Agarose gel

The primers of α-9 HPV E6 were designed by PRIMER version 5.0 and NCBI (National Center for Biotechnology Information) Primer Blast based on the reference sequences, the primers and reference sequences used for the molecular characterization analysis of α-9 HPV E6 were shown in Additional file 1: Table S1 and synthesized by TSINGKE (Chengdu, China). The PCR reaction system consists of 5 µl HPV DNA, 13.1 µl ddH₂O, 1 µl primers, 0.4 µl TransTaq DNA polymerase, 2.5 µl dNTPs, and 3 µl buffer. The reaction conditions were shown in Additional file 1: Table S1. The PCR products were visualized by gel electrophoresis in 2% agarose gel (Sangon Biotech Co., Ltd.). The target products of E6 were purified and sequenced by TSINGKE at least twice (Chengdu, China).

Following overnight fasting, a 5 ml venous blood sample was collected from the elbow, anticoagulated with EDTA Na₂ (Amresco, LLC, Solon, OH, USA) and stored at −80°C for the subsequent experiments. Genomic DNA was extracted from the blood using a TIANamp genomic DNA kit (Tiangen Biotech, Beijing, China), according to the manufacturer’s instructions, and the concentration was determined using ultraviolet spectrophotometry (UV3100; Hitachi, Ltd., Tokyo, Japan). PCR amplification of the GDF9, BMP15, FSHR and INHBB genes was performed using the primers shown in Table I, in a 25 μl reaction volume containing 1 μl DNA template, 12.5 μl PCR mix, 0.75 μl forward and reverse primers, 0.25 μl Taq DNA polymerase and 10.5 μl ddH₂O, under the PCR amplification conditions shown in Table I. The PCR was conducted on an ABI 3100 sequencer (Applied Biosystems, Foster City, CA, USA). The PCR products were confirmed using gel electrophoresis on a 2% agarose gel (Sangon Biotech Co., Ltd., Shanghai, China). All PCR reagents, primers and kits were purchased from Sangon Biotech Co., Ltd. Subsequently, the PCR products were sequenced for GDF9, the BMP15 gene protein coding region, INHBB gene exon 2 and the FSHR Ala307Thr and Ser680Asn variants at the Beijing Genomics Institute (Beijing, China) using a dideoxy chain termination method (27 (link)).