Microbiome and Metabolome Profiles in Couples Undergoing IVF and Their Association With Reproductive Outcomes in a Prospective Longitudinal Cohort Study
Conditions: Infertility, Assisted Reproductive Technology, Pregnancy Complications, Pregnancy Loss, Early, Metabolome, Microbiome
Sponsor: Karolinska Institutet

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STUDY PROTOCOL 
ClinicalTrials.gov Uploaded Document 
Microbiome and Metabolome Profiles in Couples Undergoing IVF and 
Their Association 
With Reproductive Outcomes: A Prospective Cohort Study 
Document Type 
Study Protocol 
Official Title 
Microbiome and Metabolome Profiles in Couples Undergoing IVF and 
Their Association With Reproductive Outcomes: A Prospective Cohort 
Study 
NCT Number 
[Insert NCT Number, if assigned] 
Document Date 
April 5, 2026 
 
 
 

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Study Protocol 
1. Official Title 
Microbiome and Metabolome Profiles in Couples Undergoing IVF and Their Association With 
Reproductive Outcomes: A Prospective Cohort Study 
2. Document Date 
April 5, 2026 
3. Investigators and Affiliations 
Hana Shabana 
Department of Oncology and Pathology, Laboratory of Translational Fertility Preservation, Karolinska 
Institutet, Stockholm, Sweden 
Department of Reproductive Medicine, Karolinska University Hospital, Stockholm, Sweden 
Carolyn E. Cesta 
Department of Medicine, Solna, Centre for Pharmacoepidemiology, Karolinska Institutet, Stockholm, 
Sweden 
Hanna Nilsson 
Department of Oncology and Pathology, Laboratory of Translational Fertility Preservation, Karolinska 
Institutet, Stockholm, Sweden 
Ina Schuppe Koistinen 
Department of Microbiology, Tumor and Cell Biology, Centre for Translational Microbiome Research, 
Karolinska Institutet, Stockholm, Sweden 
Lars Engstrand 
Department of Microbiology, Tumor and Cell Biology, Centre for Translational Microbiome Research, 
Karolinska Institutet, Stockholm, Sweden 
Kenny A. Rodriguez-Wallberg 
Department of Oncology and Pathology, Laboratory of Translational Fertility Preservation, Karolinska 
Institutet, Stockholm, Sweden 
Department of Reproductive Medicine, Karolinska University Hospital, Stockholm, Sweden 
4. Funding 
This study is supported by the Swedish Research Council, the Swedish Cancer Society, Radiumhemmets 
Research Funds, Stockholm Region ALF grants, and Karolinska Institutet. 
5. Conflicts of Interest 
The investigators declare no conflicts of interest. 

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6. Background and Rationale 
Infertility affects a substantial proportion of couples worldwide, and assisted reproductive technologies, 
including in vitro fertilization, are increasingly used to achieve pregnancy. Despite advances in IVF, 
implantation failure, early pregnancy loss, and adverse obstetric outcomes remain insufficiently 
understood. 
Emerging evidence suggests that the human microbiome may influence reproductive physiology through 
immune, inflammatory, and metabolic pathways. The female reproductive tract microbiome has been 
associated with implantation success and miscarriage risk, while the gut microbiome may contribute to 
systemic immune regulation through microbial metabolites. The potential contribution of the male 
reproductive microbiome to reproductive outcomes is less well characterized. 
Most existing studies are cross-sectional, focus primarily on women, or evaluate a single biological 
compartment. Prospective longitudinal studies integrating microbiome, metabolome, and immune 
markers in couples undergoing IVF remain limited. 
This study is designed to evaluate microbiome and metabolome profiles in couples undergoing IVF and to 
assess their association with reproductive outcomes. 
7. Objectives 
7.1 Primary Objective 
To evaluate the association between baseline microbiome and metabolome profiles and live birth 
following IVF treatment. 
7.2 Secondary Objectives 
1. To evaluate associations between microbiome and metabolome characteristics and time to 
pregnancy. 
2. To assess relationships between microbial profiles and fertilization rate, embryo development, 
implantation success, and clinical pregnancy. 
3. To investigate associations between microbiome signatures and miscarriage. 
4. To evaluate associations between microbiome and metabolome profiles and obstetric outcomes. 
5. To explore the contribution of the male reproductive microbiome to IVF outcomes. 
8. Study Design 
This study is a prospective observational cohort study of couples initiating IVF treatment in Stockholm 
County, Sweden. Biological samples will be collected at baseline before initiation of IVF stimulation and, 
when applicable, during early pregnancy according to the study sampling plan. Couples will be followed 
from initiation of IVF treatment until pregnancy or for up to 1 year, whichever occurs first. Pregnancies 
achieved during follow-up will be followed until completion, including miscarriage or live birth. 
Because this is an observational cohort study, no experimental intervention will be assigned as part of the 
protocol. 

