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STUDY QUESTION Is intake of sugar-sweetened beverages (SSB) or artificially sweetened beverages (ASB) associated with testicular function in young men? SUMMARY ANSWER Among young men unaware of their semen quality and reproductive hormone levels, intake of SSBs was associated with lower sperm concentration, lower total sperm count, and a lower ratio of serum inhibin-B/FSH. WHAT IS KNOWN ALREADY SSBs may adversely impact testicular function, but results are not consistent across studies. Moreover, the associations of ASB, energy-drinks or fruit juices with testicular function are unclear. STUDY DESIGN, SIZE, DURATION Young healthy men and unselected for fertility status men enrolled in a cross-sectional study between 2008 and 2017. PARTICIPANTS/MATERIALS, SETTING, METHODS A total of 2935 young (median age: 19 years) men enrolled in the study. Intake of SSBs, ASBs, fruit juices, and energy-drinks was assessed with a validated food frequency questionnaire. Testicular function was assessed through conventional semen quality parameters (semen volume, sperm concentration, total count, motility and morphology), testicular volume assessed with ultrasound, and serum reproductive hormone concentrations (total testosterone, free testosterone, E2, inhibin-B, LH, FSH, sex hormone-binding globulin) were measured. MAIN RESULTS AND THE ROLE OF CHANCE In multivariable-adjusted analyses, men in the highest category of SSB intake (median: 1.1 servings (∼220 ml)/day) had a 13.2 million/ml lower median sperm concentration (95% CI: –21.0, –5.5) than non-consumers. A similar pattern was observed with total sperm count (–28 million (95% CI: –48, –9)), serum inhibin-B (–12 pg/ml (95% CI: –21, –4)), and inhibin-B/FSH ratio (–9 (95% CI: –18, 0)). The adjusted median difference in sperm concentration and inhibin-B associated with increasing SSB intake by 1 serving (∼200ml)/day at the expense of water was –3.4 million sperm/ml (95% CI: –5.8, –1.0) and –7 pg/ml (95% CI: –11, –3), respectively. LIMITATIONS, REASONS FOR CAUTION Inferring causality is limited owing to the cross-sectional design. We adjusted for a number of potential confounders but cannot exclude that unmeasured lifestyle and behavior associated with soft drink intake is associated with testicular function in these young men. WIDER IMPLICATIONS OF THE FINDINGS In the largest study to date, intake of SSBs was associated with lower sperm concentration, total sperm count, and serum inhibin-B/FSH ratio, consistent with a direct suppressive effect of SSB intake on testicular function among otherwise healthy men, potentially affecting fertility. However, the observed association between higher SSB intake and lower semen quality does not necessarily imply a decrease in fertility. STUDY FUNDING/COMPETING INTEREST(S) Supported by research from the Danish Council for Strategic Research (2101-08-0058), Independent Research Fund Denmark (8020-00218B), European Union (212844), the Kirsten and Freddy Johansen’s Foundation (95-103-72087), the Research Fund of the Capital Region of Denmark (A6176), and the NIH (P30DK046200). The authors report no conflict of interest. TRIAL REGISTRATION NUMBER N/A.

BACKGROUND Human male infertility has a notable genetic component, including well-established diagnoses such as Klinefelter syndrome, Y-chromosome microdeletions and monogenic causes. Approximately 4% of all infertile men are now diagnosed with a genetic cause, but a majority (60–70%) remain without a clear diagnosis and are classified as unexplained. This is likely in large part due to a delay in the field adopting next-generation sequencing (NGS) technologies, and the absence of clear statements from field leaders as to what constitutes a validated cause of human male infertility (the current paper aims to address this). Fortunately, there has been a significant increase in the number of male infertility NGS studies. These have revealed a considerable number of novel gene–disease relationships (GDRs), which each require stringent assessment to validate the strength of genotype–phenotype associations. To definitively assess which of these GDRs are clinically relevant, the International Male Infertility Genomics Consortium (IMIGC) has identified the need for a systematic review and a comprehensive overview of known male infertility genes and an assessment of the evidence for reported GDRs. OBJECTIVE AND RATIONALE In 2019, the first standardised clinical validity assessment of monogenic causes of male infertility was published. Here, we provide a comprehensive update of the subsequent 1.5 years, employing the joint expertise of the IMIGC to systematically evaluate all available evidence (as of 1 July 2020) for monogenic causes of isolated or syndromic male infertility, endocrine disorders or reproductive system abnormalities affecting the male sex organs. In addition, we systematically assessed the evidence for all previously reported possible monogenic causes of male infertility, using a framework designed for a more appropriate clinical interpretation of disease genes. SEARCH METHODS We performed a literature search according to the PRISMA guidelines up until 1 July 2020 