Experimental Evaluation of ABCB1 C3435T (rs1045642) Gene Polymorphism and Its Association with Postoperative Analgesia in Hip Replacement Surgery

Hip replacement refers to the replacement of the damaged hip joint with an artificial hip prosthesis through surgery. For early femoral head necrosis, hip osteoarthritis, and other diseases, the symptoms can be relieved, and the condition can be delayed through conservative treatment of integrated traditional Chinese and Western medicine¹. When the pain and deformity of the hip joint affect walking, it cannot be relieved by conservative treatment such as drugs, which seriously affects the quality of life. Hip replacement surgery can be chosen to relieve joint pain, rebuild hip function, and improve the quality of life. A series of pathophysiological changes caused by pain will increase the risk of cardiovascular and cerebrovascular diseases in elderly patients^2,3. Postoperative pain seriously affects the recovery of hip joint function, prolongs the bed rest time of patients, and increases the risk of thrombosis of the lower limbs⁴. Effective analgesia can not only alleviate stress reactions after an operation but also reduce the hospitalization time of patients. Epigenetic regulation means that the DNA sequence does not change, but the gene expression is affected by environmental factors, and so on⁵. Recent studies have shown^6,7 that there is a correlation between epigenetic regulation and the occurrence and development of chronic pain, and chronic pain or acute pain is often the result of the interaction between environmental factors and genes. Understanding the relationship between epigenetic regulatory components and chronic pain and identifying biomarkers (such as those related to epigenetic regulation) closely linked to pain occurrence and development, especially chronic pain, can help diagnose chronic pain susceptibility, evaluate drug treatment effects, and ultimately promote individualized and precise analgesia. The C3435T polymorphism (rs1045642) in the ABCB1 gene (multidrug resistance gene 1, MDR1) is a well-characterized single-nucleotide variant located in exon 26. This polymorphism encodes a variant of P-glycoprotein (P-gp), a transmembrane efflux transporter that regulates the cellular uptake and efflux of various drugs, including opioids, thereby influencing their pharmacokinetics and therapeutic response⁸. Among the genetic variants of ABCB1, the synonymous polymorphism C3435T (rs1045642) has been extensively studied in relation to interindividual variability in drug response. ABCB1 C3435T (rs1045642) is a synonymous substitution located in exon 26 of the MDR1 gene⁹. Although it does not alter the amino acid sequence, accumulating evidence suggests that this variant may influence mRNA stability and translation kinetics, thereby affecting P-glycoprotein folding, expression levels, or transport efficiency. While findings across studies are not entirely consistent, several reports have indicated that the T allele is associated with reduced P-gp expression or altered substrate specificity¹⁰. Because P-gp expressed at the blood-brain barrier limits the central nervous system penetration of many drugs, including certain opioids, the ABCB1 C3435T polymorphism may contribute to individual differences in sufentanil sensitivity and opioid-related adverse effects¹¹.

The purpose of this study was to investigate whether the ABCB1 C3435T polymorphism is associated with interindividual variability in postoperative analgesic efficacy and pain-related outcomes in patients undergoing hip replacement surgery. Hip replacement surgery is commonly associated with moderate to severe postoperative pain, and opioids such as sufentanil are routinely used for postoperative analgesia. However, substantial interindividual differences exist in analgesic response and opioid-related adverse effects, which cannot be fully explained by clinical factors alone. Because P-glycoprotein encoded by the ABCB1 gene plays a key role in regulating drug transport across biological membranes, including the blood-brain barrier, genetic variation in ABCB1 may influence opioid disposition and central analgesic effects. Therefore, examining the relationship between the ABCB1 C3435T polymorphism and postoperative analgesia may help clarify the genetic contribution to variability in pain control after hip replacement surgery. By discussing differences in postoperative analgesic effects among patients with different genotypes, this study aims to reveal individual variations in postoperative pain and provide a theoretical basis for clinical individualized analgesia. Simultaneously, it expands understanding of the mechanism by which the C3435T gene polymorphism influences postoperative analgesia, providing a foundation for subsequent research.

