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Intrauterine insemination of cultured peripheral blood mononuclear cells prior to embryo transfer improves clinical outcome for patients with repeated implantation failures

  • Aicha Madkour (a1) (a2) (a3), Nouzha Bouamoud (a2), Noureddine Louanjli (a1) (a3), Ismail Kaarouch (a1) (a2) (a3), Henri Copin (a4), Moncef Benkhalifa (a5) and Omar Sefrioui (a1)...

Implantation failure is a major limiting factor in assisted reproduction improvement. Dysfunction of embryo–maternal immuno-tolerance pathways may be responsible for repeated implantation failures. This fact is supported by immunotropic theory stipulating that maternal immune cells, essentially uterine CD56+ natural killer cells, are determinants of implantation success. In order to test this hypothesis, we applied endometrium immuno-modulation prior to fresh embryo transfer for patients with repeated implantation failures. Peripheral blood mononuclear cells were isolated from repeated implantation failure patients undergoing assisted reproductive technology cycles. On the day of ovulation induction, cells were isolated and then cultured for 3 days and transferred into the endometrium cavity prior to fresh embryo transfer. This immunotherapy was performed on 27 patients with repeated implantation failures and compared with another 27 patients who served as controls. Implantation and clinical pregnancy were increased significantly in the peripheral blood mononuclear cell test versus control (21.54, 44.44 vs. 8.62, 14.81%). This finding suggests a clear role for endometrium immuno-modulation and the inflammation process in implantation success. Our study showed the feasibility of intrauterine administration of autologous peripheral blood mononuclear cells as an effective therapy to improve clinical outcomes for patients with repeated implantation failures and who are undergoing in vitro fertilization cycles.

Corresponding author
All correspondence to: M Benkhalifa. Reproductive Biology and Medical Cytogenetics Laboratory, Regional University Hospital & School of Medicine, Picardie University Jules Verne. Amiens. France. Tel: +33 677867390. Fax: +33 130571934. e-mail:
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Abrahams, V.M., Straszewski-Chavez, S.L., Guller, S. & Mor, G. (2004). First trimester trophoblast cells secrete Fas ligand which induces immune cell apoptosis. Mol. Hum. Reprod. 10 (1), 5563.
Ashkar, A.A., Black, J.P., Wei, Q., He, H., Liang, L., Head, J.R. & Croy, B.A. (2003). Assessment of requirements for IL 15 and INF regulatory factors in uterine NK cell differentiation and function during pregnancy. J. Immunol. 171, 2937–44.
Basu, S., Eriksson, M., Pioli, P.A., Conejo-Garcia, J., Mselle, T.F., Yamamoto, S., Wira, C.R. & Sentman, C.L. (2009). Human uterine NK cells interact with uterine macrophages via NKG2D upon stimulation with PAMPs. Am. J. Reprod. Immunol. 61, 5261
Becknell, B. & Caligiuri, M.A. (2005). Interleukin-2, interleukin-15, and their roles in human natural killer cells. Adv. Immunol. 86, 209–39.
Bulmer, J.N. & Lash, G. E. (2005). Human uterine natural killer cells: a reappraisal. Mol. Immunol. 42, 511–21.
Bulmer, J.N., Lunny, D.P. & Hagin, S.V. (1988). Immunohistochemical characterization of stromal leucocytes in nonpregnant human endometrium. Am. J. Reprod. Immunol. Microbiol. 17, 8390.
Bulmer, J.N., Longfellow, M. & Ritson, A. (1991). Leukocytes and resident blood cells in endometrium. Ann. N. Y. Acad. Sci. 622, 5768.
Challis, J.R., Lockwood, C.J., Myatt, L., Norman, J.E., Strauss, J.F.III. & Petraglia, F. (2009). Inflammation and pregnancy. Reprod. Sci. 2, 206–15.
Chaouat, G., Dubanchet, S. & Ledee, N. (2007). Cytokines: important for implantation? J. Assist. Reprod. Genet. 11, 491505.
