Anti-Inflammatory Diet and Physical Activity for Reproductive Health in Adolescent Girls (4fertil)

Participants receive a 16-week anti-inflammatory dietary intervention based on Mediterranean dietary principles. The programme includes dietary education, practical workshops, dietary recommendations, and access to educational materials through a dedicated digital platform. No additional physical activity programme is provided as part of this study arm.

A 16-week dietary education and behavior-change programme based on Mediterranean dietary principles. The intervention includes four theoretical and practical workshops, dietary recommendations, and educational materials provided through a dedicated digital platform. Participants are encouraged to increase their consumption of vegetables, fruit, whole grains, legumes, fish, nuts, olive oil, herbs, and spices and to limit red meat and sweets. The intervention does not include energy restriction.

Participants receive a 16-week anti-inflammatory dietary intervention based on Mediterranean dietary principles and additionally participate in a physical activity programme consisting of two sessions per week. The physical activity programme is delivered by the study team in the school setting and adapted to the participants' age and abilities. Educational materials are also available through a dedicated digital platform.

A 16-week dietary education and behavior-change programme based on Mediterranean dietary principles. The intervention includes four theoretical and practical workshops, dietary recommendations, and educational materials provided through a dedicated digital platform. Participants are encouraged to increase their consumption of vegetables, fruit, whole grains, legumes, fish, nuts, olive oil, herbs, and spices and to limit red meat and sweets. The intervention does not include energy restriction.

A 16-week physical activity programme consisting of two sessions per week. The sessions are delivered by the study team in the school setting and are adapted to the participants' age and abilities. Educational materials related to physical activity are also available through a dedicated digital platform.

Diet quality will be assessed using the 16-item Mediterranean Diet Quality Index for Children and Adolescents (KIDMED). Positively scored items receive +1 point and negatively scored items receive -1 point, giving a total score ranging from -4 to 12. Higher scores indicate better adherence to a Mediterranean dietary pattern. Change from baseline to post-intervention will be compared between the study arms.

Menstrual pain intensity will be assessed using a Visual Analogue Scale (VAS) ranging from 0 to 10, where 0 indicates no pain and 10 indicates the worst imaginable pain. Higher scores indicate greater menstrual pain intensity. Change from baseline to post-intervention will be compared between the study arms.

Premenstrual symptoms will be assessed using the Premenstrual Symptoms Screening Tool (PSST). The instrument evaluates 14 premenstrual symptoms and five areas of functional impairment. Each item is rated from 0 (not at all) to 3 (severe), giving a total score ranging from 0 to 57. Higher scores indicate greater severity of premenstrual symptoms and related functional impairment. Change from baseline to post-intervention will be compared between the study arms.

Menstrual cycle regularity will be assessed during a medical interview based on self-reported menstrual cycle characteristics, including usual cycle length and variability in cycle length. The assessment will be performed before and after the intervention. Changes in menstrual cycle regularity from baseline to post-intervention will be compared between the study arms.

Body weight will be measured in kilograms and height in metres. Body mass index (BMI) will be calculated as body weight divided by height squared and expressed in kilograms per square metre (kg/m²). Change in BMI from baseline to post-intervention will be compared between the study arms.

Body fat percentage will be assessed using bioelectrical impedance analysis and expressed as a percentage of total body weight. Change in body fat percentage from baseline to post-intervention will be compared between the study arms.

Fat-free mass will be assessed using bioelectrical impedance analysis and expressed in kilograms. Change in fat-free mass from baseline to post-intervention will be compared between the study arms.

Waist circumference will be measured according to a standardized anthropometric procedure and expressed in centimetres. Change in waist circumference from baseline to post-intervention will be compared between the study arms.

Resting systolic and diastolic blood pressure will be measured using a standardized procedure and expressed in millimetres of mercury (mmHg). Changes in systolic and diastolic blood pressure from baseline to post-intervention will be compared between the study arms.

Acne severity will be evaluated using the Investigator's Global Assessment (IGA). The scale provides an overall clinical assessment of acne severity, with higher grades indicating greater severity. Change in the IGA grade from baseline to post-intervention will be compared between the study arms.

