Ovarian Hyperandrogenism in Adolescent Girls and Concomitant Metabolic Changes, Oxidative Stress, Inflammation, Oral Health and Their Relation to Diet
Phase: NA
Conditions: Polycystic Ovary Syndrome
Sponsor: Poznan University of Physical Education

Study Protocol: Ovarian Hyperandrogenism in Adolescent Girls and Concomitant Metabolic Changes, Oxidative 
Stress, Inflammation, Oral Health and Their Relation to Diet 
 
 
Poznan University of Physical Education 
Dietetic Department 
61-871 Poznan, Poland 
Principal Investigator: Małgorzata Mizgier 
Phone: (+48) 61 835 50 61 
 
Poznan University of Medical Sciences 
Department of Peadiatric Dentistry 
60-812 Poznan, Poland 
Principal Invesitgators: Justyna Opydo-Szymaczek, 
Phone: (+48) 61 854 72 72 
Department of Perinatology and Gynecology 
Division of Developmental Gynecology and Sexology 
Principal Investigator: Grażyna Jarząbek Bielecka 
STUDY PROTOCOL 
Ovarian Hyperandrogenism in Adolescent Girls and Concomitant Metabolic Changes, 
Oxidative Stress, Inflammation, Oral Health and Their Relation to Diet 
 
1. Introduction 
 
Hyperandrogenism in women is a state of increased androgen production, manifested by irregular 
menstrual cycles, a more frequent incidence of infertility, metabolic syndrome, oxidative stress, and 
inflammation, as well as acne and hirsutism. In addition to ovulation disorders and the multifollicular 
ovaries structure in an ultrasound examination, hyperandrogenism is a typical clinical feature of the 
Polycystic Ovary Syndrome (PCOS), which is diagnosed in approximately 8.6% of adolescent girls. 
Excessive body weight makes it difficult to diagnose at an early stage of the disease and intensifies 
metabolic and hormonal disorders, as well as those related to oxidative stress and inflammation of the 
body. Some research studies suggest that PCOS may negatively affect oral health of women, 
increasing the risk of periodontal problems. 
The main purpose of this research was to check whether serum androgen levels and concomitant 
metabolic changes, oxidative stress, inflammation and oral health in adolescent girls with 
hyperandrogenism may be related to diet. 
The investigators aimed to identify the factors that increase the risk of being overweight and of obesity 
among adolescents with clinical features of PCOS, related to diet and DEA(Disordered Eating 
Attitudes). 
The objective was also to investigate the relationships between markers of oxidation and markers of 
Systemic Inflammation and macronutrients intake, such as carbohydrates and fiber, protein (animal, 
plant), fats and cholesterol in normal and overweight/obese girls with clinical features of PCOS. 
Additionally, the investigators would like to evaluate the relation between oral health of girls with 
PCOS (including condition of periodontium and dental caries) and their dietary habits, hormonal, 
metabolic and oxidative and inflammatory status. 
 
2. Characteristics of tested population 
The research will be carried out with the participation of 200 Caucasian adolescent girls, patients at the 
Gynecology and Obstetrics Hospital of Poznan University of Medical Sciences and Department of 
Perinatology and Gynecology, Division of Developmental Gynecology and Sexology, Poznan 
University of Medical Sciences, aged 13–18 years 
 

Study Protocol: Ovarian Hyperandrogenism in Adolescent Girls and Concomitant Metabolic Changes, Oxidative 
Stress, Inflammation, Oral Health and Their Relation to Diet 
 
 
Inclusion Criteria: 
• 
Gender: Female 
• 
Minimum Age: 13 Years 
• 
Maximum Age: 18Years 
• 
Written consent for inclusion (girls and their parents)  
• 
clinical and/or biochemical hyperandrogenism (hirsutism with moderate to severe acne, and/or 
elevation of serum total testosterone or free testosterone, 
• 
oligoovulation (based on oligomenorrhea defined as bleeding episodes occurring less than 8 
times per year or secondary amenorrhea), 
• 
polycystic ovarian picture in an ultrasound examination (at least 12 follicles in each ovary 
each measuring 2-9 mm in diameter and/or ovarian volume >10 mL). 
Exclusion Criteria: 
• 
any systemic disease, thyroid dysfunction, diabetes, congenital adrenal hyperplasia, Cushing 
syndrome, hyperprolactinemia suggestive of pituitary adenoma and androgen-secreting tumors 
• 
medications of continuous use, 
• 
the use of hormonal therapy or antibiotics in the past three months, 
• 
vitamin or supplements use, 
• 
alcohol consumption 
• 
smoking 
 
