperactivity disorder- combined; ADHD- HI, attention deficit hyperactivity disorder- hyperactivity/impulsiveness.; ADHD- I, attention deficit hyperactivity disorder- inattentive.

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percentile of the Dutch reference group is considered abnormal.29

► ADHD symptoms: the Strengths and Weaknesses of ADHD symptoms and Normal- behaviour (SWAN) rating consists of 18 items addressing attention deficit and hyperactivity/impulsiveness on a 7- point Likert scale.30 A lower the score on the rating scale, the more attention problems the child has. Cut- off score of <5% is considered abnormal.

► Composite behavioural problems, at least one of the following applies: – Abnormal SDQ (parent, child or teacher report).

Composite abnormal cognition and/or behaviour developmentAs there are many questionnaires reflecting cognition and behaviour in children, and not one outcome is the most important outcome, we included a composite outcome to make a distinction for the most affected children. This will consist of a composite of abnormal cognition and/or behaviour if the child scores abnormal on at least one of the following outcomes:

► Abnormal executive function (BRIEF parent or self- report).

► Attending special education. ► Behavioural problems (SDQ parent, child or teacher

report).

Mortality ► Mortality is defined as perinatal death (from 16

weeks of gestation) and death up to 14 years of age. Medical records and the Dutch Personal Records Database will be used to verify the number of deceased children.

Gender identity ► The Gender Identity Questionnaire for Children

covers a range of gender- typed behaviour questions in 16 items, rating on a 5- point Likert scale.31 These behaviours correspond to various features of the core phenomenology of the Diagnostic and Statis-tical Manual of Mental Disorders (DSM) diagnosis of Gender Dysphoria. Lower scores reflect more cross- gendered behaviour. The DSM threshold for diag-nostic status validated in a Dutch population is used (mean 2.21).31

General health ► In a general health questionnaire, developed by

our team and used in several previous follow- up studies,32–34 questions will be asked on health and growth (concerning child’s current and previous health, including hospital visits, medication and mental health). Health- related problems (assessed in terms of the need for medical specialist consul-tation and/or paramedical care, medication used in the past 12 months, hospital admissions and/or need for surgery) will be clustered in categories to provide insight in the range of health- related problems.

Sociodemographic information ► The general health questionnaire also addresses

demographic information on both parents and child(ren).

Educational performance at the end of primary schoolWe will collect data on the school performance at the end of primary school (cito eindtoets) in all children born in the AMPHIA trial. This analysis will be carried out within the secure microdata environment of Statistic Netherlands (Central bureau of Statistics).35 Anonymous records of the AMPHIA trial will be linked to the educational results within the secure environment and enables compar-ison between progesterone and placebo group, without linking it to the individual participant.

Sample sizeIn the AMPHIA trial, 681 children (336 mothers) were born in the progesterone group, and 674 children (335 mothers) in the placebo group. Since the follow- up is based on this randomised controlled trial, the maximum number of possible participants is fixed. We calculated the minimum number of participants that need to be assessed to find a clinically significant difference, defined as a difference of 0.5 SD, for the main outcomes in offspring.

With a sample size of at 63 per study group we would be able detect a mean difference of 0.5 SD (5.4 points for cognition (BRIEF screener)26 27 and 3.1 point for behaviour (SDQ)36 with a power of 80% and a two- sided alpha of 0.05.

Current practices show a follow- up rate between 30%–70% in follow- up studies using solely questionnaires. Therefore, even at 30% attrition of AMPHIA participants (n=407 children), we will have enough power to detect a clinically important difference of 0.5 SD in mean scores of cognitive and behavioural outcomes.

Statistical analysisDifferences in demographic characteristics of maternal and perinatal outcomes will be compared for participants of the AMPHIA follow- up between progesterone and placebo groups using unpaired t- test, Mann Whitney U test, χ2 test or Fisher’s exact test when appropriate. To estimate presence of attrition bias, we will compare base-line characteristics of follow- up participants to the group of subjects lost to follow- up. All analyses will be based on the intention- to- treat principle. For the outcomes cogni-tion, behaviour and the composite outcome we will report means of continuous scores (with SDs) as well as dichoto-mised scores on basis of the cut- off for abnormal outcome (see table 1). Our analyses will focus on the results from children assessed at follow- up (complete case analysis).37

To adjust for the correlation between children from the same pregnancy we will analyse all data comparing proges-terone versus placebo group with a general linear mixed- effects model. For the linear outcomes this will provide us

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mean differences with their corresponding 95% CI and for the dichotomous outcomes, OR and 95% CI.

