Aspirin Supplementation for Pregnancy Indicated Risk Reduction In Nulliparas (ASPIRIN)
Phase: NA
Conditions: Premature Birth
Sponsor: NICHD Global Network for Women's and Children's Health

Aspirin Protocol V 1.3 
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 Aspirin Supplementation for Pregnancy Indicated 
Risk Reduction In Nulliparas (ASPIRIN) 
ClinicalTrials.gov number: 
NCT02409680 
Protocol number: 
GN-LDA.8.1 
Lead Study Investigator(s): 
Matthew Kendall Hoffman, MD, MPH-Christiana Care 
Richard J Derman, MD, MPH-Thomas Jefferson University 
Bhala S Kodkany, MD-Jawaharlal Nehru Medical College (JNMC) 
Shivaprasad S Goudar, MD, MHPE- JNMC  
Subcommittee Members: 
Marion Koso-Thomas, MD-NICHD 
Menachem Miodovnik, MD-NICHD 
Dennis Wallace, PHD-RTI International 
Elizabeth McClure, PHD-RTI International 
Jennifer J. Hemingway-Foday, MPH-RTI International 
Robert Silver, MD-University of Utah 
Version Number: 
Version 1.3 
Version Date: 
February 21, 2019 
Funding: 
Eunice Kennedy Shriver National Institute of Child Health and Human 
Development 

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Version Tracking 
Version 
Date 
Authors 
Comments 
0.0.1 
03 Nov 2014 
MH, DW, NG, 
EMac 
Draft reviewed by WG at November 12 planning meeting 
0.0.2 
17 Dec 2014 
JHF 
Incorporated WG comments and added more detail to sections on study procedures and data forms in 
preparation for GN Investigator review 
0.1.0 
18 Dec 2014 
NG, JHF 
Minor revisions. This version will be sent to GN investigators for initial review. 
0.1.1 
10 Jan 2015 
JHF in response 
to PI/SFI review 
 
Made revisions to improve clarity and fix grammar/spelling as suggested by reviewers.  
 
Incorporated comments from R. Derman, S. Goudar, E. Liechty, F. Jaeger, C Bose, W. Carlo that need 
to be discussed further. 
 
Updated Sample Size section to consistently note a 20% reduction instead of 25%. 
 
Added secondary outcomes from the survey responses. 
 
Added comments and information from the survey responses for discussion/consideration by WG. 
0.2.0 
19 Jan 2015 
JHF and MH 
 
Finalized revisions from all reviewers. This version was circulated to GN Investigators in advance of 
the Jan SC meeting. 
0.2.1 
29 Jan 2015 
JHF and MH 
 
Revisions based on investigator feedback at SC Meeting 
0.2.2 
06 Feb 2015 
JHF 
 
Further revisions based on investigator and WG feedback 
0.2.3 
12 Feb 2015 
JHF/RTI 
 
Revisions based on internal review at RTI 
0.3 
16 Feb 2015 
Aspirin WG 
 
Updates to language re: pill packaging, anemia monitoring, sample size. Appendix 2 and 3 added.  
0.3.1 
19 Feb 2015 
NICHD 
 
Suggestions from Menachem and Marion 
0.3.2 
20 Feb 2015 
NICHD, MH, RD, 
RTI 
 
Revisions based on group review 
0.3.3 
24 Feb 
 
 
Updated based on group review 
0.3.4 
26 Feb 
NICHD, MH, BM, 
JH 
 
Updates following protocol review 
0.3.5 
05 Mar 2015 
MKT, DW, JHF 
 
Updates to Section 2.3 (study flow chart), Section 2.4.3.1 (Assigning EDD), Section 2.11 
(Risk/benefit), Section 3. (Secondary analysis, DSMB/DMC description), Section 4 (form description). 
0.4.0 
09 March 
2015 
SG, MKT, JHF 
 
Revised sample consent, updated secondary outcome to include perinatal mortality. 
1.0 
10 March 
2015 
WG 
 
Final revisions to fix consistency issues. This is the version circulated to sites for IRB/EC submission. 
1.1 
21 March 
2015 
JHF 
 
Removed “perinatal mortality and morbidity” from the Section 1.1 (Primary Hypothesis or Problem). 
 
This version was sent to the sites to replace version 1.0 for IRB submission. 
1.2 
05 Dec 2016 
NG 
 
Included ClinicalTrials.gov number, added protocol number, made hypertension exclusion explicit 
with definition, updated site information and fixed minor typographical errors. 
1.3 
21 Feb 2019 
NG 
 
Replaced ‘heart rate greater than 110 bpm’ with ‘presence of a heartbeat’ in Section 2.2.1 Inclusion 
Criteria. 
 
Added ‘(hypertensive disorders of pregnancy)’ to Preeclampsia and eclampsia in Section 2.6 
Secondary Outcomes for clarification.  
 
Included ‘Preterm, preeclampsia’ in Section 2.7 Other outcome of interest – Maternal outcomes.  

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Table of Contents 
Aspirin Investigators ..................................................................................................................................... 6 
Acronyms 
 ............................................................................................................................................ 9 
Abstract 
 .......................................................................................................................................... 10 
1. Statement of Problem ............................................................................................................................. 11 
1.1 
Primary Hypothesis or Question .......................................................................... 11 
1.2 
Secondary Hypothesis .......................................................................................... 11 
1.3 
Background and Rationale ................................................................................... 11 
1.3.1 
Biology of Aspirin in Pregnancy .............................................................. 11 
1.3.2 
Aspirin & Preterm Birth .......................................................................... 12 
1.3.3 
Aspirin & Preeclampsia ........................................................................... 12 
1.3.4 
Aspirin & Growth Restriction (Small for Gestational Age) ...................... 13 
1.3.5 
Aspirin safety .......................................................................................... 13 
1.3.6 
Summary ................................................................................................. 13 
2. Methods 
 .......................................................................................................................................... 14 
2.1 
Study Design ........................................................................................................ 14 
2.2 
Study Population .................................................................................................. 14 
2.2.1 
Inclusion Criteria ..................................................................................... 14 
2.2.2 
Exclusion Criteria .................................................................................... 14 
2.3 
Sequence of study activities ................................................................................ 14 
2.4 
Detailed Study Procedures .................................................................................. 16 
2.4.1 
Initial Screening ...................................................................................... 16 
2.4.2 
Consent ................................................................................................... 16 
2.4.3 
Ultrasound Screening ............................................................................. 17 
2.4.4 
Enrollment Procedures ........................................................................... 18 
2.4.5 
Monitoring .............................................................................................. 19 
2.5 
Primary Outcome ................................................................................................. 19 

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2.6 
Secondary Outcomes ........................................................................................... 19 
2.7 
Other Outcomes of interest ................................................................................. 20 
2.8 
Safety monitoring ................................................................................................ 20 
2.9 
Compliance Monitoring ....................................................................................... 21 
2.10 
Site Preparation ................................................................................................... 21 
2.11 
Potential Risks and Benefits to Participants ........................................................ 21 
3.  Analytical Plan ........................................................................................................................................ 23 
3.1 
S tatistical Analysis Plan ....................................................................................... 23 
3.1.1 
Primary Analysis—Risk of Preterm (<37 weeks) Birth ............................ 23 
3.1.2 
Secondary and Exploratory Analysis ....................................................... 23 
3.2 
Sample Size and Power Estimates ....................................................................... 24 
3.3 
Available Population ............................................................................................ 26 
3.4 
Projected Recruitment time ................................................................................ 26 
3.5 
Study Monitoring Plan ......................................................................................... 26 
3.5.1 
Reporting Serious Adverse Events .......................................................... 26 
3.5.2 
Method and Timing for Reporting Serious Adverse Events .................... 27 
3.5.3 
Data Monitoring Plan and Stopping Rules .............................................. 27 
3.6 
Data Management Procedures ............................................................................ 28 
3.7 
Quality Control ..................................................................................................... 29 
3.7.1 
Training ................................................................................................... 29 
3.7.2 
Study Monitoring .................................................................................... 29 
3.7.3 
Drug Quality Assurance and Monitoring ................................................ 29 
3.7.4 
Ultrasound Gestational Age Dating ........................................................ 29 

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4. Data Forms  .......................................................................................................................................... 30 
5. References 
 .......................................................................................................................................... 31 
Appendix 1. Schedule of Study procedures ................................................................................................ 35 
Appendix 2. Sample Informed Consent ...................................................................................................... 36 
Appendix 3. Outcome Definitions ............................................................................................................... 40 
Appendix 4. Study Timeline ........................................................................................................................ 42 
Appendix 5. Aspirin Protocol Steering Committee and Technical Advisors ............................................... 43 
 

 
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ASPIRIN INVESTIGATORS 
Central Team: Global Network Site 08 (Belgaum, India) 
Richard Derman, MD, MPH 
Principal Investigator 
Thomas Jefferson University 
Philadelphia, PA 
Richard.Derman@jefferson.edu  
 
Bhalchandra Kodkany, MD, MBBS 
Senior Foreign Investigator 
KLE University’s J N Medical College 
Belgaum, India 
drkodkany@jnmc.edu 
 
Shivaprasad S. Goudar MD, MHPE 
Co-Investigator 
KLE University’s J N Medical College 
Belgaum, India 
sgoudar@jnmc.edu 
 
Matthew K. Hoffman, MD, MPH 
Lead Investigator for Aspirin Protocol 
Christiana Care  
Newark, Delaware 
Mhoffman@christianacare.org 
Site Investigators 
 
Global Network Site 02 (Democratic Republic of Congo) 
Carl Bose, M.D.  
 
 
 
. 
 
 
 
Principal Investigator 
University of North Carolina School of Medicine 
 
 
 
 
Chapel Hill, North Carolina 
 
 
 
 
 
cbose@med.unc.edu  
 
Antoinette Tshefu, M.D, Ph.D., M.P.H  
Senior Foreign Investigator 
Kinshasa School of Public Health 
antotshe@yahoo.com 
 
Global Network Site 03 (Zambia) 
Wally Carlo, MD 
Principal Investigator 
University of Alabama at Birmingham 
Birmingham, Alabama 
Wcarlo@PEDS.UAB.EDU 
 

 
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Elwyn Chomba, MBChB, DCH, MRCP 
Senior Foreign Investigator 
University Teaching Hospital 
Lusaka, Zambia 
echomba@zamnet.zm 
 
Global Network Site 06 (Guatemala) 
Michael Hambidge, MD 
Principal Investigator 
University of Colorado Health Care System (UCHSC) 
Denver, Colorado 
Michael.hambidge@ucdenver.edu 
 
 
Nancy Krebs, MD 
Co-Investigator 
UCHSC 
Denver, Colorado 
Nancy.krebs@ucdenver.edu 
Ana Garces, MD, MPH 
Senior Foreign Investigator 
INCAP 
Guatemala City, Guatemala 
agarces@incap.net  
 
Global Network Site 09 (Pakistan) 
Robert L. Goldenberg, MD 
Principal Investigator 
Columbia University 
New York, New York 
 
 
 
 
 
rlg88@columbia.edu 
 
 
 
Sarah Saleem, MD 
Senior Foreign Investigator 
Aga Khan University 
Karachi, Pakistan 
Sarah.saleem@aku.edu 
 
Global Network Site 11 (Nagpur, India) 
Patricia L. Hibberd, MD, PhD 
Principal Investigator 
Boston University 
Boston, Massachusetts  
plh0@bu.edu                                                 
 
Archana Patel, MD, DNB, MSCE 
Senior Foreign Investigator 
Lata Medical Research Foundation 
Nagpur, India 
Dr_apatel@yahoo.com 

 
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Global Network Site 12 (Kenya) 
Ed Liechty, MD 
Principal Investigator 
Indiana University School of Medicine 
Indianapolis, Indiana 
eliecht@iu.edu   
 
Fabian Esamai, MBChB, MMed, PhD 
Senior Foreign Investigator 
Moi University School of Medicine 
Eldoret, Kenya 
fesamai2007@gmail.com 
National Institute of Child Health and Human Development (NICHD) 
Marion Koso-Thomas, MD, MPH 
Medical Officer, Global Network for Women’s and Children’s Health  
Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) 
kosomari@mail.nih.gov 
 
Menachem Miodovnik, MD 
Director – Global Network for Women’s and Children’s Health 
Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) 
menachemmiodovnik@mail.nih.gov  
RTI International 
Elizabeth McClure, PhD 
Principal Investigator, Data Coordinating Center 
mcclure@rti.org 
 
Dennis Wallace, PhD 
Co-Principal Investigator, Data Coordinating Center 
Senior Statistician 
dwallace@rti.org 
 
Jay Hemingway-Foday, MPH, MSW 
Protocol Manager for Aspirin Protocol 
hemingway@rti.org 
 
 

 
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ACRONYMS 
ACOG  
American Congress of Obstetricians and Gynecologists  
APS 
 
Antiphospholipid syndrome 
ASA 
 
Acetylsalicylic acid 
BP 
 
Blood pressure 
CLASP  
Collaborative Low-dose Aspirin Study in Pregnancy 
COX  
 
Cyclo-oxygenase 
CRL 
 
Crown rump length 
DCC 
 
Data Coordinating Center 
DMC 
 
Data monitoring committee 
DMS 
 
Data Management System 
DRC 
 
Democratic Republic of Congo 
EAGER  
Effects of Aspirin in Gestation and Reproduction Study 
EDD 
 
Estimated due date (or estimated date of delivery) 
ERC 
 
Ethical review Committee 
GA 
 
Gestational age 
GN 
 
Global Network for Women’s and Children’s Health Research 
Hb 
 
Hemoglobin 
JNMC  
Jawaharlal Nehru Medical College 
IRB 
 
Institutional Review Board 
IUGR 
 
Intrauterine growth restriction 
LBW 
 
Low birth weight   
LDA 
 
Low dose aspirin 
LMP 
 
Last menstrual period 
MNH 
 
Maternal and Newborn Health 
NICHD  
Eunice Kennedy Shriver National Institute of Child Health and Human Development 
NIH 
 
National Institutes of Health 
NSAID   
Non-steroidal anti-inflammatory drug 
PIH 
 
Pregnancy induced hypertension 
PMNCH 
The Partnership for Maternal, Newborn, & Child Health 
PROM   
Premature rupture of membranes 
PTB 
 
Preterm birth 
RCT 
 
Randomized controlled trial 
RTI 
 
Research Triangle Institute International 
SAE 
 
Serious adverse event 
SFI 
 
Senior Foreign Investigator 
SGA 
 
Small for gestational age 
SPTB   
Spontaneous preterm birth 
VLBW  
Very low birth weight 
WHO 
 
World Health Organization 
 
 

 
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ABSTRACT 
Background: Preterm birth (PTB) remains the leading cause of neonatal mortality and long term 
disability throughout the world.  Though complex in its origins, a growing body of evidence suggests that 
first trimester administration of low dose aspirin (LDA) holds promise to reduce the rate of PTB 
substantially. 
Hypothesis: First trimester administration of aspirin will reduce the risk of preterm birth. 
Study Design Type: Prospective randomized, placebo-controlled, double-blinded multicenter clinical 
trial. Trial will be individually randomized with one-to-one ratio (intervention/control) 
Population: Nulliparous women between the ages of 18 and 40, with a singleton pregnancy between 6 
0/7 weeks and 13 6/7 weeks gestational age (GA) confirmed by ultrasound prior to enrollment, no more 
than two previous first trimester pregnancy losses, and no contraindications to aspirin. Minors who are 
≥ 14 years of age may be enrolled if permitted by the country’s ethical guidelines.   
Intervention: Daily administration of low dose (81 mg) aspirin [also known as acetylsalicylic acid (ASA], 
initiated between 6 0/7 weeks and 13 6/7 weeks GA and continued to 36 0/7 weeks GA, compared to an 
identical appearing placebo.  Compliance and outcomes will be assessed biweekly.  
Outcomes:  
Primary outcome: To determine whether daily LDA initiated between 6 0/7 -13 6/7 weeks GA and 
continued to 36 0/7 weeks GA reduces the risk of PTB (birth prior to 37 0/7 weeks GA). 
 
Secondary outcomes of interest are the rate of preeclampsia/eclampsia, small for gestational age (SGA), 
and perinatal mortality. 
 
 

 
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1 STATEMENT OF PROBLEM 
1.1 
PRIMARY HYPOTHESIS OR QUESTION 
Our primary hypothesis is that nulliparous women with no more than two previous first trimester 
pregnancy losses who are treated with LDA daily beginning between 6 0/7 weeks and 13 6/7 weeks GA 
through 36 0/7 weeks GA will reduce the rate of preterm birth from all causes. 
1.2 
SECONDARY HYPOTHESIS  
Women who take antenatal daily LDA initiated at 6 0/7 to 13 6/7 weeks GA will have lower rates of: 
 
Small for gestational age (SGA) 
 
Eclampsia and preeclampsia 
 
Perinatal Mortality 
1.3 
BACKGROUND AND RATIONALE  
Preterm delivery, defined as delivery prior to 37 weeks 0/7 days gestation, remains the dominant cause 
of neonatal morbidity and mortality throughout the world1–3 and directly leads to 28% of neonatal 
deaths within the first seven days of life 4 .  Moreover it is responsible of up to 50% of pediatric 
neurodevelopmental disorders 5. Infants born prematurely are likewise at increased risk for a variety of 
long term medical complications such as respiratory, gastrointestinal, cardiovascular, and metabolic 
disorders 6,7. 
Compounding the issue, the risk of preterm birth is highest in developing countries where an estimated 
12% of births are preterm compared to 5-7% in developed countries 8.  Of the near 13 million preterm 
births worldwide in 2005, 11 million were in Africa and Asia 9.  Given the tremendous medical, financial 
and emotional burden of preterm birth in the developing world and the limited resources to provide 
postnatal care, any interventions with the potential to reduce the rate of preterm birth deserve 
consideration.  If one were to choose an intervention, it would be one that is widely available, 
inexpensive, and safe for the mother and fetus.  Low dose aspirin may be just such an intervention. 
1.3.1 
Biology of Aspirin in Pregnancy 
Aspirin is best known for its analgesic properties; however, it is well documented that aspirin is an anti-
inflammatory and potent inhibitor of platelet aggregation, contributing to its anti-thrombotic effects.  
Both inflammation and thrombosis have been demonstrated to be the pathways responsible for the 
majority of preterm birth, preeclampsia and fetal growth restriction. The primary biologic effects of 
aspirin are mediated by inhibition of the enzyme cyclooxygenase (Cox) 10–12, which produces substances 
known to be involved in the defined pathways  of preterm birth and placental dysfunction 
(Preeclampsia/growth restriction/stillbirth)13.  By decreasing these mediators and in turn decreasing 
inflammation and placental dysfunction due to thrombosis, aspirin may reduce the rates of the major 
obstetrical complications of preterm birth, preeclampsia and growth restriction.  

 
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Equally important to the mechanisms by which Aspirin works, may be the time frame in which it is 
initiated. Biologically it has been demonstrated that the process of preterm birth generally begins prior 
to 16 weeks 13.   Likewise placental invasion of the maternal decidua is noted to occur in the first 
trimester and underlies the pathologic processes of preeclampsia and pathologic growth restriction. 
Thus aspirin may be maximally effective if initiated in the first trimester as proposed in this study. 
1.3.2 
Aspirin & Preterm Birth 
Though the available evidence is promising, data regarding the early use of aspirin in pregnancy to 
prevent preterm birth are limited to two main sources: (1) Meta-analyses of prior trials and (2) a single 
trial of preconception aspirin (Effects of Aspirin in Gestation and Reproduction (EAGeR) Study).  Multiple 
meta- analyses have been performed showing a limited impact on preterm birth. For example Roberge, 
in a meta-analysis of LDA included 22 trials that included 11,302 women, found a 19% reduction in the 
overall rate of preterm birth (RR 0.81, 95% CI 0.71 to 0.92) 14.  When this meta-analysis was restricted to 
women who initiated aspirin prior to 16 0/7 weeks a 65% reduction was observed (6 trials including 904 
women, RR 0.35, 95% CI to 0.22 to 0.57). 
The EAGER trial shows similar trend towards risk reduction in a cohort of 1,078 women who were 
randomized to LDA prior to conception.  If only the original cohort (women with just one prior loss or 
less) is examined, there is a 55% reduction in preterm birth (RR 0.45; p-value of 0.087).  When expanded 
to include the entire cohort (includes women with more than one fetal loss), there is a 28% risk 
reduction (RR of 0.72; p value 0.260).  The lack of significant difference in this trial is not surprising as 
the primary outcome was not preterm delivery and the low risk nature of the enrolled women 
15.  Nonetheless, the point estimates of the sole prospective pre conception LDA trial (EAGeR) and the 
multiple meta-analysis of women who initiated LDA early, suggest that LDA is associated with a decrease 
in the rate of PTB between28% and 65%. 
1.3.3 
Aspirin & Preeclampsia 
Many large randomized controlled clinical trials have been conducted in both high and low risk 
populations (with regard to the development of preeclampsia). Duley and colleagues performed a 
systematic review that included 51 trials involving 36,500 women treated with antiplatelet agents for 
the prevention of preeclampsia 16. Forty-four of these trials involved the use of aspirin alone (compared 
with placebo or no treatment) while the remainder included other treatments, often in conjunction with 
aspirin.  Overall, the use of antiplatelet agents conferred a 19% reduction in the risk of preeclampsia (RR 
0.81; 95% CI, 0.74-0.96).   There was a greater risk reduction in women treated with doses greater than 
75 mg/day (RR 0.49, 95% CI 0.38-0.65) compared to lower doses (RR 0.86, 95% CI 0.79-0.93).  Others 
reviews emphasize the increased benefit from LDA in women with historical risk factors (high risk for 
preeclampsia) and suggest that focusing on at risk groups would decrease the number of women it is 
necessary to treat to prevent a single case of preeclampsia 17–19.  Like prematurity, one sees a difference 
in effect size based on the time of initiation. Roberge in a meta-analysis of 33 trials found a risk 
reduction of 0.62 (0.49 to 0.78); however, when this was restricted to women who began therapy <16 
weeks the effect size was much greater (RR 0.47 (0.36 to 0.62). Of course, the “risk: benefit” ratio also is 
influenced by risk, which appears to be quite low for LDA. Thus, treatment of low risk women may yet 
prove to be justified.  

