Prophylactic Antibiotics Prior to Embryo Transfer: a Randomized Controlled Trial
Phase: NA
Conditions: Assisted Reproductive Technology, Antibiotic Prophylaxis, Embryo Transfer
Sponsor: Washington University School of Medicine

 
 
 
 
 
 
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[Prophylactic antibiotics prior to embryo transfer: a randomized 
controlled trial] 
 
 
Principal Investigator:  Emily Jungheim, MD, MSCI 
 
 
 
 
 
 
 
Version Date/Number: 06/21/2019 
 
 

 
 
 
 
 
 
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TABLE OF CONTENTS 
 
A 
INTRODUCTION............................................................................................................................... 4 
A1 
STUDY ABSTRACT ......................................................................................................................... 4 
A2 
PRIMARY HYPOTHESIS .................................................................................................................. 4 
A3 
PURPOSE OF THE STUDY PROTOCOL.............................................................................................. 4 
B 
BACKGROUND ................................................................................................................................. 4 
B1 
PRIOR LITERATURE AND STUDIES ................................................................................................. 5 
B2 
RATIONALE FOR THIS STUDY ........................................................................................................ 5 
C 
STUDY OBJECTIVES ....................................................................................................................... 6 
C1 
PRIMARY AIM ............................................................................................................................... 6 
C2 
SECONDARY AIM .......................................................................................................................... 6 
C3 
RATIONALE FOR THE SELECTION OF OUTCOME MEASURES .......................................................... 7 
D 
INVESTIGATIONAL AGENT ........................................ ERROR! BOOKMARK NOT DEFINED. 
D1 
PRECLINICAL DATA .............................................................. ERROR! BOOKMARK NOT DEFINED. 
D2 
CLINICAL DATA TO DATE ..................................................... ERROR! BOOKMARK NOT DEFINED. 
D3 
DOSE RATIONALE AND RISK/BENEFITS ................................ ERROR! BOOKMARK NOT DEFINED. 
E 
STUDY DESIGN ................................................................................................................................. 7 
E1 
OVERVIEW OR DESIGN SUMMARY ................................................................................................ 7 
E2 
SUBJECT SELECTION AND WITHDRAWAL ...................................................................................... 7 
2.a 
Inclusion Criteria ..................................................................................................................... 7 
2.a 
Exclusion Criteria .................................................................................................................... 7 
2.b 
Ethical Considerations ............................................................................................................ 8 
2.c 
Subject Recruitment Plans and Consent Process ..................................................................... 8 
2.d 
Randomization Method and Blinding ...................................................................................... 8 
2.e 
Risks and Benefits .................................................................................................................... 8 
2.f 
Early Withdrawal of Subjects ....................................................................................................... 9 
2.g 
When and How to Withdraw Subjects ...................................................................................... 9 
2.h 
Data Collection and Follow-up for Withdrawn Subjects ......................................................... 9 
E3 
STUDY DRUG ................................................................................................................................ 9 
3.a 
Description .............................................................................................................................. 9 
3.b 
Treatment Regimen ................................................................................................................ 10 
3.c 
Method for Assigning Subjects to Treatment Groups ............................................................ 10 
3.d 
Preparation and Administration of Study Drug ..................................................................... 10 
3.e 
Subject Compliance Monitoring ............................................................................................ 10 
3.f 
Prior and Concomitant Therapy .................................................. Error! Bookmark not defined. 
3.g 
Packaging ................................................................................ Error! Bookmark not defined. 
3.h 
Blinding of Study Drug .......................................................................................................... 10 
3.i 
Receiving, Storage, Dispensing and Return ............................................................................... 10 
F 
STUDY PROCEDURES .................................................................................................................. 10 
F1 
SCREENING FOR ELIGIBILITY ........................................................................................................... 10 
F2 
SCHEDULE OF MEASUREMENTS ....................................................................................................... 10 
F3 
VISIT 1 ............................................................................................................................................ 10 

 
 
 
 
 
 