for publications in English, using search terms related to ‘male infertility’ in combination with the word ‘genetics’ in PubMed. Next, the quality and the extent of all evidence supporting selected genes were assessed using an established and standardised scoring method. We assessed the experimental quality, patient phenotype assessment and functional evidence based on gene expression, mutant in-vitro cell and in-vivo animal model phenotypes. A final score was used to determine the clinical validity of each GDR, across the following five categories: no evidence, limited, moderate, strong or definitive. Variants were also reclassified according to the American College of Medical Genetics and Genomics-Association for Molecular Pathology (ACMG-AMP) guidelines and were recorded in spreadsheets for each GDR, which are available at imigc.org. OUTCOMES The primary outcome of this review was an overview of all known GDRs for monogenic causes of human male infertility and their clinical validity. We identified a total of 120 genes that were moderately, strongly or definitively linked to 104 infertility phenotypes. WIDER IMPLICATIONS Our systematic review curates all currently available evidence to reveal the strength of GDRs in male infertility. The existing guidelines for genetic testing in male infertility cases are based on studies published 25 years ago, and an update is far overdue. The identification of 104 high-probability ‘human male infertility genes’ is a 33% increase from the number identified in 2019. The insights generated in the current review will provide the impetus for an update of existing guidelines, will inform novel evidence-based genetic testing strategies used in clinics, and will identify gaps in our knowledge of male infertility genetics. We discuss the relevant international guidelines regarding research related to gene discovery and provide specific recommendations to the field of male infertility. Based on our findings, the IMIGC consortium recommend several updates to the genetic testing standards currently employed in the field of human male infertility, most important being the adoption of exome sequencing, or at least sequencing of the genes validated in this study, and expanding the patient groups for which genetic testing is recommended.

Study question Is sperm Glutathione S-transferase Mu 3 (GSTM3) associated to sperm physiological status and male (in)fertility? Summary answer GSTM3 is expressed in human sperm, and is associated to sperm quality and fertility, which suggests it as a potential molecular biomarker for male (in)fertility. What is known already GSTM3 is an antioxidant enzyme expressed in sperm cells that is essential for an appropriate mitochondrial function, plasma membrane stability and oxidative regulation. Previous evidence suggested sperm GSTM3 to be useful as an infertility prognosis and diagnosis tool in mammalian species. In humans, expression of sperm GSTM3 has been found to be altered in mitochondrial-impaired sperm and in sperm from patients with unilateral varicocele. However, neither has this enzyme been related to semen alterations nor has it been reported to be associated to male infertility. Study design, size, duration A total of 34 semen samples from healthy, fertile men and men referred for infertility evaluation were collected, analysed and subsequently cryopreserved. Samples were classified as fertile having normal sperm parameters (NSP; n = 10); infertile with asthenoteratozoospermia (AT; n = 8) or oligoasthenoteratozoospermia (OAT; n = 8); or with idiopathic infertility (n = 8). Finally, frozen-thawed sperm samples were assessed for DNA fragmentation, and the presence, localization and relative content of GSTM3. Protein expression of GSTM3 in sperm was compared between groupss. Participants/materials, setting, methods All samples were initially evaluated by conventional sperm analysis (ejaculate volume and pH, sperm concentration, motility and morphology). Protein expression profile of GSTM3 was determined through immunoblotting and immunolocalization assays. Sperm GSTM3 was quantified using an enzyme-linked immunosorbent assay. Spearman's rank-order correlation coefficients between GSTM3 levels and sperm quality parameters were calculated. Statistical differences between groups were determined by Kruskal-Wallis followed by Mann-Whitney U tests. Main results and the role of chance GSTM3 was found to be present in human sperm cells, and to be localized along the tail. Sperm GSTM3 was positively and significantly correlated with sperm quality parameters (concentration [Rs=0.51], morphology [Rs=0.42], total [Rs=0.60] and progressive motility [Rs=0.58]; P < 0.05). Sperm GSTM3 levels were compared between sperm quality groups (NSP, AT and OAT). Sperm GSTM3 levels were found to be significantly higher (P < 0.05) in men with NSP than in those with AT or OAT (62.1, 28.1 and 20.6 ng GSTM3/mg total protein, respectively). Finally, levels of GSTM3 in sperm were compared between fertile and infertile men. Whereas fertile and idiopathic infertile men showed similar sperm GSTM3 levels (63.7 vs. 60.0 ng GSTM3/mg total protein, respectively, P > 0.05), infertile men with an altered seminogram had significantly lower (P < 0.05) levels (24.4 ng GSTM3/mg total protein). Limitations, reasons for caution The limited sample size of the present study warrants further research and clinical trials with larger sample sets before implementing this biomarker as a molecular diagnostic tool in fertility clinics. Wider implications of the findings The use of sperm GSTM3 as a novel biomarker in fertility clinics may be translated into cost-effective, non-invasive, time-saving and accurate diagnosis of men (in)fertility. Trial registration number N/A

