Eclia

Data on demographics, type of cancer, cancer treatment, and FP options were obtained. FP methods included cryopreservation of ovarian tissue, ovarian stimulation with cryopreservation of fertilized and non-fertilized oocytes, transposition of the ovaries, and GnRH analogues. Hormone analysis included anti-Müllerian hormone (AMH, Elecsys® Roche), follicle-stimulating hormone (FSH, ECLIA® Roche), and estradiol (ECLIA® Roche) concentrations at time of FP and follow-up (mean time of follow-up: 70 ± 50 months). Patients underwent transvaginal ultrasound at time of FP and follow-up to estimate antral follicle count (AFC). Patients were asked about their menstrual cycle. A regular menstrual cycle was defined as a menstrual cycle length between 24 and 35 days.

All hormones were measured in our laboratory using commercial kits. Serum insulin was assessed by electrochemiluminescence immunoassay (ECLIA) (Roche Diagnostic), with a lower detection limit of 0.2 μUI/ml (< 1.39 pmol/L). Serum total testosterone was assessed by ECLIA (Roche Diagnostic), with a lower detection limit of < 2.50 ng/dL (< 0.087 nmol/L). Δ4-androstenedione was assessed by ELISA (ng/ml; Arnika, Milan, Italy; analytical sensitivity: 0.021 ng/ml). Chemiluminescence assays were used for DHEAS (μg/dl; Immulite, Diagnostic Products, Genoa, Italy; analytical sensitivity: 15 μg/dl) and serum SHBG (nmol/l; Immulite, Diagnostic Products, Genoa, Italy; analytical sensitivity: 0.015 nmol/l). Serum glucose levels were measured using an electrochemical system (Glucocard, Menarini Diagnostics, Florence, Italy). Total cholesterol, HDL, triglycerides, AST and ALT were measured in our laboratory using standard assays. LDL cholesterol levels were calculated with Friedewald's formula.

All biochemical data were collected after overnight fasting. Fasting glucose, ALT, AST, GGT, ALP, bilirubin, and lipid levels were measured in our centralized accredited laboratory with standard methods. Serum insulin was measured by electrochemiluminescence (ECLIA, Elecsys Insulin, Roche, Milan, Italy). The sensitivity of the method was 0.4 μU/mL. The normal range (μU/mL) was 2.6-24.9. Serum GH levels were measured by immunoassay in electrochemiluminescence (ECLIA, Elecsys hGH, Roche, Milan, Italy). The lower limit of detection of the assay was 0.030 μg/L. The intra- and interassay coefficients of variation (CV) were 0.6-5.0 and 3.8-5.0%, respectively. We reported GH concentrations in μg/L of IS 98/574. Serum IGF-I levels were measured by means of a chemiluminescent immunometric assay (Immulite 2000; Diagnostic Products Corp., Los Angeles, CA) using murine monoclonal anti-IGF-I antibodies. The standards were calibrated against the World Health Organization second IS 87/518. The sensitivity was 1.9 μg/L. The intra- and interassay CVs were 2.3-3.9% and 3.7-8.1%, respectively.

Serum estradiol (E2) was measured using an electrochemiluminescence immunoassay (ECLIA; Roche Diagnostics GmbH). Two bone turnover markers, carboxy terminal telopeptide (CTX) and procollagen type I N-terminal propeptide (PINP), were measured in serum samples via ECLIA (Roche diagnostics GmbH).
Levels of inflammatory markers, including interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), interleukin-1 (IL1), interleukin-4 (IL4), interleukin-6 (IL6), interleukin-10 (IL10), interleukin-12 (IL12), interleukin-17 (IL17) and interleukin-23 (IL23), were measured. The concentrations of the serum inflammatory markers OPG, RANK and RANKL in supernatants were assessed using an enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN).

The serum PIVKA‐II levels were determined by Chemiluminescent Enzyme Immunoassay (CLEIA) (Fujirebio Diagnostics, Tokyo, Japan, the limit of quantification: 6 mAU/mL, normal value≤40mAU/mL); the serum AFP levels were determined by Immunoassay in Electrochemistry Luminescence (ECLIA) (Roche Diagnostic GmbH, Mannheim, Germany, normal value≤7 ng/mL). The PIV‐AFP status was as follows: PIV‐AFP status 1, AFP≤7 ng/mL, PIVKA‐II≤40 mAU/mL; PIV‐AFP status 2, AFP≤7 ng/mL, PIVKA‐II > 40 mAU/mL; PIV‐AFP status 3, AFP >7 ng/mL, PIVKA‐II≤40 mAU/mL; PIV‐AFP status 4, AFP >7 ng/mL, PIVKA‐II > 40 mAU/mL.