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F4 
VISIT 2 ETC. ..................................................................................................................................... 10 
F5 
SAFETY AND ADVERSE EVENTS ...................................................................................................... 11 
5.a 
Safety and Compliance Monitoring ....................................................................................... 11 
5.b 
Medical Monitoring ............................................................................................................... 11 
i 
Investigator only .................................................................................... Error! Bookmark not defined. 
ii 
Independent expert to monitor ............................................................... Error! Bookmark not defined. 
iii 
Institutional Data and Safety Monitoring Board .................................................................................... 11 
iv 
Independent Data and Safety Monitoring Board .................................... Error! Bookmark not defined. 
5.c 
Definitions of Adverse Events ................................................................................................ 12 
5.d 
Classification of Events ......................................................................................................... 12 
i 
Relationship ........................................................................................... Error! Bookmark not defined. 
ii 
Severity .................................................................................................. Error! Bookmark not defined. 
iii 
Expectedness ......................................................................................... Error! Bookmark not defined. 
5.e 
Data Collection Procedures for Adverse Events ................................................................... 12 
5.f 
Reporting Procedures ................................................................................................................ 12 
5.g 
Adverse Event Reporting Period ............................................................................................ 12 
5.h 
Post-study Adverse Event ......................................................... Error! Bookmark not defined. 
F6 
STUDY OUTCOME MEASUREMENTS AND ASCERTAINMENT ....... ERROR! BOOKMARK NOT DEFINED. 
G 
STATISTICAL PLAN ...................................................................................................................... 13 
G1 
SAMPLE SIZE DETERMINATION AND POWER ............................................................................... 13 
G2 
INTERIM MONITORING AND EARLY STOPPING ............................................................................ 13 
G3 
ANALYSIS PLAN .......................................................................................................................... 13 
G4 
STATISTICAL METHODS .............................................................................................................. 13 
G5 
MISSING OUTCOME DATA........................................................................................................... 14 
G6 
UNBLINDING PROCEDURES ................................................... ERROR! BOOKMARK NOT DEFINED. 
H 
DATA HANDLING AND RECORD KEEPING ........................................................................... 14 
H1 
CONFIDENTIALITY AND SECURITY .............................................................................................. 14 
H2 
TRAINING .............................................................................. ERROR! BOOKMARK NOT DEFINED. 
H3 
CASE REPORT FORMS AND SOURCE DOCUMENTS ................. ERROR! BOOKMARK NOT DEFINED. 
H4 
RECORDS RETENTION ................................................................................................................. 14 
H5 
PERFORMANCE MONITORING ................................................ ERROR! BOOKMARK NOT DEFINED. 
I 
STUDY MONITORING, AUDITING, AND INSPECTING ............ ERROR! BOOKMARK NOT 
DEFINED. 
I1 
STUDY MONITORING PLAN ....................................................... ERROR! BOOKMARK NOT DEFINED. 
I2 
AUDITING AND INSPECTING ....................................................... ERROR! BOOKMARK NOT DEFINED. 
J 
STUDY ADMINISTRATION .......................................................................................................... 14 
J1 
ORGANIZATION AND PARTICIPATING CENTERS ............................................................................... 14 
J2 
FUNDING SOURCE AND CONFLICTS OF INTEREST ............................................................................ 15 
J3 
COMMITTEES ............................................................................. ERROR! BOOKMARK NOT DEFINED. 
J4 
SUBJECT STIPENDS OR PAYMENTS .................................................................................................. 15 
J5 
STUDY TIMETABLE .......................................................................................................................... 15 
K 
PUBLICATION PLAN .................................................................................................................... 15 
L 
ATTACHMENTS ............................................................................................................................. 15 
L1 
TABLES ....................................................................................................................................... 15 
L2 
PATIENT EDUCATION BROCHURES ......................................... ERROR! BOOKMARK NOT DEFINED. 
L3 
SPECIAL PROCEDURES PROTOCOLS ....................................... ERROR! BOOKMARK NOT DEFINED. 
L4 
QUESTIONNAIRES OR SURVEYS ............................................. ERROR! BOOKMARK NOT DEFINED. 
M 
REFERENCES .................................................................................................................................. 16 
 

 
 
 
 
 
 
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A Introduction 
 
A1 Study Abstract 
Over 200,000 in vitro fertilization (IVF) cycles were initiated in the United States in 
2015,1 allowing thousands of infertile couples to start families.  Although most of the 
steps in IVF protocols are based on strong evidence, one, prophylactic antibiotic 
administration, is commonplace but not proven to produce benefit. This practice began 
in 1978 as a means to prevent bacterial contamination of the tip of the catheter used to 
transfer embryos into the uterus.2  Although subsequent studies showed that bacterial 
contamination of the transfer catheters had a negative impact on IVF cycle outcomes,3,4 
a more recent, robust randomized controlled trial powered to detect catheter tip 
contamination rates noted a decrease in contamination with routine administration of 
amoxicillin-clavulanate but no difference in clinical pregnancy rate.5  Current standard of 
care in our practice for a fresh IVF cycle is to administer one dose of 1 gram oral 
azithromycin (to both male and female partners) on day one of the IVF cycle start.  
Given the limited evidence for benefit and the true risk for subjecting patients to 
unnecessary treatments, we must determine whether prophylactic antibiotics actually 
improve the IVF outcome that matters most to couples: pregnancy. 
A2 Primary Hypothesis 
Our hypothesis is that withholding antibiotic prophylaxis will be non-inferior to routine 
administration. 
A3 Purpose of the Study Protocol 
The objective of this study protocol is to determine the impact on clinical pregnancy rate 
of withholding routine prophylactic antibiotic therapy during IVF. 
 
As a smaller exploratory study within our RCT we aim to assess alterations and 
influence of the vaginal microbiome of women going through a fresh IVF cycles with a 
mid-vaginal swab at three time points in 50 women (25 in each arm): at the time of 
baseline testing, prior to egg retrieval and at the time of embryo transfer. Lastly we plan 
to obtain a swab at the time of embryo transfer to assess the human virome, which has 
never been described or evaluated in the IVF population.6,7  We will correlate our 
findings with the clinical outcomes we plan to track as a part of our primary study.  We 
will consent and enroll the first 50 couples (25 in each arm) going through their first IVF 
cycle who enroll in the PAPET trial for the microbiome/virome study as well.  The male 
partners of the women enrolled in the pilot RCT will also be enrolled in the pilot RCT.  A 
small volume of the sample they produce at the time of in vitro fertilization will be 
obtained to evaluate the microbiome and virome in them in the same fashion.  
 