 
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1.3.4 
Aspirin & Growth Restriction (Small for Gestational Age) 
Low dose aspirin (LDA) also may reduce the risk of other adverse perinatal outcomes such as small for 
gestational age (SGA) fetus and late fetal death.  In 32 trials of 24,310 women, antiplatelet therapy 
conferred an 8% reduction in SGA (RR 0.92, 95% CI 0.85-1.00) in women treated with the intent to 
prevent preeclampsia 16.  A small trial showed no benefit from LDA in women treated after the diagnosis 
of SGA fetus 20.  LDA also has not been effective when started after the diagnosis of preeclampsia 21,22. 
Once again early initiation may be the key to success.  Roberge in a like meta-analysis found a modest 
impact of LDA on growth restriction (RR 0.86, 95%CI 0.75-99); however when only women who initiated 
therapy at or before 16 weeks were examined the effect size was markedly more pronounced (0.46, 
95%CI 0.33-0.66)14.  
The review by Duley and colleagues noted a 16% reduction in combined fetal, neonatal, and infant 
mortality in women taking antiplatelet therapy (RR 0.84, 95% CI 0.74-0.96)23.  Perinatal death and SGA 
fetuses were not primary end points of these trials and results should be interpreted with caution. 
1.3.5 
Aspirin safety 
Low dose aspirin is interesting as a potential therapy for reproductive disorders because it has a 
demonstrated track record of both fetal and maternal safety. Randomized controlled trials (RCTs) in 
thousands of women showed no increase in adverse fetal sequelae in doses < 150 mg per day 16,22,24. In a 
meta-analyses of 22 studies, aspirin was not associated with an overall increase in the risk of congenital 
malformations 25.  The same meta-analysis reported an increase in the risk of gastroschisis (OR 2.37; 95% 
CI, 1.44 – 3.88) in infants exposed to high-dose aspirin (325 mg per day) in the first trimester 25. This is 
biologically plausible since this malformation may be caused by vascular disruption of mesenteric vessels 
26. Results were not confirmed in a recent population based case-control study 27,28 and the association 
between aspirin use and gastroschisis remains uncertain. Indeed, aspirin is often used empirically to 
treat infertility, recurrent miscarriage and Antiphospholipid syndrome (APS). Studies regarding safety 
are difficult to compare due to different doses, duration, and timing of aspirin use (with regard to 
pregnancy).  Nonetheless, the majority of data indicates minimal fetal risk from in utero LDA exposure. 
In fact, a recent study noted that LDA was actually associated with a reduction in neurobehavioral 
difficulties in very preterm infants 29. 
1.3.6 
Summary 
We intend to study the effects of LDA in nulliparous women for several reasons.  First, although it would 
be of interest to study the effect of LDA in women at high risk for preterm birth (e.g. prior preterm 
birth), such women may undergo interventions intended to decrease their risk of preterm birth. Thus, a 
study in patients with prior preterm birth would have numerous potential confounders. Conversely, 
multiparous women with prior term births would be at very low risk for preterm birth and also be a 
suboptimal population to study. Nulliparous women appear to be an ideal population since they will not 
undergo special interventions in an attempt to avoid preterm birth.  Also, it appears that the risk of 
preterm birth in nulliparous women is higher than for the general obstetric population. In summary, 
available data suggest that LDA may be a safe, widely available and inexpensive intervention that may 
significantly reduce the risk of preterm birth. However, this possibility needs to be proven in a properly 
designed RCT with preterm birth as the primary outcome.  Such a clinical trial in a racially, ethnically and 
geographically diverse population could best be accomplished through the established infrastructure of 
the Global Network for Women’s and Children’s Health Research (Global Network or GN). 

 
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2 METHODS 
2.1 
STUDY DESIGN 
We propose a randomized, placebo-controlled, double-blinded multicenter clinical trial to assess the 
efficacy of LDA in the reduction of preterm birth.  Women will be randomized equally to receive either 
daily LDA (81 mg) or an identical appearing placebo beginning between 6 0/7 weeks and 13 6/7 weeks 
GA and continuing until 36 0/7 weeks GA or delivery. 
2.2 
STUDY POPULATION 
Studying the effect of LDA in women at very high risk for preterm birth (e.g., prior preterm birth) would 
have numerous confounders, as these women are likely to undergo interventions intended to decrease 
their risk. Conversely, multiparous women with prior term births would be at very low risk for preterm 
birth, making them a suboptimal study population. Nulliparous women appear to be the ideal 
population to study the effects of LDA since they will not undergo special interventions to avoid preterm 
birth.  Multiple studies have shown that the risk of preterm birth in nulliparous women is higher than for 
the general obstetric population.   
A total of 11,920 nulliparous women will be enrolled (5960 per group) across seven sites in sub-Saharan 
Africa, South Asia, and Latin America. For balance, each site will enroll no more than 25% of the total 
sample. To study the possible effect of LDA on anemia will enroll 500 women, with each site 
contributing at least 10% of the sample.   
2.2.1 
Inclusion Criteria 
 
Nulliparous women between 18 – 40 years of age. Minors who are ≥ 14 years of age may be enrolled 
if permitted by the country’s ethical guidelines.   
 
No more than two previous first trimester pregnancy losses  
 
No medical contraindications to aspirin; 
 
Single live intrauterine pregnancy (IUP) between 6 0/7 and 13 6/7 weeks GA corroborated by an 
early dating ultrasound and with presence of a heartbeat. 
2.2.2 
Exclusion Criteria 
 
Women prescribed daily aspirin for more than 7 days; 
 
Multiple gestations; 
 
Fetal anomaly by ultrasound (Note most fetal anomalies are not detectable by ultrasounds done at 
this early gestation. Subsequent discovery of a fetal anomaly is not viewed as an exclusion.); 
 
Hemoglobin  < 7.0 g/dl at screening; 
 
Systolic blood pressure ≥ 140 and diastolic ≥ 90 at screening; 
 
Any other medical conditions that may be considered a contraindication per the judgment of the 
site investigator (e.g., Lupus, Type 1 Diabetes, or any other known significant disease) 
2.3 SEQUENCE OF STUDY ACTIVITIES 
The sequence of study activities is described in Figure 1. A detailed schedule of study procedures is 
found in Appendix 1.  

 
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2.4 DETAILED STUDY PROCEDURES 
2.4.1 
Initial Screening 
Potential study participants will be recruited from multiple sources so as to reach a diverse study 
population that includes nulliparous women who are pregnant. To reach the maximum number of 
potential participants, clinic-based and community-based recruitment methods will be used. Sites are to 
determine the most effective method for their site.    
Upon identification, a brief assessment of eligibility will be made to determine whether the patient is 
nulliparous and pregnant in the required GA window based on last menstrual period (LMP), has no more 
than two previous 1st trimester pregnancy losses, and has no medical contraindications to aspirin or 
pregnancy. Recognizing that literacy may be a recruitment challenge, study staff will be provided with an 
obstetrical wheel to facilitate GA dating. Knowledge of exact LMP and/or GA are not absolute 
contraindications to study enrollment, as ultrasound will ultimately determine the participant’s GA.  
Nonetheless, it should be noted that the rate of screened versus enrolled participants will be tracked 
centrally and used for study monitoring.  
The initial screening will include collection of information on specific medical conditions, prior surgical 
procedures, medication use, allergy history, and outcomes of any prior pregnancies.  If a 
contraindication to participation in the trial is found, the woman will be excluded from the trial at this 
point.  If the field staff are uncertain about whether a woman is an appropriate candidate, her 
enrollment will be deferred until the study investigators can review her medical history. Women who 
meet the medical history eligibility criteria will proceed to have an ultrasound performed. Potential 
participants will be advised that ultrasound screening activities can be completed on a different day, as 
long as the woman is enrolled during the correct GA window. 
2.4.2 Consent  
Before a woman participates in any research activity, the research staff must obtain her informed consent 
to voluntarily take part in the study. Consent will be obtained from women ≥18 years of age or minors 14-
17 years of age in countries where married or pregnant minors (or their authorized representatives) are 
legally permitted to give consent.  When enrolling minors, we will follow the in-country policies for human 
research protection and the guidelines approved by the local ethical review committees (ERCs). In the 
case of pregnant minors, this may require that written consent is obtained from her parents/guardians or 
husband, with written assent from the minor.   
The research staff will give adequate opportunity to each potential participant and/or immediate family 
members to read the consent form and ask questions. Recognizing that literacy levels will vary and may 
be a challenge, the consent process will include a verbal review of the consent form. If the participant 
cannot read, the form will be read aloud to her by a person unaffiliated with the study. Alternatively, the 
Program Coordinator or a designate may read the consent, but in the presence of a witness who is 
unaffiliated with the research study. Potential participants will be given an opportunity to discuss the 
study procedures and ask questions. Fair balance will be maintained while describing the risks and 
benefits of participation in the study.  No undue pressure will be placed on the potential participant to 
enroll in the trial.  It will further be explained that lack of participation will not affect the usual and 
anticipated standard of care.  

 
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After the potential participant has read the consent form, but before she signs, the research staff will 
show her a sample study pill and confirm that she is willing and able to take the study pills as prescribed.  
Only if she is willing to commit to taking the pills will she be enrolled; otherwise, this will be recorded as 
a refusal of consent. Following review of the consent and drug administration procedures, the 
participant (or parent/guardian) will be asked to sign the form.  If the participant (or parent/guardian) is 
unable to sign her name, she will be asked to use her thumbprint to indicate written approval. In both 
cases, the unaffiliated person will also sign the consent form.  Both the research staff and the study 
participant retain signed copies of the form.  The potential participant may also take the form home to 
discuss with her family before signing. 
An eligible woman may refuse to participate in the trial at the time of recruitment. This will be recorded 
in the Screening and Recruitment Form. She may also choose to withdraw from the study at any time after 
enrollment. This will be recorded on the Withdrawal/Termination Form. Similarly, if an enrolled woman 
becomes ineligible after the time of randomization, she will be withdrawn from the study and this will be 
recorded. 
All research staff responsible for obtaining consent will be trained and certified in the protection of human 
subjects and the study-specific consent procedures. A model written informed consent form, developed 
according to the requirements of the U.S. Office of Research Protections (OHRP), is found in appendix 2.  
Each site may modify the model consent to conform to local standards, but the OHRP required elements 
must be maintained. The research sites will also be responsible for translating the consent form into the 
appropriate language(s) for their local context. 
Global Network countries with legislation regarding the need to videotape consents will comply with the 
country regulations; however this is not part of the consent form requirements. This will not be required 
by protocol but rather decided by each site so as to comply with country’s rules and regulations. 
2.4.3 Ultrasound Screening  
To be included in the study, the following three findings must be obtained via ultrasound: 
 
A single intrauterine gestation 
 
Presence of a fetal heart rate >110 beats per minute 
 
Crown rump length (CRL) 
 
Ultrasound will be assessed transabdominally in all participants consistent with local good clinical 
practice. To obtain the CRL, a total of 3 measurements should be obtained in millimeters and the 
average should be utilized. Other measurements, such as the size of the gestational sac, will not be 
used.  To determine the CRL, the sonographer is to find the mid-sagittal section of the fetus, which 
should be horizontal (at 90º to the angle of insonation). Ideally, the fetus should be in a neutral position 
(not hyperextended or flexed) and the fetal image should fill at least 30% of the monitor screen.  
Measurement of the CRL is done by placing the intersection of the calipers on the outer borders of the 
head and rump. All study sonographers will be trained to assess gestational age using CRL 
measurements and quality will be monitored as described in Section 3.7.4. The ultrasound screening 
does not need to be completed on the same day as the other screening activities, as ultrasound is likely 
to be conducted by different staff and location than initial screening.  

 
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2.4.3.1 Assigning Projected Estimated Due Date (EDD)  
It is noted that an accurate LMP will frequently be unavailable for sizable numbers of women enrolled in 
this study.  The assignment of the projected due date will be consistent with the recent direction 
provided by the NICHD consensus statement as seen in Table 1 30.  
Table 1. Guidelines for Assigning Projected Due Date  
Gestational Age 
by LMP 
Difference between 
LMP and US GA 
Basis for assigning 
projected due date 
Unknown/Not 
available 
N/A 
CRL 
< 9 0/7 weeks 
> 5 days 
CRL 
≤ 5 days 
LMP 
≥ 9 0/7 and ≤13 
6/7 weeks 
> 7 days 
CRL 
≤ 7 days 
LMP 
2.4.4 Enrollment Procedures 
2.4.4.1 Clinical Assessment 
Following confirmation of eligibility and attainment of signed informed consent, each participant will be assessed 
to determine clinical status. The assessment will consist of a brief physical examination, medical history, and 
questions about the use of other medicines or medicinal products. It will also include a baseline hemoglobin (Hb) 
measurement. If the Hb measurement is < 7.0 g/dl, she will be excluded from the study.  
2.4.4.2 Randomization Procedures  
Randomization of subjects will be carried out to obtain a 1:1 allocation ratio between the treatment and 
placebo arms.  Randomization will be stratified by site.  A computer algorithm generated by the data 
coordinating center (DCC) will create the random assignment to one of the treatment arms based on 
randomly permuted block design with randomly varied block sizes. The block sizes will be known only by 
the DCC personnel.  
2.4.4.3 Study Intervention and Comparison  
The study intervention is 81 mg of aspirin administered daily beginning between 6 0/7 weeks and 13 6/7 
weeks through 36 0/7 weeks or delivery. Though 81 mg and 75 mg doses may both be found as standard 
doses in developing countries, the 81 mg dose was selected because it is consistent with the dosage 
used in other large trials such as EAGER, is the standard in five of the seven participating sites, and 
introduces no additional risk over the 75 mg dose. In fact, Duley’s meta-analysis showed a greater risk 
reduction for the development of preeclampsia associated with proteinuria in women treated with 
doses greater than 75 mg/day (RR 0.49, 95% CI 0.38-0.65) compared to lower doses (RR 0.86, 95% CI 
0.79-0.93).23 
Following randomization, each woman will be provided with a supply of either LDA or an identical 
appearing placebo. The drug or placebo will be enteric coated pills, provided in identical, child-resistant 
packaging, with written instructions for use. To ensure that literacy levels will not affect proper use, the 
study staff will provide verbal instructions at randomization and reinforce these guidelines at 
subsequent follow-up visits. Each woman will also receive a back-up supply of medication or placebo, 
which will be maintained throughout the duration of the study.  The purpose of the backup is to bridge 

 
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circumstances wherein the woman misses or is late to a planned follow-up visit or the primary supply of 
study drug/placebo is misplaced or destroyed.  Medication compliance will be monitored and the 
drug/placebo supply will be replenished during routine study visits.  
2.4.4.4 Blinding/Masking 
Both the formulation of the medication and placebo will be procured from the same manufacturer.  The 
packaging will be standardized across sites and will be labeled as ASA 81 mg/placebo, with the 
expiration data and a unique identifier.  A certificate of authenticity will likewise be provided. 
Throughout the study, research staff and local health providers will be blinded to treatment status 
unless there is a serious adverse event potentially related to the treatment modality that requires 
unblinding for safety reasons. There will be one pharmacist at each site who will remain unmasked to 
monitor randomization, drug supply, and safety as needed.  
2.4.5 
Monitoring  
Participants will meet with study staff biweekly to monitor medical side effects and other medical co-
interventions. As this is a pragmatic trial, no limitations on local treatment will be prescribed but rather 
simply documented. An assessment of unplanned medical visits will also be made at this time. The 
routine study visits will also provide an opportunity to monitor drug compliance and exchange her 
completed drug/placebo supply for a new allotment of medication. This process will be completed until 
the beginning of 36 0/7 weeks GA or the participant delivers.   
In order to assess the development of preeclampsia, blood pressure will be monitored at the following 
time points: 
- Between 16-20 weeks 
- At 28-30 weeks 
- At 34 weeks  
- Every 2 weeks beyond that until delivery, alternating with pill monitoring visits 
 
Each visit will consist of routine blood pressure measurements using a standardized blood pressure 
instrument and protocol. If the blood pressure is found to be >140/90 and indicated by review of 
symptoms, then proteinuria will be evaluated by urine dipstick and they will be referred, as indicated. 
2.5 PRIMARY OUTCOME 
The primary outcome of this study is preterm birth, which will be defined as delivery at or after 20 0/7 
weeks and prior to 37 0/7 weeks.   This will be determined based on actual date of delivery in 
comparison to the projected EDD, independent of whether or not the preterm delivery is indicated or 
spontaneous. 
Primary outcome variable:  Delivery at < 37 0/7 weeks gestation 
2.6 SECONDARY OUTCOMES 
 
Preeclampsia and eclampsia (hypertensive disorders of pregnancy) 
 
SGA 
 
Perinatal mortality 

 
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2.7 OTHER OUTCOMES OF INTEREST  
Maternal outcomes: 
 
Vaginal bleeding 
 
Antepartum hemorrhage  
 
Postpartum hemorrhage 
 
Maternal mortality  
 
Late abortion 
 
Change in maternal hemoglobin 
 
Preterm, preeclampsia  
 
Fetal outcomes: 
 
Preterm birth <34 0/7 weeks of pregnancy  
 
Birth weight <2500g and <1500g 
 
Fetal loss 
 
Spontaneous abortion 
 
Stillbirth  
 
Medical termination of pregnancy 
 
The primary, secondary, and other outcomes are defined in Appendix 3. 
2.8 SAFETY MONITORING  
It should be noted that aspirin remains the oldest prescribed drug in human medicine. As such, the 
safety profile has been well described.  Large longitudinal studies of both adult populations and more 
specifically pregnant populations have been performed, with minimal or no side effects detected. A 
2014 systematic review of the use of LDA for the prevention of morbidity and mortality from 
preeclampsia found a 10% reduction in risk of preeclampsia, 20% reduction of Intrauterine growth 
restriction (IUGR), and a 14% reduction of preterm birth 31. Further, there was no evidence of serious 
harms associated with LDA use during pregnancy. Bleeding-related complications, such as postpartum 
hemorrhage, maternal blood loss, and neonatal intracranial or intraventricular bleeding were not found. 
The evidence on longer-term outcomes for offspring from in utero aspirin exposure (low-dose) is  
limited, but follow-up data from one large RCT is reassuring 31.  Nonetheless, it should be noted these 
studies have not assessed the safety of aspirin in pregnancy in the developing world where unique 
circumstances such as endemic anemia may be present. Therefore safety monitoring remains a strong 
focus of this project and can be divided into three distinct areas:  
1. Active surveillance of maternal side effects and medical complications associated with aspirin: 
Maternal surveillance will be composed of active assessment of unintended medical visits. 
Likewise, where obtained for clinical care, hemoglobin will be recorded as well as administration 
of both oral and intravenous iron. Likewise the incidence of postpartum hemorrhage, 
antepartum hemorrhage, cesarean delivery and maternal death due to postpartum hemorrhage 
will likewise be monitored on an ongoing basis.   
2. Evaluation of fetal side effects: If occurrences of major fetal abnormalities or fetal loss are 
discovered during ultrasound procedures or follow-up visits, they will be noted and the woman 
will be referred according to local standard of care. Likewise, stillbirth and late abortion (delivery 
between 16 and 20 weeks) will be monitored via established maternal and newborn health 

 
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registry that is conducted within the Global Network infrastructure at each site. Fetal anomalies 
and loss will also be reviewed at least twice a year by the Data Monitoring Committee (DMC) 
3. Evaluation of changes in maternal hemoglobin: Anemia will be monitored in all women, with 
sequential hemoglobin measurements at randomization and again between 26-30 weeks GA. 
Women with hemoglobin < 7.0 g/dl at randomization will be excluded from the study. 
Hemoglobin will also be measured at 4 weeks post-randomization in the first 500 available 
women who agree to participate, with changes in hemoglobin assessed by treatment group. 
Recognizing that site-specific characteristics may affect maternal hemoglobin, the 500 woman 
sample will include at least 50 women from each site. Women with hemoglobin levels of < 7.0 
g/dL or with a change of more than 3.5 g/dl when measured post-randomization will be referred 
to a health provider to receive the local standard of care.  
2.9 COMPLIANCE MONITORING 
The participant will take daily LDA from the time of randomization until 36 0/7 weeks GA or delivery. 
Drug compliance will be routinely monitored throughout this period through pill counts and the 
completion of a medicine compliance form each time the pill supply is replenished. An interval medical 
obstetrical history will also be taken during these scheduled visits. Study staff will also provide 
reminders about proper drug administration and the importance of compliance during bi-weekly visits 
to monitor side effects. 
2.10 SITE PREPARATION 
In preparation for study implementation, the sites will meet with health authorities and conduct 
community sensitization activities to ensure that study procedures are appropriate for the local context 
and to encourage commitment and engagement at the facility and community level. Site preparation 
activities will focus on: 
 
Identifying and hiring study staff; 
 
Developing site-specific procedures for safety monitoring (blood pressure, anemia) and 
procuring the necessary equipment; 
 
Exploring locally-acceptable methods to monitor and improve medication compliance. 
 