For the health- related outcomes only one predeter-mined analysis will be performed in each health- related category (≥3 admissions/medication/surgeries) to reduce the number of tests.

For the outcome of mortality the denominator needs to be changed in the analysis. In this analysis, all randomised children will be included in the denominator. In case the researchers have been able to collect data on mortality in all children (including loss to follow- up) no imputation technique is needed. In case it is not possible to collect

Table 2 WHO trial registration data set

Primary registry and trial identifying number Trial NL8933

Date of registration in primary registry 29 September 2020

Secondary identifying numbers n/a

Source(s) of monetary or material support Amsterdam Reproduction and Development Institute, V.000296.

Primary sponsor Academic Medical Center, Amsterdam, The Netherlands.

Secondary sponsor(s) n/a

Contact for public queries Drs Emilie van Limburg Stirumamphia- [email protected]

Contact for scientific queries Dr Janneke van ’t [email protected]

Public title AMPHIA follow- up.

Scientific title Long- term follow- up of children exposed in- utero to progesterone for prevention of preterm birth: follow- up of the AMPHIA trial.

Countries of recruitment The Netherlands.

Health condition(s) or problem(s) studied Multiple gestations is a strong risk factor for preterm birth, with an incidence of delivery before 37 weeks of gestation of 53% in the Netherlands. Progesterone is extensively studied as an intervention to prevent preterm birth, not only in singleton pregnancies, but also in other high risk pregnancies (such as multiple gestations). However, little is known about the long- term effect of progesterone on the fetus and the development and health of the child later in life.

Intervention(s) n/a

Key inclusion and exclusion criteria The AMPHIA follow- up study population consists of all women that participated in the AMPHIA trial (n=671) and their children (n=1355) between 11–14 years of age.

Study type Follow- up of a randomised controlled trial.

Date of first enrolment 29 September 2020

Sample size In the AMPHIA trial, 681 children (336 mothers) were born in the progesterone group, and 674 children (335 mothers) in the placebo group. Since the follow- up is based on this randomised controlled trial, the maximum number of possible participants is fixed. We calculated the minimum number of participants that need to be assessed to find a clinically significant difference, defined as a difference of 0.5 SD, for the main outcomes in offspring.With a sample size of at 94 per study group we would be able detect a mean difference of 0.5 SD (5.4 points for cognition and 3.1 point for behaviour) with a power of 80% and a two- sided alpha of 0.01 to enable multiple testing.Current practices show a follow- up rate between 30%–70% in follow- up studies using solely questionnaires. Therefore, even at 30% attrition of AMPHIA participants (n=405), we will have enough power to detect a clinical important difference of 0.5 SD in mean scores of cognitive and behavioural outcomes.

Recruitment status Open for patient inclusion.

Primary outcome(s) Cognition and behaviour in children between 11–14 years of age.

Key secondary outcomes Death (perinatal and death up to 14 years of age), gender identity and health- related problems (including information on surgery, hospital admission and medication use). Educational performance at the end of primary school for all children born to women who participated in the AMPHIA trial (regardless of participation in follow- up).

Ethics review The Medical Ethics Committee of the Amsterdam UMC confirmed that the Medical Research Involving Human Subjects Act does not apply to the AMPHIA follow- up (W20_234 # 20.268, date of confirmation 20 May 2020).

Completion date n/a

Summary results n/a

Individual patient data sharing statement n/a

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mortality outcome for all participants, multiple imputa-tion techniques will be considered (depending on the presence of sufficient data to apply this techniques).