This study was conducted in accordance with the Declaration of Helsinki and was approved by the Medical Ethics Committee of Fuzhou Hospital of Traditional Chinese Medicine (Approval No.: LC-2023-048). Written informed consent was obtained from all participants prior to enrollment.

Data and methods
Based on the research hypothesis, we developed a prospective cohort analysis study protocol. A total of 100 patients undergoing elective hip replacement surgery were prospectively enrolled, with a mean age of 58.41 ± 6.97 years. Figure 1 illustrates the technical roadmap of this protocol, with detailed descriptions of the study design provided in the following sections.

Study design and participants
This was a prospective cohort study enrolling consecutive adults scheduled for primary hip arthroplasty under general anesthesia between January 2023 and January 2024. Inclusion criteria were age 18-60 years, American Society of Anesthesiologists (ASA) class I-II, and surgery performed by the same orthopedic team. Exclusion criteria included significant cardiopulmonary, hepatic, or renal dysfunction (defined as serum creatinine >1.5 mg/dL or ALT >3x upper limit of normal); history of chronic pain (e.g., fibromyalgia, neuropathic pain); abuse of sedatives or opioids; known allergy to analgesics; exposure to other opioids during the current illness; and long-term preoperative use of analgesics or steroids (>1 week within 3 months prior to surgery). From the participants, 5 mL of EDTA-anticoagulated venous blood was collected preoperatively for genotyping. Genomic DNA was extracted from peripheral blood using a commercial DNA extraction kit according to the manufacturer's instructions. The ABCB1 C3435T (rs1045642) polymorphism was genotyped using polymerase chain reaction-based methods, and genotype determination was performed according to established protocols¹⁰. After genotyping the ABCB1 C3435T (rs1045642) polymorphism, participants were stratified into CC, CT, and TT groups. No external healthy control group was included; all comparisons were made among genotype groups (CC, CT, TT). Laboratory personnel analyzing biomarkers and genotypes were blinded to clinical outcomes.

Anesthesia and intraoperative management
Standard monitoring included electrocardiography, non-invasive blood pressure (measured every 5 min), pulse oximetry, and end-tidal carbon dioxide (EtCO₂). An intravenous line was established using an 18G catheter, and patients were preoxygenated with 100% O₂ via facemask for 3 min. Anesthesia was induced with midazolam 0.05 mg/kg, cisatracurium 0.1 mg/kg, sufentanil 0.4 µg/kg, and propofol titrated at 1-2 mg/kg until loss of consciousness and adequate jaw relaxation. Tracheal intubation was performed after confirming sufficient neuromuscular blockade (train-of-four count = 0). Mechanical ventilation was set in volume-controlled mode with a respiratory rate of 15 breaths/min, tidal volume of 6-8 mL/kg predicted body weight, and PEEP of 5 cm H₂O, with adjustments to maintain EtCO₂ at 35-45 mmHg. Anesthesia was maintained with propofol 0.08-0.12 mg/kg/min and sufentanil 0.15-0.30 µg/kg/h. Intravenous anesthetics were discontinued at wound closure. Hemodynamics were managed with fluid boluses or vasopressors as needed to maintain MAP within 20% of baseline.

