Modulating the Vaginal Microbiome After Implantation Failure A Randomized Placebo Controlled Study of Vaginal Lactobacilli Supplements
Phase: PHASE4
Conditions: Implantation Failure, Infertility, Female, Vaginal Flora Imbalance
Sponsor: Zealand University Hospital

Protocol version: 3.0 MICINT 
EudraCT number: 2018 002376-41, 16.11.2018 
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PhD protocol 2 
 
 
Modulating the Vaginal Microbiome after Implantation failure 
A randomized placebo controlled study of vaginal lactobacilli supplements 
 
Hviid MM, Macklon NS 
 
 
 
PhD student: 
Malene Meisner Hviid, MD 
Faculty of Health and Medical Science, University of Copenhagen 
Department of Gynecology and Obstetrics 
Division of Reproductive Medicine, Fertility Clinic 
Zealand University Hospital, Roskilde 
 
Sponsor and Principal Investigator:  
Professor Nicholas Stephen Macklon, MD, PhD 
Faculty of Health and Medical Science, University of Copenhagen 
Department of Gynecology and Obstetrics 
Division of Reproductive Medicine, Fertility Clinic 
Zealand University Hospital, Roskilde 
 
 
University of Copenhagen 
Faculty of Health and Medical Science 
 
 
 
 
 
 

Protocol version: 3.0 MICINT 
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Table of Content 
 
I. Setting 
 
 
 
 
 
 
 
 
          Page 
 
Sponsor and principal investigator………………………………………………………………………….. 4 
 
Study co-ordinator and co-investigator…………………………………………………………………… 4 
 
Participating clinic…………………………………………………………………………………………………… 4 
 
 
Fertility Clinic, Zealand University Hospital Køge………………………………………… 4 
 
Collaborators………………………………………………………………………………………………………….. 5 
 
 
Orkla Care A/S……………………………………………………………………………………….…… 5 
 
 
Bifodan A/S…………………………………………………………………………………………….….. 5 
 
 
ARTPred…………………………………………………………………………………………………….. 5 
IS Diagnostisc Ltd……………………………………………………………………………………….. 6 
 
 
GCP Monitor…………………………………………………………………………………………….… 6 
 
II. Introduction and Background 
 
Introduction, background, and purpose………………………………………………………………….. 7 
Research question………………………………………………………………………………………………….. 8 
 
III. Methods 
Study design…………………………………………………………………………………………………………… 8 
 
Study population…………………………………………………………………………………………………….. 8 
 
The Implantation Clinic…………………………………………………………………………………………… 8 
 
Study flowchart………………………………………………………………………………………………………. 9  
Inclusion criteria…………………………………………………………………………………………………… 10 
 
Exclusion criteria…………………………………………………………………………………………………… 10 
Laboratory tests and examinations……………………………………………………………………….. 10 
 
 
Gynecological examination of vaginal smear ………………………….……………….. 10 
 
 
Vaginal swab (ARTPred)……………………………………………………………………………. 10 
 
Side-effects, risks, and disadvantage..…………………………………………………………………… 10 
Randomization……………………………………………………………………………………………………… 11 
 
Intervention, lactobacillus and placebo………………………………………………………………… 11 
 
Active Study Drug…………………………………………………………………………………………………. 11 
 
 
Content……………………………………………………………………………………………………. 11 
 
 
Manufacturer…………………………………………………………………………………………… 11 
 
 
Packaging, labeling and handling of study drug………………………………………… 12 
 
Adverse reactions of active study drug, Vivag Plus………………………………………………… 12 
 
Registration of adverse reactions………………………………………………………………………….. 12 
 
Statistical considerations, primary endpoint, study participants and size.………..…… 13 
Study duration………………………………………………………………………………………………………. 14 
Biological material from participants……………………………………………………………………. 14 
 
Information from patient journal and research biobank…………………………………….…. 14 
 
Data collection……………………………………………………………………………………………………… 14 
 
IV. Quality Assurance 
 
Helsinki Declaration……………………………………………………………………………………….……… 15 
 
EudraCT………………………………………………………………………………………………………………… 15 
 
GCP monitor…………………………………………………………………………………………………………. 15 
 
 

