Abstract
Serval studies showed an increased uterine natural killer cell density in women with recurrent miscarriage. However, no study has previously investigated the density and clustering of major immune cells simultaneously in precisely timed endometrial specimen section of this group of women. This study aimed to investigate the profile of endometrial immune cells populations and clustering level simultaneously in women with recurrent miscarriage and compare the results to fertile controls. A total of 30 women with unexplained recurrent miscarriage and 30 fertile controls were included in this study. Endometrial biopsy was performed precisely 7 days after LH surge. The cells density was expressed as percentage of positive immune cell/total stromal cells and the clustering of different endometrial cells was measured by R language toolbox ‘spatstat’. Multiplex immunohistochemical method was employed to stain a panel of human endometrium samples simultaneously with antibodies against CD3 for T cells, CD20 for B cells, CD68 for macrophages and CD56 for uterine natural killer cells. The median CD3+, CD68+ and CD56+ cell density in the miscarriage group were significantly higher than those of the fertile controls. In addition, the clustering between CD56+ uterine natural killer cells and CD68+ macrophages in the miscarriage group was significantly increased compared with fertile controls. In conclusion, the significant change in numbers of three out of four endometrial immune cell density and a significant increase in clustering between CD68+ and CD56+ cells suggest that several immune cells and their interactions may be important in the function of the endometrium; abnormal interactions may predispose to recurrent miscarriage.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Arruvito L, Sanz M, Banham AH, Fainboim L (2007) Expansion of CD4 + CD25 + and FOXP3 + regulatory T cells during the follicular phase of the menstrual cycle: implications for human reproduction. J Immunol 178:2572–2578. https://doi.org/10.4049/jimmunol.178.4.2572
Basu S, Eriksson M, Pioli PA et al (2009) Human uterine NK cells interact with uterine macrophages via NKG2D upon stimulation with PAMPs. Am J Reprod Immunol 61:52–61. https://doi.org/10.1111/j.1600-0897.2008.00661.x
Berbic M, Schulke L, Markham R et al (2009) Macrophage expression in endometrium of women with and without endometriosis. Hum Reprod 24:325–332. https://doi.org/10.1093/humrep/den393
Biswas Shivhare S, Bulmer JN, Innes BA et al (2015) Menstrual cycle distribution of uterine natural killer cells is altered in heavy menstrual bleeding. J Reprod Immunol 112:88–94. https://doi.org/10.1016/j.jri.2015.09.001
Bulmer JN, Lash GE (2005) Human uterine natural killer cells: a reappraisal. Mol Immunol 42:511–521. https://doi.org/10.1016/j.molimm.2004.07.035
Bulmer JN, Morrison L, Longfellow M et al (1991) Granulated lymphocytes in human endometrium: histochemical and immunohistochemical studies. Hum Reprod 6:791–798
Carrington B, Sacks G, Regan L (2005) Recurrent miscarriage: pathophysiology and outcome. Curr Opin Obstet Gynecol 17:591–597
Carstens JL, De Sampaio PC, Yang D et al (2017) Spatial computation of intratumoral T cells correlates with survival of patients with pancreatic cancer. Nat Commun 8:1–13. https://doi.org/10.1038/ncomms15095
Chen X, Jiang L, Wang CC et al (2016) Hypoxia inducible factor and microvessels in peri-implantation endometrium of women with recurrent miscarriage. Fertil Steril 105:1496–1502.e4. https://doi.org/10.1016/j.fertnstert.2016.02.032
Chen X, Man GCW, Liu Y et al (2017) Physiological and pathological angiogenesis in endometrium at the time of embryo implantation. Am J Reprod Immunol 78:e12693
Chen X, Mariee N, Jiang L et al (2017) Measurement of uterine natural killer cell percentage in the periimplantation endometrium from fertile women and women with recurrent reproductive failure: establishment of a reference range. Am J Obstet Gynecol 217:680.e1–680.e6. https://doi.org/10.1016/j.ajog.2017.09.010
Disep B, Innes BA, Cochrane HR et al (2004) Immunohistochemical characterization of endometrial leucocytes in endometritis. Histopathology 45:625–632. https://doi.org/10.1111/j.1365-2559.2004.02052.x