Chernyshov, V.P., Sudoma, I.O., Dons’koi, B.V., Kostyuchyk, A.A. & Masliy, Y.V. (2010). Elevated NK cell cytotoxicity, CD158a expression in NK cells and activated T lymphocytes in peripheral blood of women with IVF failures. Am. J. Reprod. Immunol. 64, 5867.
Coughlan, C., Ledger, W., Wang, Q., Demirol, A., Gurgan, T., Cutting, R., Ong, K., Sallam, H. & Li, T.C. (2014). Recurrent implantation failure: definition and management. Reprod. Biomed. Online 28, 1438.
Croy, B.A., Chantakru, S., Esadeg, S., Ashkar, A.A. & Wei, Q. (2002). Decidual natural killer cells: key regulators of placental development. J. Reprod. Immunol. 57 (1–2), 151–68.
Croy, B.A., Esadeg, S., Chantakru, S., van den Heuvel, M., Paffaro, V.A., He, H., Black, G.P., Ashkar, A.A., Kiso, Y. & Zhang, J. (2003). Update on pathways regulating the activation of uterine natural killer cells, their interactions with decidual spiral arteries and homing of their precursors to the uterus. J. Reprod. Immunol. 59, 175–91.
Darmochwal, K.D., Leszczynska, G.B., Rolinski, J. & Oleszczuk, J. (1999). T helper 1- and T helper 2-type cytokine imbalance in pregnant women with pre-eclampsia. Eur. J. Obstet. Gynecol. Reprod. Biol. 86, 165–70.
David Dong, Z.M., Aplin, A.C. & Nicosia, R.F. (2009). Regulation of angiogenesis by macrophages, dendritic cells, and circulating myelomonocytic cells. Curr. Pharm. Des. 15, 365379.
Dons’koi, B.V., Chernyshov, V.P., Sudoma, I.O., Honcharova, I.O. & Osypchuk, D.V. (2013a). Qualitative analysis of accented CD158a receptor expression in NK-lymphocytes in women with reproductive failures. Lik. Sprava. 1, 8693.
Dons’koi, B.V., Chernyshov, V.P., Sirenko, V.Y., Strelko, G.V. & Osypchuk, D.V. (2013b). Peripheral blood natural killer cells activation status determined by CD69 upregulation predicts implantation outcome in IVF. Immunobiology 219, 167–71.
Fettback, P.B.T., Domingues, T.S., Hassun Filho, P.A., Motta, E.L.A., Serafini, P.C. & Baracat, E.C. (2009). Endometrial natural killer cells: What are they? What do they do? What do we need to know? Femina 37, 373–8.
Fukui, A., Ntrivalas, E., Gilman-Sachs, A., Kwak-Kim, J., Lee, S.K., Levine, R. & Beaman, K. (2006). Expression of natural cytotoxicity receptors and a2V-ATPase on peripheral blood NK cell subsets in women with recurrent spontaneous abortions and implantation failures. Am. J. Reprod. Immunol. 56, 312–20.
Guenther, S., Vrekoussis, T., Heublein, S., Bayer, B., Anz, D., Knabl, J., Navrozoglou, I., Dian, D., Friese, K., Makrigiannakis, A., & Jeschke, U. (2012). Decidual macrophages are significantly increased in spontaneous miscarriages and over-express FasL: a potential role for macrophages in trophoblast apoptosis. Int. J. Mol. Sci. 13, 90699080
Hanna, J., Goldman-Wohl, D., Hamani, Y., Avraham, I., Greenfield, C., Natanson-Yaron, S., Prus, D., Cohen-Daniel, L., Arnon, T.I., Manaster, I., Gazit, R., Yutkin, V., Benharroch, D., Porgador, A., Keshet, E., Yagel, S. & Mandelboim, O. (2006). Decidual NK cells regulate key developmental processes at the human fetal–maternal interface. Nat. Med. 12, 1065–74.
Heublein, S., Lenhard, M., Vrekoussis, T., Schoepfer, J., Kuhn, C., Friese, K., Makrigiannakis, A., Mayr, D. & Jeschke, U. (2012). The G-protein-coupled estrogen receptor (GPER) is expressed in normal human ovaries and is upregulated in ovarian endometriosis and pelvic inflammatory disease involving the ovary. Reprod. Sci. 19, 1197–204.