Subgroup and/or sensitivity analysesWe will analyses a composite outcome of death or survival with composite abnormal cognitive and/or behaviour development. Combining mortality data with survival of children with a severe developmental disability will help in providing the full scope of relevant outcomes from the start of randomisation until up to 14 years of age. The denominator in this analyses will be all children born in the AMPHIA trial, instead of the number of children included in follow- up. However, this outcome has some challenges due to the high probability of loss to follow- up (as is the case in many obstetrical follow- up studies after several years) and therefore the inevitable need to deal with missing data. This will be done by either applying multiple imputation techniques or, in case of a high loss to follow- up, a worst- case and best- case scenario analyses.

Furthermore, in our main analyses we evaluate a composite of children who score abnormal in at least one of the questionnaires per outcome. Additionally, we will evaluate the number of children that score abnormal in all questionnaires per outcome (eg, for the composite cognition, it will be defined as an abnormal score in the BRIEF screener parent and self- report).

In addition, we will analyse the number of children with a −1 SD score or within ‘subclinical’ range, because a mild delay in children’s development could also interfere with daily activities.

Data managementTo ensure confidentiality, a subject identification code will be used as an identifier. The key to this code is only known by the research team. Handling of the personal data will comply within the General Data Protection Regulation and The Dutch Medical Treatment Contracts Act. An electronic case report from (Castor EDC)38 linked to the unique identification code will be used for data collection and documentation. All questionnaires are filled out through the same data management system (Castor EDC), and directly linked to the individual partic-ipant by unique identification code. All data will be stored for 15 years, according to national guidelines.

DISCUSSIONEvidence on the long- term effects of in utero expo-sure to progesterone treatment is scarce. Although no harmful or beneficial effects are described in literature, research is limited up to the age of 7 years.17 Proges-terone is used for multiple indications and is increas-ingly prescribed in pregnancy. Patients have indicated the need to know more about the long- term effects of obstetrical interventions in respect to child’s behaviour and cognition.

Long- term neurodevelopmental outcome is consid-ered one of the core outcomes in studies evaluating preterm birth prevention.25 However, follow- up studies are time consuming and often beyond the scope of the original randomised controlled trial.39 40 A systematic review showed that only 16% of randomised controlled trials evaluating obstetrical interventions performed a follow- up study.40 The Dutch consortium for Healthcare Evaluation and Research in Obstetrics and Gynaecology took on this task to execute long- term follow- up of all trials performed, even though funding for this type of research is often lacking.

The use of parent and child report questionnaires has the advantage that they are relatively inexpensive and highly feasible. We use mainly internationally validated questionnaires, to increase comparability of results to previous international studies. However, not all ques-tionnaires used have a validated (English) translation. For this reason we will exclude non- Dutch participants. Nonetheless, the number of non- Dutch speaking partic-ipants in the original trial is low, but it could cause a minor selection bias in our follow- up study.

This follow- up study will add valuable information to the existing evidence of long- term effects of prenatal progesterone up to 14 years after exposure in multiple pregnancies. This data will provide information for the increasing number of women taking progesterone in pregnancy and help clinicians to counsel parents with the best available evidence on the risks and benefits of this medication.

Ethics approval and consent to participateThe AMPHIA follow- up aims to assess long- term child-hood outcomes of the AMPHIA trial (ISRCTN40512715). The Medical Ethics Committee of the Amsterdam UMC provided a waiver for the Medical Research Involving Human Subjects Act for the proposed study (W20_234 # 20.268, date of confirmation 20 May 2020) (see https:// zorg eval uati ened erland. nl/ evaluations/ amphia- follow- up for more information).

Written informed consent will be obtained from both parents prior to participation. Children ≥12 years of age have to sign their own informed consent, in addition to the parental informed consent form (see online supple-mental file 3). A copy of the informed consent form(s) will be given to parents and/or children. Participants (parents, children or teachers) can leave the study at any time for any reason if they wish to do so without any consequences.

DisseminationNo arrangements have been made concerning public disclosure. The follow- up study is registered at the Dutch Trial Registry. Date of registration 29 September 2020. For the WHO trial registration data set, see table 2.