 
B Background 
 

 
 
 
 
 
 
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B1 Prior Literature and Studies 
Limited evidence supporting a common IVF practice: Although patients are 
routinely prescribed prophylactic antibiotics during an IVF cycle to improve embryo 
transfer success rates, little evidence supports this practice. A 2012 Cochrane review 
analyzed randomized controlled trials from the literature but only identified one study 
that was suitable for inclusion.8   In that trial, 350 women were randomized to receive 
either prophylactic amoxicillin and clavulanic acid on the day before and the day of 
embryo transfer, or no antibiotic treatment.5 The primary and secondary outcomes were 
catheter contamination rate and clinical pregnancy rate, respectively.  Although the 
catheter contamination rate was significantly lower in the antibiotic group than in the 
control group (49.4% versus 62.3%, P<0.03), there was no difference in clinical 
pregnancy rate (36% versus 35.5% respectively, P=0.83).  Impact on live birth rate was 
not assessed.  A more recent retrospective analysis of 876 embryo transfers assessed 
the impact of discontinuation of routine corticosteroid and antibiotic administration 
during IVF cycles and found no significant difference in clinical pregnancy rate (56.1% 
in medicated group versus 61.5% in un-medicated group, P=0.10) or other secondary 
outcomes including clinical miscarriage and live birth rate.9  Given this limited evidence 
about the clinical usefulness of prophylactic antibiotics, a summary statement on 
embryo transfer from the American Society for Reproductive Medicine states that, “a 
recommendation for routine prophylactic antibiotics cannot be made”.10 Thus, we need 
a well-designed randomized controlled study to rigorously address this question and 
inform recommendations for the thousands of IVF cycles completed each year. 
Potential of prophylactic antibiotics to cause harm: The impact of broad-spectrum 
antibiotic exposure in the preconception period is not well described but could have the 
unintended outcome of altering the follicular and endometrial microbiome, which may 
play a significant role in reproductive competence and IVF outcomes.11,12 For example, 
a prospective study of 35 infertile patients undergoing IVF demonstrated that a 
lactobacillus-dominated endometrial environment was associated with higher rates of 
implantation (60.7% vs. 23.1%, P=0.02), pregnancy (70.6% vs. 33.3%, P=0.03), 
ongoing pregnancy (58.8% vs. 13.3%, P=0.02) and live birth (58.8% vs. 6.7%, 
P=0.002).13  Vaginal microbiota diversity has also been implicated as a causative factor 
in preterm delivery which is known to occur with higher incidience in the IVF 
population.1,14  The microbial community structure of the vagina and endometrium could 
be negatively affected by potentially unnecessary broad-spectrum antibiotic exposure.  
We intend to provide proof of concept with our smaller exploratory study evlauting the 
vaginal and endometrial microbiome and changed across an IVF cycle and as a result 
of prophylactic antibiotic exposure.  Additionally, the critical importance of avoiding the 
misuse and overuse of antibiotics is continually discussed across all fields of medicine 
because of the increasing incidence of widespread antibiotic resistance.15-17  Finally, 
most antibiotics cause negative side effects including nausea, emesis, and yeast 
vaginitis and may add unneeded stress in the already-complicated IVF treatment 
process. 
B2 Rationale for this Study 
Non-inferiority RCT: 
Current standard of care in our practice for a fresh IVF cycle is to administer one dose 
of 1 gram oral azithromycin (to both male and female partners) on day one of the IVF 
cycle start.   This practice is of unproven clinical benefit and is based on theoretical risk 
and dated, poor-quality data.2-5,8,9  Although prophylactic antibiotic treatment may 

 
 
 
 
 
 
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decrease contamination of the embryo transfer catheter tip, no studies have been 
powered to determine impact on clinical pregnancy rate, the most direct correlate with 
successful IVF outcome.2   This will be the first randomized controlled trial powered to 
detect a difference in the primary endpoint of clinical pregnancy rate. 
 
Exploratory Pilot Study: 
Profiling the vaginal and seminal microbiome and human virome in a smaller cohort of 
patients going through their first IVF cycle is an exploratory portion of this study.  
Existing studies are limited and conflicting regarding influence of the microbiome on IVF 
cycle outcomes.11-13  This would be the first study to evaluate alterations in the 
reproductive tract microbiome across the IVF cycle in response to medications and 
supraphysiologic hormone levels, and the first to evaluate the human virome in this 
patient population.6,7  We would examine differences within and between groups and 
assess for the potential impact and association clinical outcomes of those patients 
exposed to prophylactic azithromycin, and those who are not.  Optimizing the vaginal 
microbiome could serve as a potential target for therapy to help increase IVF success 
rates in the future with the goal of a full term, live birth.  
 