Identifying potential implementation challenges and developing culturally-appropriate solutions. 
 
Identifying local medicines/treatments that contain aspirin or have contraindications for its use.  
 
Educating health workers and community members on the use of aspirin in pregnancy (including 
safety of aspirin use) 
2.11 POTENTIAL RISKS AND BENEFITS TO PARTICIPANTS 
There are several potential direct and indirect benefits of this trial.  In developing countries, including 
those of Global Network partners, the rate of preterm births can be as high as 19% of all deliveries 
amounting to an estimated 15 million preterm births in 2012 1,9. Should this trial be successful and the 
rate of preterm deliveries be dropped by 20%, we could eliminate 3,750,000 of these early births per 
year. Aspirin has also been shown to decrease the incidence of stillbirth and delivery of SGA infants 
20,21,32,23. In addition to the reduction of preterm birth, there is clear evidence of maternal benefit. There 
are data supporting the use of daily LDA to stave off preeclampsia23,33,34. This is an important benefit, as 

 
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pregnancy induced hypertension (PIH) is a major contributor to maternal mortality 35. This benefit is 
described in more detail in Section 1.3.3.  
 
By reducing the number of preterm deliveries, the trial also has the potential to decrease need for 
extended and recurrent hospital stays that are frequent in this population, as well as impacting the cost 
of care for the long-lasting neurodevelopmental disorders and chronic health conditions incurred later 
on in life 5–7. Moreover, with this simple strategy, we could potentially save the lives of over 1 million 
neonates that die due to prematurity every year1. Globally, preterm delivery remains a major health 
care financial burden 1,36 
 
The efficacy and side effect profile of aspirin is well-established and thoroughly investigated. Women 
with a known adverse reaction to acetylsalicylic acid (aspirin) will be excluded from the trial. This occurs 
rarely, but an allergic reaction to the intervention medication remains a small risk. Fetal concerns are 
also limited. Though the risk of congenital malformations has been mostly negated by a meta-analysis 25, 
a conflicting case-control study demonstrated an association between high-dose aspirin use and 
gastroschisis 26. Most notably, LDA has actually been shown to improve neurologic outcomes in preterm 
infants 29. In light of such findings, the medical community acknowledges that antepartum exposure to 
low dose aspirin incurs only marginal fetal risk. Overall, the profound benefits of this intervention 
greatly outweigh the minimal risks to both mother and child. 
 
 

 
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3 ANALYTICAL PLAN 
3.1 STATISTICAL ANALYSIS PLAN 
Prior to conducting formal analyses baseline demographic characteristics and key clinical measures will 
be compared between the women in the two treatment arms using contingency table approaches for 
categorical variables and analysis of variance models and t-tests for continuous variables.  For all of 
these analyses, comparisons will be made within Global Network site and overall across the sites 
controlling for site in the models.  
3.1.1 
Primary Analysis—Risk of Preterm (<37 weeks) Birth 
The primary analysis will compare the risk of preterm birth between the two treatment arms using two 
complementary approaches.  First, the formal test of the primary hypothesis that the risk of preterm 
birth differs between the two arms will utilize a modified intention-to-treat approach based on a 
Cochran-Mantel-Haenszel test stratified by Global Network sites.  The approach is characterized as a 
modified intention to treat approach because the analysis population for the test will be all randomized 
pregnancies for which the delivery occurs after 20 weeks.  Earlier deliveries (miscarriages) will be 
considered missing completely at random for purposes of this analysis. 
In order to evaluate the sensitivity of the primary hypothesis test to the assumption by which 
miscarriages are treated as randomly missing and to control for potential confounders of the treatment 
effect, additional analyses will be conducted using a series of three generalized linear models.  These 
generalized linear models will be fit with the binary outcome of preterm (<37 week delivery) as the 
outcome measure.  The initial model will include terms for treatment, site, and a treatment by site 
interaction.  If the treatment by site interaction term is found to be significant (p<0.05), then all 
subsequent models will include the interaction term and all effects will be reported by site.  If the 
interaction term is not significant, it will be removed from the model and this initial model be used to 
estimate an unadjusted treatment effect controlling for site.  The second series of models will include 
any demographic or clinical variable found to differ significantly between the treatment arms in the 
preliminary analyses described above.  These models will be used to generate adjusted estimates of the 
treatment effect controlling for potential confounders.  The third set of models will utilize extensions of 
the generalized linear model that incorporate inverse probability weighting to evaluate the sensitivity of 
the inference to treatment of miscarriages as randomly missing 37. 
3.1.2 
Secondary and Exploratory Analysis 
This study has been designed to evaluate formally the differences between the two treatment arms for 
three secondary outcomes: the risk of perinatal mortality; the risk of eclampsia/preeclampsia, and the 
risk of a SGA delivery.  As with the primary analyses, each of these outcomes will be examined 
individually using formal tests of hypotheses that the risk differ between the two arms based on 
Cochran-Mantel-Haenszel tests stratified by Global Network sites. The analyses for each of these 3 
secondary outcomes will utilize the same modified intention-to-treat population used for the primary 
outcome.  Each of these hypothesis tests will be conducted at the 0.05 level of significance with no 
adjustment for multiple comparisons.  In addition to this set of formal hypothesis tests, model based 
analyses comparable to those conducted for the primary analyses will be used to evaluate potential 
heterogeneity of treatment effect across sites and evaluate potential confounding.  For each of the 3 

 
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outcomes, an initial model will include terms for treatment, site, and a treatment by site interaction.  If 
the treatment by site interaction term is found to be significant (p<0.05), then all subsequent models 
will include the interaction term and all effects will be reported by site.  If the interaction term is not 
significant, it will be removed from the model and this initial model be used to estimate an unadjusted 
treatment effect controlling for site.  The second series of models will include any demographic or 
clinical variable found to differ significantly between the treatment arms in the preliminary analyses 
described above.  These models will be used to generate adjusted estimates of the treatment effect 
controlling for potential confounders.   
In addition to these planned formal secondary outcome analyses, we will also conduct exploratory 
outcomes for a number of other binary outcomes.  The analytic approach for these exploratory analyses, 
will be comparable to that planned for the primary and formal secondary analyses but will focus on 
estimation of effect sizes and generation of hypotheses planned for this study are all binary outcomes, 
therefore the analytic approach will be comparable to the approach described for the primary analysis.  
Initially, contingency tables will be used to generate estimates of the risk of each outcome for the two 
treatment arms, both overall and separately by Global Network (GN) site and across all sites, and the 
relative risk associated with treatment will be estimated by site and across sites controlling for site. For 
each outcome a series of generalized linear models analogous to those described for the primary 
outcome to generate unadjusted estimates of risk, estimates of risk adjusted for potential confounders, 
and estimates of risk adjusted to account for any possible differences in risk between the arms 
associated with miscarriages. 
3.2 SAMPLE SIZE AND POWER ESTIMATES 
The risk of preterm birth < 37 weeks gestation in untreated nulliparous women was estimated to be 
about 8.0% for the EAGeR trial 38,15. Unpublished data for 2012 from the GN sites show preterm birth 
rates, ranging from 2.9% to 9.8%, while WHO estimates the range for the countries in which GN sites are 
located to be 7.7% to 16.7% 1. In developing sample size estimates we considered risks in the range of 
8% to 14% and ultimately selected sample sizes based on a conservative estimate of 8%.  Because the 
association of LDA and preterm birth may differ in international settings, we examined sample size 
requirements for reductions of 40%, 25%, and 20%. As shown in Table 2 below, the number of evaluable 
participants needed to detect a 20% reduction from a usual rate of 8% preterm births with 90% power is 
5,483 per treatment arm. To account for the loss of evaluable subjects due to miscarriages, which are 
anticipated to occur in approximately 5% of participants) and loss to follow-up (assumed to be in the 1% 
to 2% range), sample sizes were increased by 8% to obtain a final sample size of 11,920 (or 5,960 per 
treatment arm).  
The Global Network has access to approximately 68,000 births per year.  It is estimated that 40% would 
be nulliparous, yielding 23,800 potential pregnancies per year.  With an enrollment rate of one third of 
eligible women, this trial could easily be accomplished by the Global Network within an 18 month 
enrollment period. 
 
 
 
 

 
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Table 2. Evaluable Sample Size Estimates 
 
 
 
PTB rate in 
Placebo Arm 
PTB rate 
in ASA 
% 
Red 
Sample size 
per group 
PTB rate 
in ASA 
% 
Red. 
Sample size 
per group 
PTB rate 
in ASA 
% 
Red. 
Sample size 
per group 
Power=80% (α=0.05; β=0.2) 
8% 
4.8% 
40% 
918 
6.0% 
25% 
2,554 
6.40% 
20% 
4,096 
10% 
6.0% 
40% 
721 
7.5% 
25% 
2,005 
8.00% 
20% 
3,213 
12% 
7.2% 
40% 
591 
9.0% 
25% 
1,638 
9.60% 
20% 
2,625 
14% 
8.4% 
40% 
497 
10.0% 
25% 
1,377 
11.20% 
20% 
2,204 
Power=90% (α=0.05; β=0.1) 
8% 
4.8% 
40% 
1,228 
6.0% 
25% 
3,419 
6.40% 
20% 
5,483 
10% 
6.0% 
40% 
965 
7.5% 
25% 
2,683 
8.00% 
20% 
4,301 
12% 
7.2% 
40% 
790 
9.0% 
25% 
2,193 
9.60% 
20% 
3,513 
14% 
8.4% 
40% 
665 
10.5% 
25% 
1,842 
11.20% 
20% 
2,950 
 
Sample Size—Secondary Outcomes: One of the key secondary objectives of this trial is to evaluate the 
effect of low dose aspirin on perinatal mortality.  We evaluated the reduction in perinatal mortality that 
could be detected with 80% power and 90% power under the assumption that the sample size for the 
study would be based on that needed to achieve the primary aim of demonstrating a 20% reduction in 
low-birth weight infants.  The analyses were based on the assumption that the perinatal mortality rate 
in the GN population is approximately 45 perinatal deaths per 1000 deliveries (unpublished data from 
the MNH Registry for 2013).  With this estimated underlying mortality rate and 5,483 evaluable subjects 
per treatment arm required to demonstrate the 20% reduction in low birth weight prevalence as 
outlined in the primary aim, the study will have 80% power to detect a 24% reduction in perinatal 
mortality and 90% power to detect a 27% reduction in perinatal mortality. 
To determine the sample size actually needed to demonstrate the reduction in perinatal mortality 
actually expected if the intervention results in a 20% reduction in prevalence of low birth weight infants, 
we examined both the prevalence of low birth weight infants across the GN sites and the perinatal 
mortality risk in both the low birth weight and normal birth weight infants.  Based on 2013 MNH 
Registry data, the prevalence of low birth weight among all GN deliveries was 16.3% and the perinatal 
mortality rates were 20.5 per 1000 deliveries among normal birth weight infants and 162.3 per 1000 
deliveries among low birth weight infants; these values yield a weighted average perinatal mortality rate 
of 43.6 per 1000 deliveries.  If the low dose aspirin intervention is effective in reducing the prevalence of 
low birth weight deliveries by 20% (i.e. reducing the prevalence from 16.3% to 13%) and the conditional 
risk of perinatal mortality is unchanged in the low birth weight and normal birth weight cohorts, we 
would expect the resultant overall perinatal mortality rate to be reduced to 38.9 per 1000 deliveries or a 
10.8% reduction.  Detection of a reduction of this magnitude with 80% power would require an 
evaluable sample size of greater than 28,000 evaluable deliveries per treatment arm, and the study 
would need over 37,000 evaluable deliveries per arm to achieve 90% power to detect a reduction of this 
level in perinatal mortality. 
Given the sample sizes associated with the second approach, the best alternative appears to be to 
power the study to demonstrate the 20% reduction in prevalence of low birth weight deliveries with a 
commitment to generate point and interval estimates of the impact of the intervention on perinatal 

 
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mortality.  By examining the risk of perinatal mortality in both the normal and low birth weight infants in 
the two arms, the trial would provide some insight about impacts of the intervention on mortality other 
than that mediated through the effect on birth weight. 
We also conducted power analyses to examine the effect sizes that could be detected with the planned 
sample sizes for two additional secondary outcomes, prevalence of SGA infants and incidence of 
eclampsia/preeclampsia in the target population.  Generic information as well as preliminary 
information from the 2013 MNH Registry indicates that the risk of each of these events in the 
population of interest is approximately 5%. Under the assumption of 5483 evaluable participants in each 
treatment arm and an assumed Type I error rate of 0.05 for per-comparison analyses (i.e. no control for 
multiple comparisons in the analyses of these secondary outcomes), the study will have 70% power to 
detect a 20% reduction in the risk of each of these outcome measures, 85% power to detect a 24% 
reduction and 90% power to detect a 26% reduction.  Consequently, the study is reasonably powered to 
examine these secondary outcome measures. 
3.3 AVAILABLE POPULATION 
The sites of the Global Network have access to approximately 68,000 deliveries annually with 24,000 
being potentially eligible pregnancies for this study.  This diverse pregnant population is highly 
accessible and one which, in our international experience, has been eager to participate in non-invasive 
studies to promote the well-being of both mother and child.  The Global Network sites estimate that as 
few as one third and as many as one half of eligible women will enroll in this protocol, which will allow 
the study to meet recruitment goals within 18 months. 
3.4 PROJECTED RECRUITMENT TIME 
Each site will begin recruitment after their site-specific regulatory approvals are in place, the site has 
been adequately prepared, and training is completed. This will require a staggered start, as the timeline 
for these activities will vary per site. The projected timeline for this study is approximately 3 years (36 
months). This includes a 12 month preparatory phase with 3 months for training, an 18 month 
recruitment period, and 6 months for data cleaning and analysis. A detailed study timeline is found in 
Appendix 4.  
3.5 STUDY MONITORING PLAN 
3.5.1 
Reporting Serious Adverse Events 
Serious Adverse events (SAEs) will be monitored continuously using a special form that will be required 
for any event that meets the following criteria: 
 
Results in death; 
 
Is life-threatening; 
 
Requires hospitalization or prolongs existing hospitalization; 
 
Results in persistent or significant disability or incapacity; 
 
Any other serious or unexpected adverse event that the study investigator(s) feels should be 
reported. 

 
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The specific adverse events to be monitored in this trial include maternal death, upper GI bleeding 
(vomiting blood), fetal anomaly, gastroschisis, post-partum hemorrhage, or antepartum hemorrhage. 
The occurrence of any of these events will also trigger completion of the Serious Adverse Events Form.  
3.5.2 
Method and Timing for Reporting Serious Adverse Events 
The Senior Foreign Investigator (SFI) must report the following SAEs by emailing or faxing a copy of the 
form to RTI as follows: 
Within 48 hours of SFI’s notification of the event: 
 
All maternal deaths  
 
All SAEs with a definite or suspected/probable relationship to the intervention 
 
Within 7 days of SFI’s notification of the event:  
 
All life-threatening events 
 
All SAEs considered to have a probable or possible relationship to the intervention. 
 
All emailed or faxed forms should also be entered into the DMS and transmitted within 7 days as a back-
up to ensure no SAE is missed. Additional reporting procedures include: 
 
RTI will forward all SAEs to the US-based Principal Investigator (PI) and NIH for further 
assessment of relationship to study intervention.  
 
The PI and SFI will be responsible for reporting to their respective IRB and other regulatory 
authorities per their institutional policy.  
 
RTI will be responsible for reporting SAEs to the DMC bi-annually at a minimum. The frequency 
of reporting to the DMC may be increased if the reported events or interim data reviews by the 
DMC indicate that more frequent safety monitoring is needed. 
 
Any SAE considered unrelated to the intervention is not required to be reported in an expedited 
manner.  These events should be entered into the data management system and transmitted per 
routine procedures. 
3.5.3 
Data Monitoring Plan and Stopping Rules 
All the Global Network sites will report data to the Global Network Data Coordinating Center, located at 
RTI International. The data will be used to evaluate protocol adherence and site performance (e.g., 
recruitment, loss to follow-up, data quality). The DCC will provide standardized progress reports to 
NICHD and the site investigators on a monthly basis to monitor outcome variables and adverse events. 
Oversight of the trial will be handled by two principal groups with different focuses: 
1) Protocol-focused Steering Committee (SC): The SC is comprised of the Central Study Team from 
Christiana Care and Jawaharlal Nehru Medical College (JNMC), NICHD, the DCC, and investigators 
from each of the participating sites (see Appendix 5). The Central Study Team, with assistance from 
NICHD and the DCC, will have primary responsibility for overall study design, development of study 
materials and procedures, and oversight of study implementation. They will meet via conference call 
bi-weekly to monitor study progress and ensure proper implementation of the trial. The Site 
Investigators will be responsible for providing guidance on study design, developing site-specific 
implementation plans, ensuring study staff are properly trained, and providing oversight of the 

 
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21 February 2019  
study at the site level. The SC will convene via conference call at least once per quarter and will 
meet in person twice a year to discuss study design and implementation issues. Members of the 
Central Study team, NICHD, and RTI will also conduct site visits, as the budget allows, to bolster 
enthusiasm, provide hands-on training and education to the participating staff, and address site-
specific issues, if any. 
2) Data Monitoring Committee (DMC): The DMC, a standing group that monitors all NICHD-funded 
Global Network studies, will be responsible for ensuring safe and ethical treatment of study 
participants through monitoring of the study.  The membership will include, at a minimum, a 
statistician, obstetrician, pediatrician and an expert in international health. The DMC designated by 
NICHD will review the data collected at approximate 6 month intervals throughout the course of the 
study.  The DMC reports, which are prepared by the Data coordinating center, will include 
information on study enrollment rates and participant progress through the study, participant 
compliance with protocol-specified treatment regimens, protocol violations, adverse events, and 
efficacy outcomes. The focus of the DMC review will be on monitoring participant safety and study 
progress/futility but data on treatment effectiveness will also be presented to frame the DMC 
discussions on safety and futility.  However, no formal interim analysis of efficacy or effectiveness 
are planned and the DMC will not be responsible for stopping the trial for efficacy. The DMC will be 
charged with monitoring adverse events and side effects from LDA. All known associated side effects 
and specific obstetric or fetal concerns will be considered reportable to the DMC. The study will be 
reviewed by the DMC bi-annually at a minimum, but may be reviewed more frequently if concerns 
are raised about participant safety or about adequate process of the study. 
3.6 DATA MANAGEMENT PROCEDURES 
Data will be collected both prospectively and from existing clinical records, using hard copy forms or 
Android Tablets. Regardless of data capture methodology, all data will be kept confidential. Each 
participant will be assigned a unique study ID which will be used to identify the participant.  Only the 
screening log will contain the name (which is not transmitted). If hard copy forms are used, they will be 
retained in a secure location for possible editing or queries at the central data entry site.  Data will be 
entered into computers using the Data Management System (DMS) developed by RTI and the assigned 
study number.  The DMS will also allow site staff to produce project reports and backup the study 
database.  Electronic data will be transferred from each data management computer to a single 
Research Unit Data Center (RUDC) in each country, creating a complete data repository.  At least once a 
week, data will be transmitted from the RUDC to the DCC at RTI.  The data center will conduct training 
on the DMS system, as needed, and will maintain the central database for the study.  
 
Precision and accuracy of actual data collected will be checked by chart review (random 5%) and internal 
procedures using the computer program.  Monthly audits and incomplete data reports will be 
performed by a review team consisting of at least of the SFI and the country coordinator.  Data editing 
and error resolution will be performed monthly.  In addition, a sample of participants will be visited to 
confirm their participation, with procedures determined per site. These activities will be shared between 
the site and the data center.  

