Integrated Genetic and Functional Analysis of the Influence of Menstrual Hygiene Products on Female Health
Phase: NA
Conditions: Vaginal Disease, Menstrual Discomfort
Sponsor: University Hospital, Antwerp

PROTOCOL of the study 
Versie 1 – 21 december 2023 
Integrated genetic and functional analysis of the influence of menstrual 
products on intimate female health (abbreviated as ‘Luna’ project). 
Research study at Antwerp University Hospital and the University of Antwerp, in a 
collaboration between Faculty of Medicine and Health Sciences and Faculty of 
Science, Department of Bioengineering 
Inclusion/exclusion criteria 
Volunteers 
Age: 18+ 
Gender: F 
Not pregnant 
Not menopausal 
Natural menstrual cycle, copper IUD or 
combination pill 
Regular menstrual cycle 
No reproductive disease (endometriosis, PCOS,..) 
No history ofn Toxic Shock Syndroom (TSS), 
sensitivity or allergy to menstrual hygiene products 
 
Furthermore, selections will be made based on the questionnaire in order to achieve a 
balanced study population.  
  
Recruitment of participants  
The volunteers (n=500) will be recruited through the channels built up in the Isala 
project (EDGE000471), such as the website (www.isala.be), instagram 
(Isala_UAntwerp), twitter (Isala_UAntwerp) and Linkedin (Isala). Thus, a large 
community has already been built, consisting of women who are very eager to 
participate in follow-up research. These channels will be used to obtain a 
representative group. Staff and students of the UAntwerpen and the UZA will also be 
recruited through posters, ad valvas and mails. No students will be recruited who have 
yet to take exams from the relevant members of the study. The volunteers will receive 
the information about the study and a consent form during information sessions, during 
which the self-sampling kit and menstrual products can also be distributed.   

 
 
Sample collection  
Samples will be self-sampled by the selected volunteers (n = 100).  The self-sampling 
kit contains a detailed manual, information brochure and all materials needed by the 
volunteers for sample collection, storage and delivery to Prof. Lebeer's lab. Participants 
will have 3 sample collections for each menstrual product. This will consist of a vaginal, 
vulvar and groin swab, and during menstruation the menstrual blood will also be 
collected, this in the case of the cup directly and in the case of the other products as 
the product itself, this via non-transparent sterile sealed bags provided for this 
purpose. These products are a tampon, a sanitary pad, a menstrual cup and 2 types of 
menstrual underwear, 1 with a cotton lining and 1 with a bamboo lining. 
 

 
 
Sample size calculation  
Power analyses are challenging for microbiome studies because the magnitude and 
variance of effects on the microbiome are unknown and are highly individual-
dependent and different for each bacterium. However, from literature review and our 
experience with the Isala study, we assume that the sample size selected is 
reasonable. Thus, initially recruited participants (n=1500) will complete a 

comprehensive questionnaire regarding their health, lifestyle, menstrual hygiene and 
vaginal hygiene. Based on this questionnaire, a selection will be made (n = 100) of 
participants who will participate in the actual intervention study, in order to obtain a 
balanced study population, as well as based on location to make the study logistically 
feasible. 
Thus, with 50 participants per condition (half natural cycle, half combined pill) in time 
series, we can perform paired analyses that allow us to account for intra-personal 
variation and increase power. Due to the nature of the study, no wash-out period will be 
necessary during the different interventions, as this is already natural between 2 
menstruations, this also makes each participant a separate control and does not 
require varying the order of menstrual products. From our own experience with 
longitudinal studies, we take into account 5-10% dropout. If it is larger, we will recruit 
additional participants to avoid undermining the study.   
We examine the influence of menstrual products at different levels. A PERMANOVA test 
will allow us to examine the overall effect of the intervention on the composition of the 
microbiome. Furthermore, we will investigate the effect on alpha diversity, for which 
health associations are known in the vaginal microbiome. This can be examined with a 
mixed effect model. Differential abundance of individual taxa will also be tested. It has 
been found that test tools for differential abundance often do not agree in reported 
results. To compensate for this, we have developed a consensus-based way in which 
several differential abundance testing tools are used and a taxon/condition is 
considered significant only if multiple tools agree. Three tools are capable of including 
random effects for paired analysis. Finally, for the vaginal microbiome, we test the 
effect on taxa modules known to be associated with lifestyle factors. This multi-level 
approach gives us a broad view to detect effects that may not be visible at a single 
individual level. 
 