C Study Objectives 
 
C1 Primary Aim 
Specific Aim 1: Determine the effect of withholding prophylactic antibiotic 
administration on clinical pregnancy rate (defined as a gestational sac, with fetal 
pole and cardiac activity, on ultrasound).  
We hypothesize that withholding prophylactic antibiotics before embryo transfer will be 
non-inferior (absolute difference ≤15%) to routine prophylaxis in terms of clinical 
pregnancy rates. 
C2 Secondary Aim 
Specific Aim 2: Evaluate the impact of prophylactic antibiotic administration on 
miscarriage rate (pregnancy loss per clinical pregnancy) before 20 weeks.   
Our working hypothesis is that miscarriage rates will be equivalent between the two groups.   
Specific Aim 3: Determine the effect of no prophylactic antibiotic administration 
on embryo development.   
We hypothesize that embryo development, as evaluated by fertilization rate (number of 
two-pronuclei embryos per mature oocyte), blastocyst conversion rate (number of day 5 
embryos from two-pronuclei embryos), and blastocyst utilization rate (number of day 5 
embryos transferred or frozen per two-pronuclei embryos) will be equivalent between 
the two groups. 
 
Specific Aim 4:  Determine the impact of prophylactic antibiotics or withholding 
prophylaxis on the vaginal and endometrial microbiome.    
 

 
 
 
 
 
 
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We hypothesize that azithromycin will impact the vaginal microbiome.  Additionally, we 
expect to see changes across the IVF cycle that may be associated with cycle outcome 
endpoints.   
 
Specific Aim 5:  Evaluate the implications of the human virome on IVF outcomes.     
 
We hypothesize that subclinical eukaryotic viruses will be associated with negative IVF 
cycle outcomes.   
 
C3 Rationale for the Selection of Outcome Measures 
We expect to define the clinical impact of withholding antibiotic prophylaxis before 
embryo transfer as measured by markers of a successful IVF cycle. As we hypothesize 
that withholding prophylaxis will be non-inferior to routine antibiotic administration, these 
results are expected have a positive clinical impact by decreasing unnecessary broad-
spectrum antibiotic exposure and potentially creating a paradigm shift in IVF protocols. 
 
 
D Study Design  
 
D1 Overview or Design Summary 
This will be a prospective randomized controlled non-inferiority trial in which 178 
couples will receive the standard of care prophylactic antibiotics (control) and 178 will 
not receive prophylactic antibiotics (experimental). The primary outcome is clinical 
pregnancy rate. The expected primary outcome is that withholding prophylactic 
antibiotics will be non-inferior to antibiotic prophylaxis in terms of clinical pregnancy 
rate. Secondary outcomes include spontaneous miscarriage rate and embryo 
development as measured by fertilization rate, blastocyst conversion rate, blastocyst 
utilization rate, and live birth rate.   
 
D2 Subject Selection and Withdrawal  
2.a 
Inclusion Criteria  
The female partner must be aged 18–43 years and going through a fresh IVF cycle. 
The male partner must be aged 18 at minimum.  There is no upper age limit to males.  
2.a 
Exclusion Criteria  
Couples will be excluded from enrollment if they have any contraindication to antibiotic 
treatment (ie: allergy), are not intending to undergo embryo transfer (fertility 
preservation patients and oocyte donors), require use of extended antibiotic coverage 
at time of egg retrieval, are already on antibiotics for any reason (e.g., upper respiratory 
infection), have a recent history of pelvic infection, or are planning on limited 
insemination (inseminating a limited number of the eggs retrieved). 

 
 
 
 
 
 
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2.b 
Ethical Considerations  
Participation will be completely voluntary.  Patients care will not be impacted in any way 
by their decision to participate or lack thereof.  Incidence of pelvic infection following 
embryo transfer approaches zero in this low risk population therefore patients are not 
being placed at any additional undo risk.  They may be spared from experiencing 
potential adverse side effects of the oral antibiotic.   
 
2.c 
Subject Recruitment Plans and Consent Process 
Couples going through fresh IVF cycles will be identified and their charts reviewed to 
assess eligibility for inclusion. Eligible couples will be contacted via phone by a trained 
study coordinator and offered enrollment. Those who express interest will have a time 
arranged for both partners to provide written consent in the office.  If the patient or 
couple is unable to find a mutually convenient time to come into the office for consent, 
both the patient and their partner will be emailed the consent form for review.  They will 
then be consented in full over the phone and given instruction on return of the hard 
copy of the signed consents. Couples will not be randomized until receipt of the hard 
copy of their signed consents and review and signature of the research team member 
providing consent.  Couples will only be included in the study during one IVF cycle.  
Subjects going through their first IVF cycle will also have an introduction to the study 
during IVF orientation.   
 
