Two hundred and five (205) adolescents between 12–18 years-old complaining of PD [study group (dysmenorrhea group)] were compared with matched controls of the same age and ethnic characteristics (210 controls without dysmenorrhea).
Adolescents (study and controls = 415) were recruited for this comparative study from 43 schools of Aktobe-Kazakhstan from January 2021 to November 2022 after approval of the ethical committee of West Kazakhstan Medical University (WKMU), (Meeting No. 10 dated 04.10.2020).
Adolescents were included in this study after informed consent from the adolescents themselves, and their parents or legal guardian following the Helsinki Declaration.
Adolescents were evaluated thoroughly and examined at the diagnostic Youth Health Center by an adolescent gynecologist according to the hospital`s protocol.
After a thorough history, and anthropometric evaluation [i.e., weight, height, and body mass index (BMI)], a trans-abdominal pelvic ultrasound was done for the studied adolescents to exclude any pelvic pathology.
The trans-abdominal pelvic ultrasound was done by an expert sonographer blinded to the adolescents’ data, using the tarns-abdominal convex probe of the Samsung HS40 machine (Samsung Co., Korea).
Adolescents between 12–18 years old, with BMI < 30 Kg/m2, regular menstrual cycle (every 21–35 days), and complaining of PD [visual analogue scale (VAS) ≥ 5] for ≥ one year duration since their menarche were included in the study group (dysmenorrhea group = 205).
Adolescents between 12–18 years old, with BMI < 30 Kg/m2, regular menstrual cycle (every 21–35 days), without dysmenorrhea for ≥ one year duration since their menarche were included as controls (210 without dysmenorrhea).
Adolescents < 12 or > 18 years old, with BMI > 30 Kg/m2, pelvic organs anomalies (i.e., including anomalies of the genital and/or urinary tracts) or pelvic pathology (i.e., fibroid uterus and/or ovarian cyst or mass), previous pelvic surgery, neurological or psychiatric disorders, or received exogenous hormonal therapy within the last year were excluded from this study [19].
The BMI was calculated from the studied adolescents’ body weight and their height (kg/m2). The WHO considered ≤ 24.9 kg/m2 normal-weight, 25.0-29.9 kg/m2 overweight, and > 30 Kg/m2 obesity class-I [20].
The visual analogue scale (VAS) was used to assess the severity of dysmenorrhea (0 is the lowest VAS and means no pain, while 10 is the highest VAS and means unbearable pain) [21].
The blood samples collected from the studied adolescents were used for measuring the serum 25(OH)D and for VDR TaqI (rs731236) genotyping.
The serum 25(OH)D level was measured using an enzyme-linked immunosorbent assay (Ottignies-Louvain-la-Neuve, Belgium) [14]. Serum 25(OH)D between 20–40 ng/mL considered normal, while serum 25(OH)D < 20 ng/mL was considered 25(OH)D deficiency, according to the Endocrine Society guideline [22].
The TaqI genotyping was done after DNA extraction from the adolescents’ blood samples. The DNA was extracted using the standard salting out method [14].
The TaqI genotyping was performed using Polymerase Chain Reaction (PCR) and amplification of the targeted region.
Both TaqI wild-type and variant, were prepared for amplification and ran on 2% agarose gel separately as a result of the allele-specific primer design that resulted in the same product size (i.e., 148 bp) [14].
The sequences of the primers covering the targeted gene region were 50-CAGGACGCC GCGCTGATT-30 (forward primer for wild type), 50-CAGGACGCCGCGCTGATC-30 (forward primer sequence for variant allele), and 50-CCTCATTGAGGCTGCGCAG-30 (reverse common primer) [14].
The PCR was optimized after 30 cycles of 5 min. of denaturation at 94°C, 30 sec. of second denaturation at 94°C, 45 sec. of annealing at 60°C, and 45 sec. of elongation at 72°C.
The amplified product was visualized on 2% agarose gel electrophoresis under Applied Biosystems 3130/3130xl Genetic Analyzers (Applied Biosystems and Hitachi, Ltd., US) imaging system.
The presence of a single 148-bp band either for wild type or variant allele in homozygous condition indicates successful amplification. While the presence of both bands with the same size (148-bp) represented the heterozygous condition [14].
Collected data were analyzed to detect the relation between serum 25(OH)D and PD (primary outcome). The secondary outcome measures the odds of PD in Asian adolescents with TaqI polymorphism.
Sample Size
The required sample required for this study was calculated based on the total number of adolescents between 12–18 years old in Aktobe-Kazakhstan (27,972), the prevalence of dysmenorrhea among adolescents (8–83%), and using the G Power (version 3.1.9.7) for sample size (Düsseldorf; Germany), setting the probability at 0.05, power at 0.95%, and sample size at 0.5. Consequently, an effective sample size ≥ 210 in two groups (study and controls) was needed to produce an acceptable figure.
Statistical analysis
The SPSS (Statistical Package for Social Sciences) version 25 (Chicago, IL, USA) was used for analysis of the collected data. The mean and standard deviation (± SD) were used to present numerical values, while the number (n) and percentage (%) were used to present categorical values. The Student t was used for analysis of quantitative and the Chi-square (x2) test was used for analysis of qualitative data. The correlation analysis was done using the Pearson`s correlation coefficient (r) to detect the relation between serum 25(OH)D and PD. The MedCalc 20.106 (MedCalc Software Ltd, Belgium) was also used to calculate the odd ratio (OR) of PD in Asian adolescents with TaqI polymorphism. P < 0.05 considered significant.