Hill, J.A., Polgar, K. & Anderson, D.J. (1995). T-helper 1-type immunity to trophoblast in women with recurrent spontaneous abortion. J. Am. Med. Ass. 273, 1933–6.
Hutter, S., Heublein, S., Knabl, J., Andergassen, U., Vrekoussis, T., Makrigiannakis, A., Friese, K., Mayr, D. & Jeschke, U. (2013). Macrophages: are they involved in endometriosis, abortion and preeclampsia and how? J. Nippon. Med. Sch. 80, 97103.
Ideta, A., Sakai, S.I., Nakamura, Y., Urakawa, M., Hayama, K., Tsuchiya, K., Fujiwara, H. & Aoyagi, Y. (2010). Administration of peripheral blood mononuclear cells into the uterine horn to improve pregnancy rate following bovine embryo transfer. Anim. Reprod. Sci. 117, 1823.
Johnson, P.M., Christmas, S.E. & Vince, G.S. (1999). Immunological aspects of implantation and implantation failure. Hum. Reprod. 14, 2636.
Jokhi, P.P., King, A., Sharkey, A.M., Smith, S.K. & Loke, Y.W. (1994). Screening for cytokine messenger ribonucleic acids in purified human decidual lymphocyte populations by the reverse-transcriptase polymerase chain reaction. J. Immunol. 153, 4427–35.
Kachkache, M., Acker, G.M., Chaouat, G., Noun, A. & Garabedian, M. (1991). Hormonal and local factors control the immunohistochemical distribution of immunocytes in the rat uterus before conceptus implantation: effects of ovariectomy, fallopian tube section, and injection. Biol. Reprod. 45, 860–8.
Kalantaridou, S.N., Zoumakis, E., Weil, S., Lavasidis, L.G., Chrousos, G.P., Makrigiannakis, A. (2007). Reproductive corticotropin releasing hormone, implantation, and fetal immunotolerance. Crit. Rev. Clin. Lab. Sci. 44 (5–6), 461–81.
Karami, N., Boroujerdnia, M.G., Nikbakht, R. & Khodadadi, A. (2012). Enhancement of peripheral blood CD56dim cell and NK cell cytotoxicity in women with recurrent spontaneous abortion or in vitro fertilization failure. J. Reprod. Immunol. 95 (1–2), 8792.
Kimber, S.J. (2005). Leukaemia inhibitory factor in implantation and uterine biology. Reproduction 130, 131–45.
King, A. (2000). Uterine leukocytes and decidualization. Hum. Reprod. 6, 2836.
King, A., Jokhi, P.P., Smith, S.K., Sharkey, A.M. & Loke, Y.W. (1995). Screening for cytokine mRNA in human villous and extravillous trophoblasts using the reverse-transcriptase polymerase chain reaction (RT-PCR). Cytokine 7, 364–71.
Klentzeris, L.D., Li, T.C., Dockery, P. & Cooke, I.D. (1992). The endometrial biopsy as a predictive factor of pregnancy rate in women with unexplained infertility. Euro. J. Obst. Gynec. Reprod. Biol. 45, 119–24.
Lash, G.E. & Bulmer, J.N. (2011). Do uterine natural killer (uNK) cells contribute to female reproductive disorders? J. Reprod. Immunol. 88, 156–64.
Makrigiannakis, A., Minas, V., Kalantaridou, S.N., Nikas, G. & Chrousos, G.P. (2006). Hormonal and cytokine regulation of early implantation. Trends Endocrinol. Metab. 17, 178–85.
Makrigiannakis, A., Karamouti, M., Drakakis, P., Loutradis, D. & Antsaklis, A. (2008). Fetomaternal immunotolerance. Am. J. Reprod. Immunol. 60, 482–96.
Makrigiannakis, A., Petsas, G., Toth, B., Relakis, K. & Jeschke, U. (2011). Recent advances in understanding immunology of reproductive failure. J. Reprod. Immunol. 90, 96104.