2.d 
Randomization Method and Blinding 
Couples will be randomized after providing written consent.  Written consent will be 
obtained separately for both the male and female partner.  In the case of same sex 
couples, only the female undergoing embryo transfer will be consented.  Fifty couples 
going through their first IVF cycle enrolled in the noninferiority trial will also be offered 
enrollment in the pilot RCT and subsequently consented for the microbiome and virome 
profiling exploratory study.  After consent, randomization in a 1:1 ratio to prophylactic 
antibiotics or no antibiotics will be performed in a block-randomized fashion per cycle 
number via computer random number generator. Couples randomized to the antibiotic 
group will receive the current standard regimen in our clinic: 1 gram azithromycin oral 
once for both partners the day controlled ovarian stimulation is initiated with injectable 
gonadotropins (IVF cycle start). In cases of same-sex couples, only the female 
undergoing the embryo transfer receives prophylaxis. Couples randomized to the no-
antibiotic treatment group will not be prescribed oral antibiotic prophylaxis. Couples who 
agree to participate will have their charts marked indicating participation in the study. 
The couples' respective nurses will receive a message that will remain part of the 
electronic medical record regarding patient treatment group allocation so they can 
prescribe antibiotics appropriately.  Embryologists who assess the embryo outcome 
measures will be blinded to patient treatment group allocation.  The patients and their 
providers and nurses will not be blinded to treatment group allocation.  
 
2.e 
Risks and Benefits 
Potential problems with our study may be insufficient patient recruitment.  The process 
of IVF is already stressful, time and user intensive and we will have to consent both 

 
 
 
 
 
 
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partners.  Patients may be hesitant to deviate from our previous standard of care.  On 
the other hand, patients may want to avoid additional medication and may not take the 
antibiotic regardless to which arm they are randomized.  We would mitigate this risk by 
a thorough informed consent and expressing our goal is to improve our current IVF 
protocols and that the best approach is not known while emphasizing the importance of 
compliance.   Reportable severe events include development of pelvic infection 
following embryo transfer, a significant increase in spontaneous miscarriage rates or a 
note in increased contamination rates in the lab.  Risks of pelvic infection in this low risk 
population following embryo transfer is remarkably rare as stated above and we do not 
anticipate this occurring.  We anticipate miscarriage rates to be equivalent or improved 
in the non-prophylaxis group.  The lab uses genatmicin routinely in embryo culture 
media to decrease contamination rates and will continue to be closely monitored for any 
increase in contamination rates.   Reportable non-severe adverse events include yeast 
vaginitis, nausea and vomiting or other adverse reactions from antibiotic prophylaxis. 
 
Obtaining a mid-vaginal swab poses little to no risk to the patient.  The patient may 
experience some slight discomfort from one additional speculum exam at the time of 
baseline testing.  The embryo transfer catheter tip will be sequenced for comparison to 
the vaginal swab following embryo transfer and will require no additional invasive 
testing or exams.   
 
 
2.f 
Early Withdrawal of Subjects  
While loss to follow up is extremely rare in this circumstance, some patients who are 
enrolled and randomized may not make it to egg retrieval or may not get an embryo 
transfer due to either a poor response to medications or poor embryo quality.  We will 
account for these patients in our 5% drop out estimation and they will not be included in 
analysis.   
2.g 
When and How to Withdraw Subjects  
Patients can withdraw from the study at any time if they choose to do so.  Their care will 
not be impacted by their participation or lack thereof.  
2.h 
Data Collection and Follow-up for Withdrawn Subjects  
Subjects who withdraw will not be included in the final data analysis.  
 
D3 Study Drug – 
 
3.a 
Description  
Azithromycin is routinely given to both partners (in male/female couples) or to the 
embryo recipient in same sex couples or gestational carriers when they start their IVF 
cycle medications and is our current standard of care.   

 
 
 
 
 
 
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3.b 
Treatment Regimen  
1 gram oral azithromycin, once the day of IVF cycle start (when they start injectable 
gonadotropin mediciations) which is our current standard of care.   
3.c 
Method for Assigning Subjects to Treatment Groups  
Computer random number generator.   
3.d 
Preparation and Administration of Study Drug  
Azithromycine will be prepared and dispensed by the patients preferred pharmacy as is 
standard practice if they are randomized to the antibiotic prophylaxis group.   
3.e 
Subject Compliance Monitoring  
Patients will give verbal confirmation of compliance.   
3.f 
Blinding of Study Drug  
Patients will not be blinded to which group they’re in.  A placebo control will not be 
used.  
3.g 
Receiving, Storage, Dispensing and Return  
Azithromycin will be prepared and dispensed by the patients preferred pharmacy as is 
standard practice if they are randomized to the antibiotic prophylaxis group.   
 
