Cetrorelix acetate

In our study, cetrorelix acetate (450 μg/kg/day) (Sigma C5249, USA) with less side effects as a GnRH antagonist was preferred [44 (link)], and osteocalcin (Gla-OC) protein (10 μg/kg/day) (Fitzgerald 30R-3286, USA) was administered to the subjects orally [16 (link)]. Furthermore, EDS, used for adult Leydig cell elimination in postpubertal subjects, was synthesized at the Chemistry Department of Science Faculty in Cukurova University (Adana-Turkey) as specified by Jackson and Jackson (1984) [45 (link)]. Firstly, 75 mg of EDS was dissolved in 0.5 ml of dimethylsulfoxide (DMSO) where 1.5 ml of distilled water was added to prepare 2 ml of EDS solution injected intraperitoneally into the rats as 2 ml per kg.

FVB-Tg(CAG-luc,-GFP)L2G85Chco/J (stock number 008450|L2G85) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA), and were bred and maintained in specific pathogen-free facilities at the University of Edinburgh and the University of Warwick. A breeding stock of wild-type FVB mice was maintained to produce experimental cohorts of recipient mice, or bought from Charles River (UK). All experiments were permitted under licence by the UK Home Office and were approved by the University of Edinburgh and University of Warwick Animal Welfare and Ethical Review Bodies. Mice had access to food and water ad libitum. Ambient temperature and humidity were 21°C and 50%, respectively. To visualize bioluminescent endometriosis lesions, the substrate D-luciferin (1.5 mg/100 μl in PBS; Sigma-Aldrich, Dorset, UK) was injected s.c. prior to imaging. Female mice between the age of 8 and 12 weeks were used for the experiments. Mice with induced endometriosis were administered 20 mg/kg Cetrorelix acetate in dH₂O s.c. (Sigma-Aldrich) every 48 h. The dose was selected based on previous in vivo studies (Danforth et al., 2005 (link); Otto et al., 2012 (link)). For the Cetrorelix experiments, mice were randomly allocated to either vehicle or drug group, and the investigator performing von Frey testing was blinded to the experimental group.

Cell treatments were performed using GnRH purchased from SANOFI (Relefact LH-RH 0.1 mg), in the presence or in the absence of Cetrorelix acetate (C5249; Sigma-Aldrich, St. Louis, MO, USA), Ganirelix acetate (SML024; Sigma-Aldrich) or Teverelix [31 (link)]. Hemagglutinin-tagged GnRHR (GnRHR-HA)-expressing vector was previously described [58 (link)] and was used for inducing the overexpression of the receptor in cell lines.

Both gonadotropin-releasing hormone (GnRH) agonist (Lupron; Takeda Chemical Industries, Tokyo, Japan) and antagonist (cetrorelix acetate; Merck Serono, Darmstadt, Germany) protocols were used for controlled ovarian stimulation (COS). For ovarian stimulation, all patients received a recombinant follicle-stimulating hormone (FSH, Gonal-F; Merck Serono) from cycle day 3 until the diameter of the dominant follicle exceeded 18 mm, followed by a dual trigger, including 250 μg of human chorionic gonadotropin (hCG) (Ovidrel; EMD Serono, Rockland, MA, USA), and 0.2 mg of triptorelin (Decapeptyl; Ferring, Schleswig–Holstein, Germany) at 36 h before oocyte retrieval. Finally, COS procedures involving GnRH agonist and antagonist protocols, oocyte collection, and denudation were performed as previously described^{56 (link)}.