The diagnosis of male infertility is primarily based on the macroscopic analysis of semen and microscopic analysis of sperm (mainly concentration and count, motility, and morphology) through the seminogram. Although six different editions of the “WHO Manual for the Laboratory Examination and Processing of Human Semen” have been published to date, the diagnostic potential of this approach has been called into question. Additionally, recent studies report a significant decline in sperm counts and fecundability in general. Therefore, the need for alternative diagnostic approaches that effectively predict fertility in men is urgently needed. In this talk we will cover what the limitations of the current semen analysis are, what novel tests are available and more important the new horizons, ideas and research in male fertility testing. We will focus in: 1) the usefulness of sperm functionality analyses (e.g., vitality, plasma membrane and acrosome integrity, plasma membrane lipid disorder, mitochondrial membrane potential, mitochondrial peroxide and superoxide levels, intracellular levels of ROS and Ca2+, and sperm chromatin condensation, among others), 2) the clinical implications of sperm DNA fragmentation and the newest recommendations for assisted reproduction clinics, 3) the newly described monogenic causes of human male infertility, and 4) how comorbidities are related with infertility.

Study question Does the structural maintenance of sperm chromatin have an implication in assisted reproduction outcomes? Summary answer Sperm deprotamination is related to fertilizing capacity. Also, the time needed for the reduction of protamine disulphide bridges is a parameter underlying early embryo development. What is known already How male factor affects embryo development has been a controversial topic for decades, with opposite results. A meta-analysis recently showed that whereas sperm genotoxic damage has an impact on IVF success, its repercussion on ICSI remains unclear. Revealing the factors that may predict embryo development is much warranted to understand the sperm contribution in the ICSI era. Sperm DNA is preferentially protamine-condensed and necessarily needs to be switched to histone-condensed chromatin upon fertilization. This study aims to determine the degree of protamination and the disulphide bond integrity to provide new insights into the influence of chromatin resilience on embryo development. Study design, size, duration The study setting included a public university and a private sperm bank. The design was a blinded retrospective study, including samples from 51 sperm donors that were used for a total of 55 cycles conducted with donated oocytes in the fertility clinic. Sperm quality parameters, including those focused on chromatin structure, were evaluated after 0 min and 240 min post-thawing and compared to embryo outcomes. Participants/materials, setting, methods Sperm samples from donors selected by the germplasm bank were collected and used to fertilize oocytes in double-donation cycles (oocyte donation) with standard intracytoplasmic sperm injection. Cryopreserved samples were used to assess sperm viability (SYBR14/PI), incidence of thiol reduction (Dibromobimane, DBB) and chromatin deprotamination (Chromomycin A3, CMA3) through flow cytometry, at 0 min and 240 min after thawing. Positive controls after incubation with Triton-X100, DTT and DTT+NaCl were used to establish the flow cytometry thresholds. Main results and the role of chance In fresh semen samples, sperm concentration was 34.12±18.13 sperm/mL and progressive motility was 37.49±13.22% (mean±standard deviation). At 0 min and 240 min post-thawing, sperm viability was 24.80±12.12% and 18.65±10.37%, respectively, and the death rate was -1.54±1.24%/hour. Percentages of sperm with reduced thiols were 17.37±11.26% and 11.53±9.16% after 0 min and 240 min post-thawing, the reduction rate being -1.46±2.31%. Percentages of sperm with chromatin deprotamination were 21.12±9.03% and 35.34±14.10% after 0 min and 240 min post-thawing, and the deprotamination rate was 14.22±11.90%/hour. ICSI was conducted with an average of 11.86±7.89 oocytes per sperm sample, of which 8.78±5.95 were fertilized; the mean fertilization rate was 75.77±17.61%. For each sperm sample, 5.98±4.93 blastocysts at day 5 were obtained, leading to an average blastocyst rate of 65.17±22.22%. Interestingly, the deprotamination status at 0 min post-thaw was correlated to fertilization rates (Rs= -0.388; P = 0.005) and the decreasing rate of sperm with reduced thiols was correlated to blastocyst rate (Rs= -0.351; P = 0.012). Reduced thiols and deprotamination were also correlated with each other (Rs= -0.320; P = 0.022). Limitations, reasons for caution The main limitation of the present study was the sample size. More donors are thus needed in order for the promising results found in this study to be confirmed. Wider implications of the findings The research conducted herein has evidenced that the resilience of the sperm chromatin structure is related to embryo development outcomes from ICSI cycles conducted with oocyte donors. Trial registration number N/A