Author affiliations1Department of Obstetrics and Gynaecology, Amsterdam Reproduction & Development, Amsterdam UMC Location AMC, Amsterdam, The Netherlands

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2Department of Neonatology and Paediatrics, Emma Children's Hospital, Amsterdam Reproduction & Development, Amsterdam UMC Location AMC, Amsterdam, The Netherlands3Department of Paediatric Endocrinology, Emma Children's Hospital Amsterdam, Amsterdam Reproduction & Development, Amsterdam UMC Location VUmc, Amsterdam, The Netherlands4Department of Paediatrics, Emma Children’s Hospital, Amsterdam Reproduction & Development, Amsterdam UMC Location AMC, Amsterdam, The Netherlands5Child and Adolescent Studies, Utrecht University, Utrecht, The Netherlands6Department of Clinical Epidemiology, Biostatistics and Bioinformatics, Amsterdam UMC Locatie AMC, Amsterdam, The Netherlands7Department of Obstetrics and Gynaecology, Maastricht UMC, Maastricht, The Netherlands8Department of Obstetrics and Gynaecology, Amsterdam Reproduction & Development, Amsterdam UMC location VUmc, Amsterdam, the Netherlands

Acknowledgements We would like to thank the research nurses and research midwives from the Dutch Consortium for Healthcare Evaluation and Research in Obstetrics and Gynecology—NVOG Consortium 2.0 as well as the Dutch Consortium Trialbureau for their efforts. The authors thank the contributors of the AMPHIA trial (AMPHIA study group).

Collaborators AMPHIA study group: Drs. B.M.C. Akerboom, J. Olsthoorn,(Albert Schweitzer Ziekenhuis, Dordrecht); Dr. M.A. Oudijk, Dr. M.A. de Boer, Dr. J.J.H. Bakker, S.M. Ratsma,(Amsterdam UMC, Amsterdam); Dr. D.N.M. Papatsonis, J. Catineau (Amphia Ziekenhuis, Breda); Drs. N.M. van der Velde (Antonius Ziekenhuis, Sneek); Drs. D.H. Schippers (Canisius- Wilhelmina, Nijmegen); Dr. M.E.M.H. Westerhuis, K. Slobben (Catharina ziekenhuis, Eindhoven); Dr. T.E. Vogelvang, T. Voogt (Diakonessen Ziekenhuis, Utrecht); Dr. J. van de Ven (Elkerliek Ziekenhuis, Helmond); Dr. J.J. Duvekot (EramusMC, Rotterdam); Dr. W.M. van Baal, Drs. G. Meertens (Flevoziekenhuis, Almere); Dr. N. van Gemund, A. van Oosten (Sint Franciscus Gasthuis & Vlietland, Rotterdam); A. Ritman (Gelre Ziekenhuis, Apeldoor); Dr. C.A. van Meir, M. Kramer (Groene Hart Ziekenhuis, Gouda); Dr. M.R. Douma, A. van der Ster (Haga Ziekenhuis, Den Haag); Dr. J. van Eyck, N.C.W. van Rijn (Isala Klinieken, Zwolle); Dr. R.J.P. Rijnders, M. Linders (Jeroen Bosch Ziekenhuis, Den Bosch); Dr. M Sueters, M.L. Tendeloo- Klarenbeek (LUMC, Leiden); Dr. E.C. Haagen, S. Smits (Maasziekenhuis Pantein, Beugen); Dr. W.J. van Wijngaarden, E. Gortmaker, MC Haaglanden (Den Haag); Dr. H.C.J. Scheepers, T.A. van Dooren (MUMC+, Maastricht); L.E. Hamming, Dr. D.P. van der Ham, Dr. F.J. Kortweg (Martini Ziekenhuis, Groningen); Dr. J.O.E.H. van Laar, S. van Weelden (Maxima Medisch Centrum, Veldhoven); Drs. P.C.M. van der Salm (MeanderMC, Amersfoort); Dr. M.S. Post (Medisch Centrum Leeuwarden, Leeuwarde)n; Dr. J.A. Kroese, Dr. J. Sikkema, S. Arendsen- Meijer (Medisch Spectrum Twente, Enschede); Dr. N.M. Vink, Nij Smellinghe (Drachten); Dr. M. de Hundt, B. Kruitbosch- Groen (Noordwest Ziekenhuisgroep, Alkmaar); Dr. E.S.A. van den Akker, Dr. E. Moll, Dr. M.G. van Pampus, L. Videler- Sinke, F. Makhloufi (OLVG, Amsterdam); Dr. R. Scholten, G. Zijderveld (RadboudUMC, Nijmegen); Dr. H.A. Bremer, A. van der Ster (Reinier de Graaf Gasthuis, Delft); Dr. K. de Boer (Rijnstate, Arnhem); Dr. M.J. Pelink (Scheper ziekenhuis, Emmen); Dr. K.C. Vollebregt (Spaarne Gasthuis, Haarlem); F. van de Crommenacker (St. Anna Ziekenhuis, Geldrop); Dr. W.M. Klerkx, K. Swarts (St. Antonius Ziekenhuis, Nieuwegein); Dr. S.V. Koenen, L.C.M. van Egeraat (Elisabeth- TweeSteden Ziekenhuis, Tilburg); Dr. E.C. van Asbeck, J. Beutler, V. de Zeeuw (Tergooi, Blaricum); Dr. S.J. Gordijn, J.H.M. Keurentjes (UMC Groningen, Groningen); Dr. M.N. Bekker, M.E. de Reus, H.H.J. Monteiro (UMC Utrecht, Utrecht); Dr. R. van de Laar, VieCuri, Venlo; Dr. A.B. Hooker,(Zaans Medisch Centrum, Zaandam); Dr. J. Langenveld, J. Willems- Robberts (Zuyderland Medisch Centrum, Sittard).

