Food Intake and Epigenetic Alteration in the Spermatozoa of Singletons and Twins (FEASST)

The purpose of this study is to elucidate the role of acute dietary intake in male populations, and its impact on spermatozoa quality, integrity, content and epigenetic programming.

The FEASST study has been designed to determine the impact of diet on the content, quality, and genetic programming of sperm. They are specifically interested in determining if a man's diet plays a role in influencing the health of his potential, future offspring through modifications of the sperm. Previous research has highlighted that certain dietary patterns in fathers-to-be may lead to variable health outcome for their children. However, the specific effect of a man's diet has yet to be fully understood. This study will help understand the impact of a man's diet on semen quality and potential downstream implications for child health, to aid in the development of dietary guidelines for future fathers-to-be. For this study, male participants will be provided with two prescribed diets for three-week increments punctuated by a three month break. Health information and biological samples such as blood, semen, and saliva will be collected from study participants throughout the five month period. The diets will consist of a 'Processed diet', mimicking a traditional Western dietary intake pattern and an 'Unprocessed diet', based upon current dietary guidelines at quantities either adequate for the participants energetic needs or 500 calories in excess per day. Patient information and samples will be collected before and after each dietary intervention. Aspects of patient health examined at various time points include weight, body fat percentage, sperm quality, epigenetic sequencing, serum levels of metabolic parameters, and survey information surrounding health history, typical diet, physical activity patterns, and treatment adherence.

Participants within the isocaloric arm will be provided with food provisions in accordance with their nutrition needs. Participants within this arm will be further randomised to the order of first and second dietary interventions for the crossover design; unprocessed diet or processed diet.

Participants within the excess calorie arm will be provided with food provisions in accordance with their nutrition needs plus an additional 500 kilocalories per day. Participants within this arm will be further randomised to the order of first and second dietary interventions for the crossover design; unprocessed diet or processed diet.

Intervention consists of an unprocessed diet meeting the Nordic and Australian Dietary Guidelines for food groups and micronutrients, wherein participants will be supplied with food providing kilocalories (kcal) in accordance with their nutritional needs as calculated based on theSchofield equation based on their weight, activity multiplier, and age at baseline. Almost all food and beverage products provided during this treatment to fall into the category of unprocessed/minimally processed foods and processed culinary ingredients in accordance with the NOVA classification system.

Intervention consists of a processed diet based on the dietary intake pattern resembling a processed western dietary intake pattern representative of the typical intake pattern of American men aged 19- 50, wherein participants will be supplied with food providing kcals in accordance with their nutritional needs as calculated based on the Schofield equation based on their height, weight, activity multiplier, and age at baseline. Almost all food and beverage products provided during this treatment to fall into the category of processed foods and ultra-processed foods in accordance with the NOVA classification system.

Intervention consists of an unprocessed diet meeting the Nordic and Australian Dietary Guidelines for food groups and micronutrients, wherein participants will be supplied with food providing kilocalories (kcal) in accordance with their nutritional needs as calculated based on theSchofield equation based on their weight, activity multiplier, and age at baseline, plus an excess of 500 kcal per day. Almost all food and beverage products provided during this treatment to fall into the category of unprocessed/minimally processed foods and processed culinary ingredients in accordance with the NOVA classification system.

Intervention consists of a processed diet based on the dietary intake pattern resembling a processed western dietary intake pattern representative of the typical intake pattern of American men aged 19- 50, wherein participants will be supplied with food providing kcals in accordance with their nutritional needs as calculated based on the Schofield equation based on their height, weight, activity multiplier, and age at baseline, plus an excess of 500 kcal per day. Almost all food and beverage products provided during this treatment to fall into the category of processed foods and ultra-processed foods in accordance with the NOVA classification system.

Differential methylation of CpG sites within mature spermatozoa determined from extracted genomic DNA which has undergone library preperation with Ovation® RRBS Methyl-Seq (NuGEN Technologies) and sequenced on a NextSeq Illumina platform.

Measured in million sperm per mL using LensHooke X1 Pro Semen Quality Analyser and Microscopy-based methodology

Measured in precent of sperm classified as progressively motile and non-progressively motile using LensHooke X1 Pro Semen Quality Analyser and Microscopy-based methodology

Differential expression of small RNA species within mature spermatozoa performed by generation of RNA sequencing libraries using NEXTFLEX® Small RNA-Seq Kit for Illumina (PerkinElmer), according to the manufacturer's instructions, and sequenced on a NextSeq Illumina platform.

Measured in whole blood using a handheld glucometer and test strip (Contour next Blood Glucose Meter and Blood Sugar Test strip)

Systolic and Diastolic blood pressure measured in mmHg using an automatic blood pressure cuff (Omron M6 Comfort Automatic Upper Arm Blood Pressure Monitor, 22-42cm)

Peripheral blood mononuclear cell (PBMC) DNA methylation via Reduced Representation Bisulfite Sequencing

Differential methylation of CpG sites within PBMCs determined from extracted genomic DNA which has undergone library preperation with Ovation® RRBS Methyl-Seq (NuGEN Technologies) and sequenced on a NextSeq Illumina platform.

Inclusion Criteria: Male 20 to 35 years old BMI 18.5 to 30 Located within the Copenhagen region Fluent in English and/or Danish language Sperm concentration of >15 mil/mL according to the WHO's criteria for semen quality Semen volume of >1.5mL mL according to the WHO's criteria for semen quality Clinician approval for participation in study Obtained informed consent Exclusion Criteria: Self-reported history of serious or chronic illness Self-reported history of Obesity History of any food restrictions History of allergies to any food products History of disordered eating Record of current use of drugs, alcohol (>14 units per week and/or chronic binge drinking), and/or tobacco/nicotine products within the past month Current use of prescription medication Engage in > 200 minutes of vigorous aerobic exercise per week Currently actively trying to conceive a child Diagnosis of infertility or disease of the reproductive system Evidence of dysregulated metabolism, characterized by the occurrence of any one of the following: Waist circumference >102 cm Blood pressure > 130/85 mm Hg