Correlation Between Oxidative Stress and Antioxidant Status in Adolescents and Young Females with Primary Dysmenorrhea: A Case-Control Study

A total of 73 participants were enrolled, including 40 with PD and 33 in the control group Demographic characteristics of cases and controls Significance was calculated by Binary logistic regression analysis and chi squire $ ; OR: odds ratio; 95% CI: confidence interval; *p<0.05: significant, BMI: body mass index; **0.05). Early menarche (<12 years) significantly increased the risk of PD (OR: 14.76, 95% CI: 4.54-47.94; p 25, indicating higher overweight/obesity rates in the control group (OR: 9.0; 95% CI: 2.7229.75; p=0.0003). In terms of dietary habits, the case group consumed a higher amount of spicy food ( p <0.05) and fewer vegetables and fruits ( p 0.05). Subjects with PD exhibited significantly lower physical activity levels (7.5%) than controls (75.80%) (OR: 125; 95% CI: 13.34-808; p <0.05) Distribution of subjects on the basis of life style pattern and family history of PD Significance was calculated by Binary logistic regression analysis; OR: odds ratio; 95% CI: confidence interval; *p<0.05: significant, **<0.001: more significant Sedentary behavior (45% vs. 12.1%) and poor sleep quality (52.5% vs. 9.1%) were also markedly higher in the case group (p<0.01). A family history of PD (primary maternal) was reported by 60% of the PD group compared to 21.1% of controls, a statistically significant association (OR: 5.57 (95% CI: 1.9-15.87; p = 0.0013). No significant differences were found in Hb and hormone profile (S.LH, S.FSH, S.PRL, and S.TSH) between PD and control groups (all p>0.05) ( Figure 1 ). Distribution level of Biochemical assessments and Hemoglobin (Hb) in case and control ( ns = not significant) The timing of distribution onset did not differ significantly between pain severity groups. Most females with PD (65%) reported that pain started on the first day of their period. However, a pain duration ≤3 days were more common in cases with mild and severe pain (p<0.05). Lower abdominal pain and fatigue were significantly associated with pain severity (p<0.05). Comparison of VAS score for Pain severity with Primary dysmenorrhea characteristics and symptoms during menstruation in case (N=40) P0.05). MDA levels (μMol/ml) were significantly higher in the PD group (5.40 ± 1.03) compared to controls (4.46 ± 0.73; p-value=<0.001). SOD levels (U/ml ) were significantly lower in the PD gr o up (10.23 ± 2.37) compared to controls (14.41 ± 1.31; p-value = 0.0001) ( Figure 2 ). GSH levels (μMol/ml) were also significantly reduced in PD (15.77 ± 2.41) compared to controls (21.83 ± 1.44; p-value= 0.0001). Distribution of oxidative marker (MDA) and anti-oxidative markers (SOD and GSH) in case and control (** p value <0.001). In women with PD, oxidative stress marker MDA was negatively correlated with SOD (r=-0.66; p=0.001), GSH (r=-0.45; p=0.032), and BMI (r=-0.23) (all p<0.05), indicating that higher oxidative stress is associated with lower antioxidant levels and BMI ( Table IV ). Pearson’s correlation (r) analysis of oxidative stress marker MDA with antioxidative markers (SOD & GSH), Age and BMI. *: p <0.05. Furthermore, BMI showed a significant negative correlation with MDA levels in the PD group (-0.233; p=0.02). Controls exhibited weak, non-significant correlations between these markers. SOD and GSH levels demonstrated a significant positive association in the PD group (r = 0.49, p = 0.031), suggesting a coordinated antioxidant response. Age and BMI were positively correlated in both the PD (r=0.53; p=<0.011) and control groups (r=0.66; p=0.037).