 
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3.7 QUALITY CONTROL  
3.7.1 
Training 
All study personnel must participate in training on the proper implementation of study procedures and 
the ethics of conducting research with human subjects before beginning any research activity. The SFI 
and project coordinator will ensure that all study personnel receive the appropriate training, and obtain 
the required certification ensuring that they have met the training objectives. RTI will be responsible for 
developing a certification test. The SFI and project manager will be responsible for overseeing the 
certification process. 
3.7.2 
Study Monitoring 
Major monitoring responsibilities of the PI/SFI, assisted by the country coordinator, are (1) confirming 
proper IRB approval; (2) monitoring the delivery of the study intervention; (3) assessing and evaluating 
the quality of study implementation; (4) ensuring compliance with the intervention, including proper 
randomization; (5) evaluating accuracy, precision, and completeness of data collected, entered, and 
transmitted (along with the DCC); (6) ensuring that all personnel are fulfilling their obligations; (7) 
maintaining morale and enthusiasm of the staff; (8) handling ad hoc problems and maintaining 
communication; (9) ensuring inter-site consistency; and (10) proposing improvements to the monitoring 
activities. 
NICHD and the DCC staff will conduct site visits as needed. These visits will include review of individual 
participant records, including supporting data, to ensure protection of study participants, compliance 
with the protocol, and accuracy and completeness of records. The SFI/PI will make study documents 
(e.g., logbooks, data forms, staff training certificates) and pertinent hospital/clinic records readily 
available for inspection by the local IRB, site monitors, and the NICHD for confirmation of the study data. 
3.7.3 
Drug Quality Assurance and Monitoring 
The study drug manufacturer will have a Good Manufacturing Practices (GMP) designation vetted by the 
FDA and a certificate of authenticity will be provided. Each site will adapt best practice guidelines for 
drug shipment and storage to the needs and infrastructure of their local environment. Study staff will be 
trained in on the drug shipment and storage plan to ensure that best practices are maintained at all 
time. Additionally, participants will receive detailed instruction on proper storage of the study drug at 
home. Drug stability information will be maintained throughout the study. For quality assurance, a 
sample of pills from each site will be randomly selected and tested for bioavailability at multiple time 
points during the study period. A sample from each batch will be tested.  
3.7.4 
Ultrasound Gestational Age Dating 
All study sonographers will be trained to assess gestational age using CRL measurements. A sample of 
ultrasound images at each site will be reviewed by study investigators to ensure that CRL is accurately 
measured and gestational age is accurately assigned. If discrepancies are found, the sonographers will 
be retrained. Ultrasound will be used only for pregnancy dating and no other purpose. 
 
 

 
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4 DATA FORMS 
The following forms will be used for this study: 
Form Description 
Purpose 
Key Data Elements 
Data Source 
Screening Log 
To track screening and enrollment at facility 
or community level 
Contact information, screening and 
enrollment date and status 
Patient report 
Initial Screening 
and Recruitment  
To determine eligibility and record consent 
status 
Screening criteria: age, pregnancy history, 
estimated GA by LMP, consent status/date. 
Patient report, 
obstetrical wheel,  
Ultrasound 
Screening  
To confirm eligibility criteria by ultrasound. 
GA dating, assessment of fetal anomaly, 
singleton pregnancy, and fetal heart rate  
Ultrasound  
Hemoglobin (Hb) 
Monitoring   
To monitor Hb at enrollment and 26-30 GA. 
It will also capture Hb at 4 weeks post-
randomization in subset of 500 women  
Hemoglobin 
Lab test 
Clinical 
Assessment  
To assess clinical status through examination 
and patient history and provide information 
on medicine distribution/use 
Height, weight, BP, Pulse, patient-reported 
medical history, use of other medications 
Clinical exam, lab 
tests, patient report 
Biweekly 
Monitoring  
To monitor side effects, use of other 
medications, and drug compliance. 
Drug compliance, BP, side effects (nausea, 
vomiting, rash/hives, diarrhea, vaginal 
bleeding, gastritis, other), use of other 
medicines, care seeking, pregnancy 
complications. 
Pill count, patient 
report, clinical 
examination 
Blood Pressure 
Monitoring  
To monitor BP for active identification of 
pre-eclampsia  
BP, urine protein (if indicated), fetal 
movement, diagnosis of eclampsia or 
stroke. 
Clinical 
examination, 
patient report 
Unscheduled 
Emergency Visit  
To monitor unscheduled medical visits or 
medical emergencies 
Patient status, pulse, BP, reason for 
medical visit/emergency, pill count and 
patient report on drug compliance, 
presence of side effects, referrals 
Existing medical 
records, patient 
reports 
Severe Adverse 
Events  
To record fatal, life-threatening, or any 
other serious, unexpected adverse event 
Date/time of event, date/time of 
resolution, nature of adverse event, 
management of adverse event, attribution 
to study (yes/no). 
Patient reports, 
existing medical 
records 
Study Withdrawal  
To describe reasons for study termination or 
withdrawal  
Date and reason for 
termination/withdrawal.  
Patient report 
Protocol 
Deviation  
To record protocol deviations and corrective 
actions  
Date and nature of deviation/violation, 
corrective action. 
Patient report, 
study records 
Outcomes 
Maternal and Fetal outcomes will be 
documented via the GN’s established MNH 
Registry  
Preterm birth (primary outcome); maternal 
and fetal secondary outcomes. 
MNH Registry 
 
 

 
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5 REFERENCES 
1.  
March of Dimes, PMNCH, Save the Children, WHO. Born Too Soon: The Global Action Report on 
Preterm Birth. Geneva: World Health Organization; 2012. 
2.  
Mathews TJ, Menacker F, MacDorman MF. Infant mortality statistics from the 2002 period: 
linked birth/infant death data set. Natl Vital Stat Rep. 2004;53:1-29. 
3.  
Anderson RN, Smith BL. Deaths: leading causes for 2001. Natl Vital Stat Rep. 2003;52:1-85. 
4.  
Beck S, Wojdyla D, Say L, et al. The worldwide incidence of preterm birth: A systematic review of 
maternal mortality and morbidity. Bull World Health Organ. 2010;88:31-38. 
doi:10.2471/BLT.08.062554. 
5.  
Goldenberg R, Rouse D. Prevention of Premature Birth. N Engl J Med. 1998;339(5):313-320. 
6.  
McCormick MC, Richardson DK. Premature infants grow up. N Engl J Med. 2002;346(3):197-198. 
doi:10.1056/NEJM200201173460310. 
7.  
Saigal S, Doyle LW. An overview of mortality and sequelae of preterm birth from infancy to 
adulthood. Lancet. 2008;371:261-269. doi:10.1016/S0140-6736(08)60136-1. 
8.  
Valero De Bernabé J, Soriano T, Albaladejo R, et al. Risk factors for low birth weight: A review. Eur 
J Obstet Gynecol Reprod Biol. 2004;116:3-15. doi:10.1016/j.ejogrb.2004.03.007. 
9.  
Blencowe H, Cousens S, Oestergaard MZ, et al. National, regional, and worldwide estimates of 
preterm birth rates in the year 2010 with time trends since 1990 for selected countries: a 
systematic analysis and implications. Lancet. 2012;379:2162-2172. doi:10.1016/S0140-
6736(12)60820-4. 
10.  
FitzGerald G a., Oates J a., Hawiger J, et al. Endogenous biosynthesis of prostacyclin and 
thromboxane and platelet function during chronic administration of aspirin in man. J Clin Invest. 
1983;71(November 1982):676-688. doi:10.1172/JCI110814. 
11.  
Chavarría ME, Lara-González L, González-Gleason A, García-Paleta Y, Vital-Reyes VS, Reyes A. 
Prostacyclin/thromboxane early changes in pregnancies that are complicated by preeclampsia. 
Am J Obstet Gynecol. 2003;188:986-992. doi:10.1067/mob.2003.203. 
12.  
Yu Y, Cheng V, Fan J, et al. Differential impact of prostaglandin H synthase 1 knockdown on 
platelets and parturition. J Clin Invest. 2005;115:986-995. doi:10.1172/JCI200523683. 
13.  
Goldenberg RL, Culhane JF, Iams JD, Romero R. Epidemiology and causes of preterm birth. 
Lancet. 2008;371:75-84. doi:10.1016/S0140-6736(08)60074-4. 

 
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14.  
Roberge S, Nicolaides KH, Demers S, et al. Prevention of perinatal death and adverse perinatal 
outcome using low-dose aspirin: a meta-analysis. Ultrasound Obstet Gynecol. 
2013;41(January):491-499. doi:10.1002/uog.12421. 
15.  
Schisterman EF, Silver RM, Lesher LL, et al. Preconception low-dose aspirin and pregnancy 
outcomes: Results from the EAGeR randomised trial. Lancet. 2014;384:29-36. 
doi:10.1016/S0140-6736(14)60157-4. 
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Duley L, Henderson-Smart DJ, Knight M, King JF. Antiplatelet agents for preventing and treating 
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doi:10.1002/14651858.CD000492. 
17.  
Coomarasamy A, Braunholtz D, Song F, Taylor R, Khan KS. Individualising use of aspirin to prevent 
pre-eclampsia: A framework for clinical decision making. BJOG An Int J Obstet Gynaecol. 
2003;110(October):882-888. doi:10.1111/j.1471-0528.2003.03002.x. 
18.  
Coomarasamy A, Honest H, Papaioannou S, Gee H, Khan KS. Aspirin for prevention of 
preeclampsia in women with historical risk factors: A systematic review. Obstet Gynecol. 
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19.  
Heyborne KD. Preeclampsia prevention: Lessons from the low-dose aspirin therapy trials. Am J 
Obstet Gynecol. 2000;183:523-528. doi:10.1067/mob.2000.106757. 
20.  
Newnham JP, Godfrey M, Walters BJ, Phillips J, Evans SF. Low Dose Aspirin for the Treatment of 
Fetal Growth Restriction: A Randomized Controlled Trial.; 1995:370-374. 
21.  
Italian Study of Aspirin in Pregnancy. Low-dose aspirin in prevention and treatment of 
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22.  
CLASP. CLASP: a randomised trial of low-dose aspirin for the prevention and treatment of pre-
eclampsia among 9364 pregnant women. CLASP (Collaborative Low-dose Aspirin Study in 
Pregnancy) Collaborative Group. Lancet. 1994;343:619-629. doi:10.1016/0091-2182(94)90141-4. 
23.  
Duley L, Henderson-Smart DJ, Knight M, King JF. Antiplatelet agents for preventing and treating 
pre-eclampsia. Cochrane Database Syst Rev. 2007;(2):CD000492. 
doi:10.1002/14651858.CD000492. 
24.  
Knight M, Duley L, Henderson-Smart DJ, King JF. Antiplatelet agents for preventing and treating 
pre-eclampsia. Cochrane Database Syst Rev. 2000:CD000492. doi:10.1002/14651858.CD000492. 
25.  
Kozer E, Nikfar S, Costei A, Boskovic R, Nulman I, Koren G. Aspirin consumption during the first 
trimester of pregnancy and congenital anomalies: A meta-analysis. Am J Obstet Gynecol. 
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26.  
Werler M, Sheehan JE, Mitchell AA. Maternal medication use and risks of gastroschisis and small 
intestinal atresia. Am J Epidemiol. 2002;155:26-31. doi:10.1093/aje/155.1.26. 
27.  
American College of Obstetricians and Gynecologists. ACOG committee opinion no. 561: 
Nonmedically indicated early-term deliveries. Obstet Gynecol. 2013;121:911-915. 
doi:10.1097/01.AOG.0000428649.57622.a7. 
28.  
Norgard B, Puho E, Czeizel AE, Skriver M V, Sorensen HT. Aspirin use during early pregnancy and 
the risk of congenital abnormalities: a population-based case-control study. Am J Obs Gynecol. 
2005;192:922-923. http://www.ncbi.nlm.nih.gov/pubmed/15746692. 
29.  
Marret S, Marchand L, Kaminski M, et al. Prenatal low-dose aspirin and neurobehavioral 
outcomes of children born very preterm. Pediatrics. 2010;125:e29-e34. doi:10.1542/peds.2009-
0994. 
30.  
American College of Obstetricians and Gynecologists. Method for Estimating Due Date. 
Committee Opinion 611. Obs Gynecol. 2014;124:863-866. 
31.  
Henderson J, Whitlock E, O’Connor E, Senger C, Thompson J, Rowland M. Low-Dose Aspirin for 
the Prevention of Morbidity and Mortality From Preeclampsia : A Systematic Evidence Review for 
the U . S . Preventive Services Task Force. Evidence Synthesis No. 112. Rockville, MD Agency 
Healthc Res Qual. 2014;14-05207-E(112). 
32.  
Beroyz G, Casale R, Farreiros A, et al. CLASP: A randomised trial of low-dose aspirin for the 
prevention and treatment of pre-eclampsia among 9364 pregnant women. Lancet. 
1994;343:619-629. doi:10.1016/S0140-6736(94)92633-6. 
33.  
Vainio M, Kujansuu E, Iso-Mustajärvi M, Mäenpää J. Low dose acetylsalicylic acid in prevention of 
pregnancy-induced hypertension and intrauterine growth retardation in women with bilateral 
uterine artery notches. BJOG An Int J Obstet Gynaecol. 2002;109:161-167. doi:10.1111/j.1471-
0528.2002.01046.x. 
34.  
Villa PM, Kajantie E, Räikkönen K, et al. Aspirin in the prevention of pre-eclampsia in high-risk 
women: A randomised placebo-controlled PREDO Trial and a meta-analysis of randomised trials. 
BJOG An Int J Obstet Gynaecol. 2013;120:64-74. doi:10.1111/j.1471-0528.2012.03493.x. 
35.  
Steegers EAP, von Dadelszen P, Duvekot JJ, Pijnenborg R. Pre-eclampsia. Lancet. 2010;376:631-
644. doi:10.1016/S0140-6736(10)60279-6. 
36.  
Kulberg CH. The Cost of Poor Birth Outcomes. 2013. 
http://www.theplanningcouncil.org/inp/5Wellness/2LowBirthWeight/TheCostofPoorBirthOutco
mes.pdf. 

 
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37.  
Hogan JW, Lancaster T. Instrumental variables and inverse probability weighting for causal 
inference from longitudinal observational studies. Stat Methods Med Res. 2004;13:17-48. 
doi:10.1191/0962280204sm351ra. 
38.  
Abramovici A, Cantu J, Jenkins SM. Tocolytic Therapy for Acute Preterm Labor. Obstet Gynecol 
Clin North Am. 2012;39:77-87. doi:10.1016/j.ogc.2011.12.003.  
 
 
 

 
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APPENDIX 1. SCHEDULE OF STUDY PROCEDURES 
 
6 0/7- 13 6/7 
weeks GA 
Randomization 
/Enrollment 
Bi-weekly 
after 
enrollment 
4 weeks 
after 
enrollment 
monthly 
after 
enrollment 
16-20 
weeks 
GA 
26-30 
weeks 
GA 
34 
weeks 
GA 
36 weeks 
GA until 
delivery 
6 weeks 
Post-
Partum 
As 
needed 
Screening 
• Initial eligibility 
X 
 
 
 
 
 
 
 
 
 
 
• Ultrasound 
X 
 
 
 
 
 
 
 
 
 
 
Consent 
X 
 
 
 
 
 
 
 
 
 
 
Clinical Assessments 
• Physical exam 
 
X 
 
 
 
 
 
 
 
 
 
• Medical history 
 
X 
 
 
 
 
 
 
 
 
 
• Use of other 
medicines 
 
X 
X 
 
 
 
 
 
 
 
 
• Maternal Hb 
monitoring 
 
X 
 
X 
 
 
X 
 
 
 
 
• BP Monitoring 
 
X 
 
 
 
X 
X 
X 
X 
 
 
Medication Activities 
• Instructions for 
drug use 
 
X 
 
 
 
 
 
 
 
 
 
• Medication 
compliance 
 
 
X 
 
 
 
 
 
 
 
 
• Resupply 
 
 
 
 
X 
 
 
 
 
 
 
Outcome Assessment 
• Preterm Birth 
 
 
 
 
 
 
 
 
 
X 
 
• SGA 
 
 
 
 
 
 
 
 
 
X 
 
• Preeclampsia/ 
Eclampsia 
 
 
X 
 
 
 
 
 
 
X 
X 
• Other maternal 
outcomes 
 
 
X 
 
 
 
 
 
 
X 
X 
• Other fetal 
outcomes 
 
 
 
 
 
 
 
 
 
X 
X 
• Unanticipated 
medical care 
 
 
 
 
 
 
 
 
 
 
X 
• Serious Adverse 
Events 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 

 
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APPENDIX 2. SAMPLE INFORMED CONSENT 
Global Network for Women’s & Children’s Health Research 
Aspirin Supplementation for Pregnancy Indicated Risk Reduction In Nulliparas (ASPIRIN) 
 
INVESTIGATORS:  
[LIST SITE INVESTIGATORS] 
 
 
SPONSOR: 
The Eunice Kennedy Shriver National Institutes of Child Health and Human Development (NICHD) 
 
You are being asked to participate in a research study for pregnant mothers. This project is funded by 
the U.S. National Institutes of Health. This form provides you with information about the study. A 
member of the research team will describe the study to you and answer all of your questions. Please 
read the information below and ask questions about anything you don’t understand before deciding 
whether or not to take part. You may also request that the research staff read the form to you.  
 
What is the purpose of the study? 
The purpose of this study is to learn whether a daily 81 mg dose of aspirin given to pregnant women, 
beginning between 6-13 weeks of pregnancy and continuing until 36 weeks of pregnancy, can reduce 
the risk of preterm birth (when a baby is born too soon). 
 
Who will be in the study? 
A total of 11920 women will be enrolled in this study from seven sites in sub-Saharan Africa, South Asia, 
and Latin America. In [insert site name], no more than [insert max sample size] will be enrolled.  Half of 
the participants will be randomly selected to take aspirin. The other half will be randomly selected to 
take placebo, a pill that looks identical to aspirin but does not contain any medicine.  This will allow the 
researchers to compare how well aspirin works to prevent babies being born too soon.  
You qualify for this study if you are a healthy pregnant woman between 18-40 years of age, have had an 
ultrasound showing that your pregnancy is between 6-13 weeks, have never given birth before, have no 
more than 2 previous pregnancy losses, and have no known problems taking aspirin. If you qualify, you 
will be randomly assigned to either the treatment group, which will take a daily low dose of aspirin, or 
the control group, which will take a pill that looks identical to aspirin but does not have any active drug.  
 
What will happen if I join this study? 
Before participating, you will be provided with information about the study procedures and given an 
opportunity to ask questions. If you agree to participate, you will be asked to sign this form to indicate 
your consent. The research staff will perform a pregnancy test to confirm that you are pregnant and an 
ultrasound to make sure that the baby is in your womb and has a heartbeat. The ultrasound will also 
measure the size of your baby to help confirm how many weeks you have been pregnant. After the 
ultrasound, the research staff will ask you some questions about your health, do a brief medical exam, 
and conduct a hemoglobin test to make sure you are healthy enough to participate. For the hemoglobin 
test, a small sample of blood (2-3 drops) will be taken by pricking and gently squeezing your finger and 
will be used to measure the level of iron in your blood. If the iron level is too low (known as anemia), 

 
Aspirin Protocol V 1.3  
 
 
 
 
 
 
 
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21 February 2019  
you will not be able to participate. Instead, you will be referred to a health provider who will provide 
care for anemia.  
After all these procedures are completed and your eligibility is confirmed, you will be randomly assigned 
to either the treatment group that takes a daily low dose of aspirin or the control group that takes a 
daily placebo pill. The aspirin and placebo look identical so there is no way for you or the research staff 
to know your assigned group. Both groups will receive exactly the same instructions for taking the drug 
and will participate in exactly the same activities throughout the study. At the time of enrollment, you 
will be provided with verbal instructions for safe administration and storage of the drug. You may be 
asked to take your first dose while you are with the research staff. 
A member of the research team will set up bi-weekly visits to check on side effects and make sure you 
are taking the medicine properly. When needed, you will also be given a new supply of medicine. The 
research staff will conduct special tests at some of the bi-weekly visits to make sure you are not 
experiencing side effects. The special tests are: 
 
Blood pressure to assess for the development of pre-eclampsia at the following time points in 
pregnancy (1) between 16-20 weeks; (2) between 28-30 weeks; (3) at 34 weeks; (4) every 2 
weeks after that until delivery. If your blood pressure is high, you will also be asked to provide a 
small amount of urine (about 2 tablespoons) for additional testing.  
 
Hemoglobin testing to assess for anemia (low iron in the blood) at 26-30 weeks pregnancy.  
 
The research staff will choose up to 500 women total from all sites combined who will be asked 
to participate in additional hemoglobin testing 4 weeks after enrollment. 
 