Faas MM, de Vos P (2017) Uterine NK cells and macrophages in pregnancy. Placenta 56:44–52. https://doi.org/10.1016/j.placenta.2017.03.001
Flynn L, Byrne B, Carton J et al (2000) Menstrual cycle dependent fluctuations in NK and T-lymphocyte subsets from non-pregnant human endometrium. Am J Reprod Immunol 43:209–217. https://doi.org/10.1111/j.8755-8920.2000.430405.x
Baddeley A, Rubak E, Turner R (2015) Spatial point patterns - methodology and applications with R. CRC Press, Boca Raton. https://www.crcpress.com/Spatial-Point-Patterns-Methodology-and-Applications-with-R/Baddeley-Rubak-Turner/9781482210200
Givan AL, White HD, Stern JE et al (1997) Flow cytometric analysis of leukocytes in the human female reproductive tract: comparison of fallopian tube, uterus, cervix, and vagina. Am J Reprod Immunol 38:350–359
Hunt JS, Miller L, Platt JS (1998) Hormonal regulation of uterine macrophages. Dev Immunol 6:105–110. https://doi.org/10.1155/1998/87527
Jasper MJ, Tremellen KP, Robertson SA (2006) Primary unexplained infertility is associated with reduced expression of the T-regulatory cell transcription factor Foxp3 in endometrial tissue. Mol Hum Reprod 12:301–308. https://doi.org/10.1093/molehr/gal032
Jauniaux E, Farquharson RG, Christiansen OB et al (2006) Evidence-based guidelines for the investigation and medical treatment of recurrent miscarriage. Hum Reprod 21:2216–2222. https://doi.org/10.1093/humrep/del150
Johnson PM, Christmas SE, Vince GS (1999) Immunological aspects of implantation and implantation failure. Hum Reprod 14(Suppl 2):26–36. https://doi.org/10.1093/humrep/14.suppl_2.26
Kats R, Al-Akoum M, Guay S et al (2005) Cycle-dependent expression of macrophage migration inhibitory factor in the human endometrium. Hum Reprod 20:3518–3525. https://doi.org/10.1093/humrep/dei234
Lachapelle M-H, Miron P, Hemmings R, Roy DC (1996) Endometrial T, B, and NK Cells in Patients with Recurrent Spontaneous Abortion. J Immunol 156:4027–4034. https://doi.org/10.1901/jaba.2005.76-04
Lash GE, Bulmer JN, Li TC et al (2016) Standardisation of uterine natural killer (uNK) cell measurements in the endometrium of women with recurrent reproductive failure. J Reprod Immunol 116:50–59. https://doi.org/10.1016/j.jri.2016.04.290
Lash GE, Robson SC, Bulmer JN (2010) Review: functional role of uterine natural killer (uNK) cells in human early pregnancy decidua. Placenta 31:S87–S92. https://doi.org/10.1016/j.placenta.2009.12.022
Lea RG, Clark DA (1991) Macrophages and migratory cells in endometrium relevant to implantation. Baillieres Clin Obs Gynaecol 5(1):25–59
Li T-C, Rogers AW, Dockery P et al (1988) A new method of histologic dating of human endometrium in the luteal phase. Fertil Steril 50:52–60. https://doi.org/10.1016/S0015-0282(16)60008-0
Liu B, Mariee N, Laird S et al (2014) The prognostic value of uNK cell count and histological dating in the mid-luteal phase of women with reproductive failure. Eur J Obstet Gynecol Reprod Biol. https://doi.org/10.1016/j.ejogrb.2014.07.010
Liu Y, Phil M, Chen X et al (2018) Comparison of the prevalence of chronic endometritis as determined by means of different diagnostic methods in women with and without reproductive failure. Fertil Steril 109:832–839. https://doi.org/10.1016/j.fertnstert.2018.01.022
Manaster I, Mizrahi S, Goldman-Wohl D et al (2008) Endometrial NK cells are special immature cells that await pregnancy. J Immunol 181:1869–1876. https://doi.org/10.4049/jimmunol.181.3.1869
Marron K, Harrity C (2019) Endometrial lymphocyte concentrations in adverse reproductive outcome populations. J Assist Reprod Genet 36:837–846. https://doi.org/10.1007/s10815-019-01427-8
Marshall RJ, Jones DB (1988) An immunohistochemical study of lymphoid tissue in human endometrium. Int J Gynecol Pathol 7:225–235
Mei S, Tan J, Chen H et al (2010) Changes of CD4+CD25highregulatory T cells and FOXP3 expression in unexplained recurrent spontaneous abortion patients. Fertil Steril 94:2244–2247. https://doi.org/10.1016/j.fertnstert.2009.11.020
Mezheyeuski A, Bergsland CH, Backman M et al (2018) Multispectral imaging for quantitative and compartment-specific immune infiltrates reveals distinct immune profiles that classify lung cancer patients. J Pathol 244:421–431. https://doi.org/10.1002/path.5026