Contributors NES, EVJvLS, AGvW- L, MJJF, CSHA- M, JO, AvB, TJR, ACL, MvW, MAdB, RCP, EP, MAO and JvtH are member of the AMPHIA follow- up study group and were involved in conception and design of the study. NES, EVJvLS and JvtH drafted the manuscript. NES, EVJvLS, AGvW- L, MJJF, CSHA- M, JO, AvB, TJR, ACL, MvW, MAdB, RCP, EP, MAO and JvtH discussed and peer- reviewed the final design of the study. All authors edited the manuscript and read and approved the final version of the manuscript.

Funding This study is funded by Amsterdam Reproduction & Development (AR&D) Institute, V.000296.

Competing interests None declared.

Patient consent for publication Not required.

Provenance and peer review Not commissioned; externally peer reviewed.

Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer- reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.

Open access This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY- NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non- commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is noncommercial. See: http:// creativecommons. org/ licenses/ by- nc/ 4. 0/.

ORCID iDsNoor E Simons http:// orcid. org/ 0000- 0002- 9109- 0800Martijn J J Finken http:// orcid. org/ 0000- 0002- 6589- 9788Martijn A Oudijk http:// orcid. org/ 0000- 0001- 8672- 4365

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Additional file 1. SPIRIT checklist for reporting randomized trials

Section/item ItemNo

Description Adressd on page number

Administrative information

Title 1 Descriptive title identifying the study design,

population, interventions, and, if applicable, trial

acronym

1

Trial registration 2a Trial identifier and registry name. If not yet registered,

name of intended registry

3

2b All items from the World Health Organization Trial

Registration Data Set

17-18, table 2

6Protocol version 3 Date and version identifier n/a

Funding 4 Sources and types of financial, material, and other

support

3

Roles and

responsibilities

5a Names, affiliations, and roles of protocol contributors 1-2

5b Name and contact information for the trial sponsor 2

5c Role of study sponsor and funders, if any, in study

design; collection, management, analysis, and

interpretation of data; writing of the report; and the

decision to submit the report for publication, including

whether they will have ultimate authority over any of

these activities

2, 21

5d Composition, roles, and responsibilities of the

coordinating centre, steering committee, endpoint

adjudication committee, data management team, and

other individuals or groups overseeing the trial, if

applicable (see Item 21a for data monitoring

committee)