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V. Ethical Considerations 
 
Ethical considerations…………………………………………………………………………………………… 15 
 
Recruiting participants and inform consent…………….…………………………………..……….. 15 
 
Submission of results………………………………………………………………………………………….... 16 
 
Scientific ethical statement..…………………………………………………………………………….….. 16 
 
Risk assessments and alternative approaches/plans…………………………………………..… 16 
 
  
VI. Financial Aspects 
 
Economics…………………………………………………………………………………………………………….. 16 
 
Compensation to participants……………………………………………………………………….……… 16 
 
Relevant clauses in the contract………………………………………………………………….……….. 16 
 
Insurance…………………………………………………………………………………………………….……….. 17 
 
 
VII. References 
 
References……………………………………………………………………………………………………………. 17 
 
VII. Signature 
 
Sponsor/Principal investigator signature………………………………………………………………. 18 
 
 
 

Protocol version: 3.0 MICINT 
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I. Setting 
Sponsor and Principal investigator 
Professor Nicholas Stephen Macklon, MD, PhD 
Faculty of Health and Medical Science, University of Copenhagen 
Department of Gynecology and Obstetrics 
Division of Reproductive Medicine, Fertility Clinic 
Zealand University Hospital, Roskilde 
Sygehusvej 10, 4000 Roskilde 
E-mail: nism@regionsjaelland.dk 
Tel.: +45 53621645 
 
Study Coordinator and co-investigator 
 
Malene Meisner Hviid, MD 
Faculty of Health and Medical Science, University of Copenhagen 
Department of Gynecology and Obstetrics 
Division of Reproductive Medicine, Fertility Clinic 
Zealand University Hospital, Roskilde 
Sygehusvej 10, 4000 Roskilde 
E-mail: mahv@regionsjaelland.dk 
Tel.: +45 93567737 / +45 22559383 
 
 
Participating Clinic 
 
Zealand University Hospital  
Fertility Clinic, Division of Reproductive Medicine 
Lykkebækvej 14, 4600 Køge 
Tel.: +45 56631500 
 
Responsible at this clinic: 
Sponsor and principal investigator 
Professor Nicholas Stephen Macklon, MD, PhD 
E-mail: nism@regionsjaelland.dk 
Tel.: +45 53621645 
 
 
 
 
 
 
 

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Collaborators 
 
Orkla Care A/S (Licence to use study drug in Denmark) 
Industrigrenen 10 
2635 Ishøj, Denmark 
 
 
 
 
Contact: 
 
 
 
Bjarne Bloch, Medical Advisor 
 
 
 
Orkla Care A/S 
 
 
 
Industrigrenen 10, 2635 Ishøj 
 
 
 
E-mail: bjarne.bloch@orkla.dk 
 
 
 
Tel.: +45 40200833 
 
 
 
Bifodan A/S (Supplying study drug and placebo) 
Bogbinderivej 6 
3390 Hundested, Denmark 
 
 
 
 
Contact: 
 
 
 
Erik Brandsborg, Chief Scientific Officer 
 
 
 
Bifodan A/S 
 
 
 
Bogbinderivej 6, 3390 Hundested 
 
 
 
E-mail: erb@bifodan.com 
 
 
 
Tel.: +45 31637910 
 
 
 
ARTPred (supplying vaginal swabs and analyses of vaginal microbiome) 
Seringenstraat 15 
5213 GS ’s Hertogenbosch 
The Netherlands 
 
 
 
 
Contact: 
 
 
 
Joep de Mönnink, CEO 
 
 
 
Seringenstraat 15 
 
 
 
5213 GS’s Hertogenbosch, The Netherlands 
 
 
 
E-mail: joep@artpred.com 
 
 
 
Tel.: +316 2049 9729 
 
 
 
 
 
 
 

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IS Diagnostics Ltd. (Subcontractor used by ARTPred to perform analyses) 
Van der Boechorststraat 7 
1081 BT Amsterdam 
The Netherlands 
 
 
 
 
Contact: 
 
 
 
Dr. Dries Budding, CEO 
 
 
 
Van der Boechorststraat 7 
 
 
 
1081 BT Amsterdam, The Netherlands 
 
 
 
E-mail: D.Budding@vumc.nl 
 
 
 
GCP Monitor (Study monitor) 
GCP Unit Copenhagen University 
Bispebjerg Hospital, Bygning 51, 3. Sal 
Bispebjerg Bakke 23, 2400 København NV 
 
 
 
 
Contact: 
 
 
 
Jeanette Blom 
 
 
 
Bispebjerg Hospital, Bygning 51, 3. Sal 
Bispebjerg Bakke 23, 2400 København NV 
E-mail: Jeanette.blom@regionh.dk 
Tel.: +45 35313893 
 
 
 

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II. Introduction and Background 
 