McMaster, M.T., Newton, R.C., Dey, S.K. & Andrews, G.K. (1992). Activation and distribution of inflammatory cells in the mouse uterus during the preimplantation period. J. Immunol. 148, 1699–705.
Miyazaki, S., Tsuda, H., Sakai, M., Hori, S., Sasaki, Y., Futatani, T., Miyawaki, T. & Saito, S. (2003). Predominance of Th2-promoting dendritic cells in early human pregnancy decidua. J. Leukoc. Biol. 74, 514–22.
Mor, G., Cardenas, I., Abrahams, V. & Guller, S. (2011). Inflammation and pregnancy: the role of the immune system at the implantation site. Ann. N. Y. Acad. Sci. 1221, 80–7.
Nagamatsu, T. & Schust, D.J. (2010). The contribution of macrophages to normal and pathological pregnancies. Am. J. Reprod. Immunol. 63, 460–71.
Nakayama, T., Fujiwara, H., Maeda, M., Inoue, T., Yoshioka, S., Mori, T. & Fujii, S. (2002). Human peripheral blood mononuclear cells (PBMC) in early pregnancy promote embryo invasion in vitro: hCG enhances the effects of PBMC. Hum. Reprod. 17, 207–12.
Oh, M.J. & Croy, B.A.A. (2008). A map of relationships between uterine natural killer cells and progesterone receptor expressing cells during mouse pregnancy. Placenta. 29, 317–23.
Okitsu, O., Kiyokawa, M., Oda, T., Miyake, K., Sato, Y. & Fujiwara, H. (2011). Intrauterine administration of autologous peripheral blood mononuclear cells increases clinical pregnancy rates in frozen/thawed embryo transfer cycles of patients with repeated implantation failure. J. Reprod. Immunol. 92 (1–2), 82–7.
Pandey, M.K., Rani, R. & Agrawal, S. (2005). An update in recurrent spontaneous abortion. Arch. Gynecol. Obstet. 272, 95108.
Parham, P. (2004). NK cells and trophoblasts: partners in pregnancy. J. Exp. Med. 200, 951–5.
Perricone, R., De Carolis, C., Giacomelli, R., Guarino, M.D., De Sanctis, G. & Fontana, L. (2003). GM-CSF and pregnancy: evidence of significantly reduced blood concentrations in unexplained recurrent abortion efficiently reverted by intravenous immunoglobulin treatment, Am. J. Reprod. Immunol. 50, 232–7.
Raghupathy, R., Makhseed, M., Azizieh, F., Hassan, N., Al-Azemi, M. & Al-Shamali, E. (1999). Maternal Th1- and Th2-type reactivity to placental antigens in normal human pregnancy and unexplained recurrent spontaneous abortions. Cell. Immunol. 196, 122–30.
Rivera, R., Yacobson, I., & Grimes, D. (1999). The mechanism of hormonal contraceptives and intrauterine contraceptive devices. Am. J. Obstet. Gynecol. 181, 1263–69.
Sacks, G., Yang, Y., Gowen, E., Smith, S., Fay, L. & Chapman, M. (2012). Detailed analysis of peripheral blood natural killer cells in women with repeated IVF failure. Am. J. Reprod. Immunol. 67, 434–42.
Saito, S., Shima, T., Nakashima, A., Shiozaki, A., Ito, M. & Sasaki, Y. (2007). What is the role of regulatory T cells in the success of implantation and early pregnancy? J. Assist. Reprod. Gen. 24, 379–86.
Salamonsen, L.A. (2003). Tissue injury and repair in the female human reproductive tract. Reprod. 125, 301–11.
Sanford, T.R. & Wood, G.W. (1992). Expression of colony-stimulating factors and inflammatory cytokines in the uterus of CD1 mice during days 1 to 3 of pregnancy. J. Reprod. Fert. 94, 213–20.
Schulten, R.J., Lobl, R.T. & Ward, P. (1975). Neutrophils and the mechanism of IUD action in rats. Fertil. Steril. 26, 131–6.
Segerer, S., Kammerer, U., Kapp, M., Dietl, J. & Rieger, L. (2009). Upregulation of chemokine and cytokine production during pregnancy. Gynecol. Obstet. Invest. 67:145–50.