21

BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) BMJ Open

doi: 10.1136/bmjopen-2021-053066:e053066. 11 2021;BMJ Open, et al. Simons NE

Introduction

Background and

rationale

6a Description of research question and justification for

undertaking the trial, including summary of relevant

studies (published and unpublished) examining

benefits and harms for each intervention

6-7

6b Explanation for choice of comparators 6-7

Objectives 7 Specific objectives or hypotheses 7

Trial design 8 Description of trial design including type of trial (eg,

parallel group, crossover, factorial, single group),

allocation ratio, and framework (eg, superiority,

equivalence, noninferiority, exploratory)

7

Methods: Participants, interventions, and outcomes

Study setting 9 Description of study settings (eg, community clinic,

academic hospital) and list of countries where data will

be collected. Reference to where list of study sites can

be obtained

8

Eligibility criteria 10 Inclusion and exclusion criteria for participants. If

applicable, eligibility criteria for study centres and

individuals who will perform the interventions (eg,

surgeons, psychotherapists)

8

Interventions 11a Interventions for each group with sufficient detail to

allow replication, including how and when they will be

administered

8-9

11b Criteria for discontinuing or modifying allocated

interventions for a given trial participant (eg, drug dose

change in response to harms, participant request, or

improving/worsening disease)

n/a

11c Strategies to improve adherence to intervention

protocols, and any procedures for monitoring

adherence (eg, drug tablet return, laboratory tests)

n/a

11d Relevant concomitant care and interventions that are

permitted or prohibited during the trial

n/a



Outcomes 12 Primary, secondary, and other outcomes, including the

specific measurement variable (eg, systolic blood

pressure), analysis metric (eg, change from baseline,

final value, time to event), method of aggregation (eg,

median, proportion), and time point for each outcome.

Explanation of the clinical relevance of chosen efficacy

and harm outcomes is strongly recommended

10-13

Participant

timeline

13 Time schedule of enrolment, interventions (including

any run-ins and washouts), assessments, and visits for

participants. A schematic diagram is highly

recommended

Appendix 2

Sample size 14 Estimated number of participants needed to achieve

study objectives and how it was determined, including

clinical and statistical assumptions supporting any

sample size calculations

13-14

Recruitment 15 Strategies for achieving adequate participant

enrolment to reach target sample size

9

Methods: Assignment of interventions (for controlled trials)

Allocation:

Sequence

generation

16a Method of generating the allocation sequence (eg,

computer-generated random numbers), and list of any

factors for stratification. To reduce predictability of a

random sequence, details of any planned restriction

(eg, blocking) should be provided in a separate

document that is unavailable to those who enrol

participants or assign interventions

n/a

Allocation

concealment

mechanism

16b Mechanism of implementing the allocation sequence

(eg, central telephone; sequentially numbered,

opaque, sealed envelopes), describing any steps to

conceal the sequence until interventions are assigned

n/a

Implementation 16c Who will generate the allocation sequence, who will

enrol participants, and who will assign participants to

interventions

n/a



Blinding

(masking)

17a Who will be blinded after assignment to interventions

(eg, trial participants, care providers, outcome

assessors, data analysts), and how

9

17b If blinded, circumstances under which unblinding is

permissible, and procedure for revealing a participant’s

allocated intervention during the trial

9

Methods: Data collection, management, and analysis

Data collection

methods

18a Plans for assessment and collection of outcome,

baseline, and other trial data, including any related

processes to promote data quality (eg, duplicate

measurements, training of assessors) and a

description of study instruments (eg, questionnaires,

laboratory tests) along with their reliability and validity,

if known. Reference to where data collection forms can

be found, if not in the protocol

9

18b Plans to promote participant retention and complete

follow-up, including list of any outcome data to be

collected for participants who discontinue or deviate

from intervention protocols

9

Data

management

19 Plans for data entry, coding, security, and storage,

including any related processes to promote data

quality (eg, double data entry; range checks for data

values). Reference to where details of data

management procedures can be found, if not in the

protocol

15

Statistical

methods

20a Statistical methods for analysing primary and

secondary outcomes. Reference to where other details

of the statistical analysis plan can be found, if not in

the protocol

14

20b Methods for any additional analyses (eg, subgroup and

adjusted analyses)

