Association between serum vitamin C and HPV infection in ... - BioMed Central

Data sources and study population

This cross-sectional study was restricted to women aged 18–59 years who completed HPV tests in the National Health and Nutrition Examination Survey (NHANES) from 2003 to 2006. Responses coded as "don't know", "refused," "inadequate" or "missing" in the original NHANES data were treated as missing. Participants with missing HPV data, covariates, or VC levels were excluded. The NHANES is a nationally representative health survey in the US designed and administered by the National Center for Health Statistics (NCHS) at the Centers for Disease Control and Prevention (CDC). It is an ongoing survey that uses a complex multistage sampling design to obtain a representative sample of the US population during each collection cycle. It collected information on demographic indicators and health outcomes through interviews, face-to-face examinations, and laboratory tests. Each year, the NHANES examines approximately 5,000 participants per round, with participants located in different counties in the US. A computerized process randomly selects some, all, or no household members. Complete details regarding the NHANES study design, recruitment, procedures, and demographic characteristics can be accessed through the CDC website (https://www.cdc.gov/nchs/nhanes/index.htm). Briefly, NHANES study sampling consisted of a four-stage design with oversampling of some subgroups to improve precision. The NCHS ethics review board has approved the NHANES protocol. Written informed consent was obtained from all participants. The original study protocol was accessible on the website of the Ethics Review Board of the NCHS (https://www.cdc.gov/nchs/nhanes/irba98.htm) and was approved by the Ethical Review Committee (Protocol #98–12 and Protocol #2005–06). Furthermore, the NHANES covers interviews and medical examinations with a focus on various health and nutrition measurements, and is the main program of the NCHS. More detailed information can be found in the official NHANES website (https://www.cdc.gov/nchs/nhanes/).

Measurement and classification of VC

Serum VC was collected and measured using isocratic high-performance liquid chromatography (HPLC) with electrochemical detection at 650 mV. Peak area quantification was based on a standard curve generated from three different concentrations of an external standard (0.025, 0.150, and 0.500 mg/dL). The quality assurance and quality control protocols utilized by the NHANES met the 1988 Clinical Laboratory Improvement Act mandate. Serum VC levels were modeled and analyzed in continuous and categorical forms. We categorized serum VC levels according to a prior study [22], as follows: deficiency and hypovitaminosis (0–23.99 µmol/L), inadequate (24–49.99 µmol/L), adequate (50–69.99 µmol/L), and saturating (≥ 70 µmol/L) based on participant plasma levels.

Detection and classification of HPV infection

HPV infection was measured based on HPV genotyping using deoxyribonucleic acid (DNA) extracted from self-collected vaginal swabs. The DNA extracts used for the linear array HPV test were stored at  −20 °C for temporary storage and at −80 °Cfor long-term storage. The NHANES performed Roche Linear Array HPV genotyping tests for self-collected vaginal swab specimens and reported the results of HPV DNA detection tests for 37 HPV types. The HPV polymerase chain reaction summary variable indicates that if at least one HPV type is positive, the sample is negative. More information on HPV measurements can be found on the website (https://wwwn.cdc.gov/Nchs/Nhanes/2003-2004/L37SWA_C.htm#LBDHPCR).

Covariates

The present study considered age, race/ethnicity, education, marital status, poverty income ratio (PIR), health condition, health insurance, smoking status, alcohol consumption, first age, body mass index (BMI), and levels of serum folate, albumin, α-carotene, and vitamin A, E, and D. Age was considered a continuous variable (18– 59 years). Participants self-reported race/ethnicity and were divided into five categories: Mexican American, other Hispanics, non-Hispanic white, non-Hispanic black, and other races. Education was categorized as high school graduate or lower, some college, and college graduate or above [23]. Marital status was recorded as married or living with a partner, never married, and widowed, divorced, or separated. PIR, the ratio of family income to the poverty threshold, ranged from zero to five. Participants' self-reported health condition was classified into two categories: poor and fair were referred to as "poor"; good, very good and excellent were referred to as "good". Participants reported their health insurance coverage ('yes' or 'no') from any source (e.g., private individual insurance, employer provided, Medicare, Medicaid, and Veteran's Administration). Smokers were defined in the questionnaire as those who smoked more than 100 cigarettes per day. Consumption of at least 12 alcoholic beverages in any year was defined as alcohol consumption. The first age was defined as the age when the participants first had vaginal, anal, or oral sex. The number of partners was defined as the number of males with whom the participants have had vaginal, anal, or oral sex with in their lifetime. BMI was calculated for all participants by dividing the weight (kg) by the squared height (m²). The laboratory data included serum folate (nmol/L), albumin (g/L), α-carotene (µmol/L), vitamin A (µmol/L), vitamin E (µmol/L), and vitamin D (nmol/L) levels.

Statistical analysis

All analyses were performed using the statistical software package R-4.0.2 (http://www.R-project.org, The R Foundation) and Free Statistics software version 1.7. We used the Medical Examination Center examination sampling weights provided by the NCHS to account for the unequal probability of selection and non-response. All estimates shown were weighted using these sampling weights, except when reporting the sample size by demographic characteristics. Descriptive statistics (sample sizes and weighted proportions) were computed along with mean serum VC levels and weighted prevalence of categorical serum VC levels. We estimated the crude odds ratios (ORs) and 95% confidence intervals (CIs) between serum VC levels and HPV infection using weighted logistic regression. Baseline characteristics were analyzed using means, standard errors (SE), percentages, or frequencies. Continuous variables were compared using analysis of variance for normally distributed variables and non-parametric tests for non-conformity to normal distribution. Categorical variables were analyzed using the chi-squared test. We adjusted the p-values of the multiple tests for a large number of tests using Bonferroni correction. The effect of VC on HPV infection was evaluated using multiple logistic regression models as follows: Model I: No adjustment; Model II: Adjusted for age, race/ethnicity, PIR, alcohol, smoking, BMI, education, and health condition; Model III: Adjusted for the variables in Model II plus first age and partner number; Model IV: Adjusted for the variables in Model III plus vitamin A level, health insurance, and marital status. Additionally, age was divided into two groups (< 25 years and ≥ to 25 years), and subgroup analysis was performed. Statistical significance was set at P < 0.05.