Epidermal growth factor (egf)

All the laboratory culture dishes used for the current study were purchased from SPL, Lifesciences unless stated otherwise. The dishes used for molecular analysis were purchased from Axygen (Corning, USA). Insulin-transferrin-selenium, phosphate buffer saline (PBS), epidermal growth factor (EGF) and Minimum Essential Medium Eagle–Alpha Modification (α-MEM) were purchased from PanEco (Russia). Fetal calf serum (FCS) was purchased from Hyclone (Thermo-Fischer, USA). Human Tubal Fluid was purchased from Irvine Scientific (USA). Gentamycin and ascorbic acid were purchased from Dalhimfarm (Russia). Human chorionic gonado-tropin (hCG) and follicle stimulating hormone (FSH) were purchased from Merck Serono (Switzerland). All other reagents were purchased from Sigma-Aldrich (USA) unless stated otherwise.

The cytotoxicity analysis of EOs on 10⁴ MRC-5 cells/well was performed in MEM-supplemented medium at 37 °C and 5% CO₂ seeded onto the test plates for 72 h. Cell viability was assessed fluorometrically after the addition of resazurin as described above. Cytotoxicity was also studied using MCF-10A cells cultured in medium supplemented with 5% donor horse serum (BioSera, Nuaille, France), 20 ng/mL epidermal growth factor (PanEco, Moscow, Russia), 0.5 µg/mL hydrocortisone (ChemCruz, Dallas, TX, USA), and 10 µg/mL insulin (PanEco) at 37 °C, 5% CO₂ and 80–85% humidity). Briefly, 60 × 10³ MCF-10A cells were seeded into 24-well plates in 900 μL of the medium, and then, plates were incubated for 24 h at 37 °C and 5% CO₂. Then, different concentrations of EOs were added, and the plates were incubated for 48 h under the same conditions. Cell viability was then measured by the MTT method as described above for cancer cell lines. Finally, in the model of PMM, 3 × 10⁵ cells/mL was treated with different concentrations of EOs over 48 h in the same conditions. Then, 15 μL of MTT solutions were added to each well, and, after 4 h of additional incubation in the same conditions, the supernatant was discarded, formazan crystals were dissolved with 100 μL of DMSO and the absorbance was obtained as described above.

Cetuximab (Erbitux), solution 5 mg/ml, was purchased from Merck, stored at 4 °C; erlotinib, dry powder, was purchased from Sigma-Aldrich, stored at -20 °C as 10 mM solution in DMSO. EGF, dry powder, was purchased from PanEco, stored at -20 °C.
Peripheral blood samples from seven unrelated healthy 23–64 years old female donors were collected in two 8-ml vacuette tubes containing pro-coagulant and gel (Greiner), and serum was prepared within 3–4 h upon blood collection: tubes were centrifuged at 2500 rpm for 15 min, sera were aliquoted and stored at -75 °C. For all the human biomaterials investigated, informed written consents to participate in this study and to communicate the results in the form of a scientific report were collected from the corresponding donors. The procedure of taking human materials, the consent procedure, and the design of this study were approved by the ethical committee of the Vitamed Clinical Center, Moscow.