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9. Study Population 
9.1 Inclusion Criteria 
• 
Women aged 18 to 40 years undergoing IVF treatment 
• 
Male partners aged 18 to 56 years 
• 
Ability to provide informed consent 
• 
Ability to complete study questionnaires 
9.2 Exclusion Criteria 
• 
Pregnancy at the time of enrollment 
• 
Systemic antibiotic use within the previous 4 weeks 
• 
Active genital infection at the time of sampling 
10. Sample Size 
The study aims to enroll 500 couples undergoing IVF treatment. In addition, 10 sperm donors and 10 
oocyte donors will be included as reference groups for microbiome comparisons. 
11. Study Procedures and Biological Sampling 
11.1 Female Participants 
• 
Rectal swab 
• 
Vaginal swab 
• 
Peripheral blood sample 
11.2 Male Participants 
• 
Semen sample 
• 
Rectal swab 
11.3 Timing of Sample Collection 
For the main IVF cohort, samples will be collected at baseline before initiation of IVF stimulation. 
Additional sample collection may occur during early pregnancy, when applicable and according to the 
study sampling plan. 
12. Microbiome and Metabolome Analyses 
Microbial composition will be characterized using shotgun metagenomic sequencing to identify bacterial, 
viral, fungal, and archaeal communities. Metabolomic profiling will be performed using mass 
spectrometry-based analytical platforms. 
Sequencing data will undergo quality control procedures, removal of human reads, and taxonomic 
classification using established bioinformatics pipelines. 
Immune marker analyses in blood samples will be performed using laboratory-based immunologic 
assays, as specified in the analytical plan. 

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13. Data Sources 
• 
Participant questionnaires 
• 
Clinical IVF records 
• 
Biological sample analyses 
• 
Linkage with Swedish national health registers, including the Swedish Pregnancy Register 
14. Outcomes 
Planned study outcomes include reproductive and laboratory outcomes such as: 
• 
Live birth 
• 
Time to pregnancy 
• 
Fertilization rate 
• 
Embryo development 
• 
Implantation success 
• 
Clinical pregnancy 
• 
Miscarriage 
• 
Obstetric outcomes 
• 
Female and male microbiome profiles 
• 
Metabolomic profiles 
• 
Immune marker levels in female blood samples 
• 
Semen parameters, including sperm concentration 
Detailed outcome titles, descriptions, and time frames should be entered in the ClinicalTrials.gov record 
in the Outcome Measures section. 
15. Statistical Analysis 
Descriptive statistics will be used to summarize participant characteristics. 
Associations between microbiome and metabolome profiles and reproductive outcomes will be 
evaluated using multivariable regression models adjusted for relevant confounders, including maternal 
age, body mass index, smoking status, infertility diagnosis, and medication exposure. 
Microbiome diversity metrics, including alpha diversity and beta diversity, will be calculated. Differential 
abundance analyses will be performed using compositional statistical approaches with correction for 
multiple testing. 
Exploratory analyses may assess relationships between microbiome composition, metabolomic profiles, 
immune markers, and reproductive outcomes. 
16. Ethical Considerations 
The study has been approved by the Swedish Ethical Review Authority, Dnr 2024-07146-02. 
Written informed consent will be obtained from all participants before enrollment. 
No names or identifying details of research participants are included in this uploaded protocol document. 

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17. Dissemination 
Results will be disseminated through scientific conferences and peer-reviewed publications. 
18. Graphic Abstract of the Protocol