Introduction, background and purpose 
Infertility affects around 1 in 6 couples and is recognized as a public health issue by the 
World Health Organization. In 33% of couples no cause is found, but it is becoming clear that 
undiagnosed endometrial dysfunction may be a significant factor(Malchau et al., 2017). IVF 
offers an effective treatment for many, but only 50% will achieve a pregnancy and 8-10% will 
suffer recurrent implantation failure (RIF) despite the transfer of high quality embryos (Koot 
et al., 2012; Koot and Macklon, 2013). However, therapies aimed at assisting implantation or 
treating endometrial infertility remains largely empirical and ineffective (Hviid and Macklon, 
2017). This reflects in part the lack of validated means of assessing and diagnosing 
endometrial functional defects. It is clear that several etiologies may underlie implantation 
failure and there is an urgent need to develop means of diagnosing specific endometrial 
functional abnormalities (Robertson et al., 2016). Only then can targeted therapies be 
offered. 
In recent years, a number of novel insights into factors governing endometrial function have 
emerged and provide potential new avenues for intervention. One putative marker of 
endometrial receptivity that has recently attracted interest is the profile of bacteria (termed 
the ‘microbiome’) present in the vagina or endometrium. Disruption of the vaginal 
microbiome by overgrowth of specific commensal strains has long been associated with 
symptoms of itching and discharge. The diagnosis of bacterial vaginosis continues to be 
based on the recognition of histological markers in vaginal swabs. (Mastromarino et al., 
2013). However in recent years, more sensitive and specific tests based on RNA 
identification of individual bacteria have enabled a more precise and quantitative 
assessment of the bacteria present.  These technologies are shedding new light on the 
microbiome within different organs of the body and their impact on health (Lamont et al., 
2011). In the field of reproductive medicine, a close correlation has recently been reported 
between the vaginal and endometrial microbiome and the chance of conceiving through IVF 
(Hyman et al., 2012; Moreno et al., 2017) and our group is currently testing the hypothesis 
that disruption of the vaginal and endometrial microbiome may be associated with recurrent 
implantation failure after IVF treatment.  
Recent studies have indicated that a vaginal microbiome consisting of < 90% lactobacilli is 
associated with failure of IVF treatment (Data submitted for publication, ART PRED, 
Amsterdam) However, it remains unclear whether interventions designed to alter the 
microbiome are effective in changing the suboptimal microbiome diagnosed with these new 
tests, or how stable any induced change in microbiome might be. These questions are of 
particular importance to women planning IVF treatment, as an effective treatment may alter 
their prognosis in subsequent cycles. A number of approaches have been proposed, such as 
treatment with antibiotics and the use of probiotics. Vivag Plus (Bifodan, Hundested, 
marketed by Orkla, Ishøj) is a vaginal probiotic capsule containing lactobacilli species 
effective in relieving symptoms of bacterial vaginosis (Lægemiddelstyrelsen, 2017). It is 
hypothesized that this probiotic may also be effective in restoring lactobacilli dominance in 
the vaginal microbiome in women shown to have a profile associated with implantation 
failure. To test this hypothesis, the following research questions will be addressed:  

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Research questions 
1. Compared with a placebo vaginal capsule, does the application of a lactobacilli 
loaded capsule for 10 days change a lactobacillus poor or by predictive vaginal test 
otherwise unfavorable vaginal microbiome, to a lactobacillus rich or by predictive 
vaginal test otherwise favorable microbiome in the same menstrual cycle as 
intervention?  
2. Is the change in the microbiome still present one month after the intervention?  
From this, our primary hypothesis is that compared with placebo, use of lactobacillus loaded 
vaginal capsule is associated with a significant increase in the proportion of women who 
demonstrate a shift from unfavorable to favorable vaginal microbiome.  
III. Methods 
Study design and population 
74 women diagnosed with suboptimal microbiome from either vaginal swabs or endometrial 
fluid sampling will be invited to participate in our double-blinded, randomized, controlled 
pilot study offering lactobacillus treatment to an intervention group of approximately 37 
participants. The control group will receive placebo treatment with a placebo vaginal 
capsule. Information on medical and fertility history will be collected from patient journal 
after signed written consent from participants. Information on sexual life will be collected 
when participating in the study. Timing of last intercourse has shown to have an effect on 
the vaginal microbiome. 
 
