Participants and Tissue Collection

Eligible participants (n = 45) were women aged between 25–45 years, attending for gynaecological procedures or fertility clinics from August 2012 to December 2013. Baseline demographics and fertility characteristics were collected for all patients. There were two independent groups of cases. The first were women who had suffered with recurrent implantation failure (women with failure of clinical pregnancy following the transfer of three or more good quality fresh or frozen embryos transferred over two or more IVF or ICSI cycles) (n = 15). The second group were women who had undergone recurrent miscarriage (women who had suffered three or more spontaneous pregnancy losses at fewer than 24 completed weeks of pregnancy) (n = 15). The third group, who served as controls, were women who were attending for elective procedures for non-endometrial pathology (n = 15) and has had at least one full term pregnancy ending in live birth without a history of recurrent implantation failure or recurrent miscarriage as previously described. Endometrial biopsies were taken by suction curette (Pipelle device, Laboratoire CCD, Paris, France), washed in sterile saline, divided into uniform size (10mm lengths), snap frozen and stored at 80 °C in separate aliquots.

Total RNA was extracted from endometrial biopsies using the TRIzol (Thermo Fisher Scientific, USA). The A260/280 ratio of each sample was measured using mass spectrometry (NanoDrop; Thermo Fisher Scientific USA) and the total RNA concentration for each sample was calculated. Samples were stored at −80 °C until use. Total RNA was reverse transcribed to produce cDNA (Precision nanoScript RT kit; Primerdesign Ltd, UK) with Oligo-dT primers. The mRNA expression of the 10 genes listed in Table 3 were measured by qRT-PCR using primers and probes designed and made by Primerdesign Ltd (Southampton, UK). These sets of genes are from the geNorm Plus Reference Gene Selection kits. All samples were measured in duplicate using a LightCycler 480 Instrument (Roche Diagnostics, Germany). The optimised cycle parameters were 95 °C for 2 min, followed by 40 cycles of 95 °C for 15 seconds, 60 °C for 60 s.