3. Methods 
 
3.1. Evaluation of anthropometric indices and body composition 
 
3.1.1. Anthropometric Assessment  
 
Body mass index BMI:  derived from measured body height (m) and body weight (kg) as body weight 
/ height². Weight will be measured to the nearest 0.1 kg using digital medical scales. Height will be 
measured with a stadiometer attached to the scales.  
Diagnosis and classification of overweight and obesity will base on BMI, according to World Health 
Organisation (WHO) for children aged 5–19 years overweight and obesity correspond to BMI-for-age 
greater than 1 standard deviation and 2 standard deviations above the WHO growth reference median, 
respectively. 
Waist circumference (WC) will be measured to the nearest 0.1 mm using Gulick anthropometric tape 
between the lower border of rib margin and the upper border of iliac crest (WC-mid). 
 
3.1.2. Body Composition Assessment 
All measurements will  be conducted after a 12 h fast. Body composition will be assessed with a Tanita 
MC 780 body composition analyzer, which uses bioelectrical impedance analysis (BIA method). In this 
model, the accuracy of measurement for the individual components, including adipose tissue, is 100 g. 
The measurements of fat mass (FM) will be expressed as a percentage (%) and kilograms (kg).  
 
3.2. Biochemical parameters 
 
Blood samplings performed in the early follicular phase (days 3–5). 
Hormonal and biochemical parameters, including follicle-stimulating hormone (FSH), luteinizing 
hormone (LH), total testosterone, androstendione, estradiol, dehydroepiandrosterone (DHEA-S), Sex 
Hormone Binding Globulin (SHBG), fasting glucose and fasting insulin, total cholesterol (TC), high-
density lipoprotein cholesterol (HDL-C), and triglycerides (TG), 17 OH-Progesterone, Prolactin, 
Cortisol, Thyroid-stimulating hormone (TSH),  will be measured in the morning after overnight fasting. 

Study Protocol: Ovarian Hyperandrogenism in Adolescent Girls and Concomitant Metabolic Changes, Oxidative 
Stress, Inflammation, Oral Health and Their Relation to Diet 
 
 
Biochemical analyses will be performed in the central hospital laboratory of the Gynecology and 
Obstetrics Hospital of Poznan University of Medical Sciences. 
Serum concentrations of the inflammation markers: Interleukin 1 (IL-1), Interleukin 6 (IL-1), Tumor 
Necrosis Factor (TNF-α), C-reactive protein (CRP) and the markers of oxidation: malondiaeldehyde, 
total antioxidant capacity will be measured with a commercial ELISA assay kits in the Department of 
Clinical Biochemistry and Laboratory Medicine, Poznan University of Medical Sciences .  
 
3.3. Saliva assays 
Samples of unstimulated saliva will be taken with a use of a saliva cotton roll commercial collection 
device at the dental office (University Center of Stomatology and Specialist Medicine). Biochemical 
analysis of the salivary TNF-alfa, IL-6, IL-1β, and testosterone will be carried out with the use of 
commercial ELISA assay kits.  
 
3.4. Dental examinations 
The state of oral hygiene will be evaluated according to the Silness-Löe plaque index (PI) on the six 
index teeth: 16, 12, 24, 36, 32, 44. The gingival health will be assessed with the use of the Gingival 
Index (GI) by Löe and Silness on the same index teeth. The probing depth (PD) measurements will be 
done using a Williams manual periodontal probe. Bleeding upon probing score (BOP%) will be assessed 
as the proportion of bleeding sites when stimulated by a periodontal probe (the measurements recorded 
on all teeth present. Dental caries experience will be presented as a number of teeth with decay (D), 
missing due to decay (M), and filled due to decay (F) (DMT index). 
 
3.5. Assessment of subgingival microflora 
Samples of dental plaque will be collected from each patient by inserting paper points into buccal sites 
of gingival sulci. A real-time PCR method for the identification of 9 subgingival microorganisms: 
Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Treponema 
denticola, Fusobacterium nucleatum, Prevotella intermedia, Peptostreptococcus micros, Eubacterium 
nodatum, and Capnocytophaga gingivalis, as well as total subgingival bacterial count. 
 