E Study Procedures  
 
E1 Screening for Eligibility 
Couples will be screened for eligibility by their primary physician, the primary 
investigator, Dr. Jungheim or the Research Project Coordinator.  If they meet the 
inclusion and exclusion criteria they will be offered enrollment.   
E2 Schedule of Measurements 
Outcomes measures are routinely tracked by our clinical to report to the Society of 
Assisted Reproductive Technology (SART).   Patients who participate in the study will 
not be required to make any additional visits beyond consent.   
E3 Visit 1  
Outcome measures for specific aim 3 will be tracked from the patients initial oocyte 
retrieval and embryo transfer visit.  The lab routinely tracks this data and no additional 
query or visits will be required in term of embryo development data. 
E4 Visit 2 etc.  
Specific Aim 1 will be addressed and tracked when patients come in for their viability 
ultrasound at 6-7 weeks gestation which is routine practice in our clinic.  Clinical 
pregnancy is defined as a gestational sac, with fetal pole and cardiac activity, on 

 
 
 
 
 
 
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ultrasound.  No additional visits will be required.  Patients will continue to be tracked 
once they are released from our practice to their primary OBGYN to track miscarriage 
rate (spontaneous loss prior to 20 weeks) and subsequent live birth outcomes.  
 
Schedule of Visits for Microbiome/Virome Study: 
Outcome measures will be assessed the same as th above aforementioned schedule of 
measurements for the noninferiority trial.  Collection of specimens for the exploratory 
study will occur as described below:  
 
Visit 1-  
 
Women enrolled in the smaller exploratory study will have a midvaginal swab obtained 
when they get their baseline testing prior to ultrasound.  This visit is already required in 
the IVF cycle and will not require an additional visit to collect the specimen.  
 
Visit 2- 
 
The second specimen will be obtained with a midvaginal swab prior to egg retrieval.  
This visit is already required in the IVF cycle and will not require an additional visit to 
collect the specimen. 
 
Visit 3- 
 
The third and final specimen will be obtained via midvaginal swab prior to embryo 
transfer.  This visit is already required in the IVF cycle and will not require an additional 
visit to collect the specimen.  When the male partner produces a sample for use for IVF 
at this time, a 100 microliter volume will be obtained and frozen separately.  No 
additional visit will be required for them.   
 
All specimen swabs collected for microbiome and virome profiling will be flash frozen at 
-80 celsius until all specimens have been collected and can be batch analyzed at the 
same time. 
 
E5 Safety and Adverse Events  
 
5.a 
Safety and Compliance Monitoring 
As administration of 1 gram oral Azithromycin is standard of care in our practice no 
additional risk will be incurred from prescribing the antibiotic prophylaxis.  Patients will 
be asked regarding compliance with medication administration and if any adverse side 
effects were incurred including yeast vaginitis, nausea and vomiting or other adverse 
reactions from antibiotic prophylaxis.  
5.b 
Medical Monitoring  
i 
Institutional Data and Safety Monitoring Board 
In addition to principle investigator review of adverse events, a data and safety 
monitoring board (DSMB) will be established.  This will include Ann Pitcavage, Joan 

 
 
 
 
 
 
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Riley, PhD, Emily Jungheim, MD, MSCI and Nandini Raghuraman, MD and Lindsay 
Kuroki, MD. The designated group will meet once enrollment reaches 50% and then 
every 6 months thereafter to review occurrence of adverse events in addition to 
assessing recruitment progress and will review for futility of the trial in the context of lack 
of adequate recruitment.  
 
 
5.c 
Definitions of Adverse Events 
Severe adverse events: pelvic infection (endometritis, cervicitis or pelvic inflammatory 
disease) as diagnosed by cervical culture and physical exam, spontaneous miscarriage 
(pregnancy loss prior to 20 weeks gestation) or increased contamination rates in the lab 
which is routinely monitored for by our embryologists.   
Non-severe adverse events include adverse effects of antibiotic administration including 
yeast vaginitis, abdominal pain, diarrhea, nausea and vomiting. 
5.d 
Classification of Events 
Reportable severe events include development of pelvic infection following embryo 
transfer, a significant increase in spontaneous miscarriage rates or a note in increased 
contamination rates in the lab.  Risks of pelvic infection in this low risk population 
following embryo transfer approaches zero and we do not anticipate this occurring.  We 
anticipate miscarriage rates to be equivalent or improved in the non-prophylaxis group 
but will be closely tracked for any increase in incidence.  The lab uses genatmicin 
routinely in embryo culture media to decrease contamination rates and will continue to 
be closely monitored as is standard for quality control measures in our lab for any 
increase in contamination rates.   
Reportable non-severe adverse events include yeast vaginitis, nausea and vomiting or 
other adverse reactions from antibiotic prophylaxis which the patients will report to their 
nurses. 
5.e 
Data Collection Procedures for Adverse Events 
In addition to principle investigator review of adverse events, a data and safety 
monitoring board (DSMB) will be established.  This will include Ann Pitcavage, Joan 
Riley, PhD, Nandini Raghuraman, MD and Lindsay Kuroki, MD and Emily Jungheim, 
MD, MSCI.  The designated group will meet once enrollment reaches 50% and then 
every 6 months thereafter.  Adverse events will be noted in the patients chart and in the 
redcap database for complete capture and tracking.  Data will be analyzed if a noted 
increase in severe adverse events is noted or otherwise once enrollment reaches 50% 
and then every 6 months thereafter when the DSMB meets.   
5.f 
Reporting Procedures 
Patients will be instructed to report any adverse symptoms to their respective nurse or 
doctor.  These events will be immediately reported to the PI or research project 
coordinator.   
5.g 
Adverse Event Reporting Period 
Until study completion.  
 