Study question Is men’s adherence to the Mediterranean diet (MD) associated with sperm parameters and couples’ outcomes of assisted reproductive technologies (ART)? Summary answer Higher men’s adherence to the MD was associated with an increased probability of clinical pregnancy and live birth among couples undergoing ART. What is known already There is growing literature supporting the hypothesis that some nutrients, foods, and dietary patterns may be related to sperm quality and fertility. However, no previous studies analyzed the relationship of men’s adherence to the Mediterranean diet with sperm parameters and ART outcomes in the same cohort. Study design, size, duration The Environmental and Reproductive Health (EARTH) Study is a prospective preconception cohort of couples seeking fertility treatment at the Massachusetts General Hospital Fertility Center (Boston, USA). This analysis includes 314 men and their female partner, who underwent 791 ART cycles (2007–2020). Diet intake was measured by a validated semi-quantitative food frequency questionnaire completed prior to ART. Men’s adherence to the MD was estimated using the Trichopoulou score. Participants/materials, setting, methods Primary outcomes included: conventional sperm parameters (volume, sperm count, concentration, motility, and morphology), and ART outcomes (implantation, clinical pregnancy, and live birth). We estimated the marginal means and 95% confidence interval (95%CI) for semen parameters and the probability of ART (95%CI) by employing generalized linear mixed models while adjusting for several potential confounders. Sensitivity analyses by changing the cut-off points of adherence to the MD were tested. Main results and the role of chance At baseline, men had a median (IQR) age of 35.6 (32.6, 38.8) years and a BMI of 26.7 (24.0, 29.4) kg/m2. Female partner age median (IQR) was 35.0 years (32.0, 38.0) and BMI 23.3 (21.3, 26.6) kg/m2. Couples were mostly white and had never smoked. Men’s adherence to the MD was not associated with seminal parameters in the multivariable-adjusted models but it was related to a higher probability of clinical pregnancy and live birth. The predicted marginal proportions and confidence intervals among men in the lowest compared with the highest quartile of adherence to the MD were 0.25 (0.14, 0.40), 0.55 (0.41, 0.68) for clinical pregnancy (P-trend=0.005), and 0.19 (0.10, 0.32), 0.42 (0.30, 0.55) for live birth (P-trend=0.014). Male partner MD dietary pattern scores were unrelated to the probability of implantation. Sensitivity analyses using tertiles and quintiles of men’s adherence to the MD showed similar associations. Limitations, reasons for caution Although we have adjusted our models by several potential confounding factors, residual confounding cannot be ruled out, and therefore our results should be interpreted with caution. The findings may not be generalizable to other populations or couples attempting conception without ART. Wider implications of the findings: According to our knowledge, this is the first study suggesting that adherence to MD could be suitable dietary guidance for men whose partners are undergoing infertility treatment. Trial registration number Not applicable

Study question Is men’s diet associated with assisted reproductive technology (ART) outcomes? Summary answer An empirical dietary score representing the overall effect of men’s diet on semen quality was unrelated to ART outcomes. What is known already Multiple studies have related various aspects of men’s diet to semen quality. Generally, healthier foods, such as fish, vegetables, and fruits, have been related to better semen quality, whereas unhealthy foods, like processed and red meats, have had the opposite relationship. Nevertheless, while bulk semen parameters are important biomarkers of male fertility and a diagnostic cornerstone for male factor infertility, they are imperfect predictors of a couple’s fertility. Study design, size, duration Couples presenting to the Massachusetts General Hospital Fertility Center between April 2007 and April 2018 were invited to participate in the Environment and Reproductive Health (EARTH) study, a prospective cohort study. Men’s diet was assessed with a previously validated food frequency questionnaire. A dietary score reflecting the overall relation of men’s food intake with semen quality parameters was empirically derived using reduced rank regression (RRR). The resulting dietary score was related to ART outcomes. Participants/materials, setting, methods We used information from 349 men (908 semen samples) to derive the empirical diet pattern and data from 231 couples (407 ART cycles). The primary outcome