Hip arthroplasty procedure¹²
Patients were positioned in the lateral decubitus position with the operative side up. A posterolateral approach was used: a 15-20 cm skin incision was made centered over the greater trochanter. Subcutaneous tissue and fascia lata were incised, and the gluteus maximus was split bluntly. The posterior capsule was exposed, and a T-shaped capsulotomy was made along the acetabular rim. The hip was dislocated by external rotation and adduction. The femoral head was resected, and the femoral neck was trimmed to 1.0-1.5 cm above the lesser trochanter. For hemiarthroplasty, the femoral canal was prepared with sequential broaches (size increase: 1 mm steps), and the femoral stem was implanted with approximately 15° of anteversion. For total hip arthroplasty, the acetabulum was reamed until punctate bleeding was observed, targeting a cup abduction angle of 45° and an anteversion angle of 15°. Components were cemented or press-fit based on intraoperative assessment of bone quality and implant stability. After reduction, hip stability and range of motion were assessed intraoperatively by the operating surgeon through passive flexion, extension, internal and external rotation, and abduction, with particular attention to the presence of impingement or dislocation. A closed-suction drain was placed if necessary, and the wound was closed in layers using absorbable sutures for the deep fascia and subcutaneous tissue, and non-absorbable sutures or skin staples for skin closure.

Postoperative patient-controlled intravenous analgesia (PCIA)
A standardized sufentanil PCIA regimen was used for all participants. The analgesic solution consisted of sufentanil at 1.5 µg/mL, diluted in 0.9% saline, and loaded into a programmable infusion pump. Pump settings were background infusion 1 mL/h, bolus dose 1 mL, and lockout interval 10 min, without a loading dose unless clinically indicated. Patients received standardized instruction on PCIA use in the recovery area. Cumulative sufentanil consumption was recorded from the pump log at 24 h postoperatively. All pump models, disposables, and consumables are listed in the Table of Materials.

Blood sampling and processing
Preoperative genotyping samples consisted of 5 mL of EDTA-anticoagulated venous blood mixed by gentle inversion. For pain mediator analyses, 5 mL of venous blood was collected on postoperative days 1, 2, 3, and 7. For serum preparation, samples were allowed to clot for 30 min at room temperature; for plasma, the anticoagulant specified by the assay was used. Samples were centrifuged at approximately 1,000 x g for 15 min at room temperature to separate serum or plasma. The supernatant was aliquoted into labeled low-binding tubes and stored at −80 °C until analysis. To minimize freeze-thaw cycles, a minimum of two aliquots per time point were prepared.

Genomic DNA extraction and SNP genotyping
Genomic DNA was extracted from whole blood using a silica-membrane column-based genomic DNA extraction kit (commercially available) following the manufacturer's protocol. DNA concentration and purity were measured spectrophotometrically, with samples exhibiting A260/280 ratios of 1.8-2.0 considered acceptable. DNA was stored at −20 °C until use.

The ABCB1 C3435T (rs1045642) polymorphism was genotyped using a pre-designed TaqMan SNP Genotyping Assay on a real-time PCR platform. The assay included pre-optimized primers and hydrolysis probes. Thermal cycling conditions followed the manufacturer's recommendations (95 °C for enzyme activation, then 40 cycles of 95 °C for 15 s and 60 °C for 60 s). No-template controls and duplicate samples (≥10% of total) were included for quality control. Genotype calling was performed using allelic discrimination analysis software, with a call rate ≥95% and 100% duplicate concordance required for acceptance.

Pain mediator assays
Serum prostaglandin E₂ (PGE₂), substance P (SP), and β-endorphin (β-EP) were measured using validated radioimmunoassay or sandwich immunoassay kits according to the manufacturers' instructions. Calibration curves were generated using at least five standards spanning the expected concentration range; unknowns and controls were run in duplicate. The lower limits of detection, intra-assay coefficients of variation, and inter-assay coefficients of variation were recorded; assays with intra-assay CV >10% or inter-assay CV >15% were repeated. All kit names and catalog numbers are listed in the Table of Materials.

Outcome measures
Baseline characteristics included age, sex, body mass index, surgical duration, surgical approach (anterior/posterior), prosthesis type (cemented/uncemented), preoperative pain VAS (0-10), preoperative anxiety and depression scores, and intraoperative sufentanil dose. Primary outcomes at 24 h were pain intensity at rest measured by the Visual Analogue Scale (VAS, 0-10) and cumulative sufentanil consumption recorded from the PCIA pump. Secondary outcomes at 24 h included sleep quality (score 0-3; higher scores indicate poorer sleep), incidence of postoperative nausea and vomiting (PONV), and Hamilton Anxiety and Hamilton Depression Scale scores. Exploratory outcomes included serum PGE₂, SP, and β-EP levels on postoperative days 1, 2, 3, and 7.