 
 
 
 
 
 
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F Statistical Plan  
 
F1  Sample Size Determination and Power 
Given the conflicting evidence toward benefit of routine antibiotic prophylaxis and IVF 
outcomes, a non-inferiority approach was selected to test our hypothesis that no 
prophylaxis is not inferior to routine antibiotic administration.  A total sample size of 356 
patients (178 per arm) was determined based on an average baseline clinical 
pregnancy rate of 41% within our practice in this population with a non-inferiority margin 
of 15%, 80% power, and using a one sided alpha=0.025 while estimating a 5% drop out 
rate.   Our practice completed approximately 500 fresh IVF cycles in 2016.  Our 
exclusion criteria are broad and we do not anticipate patient recruitment to be difficult.  
We believe 356 patients is feasible and estimate a 14 month timeline to complete 
recruitment if we enroll 7 patients at minimum per week. 
 
F2  Interim Monitoring and Early Stopping 
Interim analysis will be performed according to the Haybittle-Peto approach at 50% 
enrollment and 75% enrollment.  This study will be considered safe to continue if there 
is an absence in trend toward severe adverse maternal, pregnancy or laboratory 
outcomes in either group.  The DSMB will also assess recruitment progress and will 
review for futility of the trial in the context of lack of adequate recruitment.  Data will be 
formally analyzed at 50% enrollment and then every 6 months thereafter to assess for 
early termination of the study.  
F3  Analysis Plan 
See Table 1 for timetable of patient enrollment and analysis.  Our practice is required to 
track patient demographic data and cycle outcome measures to report to the Society for 
Assisted Reproductive Technology. Data are recorded and tracked through Research 
Electronic Data Capture (REDCap). Data from enrolled patients will be exported and 
tracked in a separate secure REDCap dataset from our existing dataset. All of our 
outcome measures are routinely tracked, and no additional data query will be required 
to address potential confounders as listed in Table 2.   
F4 Statistical Methods  
Intention-to-treat and per protocol analyses will be performed. Chi-squared and Fisher’s 
exact tests will be used to compare categorical variables as appropriate. Kolmogorov-
Smirnov test will be initially used to assess the distribution pattern of continuous 
variables. Normally distributed variables will be analyzed via Student’s t-test. Non-
normally distributed continuous variables will be analyzed with the Mann-Whitney U 
test.  The primary analysis is based on difference in clinical pregnancy rate between the 
two groups.  The sample size estimation for the study is based on this comparison with 
a non-inferiority approach as described above with an absolute difference of 15% or 
less to be considered non-inferior.  Secondary analyses will include spontaneous 
miscarriage rate and embryo development. A subsequent interval analysis will assess 
live birth rate. Pearson’s correlation coefficients will be used to quantify the association 

 
 
 
 
 
 
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14
between the two groups and embryo development.  P<0.05 will be considered 
statistically significant.  
Each microbiota sample will have the taxon indicated as relative abundance.  To 
assess the diversity across an IVF cycle, after log transformation, linear mixed 
regression will be applied.  Wilcoxon rank sum will be used to measure for difference in 
richness and diversity with cinical outcomes of clinical pregnancy rate, miscarriage rate 
and embryo outcomes.  
IBM® SPSS® statistics version 23 will be used for all analyses. 
 
F5  Missing Outcome Data 
 
All outcome data is required to be tracked by SART and no additional query will be 
required.  We do not anticipate any issues with missing outcome data.   
 
G Data Handling and Record Keeping  
 
G1 Confidentiality and Security 
Our practice is required to track patient demographic data and cycle outcome measures 
to report to the Society for Assisted Reproductive Technology (SART).  Data is currently 
recorded and tracked through Research Electronic Data Capture (REDCap).  REDCap 
is a secure web-based application designed to support data capture for research 
studies, providing 1) an intuitive interface for validated data entry; 2) audit trails for 
tracking data manipulation and export procedures; 3) automated export procedures for 
seamless data downloads to common statistical packages; and 4) procedures for 
importing data from external sources.18  Enrolled patients will be exported and tracked in 
a separate REDCap data set from our existing dataset.   
 
G2 Records Retention 
Study recods will be stored and tracked through REDCap as noted in G1.  Signed 
consents will be kept in a locked cabinet in a locked office.  Data will be retained until 
study enrollment and analysis is complete.   
 
 
H Study Administration 
 
H1 Organization and Participating Centers 
Washington University Department of Reproductive Endocrinology and Infertility.  

 
 
 
 
 
 
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15
H2 Funding Source and Conflicts of Interest 
The PI, Ashley Eskew, MD is a recipient of funding from NIH 5T32HD055172 as a part 
of her Masters of Science and Clinical Investigation clinical research track. There are no 
conflicts of interest to report.   
H3 Subject Stipends or Payments  
Not applicable.  
H4  Study Timetable 
See Table 1.  
 
