RUBIC (ReproUnion Biobank and Infertility Cohort): A binational clinical foundation to study risk factors, life course, and treatment of infertility and infertility-related morbidity.

Infertility is a significant public health problem affecting 15%–25% of all couples during their reproductive life span. 1 , 2 Approximately one third of couple infertility is attributed to female factors, including disorders of the ovary, fallopian tubes, uterus, or cervix. Another third is a result of male reproductive issues related primarily to semen quality. The last third is accounted for by combined female and male factors or cases without any identifiable cause of infertility. 3 Failure to conceive can have severe psychological, social, and economic consequences for the affected individuals, couples, and families. 4 , 5 Infertility also adds to the basic challenges of sustaining a society that is able to provide for its aging population. 6 Medically assisted reproduction (MAR) has greatly expanded in recent decades, and today more than 10% of children in Denmark are born after MAR. 7 Over 50% of these children are born after assisted reproductive technology treatment (ART; i.e., the in vitro treatment methods where the egg, semen, and the embryo are managed in the laboratory). In 2018, 4.3% of children in Sweden and 6.5% in Denmark were born after ART. 8 , 9 At present, most treatment options bypass natural reproduction barriers by enhancing ovulation and/or increasing the chance of oocyte/sperm interaction, for example, intrauterine insemination (IUI), in vitro fertilization (IVF), or intracytoplasmic sperm injection (ICSI), whereas only few treatments target the underlying cause of infertility. Despite the availability of increasingly advanced diagnostic and therapeutic techniques, approximately 30% of infertile couples do not obtain a live birth after fertility treatment. 10 For these couples, there are currently no further options to increase their chances of a successful pregnancy and live birth. Health behavioral factors such as postponement of childbearing to an older age, smoking, and obesity play important roles in increasing the risk of infertility. 6 , 11 , 12 However, the interplay between and relative contributions of health behavior, psychosocial, environmental, genetic, endocrine, metabolic, immunologic, and epigenetic factors are still poorly understood. Furthermore, most studies focus primarily on the female or the male part of the infertile couple rather than the couple as a whole. In addition, our insight into the underlying mechanisms and the contribution of pathogenic processes at different stages of life to subsequent impairment of fertility remains limited. Male infertility has been suggested to be a proxy of general health status as men presenting with infertility have a higher comorbidity burden. 13 But also in longitudinal studies, male infertility has been linked to an increased risk of several major non‐communicable diseases such as metabolic, cardiovascular, and neurological diseases and cancer. 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 The association between female reproductive function and long‐term health is less clear, although studies have shown that (1) irregular and long menstrual cycles are associated with greater risk of premature mortality 22 ; (2) the risk of ovarian cancer is higher in women suffering from endometriosis compared to an age‐matched background population 23 ; and (3) the risk of breast cancer is higher in women initiating fertility treatment than in women not requiring fertility treatment, especially after age 40. 24 Although overall morbidity and mortality do not seem to be increased in all women suffering from infertility, 25 , 26 , 27 a diagnosis of infertility in subgroups of women and in all men may serve as an early marker of subsequent morbidity and decreased life expectancy. The associations between reproductive function and healthy aging warrant further clarification considering endocrine, immunological, molecular, developmental, environmental, and genetic mechanisms are potentially shared. The ReproUnion Biobank and Infertility Cohort (RUBIC) has been initiated to examine the causes and underlying mechanisms of infertility, improve reproductive outcomes for women and men, and ultimately provide the scientific foundation to facilitate successful pregnancies leading to live births of healthy children. Reaching beyond reproduction, our additional ambition is to promote current and long‐term health and quality of life among infertile women and men. Our scientific strategy relies on the following three specific targets. Access to detailed information on health behaviors, medical and prescription history, occupational exposure history of both partners and their parents, as well as exposure to environmental pollutants, combined with genetic and epigenetic analyses will allow us to study a wide range of suspected risk factors for infertility, early embryonic loss, and adverse pregnancy outcomes. Through the identification and ranking of modifiable risk factors, we will be able to design targeted strategies to mitigate the risk factors with the possibility of preventing infertility in the future. Understanding the mechanisms through which these factors affect fertility may also allow the development of more targeted treatments. We will utilize an unbiased multiomics approach to develop informative biomarkers to diagnose subfertility and predict fecundity and response to treatment. The results will be integrated through systems medicine tools to identify biomarkers that are causally linked to infertility. Given the complexity of the condition, it is likely that a single biomarker will not be adequate. Algorithms will be developed combining markers from different omics data and surrogates of health, which will allow the diagnosis of subfecundity caused by diverse etiologies. Improved biomarkers of fecundity will provide the foundation for precision reproductive medicine, thereby enabling the identification of couples in need of treatment and enabling the selection of the most relevant treatment for each couple to increase chances of pregnancy success and reduce the burden of treatment. Through linkage to data from national health registers, we will identify comorbidity among infertile women and men prior to and at the time of fertility work‐up, and follow the physical and mental health status of participants prospectively, both through the registries and—funding permitted—through repeated health assessments. Psychosocial and sexual consequences of infertility will be assessed using self‐reported questionnaire data. By identifying the subgroups that are at increased risk for specific diseases, we will be able to search for reliable biomarkers and risk factors that will help identify the at‐risk people for application in early screening programs. This approach will enable the design of preventive strategies to reduce morbidity as well as reduce the adverse psychosocial and sexual consequences of infertility. Efforts may include counseling at the time of fertility treatment and clinical follow‐up that will contribute to healthy aging for infertile couples. RUBIC is a public health initiative of the ReproUnion Consortium ( www.reprounion.eu )—an interdisciplinary collaboration between Danish and Swedish partners at public hospitals and universities, and the healthcare industry. The planning of the cohort has been guided by a voluntary external advisory board of experts from other European countries and the United States. In addition, a binational board with representatives of the ReproUnion stakeholders will be established and take responsibility for future optimal utilization of the collected data and biomaterial. The Medicon Valley Alliance ( www.mva.org ) professionally manages the collaboration.