To be sure that you do not have more than the normally expected hospital or clinic visits during 
your pregnancy as a result of study participation, the research staff will ask you about 
unexpected visits and will need as much information as you can give them. They will also collect 
information about the reason for your unexpected visit from your clinical record.  
The local research staff have been selected because of their skills, knowledge, and familiarity with your 
community. They will begin bi-weekly visits two weeks after enrollment and will continue these visits 
every two weeks until you deliver. The research staff are here to support you during the study and 
should be contacted between visits if you have any questions or concerns. 
What are the risks and discomforts 
Aspirin has been used for many years to safely treat adults and children. Extensive research shows that 
the risks of this intervention are minimal. There is a small but rare risk of allergic reactions; therefore, 
you will not be able to participate if you have had a bad reaction to aspirin in the past or if you have a 
bad reaction while in the study. Aspirin use in pregnancy has not been well-studied in developing 
countries with high rates of anemia, although studies of pregnant women in high-income countries do 
not suggest an increased risk. Nonetheless, hemoglobin levels will be monitored during the study and 
women who are very anemic at the time of enrollment will be excluded from the study. If you become 
very anemic during the study, you will be referred to your health provider for care and treatment. 
Additionally, pregnant women may be at risk for vaginal bleeding, but this risk has also not been 
supported by recent studies. A commonly reported side effect of aspirin use is gastric discomfort (an 
upset stomach); however, you will be given pills with a special coating which will help prevent the 
stomach discomfort that aspirin causes in some people. Any discomfort or side effect should be 
reported to your health provider and the research staff for further assessment. Based on this 
assessment, the research staff may decide to stop giving you the study drug.  

 
Aspirin Protocol V 1.3  
 
 
 
 
 
 
 
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21 February 2019  
For babies, there is limited evidence that aspirin use in pregnancy may increase the risk of gastroschisis, 
a birth defect of the abdominal (belly) wall. Fetal anomalies (birth defects) will be monitored throughout 
the study. 
You may feel temporary discomfort or pain when your blood is taken with a finger stick. To minimize 
this, we will ensure research staff are well trained in the procedure.  
Another possible risk of participating in this study is that your name and personal information may be 
seen by persons who are not part of the project. To prevent this, you will be given an identification 
number that will be used in place of your name on all study documents.  
What are the benefits of participating? 
You will not receive any money from participating in this study, but your participation may provide 
important information that can be used in the future to prevent babies from being born too soon. Also, 
there is existing evidence that the use of aspirin in pregnancy can improve the infant’s neurological 
outcomes. 
If new information about the benefits or risks of aspirin use in pregnancy becomes available during this 
study, this information will be given to you by [Insert name of Senior Investigator] or his/her staff.  
Will I have to pay for anything? 
It will not cost you anything to be in the study. 
 
Is my participation voluntary? 
Taking part in this study is voluntary. You have the right to refuse to participate or to withdraw your 
participation at any time.  If you refuse or decide to withdraw, you will not lose any benefits or rights to 
which you are entitled. These actions will not have any negative effect on the health care you receive 
from your local health providers. You will still receive your normal medical care.   
 
Can I be removed from this study?  
You will be withdrawn from the study if the research staff thinks that your participation may cause you 
harm. The research staff may also remove you from the study for other reasons at their discretion. Also, 
the sponsor may stop the study at any time. 
 
What will happen if you are injured by this research? 
Although the risk of injury is expected to be very low, all research involves a chance that something bad 
might happen to you. Despite all safety measures, your participation could result in a reaction or injury. 
If you or your infant is injured as a result of your participation, you will be provided with emergency care 
by the study and referred to a doctor for ongoing care, if needed. Ongoing care will not be paid for by 
the study. [Insert name of Research Institution] and NICHD have not set aside funds to pay you for any 
such reactions, injuries or related medical care. However, by signing this form, you do not give up any of 
your legal rights.  
What should you do if you have additional questions? 
If you have questions about this study or a project-related injury, you should contact [investigator 
contact]. If you have questions about your or your baby’s rights as a project participant, please contact 
[insert ethics committee contact]. 
If you have any questions about the study, please call [insert senior investigator]. 

 
Aspirin Protocol V 1.3  
 
 
 
 
 
 
 
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21 February 2019  
Agreement to be in this study 
I have read this paper about the study or it was read to me.  I understand the possible risks and benefits 
of this study.  I know that being in this study is voluntary and I choose to be in this study. I understand I 
will get a copy of this consent form. 
 
Signature (or thumbprint): 
 
 
 
 
________ 
Date: 
 
______  
 
 
 
 
(Mother) 
 
Print Name: 
 
 
 
 
 
 
 
__ 
 
 
 
 
(Mother) 
 
Signature (or thumbprint): 
 
 
 
 
_________ 
Date: 
 
______  
 
 
 
 
(Parent/Guardian/Husband) 
 
Print Name: 
 
 
 
 
 
 
 
__ 
 
 
 
 
(Parent/Guardian/Husband) 
 
 

 
Aspirin Protocol V 1.3  
 
 
 
 
 
 
 
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21 February 2019  
APPENDIX 3. OUTCOME DEFINITIONS 
PRIMARY OUTCOME: 
 
Preterm Birth: delivery at or after 20 0/7 weeks and prior to 37 0/7 weeks.  
- WHO definition: babies born before 37 completed weeks of pregnancy.  
- ACOG definition: Birth occurs between 20 weeks and 37 weeks of pregnancy 
(http://www.acog.org/~/media/For%20Patients/faq087.pdf) 
 
SECONDARY OUTCOMES: 
 
Preeclampsia: characterized by hypertension and proteinuria occurring after the 20th week of 
pregnancy. Hypertension is a blood pressure of 140/90 mm Hg or above. In severe pre-eclampsia 
the diastolic blood pressure is usually 110 mm Hg or above and there may also be one or more of 
the following symptoms: severe headache, blurred vision, nausea and/or vomiting, abdominal pain 
and a diminished urinary output, i.e., oliguria. Early onset Preeclampsia develops before 34 weeks 
of gestation. 
 
 
Eclampsia: Very serious complication of pregnancy characterized by convulsions and coma. It may 
be preceded by signs of pre-eclampsia or the onset may be rapid and sudden. Eclamptic fits can 
occur in pregnancy, labour or soon after delivery.  
 
 
Small for gestational age: newborns with weights below the 10th percentile for gestational age. 
 
 
Perinatal mortality: The number of stillbirths and deaths in the first week of life per 1,000 live 
births, the perinatal period commences at 22 completed weeks (154 days) of gestation and ends 
seven completed days after birth. 
 
MATERNAL OUTCOMES OF INTEREST 
 
Vaginal bleeding: Bleeding during pregnancy (i.e., WHO generally defines as ‘light’ or ‘heavy’ 
bleeding based on clinical symptoms) 
 
 
Antepartum hemorrhage: Bleeding from the genital tract at any time after the 22nd week of 
pregnancy and before the birth of the baby. There are two main causes of antepartum 
haemorrhage, placenta praevia and abruption placentae. 
 
 
Postpartum hemorrhage: Blood loss of 1000 ml or more from the genital tract after delivery and up 
to six weeks post-delivery.  Common causes include atony (poor muscle tone) of the uterus or 
trauma to the genital tract, e.g. tears of the vagina, cervix, or lower segment of the uterus.  
 
 
Maternal mortality: the death of a woman during pregnancy (i.e. conception to delivery) and the 
puerperium (i.e. up to 42 days after delivery).  
 
 
Change in maternal hemoglobin: Hemoglobin level <7.0 gm/dL or a 3.5 gm/dL decrease between 
measurements.   
 
FETAL OUTCOMES OF INTEREST 
 
Preterm birth <34 0/7 weeks of pregnancy: live birth before 34 0/7 weeks of pregnancy are 
completed.  

 
Aspirin Protocol V 1.3  
 
 
 
 
 
 
 
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21 February 2019  
 
 
Birth weight <2500g and Birth weight <1500g: Birth weight is defined as the first weight of the 
fetus or newborn obtained after birth. For live births, birth weight should preferably be measured 
within the first hour of life before significant postnatal weight loss has occurred, recorded to the 
degree of accuracy to which it is measured (WHO). Birth weight <2500 g is defined as low birth 
weight, whereas birth weight <1500 g is defined as very low birth weight.  
 
 
Fetal Loss: Spontaneous loss ≥ 16 weeks GA plus perinatal mortality. 
 
 
Late abortion: Spontaneous fetal loss ≥ 16 weeks. 
 
 
Spontaneous abortion: Premature expulsion of a non-viable fetus from the uterus at < 20 weeks 
gestation. 
 
 
Stillbirth: Birth of a baby that shows no signs of life at birth (no gasping, breathing, heart beat or 
movement). (WHO).  It may be a macerated or fresh stillbirth. The signs of maceration are 
discoloration and peeling of the skin leaving areas of raw tissue. The skull is usually soft, as the brain 
has become soft. The umbilical cord is usually stained a dark red or black. The amniotic fluid is 
usually darkly stained. Maceration is the result of the infant being dead for at least 12 hours. 
Macerated stillborn infants are assumed to have died before the onset of labor. Fresh stillbirths 
show no sign of maceration and have usually died during labor or shortly before the onset of labor.  
 
 
Medical termination of pregnancy: an operation or other procedure to terminate pregnancy before 
the fetus is viable.  
 
NOTE: (WHO) at the end of the definition means the definition is from the 2006 WHO guide titled: 
Integrated Management of Pregnancy and Childbirth Pregnancy, Childbirth, Postpartum and Newborn 
Care: A guide for essential practice. 
 
 

 
Aspirin Protocol V 1.3  
 
 
 
 
 
 
 
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21 February 2019  
APPENDIX 4. STUDY TIMELINE 
 
 
 
 
YEAR 1 
YEAR 2 
YEAR 3 
Q1 
Q2 
Q3 
Q4 
Q1 
Q2 
Q3 
Q4 
Q1 
Q2 
Q3 
Q4 
Preparatory Activities 
 
 
 
 
 
 
 
 
 
 
 
 
 
Document Development (Protocol, forms, etc.) 
 
 
 
 
 
 
 
 
 
 
 
 
 
Procurement of drug and other materials 
 
 
 
 
 
 
 
 
 
 
 
 
 
Approvals (IRB, ERC, Drug Authorities, etc.) 
 
 
 
 
 
 
 
 
 
 
 
 
 
Site Preparation 
 
 
 
 
 
 
 
 
 
 
 
 
 
Training 
 
 
 
 
 
 
 
 
 
 
 
 
Recruitment 
 
 
 
 
 
 
 
 
 
 
 
 
Data Cleaning and Analysis  
 
 
 
 
 
 
 
 
 
 
 
 

 
Aspirin Protocol V 1.3  
 
 
 
 
 
 
 
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21 February 2019  
APPENDIX 5. ASPIRIN PROTOCOL STEERING 
COMMITTEE AND TECHNICAL ADVISORS 
Central Study Team: Global Network Site 08 (Belgaum, India) 
Richard Derman, MD, MPH 
Principal Investigator 
Thomas Jefferson University 
Philadelphia, PA 
Richard.Derman@jefferson.edu  
 
 
 
Bhalchandra Kodkany, MD, MBBS 
Senior Foreign Investigator 
KLE University’s J N Medical College 
Belgaum, India 
drkodkany@jnmc.edu 
 
Shivaprasad S. Goudar MD, MHPE 
Co-Investigator 
KLE University’s J N Medical College 
Belgaum, India 
sgoudar@jnmc.edu 
 
Matthew K. Hoffman, MD, MPH 
Lead Investigator for Aspirin Protocol 
Christiana Care  
Newark, Delaware 
Mhoffman@christianacare.org 
 
Mrityunjay C. Metgud, MD 
Aspirin Protocol Country Coordinator, 
KLE University’s J N Medical College 
Belgaum, India 
metm67@gmail.com  
 
Frances Jaeger, MA, DrPh 
Co-Investigator 
Thomas Jefferson University 
Philadelphia, PA 
Frances.Jaeger@jefferson.edu 
National Institute of Child Health and Human Development (NICHD) 
Marion Koso-Thomas, MD, MPH 
Medical Officer, Global Network for Women’s and Children’s Health  
Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) 
kosomari@mail.nih.gov 
 
Menachem Miodovnik, MD 
Director – Global Network for Women’s and Children’s Health 
Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) 

Aspirin Protocol V 1.3  
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21 February 2019  
menachem.miodovnik@nih.gov 
RTI International 
Elizabeth McClure, PhD 
Principal Investigator, Data Coordinating Center 
mcclure@rti.org 
Dennis Wallace, PhD 
Co-Principal Investigator, Data Coordinating Center 
Senior Statistician 
dwallace@rti.org 
Jay Hemingway-Foday, MPH, MSW 
Protocol Manager for Aspirin Protocol 
hemingway@rti.org 
Site Investigators 
Global Network Site 02 (Democratic Republic of Congo) 
Carl Bose, M.D.  
 
 
 
. 
Principal Investigator 
University of North Carolina School of Medicine 
 
Chapel Hill, North Carolina 
cbose@med.unc.edu  
Antoinette Tshefu, M.D, Ph.D., M.P.H 
Senior Foreign Investigator 
Kinshasa School of Public Health 
antotshe@yahoo.com 
Adrien Lokangaka, M.D., M.P.H. 
Aspirin Protocol Country Coordinator 
Kinshasa School of Public Health 
adrinloks@yahoo.fr 
Global Network Site 03 (Zambia) 
Wally Carlo, MD 
Principal Investigator 
University of Alabama at Birmingham 
Birmingham, Alabama 
Wcarlo@PEDS.UAB.EDU 
Elwyn Chomba, MBChB, DCH, MRCP 
Senior Foreign Investigator 
University Teaching Hospital 
Lusaka, Zambia 
echomba@zamnet.zm 
Cindy Chirwa, MD 
Aspirin Protocol Country Coordinator 
Lusaka, Zambia 

Aspirin Protocol V 1.3  
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21 February 2019  
Cindychirwa78@gmail.com 
Global Network Site 06 (Guatemala) 
Michael Hambidge, MD 
Principal Investigator 
University of Colorado Health Care System (UCHSC) 
Denver, Colorado 
Michael.hambidge@ucdenver.edu 
Nancy Krebs, MD 
Co-Investigator 
UCHSC 
Denver, Colorado 
Nancy.krebs@ucdenver.edu 
Ana Garces, MD, MPH 
Senior Foreign Investigator 
FANCAP 
Guatemala City, Guatemala 
agarces@fancap.org 
Lester Figueroa, MD 
Aspirin Protocol Country Coordinator 
FANCAP  
Guatemala City, Guatemala 
lesterfigueroa@me.com 
Global Network Site 09 (Pakistan) 
Robert Goldenberg, MD 
Principal Investigator 
Columbia University 
New York, New York 
rlg88@columbia.edu 
 
Sarah Saleem, MD 
Senior Foreign Investigator 
Aga Khan University 
Karachi, Pakistan 
Sarah.saleem@aku.edu 
Global Network Site 11 (Nagpur, India) 
Patricia L. Hibberd, MD, PhD 
Principal Investigator 
Boston University 
Boston, Massachusetts  
plh0@bu.edu       

Aspirin Protocol V 1.3  
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21 February 2019  
Archana Patel, MD, DNB, MSCE 
Senior Foreign Investigator 
Indira Gandhi Government Medical College 
Nagpur, India 
Dr_apatel@yahoo.com 
Prabir B. Das, MD 
Aspirin Protocol Country Coordinator 
Lata Medical Research Foundation (LMRF) 
prabir_das23@rediffmail.com 
Global Network Site 12 (Kenya) 
Ed Liechty, MD 
Principal Investigator 
Indiana University School of Medicine 
Indianapolis, Indiana 
eliecht@iu.edu  
Fabian Esamai, MBChB, MMed, PhD 
Senior Foreign Investigator 
Moi University School of Medicine 
Eldoret, Kenya 
fesamai2007@gmail.com 
Technical Advisory Group 
Robert M. Silver, MD 
University of Utah Health Sciences Center 
bob.silver@hsc.utah.edu 
Enrique Schisterman, Ph.D 
Chief and Senior Investigator 
Epidemiology Branch, DIPHR 
Eunice Kennedy Shriver National Institute of Child Health and Human Development 
schistee@mail.nih.gov 

 Global Network:  Statistical Analysis Plan, ASPIRIN Study  
Version 3.0 3/18/2019; Page i  
STATISTICAL ANALYSIS PLAN 
Aspirin Supplementation for Pregnancy Indicated Risk Reduction In 
Nulliparas (ASPIRIN)
The ASPIRIN Study (ClinicalTrials.gov Number: NCT02409680)
SAP VERSION: 
Version 3 
SAP DATE: 
March 18, 2019 
SPONSOR: 
NICHD 
PREPARED BY: 
RTI International 
3040 Cornwallis Rd 
Research Triangle Park, NC 27709-2104 
AUTHOR (S): Dennis Wallace, Beth McClure, Tracy Nolen 

 Global Network:  Statistical Analysis Plan, ASPIRIN Study  
 
 
   
Version 3.0 3/18/2019; Page ii   
 
 

 Global Network:  Statistical Analysis Plan, ASPIRIN Study  
 
 
   
Version 3.0 3/18/2019; Page 3   
 
Contents 
1. 
BACKGROUND AND PROTOCOL HISTORY 
6 
2. 
PURPOSE OF THE ANALYSES 
6 
3. 
STUDY OBJECTIVES AND OUTCOMES 
7 
3.1 
PRIMARY HYPOTHESIS AND ASSOCIATED OUTCOMES 
7 
3.2 
SECONDARY HYPOTHESES AND ASSOCIATED OUTCOMES 
7 
3.3 
OTHER OUTCOMES OF INTEREST IN THIS STUDY 
8 
4. 
STUDY METHODS 
8 
4.1 
OVERALL STUDY DESIGN 
8 
4.2 
STUDY POPULATION 
10 
4.3 
STUDY ARM ASSIGNMENT AND RANDOMIZATION 
11 
4.4 
MASKING AND DATA LOCK 
11 
5. 
ANALYSIS POPULATIONS 
12 
6. 
SAMPLE SIZE DETERMINATION 
13 
6.1 
PRIMARY OUTCOME 
13 
6.2 
SECONDARY OUTCOMES 
13 
7. 
STATISTICAL / ANALYTICAL ISSUES 
14 
7.1 
GENERAL RULES 
14 
7.2 
ADJUSTMENTS FOR COVARIATES 
15 
7.3 
MISSING DATA APPROACHES: 
15 
7.4 
MULTICENTER STUDIES 
15 
7.5 
MULTIPLE COMPARISONS AND MULTIPLICITY 
16 
7.6 
MASKED DATA REVIEW 
16 
8. 
STUDY PARTICIPANTS, TREATMENT EXPOSURE AND COMPLIANCE 
16 
8.1 
PARTICIPANT DISPOSITION 
16 

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8.2 
STUDY TREATMENT EXPOSURE AND COMPLIANCE 
16 
8.3 
PROTOCOL DEVIATIONS 
17 
8.4 
DEMOGRAPHIC AND BASELINE CHARACTERISTICS 
17 
9. 
EFFICACY ANALYSES 
17 
9.1 
OVERVIEW OF EFFICACY ANALYSIS METHODS 
17 
9.2 
DEFINITION OF ANALYSIS VARIABLES 
17 
9.4 
PRIMARY ANALYSIS 
26 
9.5 
SECONDARY OUTCOME ANALYSES 
26 
9.6 
ANALYSES OF OTHER OUTCOMES OF INTEREST 
27 
10. 
SAFETY ANALYSIS: 
27 
11. 
INTERIM ANALYSES AND DATA MONITORING 
27 
12. 
CHANGES TO THE ANALYSES PLANNED IN THE PROTOCOL 
28 
13. 
REFERENCES 
28 
14. 
ATTACHMENTS 
29 
 
 
 

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LIST OF ABBREVIATIONS 
Acronym 
Definition 
DCC 
Data Coordinating Center 
DMC 
Data Monitoring Committee 
DRC 
Democratic Republic of the Congo 
EmONC 
Emergency Obstetric and Neonatal Care 
GA 
Gestational Age 
GEE 
Generalized Estimating Equation 
GN 
Global Network 
ITT 
Intention to Treat 
LMP 
Last menstrual period 
MDG 
Millennium Development Goals 
mITT 
Modified intention to treat 
MNH 
Maternal and Neonatal Health 
PP 
Per protocol 
RCT 
Randomized Clinical Trial 
SAP 
Statistical Analysis Plan 
UN 
United Nations 
 
 
 
 