The Implantation Clinic 
Since April 2017 Zealand University Hospital has been offering additional diagnostic work-up 
to women experiencing recurrent implantation failure (RIF= more than three consecutive 
transfers of high quality embryos). Patients demonstrating a suboptimal microbiome, will be 
invited to participate in the study. The results from the vaginal- and/or endometrial 
microbiome, are both part of the RIF-study and not part of diagnostic work-up. Information 
on microbiome from the RIF-study will be given to investigators in the present microbiome 
intervention study.  
Additional subjects will be recruited from women attending the Fertility who have revealed 
a suboptimal vaginal microbiome following testing by their clinician and who meet the 
criteria for inclusion in the study. 
The following flowchart gives an overview over the complete study.  
 
 
 
 
 
 
 
 
 

Protocol version: 3.0 MICINT 
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Study flowchart 
Figure 1: Overall study flow chart 
 
 
 
 
 
 
 
 
 
 
   
 
 
 
 
 
 
 
 
 
 
Figure 2  
Cycle with intervention (CD=cycle day) 
 
CD1                      CD4-7  
 
    CD 14-17  
 
        CD 21-25 
 
 
 
              10 days with treatment 
                Intervention/placebo                                       Vaginal microbiome 
 
Cycle after intervention:  
Re-test vaginal microbiome 
 
                    
 
       CD 21-25 
 
 
 
 
 
 
 
              
                 Vaginal microbiome   
 
 
 
 
 
 
 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
Microbiome suboptimal no. 74 
Intervention group no. 37 
Control group no. 37 
Treatment with lactobacilli + 
test the vaginal microbiome 
Re-test vaginal microbiome 
after one months. 
Re-test vaginal microbiome 
after one months. 
Treatment with placebo +  
test the vaginal microbiome 

Protocol version: 3.0 MICINT 
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Participant inclusion- and exclusion criteria 
Patients diagnosed with suboptimal microbiome 
• 
Age < 43 years 
• 
Non-smoker 
• 
Vaginal and/or endometrial microbiome suboptimal (<90% dominated lactobacilli) 
• 
Attending Implantation Clinic, patient or control, and agreed to be contacted in case 
of other research study 
• 
Patients attending fertility treatment in the Fertility Clinic in Køge and who have 
revealed a suboptimal microbiome following testing by their clinician, and who meet 
the criteria for inclusion in the study.  
Exclusion criteria 
• 
Age < 18 years 
• 
Use of oral antibiotics between diagnose and inclusion 
• 
Use of oral or vaginal probiotics with lactobacilli between diagnosis and inclusion 
• 
Pregnant at enrollment, since inclusion will happen after menstruation, pregnancy is 
not possible at the time of inclusion 
• 
Allergy towards any of the content in the tablets 
 
Laboratory tests and examinations 
1. Gynecological examination: At all visits, the participants will undergo gynecological 
examination.  
2. Vaginal microbiome: During the examination, a swab with vaginal secretion will be 
taken. In collaboration with Artpred (Amsterdam, The Netherlands) the vaginal 
microbiome will be identified by deep sequencing of the IS region of the 16SRNA 
gene using validated protocols. Samples will be collected using special swabs in 
fornix posterior in the vagina. After sampling the material will be inserted in a 
specialized buffer and saved for deep sequencing and qPCR. IS diagnostics will 
perform analyses on behalf of ARTPred.  
Side effects, risks, and disadvantages 
 
Side effects 
Risks 
Disadvantage 
Gynecological 
examination 
No 
No 
Requires presence 
Vaginal swab  
No 
No 
Requires presence 
Placebo 
supplements 
No 
No 
Must be applied vaginally, increased 
vaginal discharge may occur 
Vivag Plus 
supplement 
No side effects registered 
No 
Must be applied vaginally, increased 
vaginal discharge may occur 
Security measures include, sampling only collected by clinicians trained in the procedure. 
Participants will be informed about the procedure by phone before coming to the clinic.  

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Tests will be taken before treatment, after treatment, and one months after intervention or 
placebo at cycle day 21-25. 
 
Randomization 
Study participants will be randomized after inclusion and written consent forms have been 
signed. The study will be double-blinded. The manufacturer will distribute the packages 
unlabeled to Region Hovedstadens Apotek. The randomization and labeling will be 
orchestrated by the pharmacy using a validated computer based randomization program. 
The clinician and the participant will be blinded to the content of the packages. All packages 
will contain a total of 10 vaginal capsules and duration of treatment will be ten days. The 
duration of treatment is based on two previous studies, describing prolonged cure rate after 
treatment for ten days(Stray-pedersen et al., 2008; Pendharkar et al., 2015). 
 
