From October 2016 to September 2018, 280 women living in Minas Gerais State and attended at Basic Health Units of Ouro Preto, and State Specialized Care Center of Itabirito were selected for this study.
Inclusion criterion was age ≥18 years old. Exclusion criteria were pregnancy in a period <6 months, history of neoplasia, and presence of cervical atypia in glandular cells.
At the first meeting (T1), an interview was performed for information about sociodemographic and behavioral characteristics, and cervical sample was collected for cytological analysis, genetic polymorphism evaluation, and HPV detection.
After six months of follow-up (T2), 164 women performed the second cytological analysis. From this group, 50 participants presenting normal cytology at T1 and T2, and eight women with normal cytology at T1 and presence of pre-neoplastic lesion at T2 were excluded (Figure 1). Thus, sample group (n=106) was divided into:
- Remission (n=60): presence of pre-neoplastic lesion at T1, and normal cytology at T2;
- Persistence (n=46): pre-neoplastic lesion detected at T1 and T2.
Presence of pre-neoplastic lesion was considered when Atypical Squamous Cells of Undetermined Significance (ASC-US), Low Grade Squamous Intraepithelial Lesion (LSIL), High Grade Squamous Intraepithelial Lesion (HSIL), or Atypical Squamous Cell – cannot exclude HSIL (ASC-H) were detected at two times.
Research Ethics Committee of Federal University of Ouro Preto approved this study (CAAE 57187316.7.0000.5150, and CAAE 88479718.0.0000.5150).
Cervical samples were obtained through conventional double collection by health care professionals, using Ayre spatulas for ectocervical sample, and cylindrical brushes for endocervical sample. After confection of cervical smear for cytological analysis, brush was conditioned in Phosphate-Buffered Saline (PBS) pH 7.2, and stored at -80°C for genetic polymorphisms evaluation.
Cervical smears were stained according to Papanicolaou method, and samples were evaluated based on cytomorphological criteria described in Bethesda System 2014 for reporting cervical cytological diagnoses (Nayar and Wilbur, 2015). All samples were evaluated by two cytopathologists. In case of disagreement between results, a third professional evaluated the sample. The analyses were performed in Laboratório de Análises Clínicas (LAPAC) from Federal University of Ouro Preto.
DNA extraction from cervical samples was performed with illustra blood genomicPrep Mini Spin™ Kit (GE Healthcare, Chicago, Illinois, USA). Evaluation of quality and integrity of DNA was performed by amplification of β-actin gene (42).
HPV detection was performed by conventional Polymerase Chain Reaction (PCR) with MY09/MY11 primers, as described by Miranda et al. (2013). For positive samples, HPV genotype was analyzed by Restriction Fragment Length Polymorphism (RFLP) (42). HPV negative samples were also analyzed by conventional PCR with GP5+/GP6+ primers (43).
MTHFR C677T (rs1801133), MS A2756G (rs1805087), MTRR A66G (rs1801394), and TS3’UTR (rs151264360) polymorphisms were evaluated by PCR-RFLP (14, 35, 44, 45). TSER (rs34743033) was evaluated by PCR (46).
Sequences of primers, restriction enzymes, and PCR protocols were presented in Supplementary Tables 1, 2, and 3.
Table 3 shows the size of DNA fragments that characterize the genotypes of polymorphisms analyzed.
GRS was calculated to evaluate the presence of all polymorphisms simultaneously, as described by Tomita et al. (2013). Presence of heterozygosity or polymorphic homozygotes received one or two points, respectively. Non-polymorphic homozygotes were not scored (zero) (13). Thus, the higher the GRS, the higher the frequency of genetic polymorphisms.
Data were tabulated by Microsoft Office Excel™ (Microsoft, Redmond, Washington, USA), and analyzed by Statistical Package for the Social Sciences™ 17.0 (International Business Machines, New York, USA).
Descriptive statistics were performed to evaluate the frequency of genotypes. Allelic frequency was calculated by Genepop software (47). HWE of genotypic frequencies was calculated by HWE calculator including analysis for ascertainment bias (48).
Chi-square was used for comparison between groups. Binary logistic regression was used to calculate the relative risk (Odds Ratio), with a 95% confidence interval.
p values <0.05 were considered as evidence of statistically significant association.