Minimally invasive uterine aspiration 24 hours ahead of embryo transfer characterizes the compromised RIF uterine microenvironment and is predictive of reproductive outcome

Abstract

Repeat implantation failure (RIF) is particularly challenging to treat in ART, resulting in limited success even when adequate preparation of the endometrium is established and a transfer is performed with a high grade euploid blastocyst. The objective of this study was to utilize a multidisciplinary approach to decipher the complexity of RIF through investigations of the maternal molecular components ahead of an embryo transfer. Research study. Patients were recruited with IRB consent 24 hours prior to a programmed frozen embryo transfer (FET) with a euploid blastocyst. Uterine secretions were collected by gentle aspiration (∼2-5ul) under ultrasound guidance and grouped according to reproductive outcomes: Failed euploid FET (RIF patients, ≥3 prior IVF failures) and Positive live birth FET (maternally age-matched patients; mean 36.6 ±3.8 years). Total and small RNA (n = 22) was isolated for sequencing on the NovaSEQ 6000 (Illumina). Reads were aligned to hg38 using GSNAP and analyzed with edgeR (FDR cutoff of 5%; P<0.01). Metabolite analysis (n = 20) was performed by UHPLS-MS (Thermo) using MassMatrix and Maven (Princeton Univ). Proteomic analysis (n = 6) involved FASP digestion and LC-MS/MS, with protein identifications generated by Mascot (v 2.6) and Scaffold (v 4.8.9) (α of 0.05; fold change >1.5 or <0.5). A unique uterine microenvironment was observed for RIF patients and negative implantation outcomes 24 hours prior to an embryo transfer (P<0.05). An interplay of several biological processes were evident in RIF failed aspirates with a focused interest on 13 significantly reduced transcripts, 7 significantly increased maternal miRNAs, 12 significantly decreased amino acids and 16 proteins of significantly altered abundance (P<0.05). Specific examples included decreased expression of PLA2G4D (P<0.0001) which regulates the eicosanoid pathway, thereby impacting downstream synthesis of prostaglandins like PGE2. Decreased expression of TET1 (P<0.0001), an epigenetic regulator required for DNA methylation. Increased expression of miR-17, a known negative regulator of VEGFA, required for successful implantation (P<0.01). Decreased quantities of arginine, essential for blastocyst activation and trophectoderm motility (P<0.05). Lastly, an increased abundance of SERPING1, a protein associated with inflammation, which regulates complement activation (P<0.05). Analysis of uterine secretions 24 hours prior to FET, allowed for an in-depth molecular characterization of the compromised RIF uterine microenvironment and was predictive of reproductive outcome. The negative influence on key miRNAs and gene transcription levels, in addition to altered amino acid and protein concentrations, were all identified as critical contributors to poor RIF outcomes. Ongoing investigations into the relationships of these molecular networks will lead to the possibility of more effective clinical interventions for this difficult patient population.