We will combine self‐reported and clinical data with biomarkers and other biological readouts. All questionnaire and clinical data are administered in Research Electronic Data Capture (REDCap), a secure web application for building and managing online surveys and databases hosted by The Capital Region of Denmark and Lund University. 29 , 30 A detailed web‐based questionnaire specifically for this project has been developed in collaboration with public health experts within questionnaire development. Validated scales previously used either in population‐based studies or in studies of fertility patients have been applied whenever possible. The areas covered in the questionnaire along with selected references are presented in Table  1 . The questionnaire is identical for male and female participants for most items. Only questions regarding reproductive history, health, and sexuality are sex specific. Study participants are asked to complete the questionnaire prior to the clinical visit. Areas covered in the RUBIC questionnaire with selected references Years together with partner, cohabitation status Living together with children Previous pregnancies and outcomes Previous fertility treatment and outcomes Duration of current attempt to conceive Reasons for seeking fertility treatment 1 , 34 Communication regarding childlessness (with partner and others) 35 Infertility‐specific self‐efficacy Marital benefit (strengthened relationship because of infertility) 36 Infertility‐specific stress 37 , 38 , 39 General health (self‐rated SF‐12v2) 40 , 41 Specific diseases (somatic and mental), recent fever (men only) Chemotherapy Gastrointestinal symptoms and stool form (The Bristol Stool Form Scale [BSCS]) 42 Reproductive health including genital diseases and surgery Pubertal timing Medication Dietary supplements Muscle enhancing supplements (men only) General quality of life 43 , 44 Sleep Stress (perceived stress 45 , 46 and stressful life events, modified from 47 ) Depressive symptoms (Mental Health Inventory [MHI]‐5, Short Form‐36) 48 , 49 , 50 , 51 Anxiety (Generalized Anxiety Disorder [GAD‐7]) 52 , 53 , 54 , 55 Sexual satisfaction 56 Sexual orientation 57 Sexual problems Physical activity (International Physical Activity Questionnaire [IPAQ]), 58 sedentary behavior Tobacco smoking habits (different products, current and previous habits) E‐cigarette use Alcohol intake Drugs Dietary habits Use of personal care products Family (origin, parental age, siblings, adoption) Fetal life and birth Parents’ reproductive history Chronic diseases in the family (gender specific) Education Work (employment status, specific job title, working conditions, and exposures related to specific occupations) Psychosocial working environment Work–life balance Note : For inquiries about the questionnaire, please contact Lærke Priskorn: [email protected]. Both individuals within the infertile couple undergo a standardized clinical investigation including genital ultrasound and a dual‐energy X‐ray absorptiometry (DXA) scan. In addition, men are subjected to a complete andrological work‐up and women will undergo a complete gynecological examination. A full overview of the applied physical examinations and radiological modalities is presented in Table  2 . Overview of the physical and radiological examinations in RUBIC Height (standing and sitting) Weight Body mass index Arm span Waist and hip circumference Blood pressure and heart rate Ankle–Brachial pressure index Respiratory capacity (spirometry) Whole‐body bone mineral density Regional bone mineral density Body composition (fat distribution, lean mass) Testis size (Prader's orchidometer), consistency, and position Scrotal and penile abnormalities Varicocoele Pubic hair distribution Gynecomastia Ultrasound: testis size, echoic pattern, and presence of testicular microlithiasis and epididymal abnormalities Anatomical abnormalities Ovarian and uterine anatomy (transvaginal ultrasound) Antral follicle count (transvaginal ultrasound) The following biological samples are collected before fertility treatment: blood (whole blood, serum, and plasma), urine, saliva, rectal swab, feces, and hair from both sexes, and additionally an endometrial biopsy and vaginal swab in women, and semen in men. Furthermore, during fertility treatment, ovarian follicular fluid and granulosa and cumulus cells are collected in women and semen is collected in men. Some of the biological samples will be analyzed as part of the routine fertility work‐up, whereas others will be stored at –80°C in the Copenhagen Hospital Biobank (for Danish participants) or Regionalt biobankscentrum, Södra sjukvårdsregione n (for Swedish participants) for future scientific projects (Table  3 ). Overview of