Michimata T, Ogasawara MS, Tsuda H et al (2002) Distributions of endometrial NK cells, B cells, T cells, and Th2/Tc2 cells fail to predict pregnancy outcome following recurrent abortion. Am J Reprod Immunol 47:196–202. https://doi.org/10.1034/j.1600-0897.2002.01048.x
Moffett-King A (2002) Natural killer cells and pregnancy. Nat Rev Immunol 2:656–663. https://doi.org/10.1038/nri886
Moffett A, Shreeve N (2015) First do no harm: uterine natural killer (NK) cells in assisted reproduction. Hum Reprod 30:1519–1525. https://doi.org/10.1093/humrep/dev098
Nakagawa K, Kwak-Kim J, Hisano M et al (2019) Obstetric and perinatal outcome of the women with repeated implantation failures or recurrent pregnancy losses who received pre- and post-conception tacrolimus treatment. Am J Reprod Immunol 82:1–8. https://doi.org/10.1111/aji.13142
Nedvetzki S, Sowinski S, Eagle RA et al (2007) Reciprocal regulation of human natural killer cells and macrophages associated with distinct immune synapses. Blood 109:3776–3785. https://doi.org/10.1182/blood-2006-10-052977
Noyes RW, Hertig AT, Rock J (1950) Dating the endometrial biopsy. Fertil Steril 1:3–25. https://doi.org/10.1016/S0015-0282(16)30062-0
Nyborg KM, Kolte AM, Larsen EC, Christiansen OB (2014) Immunomodulatory treatment with intravenous immunoglobulin and prednisone in patients with recurrent miscarriage and implantation failure after in vitro fertilization/intracytoplasmic sperm injection. Fertil Steril 102:1650–1655.e1. https://doi.org/10.1016/j.fertnstert.2014.08.029
Parra ER, Villalobos P, Behrens C et al (2018) Effect of neoadjuvant chemotherapy on the immune microenvironment in non-small cell lung carcinomas as determined by multiplex immunofluorescence and image analysis approaches. J Immunother Cancer 6:1–11. https://doi.org/10.1186/s40425-018-0368-0
Quenby S, Bates M, Doig T et al (1999) Pre-implantation endometrial leukocytes in women with recurrent miscarriage. Hum Reprod 14:2386–2391. https://doi.org/10.1093/humrep/14.9.2386
Quenby S, Kalumbi C, Bates M et al (2005) Prednisolone reduces preconceptual endometrial natural killer cells in women with recurrent miscarriage. Fertil Steril 84:980–984. https://doi.org/10.1016/j.fertnstert.2005.05.012
Quenby S, Nik H, Innes B et al (2009) Uterine natural killer cells and angiogenesis in recurrent reproductive failure. Hum Reprod 24:45–54. https://doi.org/10.1093/humrep/den348
Ramhorst R, García V, Agriello E et al (2003) Intracellular expression of CD69 in endometrial and peripheral T cells represents a useful marker in women with recurrent miscarriage: modulation after allogeneic leukocyte immunotherapy. Am J Reprod Immunol 49:149–158. https://doi.org/10.1034/j.1600-0897.2003.00021.x
Rao A, Kalluri R, Barua S et al (2017) Spatial computation of intratumoral T cells correlates with survival of patients with pancreatic cancer. Nat Commun 8:15095. https://doi.org/10.1038/ncomms15095
Renaud SJ, Graham CH (2008) The role of macrophages in utero-placental interactions during normal and pathological pregnancy. Immunol Invest 37:535–564
Ripley BD (1976) The second-order analysis of stationary point processes. J Appl Prob 266:255–266
Robson A, Harris LK, Innes BA et al (2012) Uterine natural killer cells initiate spiral artery remodeling in human pregnancy. FASEB J Off Publ Fed Am Soc Exp Biol 26:4876–4885. https://doi.org/10.1096/fj.12-210310
Royal College Obstetricians and Gynaecologists (RCOG) (2011) The investigation and treatment of couples with recurrent first-trimester and second-trimester miscarriage. R Coll Obstet Gynaecol. https://doi.org/10.4103/2230-8210.107834
Saito S, Miyazaki S, Sasaki Y (2006) Th1/Th2 balance of the implantation site in humans BT. In: Mor G (ed) Immunology of pregnancy. Springer, New York, pp 37–48
Salamonsen LA, Zhang J, Brasted M (2002) Leukocyte networks and human endometrial remodelling. J Reprod Immunol 57:95–108. https://doi.org/10.1016/S0165-0378(02)00011-6
Shimada S, Iwabuchi K, Kato EH et al (2003) No difference in natural-killer-T cell population, but Th2/Tc2 predominance in peripheral blood of recurrent aborters. Am J Reprod Immunol 50:334–339. https://doi.org/10.1034/j.1600-0897.2003.00059.x