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1. BACKGROUND AND PROTOCOL HISTORY 
Background and Rationale 
Preterm delivery, defined as delivery prior to 37 weeks 0/7 days gestation, remains the dominant 
cause of neonatal morbidity and mortality throughout the world, (March of Dimes, 2012, Matthews, 
2004, Anderson, 2003) and directly leads to 28% of neonatal deaths within the first seven days of life. 
(Beck, 2010) Moreover, it is responsible of up to 50% of pediatric neurodevelopmental disorders. 
(Goldenberg, 1998) Infants born prematurely are likewise at increased risk for a variety of long-term 
medical complications such as respiratory, gastrointestinal, cardiovascular, and metabolic disorders. 
(McCormack, 2002, Saigal, 2008) 
Compounding the issue, the risk of preterm birth is highest in developing countries where an 
estimated 12% of births are preterm compared to 5-7% in developed countries. (Valero De Bernabé, 
2004) Of the near 13 million preterm births worldwide in 2005, 11 million were in Africa and Asia. 
(Blencowe, 2010) Given the tremendous medical, financial and emotional burden of preterm birth in 
the developing world and the limited resources to provide postnatal care, any interventions with the 
potential to reduce the risk of preterm birth deserve consideration.  If one were to choose an 
intervention, it would be one that is widely available, inexpensive, and safe for the mother and fetus.  
Low dose aspirin may be just such an intervention. 
We intend to study the effects of LDA in nulliparous women for several reasons.  First, although it 
would be of interest to study the effect of LDA in women at high risk for preterm birth (e.g. prior 
preterm birth), such women may undergo interventions intended to decrease their risk of preterm 
birth. Thus, a study in patients with prior preterm birth would have numerous potential confounders. 
Conversely, multiparous women with prior term births would be at very low risk for preterm birth and 
also be a suboptimal population to study. Nulliparous women appear to be an ideal population since 
they will not undergo special interventions in an attempt to avoid preterm birth.  Also, it appears that 
the risk of preterm birth in nulliparous women is higher than for the general obstetric population. In 
summary, available data suggest that LDA may be a safe, widely available and inexpensive 
intervention that may significantly reduce the risk of preterm birth. However, this possibility needs to 
be proven in a properly designed RCT with preterm birth as the primary outcome.  Such a clinical 
trial in a racially, ethnically and geographically diverse population could best be accomplished 
through the established infrastructure of the Global Network for Women’s and Children’s Health 
Research (Global Network or GN). 
Protocol History 
The protocol was initiated in March 2016 under Protocol version 1.1.  Protocol amendment 1.2 was 
generated in December 2016.  
This study completed randomization during the summer of 2018, with follow-up completed 
approximately 9 months after the last participant is randomized.  No formal interim analyses were 
planned nor completed for this study and as such, the first formal assessment for efficacy is planned 
for after completion of primary follow-up. 
2. PURPOSE OF THE ANALYSES 
This statistical analysis plan (SAP) contains detailed information about statistical analyses to be 
performed to assess the primary and secondary hypotheses outlined in the protocol (i.e. to evaluate 

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whether women who are treated with LDA daily beginning between 6 0/7 weeks and 13 6/7 weeks 
GA through 36 0/7 weeks GA will have a reduced risk of preterm birth from all causes, and whether 
these women will have reduced risk of small for gestational age (SGA) deliveries, reduced risk of 
eclampsia and preeclampsia during pregnancy, and reduced risk of infants who experience perinatal 
mortality. The results of these analyses will be included in the study manuscript(s). Additional 
exploratory analyses may be performed to support further manuscript development.  These analyses 
will not require an update to the SAP, but abbreviated analysis plans will be prepared prior to 
conducting those analyses. 
 
3. STUDY OBJECTIVES AND OUTCOMES 
3.1 Primary Hypothesis and Associated Outcomes 
Our primary hypothesis is that nulliparous women with no more than two previous first trimester 
pregnancy losses who are treated with LDA daily beginning between 6 0/7 weeks and 13 6/7 weeks 
GA through 36 0/7 weeks GA will reduce the risk of preterm birth from all causes.   
The primary outcome of this study is preterm birth, which will be defined as delivery at or after 20 
0/7 weeks and prior to 37 0/7 weeks.   This will be determined based on actual date of delivery in 
comparison to the projected EDD, independent of whether or not the preterm delivery is indicated or 
spontaneous. 
3.2 Secondary Hypotheses and Associated Outcomes 
The study will also test three secondary hypotheses:  
 
Infants of women who take antenatal daily LDA initiated at 6 0/7 to 13 6/7 weeks GA will be 
at lower risk of being small for gestational age (SGA). 
 
Women who take antenatal daily LDA initiated at 6 0/7 to 13 6/7 weeks GA will have lower 
risk of: eclampsia and preeclampsia. Note that this hypothesis will be operationalized as 
evidence of hypertensive disorders of pregnancy as defined in more detail in Sections 9 and 
12. 
 
Infants of women who take antenatal daily LDA initiated at 6 0/7 to 13 6/7 weeks GA will be 
at lower risk of perinatal mortality. 
 
These secondary hypotheses will be tested using the following outcomes.  
• 
Small for gestational age infants include newborns with weights below the 10th percentile for 
gestational age, as defined by Intergrowth Standards.   
• 
Risk of Pregnancy Induced Hypertension (eclampsia and/or preeclampsia) defined as a binary 
outcome of any of new onset hypertension that occurs beyond 20 weeks gestational age, 
preeclampsia, or eclampsia happening between initiation of study dosing and participant 
completion of the study at 6 weeks after delivery. Eclampsia is a serious complication of 
pregnancy characterized by convulsions and coma. It may be preceded by signs of pre-
eclampsia or the onset may be rapid and sudden and can occur in pregnancy, labor or soon 
after delivery. Preeclampsia is characterized by hypertension and proteinuria occurring after 
the 20th week of pregnancy.  See Sections 9 and 12 for more detail. 

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• 
Perinatal mortality is a binary outcome that includes any stillbirth occurring after 20 weeks 
pregnancy and any early neonatal death that occurs within 7 days after delivery.  
3.3 Other Outcomes of Interest in this Study 
Study analyses will examine a number of additional maternal and fetal or neonatal outcomes to assess 
whether use of LDA daily beginning between 6 0/7 weeks and 13 6/7 weeks has an effect on those 
outcomes.  Specific maternal and fetal/neonatal outcomes include: 
Maternal outcomes: 
 
Vaginal bleeding 
 
Antepartum hemorrhage  
 
Postpartum hemorrhage 
 
Maternal mortality  
 
Late abortion 
 
Preterm, preeclampsia  
 
Fetal/Neonatal outcomes: 
 
Preterm birth <34 0/7 weeks of pregnancy  
 
Birth weight <2500g and <1500g 
 
Fetal loss 
 
Spontaneous abortion 
 
Stillbirth  
 
Medical termination of pregnancy 
Detailed definitions of these outcomes are included in Section 9. Change in maternal hemoglobin is 
an other/exploratory outcome collected for this study.  Analyses of these data will be performed for a 
secondary manuscript and are not covered under this analysis plan. 
4. STUDY METHODS 
4.1 Overall Study Design 
The ASPIRIN trial was conducted as a randomized, placebo-controlled, double-blinded multicenter 
clinical trial to assess the efficacy of LDA in the reduction of preterm birth.  Women were 
randomized equally to receive either daily LDA (81 mg) or an identical appearing placebo beginning 
between 6 0/7 weeks and 13 6/7 weeks GA and continuing until 36 0/7 weeks GA or delivery. 
The study intervention is 81 mg of aspirin administered daily beginning between 6 0/7 weeks and 13 
6/7 weeks through 36 0/7 weeks or delivery. Following randomization, each woman was to be 
provided with a supply of either LDA or an identical appearing placebo. The drug or placebo were 
enteric coated pills, provided in identical, child-resistant packaging, with written instructions for use. 
To ensure that literacy levels did not affect proper use, the study staff was to provide verbal 
instructions at randomization and reinforce these guidelines at subsequent follow-up visits. Each 
woman was to also receive a back-up supply of medication or placebo, which will be maintained 
throughout the duration of the study.  The purpose of the backup is to bridge circumstances wherein 
the woman misses or is late to a planned follow-up visit or the primary supply of study drug/placebo 
is misplaced or destroyed.  Medication compliance was to be monitored, and the drug/placebo supply 
was to be replenished during routine study visits. 

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A study schematic is shown below 
 

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decrease their risk. Conversely, multiparous women with prior term births would be at very low risk 
for preterm birth, making them a suboptimal study population. Nulliparous women appear to be the 
ideal population to study the effects of LDA since they will not undergo special interventions to avoid 
preterm birth.  Multiple studies have shown that the risk of preterm birth in nulliparous women is 
higher than for the general obstetric population.   
Inclusion criteria 
• 
Nulliparous women between 18 – 40 years of age. Minors who are ≥ 14 years of age may be 
enrolled if permitted by the country’s ethical guidelines.   
• 
No more than two previous first trimester pregnancy losses  
• 
No medical contraindications to aspirin; 
• 
Single live intrauterine pregnancy (IUP) between 6 0/7 and 13 6/7 weeks GA corroborated by 
an early dating ultrasound and with a fetal heart rate detected. 
Exclusion criteria 
• 
Women prescribed daily aspirin for more than 7 days; 
• 
Multiple gestations; 
• 
Fetal anomaly by ultrasound (Note most fetal anomalies are not detectable by ultrasounds 
done at this early gestation. Subsequent discovery of a fetal anomaly is not viewed as an 
exclusion.); 
• 
Hemoglobin < 7.0 g/dl at screening; 
• 
Systolic blood pressure ≥ 140 and diastolic ≥ 90 at screening; 
• 
Any other medical conditions that may be considered a contraindication per the judgment of 
the site investigator (e.g., Lupus, Type 1 Diabetes, or any other known significant disease) 
4.3 Study Arm Assignment and Randomization 
Randomization of subjects was carried out to obtain a 1:1 allocation ratio between the treatment and 
placebo arms.  Randomization was stratified by site.  A computer algorithm generated by the data 
coordinating center (DCC) was created the random assignment to one of the treatment arms based on 
randomly permuted block design with randomly varied block sizes. The block sizes were known only 
by the DCC personnel.  A total of 11,920 nulliparous women were to be enrolled (5960 per group) 
across seven sites in sub-Saharan Africa, South Asia, and Latin America. For balance, each site was 
to enroll no more than 25% of the total sample.  
To study the possible effect of LDA on anemia, an ancillary study was to enroll at least 500 women to 
obtain hemoglobin measure at 4 weeks after initiation of therapy, with each site contributing at least 
75 women to that sample.  Some sites ultimately collected hemoglobin 4-weeks post randomization 
for all participants. In addition, hemoglobin measures were to be obtained from all women at 26 to 30 
weeks gestational age. The analyses of this ancillary study will be described in a separate analysis 
plan document and will not be included as part of the primary study publication. 
4.4 Masking and Data Lock 
Both the formulation of the medication and placebo were procured from two manufacturers, with one 
manufacturer supplying materials for Pakistan participants and the other manufacturer supplying 

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materials for the participants at the other sites.  The packaging was standardized across sites and 
labeled as ASA 81 mg/placebo, with the expiration data and a unique identifier.  A certificate of 
authenticity was to likewise be provided. Manufacturing details are maintained in the study file. 
Throughout the study, research staff and local health providers were masked to treatment status unless 
there was a serious adverse event potentially related to the treatment modality that requires 
unmasking for safety reasons. There was one pharmacist at each site who was to remain unmasked to 
monitor randomization, drug supply, and safety as needed. 
Data Lock: Data will be locked at the site level with final analysis data sets generated and locked 
once data from all sites have been received and all queries processed.  For each site, data collection 
for the protocol will stop approximately 9 months after the last pregnant woman was randomized into 
the study at that site. Site will transmit all data forms to the site within three months of this date, and 
data will be locked three to six months post last follow-up visit. Note that other than for the cases on 
individual unmasking described above, which are anticipated to be minimal, the study will not be 
unmasked until after data lock. 
5. ANALYSIS POPULATIONS 
Safety (SAF) Population 
The safety population will comprise all randomized participants who received any study treatment 
grouped by actual treatment received, irrespective of amount or duration of treatment received.  
Unless specified otherwise, data for this population will be analyzed as available (i.e. irrespective of 
protocol deviation occurrence, subsequent study participation termination, or study drug withdrawal). 
Modified Intention to Treat (mITT) Population  
The primary analysis population will be the modified intention to treat population, which 
includes all eligible, randomized participants who provide any post-baseline outcome data and who 
deliver at 20 weeks gestational age or greater.  All participants will be assigned to the arm to which 
they were randomized irrespective of treatment received. Unless specified otherwise, data for this 
population will be analyzed as available (i.e. irrespective of protocol deviation occurrence, 
subsequent study participation termination, or study drug withdrawal). This population will also be 
used for many of the secondary analyses. Note that this population represents a change from the 
protocol, which indicates that the ITT population will be used for the primary analyses. This change 
was made prior to any unmasking. 
Intention to Treat (ITT) Population  
The intention to treat population will include all eligible, randomized participants.  All participants 
will be assigned to the arm to which they were randomized irrespective of treatment received. Unless 
specified otherwise, all participants will be included in analyses using this population (with data 
imputed as needed and irrespective of protocol deviation occurrence, subsequent study participation 
termination, or study drug withdrawal). This population will be used to conduct sensitivity analyses 
for the primary outcome. 
Per Protocol (PP) Population  

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This population will be used to conduct sensitivity analyses for the primary outcome. This population 
will exclude all or part of the data obtained from any eligible, randomized participants who deliver at 
20 weeks gestational age or greater that did not receive at least 90% of the full amount of intended 
randomized study therapy or are considered to have substantially deviated from the protocol in a 
manner that may impact study outcome or treatment receipt.  The population will also exclude 
individuals who were randomized after 10 weeks, 6 days gestational age.  Participants will be 
grouped by actual treatment received. Treatment receipt reasons and substantial deviations that will 
lead to exclusion include:  
• 
Documented receipt of aspirin while on study outside of assigned study drug as identified by 
a reported protocol deviation 
• 
Not receiving treatment after randomization: All data from these participants will be 
excluded. 
• 
Receipt of less than 90% of planned total doses after randomization where the number of 
planned doses will be calculated as those expected between randomization and the earliest of 
end of pregnancy and 36 0/7 weeks GA irrespective of if study drug administration was 
prematurely discontinued for any reason prior to that point: All data from these participants 
will be excluded. 
Receiving more than the planned total doses of study drug will generally NOT be an exclusion 
criterion for the PP population. Additional exclusion reasons may be identified after completion of 
participant enrollment.  Any participant deviations that will exclude participants or their data from the 
PP analyses will be identified by the study team, prior to review of efficacy and safety data, as a part 
of a masked data review to reduce the opportunity for introducing of bias and will be documented in 
an addendum to this analysis plan.  
6. SAMPLE SIZE DETERMINATION 
6.1 Primary Outcome 
The risk of preterm birth < 37 weeks gestation in untreated nulliparous women was estimated to be 
about 8.0% for the EAGeR trial (Abramovitch, 2012 and Schusterman, 2014). Unpublished data for 
2012 from the GN sites shows preterm birth rates, ranging from 2.9% to 9.8%, while WHO estimates 
the range for the countries in which GN sites are located to be 7.7% to 16.7% 1. In developing sample 
size estimates we considered risks in the range of 8% to 14% and ultimately selected sample sizes 
based on a conservative estimate of 8%.  Because the association of LDA and preterm birth may 
differ in international settings, we examined sample size requirements for reductions of 40%, 25%, 
and 20%. As shown in the protocol, the number of evaluable participants needed to detect a 20% 
reduction from a usual rate of 8% preterm births with 90% power is 5,483 per treatment arm. To 
account for the loss of evaluable subjects due to miscarriages, which are anticipated to occur in 
approximately 5% of participants) and loss to follow-up (assumed to be in the 1% to 2% range), 
sample sizes were increased by 8% to obtain a final sample size of 11,920 (or 5,960 per treatment 
arm). 
6.2 Secondary Outcomes 
One of the key secondary objectives of this trial is to evaluate the effect of low dose aspirin on 
perinatal mortality.  We evaluated the reduction in perinatal mortality that could be detected with 
80% power and 90% power under the assumption that the sample size for the study would be based 

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on that needed to achieve the primary aim of demonstrating a 20% reduction in low-birth weight 
infants.  The analyses were based on the assumption that the perinatal mortality rate in the GN 
population is approximately 45 perinatal deaths per 1000 deliveries (unpublished data from the MNH 
Registry for 2013).  With this estimated underlying mortality rate and 5,483 evaluable subjects per 
treatment arm required to demonstrate the 20% reduction in low birth weight prevalence as outlined 
in the primary aim, the study will have 80% power to detect a 24% reduction in perinatal mortality 
and 90% power to detect a 27% reduction in perinatal mortality. 
To determine the sample size actually needed to demonstrate the reduction in perinatal mortality 
actually expected if the intervention results in a 20% reduction in prevalence of low birth weight 
infants, we examined both the prevalence of low birth weight infants across the GN sites and the 
perinatal mortality risk in both the low birth weight and normal birth weight infants.  Based on 2013 
MNH Registry data, the prevalence of low birth weight among all GN deliveries was 16.3% and the 
perinatal mortality rates were 20.5 per 1000 deliveries among normal birth weight infants and 162.3 
per 1000 deliveries among low birth weight infants; these values yield a weighted average perinatal 
mortality rate of 43.6 per 1000 deliveries.  If the low dose aspirin intervention is effective in reducing 
the prevalence of low birth weight deliveries by 20% (i.e. reducing the prevalence from 16.3% to 
13%) and the conditional risk of perinatal mortality is unchanged in the low birth weight and normal 
birth weight cohorts, we would expect the resultant overall perinatal mortality rate to be reduced to 
38.9 per 1000 deliveries or a 10.8% reduction.  Detection of a reduction of this magnitude with 80% 
power would require an evaluable sample size of greater than 28,000 evaluable deliveries per 
treatment arm, and the study would need over 37,000 evaluable deliveries per arm to achieve 90% 
power to detect a reduction of this level in perinatal mortality. 
Given the sample sizes associated with the second approach, the best alternative appears to be to 
power the study to demonstrate the 20% reduction in prevalence of low birth weight deliveries with a 
commitment to generate point and interval estimates of the impact of the intervention on perinatal 
mortality.  By examining the risk of perinatal mortality in both the normal and low birth weight 
infants in the two arms, the trial would provide some insight about impacts of the intervention on 
mortality other than that mediated through the effect on birth weight. 
We also conducted power analyses to examine the effect sizes that could be detected with the planned 
sample sizes for two additional secondary outcomes, prevalence of SGA infants and incidence of 
eclampsia/preeclampsia in the target population.  Generic information as well as preliminary 
information from the 2013 MNH Registry indicates that the risk of each of these events in the 
population of interest is approximately 5%. Under the assumption of 5483 evaluable participants in 
each treatment arm and an assumed Type I error rate of 0.05 for per-comparison analyses (i.e. no 
control for multiple comparisons in the analyses of these secondary outcomes), the study will have 
70% power to detect a 20% reduction in the risk of each of these outcome measures, 85% power to 
detect a 24% reduction and 90% power to detect a 26% reduction.  Consequently, the study is 
reasonably powered to examine these secondary outcome measures. 
7. STATISTICAL / ANALYTICAL ISSUES 
7.1 General Rules 
All statistical computations will be performed and data summaries will be created using SAS 9.3 or 
higher. If additional statistical packages are required, these will be discussed in the study report. For 
summaries of study data, categorical measures will be summarized in tables listing the frequency and 
the percentage of participants in each study arm; continuous data will be summarized by presenting 

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mean, standard deviation, median, minimum, and maximum; and ordinal data will be summarized by 
only presenting median and the limits of the interquartile range. 
7.2 Adjustments for Covariates  
As this is a randomized trial and the randomization process was not compromised during execution, 
the treatment effect estimate from trial is unbiased, even without adjusting for any baseline 
covariates.  Therefore, no adjustments for covariates other than site are planned for the primary 
analysis.  However, baseline clinical and demographic variables that are known predictors of neonatal 
mortality and maternal outcomes will be included in secondary analyses of intervention efficacy.   
7.3 Missing Data Approaches:  
The study is likely to have two primary sources of missing data.  Since the primary interest is in 
delivery outcomes for women who deliver after 20 weeks, the first primary source for missing data is 
early pregnancy losses due to miscarriage or medical termination of pregnancy (MTP) prior to 20 
weeks gestational age.  The second primary reason for missing data will be participant loss to follow-
up.  Each of these scenarios is treated separately below. 
Women are being enrolled in the ASPIRIN trial when they are between 6 0/7 weeks and 13 6/7 weeks 
gestational age.  Depending on the distribution of study participants over this gestational age range 
we anticipate that between 2% and 8% of the women in the study will have a pregnancy loss prior to 
20 weeks.  For the primary outcome of preterm birth, these women will be excluded from the 
analyses and hence be considered missing at random.  However, because we recognize that these 
women may not have the same risk of preterm delivery as those followed past 20 weeks, we will 
generate two additional sensitivity analyses to assess whether inference is robust to this assumption.  
For the first analyses, these early terminations will be treated as preterm deliveries in a full ITT 
analysis.  For the second approach, the mITT population will be used with inverse probability 
weighting used to account for the individuals lost prior to 20 weeks.  
Based on the historical data available from the MNH registry, we anticipate that the rate of missing 
responses for maternal and neonatal morbidity and mortality measures are likely to be 2% or less.  
Although we do not expect this rate to change over the course of this trial, we will examine data from 
both arms to ensure that the missing rate is comparable in the participants assigned to the Aspirin and 
Placebo treatment arms.  We will examine patterns of missing data by arm and site. If more than 2% 
of data in either or both arms are missing, we will conduct sensitivity analysis on the effect of the 
missing on the inference by using use multiple imputation procedures.  Multiple imputation assumes 
that the mechanism that caused the missing is independent of the value of the unobserved data (i.e. 
data are missing at random – MAR). Although there is no reason to believe that the missing data on 
the use would not be MAR, multiple imputation processes have been shown to be robust even if the 
missing mechanism is dependent on the values of the unobserved data, i.e. even is the missing data 
are non-ignorable non-response. The results of all sensitivity analyses will be presented in the final 
report. 
7.4 Multicenter Studies 
For this multicenter study, randomization of study participants was stratified within site.  
Consequently, for all test-based analyses of treatment effect and model-based primary and secondary 
analyses, site will be included as a fixed effect in the models.  As an ancillary analysis associated with 
the primary outcome (risk of premature delivery) as well as secondary outcomes, we will examine 