Intervention: Lactobacillus treatment 
The lactobacillus treatment will consist of Vivag Plus vaginal supplements. The vaginal 
capsules consist of cultures of Lactobacillus gasseri and Lactobacillus rhamnosus distributed 
and produced by Bifodan, approved for vaginal use and treatment of suboptimal vaginal 
bacteria. The vaginal capsule will be administered in the following dose; one tablet every 
night before sleep after menstruation, repeated for ten days. Each participant will receive a 
total of 10 vaginal capsules at inclusion. See figure above for details.  
 
Placebo 
Participants randomized to receive placebo, will receive supplements similar to the Vivag 
Plus, without the active substance; Lactobacillus gasseri and Lactobacillus rhamnosus. 
Placebo treatment is provided and developed by Bifodan. Placebo will be taken in the exact 
same dose as the intervention drug, Vivag Plus. Each participant will receive a total of 10 
vaginal capsules at inclusion. See figure above for details. 
 
Participants treated with oral antibiotics during the trial period will be excluded from the 
study. If pregnancy occurs during the study period, the participant will be followed through 
the rest of the study. The study drug or placebo is safe and harmless to use during 
pregnancy. 
 
Active study drug 
 
Content 
Hard vaginal gelatine capsule, with more than 108 CFU of Lactobacillus gasseri EB01 
DSM14869 and more than 108 CFU of Lactobacillus rhamnosus PB01 DSM14870.  
 
Manufacturer 
The manufacturer will produce, in anonymous packages according to good manufacturing 
practice (GMP), both active study drug and placebo.  
 
 
 

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Packaging, labeling, and handling of study drug 
Bifodan A/S will produce the active study drug and placebo and distribute the drugs to the 
pharmacy. The Capital Region pharmacy will make packages, randomization, randomization 
envelopes, and distribute them to the study coordinator. The randomization envelopes will 
be used in case of the need to “de-code” an individual included participant. The study co-
investigator will perform immediately de-coding for each participant when necessary, and 
without interference from the primary investigator. The text on the study drug will be made 
according to GMP. Distribution, inventory and return of excess medicine will be registered 
throughout the study. Participants will be asked to return the empty packages as well as 
excess medicine after treatment. They will be asked to bring the packages at the first visit 
after treatment. All handling of study drug will be done according to GMP. Please see 
labeling example in amendment. 
 
Adverse reactions of the active intervention, Vivag Plus. 
There are no known side effects or adverse reactions known to the study drug. See The 
Danish Summary of Product Characteristics, “produktresume”. 
 
Registration of adverse events and/or reactions 
There will be continuous monitoring and registration of compliance to the intervention, 
throughout the study. Participants will be asked to bring any excess study drugs at their first 
visit to the clinic, after the intervention. All excess study drugs will be registered in the CRF. 
Excess study drugs, handed back to the clinic will be destroyed. If participants forget to bring 
the excess study drug, registration of compliance will be done according to the participant’s 
memory. Rescue medicine is not applicable. Effect of treatment will be monitored 
throughout the primary and secondary end points. Throughout the study participants will be 
questioned about adverse events (AE=an unexpected medical event) and adverse reactions 
(AR= an unexpected/unintended response to a medical product) at every visit to the clinic. 
AE and AR will be registered in the trial clinical case reports and reported to the Sponsor 
within 24 hours.. All serious AE and serious AR (results in death, is life threatening, causes 
hospitalisation, or extension of hospital stay, results in disability or reduced incapacity) will 
be registered and investigator will report this to sponsor/principal investigator immediately 
and to the Danish Medicines Agency and the Ethical Committee, according to 
Lægemiddelloven §89. All serious AE will be assessed for causality before classified as a 
serious AR. Sponsor/principal investigator must also report suspected serious adverse 
reactions (SUSARs) immediately to The Danish Medicines Agency and ethics committee. All 
information regarding any SUSAR that is deadly or life threatening will be reported 
immediately and at the latest within 7 days after sponsor/principal investigator has been 
informed of the SUSAR. The sponsor/principal investigator reports all relevant information 
on sponsors and investigators follow up of the report within 8 days after the report. The 
sponsor must report all other SUSARs within 15 days of being informed of it. Every report 
should include comments on any implications and consequences for the trial.  
The Danish Summary of Product Characteristics (Produktresume) is the reference document 
for evaluating whether a SAR is expected or suspected unexpected (SUSAR). 
 