Hirsutism severity will be assessed using the modified Ferriman-Gallwey score. Terminal hair growth in nine androgen-sensitive body areas is rated from 0 to 4, giving a total score ranging from 0 to 36. Higher scores indicate greater hirsutism severity. Change from baseline to post-intervention will be compared between the study arms.

Physical activity will be assessed using the International Physical Activity Questionnaire (IPAQ). The questionnaire evaluates walking and moderate- and vigorous-intensity physical activity performed during the previous seven days. Total physical activity will be expressed as metabolic equivalent minutes per week (MET-minutes/week), calculated according to the IPAQ scoring protocol. Higher values indicate a higher level of physical activity. Change from baseline to post-intervention will be compared between the study arms.

Salivary estrogen concentration will be assessed before and after the intervention. The outcome is the change in salivary estrogen concentration from baseline to immediately after the 16-week intervention.

Salivary testosterone concentration will be assessed before and after the intervention. The outcome is the change in salivary testosterone concentration from baseline to immediately after the 16-week intervention.

Salivary progesterone concentration will be assessed before and after the intervention. The outcome is the change in salivary progesterone concentration from baseline to immediately after the 16-week intervention.

Salivary prolactin concentration will be assessed before and after the intervention. The outcome is the change in salivary prolactin concentration from baseline to immediately after the 16-week intervention.

Salivary cortisol concentration will be assessed before and after the intervention. The outcome is the change in salivary cortisol concentration from baseline to immediately after the 16-week intervention.

Salivary dehydroepiandrosterone concentration will be assessed before and after the intervention. The outcome is the change in salivary dehydroepiandrosterone concentration from baseline to immediately after the 16-week intervention.

Salivary interleukin-17A concentration will be assessed before and after the intervention. The outcome is the change in salivary interleukin-17A concentration from baseline to immediately after the 16-week intervention.

Salivary interleukin-1 beta concentration will be assessed before and after the intervention. The outcome is the change in salivary interleukin-1 beta concentration from baseline to immediately after the 16-week intervention.

Salivary matrix metalloproteinase-8 concentration will be assessed before and after the intervention. The outcome is the change in salivary matrix metalloproteinase-8 concentration from baseline to immediately after the 16-week intervention.

Salivary tumour necrosis factor alpha concentration will be assessed before and after the intervention. The outcome is the change in salivary tumour necrosis factor alpha concentration from baseline to immediately after the 16-week intervention.

Salivary malondialdehyde concentration will be assessed before and after the intervention. The outcome is the change in salivary malondialdehyde concentration from baseline to immediately after the 16-week intervention.

Salivary total antioxidant capacity will be assessed before and after the intervention. The outcome is the change in salivary total antioxidant capacity from baseline to immediately after the 16-week intervention.

Salivary 8-hydroxy-2'-deoxyguanosine concentration will be assessed before and after the intervention. The outcome is the change in salivary 8-hydroxy-2'-deoxyguanosine concentration from baseline to immediately after the 16-week intervention.

Salivary secretory immunoglobulin A concentration will be assessed before and after the intervention. The outcome is the change in salivary secretory immunoglobulin A concentration from baseline to immediately after the 16-week intervention.

Oral microbiome composition and diversity will be assessed using 16S rRNA gene sequencing. Changes from baseline to post-intervention will be compared between the study arms.

Dental status will be assessed using the Decayed, Missing and Filled Teeth Index before and after the intervention. The outcome is the change in the index from baseline to immediately after the 16-week intervention.

Dental plaque will be assessed using the Plaque Index before and after the intervention. The outcome is the change in the Plaque Index from baseline to immediately after the 16-week intervention.

Approximal dental plaque will be assessed using the Approximal Plaque Index before and after the intervention. The outcome is the change in the Approximal Plaque Index from baseline baseline to immediately after the 16-week intervention.

Gingival health will be assessed using the Gingival Index before and after the intervention. The outcome is the change in the Gingival Index from baseline to immediately after the 16-week intervention.

Gingival bleeding will be assessed using bleeding on probing before and after the intervention. The outcome is the change in bleeding on probing from baseline to immediately after the 16-week intervention.