future analyses based on biobank material of biological samples Granulosa and cumulus cells: Genomics, epigenomics, transcriptomics, proteomics, and cell‐free DNA/RNA analysis Follicular fluid: Hormone analysis, proteomics, and cell‐free DNA/RNA analysis For both men and women, several basic health markers will be assessed as part of the clinical work‐up, including anemia, inflammatory markers, and lipid profiles. For men, markers of liver, kidney, and thyroid function are measured as well. Additionally, the SARS‐CoV‐2 antibody status will be assessed, highlighting the ability to add additional tests in response to emerging threats. In men, morning and (preferably) fasting blood samples will be analyzed for serum levels of testosterone, sex hormone‐binding globulin (SHBG), luteinizing hormone (LH), follicle‐stimulating hormone (FSH), estradiol, and inhibin B. In women, serum levels of FSH, LH, anti‐Müllerian hormone, SHBG, testosterone, and progesterone will be measured. Male participants will be asked to deliver two semen samples (2–5 days abstinence period) prior to the physical examination. Assessment of semen volume, sperm concentration, total sperm count, motility, vitality, and morphology is subsequently performed according to the World Health Organization's recommendations. 31 In addition, the acrosomal status, 32 mixed antiglobulin reaction, fructose, antisperm antibodies, and the leukocyte concentration are assessed and an aliquot stored for subsequent sperm DNA fragmentation analysis (Sperm Chromatin Structure assays). The remaining sample is purified by gradient centrifugation and seminal plasma and the purified spermatozoa are stored for later investigations. Samples are collected and stored for future broad‐spectrum analyses of exposure biomarkers for environmental chemicals with known or suspected endocrine disrupting abilities or effect biomarkers related to exposure to endocrine‐disrupting chemicals. Primary specimen matrices for toxicological analyses are urine, serum, ethylenediaminetetraacetic acid whole blood, seminal plasma, and hair. Field‐blank samples will be included. Y‐chromosome microdeletion analyses and karyotype will be assessed in all included men with sperm concentration below 5 mill./ml. Currently, in Denmark, the analysis of Y‐chromosome microdeletions is performed on all infertile men. Also, karyotyping is performed routinely in women with premature ovarian failure. DNA will be isolated from peripheral blood lymphocytes. Specimen collection for various omics analyses includes blood, isolated spermatozoa, seminal plasma, fecal samples/rectal swabs, follicle cells, vaginal swabs, endometrial biopsy, and hair. The following omics are intended but may change as novel technological options evolve: ‐ Microbiome analysis of saliva, ejaculate, vaginal swabs, and fecal samples/rectal swabs; ‐ RNA sequencing of serum, seminal plasma, and purified sperm cells; ‐ DNA methylation analysis of purified sperm and follicular cells; ‐ Whole‐genome sequencing of DNA isolated from blood and spermatozoa; ‐ Proteomics analysis of serum, seminal plasma, spermatozoa, and hair. Microbiome analysis of saliva, ejaculate, vaginal swabs, and fecal samples/rectal swabs; RNA sequencing of serum, seminal plasma, and purified sperm cells; DNA methylation analysis of purified sperm and follicular cells; Whole‐genome sequencing of DNA isolated from blood and spermatozoa; Proteomics analysis of serum, seminal plasma, spermatozoa, and hair. All women—whether they become pregnant naturally, through MAR, or not at all—will be followed in the medical birth registers to obtain information on potential childbirth. Furthermore, several scientific questions regarding causes of infertility and infertility‐related long‐term morbidity and mortality will be answered through the linkage of information from RUBIC to the Danish and Swedish national registers using the unique civil registration number. Most of these national registers were established relatively early in the 20 th century and represent a unique source of perinatal, childhood, and adult health and socioeconomic characteristics of the infertile couples, their parents, and their children. The extent and quality of this information is the key to adequately addressing potential transgenerational reproductive effects of exposures. The main registers and a brief description of their content are listed in Table  4 . Overview of national registers and potential information available for linkage to the RUBIC participants using the unique Danish and Swedish personal identification numbers DK: 1968 SE: 1968 DK: 1973 SE: 1973 DK: 1977 SE: 1987 DK: 1943 SE: 1958 DK: 1946 SE: 2001 DK: 1994 SE: 2007 DK: 1970 SE: 1961 DK: 1994 SE: 2005