This Case-Control study was conducted in the Department of Obstetrics and Gynaecology in collaboration with the Department of Biochemistry at King George's Medical University (KGMU), Lucknow, India. After obtaining institutional ethical clearance, ethical clearance was obtained from the institutional ethics committee of KGMU, Lucknow, India (Reference no 571/Ethics/2024, dated 19/6/24, ref. code: XXI -PGTSC -IIA/P72). Informed written consent was obtained from all participants. A total of 73 Young women and adolescents (40 cases and 33 controls) were enrolled. The participants were divided into two groups: Cases (with PD) and Controls (without PD). A Standardized questionnaire was used to gather demographic and baseline information, including age of menarche, duration of PD, and premenstrual history. Menstrual pain severity was assessed using the visual analogue scale (VAS) [ 12 ].. Height, Weight, and BMI were also recorded. Prior to the interview, written informed consent was obtained from all participants (and their guardians for minors) after a detailed explanation of the study. Every participant underwent a detailed history taking at the outpatient department. Unmarried adolescents and young women (11- to 24-year-olds) Complaint of primary dysmenorrhea (manifesting symptoms like premenstrual pain, low back ache, nausea, vomiting, headache, and low moods). Individuals who have given written consent. Criteria for Inclusion for control: - Unmarried healthy adolescent females, age-matched (18 to 24 years) to the case group, with no menstrual pain and no use of analgesics during menses. Individuals who did not provide consent. Individuals with secondary dysmenorrhea (associated with conditions like endometriosis, pelvic inflammatory diseases, leiomyoma, or interstitial cystitis). Participants with any endocrine disorder or those who had undergone major surgery. The prevalence of PD among adolescent girls and young women in India ranges from 50% to 88%, with a pooled estimate of 70.2% [ 11 ].. For an unmatched case-control study assessing oxidative stress (e.g., MDA positivity; p₁=0.73 in cases, p₀=0.40 in controls; 33% difference observed in previous study [ 10 ].; Z α /2 = 1.96; Z β = 0.84 (80% power) [ 10 ]., the formula for sample size per group (n) is: n = ( z α / 2 + z β ) 2 × [ p 1 ( 1 − p 1 ) + p 0 ( 1 − p 0 ) ( p 1 − p 0 ) 2 This yields n ≈ 30 per group. This was adjusted upward by 10-20% to account for potential non-response or incomplete data, leading to a target of ~35-36 per group. For feasibility, we enrolled 40 cases and 33 controls (a total of 73). On day 2-3 of the menstrual cycle, 5ml venous blood was collected in an EDTA vial for oxidative stress and antioxidant assays. After collection, samples were transported to the Department of Biochemistry laboratory within 30-60 minutes. Samples were centrifuged at 2000 RPM for 15 minutes, and the plasma supernatant was transferred to another centrifuged tube and stored in a deep freezer at -80 °C until analysis. Menstrual pain severity was assessed by using the VAS. Pain levels were categorized as mild (VAS 1-3: pain did not disrupt daily activities, no pain medication required), moderate (VAS 4-6: pain slightly affected daily activities, pain medication required), or severe (VAS 7-10: persistent pain with limited relief from pain medication [ 12 ].. Mental well-being was assessed through interview questions regarding perceived stress, anger, anxiety, irritability, and depression in daily life [ 13 ].. This was not a validated scale, which is acknowledged as a limitation. Sleep quality was determined by asking participants about the presence of insomnia or other sleep disturbances, and was categorized based on self-reported average sleep duration [14]. Family history of dysmenorrhea in the participant’s mother was recorded. Dietary history was obtained by using the 24-hour recall method [15] to assess whether the patients had adequate intake of fruits and vegetables and whether there was excessive consumption of junk food or processed food on the same day. Plasma MDA levels were quantified using the Elabscience® MDA Colorimetric Assay Kit (Catalog No. E-BC-K025-S) on a microplate reader. SOD activity was assessed with the Total Superoxide Dismutase (T-SOD) Activity Assay Kit (Catalog No. E-BC-K020-M). Plasma GSH concentrations were measured using the Cayman Chemical Glutathione Assay Kit (Item No. 703002). All samples were randomized and processed blinded across plates. The Kolmogorov-Smirnov test was used to assess the normality of continuous data. Descriptive statistics for numerical data of oxidative stress are presented as mean and standard deviation. Dichotomous variables are presented as frequency (N)/percentage (%) and were analyzed using binary logistic regression and Chi-square tests. An unpaired Student t-Test was used to compare the means between the case and control groups. Pearson’s correlation coefficient was used to assess the correlation between variables. All statistical analyses were performed using SPSS version 22, and a p-value <0.05 was set as the threshold for statistical significance.