Simon, C., Moreno, C., Remohi, J. & Pellicer, A. (1998). Cytokines and embryo implantation. J. Reprod. Immunol. 39, 117–31.
Stewart, C.L., Kaspar, P., Brunet, L.J., Bhatt, H., Gadi, I., Köntgen, F. & Abbondanzo, S.J. (1992). Blastocyst implantation depends on maternal expression of leukemia inhibitory factor. Nature 359, 76–9.
Tabiasco, J., Rabot, M., Aguerre-Girr, M., El Costa, H., Berrebi, A., Parant, O., Laskarin, G., Juretic, K., Bensussan, A., Rukavina, D. & Le Bouteiller, P. (2006). Human decidual NK cells: unique phenotype and functional properties. Placenta 27, 34–9.
Toth, B., Haufe, T., Scholz, C., Kuhn, C., Friese, K., Karamouti, M., Makrigiannakis, A. & Jeschke, U. (2010). Placental interleukin-15 expression in recurrent miscarriage. Am. J. Reprod. Immunol. 64, 402–10.
Toth, B., Würfel, W., Germeyer, A., Hirv, K., Makrigiannakis, A. & Strowitzki, T. (2011). Disorders of implantation are there diagnostic and therapeutic options? J. Reprod. Immunol. 90, 117–23.
Vassiliadis, S., Ranella, A., Papadimitriou, L., Makrygiannakis, A. & Athanassakis, I. (1998). Serum levels of pro- and anti-inflammatory cytokines in non-pregnant women, during pregnancy, labor and abortion. Mediat. Inflamm. 7, 6972.
Wegmann, T.G., Lin, H., Guilbert, L. & Mosmann, T.R. (1993). Bidirectional cytokine interactions in the maternal–fetal relationship: is successful pregnancy a TH2 phenomenon? Immunol. Today 14, 353–6.
Wu, L., Luo, L.H., Zhang, Y.X., Li, Q., Xu, B., Zhou, G.X., Luan, H.B. & Liu, Y.S. (2014). Alteration of Th17 and Treg cells in patients with unexplained recurrent spontaneous abortion before and after lymphocyte immunization therapy. Reprod. Biol. Endocrino. 12, 74.
Yoshioka, S., Fujiwara, H., Nakayama, T., Kosaka, K., Mori, T. & Fujii, S. (2006). Intrauterine administration of autologous peripheral blood mononuclear cells promotes implantation rates in patients with repeated failure of IVF–embryo transfer. Hum. Reprod. 21, 3290–4.
Yu, N., Yang, J., Guo, Y., Fang, J., Yin, T., Luo, J., Li, X., Li, W., Zhao, Q., Zou, Y. & Xu, W. (2014). Intrauterine administration of peripheral blood mononuclear cells (PBMCs) improves endometrial receptivity in mice with embryonic implantation dysfunction. Am. J. Reprod. Immunol. 71, 2433.
Zhou, J., Wang, Z., Zhao, X., Wang, J., Sun, H. & Hu, Y. (2012). An increase of Treg cells in the peripheral blood is associated with a better in vitro fertilization treatment outcome. Am. J. Reprod. Immunol. 68,100–6.
Zhylkova, I., Feskov, A., Feskova, I., Somova, O. & Chumakova, N. (2010). Influence of peripheral blood mononuclear cells intrauterine transfer on implantation rates in patients with unsuccessful IVF cycles. Hum. Reprod. 25, P–257.
Ziebe, S., Loft, A., Povlsen, B.B., Erb, K., Agerholm, I., Aasted, M., Gabrielsen, A., Hnida, C., Zobel, D.P., Munding, B., Bendz, S.H. & Robertson, S.A. (2013). A randomized clinical trial to evaluate the effect of granulocyte–macrophage colony-stimulating factor (GM-CSF) in embryo culture medium for in vitro fertilization. Fertil. Steril. 99, 1600–9.
Zoumakis, E., Kalantaridou, S.N., Makrigiannakis, A. (2009). CRH-like peptides in human reproduction. Curr. Med. Chem. 16, 4230–5.
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