Infliximab

FF was obtained from 16 women undergoing oocyte retrieval (for reasons other than hormonal infertility) who provided written informed consent, following approval by the institutional ethics committee. It was processed as described before and pooled to overcome batch effects [13] (link). Serum-supplemented culture medium was used as control for FF, hence an equivalent concentration of 1% Ultroser G (PALL Life Sciences, Cergy-Saint-Christophe, France) was added to the FF [13] (link). β-estradiol (Sigma Aldrich, St. Louis, MO, USA) was used at a final concentration of 10 nM for either 4 or 24 h. Fulvestrant (Sigma Aldrich, St. Louis, MO, USA) was used at a final concentration of 100 nM. Cells were exposed to the antagonist 24 h before exposure to FF pool and throughout 4 h of incubation with FF. TNFα (Sigma Aldrich, St. Louis, MO, USA) was used at a final concentration of 5 ng/ml for 4 h. Infliximab (Janssen Biotech, Titusville, NJ, USA) was used at a final concentration of 10 μg/ml for 4 h, and was mixed with the FF pool 1 h prior to the application on the cells, in order to achieve complete TNFα blockade.

Aspirin, celecoxib, and actinomycin d were purchased from Sigma Aldrich. Heparin was obtained from Fuso Pharmaceutical Industries. Ozagrel, 12-HHT, LY255283, and CAY10583 were purchased from Cayman Chemical Company. Methyl cellulose 400 was purchased from Wako Pure Chemical Industries. Recombinant human TNF was obtained from PeproTech. The human TNF-neutralizing antibody Infliximab was purchased from Janssen Biotech. The mouse TNF-neutralizing antibody D2H4 was purchased from Cell Signaling Technology. MMP-9 inhibitor I (an MMP-9–specific reagent) and MMP inhibitor II (a broad MMP inhibitor) were purchased from EMD Millipore.

Cell viability was evaluated using a Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan). Cells were seeded in 96-well plates and treated with infliximab (Janssen Pharmaceuticals, Inc., Horsham, PA, USA) and etanercept (Enbrel^®; Wyeth Pharmaceuticals; Pfizer, Inc., New York, NY, USA) in gradient concentrations (2, 4, 8, 16 or 32 µg/ml) for 48 h. Cells were incubated with CCK-8 solution for 1 h at 37°C, then absorbance at 450 nm was measured using an MRX II microplate reader (Dynex Technologies, Inc., Chantilly, VA, USA). Relative cell viability was calculated as a proportion of isotype controls according to the following formula: (Treatment - blank)/(control - blank) (22 (link)).

NSG mice were obtained from Charles River Laboratories [Sulzfeld, Germany]. Mice were kept under specific pathogen-free conditions in individually ventilated cages in a facility controlled according to the Federation of Laboratory Animal Science Association [FELASA] guidelines. Following engraftment on Day 1, mice were presensitised by rectal application of 150 µL of 10% ethanol on Day 8 using a 1-mm cat catheter [Henry Schein, Hamburg, Deutschland]. The catheter was lubricated with Xylocain©Gel 2% [AstraZeneca, Wedel]. Rectal application was performed under general anaesthesia using 4% isoflurane. Post-application mice were kept at an angle of 30° to avoid ethanol dripping. On Day 15, the mice were challenged by rectal application of 50% ethanol following the protocol used on Day 8. For the drug treatments, DES1 [D. E. Shaw Research, New York, USA] and tofacitinib [Merck KGaA, Darmstadt, Germany] were administered daily over one period of 3 days and a second period of 4 days [Days 7–9 and 14–17, respectively], such that each period encompassed one of the two ethanol challenges [on Days 8 and 15]. The dosages were 5 mg/kg/day for DES1 and 5 mg/kg/day for tofacitinib. The dosage of infliximab [Janssen Biotech, Horsham, Pennsylvania, USA] was 6 mg/kg/day, and treatment was limited to Days 7 and 14 only. All therapeutics were dissolved in PBS and administered intraperitoneally.