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The ethics committee and the Danish Medicines Agency will receive yearly reports on 
security, and lists of serious AR. Reports will be send by the sponsor/principal investigator. 
 
Statistical considerations, primary endpoint, study participants, and size  
Within the clinical context in which IVF treatment is carried out, it is important to ascertain 
whether the modulation in microbiome caused by an intervention remains long enough to 
impact on the outcome of a subsequent treatment. In this regard, the key question becomes 
whether or not the impact of the intervention is still detectable one months after the 
intervention. The primary endpoint will therefore be the proportion of participants who 
demonstrate lactobacillus rich microbiome at one month follow up after randomization.  
The planned size and duration of this study are based on the following considerations.  
1. It is assumed that 80% of women randomized to the intervention will demonstrate a 
lactobacillus dominant vaginal microbiome on retesting later in the cycle, whereas 
20% of those randomized to the placebo will do so, reflecting the impact of other 
modulating factors independent of the intervention.  
2. Its assumed that subsequent spontaneous conversions between lactobacillus 
dominant and non-dominant profiles will occur in both groups at a similar rate, and 
it is estimated that this will occur in 20% of women. 
3. Given these figures, after the intervention/placebo, it is estimated that 64% of those 
randomized to the intervention will show a lactobacillus dominant microbiome, one 
months following the intervention compared to 36% of those receiving the control 
intervention.  In order to show this difference in proportions with 80% power with p 
value less than 0.05, 37 patients in in each arm will be required. 
4. Previous studies have reported that around 40% of women embarking on IVF 
treatment have a lactobacillus poor microbiome and that this can be associated with 
a poor outcome.  This percentage with this microbiome profile is therefore likely to 
be higher in the study population consisting largely of women with a history of 
repeated IVF failures. We therefore anticipate that with the current referral rate to 
the Implantation Clinic and from the patients undergoing treatment in our Fertility 
Clinic diagnosed with a sub-optimal microbome, 37 women and 37 controls can be 
recruited within a period of one year.     
 
Differences between the intervention and control group in the primary endpoint will be 
tested for significance using the chi2 test. The significance level will be set at a p-value less 
then 0.05. 
 
Participants dropping out before the primary endpoint has been reached, will be replaced by 
additional recruits. However, it is anticipated that the drop-out will be <5%. Participants 
dropping out of the study at any point will be followed until the end of the study period for 
the individual participant. Participants failing to complete intervention with less then 50% of 
the study trial medicine, will be replaced. 
The end of the study is defined as inclusion and test of the last participants one months after 
the first vaginal examination after intervention. See Figure 2. End of Trial for each 
participants will be the last visit to the clinic (third visit). 

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Study duration 
Participant recruitment: September 3rd 2018 – December 31st 2019 
Data Analysis: January 1st 2020 – February 1st 2020 
Publication of results: March 1st 2020 – May 1st 2020 
 
Biological material from participants 
Microbiome material from the vagina 
Size: One swab imbedded with bacteria from the top of the vagina. 
Purpose: deep sequencing 16SRNA and qPCR from the bacteria to verify any suboptimal 
microbiome. 
Analyzing: The vaginal swab will be send to be analysed abroad by ARTPred, in Holland. The 
samples will be stored and sent in a frozen state (-80 degrees or on dry ice respectively) in 
small batches of 10-30 samples. Storage of samples will take place in our fertility lab in Køge, 
where also sample collection will take place. Samples will be kept pseudo- anonymized and 
will be fully anonymized after the study is complete.  
Analysis: ARTPred, The Netherlands will perform analyses. All samples will be sent pseudo-
anonymized and in line with data protection legislation and the law of data protection.  
 
Information from patient journals and research biobank 
Information on medical and reproductive history will be collected from patient journal. All 
medical and fertility information from journals will be used within the study and in our 
Implantation Clinic. No personal information will be sent abroad. Samples sent will be 
identified with a participant number and date of sample collection. The participants will 
admit to use of journal medical- and reproductive history via consent forms. The Danish law 
on data protection will be followed. 
A research biobank will be made to store the samples, between collection and analyses. 
Permission from the Data Protection Agency will be made simultaneously. All samples will 
be destroyed after analyses. 
 