Analysis of samples  
Laboratory of Applied Microbiology and Biotechnology Prof. Sarah Lebeer, Faculty of 
Science, Department of Bioengineering, UAntwerpen  
 
Part 1: Microbiome Analysis  
Microbial DNA and RNA will be isolated from swabs and menstrual blood (collected via 
menstrual cups and extracted from the other menstrual products) via a commercial 
extraction kit such as the Qiagen PowerFecal DNA kit and the Qiagen RNeasy kits (cfr. 
Human Microbiome Project). The isolated DNA and RNA (converted to cDNA) will then 
be analyzed via shotgun sequencing and (meta)transcriptomics. Shotgun sequencing 

and (meta)transcriptomics allow to study the functional profiles and composition of 
these microbial communities. Combined, these techniques will provide valuable 
insights into better understanding the role of microbial communities in the context of 
health and the influence by menstrual products. The Illumina sequencing platform will 
be used.  
Milestone: Identification of the (functional) microbiome composition of swabs and 
menstrual blood, with the aim of mapping the influence of menstrual products 
 
Part 2: Quantitative analysis of concentration of specific microorganisms  
Using quantitative real-time polymerase chain reaction (qPCR), species- or genus-
specific primers will be used to estimate the concentration of specific microorganisms. 
In qPCR, the amount of a specific piece of DNA is measured in “real time. This allows 
the initial concentration of this specific DNA to be determined. The results then provide 
a picture of the concentration of different microorganisms in a sample. For processing 
these data, the “Minimum Information for Publication of Quantitative Real-Time PCR 
Experiments” (MIQE) guidelines will be used.  
Milestone: Absolute abundance of microorganisms in swabs and menstrual blood, with 
the aim of mapping the influence of menstrual products 
 
Part 3: Identification and quantification of metabolites from swabs or menstrual blood 
via a cup  
In addition to the identification and quantification of specific microorganisms from 
swabs and menstrual blood, it is also interesting to look at what metabolites are 
produced by these microorganisms, and whether any toxins or allergens might be found 
in the fluid/blood. These could be from menstrual products. This can be found out using 
metabolomics. Specifically, liquid chromatography and mass spectrometry will be 
applied to supernatant solutions that will be further prepared by extraction with a cold 
organic solvent after addition of an internal standard mixture. The MassHunter 
Quantitative/Qualitative Analysis Software will then be used for identification and 
quantification of metabolites. Finally, the Mass Profiler Professional will be used for 
peak alignment, allowing metabolite annotation and statistical analysis.   
  
Milestone: Identification and quantification of metabolites from swabs and menstrual 
blood, with the goal of mapping the influence of menstrual products 
 

Part 4: Cultivating microorganisms from swabs and menstrual blood   
Besides studying the composition of the microbiome through DNA sequencing 
technology, studying a microbiome through culture techniques remains a relevant way. 
Namely, low abundant members that might be missed by DNA sequencing techniques 
can be found, as well as individual strains examined in detail. This may be relevant to 
find differences in niche-specific strains adapted to conditions on the skin, in the 
vagina and specifically occurring in menstrual blood. Furthermore, this is expected to 
isolate a large proportion of lactic acid bacteria, which are of interest because they 
have a GRAS status (“Generally Recognized As Safe”) and numerous health effects 
have been extensively described. Consequently, we will plate swabs and menstrual 
blood from the volunteers on both selective media for lactic acid bacteria, e.g. MRS (de 
Man Rogosa and Sharpe) medium and blood agar, and rich general media for other 
bacteria and fungi such as nutrient agar and yeast extract peptone medium (YPD). For 
this purpose, swabs or menstrual blood will be dissolved in PBS (Phosphate Buffered 
Saline) and after thorough mixing, a dilution series will be plated out. Several colonies 
will be highlighted and further identification at species level will be done via colony PCR 
with universal primers for the 16S rRNA gene (e.g. 8F-1525R) for bacteria and ITS 
primers for fungi, followed by Sanger sequencing and alignment against a reference 
genome (e.g. BLAST and EZBioCloud).   
Milestone: Cultivation of niche-specific microorganisms. 
 
Part 5: Identification of pro-inflammatory cytokines in swabs and menstrual blood  
Besides changes in the microbiome composition due to menstrual products, it is also 
important to look at possible effects on the immune system of the participants. To 
investigate this, various immunoassays will be performed on the samples, including 
ELISAs for antibodies and pro-inflammatory cytokines, chemokines and growth factors 
(e.g.  osteopontin, TGF-b superfamily, VEGF, CXCL10, CCL3, GM-CSF, IL-1β, IL-2, IL-6, 
IL-8, IL-12, IL-15, IL-18, XCL1, TNF, IFN-γ, perforin and granzyme etc.) In addition, 
relevant immune cell populations (e.g. neutrophils, T-cells, ...) and cell-surface 
receptors will be quantified using flow cytometry.   
 Milestone: Identification of differentially expressed immune factors, such as cytokines, 
antibodies and surface molecules.   
Part 6: isolation of MAIT cells  
A unique goal in this study is the isolation and quantification of white blood cells in 
menstrual blood, also called “menstrual blood mononuclear cells” or MBMCs. To this 
end, the blood samples (obtained using the vaginal cup) are processed in analogy to 
standard blood samples (separation based on a density gradient (e.g. Sepmate tube) 

and freezing). The MBMCs are used purely for the quantification of immune responses 
after co-incubation with microbial (vaginal) isolates: the quantification of cell-surface 
receptors (via e.g. flow cytometry, ELISA, fluorescence microscopy), 
cytokines/chemokines and gene expression. In no case will the material be used for 
elaboration of cell lines.   
Milestone: Isolation of MBMCs with enrichment of MAIT cell subpopulation for studying 
immune responses (cell surface receptors, cytokine production, gene expression of 
immune signaling).