Smith SD, Dunk CE, Aplin JD et al (2009) Evidence for immune cell involvement in decidual spiral arteriole remodeling in early human pregnancy. Am J Pathol 174:1959–1971. https://doi.org/10.2353/ajpath.2009.080995
Stephenson MD, Kutteh WH, Purkiss S et al (2010) Intravenous immunoglobulin and idiopathic secondary recurrent miscarriage: a multicentered randomized placebo-controlled trial. Hum Reprod 25:2203–2209. https://doi.org/10.1093/humrep/deq179
Stirrat GM (1990) Recurrent miscarriage I: definition and epidemiology. Lancet 336:673–675
Tang AW, Alfirevic Z, Quenby S (2011) Natural killer cells and pregnancy outcomes in women with recurrent miscarriage and infertility: a systematic review. Hum Reprod 26:1971–1980
Tang AW, Alfirevic Z, Turner MA et al (2013) A feasibility trial of screening women with idiopathic recurrent miscarriage for high uterine natural killer cell density and randomizing to prednisolone or placebo when pregnant. Hum Reprod 28:1743–1752. https://doi.org/10.1093/humrep/det117
Tuckerman E, Laird SM, Prakash A, Li TC (2007) Prognostic value of the measurement of uterine natural killer cells in the endometrium of women with recurrent miscarriage. Hum Reprod 22:2208–2213. https://doi.org/10.1093/humrep/dem141
Van Mourik MSM, Macklon NS, Heijnen CJ (2009) Embryonic implantation: cytokines, adhesion molecules, and immune cells in establishing an implantation environment. J Leukoc Biol 85:4–19
Vivier E, Tomasello E, Baratin M et al (2008) Functions of natural killer cells. Nat Immunol 9:503–510. https://doi.org/10.1038/ni1582
Yan Y, Bao S, Sheng S et al (2017) Insulin resistance in patients with recurrent pregnancy loss is associated with lymphocyte population aberration. Syst Biol Reprod Med 63:397–404. https://doi.org/10.1080/19396368.2017.1378936
Yang HL, Zhou WJ, Chang KK et al (2017) The crosstalk between endometrial stromal cells and macrophages impairs cytotoxicity of NK cells in endometriosis by secreting IL-10 and TGF-β. Reproduction. https://doi.org/10.1530/REP-17-0342
Yin Y, Han X, Shi Q et al (2012) Adoptive transfer of CD4+CD25+ regulatory T cells for prevention and treatment of spontaneous abortion. Eur J Obstet Gynecol Reprod Biol 161:177–181. https://doi.org/10.1016/j.ejogrb.2011.12.023
Acknowledgements
The authors wish to thank Mr. Guangcheng ZHANG (PhD candidate) in Department of Mathematics, Nanjing University for his assistance with the analysis of the spatial distribution analysis of four immune cells in endometrium using R language, and to thank research nurse Dr. Wing-Ching CHEUNG in Department of Obstetrics and Gynaecology, The Chinese University of Hong Kong for recruiting patients. We also wish to express our gratitude to Professor Susan Laird, Head of Department of Biosciences and Chemistry at Sheffield Hallam University, for reviewing this article and making suggestions to improve it.
Funding
This study was supported by Hong Kong Obstetrical and Gynecological Trust Fund in 2018 and Hong Kong Health and Medical Research Fund (04152786).
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interests.
Ethical approval
Approvals from Joint Chinses University of Hong Kong-New Territories East Cluster Clinical Research Ethics Committee was obtained for this study.
Informed consent
All the endometrial biopsies were collected with the informed consent of the participants.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
10735_2020_9908_MOESM1_ESM.tiff
Supplementary file1 Supplementary Fig 1 Graphical representation of the AUC levels based on an L-function calculated for the endometrial immune cells clustering level. (TIFF 671 kb)
Rights and permissions
About this article
Cite this article
Zhao, Y., Chen, X., Zhang, T. et al. The use of multiplex staining to measure the density and clustering of four endometrial immune cells around the implantation period in women with recurrent miscarriage: comparison with fertile controls. J Mol Hist 51, 593–603 (2020). https://doi.org/10.1007/s10735-020-09908-2
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10735-020-09908-2