14-15

20c Definition of analysis population relating to protocol

non-adherence (eg, as randomised analysis), and any

statistical methods to handle missing data (eg, multiple

imputation)

14-15



Methods: Monitoring

Data monitoring 21a Composition of data monitoring committee (DMC);

summary of its role and reporting structure; statement

of whether it is independent from the sponsor and

competing interests; and reference to where further

details about its charter can be found, if not in the

protocol. Alternatively, an explanation of why a DMC is

not needed

n/a

21b Description of any interim analyses and stopping

guidelines, including who will have access to these

interim results and make the final decision to terminate

the trial

n/a

Harms 22 Plans for collecting, assessing, reporting, and

managing solicited and spontaneously reported

adverse events and other unintended effects of trial

interventions or trial conduct

n/a

Auditing 23 Frequency and procedures for auditing trial conduct, if

any, and whether the process will be independent from

investigators and the sponsor

n/a

Ethics and dissemination

Research ethics

approval

24 Plans for seeking research ethics

committee/institutional review board (REC/IRB)

approval

3

Protocol

amendments

25 Plans for communicating important protocol

modifications (eg, changes to eligibility criteria,

outcomes, analyses) to relevant parties (eg,

investigators, REC/IRBs, trial participants, trial

registries, journals, regulators)

n/a

Consent or

assent

26a Who will obtain informed consent or assent from

potential trial participants or authorised surrogates,

and how (see Item 32)

9

26b Additional consent provisions for collection and use of

participant data and biological specimens in ancillary

studies, if applicable

n/a



Confidentiality 27 How personal information about potential and enrolled

participants will be collected, shared, and maintained

in order to protect confidentiality before, during, and

after the trial

15

Declaration of

interests

28 Financial and other competing interests for principal

investigators for the overall trial and each study site

15

Access to data 29 Statement of who will have access to the final trial

dataset, and disclosure of contractual agreements that

limit such access for investigators

15

Ancillary and

post-trial care

30 Provisions, if any, for ancillary and post-trial care, and

for compensation to those who suffer harm from trial

participation

n/a

Dissemination

policy

31a Plans for investigators and sponsor to communicate

trial results to participants, healthcare professionals,

the public, and other relevant groups (eg, via

publication, reporting in results databases, or other

data sharing arrangements), including any publication

restrictions

17

31b Authorship eligibility guidelines and any intended use

of professional writers

n/a

31c Plans, if any, for granting public access to the full

protocol, participant-level dataset, and statistical code

18

Appendices

Informed consent

materials

32 Model consent form and other related documentation

given to participants and authorised surrogates

22, additional

file 3

Biological

specimens

33 Plans for collection, laboratory evaluation, and storage

of biological specimens for genetic or molecular

analysis in the current trial and for future use in

ancillary studies, if applicable

n/a

*It is strongly recommended that this checklist be read in conjunction with the SPIRIT 2013

Explanation & Elaboration for important clarification on the items. Amendments to the

protocol should be tracked and dated. The SPIRIT checklist is copyrighted by the SPIRIT

Group under the Creative Commons “Attribution-NonCommercial-NoDerivs 3.0 Unported” license



http://www.creativecommons.org/licenses/by-nc-nd/3.0/

Additional file 2. Schematic diagram of schedule of enrolment, interventions, and

assessments of the women participating in AMPHIA trial and children participating in

AMPHIA follow-up

STUDY PERIOD

Original AMPHIA - women

STUDY PERIOD

AMPHIA Follow-up - children

Enrolment

original trial

Allocation

original trials

Outcomes

original

trials

Enrolment

follow-up

study

Assessment

follow-up

study

Close-out

TIMEPOINT t = 0

Pregnancy,

childbirth and

neonatal

period

After 11 – 14

years

Age 11-14

years

Age 11-14

years

ENROLMENT:

Eligibility screen X X

Informed consent X X X

Allocation X

INTERVENTIONS:

17-OHPC X

Placebo X

ASSESSMENTS:

Baseline variables X X X

Outcome variables X X X



AMPHIA Follow-up

Informed consent, version 02-09-2020

Zie andere zijde (z.o.z.)