This case-control study investigated the association between oxidative stress and primary dysmenorrhea in adolescents and young women. Our primary finding is a significant pro-oxidant state in participants with PD, characterized by elevated plasma MDA and diminished levels of the enzymatic and non-enzymatic antioxidant SOD and GSH. The significant negative correlations between MDA and both SOD and GSH in the PD group further support an oxidative imbalance, where increased lipid peroxidation is coupled with a depleted antioxidant defense system. Interestingly, BMI showed a negative correlation with MDA in the PD group, suggesting that within this group, those with a higher BMI had lower oxidative stress, a finding that warrants further investigation. The demographic findings are consistent with previous research. Early menarche was a strong risk factor for PD, echoing studies by Omidvar and Begum [ 11 ].. The association of PD with a normal BMI, as opposed to the higher BMI observed in controls, has also been reported by others [ 16 - 19 ]. and may reflect pain perception or metabolic profiles. Our finding with lifestyle factors aligns with literature, linking low physical activity, poor sleep, and certain dietary habits (high spicy food, low fruits/vegetable intake) to PD [ 10, 19-21 ]. The strong family link (OR: 5.57) corroborated the well-documented genetic predisposition to PD [ 22 - 24 ].. Our biochemical results are in agreement with a systematic review [ 10, 25 ] and other individual studies that have reported elevated MDA and reduced SOD and GSH in women with PD [ 9 , 26 , 27 ]. The lack of difference in hormonal profiles between groups is consistent with studies that PD is more related to local uterine factors and prostaglandin production than systemic hormonal imbalance [ 25 ].. The observed oxidative imbalance provides a biologically plausible mechanism for PD pathogenesis. Uterine ischemia-reperfusion during painful contractions likely generates ROS, leading to lipid peroxidation (reflected in terms of MDA) and consumption of antioxidant reserves (SOD and GSH) [ 7 - 8 , 28 ]. Oxidative stress can exacerbate inflammation and pain. These findings suggest that antioxidant supplementation or lifestyle modifications that boost antioxidant capacity (for example, increases row fruits/vegetable intake and physical activity) could be promising adjunctive therapies for managing PD symptoms. The modifiable nature of the associated lifestyle factors also highlights important targets for public health interventions and patients’ education. This study has several limitations that should be considered. First, the sample size is relatively small, and the extremely wide confidence interval for some odds ratios (e.g. OR for physical activity: 13.34-808) suggest possible sparse data bias or overfitting, and these estimates should be interpreted with caution. Second, the cross-sectional, case-control design precludes any inference of causality. Third, the study was conducted at a single tertiary care center, which may limit the generalizability of the findings. Fourth, data on life style factors, diet, sleep and mental health were collected via self-reported and were not based on validatory questionnaires, introducing the potential recall bias. Future research should prioritize larger, multicentric, prospective cohort studies to established temporal relationship. Furthermore, interventional trials examining the efficacy of antioxidant therapies or structured lifestyle programs are needed to confirm their therapeutic potential in PD. In conclusion, this study demonstrated a significant association between PD and oxidative stress, marked by increased lipid peroxidation and decreased antioxidant defenses. These finding, along with the identification of several modifiable lifestyle risk factors, underscore the complex interplay of biological and behavioral element in PD and point towards oxidative stress as a key target for future interventions. Ethical consideration: Ethical clearance was obtained from the institutional ethics committee of KGMU, Lucknow, India (Reference no 571/Ethics/2024, dated 19/6/24, ref. code: XXI -PGTSC -IIA/P72). Informed written consent was obtained from the entire participant. Source of funding: None Declaration of completing interest: None Acknowledgement: The authors would like to thanks the participants who made this study possible.

Dysmenorrhea, characterized by painful and distressing menstruation, is commonly classified into primary dysmenorrhea (PD), which occurs in the absence of underlying pelvic pathology, and secondary dysmenorrhea, which is associated with identifiable conditions [ 1 ].. PD predominantly affects adolescents and young women, imposing a significant burden on quality of life, academic performance, and distress [ 2 , 3 ].. The pathophysiology of PD is complex, but it is primarily linked to increased endometrial prostaglandin production during ovulatory cycles. This leads to heightened uterine contractility, vasoconstriction, and ischemia, resulting in pain [ 3 - 4 ]. A growing body of evidence suggests that oxidative stress may play a crucial role in this process. Ischemia-reperfusion injury from uterine contractions can generate reactive oxygen species (ROS), leading to a state of oxidative stress when the body’s antioxidant defenses are overwhelmed [ 5 - 6 ].. Key biomarkers of oxidative stress include malondialdehyde (MDA), a product of lipid peroxidation, which reflects cellular damage. The body’s primary enzymatic antioxidant defenses include superoxide dismutase (SOD), which catalyzes the dismutation of superoxide radicals, and non- enzymatic antioxidants like glutathione (GSH), which help neutralize ROS [ 7 - 8 ]. Previous studies have reported elevated MDA and reduced antioxidant levels in women with PD, suggesting an imbalance in the oxidant-antioxidant system [ 9 - 10 ].. While the link between oxidative stress and PD is plausible, data from north Indian populations remain limited, and the interplay between these biochemical markers and lifestyle factors is not fully understood [ 11 ].. Therefore, this case-control study aims to investigate the association between PD and oxidative stress by comparing plasma levels of MDA, SOD, and GSH in adolescents and young females with and without PD, and to explore their correlation with demographic and lifestyle factors.