Data collection 
Case report forms (CRF) are used to collect data and participant information regarding 
history of fertility treatment, menstrual cycle, age, BMI, last menstruation, sexual history, 
and results from tests. All in line with Good Clinical Practice (GCP). At every participant visit a 
CRF will be filled in electronically using “Easytrial”, with the required information to meet 
the endpoints. An access to the above will be provided for inspection from the GCP-monitor, 
The Danish Medicines Agency or the Danish Data Protection Agency. Data collection will be 
performed in accordance with the guidelines of CONSORT, in order to achieve transparent 
reporting of trials. All collected CRF will be kept in Easytrial and will be deleted 5 years and 
one months after inclusion of the last participant. After this period of time, data will be 
anonymized.    
 
 
 
 

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IV. Quality Assurance 
 
Helsinki Declaration 
The study will be carried out in accordance with the Helsinki Declaration, EU Directive on 
GCP and ICH-GCP guidelines after approval by the Regional Ethics Committee, Danish 
Health and Medicines Agency and the Danish Data Protection Agency. The law of 
personal data treatment will be complied at all times. 
All elements in the trial are planned and conducted in accordance with current Danish 
legislation and the ICH GCP Guidelines. 
 
EudraCT 
The study will be registered at EudraCT and Clinicaltrials.gov. 
 
GCP monitor 
The Danish Medicine Agency and a GCP monitor from The GCP Unit at Copenhagen 
University will monitor the project and make sure the study comply with applicable 
legislation as stated above. 
The Sponsor/Principal Investigator will also monitor the study to do quality control. 
In order to complete the above, the relevant authorities will be given access to the study 
participant’s patient charts regarding fertility, and all other study material. The study 
participants will be well-informed about this before joining the study. 
 
V. Ethical Considerations 
 
Ethical considerations  
The patients in this study will already have undergone the initial work-up leading to the 
diagnosis suboptimal microbiome. At present no treatment has been shown to effectively 
treat or change the unfeasible microbiome present in the vagina and/or endometrium. 
Studies show that a Lactobacillus loaded environment in the female reproductive tract is 
important in order to have successful artificial reproductive treatment. The tests performed 
in this study are non-invasive and can be performed during fertility treatment, however our 
participants will be advised not to undergo fertility treatment during the study period, 
because of the hormonal effect from fertility treatment on the bacteria in the vagina. 
Clouding the results collected. 
 
Recruiting participants and informed consent 
Participants undergoing endometrial profiling in our Implantation Clinic and diagnosed with 
suboptimal vaginal and/or endometrial microbiome will be invited to participate, when they 
are informed about their work-up in the clinic. Investigation of the vaginal microbiome will 
be offered to patients undergoing fertility treatment in our Fertility Clinic. The vaginal swab 
will be performed at a routine visit in the clinic. This will provide the patients and the 
clinician with a prediction of the outcome of fertility treatment. Patients with a sub-optimal 
microbiome will hereafter be invited to participate in the present randomized controlled 
study. This can be done by phone or at a planned consultation. Before enrollment the 
participants will receive study information and “Participants rights when participating in a 
research project” by email or handed out during consultation. Hereafter, a consultation will 
be performed where the oral information will take place and a printed study information 

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and “Participants rights when participating in a research project” will be given. The oral 
information will be given according to guidelines in the field. See attached material. The oral 
information will be given by PhD-student Malene Meisner Hviid or health personal who have 
received the proper training and education. The consultation will take place in one of the 
clinics assigned rooms and behind closed doors. Before the participants consultation in the 
clinic they are informed about their right to an assessor. The informed consent will be 
collected after a suitable time of consideration.  
 
Submission of results 
All results, positive, negative and inconclusive will be registered and available from 
clinicaltrialsregister.eu or clinicaltrials.gov. 
The principal investigator will submit the results in EudraCT (European Clinical Trials 
Database) after the study is complete (within one year). 
 
Scientific ethical statement 
It is our believe that results collected after completion of this study will exceed the possible 
side effects and risks connected to participation in the study. In addition to this, obtaining 
new knowledge in the field will help and guide women in the future suffering from 
suboptimal vaginal and/or endometrial microbiome. Without the possibility to conduct 
research trials of this character, no progress in the research field on microbiome in the 
female reproductive tract will be made.  
 
Risk assessment and alternative approaches/plans 
The primary risks of this pilot randomized controlled study reside in achieving the numbers 
of participants desired in order to complete the study. 
 
