Data sources and study population
This cross-sectional study was limited to women ages 18 to 59 who completed HPV testing in the National Health and Nutrition Examination Survey (NHANES) from 2003 to 2006. Responses coded “don’t know”, “denied”, “insufficient”, or “missing” in the original NHANES data were treated as missing. Participants with missing HPV data, covariates, or VC values were excluded. The NHANES is a nationally representative health survey in the United States developed and administered by the National Center for Health Statistics (NCHS) at the Centers for Disease Control and Prevention (CDC). It is a continuous survey that uses a complex multi-level sampling design to obtain a representative sample of the US population each survey cycle. It collected information on demographic indicators and health outcomes through interviews, personal examinations and laboratory tests. Each year, NHANES screens approximately 5,000 participants per round, with participants located in various counties across the United States. A computerized process randomly selects some, all, or none of the household members. Full details on the NHANES study design, recruitment, procedures, and demographics are available on the CDC website (https://www.cdc.gov/nchs/nhanes/index.htm). Briefly, the sampling of the NHANES study consisted of a four-stage design with oversampling of some subgroups to improve accuracy. The NCHS Ethics Review Board has approved the NHANES protocol. Written informed consent was obtained from all participants. The original study protocol was available on the NCHS Ethics Review Board website (https://www.cdc.gov/nchs/nhanes/irba98.htm) and was approved by the Ethical Review Committee (Protocol #98-12 and Protocol #2005 -06). In addition, the NHANES includes interviews and medical examinations focusing on various health and nutritional measurements and is the main program of the NCHS. For more detailed information, visit 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. The quantification of the peak area was based on a standard curve constructed from three different concentrations of an external standard (0.025, 0.150 and 0.500 mg/dL). The quality assurance and quality control protocols used by NHANES met the mandate of the Clinical Laboratory Improvement Act 1988. Serum VC levels were modeled and analyzed in a continuous and categorical form. We categorized serum VC levels according to a previous study as follows: deficiency and hypovitaminosis (0-23.99 µmol/l), insufficient (24-49.99 µmol/l), adequate (50-69.99 µmol/l) and saturation (≥ 70 µmol/l) based on the plasma levels of the participants.
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 interim storage and at -80°C for long-term storage. NHANES performed Roche Linear Array HPV genotyping tests on self-collected vaginal swab specimens and reported HPV DNA detection test results for 37 HPV types. The HPV polymerase chain reaction summary variable indicates that the sample is negative if at least one HPV type is positive. Visit the website (https://wwwn.cdc.gov/Nchs/Nhanes/2003-2004/L37SWA_C.htm#LBDHPCR) for more information on HPV measurements.
The present study took into account age, race/ethnicity, education, marital status, poverty income rate (PIR), health status, health insurance, smoking status, alcohol consumption, early age, body mass index (BMI), and levels of serum folate, albumin, a-carotene and vitamins A, E and D. Age was considered as a continuous variable (18-59 years). Participants indicated their race/ethnicity and were grouped into five categories: Mexican, other Hispanic, non-Hispanic white, non-Hispanic black, and other race. Education was categorized as high school graduate or below, some college grads, and college graduate or above . Marital status was recorded as married or cohabiting, never married and widowed, divorced or separated. PIR, the ratio of family income to the poverty line, ranged from zero to five. Participants’ self-reported health was divided into two categories: poor and fair were labeled “poor”; good, very good and excellent were rated as “good”. Participants reported their health insurance coverage (“yes” or “no”) from any source (eg, private individual insurance, employer-provided, Medicare, Medicaid, and Veteran’s Administration). The questionnaire defined smokers as people who smoked more than 100 cigarettes per day. Alcohol consumption was defined as the consumption of at least 12 alcoholic beverages per year. First age was defined as the age at which participants had vaginal, anal, or oral sex for the first time. Number of partners was defined as the number of men with whom participants had vaginal, anal, or oral sex in their lifetime. BMI was calculated for all participants by dividing weight (kg) by height (m) squared2). 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). L) levels.
All analyzes 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 sample weights provided by the NCHS to account for the unequal likelihood of selection and non-response. All estimates shown have been weighted with these sample weights, except where sample size is specified by demographic characteristics. Descriptive statistics (sample sizes and weighted proportions) were calculated along with mean serum VC levels and weighted prevalence of categorical serum VC levels. We estimated the raw odds ratios (ORs) and 95% confidence intervals (CIs) between serum VC levels and HPV infection using a weighted logistic regression. Baseline characteristics were analyzed using means, standard errors (SE), percentages, or frequencies. Continuous variables were compared to the normal distribution using analysis of variance for normally distributed variables and nonparametric tests for nonconformity. Categorical variables were analyzed using the chi-square test. We adjusted the p-values of the multiple tests for a large number of tests using Bonferroni’s correction. The effect of VC on HPV infection was assessed using several logistic regression models as follows: Model I: no adjustment; Model II: Adjusted for age, race/ethnicity, PIR, alcohol, smoking, BMI, education and health status; 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 levels, health insurance, and marital status. In addition, age was divided into two groups (< 25 years and ≥ up to 25 years) and a subgroup analysis was carried out. Statistical significance was set at P< 0.05.