RUBIC is established as an add‐on to the routine fertility management at Copenhagen University Hospital departments in the Capital Region of Denmark and Lund University‐affiliated Reproductive Medicine Centre in Region Skåne in Sweden. The participating sites in each of these regions provide reproductive care to 600 new couples annually (Figure  1 ). In Denmark and Sweden, the healthcare sectors are public and tax financed, which means that fertility treatment with IUI, IVF, and ICSI is reimbursed for couples without joint children. About half of fertility treatments are conducted in the public sector. Hence, the distribution of socioeconomic status of these couples is similar to that of the general population. 28 Map of departments within the Sound Region that are participating in cohort recruitment and examinations In the Capital Region of Denmark, 2500 couples will be recruited in total from the Danish public fertility departments at Rigshospitalet, Herlev Hospital, Nordsjællands Hospital Hillerød, and Hvidovre Hospital, and from the Department of Growth and Reproduction at Rigshospitalet. In Sweden, 2500 couples will be recruited from the Reproductive Medicine Centre at Skåne University Hospital in Malmö. As the project is embedded in the routine treatment of fertility patients in Denmark and Sweden, minor logistic differences in procedures are accepted in the two countries. Inclusion criteria for participation are as follows: being part of a couple of opposite biological sex; incident referral for the couple (the partners may have been examined or treated individually before); referral for infertility investigation or fertility treatment; age (depending on national regulations): women: age 18–41 years in Denmark and 24–39 years in Sweden; men: 18 years or older in Denmark (no upper age limit) and 24–53 years in Sweden. being part of a couple of opposite biological sex; incident referral for the couple (the partners may have been examined or treated individually before); referral for infertility investigation or fertility treatment; age (depending on national regulations): women: age 18–41 years in Denmark and 24–39 years in Sweden; men: 18 years or older in Denmark (no upper age limit) and 24–53 years in Sweden. Exclusion criteria for participation are as follows: inability to understand study information given in Danish or Swedish; surgical sterilization; HIV or hepatitis A, B, or C. inability to understand study information given in Danish or Swedish; surgical sterilization; HIV or hepatitis A, B, or C. The enrollment started in June 2020 and based on the current numbers of annual referrals the recruitment period is expected to last 5–8 years. All eligible patients are asked to participate in the project, and they receive detailed oral and written information prior to giving their written informed consent. A project website has been developed ( www.rubic.nu ) for potential participants to obtain further information. After obtaining informed consent, the participants are asked to complete an extensive questionnaire, and they undergo physical examination and collection of biological samples. Project procedures are the same for all enrolled couples even if the cause of infertility has been identified previously in one or both partners. In Sweden, both women and men are examined at one clinic. In Denmark, the women are examined at one of the four participating fertility clinics, whereas the men attend the Department of Growth and Reproduction at Rigshospitalet. The recruitment flow and data collection processes are depicted in Figure  2 . Data on sex, age, body mass index, postal code, and infertility diagnosis are obtained for participants as well as non‐participants for the assessment of potential selection bias. Recruitment flow and data collection in the ReproUnion Biobank and Infertility Cohort (RUBIC)