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descriptively whether the treatment effects vary across sites; however, these analyses of site 
differences in treatment effect are considered to be descriptive because sample sizes are likely 
inadequate to provide adequate power to detect site by treatment interaction effects.    
7.5 Multiple Comparisons and Multiplicity 
There is only one formal hypothesis test for this study. As such, a statistical test will be conducted at a 
5% type I error rate (two-sided) for the primary efficacy measure, and no adjustments for multiplicity 
will be made. All analyses of secondary outcomes are exploratory in nature; therefore, p-values and 
confidence intervals are provided for descriptive purposes only. All p-values provided for any 
baseline and demographic characteristics and safety parameters will be for descriptive purposes only. 
As such, unless otherwise specified, p-values presented will be on a per analysis basis, with no further 
control for multiple tests.  
7.6 Masked Data Review 
A masked data review process will be performed by members of the protocol team prior to final data 
lock to define study analysis populations and to adjudicate key outcome variables.  Key questions that 
will be addressed during this review will be definition of individuals to be included in the per protocol 
population, adjudication of the primary outcome variable and selected secondary outcomes for this 
study.  Detailed documentation of the masked data review and resulting decisions will be described in 
a SAP addendum.  
8. STUDY PARTICIPANTS, TREATMENT EXPOSURE AND COMPLIANCE 
8.1 Participant Disposition   
Participant eligibility status will be summarized by study arm and overall disposition of study 
participants will be described using a standard CONSORT diagram. The number of participants 
randomized; completing or discontinuing from study therapy; and completing the study follow-up 
will be summarized by study arm. Reasons for study withdrawal will also be summarized by 
treatment arm to assess whether they are balanced across treatment arm and to assure that any 
imbalances are unlikely to affect inference. 
8.2 Study Treatment Exposure and Compliance 
Each participant will take daily LDA or placebo from the time of randomization until 36 0/7 weeks 
GA or delivery. Drug compliance will be routinely monitored throughout this period through pill 
counts and the completion of a medicine compliance form each time the pill supply is replenished. An 
interval medical obstetrical history will also be taken during these scheduled visits. Study staff will 
also provide reminders about proper drug administration and the importance of compliance during bi-
weekly visits to monitor side effects. 
• 
As a part of the analyses, characteristics of study drug exposure will be summarized by study 
arm. Characteristics that will be included in this analysis summary include  
o Gestational age at initiation of first dose treated as a binary measure before and after 
10 weeks. 
o Gestational age when last dose was taken 
o Number of individuals with dose withheld under clinical direction 
o Percentage of possible days on which dose was taken:  

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 
As a measure of adherence where the number of planned/possible doses 
excludes any periods where the participants was asked by a health provider 
or study staff to stop taking the study medication as a result of a medication 
side effect or unexpected medical event 
 
As a measure of exposure where the number of planned/possible doses 
includes all days between randomization and earliest of end of pregnancy and 
36 0/7 weeks GA 
o Summarize the percent of doses missed for planned doses prior to 16 weeks GA vs. 
after 16 weeks GA 
8.3 Protocol Deviations 
Protocol deviations will be summarized by treatment arm and by site with information such as type of 
deviation, time of occurrence, and reason. Incidence rate of protocol deviations will also be 
summarized overall and for each protocol deviation category by treatment arm. Incidence rate of 
protocol deviations will be calculated as: number of deviations divided by the number of participant 
weeks at the site. The comments section will be reviewed to examine overarching themes, and these 
will be included in the final report. 
8.4 Demographic and Baseline Characteristics 
Demographic and baseline clinical characteristics for the study participants will be summarized by 
treatment group using the general analysis rules describe above for the mITT, ITT, PP and SAF 
populations.  Variables of interest include maternal age, maternal education, gravidity, maternal 
weight and BMI, previous pregnancy loss, number of antenatal care visits, delivery attendant, 
delivery location, and type of delivery.  
9. EFFICACY ANALYSES 
9.1 Overview of Efficacy Analysis Methods 
The primary efficacy analyses will be performed using the mITT population.  All other efficacy 
analyses will be performed either using the mITT, ITT or per-protocol population as detailed below. 
The data will be summarized by treatment arm, overall and by country.  All model-based analyses 
and test-statistics examining the treatment effect will adjust for GN site as a randomization variable. 
Additional details are presented in the sections below. 
9.2 Definition of Analysis Variables 
The following table defines each of the effectiveness and process analysis variables. 
 
 

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Variable 
Type 
Definition 
Primary Outcome  
Preterm birth 
Binary 
The unit of analysis is the pregnancy delivery with the outcome classified as “Yes” if the 
delivery is classified as preterm and “No” if not.  Preterm is defined as delivery at or 
after 20 0/7 weeks and prior to 37 0/7 weeks and includes both live births and stillbirths. 
This definition is consistent with 
- 
WHO definition: babies born before 37 completed weeks of pregnancy.  
- 
ACOG definition: Birth occurs between 20 weeks and 37 weeks of pregnancy  
Analysis Population: mITT  
Secondary Outcomes 

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Variable 
Type 
Definition 
Preeclampsia and eclampsia 
(or more appropriately 
hypertensive disorders of 
pregnancy) 
Binary 
The unit of analysis is the woman randomized to the trial, but the outcome will be 
defined by the characterization of evidence of hypertensive disorder, including either 
preeclampsia or eclampsia occurring during the pregnancy. The algorithm for defining 
this endpoint is included as an addendum to this SAP in 
HypertensionAlgortihm_V1.1_10052018 (amendments to this algorithm may occur as 
part of the endpoint adjudication process and will be documented in new versions of this 
addendum accordingly). 
• 
Briefly, any of the following will count as evidence of hypertensive disease:  
o Any reported SAE of preeclampsia or eclampsia. 
o MNH registry report of hypertensive disease, preeclampsia or eclampsia. 
o Reports of elevated blood pressures that meet criteria based on the ACOG 
2013 “Hypertension in Pregnancy” Task Force Report at any point after 20 
weeks GA.   
 
The only case that will programmatically qualify for evidence of 
hypertension is: Any individual with 2 consecutive timepoints with 
≥ 140 mm Hg systolic or ≥ 90 mm Hg diastolic where those 
timepoints occur more than a week (7 days) apart. The criteria (e.g. 
elevated systolic or elevated diastolic) must be consistent for the two 
consecutive visits. 
• 
For individuals that do meet the above criteria 
o If they have less no reports of elevated blood pressure or a single report of 
elevated blood pressure that is followed by a normal value, they will not be 
considered to have evidence of hypertensive disease. 
o If they have any other reports of elevated blood pressure, their outcome 
classification will be adjudicated in a masked manner by clinical experts. 
Will excluded any MTP but include withdrawals and terminations as data are available. 
Analysis Population: mITT 
. 

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Variable 
Type 
Definition 
Small for Gestational Age 
Binary 
The unit of analysis is the infant delivered from each individual pregnancy.  The outcome 
will be classified as “Yes” if the infant is classified as SGA, defined as a newborn with 
weight below the 10th percentile for gestational age” and “No” otherwise.  Note that 
growth curves appropriate for each GN site based on Intergrowth standards will be used 
to define the expected gestational age distribution.  
For any outcomes involving birth weight, the reported weight will only be used to 
calculate that outcome if collected within 4 days of birth.  Any weight captured after 4 
days will be excluded. 
Analysis Population: mITT 
Perinatal Mortality 
Binary 
The unit of analysis is the infant delivered from each individual pregnancy.  The outcome 
will be classified as “Yes” if the infant is a fresh or macerated stillbirth or a death in the 
first week of life; the perinatal period commences at 20 completed weeks (154 days) of 
gestation and ends seven completed days after birth.  Pregnancies terminated prior to 20 
completed weeks of gestation will be treated as missing for this outcome. 
Analysis Population: mITT 

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Variable 
Type 
Definition 
Other Outcomes of Interest—Maternal Outcomes  
Preterm, preeclampsia 
Binary 
The unit of analysis is the woman randomized to the trial who delivers after 20 weeks 
gestational age.  The outcome will be defined as delivered at less than 34 weeks GA 
AND also meeting the criteria for the endpoint “Preeclampsia and eclampsia (or more 
appropriately hypertensive disorders of pregnancy).”  
Analysis population: mITT 
Vaginal Bleeding 
Binary 
The unit of analysis will be individual woman.  Per the protocol, Vaginal Bleeding is 
defined as: “Bleeding during pregnancy (i.e., WHO generally defines as ‘light’ or 
‘heavy’ bleeding based on clinical symptoms).”  This outcome will be defined based on 
Question 2e of Form ASP06 and will be classified as “Yes” if the response to this 
question is Yes at any bi-weekly visit, and No if all completed biweekly visits have either 
a No or a DK response. Any woman with at least one biweekly visit form will be 
included in the denominator; the outcome will be classified as missing if a subject has no 
biweekly visit forms.  
Analysis population: ITT 
Antepartum Hemorrhage 
Binary 
The unit of analysis will be individual woman with a delivery after 22 weeks.  Per the 
protocol, antepartum hemorrhage is defined as:” Bleeding from the genital tract at any 
time after the 22nd week of pregnancy and before the birth of the baby. There are two 
main causes of antepartum hemorrhage, placenta previa and abruption placentae.”  This 
outcome will be defined based on Question A8b from Form MN02 from the MNH 
Registry.  The event is defined as follows in the MNH Registry Manual of Operations: 
“Defined as blood loss of > 1000 cc of blood prior to delivery.”  
Analysis population: mITT  

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Variable 
Type 
Definition 
Postpartum Hemorrhage 
Binary 
The unit of analysis will be individual woman.  Per the protocol, postpartum hemorrhage 
is defined as:” Blood loss of 1000 ml or more from the genital tract after delivery and up 
to six weeks post-delivery.  Common causes include atony (poor muscle tone) of the 
uterus or trauma to the genital tract, e.g. tears of the vagina, cervix, or lower segment of 
the uterus.”  This outcome will be defined based on Question A8c from Form MN02 or 
Question B7 from Form MN03 from the MNH Registry.  The event is defined as follows 
in the MNH Registry Manual of Operations: Defined as blood loss of > 1000 cc of blood 
after delivery prior to six-week visit.  
Analysis population: Safety 
Maternal mortality through 
42 days 
Binary 
The unit of analysis will be women randomized.  Per the protocol, maternal mortality is 
defined as: “the death of a woman during pregnancy (i.e. conception to delivery) and the 
puerperium (i.e. up to 42 days after delivery).” The event will be defined as “Yes” if the 
woman dies prior to 42 days after delivery and “No” otherwise.  This outcome measure 
will be obtained from forms MN02 and MN03 in the MNH Registry.  If the 42-day 
outcome is missing and the woman was alive at the last known follow-up, this outcome 
will be treated as missing.  
Analysis population: ITT 
Late Abortion  
 Binary 
The unit of analysis will be individual woman.  Per the protocol, late abortion is defined 
as: “Spontaneous fetal loss ≥ 16 weeks.”  This outcome will be defined based on a 
response of 4 for Question A7 from Form MN02 from the MNH registry and will be 
classified as “Yes” if that response is listed as the Mode of Delivery and the pregnancy 
has a GA of 16 weeks or greater and “No” otherwise.  Per the MNH Registry Manual of 
Operations, that mode of delivery should be used if “A delivery prior to 20 weeks 
gestation (or <500 g). Miscarriage is also known as spontaneous abortion.” If the 
delivery is after 20 weeks and is classified as a miscarriage on the form, the outcome will 
be reclassified as a stillbirth. Individuals with loss prior to 16 weeks will be excluded 
from the analyses.  
Analysis population: ITT 

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Variable 
Type 
Definition 
Other Outcomes of Interest—Fetal Outcomes 
Preterm birth <34 0/7 weeks 
of pregnancy 
Binary 
The unit of analysis will be the individual delivered fetus or infant (either live birth or 
stillbirth); note that per the protocol, the event is defined as: “live birth before 34 0/7 
weeks of pregnancy are completed,” so this definition represents a departure from the 
protocol.  This will be defined as a secondary variable in the analysis data set based on 
gestational age as determined from ASP forms and neonatal outcome as determined from 
Form MN02 from the MNH Registry.  
Analysis population: mITT 
Birth weight < 2500g 
Binary 
The unit of analysis will be the individual infant and per the protocol, the event is defined 
as: “Birth weight is defined as the first weight of the fetus or newborn obtained after 
birth. For live births, birth weight should preferably be measured within the first hour of 
life before significant postnatal weight loss has occurred, recorded to the degree of 
accuracy to which it is measured (WHO). Birth weight <2500 g is defined as low birth 
weight” This outcome measure will be a derived measure obtained using the birth weight 
information provided on Questions C3-C5 from Form MN02 from the MNH Registry.  If 
the actual measure is obtained, the binary variable will be generated from Question C3; 
otherwise it will be based on the response to Question C5.  
For any outcomes involving birth weight, the reported weight will only be used to 
calculate that outcome if collected within 4 days of birth.  Any weight captured after 4 
days will be excluded. 
Analysis population: mITT 

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Variable 
Type 
Definition 
Birth weight < 1500g 
Binary 
The unit of analysis will be the individual infant and per the protocol, the event is defined 
as: “Birth weight is defined as the first weight of the fetus or newborn obtained after 
birth. For live births, birth weight should preferably be measured within the first hour of 
life before significant postnatal weight loss has occurred, recorded to the degree of 
accuracy to which it is measured (WHO). Birth weight <1500 g is defined as very low 
birth weight.” This outcome measure will be a derived measure obtained using the birth 
weight information provided on Questions C3-C5 from Form MN02 from the MNH 
Registry. If the actual measure is obtained, the binary variable will be generated from 
Question C3; otherwise it will be based on the response to Question C5.  
For any outcomes involving birth weight, the reported weight will only be used to 
calculate that outcome if collected within 4 days of birth.  Any weight captured after 4 
days will be excluded.  
Analysis population: mITT 
Fetal Loss 
Binary 
The unit of analysis will be the individual delivered fetus or infant and per the protocol, 
the event of fetal loss is defined as: “Spontaneous loss ≥ 16 weeks GA plus perinatal 
mortality.” This binary outcome measure will be defined in the analysis data set based on 
gestational age as measured via the ASP forms and mortality outcomes as determined 
from Forms MN02 and MN03 from the MNH Registry.  Any death of an infant greater 
than 16 weeks GA and prior to 7 days post-delivery will be classified as “Yes” and live 
births classified as alive at 7 days will be classified as “No.”  Any fetal loss (including 
MTP or miscarriage) that occurs prior to 16 weeks will be treated as a missing value.  
Analysis population: ITT 

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Variable 
Type 
Definition 
Spontaneous abortion 
Binary 
The unit of analysis is the individual pregnancy.  Per the protocol, the binary outcome of 
spontaneous abortion is defined as: “Premature expulsion of a non-viable fetus from the 
uterus at < 20 weeks gestation.”  This outcome will be defined based on Questions A7-4 
or C1 from Form MN02 from the MNH registry and will be classified as “Yes” if the 
response to A7-4 is listed as the Mode of Delivery or the response to C1. Fetal/Neonatal 
outcome is answered 1. Miscarriage and “No” otherwise.  Per the MNH Registry Manual 
of Operations, that mode of delivery should be used if “A delivery prior to 20 weeks 
gestation (or <500 g). Miscarriage is also known as spontaneous abortion.”  
Analysis population: ITT 
Stillbirth 
Binary 
The unit of analysis will be the individual infant that delivers at 20 weeks gestational age 
or greater during the specified analysis period. The event will be classified as “Yes” if 
the infant is classified as a non-macerated still birth or with maceration status unknown 
via Forms MN02 and MN05 in the MNH Registry and “No” if the baby is born alive as 
specified via Form MN02 in the MNH Registry.  Note that infants classified as 
Macerated still births are not included in this population. Also note that deliveries 
classified as miscarriages but that have GA of 20 weeks or greater will be reclassified as 
stillbirths.  
Analysis population: mITT 
Medical Termination of 
Pregnancy (MTP) 
Binary 
The unit of analysis is the individual pregnancy.  Per the protocol, the binary outcome of 
MTP is defined as: “an operation or other procedure to terminate pregnancy before the 
fetus is viable.”  This outcome will be defined based on Question A7-5 or Question from 
Form MN02 from the MNH registry and will be classified as “Yes” if  the response 5 is 
listed as the Mode of Delivery on Question A7 or if Question C1. Fetal/neonatal outcome 
is answered 2. MTP and “No” otherwise.  Per the MNH Registry Manual of Operations, 
that mode of delivery should be used if “This response includes any medical termination 
of pregnancy, including abortion (therapeutic abortion, medical abortion, etc.).”  
Analysis population: ITT 
 

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9.4 Primary Analysis 
The primary analysis will compare the risk of preterm birth between the two treatment arms using 
two complementary approaches.  First, the formal test of the primary hypothesis that the risk of 
preterm birth differs between the two arms will utilize a modified intention-to-treat approach based 
on a Cochran-Mantel-Haenszel test stratified by Global Network sites.  The approach is characterized 
as a modified intention to treat approach because the analysis population for the test will be all 
randomized pregnancies for which the delivery occurs after 20 weeks.  Earlier deliveries 
(miscarriages) will be considered missing completely at random for purposes of this analysis. 
In order to evaluate the sensitivity of the primary hypothesis test to the assumption by which 
miscarriages are treated as randomly missing and to control for potential confounders of the treatment 
effect, additional analyses will be conducted using a series of three generalized linear models.  These 
generalized linear models will be fit with the binary outcome of preterm (<37-week delivery) as the 
outcome measure.  The initial model will include terms for treatment, site, and a treatment by site 
interaction.  If the treatment by site interaction term is found to be significant (p<0.05), then all 
subsequent models will include the interaction term and all effects will also be reported by site, 
although formal inference will be based on the average effect across sites.  If the interaction term is 
not significant, it will be removed from the model and this initial model be used to estimate an 
unadjusted treatment effect controlling for site.  The second series of models will include any 
demographic or clinical variable found to differ significantly between the treatment arms in the 
preliminary analyses described above.  These models will be used to generate adjusted estimates of 
the treatment effect controlling for potential confounders.  The third set of models will utilize 
extensions of the generalized linear model that incorporate inverse probability weighting to evaluate 
the sensitivity of the inference to treatment of miscarriages as randomly missing (Hogan, 2004). As 
described in Section 7.3, we will also conduct sensitivity analyses using the full ITT population; for 
these analyses, missing data due to loss to follow-up will be handled using multiple imputation, and 
these early terminations will be treated as preterm deliveries. 
9.5 Secondary Outcome Analyses  
This study has been designed to evaluate the differences between the two treatment arms for three 
secondary outcomes: the risk of perinatal mortality; the risk of eclampsia/preeclampsia, and the risk 
of a SGA delivery.  As with the primary analyses, each of these outcomes will be examined 
individually using descriptive tests of hypotheses that the risk differs between the two arms based on 
Cochran-Mantel-Haenszel tests stratified by Global Network sites. The analyses for each of these 3 
secondary outcomes will utilize the same modified intention-to-treat population used for the primary 
outcome.  Each of these hypothesis tests will be conducted at the 0.05 level of significance with no 
adjustment for multiple comparisons.  In addition to this set of hypothesis tests, model-based analyses 
comparable to those conducted for the primary analyses will be used to evaluate potential 
heterogeneity of treatment effect across sites and evaluate potential confounding.  For each of the 3 
outcomes, an initial model will include terms for treatment, site, and a treatment by site interaction.  If 
the treatment by site interaction term is found to be significant (p<0.05), then all subsequent models 
will include the interaction term and all effects will be reported by site; because the study will lack 
power to detect site-specific effects, all p-values will be reported for the average effect and site effects 
will include point and interval estimates.  If the interaction term is not significant, it will be removed 
from the model and this initial model be used to estimate an unadjusted treatment effect controlling 
for site.  The second series of models will include any demographic or clinical variable found to differ 
significantly between the treatment arms in the preliminary analyses described above.  These models 