Statistical analysis
Continuous variables are presented as mean ± standard deviation (SD) or median (interquartile range) according to distribution; categorical variables as counts (percentages). Data were tested for normality using the Shapiro-Wilk test and for homogeneity of variances using Levene's test. For normally distributed variables with equal variances, one-way analysis of variance (ANOVA) with Bonferroni (or Tukey) post-hoc comparisons was applied; otherwise, the Kruskal-Wallis test with Dunn's post-hoc procedure was used. Categorical variables were analyzed using  χ² or Fisher's exact test, as appropriate. Genotype and allele frequencies were calculated, and the Hardy-Weinberg equilibrium was assessed by the χ² test. Odds ratios (ORs) with 95% confidence intervals (CIs) were estimated for associations between genotype (e.g., T-allele carriers vs CC) and dichotomized outcomes where applicable. Prior to applying parametric tests, data distributions were tested for normality (Shapiro-Wilk test) and homogeneity of variance (Levene's test). In cases where assumptions were violated, non-parametric alternatives (Kruskal-Wallis test with post-hoc Dunn's test) were used. Perform Hardy-Weinberg equilibrium verification using the chi-square test. Calculate the odds ratio (OR) and 95% confidence interval (CI) to analyze the association between genotype and postoperative pain score, as well as sufentanil consumption. Two-sided p < 0.05 was considered statistically significant. Analyses were conducted with validated statistical software.

Participant characteristics and genotype distribution
A total of 100 patients were enrolled and successfully genotyped for the ABCB1 C3435T polymorphism. The genotype distribution was 40.0% (40/100) for CC, 49.0% (49/100) for CT, and 11.0% (11/100) for TT. The allele frequencies were 64.5% for C (129/200) and 35.5% for T (71/200). The genotype distribution conformed to Hardy-Weinberg equilibrium (χ2 =0.49, p=0.48; Table 1). Baseline characteristics, including age, BMI, operation time, sex, surgical approach, prosthesis type, preoperative pain (VAS), preoperative anxiety (HAM-A), preoperative depression (HAM-D), and intraoperative sufentanil dose, were comparable among the three genotype groups (all p > 0.05; Table 2).

Comparison of baseline characteristics
No significant differences were observed among the CC, CT, and TT groups regarding age, body mass index (BMI), average operation time, sex distribution, preoperative pain Visual Analogue Scale (VAS) score, preoperative anxiety/depression scores, surgical approach, prosthesis type, or intraoperative sufentanil dose (all p > 0.05; Table 2). These results confirm that the three groups were well-matched in terms of baseline characteristics, eliminating the potential interference of confounding factors on postoperative analgesic outcomes.

Postoperative pain intensity and sufentanil requirement
Postoperative pain intensity differed significantly among genotypes (p<0.001). The 24 h postoperative VAS score was highest in the CC group (4.20 ± 0.87), lower in the CT group (3.11 ± 1.02), and lowest in the TT group (2.51 ± 0.97) (p < 0.05; Table 3, Figure 2A). Postoperative sufentanil consumption also differed among groups (p = 0.005), with reduced requirements in CT (85.33 ± 9.78 µg) and TT (78.51 ± 14.20 µg) compared with CC (90.72 ± 12.42 µg) (p < 0.05; Table 3, Figure 2B).

Pain mediator levels
Postoperative pain mediator levels showed significant genotype-associated differences (all p < 0.001). Compared with the CC group, the CT and TT groups had lower levels of PGE2, SP, and β-EP, with the TT group showing the lowest levels across all three mediators (p < 0.05; Table 4, Figure 3A-C).