All authors made substantial contributions to conception and design and participated in discussions of manuscript content. The two joint first authors drafted the manuscript. All authors have reviewed several versions of the manuscript critically and have given final approval of the version to be published.

The research protocol, questionnaires, consent forms, and recruitment posters and pamphlets have been reviewed and approved by the Regional Committee on Health Research Ethics for the Capital Region of Denmark (women: H‐19042776, men: H‐19044889) and the Swedish Ethical Review Authority (2019‐04631). The study is performed according to the Declaration of Helsinki, and all participating couples provide their informed signed consent to participate, following receipt of written and oral information. The participants will be informed about the outcome of their infertility work‐up, and the traditional metabolic and cardiovascular risk factors will be discussed with them. The planned data collection, clinical examinations, and analyses (not already included in routine infertility examination) are not considered questionable from an ethical point of view. The planned analyses of specific genes included in our study are limited to common genetic variants. For whole‐genome sequencing and the more general transcriptomic, epigenomic, and microbiomic analyses, the participants will be informed about the analyses and associated risk of incidental findings with potential consequences for their health. We will also inform the participants about the option of genetic counseling. A board of experts will be consulted about clinical implications of incidental genetic findings and we will disclose potential findings to participants, unless they specifically have waived this option. Additional biomarkers included in the protocol are not specific for prediction of any specific disease and, thus, are not reported to the participants. Potential use of the collected data and biological material for purposes other than those defined in the research plan will only take place after additional approval by the relevant ethics committees.

This article is part of the ReproUnion collaborative study, co‐financed by EU Interreg ÖKS, Capital Region of Denmark, Region Skåne, and Ferring Pharmaceuticals. None of the funders have any role in the study design, data collection, analysis or interpretation of data, or publication decisions.

