Advia 2400 chemistry system

Plasma determinations were performed at the laboratory of Nephrology at the Gómez‐Ulla hospital and at the Biochemistry Laboratory at Fundación Jiménez Díaz. The investigators who performed the laboratory studies were unaware of clinical data. Plasma calcidiol levels were quantified by chemiluminescent immunoassay (CLIA) on the LIAISON XL analyser (LIAISON 25OH‐Vitamin D total Assay DiaSorin, Saluggia, Italy), FGF23 was measured by an enzyme‐linked immunosorbent assay which recognizes epitopes within the carboxyl‐terminal portion of FGF‐23 (Human FGF23, C‐Term, Immutopics Inc, San Clemente, CA), klotho levels by ELISA (Human soluble alpha klotho assay kit, Immuno‐Biological Laboratories Co., Japan), intact parathormone was analysed by a second‐generation automated chemiluminescent method (Elecsys 2010 platform, Roche Diagnostics, Mannheim, Germany), phosphate was determined by an enzymatic method (Integra 400 analyser, Roche Diagnostics, Mannheim, Germany), and high‐sensitivity C‐reactive (hs‐CRP) protein was assessed by latex‐enhanced immunoturbidimetry (ADVIA 2400 Chemistry System, Siemens, Germany). Lipids, glucose, and creatinine determinations were performed by standard methods (ADVIA 2400 Chemistry System, Siemens, Germany). The estimated glomerular filtration rate (eGFR) was calculated using the Chronic Kidney Disease Epidemiology Collaboration (CKD‐EPI) equation.

Of the two ovaries removed, one was fixed in 10% formaldehyde solution for histological examination, while the second was dry stored at −80°C after wrapping in aluminum foil for biochemical assays. Tumor necrosis factor-alpha (TNF-α), total antioxidant status (TAS), total oxidant status (TOS), and malondialdehyde (MDA) levels were determined in the supernatant following their respective methodology. Blood samples were centrifuged at 3,000 rpm for 10 min to separate the serum, which were then placed into Eppendorf tubes and stored at −80°C. Anti-mullerian hormone (AMH) assay was performed using rat AMH ELISA kits (Elabscience, catalogue no: E-EL-R0640). For TNF-α estimations, rat TNF-α ELISA kit (Boster Immuno leader, Boster Biological Technology Co., Ltd. Pleasanton, CA, USA, catalogue no; EK0526) was utilized. The ovarian tissues were prepared for homogenization by washing each tissue with cold saline. The resultant supernatants were removed and stored in sterile tubes at −80°C for tissue TAS and TOS analyses. An autoanalyzer (Siemens Advia 2400 Chemistry System, Siemens, Tokyo, Japan) and Reel Assay Total Antioxidant Status Test Kits were used for TAS and TOS determinations in accordance with standard kit procedures at the Central Laboratory of Firat University Hospital. All blood and tissue samples were stored and analyzed in the Firat University Biochemical Laboratory.