 
3.6. Nutrition Evaluation 
Nutrition evaluation will be conducted by continuous recording of all consumed foodstuffs, dishes and 
beverages, specifying home measures and weight (using “The Album of photographs of products and 
dishes”). The method used in this study to assess eating habits will be the qualitative-quantitative method 
of current quotation (3-day food record). Data will be analysed using The Aliant computer program 
(Cambridge Diagnostics). 
In each case, the analyzed daily food intake will be compared with the current, individual daily 
nutritional and energy requirement in accordance with the Human Nutrition Standards of the Food and 
Nutrition Institute, taking into account the availability of energy at the level recommended for girls. 
3.7. Disordered Eating Attittudes 
Eating behavior will be assessed with a standard Eating Attitudes Test-26 (EAT-26), a self-administrated 
questionnaire. The questionnaire consists of 26 questions related to attitudes and beliefs, behavior 
related to eating, the perception of appearance, and body weight.  
The participant replies to a question by selecting one response: always, usually, often, sometimes, rarely, 
never. In the first 25 questions, the answer ‘always’ is worth 3 points, ‘usually’, 2 points, and ‘often’, 1 
point. In question 26, the answer ‘never’ is worth 3 points, ‘rarely’, 2 points, and ‘sometimes’, 1 point. 
A total score of 20 or more points indicates DEA (Disordered Eating Attittudes). 
3.8. The Data From The Child’s Health Book 
The data were collected from the child’s health book regarding newborn body weight and term of 
delivery. The incidence of macrosomia (body weight over 4000 g) or low birth weight, LBW (body 
weight less than 2500 g) was assessed on the basis of the newborn’s body weight. The full term of 

Study Protocol: Ovarian Hyperandrogenism in Adolescent Girls and Concomitant Metabolic Changes, Oxidative 
Stress, Inflammation, Oral Health and Their Relation to Diet 
 
 
delivery (term births) will be assessed for deliveries that occur between 38–42 weeks of pregnancy, 
while preterm births will be defined as those deliveries between 23 weeks and 37 weeks. 
 
3.9. Evaluation of physical activity 
For this purpose the International Physical Activity Questionnaire will be applied, 
including records of the type and specific character of the training and activity, its duration, 
intensity and frequency. 
 
3.10. Statistical analysis 
 
The analyses will be performed in the PQStat v1.8.0 program and the Statistica software version 
12 (StatSoft. Inc. 2014, Tulsa, USA) with significance taken as p < 0.05. Descriptive statistics 
of groups will be presented as mean and standard deviation, median and quartiles, as well as 
numbers and percentages, depending on the data type. The normal distribution of the data will 
be verified using the Shapiro–Wilk test, and the comparison of the variance of variables 
between the groups will be performed using the Fisher–Snedecor test. Quantitative data 
describing two independent groups and meeting the normal distribution will be compared using 
the Student’s t-test or the Student’s t-test with the Cochran–Cox correction, when the variances 
of the groups being compared are significantly different. The Mann–Whitney test will be used 
when the assumption of normal distribution is not met. The dichotomous data will be compared 
with the chi-square test or the Fisher’s exact test when the Cochran conditions for using the chi-
square test is not met. For the ordered category data, the Cochran–Armitage trend test will be 
used. Pearson correlation coefficient (for normally distributed variables) and Spearman’s rank 
correlation (when the assumption of normality is not met) will be calculated to evaluate 
association between two quantitative variables. 
Logistic regression (univariate and multivariate) will be carried out to identify the independent 
predictors of dichotomous outcomes. A multiple linear regression analysis model will be built 
to explain the selected  dependent quantitative variables. 
 
5. Selected literature references: 
 
1. Naz, M.S.G.; Tehrani, F.R.; Majd, H.A.; Ahmadi, F.; Ozgoli, G.; Fakari, F.R.; Ghasemi, V. 
The prevalence of polycystic ovary syndrome in adolescents: A systematic review and meta-
analysis. Int. J. Reprod. Biomed. 2019, 17, 533–542, doi:10.18502/ijrm.v17i8.4818. 
2. The Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group. Revised 2003 
consensus on diagnostic criteria and long-term health risks related to polycystic ovary 
syndrome (PCOS) The Rotterdam ESHRE/ASRM-sponsored PCOS consensus workshop 
group. Hum. Reprod. 2004, 19, 41–47, doi:1093/humrep/deh098. 
3. World Health Organization. Growth Reference 5-19. BMI-for-Age for Girls. 2007. Available 
online: https://www.who.int/growthref/bmifa_girls_5_19years_z. pdf?ua=1 (accessed on 21 
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Study Protocol: Ovarian Hyperandrogenism in Adolescent Girls and Concomitant Metabolic Changes, Oxidative 
Stress, Inflammation, Oral Health and Their Relation to Diet 
 
 
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