The study protocol was approved by the Institutional Review Board of the Department and was conducted. in accordance with the 1975 Declaration of Helsinki.

We studied the clinical files of 230 patients who, from April 2015 to April 2017, underwent LEEP for CIN2+ at the Colposcopy Outpatient Service of the Gynaecological/Obstetrics Unit at the Policlinico Universitario, Catania (University of Catania, Italy) and who satisfied the following inclusion criteria:

a) Patients with histological diagnosis of CIN2+; b) Patients who had an HPV test before and after treatment; c) Patients who had no anti-HPV vaccination; d) Patients without pathologies of the immune system; e) Patients who had completed at least two-years of follow-up.

Moreover, patients were excluded if they were HPV negative or had any suspicion of infiltrating neoplastic pathologie.

Only 192 patients satisfied the inclusion criteria; their clinical data was collected, of which: patient’s age, type of pathology, HPV strain, treatment method used, resection margins, follow-up cervical histology, date of follow-up, pretreatment viral genotype, post-treatment HPV genotype, and recurrent cases.

All the patients underwent conservative surgical treatment of cone excision (conization) electrosurgery with conization (LEEP) of 20 mm width and 12, 15 or 20 mm depth (Utah Medical Products, Midvale, Utah, USA).

The histological examination of the cervical cone established a definitive histological diagnosis and evaluated the extension of the cone margins, defined as positive if the distance between the CIN lesion and the margin of the resection was less than 1 mm.

The follow-up was carried out every 6 months during the first year after treatment and then once to year (At the moment of excisional treatment (T0) and the successive follow-ups at 6 (T1), 12 (T2), 18 (T3) and 24 (T4) and 30 (T5) months). Abnormal results during follow-up were confirmed histologically and considered recurrent high-grade intraepithelial cervical lesions (CIN2/CIN3 or CIS).

At each follow-up examination the patients were examined by conventional Pap test, HPV test and colposcopy, moreover, cytological eso-endocervical samples were taken and placed in ThinPrep Solution. The samples were sent to the laboratory for extraction of total DNA for the genotyping of viral DNA by means of genetic amplification followed by hybridization with genotype-specific probes able to identify most HPV genotypes of the genital region [28 genotypes of high-risk HPV (16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, 82), low-risk (6, 11, 40, 43, 44, 54, 70) and not defined risk (69,71,74)].

The commercial method used was NucliSenseasy MAG system bio Merieux SA, Marct l’Etoile, France.

The HPV genotype was classified as follows: 1) negative, 2) HPV 16, 3) hr HPV (HPV18, 31, 33, 45 and other hr HPV).

Colposcopy was carried out using a Zeiss OPM1F colposcope (Carl Zeiss, Jena, Germany) and applying acetic acid and a Lugol iodine solution. Any colposcopic abnormality was classified based on the nomenclature proposed by the International Federation for Colposcopy and Cervical Pathologies (IFCPC) in 3 grades of increasing anomalies based on severity: Zone of Abnormal Transformation (AnTZ) grade 1 (AnTZ1) and grade 2 (AnTZ2), or cancer. We evaluated the visibility of the squamo-columnar junction and specific biopsies were taken from the portio and/or endocervical curettage to guide the diagnosis in cases of an abnormal Pap test or suspicion of residual/recurrent disease at colposcopy.

Statistical analysis

Statistical analysis was performed by using the SPSS software package for Windows (version 15.0, SPSS, Chicago, IL, USA). Descriptive statistics are expressed as frequency, arithmetic mean, standard deviation (S.D.) and percentages.

We calculated significance (P < 0.5) with the Easy Fischer Test.

We calculated the Odds Ratio (OR) of women with peristent HPV 16 infection and positive margin, to have a recurrence.