Endometrial Immune Profile in Recurrent Implantation Failure

Recurrent implantation failure (RIF) is a phenomenon specific to assisted reproductive technology, defined using the ESHRE threshold where the calculated cumulative chance of pregnancy exceeds 60 percent yet pregnancy does not occur without a known cause. True incidence is estimated at around 5 percent. While embryonic factors affecting implantation have been substantially addressed through developments such as preimplantation genetic testing for aneuploidy (PGT-A), there remains a lack of robust research into the uterine environment itself, hampered by inconsistent definitions and investigations without established evidence of benefit.

The immune system is known to mediate implantation by balancing pro-inflammatory and anti-inflammatory activity, both to permit implantation and to avoid rejection of the embryo. This study characterises the local, tissue-resident immune cell populations of the endometrium, examining not only the number of each immune cell type present but also their phenotype and function, using surface protein expression, transcriptomic profiling, and T cell receptor sequencing to assess activation state, functional subtype, and clonal expansion within each population.

Recruitment: RIF participants are identified at King's Fertility through internal database searches and clinical record review. Control participants who have had a spontaneous pregnancy are identified through review of 36 week gestation scan appointments at the Harris Birthright Centre, King's College Hospital, between 12 and 36 months prior. Eligible individuals are approached by a member of their direct care team by email and invited to a consent appointment, which can take place remotely.

Study procedure: The mid-luteal phase is identified using twice daily home urinary ovulation prediction tests from day 9 of the menstrual cycle. Participants attend a study visit 7 days after the luteinising hormone (LH) surge, where transvaginal ultrasound and venepuncture are used to confirm a corpus luteum and serum progesterone level consistent with the mid-luteal phase. An endometrial biopsy is then performed at the same visit under transabdominal ultrasound guidance. Where a participant consents to optional future use of samples, an additional blood sample of up to 18ml is collected on the same day, bringing total blood volume to up to 25ml where this option is taken. All procedures take place at King's Fertility, First Floor, The Fetal Medicine Research Institute, 16 to 20 Windsor Walk, Denmark Hill, London SE5 8BB.

Sample processing and analysis: Endometrial tissue and blood samples are transported to the King's College London Immunoregulation Laboratory, Guy's Campus, where endometrial biopsies are immediately cryopreserved and blood is processed to isolate cells from plasma before cryopreservation. Conventional flow cytometry and fluorescence activated cell sorting (FACS) are used for cell sorting and characterisation. CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by sequencing) allows simultaneous analysis of transcriptomic and surface protein expression profiles. Single cell RNA sequencing on the 10x Genomics platform, including T cell receptor (TCR) sequencing, is performed to assess immune cell phenotype and clonal expansion. Raw sequencing data are processed using CellRanger and analysed in Seurat (R environment), producing transcriptomic profile, surface protein expression, and TCR repertoire data. Differential expression and clonality analyses compare immune cell phenotypes between the RIF and control groups, integrating gene expression, surface phenotype, and lineage.

Translational aim: This work is intended to build a high resolution reference atlas of the non-receptive endometrium, allowing stratification of individuals affected by RIF according to underlying immune aetiology, and to identify immune cells or pathways that could inform future immune modulating therapeutic approaches, whether through upregulation or downregulation of a specific immune pathway.