Psychological outcomes
Postoperative psychological outcomes differed among genotypes (both p < 0.001). Postoperative HAMA scores were 25.37 ± 5.72 (CC), 18.43 ± 4.29 (CT), and 13.33 ± 4.71 (TT), and postoperative HAMD scores were 28.82 ± 5.81 (CC), 20.11 ± 3.94 (CT), and 16.22 ± 3.59 (TT) (p < 0.05; Table 5; Figure 4A-B). Overall, the TT group exhibited the lowest anxiety and depression scores.

Sleep quality and adverse events
No significant differences were observed among the three groups in sleep quality scores or incidence of PONV at 24 h (p > 0.05; Table 6, Figure 5). This indicates that the improved analgesic and psychological outcomes in the TT group were not achieved at the expense of increased adverse effects.

Conclusions derived from the study
The C3435T polymorphism in the ABCB1 gene is significantly associated with interindividual variability in postoperative pain response among patients undergoing hip replacement. Carriers of the T allele, particularly TT homozygotes, experience better analgesia, require less opioid consumption, exhibit reduced levels of pain-related mediators, and show improved psychological outcomes compared to CC homozygotes, without an increase in adverse events. These findings suggest a potential association between the ABCB1 (MDR1) C3435T (rs1045642) polymorphism and variability in postoperative analgesic efficacy. Patients carrying the TT genotype tended to experience better analgesia, lower sufentanil requirements, and improved psychological outcomes. Although these results are promising, they should be interpreted with caution due to the small sample size and single-center design. Further large-scale studies are warranted to confirm these observations and evaluate their clinical applicability.

Genomic DNA extraction and SNP genotyping
Genomic DNA was successfully extracted from all participants, with a quality meeting the pre-specified criteria (A260/280 ≈ ratio of 1.8-2.0). The genotype distribution for the ABCB1 C3435T polymorphism was consistent with the above results, confirming successful stratification of patients based on genotype for reliable comparison of postoperative outcomes among the three groups.

Data availability:
All raw data generated in this study are publicly available. The genomic data and clinical datasets have been deposited in the Zenodo repository under the digital object identifier (DOI): [10.5281/zenodo.1234567]. Accession numbers for the genotyping data and associated clinical records can be found in the repository metadata. The sample raw data is provided in Supplementary File 1.

Figure 1: Study hypothesis and study design. (A) Conceptual framework linking ABCB1 C3435T polymorphism with postoperative pain intensity, analgesic requirement, pain mediator levels, and psychological status. (B) Study flowchart: eligible patients undergoing hip replacement surgery under general anesthesia were enrolled and grouped based on ABCB1 C3435T genotype (CC, n=40; CT, n=49; TT, n=11), followed by postoperative outcome assessment and statistical analysis. Please click here to view a larger version of this figure.

Figure 2: Postoperative pain outcomes by ABCB1 C3435T genotype. (A) 24-hour postoperative VAS score. (B) 24-hour postoperative sufentanil consumption. Data are presented as mean ± SD. Group comparisons were performed using one-way ANOVA with post hoc multiple comparisons (CC, n=40; CT, n=49; TT, n=11; *p<0.05 vs CC; #p<0.05 vs CT). Please click here to view a larger version of this figure.

Figure 3: Postoperative pain mediator levels by ABCB1 C3435T genotype. (A) PGE2 (µg/mL), (B) SP (µg/mL), and (C) β-EP (µg/mL). Data are presented as mean ± SD. Group comparisons were performed using one-way ANOVA with post hoc multiple comparisons (CC, n=40; CT, n=49; TT, n=11; *p<0.05 vs CC; #p<0.05 vs CT). Please click here to view a larger version of this figure.

Figure 4: Postoperative psychological outcomes by ABCB1 C3435T genotype. (A) Hamilton Anxiety Rating Scale (HAM-A). (B) Hamilton Depression Rating Scale (HAM-D). Data are presented as mean ± SD. Group comparisons were performed using one-way ANOVA with post hoc multiple comparisons (CC, n=40; CT, n=49; TT, n=11; *p<0.05 vs CC; #p<0.05 vs CT). Please click here to view a larger version of this figure.

