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Endometrial lymphocyte concentrations in adverse reproductive outcome populations

  • Reproductive Physiology and Disease
  • Published:
Journal of Assisted Reproduction and Genetics Aims and scope Submit manuscript

Abstract

Purpose

The uterine immunophenotype is relatively poorly understood, with most studies reporting proportions/percentages. A novel technique to calculate local endometrial lymphocyte concentrations is described, and used to compare results between aetiological subgroups such as repeated implantation failure (RIF) and recurrent pregnancy loss (RPL) with male-factor controls.

Methods

455 patients had an endometrial biopsy performed. Background history on initial presentation was used to subdivide the population into RIF (n = 149), RPL (n = 121), primary (n = 76) and secondary infertility (n = 80). A control group was identified comprising male factor infertility aetiology with all female investigations normal (n = 29). Endometrial Tissue was assessed using a comprehensive multi-parameter panel. Lymphocyte subpopulations were calculated using flowcount flurospheres and a mathematical correction applied to determine concentrations per milligram of tissue, based on original biopsy weight and volumetric dilutions.

Results

The flow cytometry technique was successful in determining population centiles for concentrations of endometrial lymphocyte subsets. Distinct differences were noted across the patient groups. Th2 concentrations were significantly higher in the controls (p = 0.0002). All RPL/infertile populations had increased concentrations of peripheral type NK’s (p = 0.016) and B cells (p = 0.045). Relative to male factor controls, CD4+ and CD8+ T lymphocyte populations were increased in RPL patients, and reduced in those with a history of RIF. Th1 concentrations were elevated in the adverse outcome groups (p = 0.032). Concentration centiles alone do not appear to accurately predict outcome with subsequent treatment.

Conclusions

Endometrial biopsy analysis by flow cytometry can provide detailed analysis of constituent lymphocyte subsets by concentration as well as proportion. This novel approach provides additional independent data to further assess the significance of endometrial changes in the setting of reproductive failure.

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Abbreviations

PGT-A:

Preimplantation genetic analysis-aneuploidy

RPL:

Recurrent pregnancy loss

RM:

Recurrent miscarriage

RIF:

Repeat implantation failure

ART:

Assisted reproductive technologies

AMH:

Anti-Mullerian hormone

AFC:

Antral follicle count

HRT:

Hormone replacement therapy

uNK:

Uterine type natural killer cells

pNK:

Peripheral blood type natural killer cells

CD:

Cluster of differentiation

Th1:

T helper type 1 (pro-inflammatory)

Th2:

T helper type 2 (anti-inflammatory)

TNFa:

Tumour necrosis factor alpha

GCSF:

Growth colony stimulating factor

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Author information

Authors and Affiliations

  1. Sims IVF Clinic, Clonskeagh Road, Clonskeagh, Dublin 14, Ireland

    Kevin Marron

  2. RCSI, Beaumont Hospital, Dublin, Ireland

    Conor Harrity

Authors
  1. Kevin Marron
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  2. Conor Harrity
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Corresponding author

Correspondence to Kevin Marron.

Ethics declarations

Advanced approval for the study was obtained from the clinic’s institutional review board, with individual written patient informed consent for the biopsy procedure and subsequent analysis taken, and recorded in the medical chart.

Informed consent

Informed consent was obtained from all individual participants included in the study.

Conflict of interest

The authors declare that they have no conflict of interest.

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Electronic supplementary material

ESM 1

Co-localisation of CD45 lymphocytes with 7-AAD illustrating only live endometrial derived cells are analysed in the lymphocytes gate (JPG 460 kb)

ESM 2

Illustration of mathematical adjustment accounting for biopys weight and dilution in conjunction with flow count fluorospheres to allow the cells to be expressed in a standardised mg format (DOCX 26 kb)

ESM 3

Fluorophores used, detectors and NAVIOS™cytometer compensation settings (DOCX 615 kb)

ESM 4

Flow cytometer images illustrating, in one individual RPL patient, the gating strategy for, in the first instance, (A) dissociated CD45+ lymphocytes separated from the other stromal, epithelial and morphonuclear cells of the endometria. And the cell types investigated, (B) Natural killer Tcells (CD3+ CD56+), (C) peripheral type natural killer cells (CD16+ CD56dim+), uterine natural killer cells (CD16- CD56bright) and (D) CD57+ natural killer cells entirely associated with the CD56dim NK’s (JPG 1485 kb)

ESM 5

broadens the markers into (A) B-cells (CD19+), (B) CD4+ and CD8+ cells, as they appear in the broad lymphocyte gate and (C) as they appear in a CD3+ Tcell gate. Panel D shows the CD4+ subtypes in their various subclasses. Gating here is more difficult and based on individual FMO findings. Panel E illustrates T regulatory cells (CD3+, CD4+ CD25+ CD127dim) (JPG 1457 kb)

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Marron, K., Harrity, C. Endometrial lymphocyte concentrations in adverse reproductive outcome populations. J Assist Reprod Genet 36, 837–846 (2019). https://doi.org/10.1007/s10815-019-01427-8

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  • DOI: https://doi.org/10.1007/s10815-019-01427-8

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