I Publication Plan  
We plan on presenting our findings at national meetings including those for American Society for 
Reproductive Medicine and Society for Reproductive Investigations.   We plan to publish our 
results in journals that have a high impact among reproductive endocrinologists within 6 months of 
completing our data collection.   
 
J  Attachments  
 
J1  Tables  
 
Figure 1: Timetable (months) 
 
 
 
 
 
 
 
 
 
 
 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 11 12 13 
14 
15 
16 
17 
18 
19 
 
Patient  
Recruitment 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
 
  Analysis  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
X 
X 
 
 
 
 Manuscript 
  Composition 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
X 
X 
 
 
 
 
 
Table 2. Potential confounders 
Patient history  
Cycle level factors 
Cycle outcome 

 
 
 
 
 
 
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_ 
 
16
 Demographic data: Maternal age, 
race, ethnicity 
 Medical history: BMI, tobacco use 
 Obstetric history: gravidity, parity  
 Infertility diagnosis  
 Duration of infertility  
 Number of prior IVF cycles 
 Baseline labs: Follicle stimulating 
hormone or antimullerian hormone 
level, antral follicle count   
 Total amount of gonadotropin 
administered 
 Stimulation protocol 
 Number oocytes collected 
 Number mature oocytes 
 Insemination method (conventional vs. 
intracyto-plasmic sperm injection 
[ICSI] vs. partial ICSI) 
 Number oocytes normally fertilized 
(two pronuclei) 
 Day of embryo transfer 
 Number of embryos transferred 
 Number of embryos frozen 
 Clinical pregnancy rate 
 Spontaneous 
miscarriage rate 
 Live birth rate  
 
 
 
 
 
K References 
 
1. 
www.sart.org  
2. 
Czernobilsky B. Endometritis and infertility. Fertil Steril.1978;30:119-130. 
3. 
Fanchin R, Harmas A, Benaoudia F, Lundkvist U, Olivennes F, Frydman R. 
Microbial flora of the cervix assessed at the time of embryo transfer adversely affects in 
vitro fertilsation outcome. Fertil Steril.1990;5:866-870 
4. 
Salim R, Ben-Shlomo I, Colodner R, Keness Y, Shalev E. Bacterial colonization 
of the uterine cervix and success rates in assisted reproduction: results of a prospective 
survey. Hum Reprod. 2002;17:337-340. 
5. 
Brook N, Khalaf Y, Coomarasamy A, Edgeworth J, Braude P. A randomized 
controlled trial of prophylactic antibiotics (co-amoxiclav) prior to embryo transfer. Hum 
Reprod 2006;21:2911-5, Level I.  
6. 
Wylie KM, Weinstock GM, Storch GA. Virome genomics: a tool for defining the 
human virome. Curr opin Microbiol 2013;16(4):479-484. 
7. 
Wylie TN, Wylie KM, Herter BN, Storch GA. Enhanced cirome sequencing using 
targeted sequence capture.  Genome Research. 2015;25:1910-1920. 
8. 
Kroon B, Hart, RJ, Wong BM, Ford E, Yazdani A. Antibiotics prior to embryo 
transfer in ART. Cochrane Database Syst Rev 2012:Cd008995, Level III. 
9. 
Kay L, Bartels C, Bartolucci A, Engmann L, Nulsen J, Benadiva C. Old habits 
die hard: retrospective analysis of outcomes with use of corticosteroids and antibiotics 
before embryo transfer. Fertil Steril. 2017;107:1336-40. 

 
 
 
 
 
 
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17
10. 
Practice Committee of the American society for Reproductive Medicine. 
Performing the embryo transfer: a guideline. Fertil Steril 2017;107:882-96. 
11. 
Franasiak JM, Scott RT. Reproductive tract microbiome in assisted reproductive 
technologies. Fertil Steril. 2015;104:1364–71 
12. 
Fox C, Eichelberger K. Maternal microbiome and pregnancy outcomes. Fertil 
Steril. 2015;104:1358–63. 
13. 
Moreno I, Codoner FM, Vilella F, Valbuena D, Martinez-Blanch JF, Jimenez-
Almazan J, Alonso R, Alama P, Remohi J, Pellicer A, Ramon D, Simone C. Evidence 
that the endometrial microbiota has an effect on implantation success or failure. AJOG. 
2015: 215;684-703. 
14. 
Stout MJ, Zhou Y, Wylie KM, Tarr PI, Macones GA, Tuuli MG. Early pregnancy 
vaginal microbiome trends and preterm birth.  AJOG. 2017. 217;3:356e1-356e18. 
15. 
Ledger WJ, Blaser MJ. Are we using too many antibiotics during pregnancy? Br 
J Obstet Gynaecol 2013;120:1450. 
16. 
Ramasethu J, Kawakita T. Antibiotic stewardship in perinatal and neonatal care. 
Seminars in Fetal & Neonatal Medicine. In press, 2017: 
http://dx.doi.org/10.1016/j.siny.2017.07.001.  
17. 
Nambudiri VE. More than skin deep- the costs of antibiotic overuse. A teachable 
moment. JAMA. 2014;174:1724-1725. 
18. 
https://www.project-redcap.org