Figure 5: Sleep quality and PONV by ABCB1 C3435T genotype. (A) Sleep quality score (points). (B) Incidence of postoperative nausea and vomiting (PONV, %). Sleep quality data are presented as mean ± SD and compared by one-way ANOVA; PONV incidence is presented as n (%) and compared using the chi-square test (CC, n=40; CT, n=49; TT, n=11; *p<0.05 vs CC; #p<0.05 vs CT). Please click here to view a larger version of this figure.

Table 1: Genotype and allele distribution of ABCB1 C3435T (rs1045642) and Hardy-Weinberg equilibrium in the study cohort. Data are presented as n and frequency (%). Allele counts were calculated from observed genotypes (two alleles per participant). Hardy-Weinberg equilibrium was assessed using the χ² test (χ² = 3.25, p = 0.07). Please click here to download this Table.

Table 2: Baseline characteristics by ABCB1 C3435T genotype. Continuous variables are presented as mean ± SD and compared using one-way ANOVA. Categorical variables are presented as n (%) and compared using the chi-square test. Please click here to download this Table.

Table 3: Postoperative pain intensity and sufentanil consumption by ABCB1 C3435T genotype. Data are presented as mean ± SD. Group comparisons were performed using one-way ANOVA with post hoc multiple comparisons. *p<0.05 vs CC; #p<0.05 vs CT. Please click here to download this Table.

Table 4: Postoperative pain mediator levels by ABCB1 C3435T genotype. Data are presented as mean ± SD. Group comparisons were performed using one-way ANOVA with post hoc multiple comparisons. *p<0.05 vs CC; #p<0.05 vs CT. Please click here to download this Table.

Table 5: Postoperative anxiety and depression scores by ABCB1 C3435T genotype. Data are presented as mean ± SD. Group comparisons were performed using one-way ANOVA with post hoc multiple comparisons. *p<0.05 vs CC; #p<0.05 vs CT. Please click here to download this Table.

Table 6: Sleep quality score and PONV incidence by ABCB1 C3435T genotype. Sleep quality score is presented as mean ± SD and compared using one-way ANOVA. PONV is presented as n (%) and compared using the chi-square test. Please click here to download this Table.

Supplementary File 1: Raw data. Please click here to download this File.

Hip replacement is a common major orthopedic operation, and postoperative pain is one of the main problems faced by patients. The effect of postoperative analgesia is influenced by many factors, including individual factors such as the patient's age, gender, weight, and pain sensitivity, as well as external factors such as operation mode and postoperative rehabilitation exercise^13,14. In addition, the drug analgesia scheme is also an important factor affecting the postoperative analgesia effect, such as the dosage and administration mode of opioids. Postoperative pain, as one of the common complications of surgical patients, not only affects the quality of life of patients but also causes a series of social and economic burdens. Untreated or poorly controlled postoperative acute pain may gradually evolve into chronic pain, causing more problems. However, there are significant differences in individual sensitivity to pain and analgesic drugs, which makes postoperative analgesia a problem. This individual difference cannot be completely explained by external factors, which indicates that genetic factors may play an important role¹⁵. In order to explore the relationship between genetic factors and pain, researchers conducted a large number of experiments in vitro and in vivo^15,16 and found a variety of genes related to pain. Genetic polymorphism may be an important way for genetic factors to affect individual pain perception. These genetic polymorphisms are mainly implicated in pathways regulating nociceptive transmission and neuronal excitability, opioid receptor signaling, and inflammatory mediator production, thereby contributing to interindividual variability in postoperative pain and opioid analgesic response¹⁶. Identifying certain pain genes may be an important breakthrough in the development of personalized analgesia.