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will be used to generate adjusted estimates of the treatment effect controlling for potential 
confounders.   
9.6 Analyses of Other Outcomes of Interest 
In addition to these planned formal primary and secondary outcome analyses, we will also conduct 
exploratory outcomes for a number of other binary outcomes.  The analytic approach for these 
exploratory analyses, will be comparable to that planned for the primary and secondary analyses but 
will focus on estimation of effect sizes and generation of hypotheses, therefore the analytic approach 
will be comparable to the effect estimation approach described for the primary analysis.  Initially, 
contingency tables will be used to generate estimates of the risk of each outcome for the two 
treatment arms, both overall and separately by Global Network (GN) site and across all sites, and the 
relative risk associated with treatment will be estimated by site and across sites controlling for site. 
For each outcome a series of generalized linear models analogous to those described for the primary 
outcome will be used to generate unadjusted estimates of risk, estimates of risk adjusted for potential 
confounders, and estimates of risk adjusted to account for any possible differences in risk between the 
arms associated with miscarriages. 
10. SAFETY ANALYSIS:  
No formal safety analyses are planned as a part of this study, although rates of pregnancy 
complications identified by ultrasound will be summarized as will SAEs and hospitalizations.  No 
formal hypothesis tests will be conducted, but descriptive p-values generated using Cochran-Mantel-
Haenszel statistics with stratification by GN site will be generated.  
11. INTERIM ANALYSES AND DATA MONITORING 
No formal interim efficacy analyses were planned nor conducted for the study.  Because the study 
was planned with an enrollment period of 24 months with the duration between randomization and 
determination of study endpoint being 6 to 8 months, preliminary analyses indicated that all 
participants would have completed randomization well before sufficient data could be accumulated 
for an interim efficacy analysis. 
While no formal efficacy analysis was planned, the Data Monitoring Committee (DMC) reviewed the 
study at periodic intervals, to evaluate whether any safety or study progress issues warrant possible 
study stopping.  These periodic reviews were based on DMC reports prepared by the Data 
coordinating center, which included information on study enrollment rates and participant progress 
through the study, participant compliance with protocol-specified treatment regimens, protocol 
violations, adverse events, and efficacy outcomes. The focus of the DMC review was on monitoring 
participant safety and study progress/futility but data on treatment effectiveness was also presented to 
frame the DMC discussions on safety and futility.   
The primary focus of the DMC was on the safety of study participants and the DMC monitored 
adverse events and side effects from LDA. All known associated side effects and specific obstetric or 
fetal concerns were considered reportable to the DMC.  
The DMC also reviewed the study progress for study progress and futility, with futility for these 
analyses defined in terms of inability to achieve study enrollment goals.  To effectively complete the 
study within the parameters defined by NICHD and the Global Network Steering Committee, the goal 

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of study was to complete enrollment in a 24-month period, and enrollment be completed within 30 
months.  Consequently, cumulative enrollment for the study should at worst case achieve a minimum 
of 20%, 40%, 60%, and 80% of total enrollment at the 6, 12, 18, and 24-month DMC reviews starting 
from the time the study is first opened to enrollment.  Should the study have failed to achieve at least 
90% of the cumulative target at any review, the DMC would have requested from the protocol 
committee an action plan for increasing enrollment that should be delivered to the DMC for review 
within a 1-month period.  Should the DMC have considered the plan inadequate or should the plan 
not result in increased enrollment by the next review, the DMC would have considered 
recommending to the NICHD closing the study for futility.  
12. CHANGES TO THE ANALYSES PLANNED IN THE PROTOCOL 
Additional sensitivity analyses beyond those included in the original protocol have been included in 
Sections 7.3 and 9 to account for loss-to-follow-up and to account for early pregnancy losses.  Also, 
one secondary outcome variable has been modified and on secondary outcome variable has been 
added.  The outcome of risk of preeclampsia or eclampsia has been modified to clarify that this 
outcome is the risk of a hypertensive disorder of pregnancy.  This modification was made to clarify 
the true variable of interest, reflecting the data collection process used in the MNH registry.  The 
outcome of preterm, preeclampsia has been added to the secondary outcome list to reflect additional 
research that was reported on this outcome after the study was launched.  
 
13. REFERENCES 
. 
Abramovici A, Cantu J, Jenkins SM. Tocolytic Therapy for Acute Preterm Labor. Obstet Gynecol 
Clin North Am. 2012; 39:77-87. doi: 10.1016/j.ogc.2011.12.003.  
Anderson RN, Smith BL. Deaths: leading causes for 2001. Natl Vital Stat Rep. 2003;52:1-85.  
Beck S, Wojdyla D, Say L, et al. The worldwide incidence of preterm birth: A systematic review of 
maternal mortality and morbidity. Bull World Health Organ. 2010;88:31-38.  
Blencowe H, Cousens S, Oestergaard MZ, et al. National, regional, and worldwide estimates of 
preterm birth rates in the year 2010 with time trends since 1990 for selected countries: a systematic 
analysis and implications. Lancet. 2012;379:2162-2172. doi:10.1016/S0140-6736(12)60820-4. 
Goldenberg R, Rouse D. Prevention of Premature Birth. N Engl J Med. 1998;339(5):313-320.  
Hogan JW, Lancaster T. Instrumental variables and inverse probability weighting for causal inference 
from longitudinal observational studies. Stat Methods Med Res. 2004;13:17-48. 
doi:10.1191/0962280204sm351ra. 
Liang KY and Zeger SL. Longitudinal Data Analysis Using Generalized Linear Models. Biometrika. 
1986:73(1): 13-22. 
March of Dimes, PMNCH, Save the Children, WHO. Born Too Soon: The Global Action Report on 
Preterm Birth. Geneva: World Health Organization; 2012. 

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Mathews TJ, Menacker F, MacDorman MF. Infant mortality statistics from the 2002 period: linked 
birth/infant death data set. Natl Vital Stat Rep. 2004;53:1-29 
Maine D, Rosenfield A, McCarthy J, Kamara A, Lucas AO: Safe Motherhood Programs: options and 
issues. New-York: Columbia University; 1991. 
McCormick MC, Richardson DK. Premature infants grow up. N Engl J Med. 2002;346(3):197-198. 
doi:10.1056/NEJM200201173460310. 
McClure EM, Pasha O, Goudar SS, et al; Global Network Investigators. Epidemiology of stillbirth in 
low-middle income countries: a Global Network Study. Acta Obstet Gynecol Scand. 
2011;90(12):1379-85. 
Oestergaard MZ, Inoue M, Yoshida S, Mahanani WR, Gore FM, Cousens S, Lawn JE, Mathers CD; 
United Nations Inter-Agency Group for Child Mortality Estimation and the Child Health 
Epidemiology Reference Group. Neonatal mortality levels for 193 countries in 2009 with trends since 
1990: a systematic analysis of progress, projections, and priorities. PLoS Med. 2011;8(8):e1001080. 
Schisterman EF, Silver RM, Lesher LL, et al. Preconception low-dose aspirin and pregnancy 
outcomes: Results from the EAGeR randomised trial. Lancet. 2014;384:29-36. doi:10.1016/S0140-
6736(14)60157-4. 
Saigal S, Doyle LW. An overview of mortality and sequelae of preterm birth from infancy to 
adulthood. Lancet. 2008;371:261-269. doi:10.1016/S0140-6736(08)60136-1. 
Valero De Bernabé J, Soriano T, Albaladejo R, et al. Risk factors for low birth weight: A review. Eur 
J Obstet Gynecol Reprod Biol. 2004;116:3-15. doi:10.1016/j.ejogrb.2004.03.007. 
14. ATTACHMENTS 
 
Table 1  
Participant Distribution 
Table 2  
Baseline Characteristics 
Table 3  
Patient Medication Compliance 
Table 4  
Reasons for Noncompliance 
Table 5  
Primary and Secondary Efficacy Outcomes 
Table 6  
Key Safety (SAE) Outcomes  
Table 7   
Protocol Deviations 
Figure 1 
Consort Diagram 
 

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Table 1. Participant Distribution  
GN Site 
Screened 
N 
Consented 
N (%) 
Eligible 
N (%) 
Randomized 
N (%) 
Analysis Population Membership 
N (%) 
Withdrew 
consent 
N (%) 
Study Terminations - N (%) 
Safety 
MITT 
ITT  
PP 
 
Terminated 
Participant 
moved away 
from study 
area 
Investigator 
terminated 
participant 
GN02: DRC 
 
 
 
 
 
GN03: Zambia 
 
 
 
 
 
GN06: Guatemala 
 
 
 
 
 
GN08: India (Belagavi) 
 
 
 
 
 
GN09: Pakistan 
 
 
 
 
 
GN11: India (Nagpur) 
 
 
 
 
 
GN12: Kenya 
 
 
 
 
 
Total 
 
 
 
 
 
NOTE: Analysis population membership % will be out of randomized. 
 
 
 

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Table 2: Baseline Characteristics – For ITT and MITT Populations 
 
Variable 
Placebo 
Aspirin 
Total 
Randomized, N 
 
 
 
Maternal Age, N (%) 
 
 
 
< 20 
 
 
 
20-29 
 
 
 
> 29 
 
 
 
Median (P25, P75) 
 
 
 
Projected GA at Enrollment, N (%) 
 
 
 
6,0 - 7,6 
 
 
 
8,0 - 9,6 
 
 
 
10,0 - 10,6 
 
 
 
11,0-11,6 
 
 
 
12,0 - 13,6 
 
 
 
Median (P25, P75) 
 
 
 
Maternal Education, N (%) 
 
 
 
No formal 
 
 
 
Primary 
 
 
 
Secondary 
 
 
 
University+ 
 
 
 
Maternal Height 
 
 
 
Mean (StdDev) 
 
 
 
Median (Min, Max) 
 
 
 
Maternal Weight 
 
 
 
Mean (StdDev) 
 
 
 
Median (Min, Max) 
 
 
 
BMI 
 
 
 

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Variable 
Placebo 
Aspirin 
Total 
Mean (StdDev) 
 
 
 
Median (Min, Max) 
 
 
 
Number of Antenatal Care Visits 
 
 
 
Median (P25, P75) 
 
 
 
Delivery Attendant, N (%) 
 
 
 
Physician 
 
 
 
Nurse/Nurse midwife 
 
 
 
TBA 
 
 
 
Family/Self/Other 
 
 
 
Delivery Location, N (%) 
 
 
 
Hospital 
 
 
 
Clinic/Health Center 
 
 
 
Home/Other 
 
 
 
Delivery Mode, N (%) 
 
 
 
Vaginal 
 
 
 
C-Section 
 
 
 
Miscarriage 
 
 
 
MTP 
 
 
 
 
 
 
 

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Table 3: Participant Medication Exposure and Compliance – For MITT and ITT Populations 
 Compliance/Exposure Parameter 
Placebo (N = XXXX) 
Aspirin (N = XXXX) 
No Drug 
 
 
Gestational age at first dose (weeks) 
 
 
Mean (StdDev) 
 
 
Median (Min, Max) 
 
 
Gestational age at last dose (weeks) 
 
 
Mean (Std Err) 
 
 
Min, Max 
 
 
Total number of days doses taken 
 
 
Mean (Std Err) 
 
 
Min, Max 
 
 
Level of Adherence: N (%) 
 
 
0% – 30% 
 
 
30% – 50% 
 
 
50% – 70% 
 
 
70% – 90% 
 
 
>90% 
 
 
Level of Exposure: N (%) 
 
 
0% – 30% 
 
 
30% – 50% 
 
 
50% – 70% 
 
 
70% – 90% 
 
 
>90% 
 
 
NOTE: Summaries of gestational age at first and last dose excludes individuals who never initiated treatment while such individuals are included in summaries of 
all other parameters. 
 
 

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Table 3a: Medication Compliance by Site – MITT Population 
 
 
Level of compliance  
GN Site 
Enrolled 
N 
Total 
Dosed 
N 
< 30% 
N (%) 
30-50% 
N (%) 
50-70% 
N (%) 
70-90% 
N (%) 
> 90% 
N (%) 
GN02: DRC 
 
 
 
 
 
 
GN03: Zambia 
 
 
 
 
 
 
GN06: Guatemala 
 
 
 
 
 
 
GN08: India (Belagavi) 
 
 
 
 
 
 
GN09: Pakistan 
 
 
 
 
 
 
GN11: India (Nagpur) 
 
 
 
 
 
 
GN12: Kenya 
 
 
 
 
 
 
 
 
 
 

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Table 4: Reasons for Medication Noncompliance – MITT Population 
 
Measures of Non-Compliance 
Placebo  
 
Aspirin  
 
Number of Biweekly Visits N 
 
 
Visits where pills not taken as instructed, N (%) 
 
 
Reasons for Noncompliance, N (%) 
 
 
Another person took pills 
 
 
Pills were lost 
 
 
Pills made me feel sick 
 
 
Forgot to take pills 
 
 
Travelled and did not bring pills 
 
 
Didn’t understand instructions for pills 
 
 
Pills were destroyed 
 
 
Other 
 
 
 
 
 
 

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Table 5a: Primary and Secondary Efficacy Outcomes  
 
Placebo  
N = XXXX 
 
Aspirin  
N = XXXX 
 
p-value 
Unadjusted RR 
(95% CI) 
Adjusted RR 
(95% CI) 
Primary Outcome, N included 
Preterm Delivery, N (%) 
 
 
 
Secondary Efficacy Outcomes 
Hypertensive Disorders, N (%) 
 
 
 
Small for Gestational Age, N (%) 
 
 
 
Perinatal Mortality, N (%) 
 
 
 
Other Maternal Outcomes of Interest 
Vaginal bleeding, N (%) 
 
 
 
Antepartum hemorrhage, N (%) 
 
 
 
Postpartum Hemorrhage, N (%) 
 
 
 
Maternal mortality through 42 days, N (%) 
 
 
 
Late Abortion, N (%)  
 
 
 
Preterm Preeclampsia, N (%) 
 
 
 
Other Fetal Outcomes of Interest 
Preterm birth <34 0/7 weeks of pregnancy, N (%) 
 
 
 
Birth weight < 2500g, N (%) 
 
 
 
Birth weight < 1500g, N (%) 
 
 
 
Fetal Loss, N (%) 
 
 
 
Spontaneous abortion, N (%) 
 
 
 
Stillbirth, N (%) 
 
 
 
Medical Termination of Pregnancy (MTP), N (%) 
 
 
 
 
NOTE: Table contains outcomes summarize using MITT population as well as outcomes summarized using ITT population.  Final table will clearly identify which 
population is used for each outcome.  Birth weight will be summarized both including and excluding estimated weights. Adjusted RR will not be presented in 
primary paper.  Footnotes will be included to describe p-value is from CMH test as well as models used to obtain RR estimates. 

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Table 5b: Primary and Secondary Efficacy Outcomes by Site 
NOTE: Table will also include fetal outcomes of interest. 
Outcome Measure 
Risk of Outcome 
Placebo  
N = XXXX 
 
Aspirin  
N = XXXX 
 
RR 
(95% CI) 
p-Value 
Preterm Delivery, N (%) 
GN01: DRC 
 
 
GN03: Zambia 
 
 
GN12: Kenya 
 
 
GN06: Guatemala 
 
 
GN08: Belagavi 
 
 
GN11: Nagpur 
 
 
GN09: Pakistan 
 
 
Hypertensive Disorders, N (%) 
GN01: DRC 
 
 
GN03: Zambia 
 
 
GN12: Kenya 
 
 
GN06: Guatemala 
 
 
GN08: Belagavi 
 
 
GN11: Nagpur 
 
 
GN09: Pakistan 
 
 
Small for Gestational Age 
GN01: DRC 
 
 
GN03: Zambia 
 
 
GN12: Kenya 
 
 
GN06: Guatemala 
 
 
GN08: Belagavi 
 
 
GN11: Nagpur 
 
 

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Outcome Measure 
Risk of Outcome 
Placebo  
N = XXXX 
 
Aspirin  
N = XXXX 
 
RR 
(95% CI) 
p-Value 
GN09: Pakistan 
 
 
Perinatal Mortality 
GN01: DRC 
 
 
GN03: Zambia 
 
 
GN12: Kenya 
 
 
GN06: Guatemala 
 
 
GN08: Belagavi 
 
 
GN11: Nagpur 
 
 
GN09: Pakistan 
 
 
Preeclampsia 
GN01: DRC 
 
 
GN03: Zambia 
 
 
GN12: Kenya 
 
 
GN06: Guatemala 
 
 
GN08: Belagavi 
 
 
GN11: Nagpur 
 
 
GN09: Pakistan 
 
 
Eclampsia 
GN01: DRC 
 
 
GN03: Zambia 
 
 
GN12: Kenya 
 
 
GN06: Guatemala 
 
 
GN08: Belagavi 
 
 
GN11: Nagpur 
 
 
GN09: Pakistan 
 
 
 

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NOTE: Table contains outcomes summarize using MITT population as well as outcomes summarized using ITT population.  Final table will clearly identify which 
population is used for each outcome.  Birth weight will be summarized both including and excluding estimated weights. Footnotes will be included to describe p-
value and RR is from unadjusted model that includes a site by treatment group interaction term.  The p-value for the interaction term will also be presented. 
 
 
 

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Table 6:  Serious Adverse Event Summary 
Variable 
Placebo 
N (%) 
Aspirin 
N (%) 
Relative Risk 
RR (95% CI) 
p-Value 
Randomized, N 
 
 
 
Participants with at least one SAE1 
 
 
 
Maternal death1, N (%) 
 
 
 
Fetal loss after 20 weeks1, N (%) 
 
 
 
Fetal anomaly1, N (%) 
 
 
 
Antepartum hemorrhage1, N (%) 
 
 
 
Post-partum hemorrhage1, N (%) 
 
 
 
Preeclampsia/Eclampsia1, N (%) 
 
 
 
Upper GI bleeding1, N (%) 
 
 
 
Gastroschisis, N (%) 
 
 
 
At least one Other SAE1, N (%) 
 
 
 
The denominator for this table is any woman included in the safety population. 
 
 

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Table 7a: Summary of Protocol Deviations by Treatment Arm 
Variable 
Arm 1 
Arm 2 
Total 
Randomized, N (%) 
XXXX 
XXXX 
XXXX 
At least one protocol deviation, N (%) 
XXX (Y.Y) 
XXX (Y.Y) 
XXX (Y.Y) 
Protocol deviations, N 
XX 
XX 
XX 
Study participant enrolled but did not meet inclusion criteria 
XX 
XX 
XX 
Study participant enrolled but met criteria requiring exclusion 
XX 
XX 
XX 
Study participant enrolled twice 
XX 
XX 
XX 
Medication card was lost or destroyed 
XX 
XX 
XX 
The study participant was incorrectly randomized 
XX 
XX 
XX 
The randomization assignment was revealed to study personnel, participant or both 
XX 
XX 
XX 
Other deviation 
XX 
XX 
XX 
Protocol Deviation Rate (N/Person Month), Mean (StdDev) 
X.X 
X.X 
X.X 
 
 
 

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Table 7b: Summary of Protocol Deviations by Site 
Variable 
GN02 
GN03 
GN06 
GN08 
GN09 
GN11 
GN12 
Randomized, N (%) 
XXXX 
XXXX 
XXXX 
XXXX
XXXX
XXXX
XXXX
At least one protocol deviation, N (%) 
XXX (Y.Y) 
XXX (Y.Y) 
XXX (Y.Y) 
XXX (Y.Y)
XXX (Y.Y)
XXX (Y.Y)
XXX (Y.Y)
Protocol deviations, N 
XX 
XX 
XX 
XX
XX
XX
XX
Study participant enrolled but did not meet inclusion criteria 
XX 
XX 
XX 
XX
XX
XX
XX
Study participant enrolled but met criteria requiring exclusion 
XX 
XX 
XX 
XX
XX
XX
XX
Study participant enrolled twice 
XX 
XX 
XX 
XX
XX
XX
XX
Medication card was lost or destroyed 
XX 
XX 
XX 
XX
XX
XX
XX
The study participant was incorrectly randomized 
XX 
XX 
XX 
XX
XX
XX
XX
The randomization assignment was revealed to study personnel, 
participant or both 
XX 
XX 
XX 
XX
XX
XX
XX
Other deviation 
XX 
XX 
XX 
XX
XX
XX
XX
Protocol Deviation Rate (N/Person Month), Mean (StdDev) 
X.X 
X.X 
X.X 
X.X
X.X
X.X
X.X