By establishing RUBIC as an add‐on to the public routine fertility management, an existing setup is utilized, and the burden of participation is diminished as part of the participants would go through part of the examinations anyhow as part of their fertility work‐up. This reduces the logistic challenges and increases the chance of successful inclusion. Including infertile couples rather than either the female or male part is a major strength of the current study as most studies so far focus on one of the genders only. Furthermore, the large cohort will enable studies of relatively rare exposures and outcomes and allow for more complex associations to be studied. Sweden and Denmark have long traditions regarding health and demographic registers with valid and nearly complete information on an individual level. This expands the usefulness of the collected data, allowing for studies reaching back in time and for later follow‐up of the couples included in RUBIC. Many of the planned studies rely on internal comparisons using partners from couples with exclusively female or male factor infertility as controls. However, for the central purpose of studying couple fertility per se, external comparison groups of fertile couples or couples with unknown fecundity are needed. The extensive Danish and Swedish registries allow for easy identification of such external comparison groups in the size and with selection criteria suitable for the specific study in question. Provided additional funding will be obtained, we still have the option of future inclusion of a cohort of proven fertile couples and/or first pregnancy planners. Another potential limitation is the source population covering the greater Copenhagen area in Denmark and Southern Sweden. Although both locations encompass urban and rural areas, our results cannot necessarily be generalized to the national level. This relates specifically to the prevalence of different causes of infertility and the exposure levels that might differ between the source population and the national level, whereas results from the biological associations studied are expected to be generalizable. Lastly, the study has been initiated without having secured funding for the entire study period, which is a potential obstacle for reaching the aimed population size. The major strengths, weaknesses, opportunities, and threats of RUBIC have been summarized in Table  5 . Overview of study strengths, weaknesses, opportunities and threats (SWOT analysis) *Binational clinical foundation *Recruitment: add‐on to routine fertility management *Ability to assess couple infertility *Size: allows for studies of rare exposures and outcomes *All‐encompassing types of biospecimens, analyses, and questionnaire data *Combination of biobank and register data *Joint binational management of biobank and register data *Multidisciplinary approach *International consortium on causes and consequences of infertility *Lack of external reference group *Yet incomplete standardization between countries *Limited ethnic, geographical, and social diversity *Exclusion of couples with “mild” infertility, not handled by tertiary referral centers *Long‐term follow‐up of infertile subjects *Register data allow for three‐generation studies *Well‐defined standard operating procedures and routines for data collection can be exported to other centers *Comparison of study populations with background population based on register data *Development of new routines for multinational data handling and biobank management *Research tool for specialties outside the field of reproduction *Basis for new clinical trials and development of new diagnostic tests *Triple‐ or Quadruple Helix scientific collaboration *Lack of funding for primary data collection and/or follow‐up *Pandemic outbreaks affecting infertility treatment and course *Changes in referral patterns for public healthcare service *New regulations for international exchange of data and biological material

The Scientific Board welcomes collaboration on research projects using data from RUBIC or available background material from the project to conduct parallel studies with similar setups. For external use of the collected data, including biobank material, scientific applications will be reviewed by the board of RUBIC. More information on how to apply, administration fees, and how to find relevant background documents can be found on the ReproUnion webpage ( www.ReproUnion.eu ). The Scientific Ethical Committees in Denmark and Sweden have approved the use of biological material from RUBIC participants for specified research purposes described in the present paper. Thus, use of material for additional purposes will require further ethical approval aside from the approval granted by the RUBIC board.

Data will be stored nationally on secure servers according to the regulations of the Danish and Swedish Data Protection Agencies (Record number Danish women P‐2020‐69; Danish men P‐2020‐68, and Swedish women/men Record number: 2019–04631 and amendment 2020–02082). Pseudonymized data will be utilized for compiled analyses according to European General Data Protection Regulation guidelines. Code keys linking the national personal number to the specific study code will be kept at respective centers involved in recruitment of study subjects and will not be exchanged between the participating institutions.