was the probability of live birth per treatment cycle; secondary outcomes were semen quality, and fertilization, implantation, and clinical pregnancy rates. We evaluated the association between the dietary score and these outcomes using logistic generalized linear mixed models to account for repeated cycles while adjusting for confounders. Main results and the role of chance Men had a median baseline age and BMI of 36.8 years and 26.9 kg/m2, respectively. The empirical diet pattern was significantly associated with all semen parameters. One standard deviation increase in the empirical diet pattern was associated with lower volume (–0.10 standard units [95% CI: –0.17 to –0.04]) and to higher sperm total sperm count (0.13 standard units [0.06 to 0.20]), concentration (0.17 standard units [0.10 to 0.24]), total motility (0.14 standard units [0.07 to 0.20]), progressive motility (0.08 standard units [0.01 to 0.15]), and normal morphology (0.18 standard units [0.11 to 0.25]). Couples with men in the lowest quartile of the empirical score were more likely to have a diagnosis of male infertility than couples with men in the highest quartile (49% vs 24%). Despite the association with semen parameters, the empirical diet score was not related to any clinical outcome of infertility treatment with ART. The adjusted probabilities of implantation, clinical pregnancy and live birth in the lowest and highest quartile of the empirical score were 0.62 (0.50–0.73) and 0.55 (0.45–0.66), 0.57 (0.46–0.69) and 0.50 (0.40–0.61), and 0.49 (0.37–0.62) and 0.36 (0.25–0.48), respectively. Analyses excluding couples with a diagnosis of male factor infertility yielded similar results. Limitations, reasons for caution We evaluated the relationship only among couples presenting to a fertility center and therefore it is unclear whether findings can be generalized to couples trying to conceive without ART. Wider implications of the findings: Given ART is a robust intervention including stringent sperm selection procedures, any effect that empirical diet may have on a couple’s chances of conceiving through assisted reproduction is unlikely to reflect the effect of these factors on bulk semen quality parameters. Trial registration number The project was funded by ES009718, ES022955, ES026648, and ES000002 from the National Institute of Environmental Health Sciences, and P30DK46200 from the National Institute of Diabetes and Digestive and Kidney Diseases

Background Infertility is a global public health issue, affecting 15% of all couples of reproductive age. Male factors, including decreased semen quality, are responsible for ~25% of these cases. The dietary pattern, the components of the diet and nutrients have been studied as possible determinants of sperm function and/or fertility. Objective and rationale Previous systematic reviews have been made of the few heterogeneous low-quality randomized clinical trials (RCTs) conducted in small samples of participants and investigating the effect of specific nutrients and nutritional supplements on male infertility. However, as yet there has been no systematic review of observational studies. Search methods A comprehensive systematic review was made of the published literature, from the earliest available online indexing year to November 2016, in accordance with the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses. We have included cross-sectional, case-control and prospective and retrospective studies in which fertile/infertile men were well defined (men with sperm disorders, sperm DNA damage, varicocele or idiopathic infertility). The primary outcomes were semen quality or fecundability. With the data extracted, we evaluated and scored the quality of the studies selected. We excluded RCTs, animal studies, review articles and low-quality studies. Outcomes A total of 1944 articles were identified, of which 35 were selected for qualitative analysis. Generally, the results indicated that healthy diets rich in some nutrients such as omega-3 fatty acids, some antioxidants (vitamin E, vitamin C, β-carotene, selenium, zinc, cryptoxanthin and lycopene), other vitamins (vitamin D and folate) and low in saturated fatty acids and trans-fatty acids were inversely associated with low semen quality parameters. Fish, shellfish and seafood, poultry, cereals, vegetables and fruits, low-fat dairy and skimmed milk were positively associated with several sperm quality parameters. However, diets rich in processed meat, soy foods, potatoes, full-fat dairy and total dairy products, cheese, coffee, alcohol, sugar-sweetened beverages and sweets have been detrimentally associated with the quality of semen in some studies. As far as fecundability is concerned, a high intake of alcohol, caffeine and red meat and processed meat by males has a negative influence on the chance of pregnancy or fertilization rates in their partners. Wider implications Male adherence to a healthy diet could improve semen quality and fecundability rates. Since observational studies may prove associations but not causation, the associations summarized in the present review need to be confirmed with large prospective cohort studies and especially with well-designed RCTs.