VI. Financial Aspects 
 
Economics 
Professor Nick Macklon initiated the project with funds from “Future Research Fonds” 
receiving Ph.D salary for three years, 1,500,000.00 mil DKK and Savværksejer Jeppe Juhls og 
Hustrus Ovita Juhls legat, 250.000 DKK. 
ARTPred in the Netherlands provide the analysis of bacterial swaps, 100 euro pr. test. Orkla 
Care will provide the vaginal supplements Vivag Plus and the Placebo supplements, 
produced by Bifodan. Professor Nick Macklon and PhD-student Malene Meisner Hviid have 
no financial interest in the companies supporting the project with analysis and treatment. 
Compensation to participants 
No financial support will be given to participants in this double-blinded randomized 
controlled study. 
 
Relevant clauses in the contract 
There are no clauses made between scientists and companies involved in this protocol. 
 

Protocol version: 3.0 MICINT 
EudraCT number: 2018 002376-41, 16.11.2018 
17
Insurance 
All participants in the trial are covered under “Patient insurance.”  
 
VII. References 
 
References 
Hviid MM, Macklon N. Immune modulation treatments-where is the evidence? Fertil Steril 
[Internet] 2017;107:1284–1293. 
Hyman RW, Herndon CN, Jiang H, Palm C, Fukushima M, Bernstein D, Vo KC, Zelenko Z, Davis 
RW, Giudice LC. The dynamics of the vaginal microbiome during infertility therapy with 
in vitro fertilization-embryo transfer. J Assist Reprod Genet 2012;29:105–115. 
Koot YEM, Macklon NS. Embryo implantation. Curr Opin Obstet Gynecol [Internet] 
2013;25:274–279. 
Koot YEM, Teklenburg G, Salker MS, Brosens JJ, Macklon NS. Biochimica et Biophysica Acta 
Molecular aspects of implantation failure ☆. BBA - Mol Basis Dis [Internet] 
2012;1822:1943–1950. Elsevier B.V. 
Lægemiddelstyrelsen. Produktresumé Vivag Plus_. 2017; 
Lamont RF, Sobel JD, Akins RA, Hassan SS, Chaiworapongsa T, Kusanovic JP, Romeroa R. The 
vaginal microbiome: New information about genital tract flora using molecular based 
techniques. BJOG An Int J Obstet Gynaecol 2011;118:533–549. 
Malchau SS, Henningsen AA, Loft A, Rasmussen S, Forman J, Nyboe Andersen A, Pinborg A. 
The long-term prognosis for live birth in couples initiating fertility treatments. Hum 
Reprod [Internet] 2017;1–11Available from: 
https://academic.oup.com/humrep/article-lookup/doi/10.1093/humrep/dex096. 
Mastromarino P, Vitali B, Mosca L. Bacterial vaginosis : a review on clinical trials with 
probiotics. 2013;229–238. 
Moreno I, Codoñer FM, Vilella F, Valbuena D, Martinez-blanch JF, Jimenez-almazán J, Alonso 
R, Alamá P, Remohí J, Pellicer A, et al. Report of Major Impact Evidence that the 
endometrial microbiota has an effect on. Am J Obstet Gynecol [Internet] 
2017;215:684–703. Elsevier Inc. 
Pendharkar S, Brandsborg E, Hammarström L, Marcotte H, Larsson P. Vaginal colonisation by 
probiotic lactobacilli and clinical outcome in women conventionally treated for 
bacterial vaginosis and yeast infection. BMC Infect Dis [Internet] 2015;1–12. BMC 
Infectious DiseasesAvailable from: http://dx.doi.org/10.1186/s12879-015-0971-3. 
Robertson SA, Jin M, Yu D, Moldenhauer LM, Davies MJ, Hull ML, Norman RJ. Corticosteroid 
therapy in assisted reproduction – immune suppression is a faulty premise Immune 
Cell Regulation of Peri-Implantation. Hum Reprod 2016;31:2164–2173. 
Stray-pedersen B, Ryttig KR, Larsen S. BMC Women ’ s Health with bacterial vaginosis reduce 
the recurrence rate ; a 6-month ,. 2008;8:1–8. 
 

Protocol version: 3.0 MICINT 
EudraCT number: 2018 002376-41, 16.11.2018 
18
VII. Signature 
 
Sponsor/ Principal investigator 
 
 
     07/08/2018 
----------------------------                      ------------------------------------------------------------------------------ 
            Date 
 
 
 
 
Principal investigator 
 
 
Faculty of Health and Medical Sciences, University of Copenhagen 
Department of Gynecology and Obstetrics 
Division of Reproductive Medicine 
Region Zealand University Hospital Roskilde 
Sygehusvej 10 
4000 Roskilde 
Email: nism@regionsjaelland.dk 
Tel.: +45 47324007