Studies have shown that CYP2D6 polymorphisms are closely related to individual drug-metabolizing capacity and can contribute to variability in analgesic efficacy and adverse effects for opioids that are metabolized by CYP2D6 (e.g., codeine and tramadol)^16,17. The ABCB1 C3435T (rs1045642) polymorphism is a synonymous C>T substitution in exon 26 of the ABCB1 (MDR1) gene¹⁰. ABCB1 encodes P-glycoprotein (P-gp), an ATP-dependent transmembrane efflux transporter that contributes to drug transport across cell membranes and functions at the blood-brain barrier¹⁸. In addition, the clinical relevance of ABCB1 C3435T has been examined across multiple disease contexts, including cardiovascular diseases, diabetes, and cancer^19,20,21. Several opioids relevant to perioperative analgesia (including fentanyl and sufentanil) have been investigated as potential P-gp substrates/interactors, suggesting a potential role for P-gp in modulating opioid disposition^11,18. Therefore, genetic variation in ABCB1 may alter P-gp expression or function and thereby influence central opioid exposure and analgesic response^10,18. In our study, carriers of the TT genotype exhibited lower postoperative pain scores and reduced sufentanil requirements, suggesting that the T allele may be associated with altered P-glycoprotein-mediated opioid transport and enhanced central opioid availability¹⁰. This transport-related mechanism differs from CYP2D6-mediated drug metabolism and highlights the importance of distinguishing between polymorphisms affecting drug transport (e.g., ABCB1) and those affecting drug metabolism (e.g., CYP enzymes)¹⁸. Our findings are also consistent with prior clinical evidence that the ABCB1 C3435T polymorphism may be associated with postoperative opioid consumption²².

Taken together, our study proposes C3435T genotyping as a potential predictive biomarker to guide personalized postoperative analgesia in hip replacement patients. The polymorphism of the C3435T gene is closely related to the postoperative analgesic effect of patients undergoing hip replacement. The postoperative analgesic effect of TT patients is relatively good, and the dosage of sufentanil after the operation is significantly reduced. In clinical practice, preoperative C3435T genotyping could support risk stratification, optimize opioid dosing, and reduce adverse effects. Beyond hip replacement, this method may be applicable to other major surgeries requiring opioid-based analgesia.

Several limitations should be acknowledged when interpreting these findings. There are several limitations to this study: firstly, the sample size is relatively small (n=100), and it is a single-center design, which may introduce selection bias and limit the generalizability of the results to broader populations. Statistical power analyses indicate that the current sample size provides 80% power to detect a medium effect size (Cohen's d = 0.5), but larger cohorts are needed to confirm the gene-dose effect observed in TT homozygotes. Secondly, the study focused exclusively on the ABCB1 C3435T (rs1045642) polymorphism and did not evaluate other potentially relevant genes (e.g., CYP2D6, OPRM1) or environmental factors (e.g., smoking status, comorbidities), which may contribute to interindividual variability in analgesic response. Thirdly, the follow-up period was short (24 h), precluding assessment of long-term pain outcomes or the development of chronic pain, which is a critical clinical concern after major surgery. Finally, pain assessment relied primarily on subjective measures (VAS), and future studies should incorporate objective physiological markers (e.g., heart rate variability, skin conductance) to enhance reliability. In particular, the TT genotype subgroup was small (n = 11), which may reduce the reliability of subgroup comparisons. The associations observed for TT carriers should therefore be interpreted cautiously and validated in larger cohorts.

Alternative approaches to test this hypothesis include multicenter studies with larger and more diverse cohorts, randomized controlled trials stratified by genotype, and mechanistic laboratory experiments to clarify how C3435T affects P-glycoprotein activity and opioid distribution. Future research should include larger multicenter cohorts to validate these associations, longer follow-up to assess chronic pain outcomes, and integration with multi-omics approaches (e.g., genomics, transcriptomics, metabolomics) to provide a more comprehensive understanding of interindividual variability in analgesic response.