The proposed study has ample statistical power for evaluating associations within the three main objectives described in this paper. The included power calculations illustrated in Figure  3A–C all address the power with which a given association can be detected at different exposure and outcome prevalence levels when assuming an odds ratio of 1.5 and a two‐sided test at a significance level (alpha) of 5%. In all figures, the dotted lines represent the exposure and outcome prevalence with a power of 80%. The inclusion of a study population of 5000 women and men (Figure  3A ) will enable the detection of an odds ratio of 1.5 with a significance level of 5% for an exposure with a prevalence of 6% and an outcome with a prevalence of 20% with a statistical power of 80% assuming that the entire population can be included in the given analysis (blue line, Figure  3A ). Studies of more prevalent exposures and outcomes of, for example, 17% and 15%, respectively, can be initiated halfway through data collection (i.e., 2500 couples, cf. Figure  3B ) with the same statistical power of 80% (green line, Figure  3B ). Examples of studied associations including only subpopulations are illustrated in Figure  3C . If, for example, only the lowest and highest 15% for a given outcome are included, an odds ratio of 1.5 with a significance level of 5% and a power of 80% can be detected for an exposure prevalence of 15% (red line, Figure  3C ). For all analyses, the statistical power will be even higher when exposures and/or outcomes are modeled as continuous variables. Graphical presentation of calculated power to detect an odds ratio of 1.5 with a significance level of 5% for a given exposure at different outcome prevalences. Panels A and B are based on the assumption that the entire study population of 5000 and 2500 women or men, respectively, are included in the analyses, and the outcome prevalences are depicted in 5% intervals ranging from 5% to 30% (cases) with corresponding controls ranging from 95% to 70% (e.g., the black line represents 5% cases and 95% controls and the purple line 30% cases and 70% controls). Calculations in panel C are based on a study population of 5000 women and men of which only part of the population belongs to one of the two categories (i.e., black line representing 33% vs. 33% ≈ 1650 vs. 1650 women or men). In all figures, the dotted lines illustrate the exposure prevalence that can be studied with a power of 80%

RUBIC has the potential to become a unique worldwide scientific resource within the field of reproduction as the largest collection of clinical and biological material on infertile couples. This extensive data collection will enable us to elucidate new causes of infertility as well as the causes of known infertility diagnoses such as low semen quality, polycystic ovary syndrome, premature ovarian insufficiency, and endometriosis and offer leads for novel therapeutic targets. Our extensive data collection on both infertile women and men has a tremendous developmental potential and the generated data will provide the basis for several future national and international studies on disease mechanisms, targeted interventions, and treatments for specific patient groups. RUBIC will also enable future detailed and targeted investigational follow‐up studies that may aid in describing the natural course of certain infertility phenotypes, and in validating newly developed biomarkers. New prevention strategies can be tailored to the profiles and needs of specific patient groups to avoid future morbidity. The infertility cohort is unique because both women and men are included, enabling us to examine combinations of and interactions between contributory causes of disease within each partner. Despite their close geographical proximity, populations in Denmark and Sweden exhibit notable differences in disease prevalence and life expectancy and comparisons of reproductive determinants in these two countries are of considerable scientific interest. 33 We are planning to include the following additional cohorts relevant for the scope of RUBIC: (a) first pregnancy planners and proven fertile couples as controls for the infertility cohort and (b) offspring conceived by enrolled couples (by means of assisted reproductive technology or natural conception) to study potential transgenerational effects of different internal and external factors.

SB has received a research grant from NIA, NICHD, PCORI, AbbVie, MIB, and FPT as well as consulting fees from Aditum. MLE is advisor to Sandstone Diagnostics, Dadi, Hannah, Underdog, and Roman. AG has received lecture and consultant fee from Besins Healthcare. The rest of the authors declare no potential conflict of interest.

The questionnaire was translated from Danish to Swedish and back‐translated to ensure comparability of the Danish and Swedish versions. Standard operating procedures have been composed for all clinical examinations and collection of biospecimens to secure standardization of the data collection between the participating clinics. Furthermore, examination workshops will be carried out regularly during the study period. Same is true for assessment of standard semen parameters. The biochemical analyses that are part of the routine fertility work‐up will be handled locally as immediate analysis is necessary, meaning that some systematic differences are to be expected and must be handled in the statistical analyses. However, samples will be exchanged between the sites in order to check the comparability of results delivered by different laboratories. All strictly research‐related planned analyses will all be conducted together for Swedish and Danish samples in the same laboratory. Minor differences in the processing of biomaterial might exist and thus field‐blank samples are collected recurrently to enable us to take any contamination from the processing or storage into account when conducting toxicological analyses.