Informed consent form – parent(s) This informed consent form needs to be signed by both parents/guardians.

AMPHIA Follow-up

I have read the information sheet. I was also able to ask questions. My questions have been

answered to my satisfaction. I have had enough time to decide whether me and my

child(ren) will participate in this study.

I know that participation is voluntary. I know that me and my child(ren) may decide at any

time not to participate after all or to withdraw from the study.

I know that some people will be able to access this person’s personal data. These people are

listed in the information sheet.

I give permission for information to be requested from my gynaecologist about my

pregnancy and delivery.

I give permission to fill out questionnaires about my child(ren).

I give permission that the research team will contact the teacher(s) of my child(ren) and that

they fill out questionnaires.

I give permission for information (regarding myself or my child(ren)) to be requested from my

general practitioner or the GGD (‘consultatiebureau’).

I know that it’s possible to be contacted in the future (by post or telephone) after this study

for another follow-up research project, if I give consent for this.

I consent to my data and data regarding my child(ren) being stored at the research location

for another 15 years after this study.

I agree to participation in this study

Date:____ - ____ - _________

Parent 1 / guardian

First and last name parent 1 / guardian: ………………………………………………………………………………………………

Signature parent 1 / guardian:

Parent 2 / guardian

First and last name parent 2 / guardian: ………………………………………………………………………………………………

Signature parent 2 / guardian:



□ not applicable, because: ……………………………………………………………………

First and last name child 1: …………………………………………………………………………………….

Date of birth child 1: ...…………………………………………………………………………………………

First and last name child 2: …………………………………………………………………………………….

Date of birth child 2: ...…………………………………………………………………………………………

To be signed by the AMC investigator:

I declare that I have fully informed this/these person(s) about this study.

Name of investigator (or his/her representative):………………………………………………………………

Date:____ - ____ - _________

Signature:

The study subject will receive the full information sheet, together with a copy of the signed consent

form.



AMPHIA Follow-up

Informed consent, version 02-09-2020

Zie andere zijde (z.o.z.)

Informed consent – children 12 year and older

AMPHIA Follow-up

I have read the information sheet. I was also able to ask questions. My questions have been

answered to my satisfaction. I have had enough time to decide whether I will participate in

this study.

I know that participation is voluntary. I know that I can decide at any time not to participate

after all or to withdraw from the study.

I agree to fill out questionnaires

I agree that my parent(s) fill out questionnaires about me.

I agree that the research team reached out to my teacher(s) and that the teacher(s) fill out

questionnaires about me.

I know that some people will be able to access this person’s personal data. These people are

listed in this information sheet.

I know that it’s possible to be contacted in the future (by post or telephone) after this study

for another follow-up research project, if I give consent for this.

I consent to my data being stored at the research location for another 15 years after this

study.

I have read all above and the information letter and want to participate with this research.

Date:____ - ____ - _________

First and last name child:………………………………………………………………………………………

Date of birth child: ………………………………………………………………………………………

Signature child: ………………………………………………………………………………………

My parent(s)/guardian(s) also agree with participation in this study and sign informed consent on a

separate form.

- I am □ the first born child

□ the second born child



To be signed by the AMC investigator:

I declare that I have fully informed this/these person(s) about this study.

Name of investigator (or his/her representative):………………………………………………………………

Date:____ - ____ - _________

Signature:

The study subject will receive the full information sheet, together with a copy of the signed consent

form.



Additional file 4. GRIPP2 short form

Section and topic Item Reported on page No

1: Aim Report the aim of PPI in the study 8-9

2: Methods Provide a clear description of the methods used for PPI in the

study 9

3: Study results Outcomes—Report the results of PPI in the study, including both

positive and negative outcomes 9

4: Discussion and conclusions Outcomes—Comment on the extent to which PPI influenced the

study overall. Describe positive and negative effects NA

5: Reflections/critical perspective

Comment critically on the study, reflecting on the things that went

well and those that did not, so others can learn from this

experience

NA



open access protocol long-term follow-up of children

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