Study question What is the most reliable normalization strategy for sperm microRNA (miRNA) quantitative Reverse Transcription Polymerase Chain Reactions (qRT-PCR) using singleplex assays? Summary answer The use of the average expression of hsa-miR-100-5p and hsa-miR-30a-5p as sperm miRNA qRT-PCR data normalizer is suggested as an optimal strategy. What is known already Mean-centering methods are the most reliable normalization strategies for miRNA high-throughput expression analyses. Nevertheless, specific trustworthy reference controls must be established in singleplex sperm miRNA qRT-PCRs. Study design, size duration Cycle threshold (Ct) values from previously published sperm miRNA expression profiles were normalized using four approaches: (i) Mean-Centering Restricted (MCR) method (taken as the reference strategy); (ii) expression of the small nuclear RNA RNU6B; (iii) expression of four miRNAs selected by the Concordance Correlation Restricted (CCR) algorithm: hsa-miR-100-5p, hsa-miR-146b-5p, hsa-miR-92a-3p and hsa-miR-30a-5p; (iv) the combination of two of these miRNAs that achieved the highest proximity to MCR. Participants/materials, setting, methods Expression profile data from 736 sperm miRNAs were taken from previously published studies performed in fertile donors (n = 10) and infertile patients (n = 38). For each tested normalizer molecule, expression ubiquity and uniformity across the different samples and populations were assessed as indispensable requirements for being considered as valid candidates. The reliability of the different normalizing strategies was compared to MCR based on the set of differentially expressed miRNAs (DE-miRNAs) detected between populations, the corresponding predicted targets and the associated enriched biological processes. Main results and the role of chance All tested normalizers were found to be ubiquitous and non-differentially expressed between populations. RNU6B was the least uniformly expressed candidate across samples. Data normalization through RNU6B led to dramatically misguided results when compared to MCR outputs, with a null prediction of target genes and enriched biological processes. Hsa-miR-146b-5p and hsa-miR-92a-3p were more uniformly expressed than RNU6B, but their results still showed scant proximity to the reference method. The highest resemblance to MCR was achieved by hsa-miR-100-5p and hsa-miR-30a-5p. Normalization against the combination of both miRNAs reached the best proximity rank regarding the detected DE-miRNAs (Area Under the Curve = 0.8). This combination also exhibited the best performance in terms of the target genes predicted (72.3% of True Positives) and their corresponding enriched biological processes (70.4% of True Positives). Large scale data Not applicable. Limitations, reasons for caution This study is focused on sperm miRNA qRT-PCR analysis. The use of the selected normalizers in other cell types or tissues would still require confirmation. Wider implications of the findings The search for new fertility biomarkers based on sperm miRNA expression using high-throughput assays is one of the upcoming challenges in the field of reproductive genetics. In this context, validation of the results using singleplex assays would be mandatory. The normalizer strategy suggested in this study would provide a universal option in this area, allowing for normalization of the validated data without causing meaningful variations of the results. Instead, qRT-PCR data normalization by RNU6B should be discarded in sperm-miRNA expression studies. Study funding/competing interests This work was supported by the 2014/SGR00524 project (Agencia de Gestio d'Ajuts Universitaris i de Recerca, Generalitat de Catalunya, Spain) and UAB CF-180034 grant (Universitat Autonoma de Barcelona). Celia Corral-Vazquez is a recipient of a Personal Investigador en Formacio grant UAB/PIF2015 (Universitat Autonoma de Barcelona). The authors report no conflict of interest.

The PREDIMED (in Spanish: PREvención con DIeta MEDiterránea) primary prevention trial1–3 reported in 2013 that long-term adherence to an energy-unrestricted Mediterranean diet (MedDiet), supplemented with either extra-virgin olive oil (EVOO) or nuts, reduced cardiovascular disease (CVD). PREDIMED showed a 30% relative reduction in the composite cardiovascular primary endpoint (stroke, myocardial infarction or cardiovascular death).2,,3 However, the PREDIMED trial tested only the composition of the diet, but did not test other lifestyle interventions (i.e. energy reduction, increased physical activity (PA) and behavioural modification) frequently applied in the context of the current unprecedented obesity pandemic.4,,5 With the exception of the null results of the Look-AHEAD trial,6 and the successful results of the EXERDIET-HTA study that found improvements in blood pressure, cardio-respiratory fitness and body composition (though they encouraged adherence to an hypocaloric DASH diet, instead of a Mediterranean diet),7 lifestyle interventions including such components have never been tested in long-term randomized trials using hard cardiovascular events as endpoints.