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Immunomodulators to treat recurrent miscarriage
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EURO-8674; No. of Pages 4 European Journal of Obstetrics & Gynecology and Reproductive Biology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Invited editorial
Immunomodulators to treat recurrent miscarriage
A B S T R A C T
Keywords: Recurrent miscarriage Immune system Therapy Review
Recurrent miscarriage is a reproductive disorder affecting many couples. Although several factors are associated with recurrent miscarriage, in more than 50% of the cases the cause is unknown. Maladaptation of the maternal immune system is associated with recurrent miscarriage and could explain part of its pathophysiology. Modulating the maternal immune system toward pregnancy tolerance could benefit pregnancy outcome. Although there is a clear scientific rationale that modulating the maternal immune system could benefit recurrent miscarriage, only a few studies suggest possible beneficial effects of immune modulators as a therapy for recurrent miscarriage. Therapies skewing the maternal immune response to a tolerating regulatory T cell rich environment seem especially promising; however, more research is needed to find effective and safe maternal immune modulators for reproductive pathologies as recurrent miscarriage. Moreover, the possible side effects on maternal, fetal, and neonatal immune function are essentially unknown, and its elucidation is crucial before any possible therapeutic strategies could be clinically implemented. ß 2014 Elsevier Ireland Ltd. All rights reserved.
Introduction Recurrent miscarriage is a reproductive disorder distressing many couples. Depending on the definition used, it is estimated that about 1–3% of couples seeking to reproduce are affected by it [1,2]. In contrast to a sporadic miscarriage which is often caused by a conceptus having structural malformations or chromosomal abnormalities, the causes of recurrent miscarriage are far from completely understood [1]. Although some factors are clearly associated with recurrent miscarriage, most cases are idiopathic, and no possible cause is evident. The maternal immune system plays an important role in the success of pregnancy. Optimal regulation of the maternal immune system is essential to tolerate the allogeneic fetus. Improper regulation leads to several reproductive disorders, such as recurrent miscarriage [1]. The complex mechanisms behind the tolerance toward the fetus are not fully elucidated [3]. The important regulating role of the maternal immune system gives the female reproductive system the possibility to accommodate or reject a conceptus depending on maternal, environmental, and fetal factors [4]. Since the maternal immune system is implicated in the pathogenesis of recurrent miscarriage, modulation of the maternal immune system holds potential, and gives hope for treatment and prevention of recurrent miscarriage. Immune modulators being
able to skew the maternal immune system toward fetal tolerance could benefit pregnancy outcome. However, skewing the maternal immune system toward tolerance could also result in tolerance of fetuses which would have been rejected rightfully by the maternal quality control system. Moreover, modulation of a pregnancy immune environment could very well influence fetal and neonatal development and have a role in the priming of the fetal immune system [5]. Furthermore, modulation of the maternal immune system could cause long term effects, causing the mother to be vulnerable for immune associated diseases in later live. Although no evidence of long term maternal or fetal effects has been found yet, caution is essential before implying immune modulating therapies in clinical settings without a proven and clear beneficial effect, and without evidence of safety. This short review aims to examine and highlight some of the possibilities of modulation of the maternal immune system as a treatment option for recurrent miscarriage. Methods For this review, papers were included using combinations of the following search terms in Pubmed: ‘recurrent miscarriage, recurrent abortions, deciduas, immune, placenta, pregnancy, abortion, regulatory T cells, Treg cells, paternal, NK cells, cytokines, Th17 cells, Th1 cells, progesterone, G-CSF, GM-CSF, (immune)
http://dx.doi.org/10.1016/j.ejogrb.2014.07.038 0301-2115/ß 2014 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: Prins JR, et al. Immunomodulators to treat recurrent miscarriage. Eur J Obstet Gynecol (2014), http:// dx.doi.org/10.1016/j.ejogrb.2014.07.038
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therapy, and prevention’. Systematic reviews were used for additional search through their reference list. Most (immune) therapies for recurrent miscarriage have been reviewed separately recently in meta-analyses, these Cochrane meta-analyses were also used in this review. Results Recurrent miscarriage Recurrent miscarriage is a disorder which is not consistently defined within existing literature. Most studies define recurrent miscarriage as three or more consecutive miscarriages before 20 weeks of gestation [1,6]. Depending on the definition it is estimated that 1–3% of couples seeking to reproduce are affected by it [1,2]. Although several causes are associated with recurrent miscarriage including anatomical anomalies, genetic abnormalities, thrombophilia, lifestyle, and hormonal dysfunction, in more than 50% of the couples suffering from recurrent miscarriage no such associations are found [1,7,8]. Most likely, a part of these unexplained miscarriages is caused by malfunctioning of maternal immune regulation. Immunology during pregnancy It is known that regulation of the maternal immune system is essential to prevent rejection of the fetus. Several mechanisms are involved in the induction of maternal tolerance, and as adequate regulation is important, most likely redundant mechanisms prevent rejection of the fetus. Besides the immune changes seen locally at the fetal maternal interface, peripheral immune responses are also altered during pregnancy [9]. Regulatory T (Treg) cells are a key player within the regulation of maternal immune tolerance [10]. Lower numbers and altered function of Treg cells have been associated with adverse pregnancy outcomes in both human and animal studies, and it has been shown that adequate numbers and function of Treg cells are essential for pregnancy success [10]. During pregnancy the balance of T cell subsets is essential for preventing fetal rejection, for example increased levels of Th1 and Th17 related cells and cytokines are associated with adverse pregnancy outcomes. Depending on the cytokine environment, naı¨ve T cells will differentiate to a T cell subset. Other immune cells which are important during pregnancy are NK cells and macrophages [3]. NK cells are regarded as important in the regulation of vascularization and invasion processes during pregnancy [11]. All immune cells work together to ensure fetal tolerance. A good example of this is the recent finding that NK cells are able to inhibit the Th17 cells, and with that skew the maternal immune system toward tolerance [12]. The mechanisms causing the shift of the maternal immune system toward fetal tolerance are not fully known, but most likely endogenous and exogenous factors, including exposure to semen, hormones, and lifestyle, modulate the maternal immune system toward tolerance [10]. The precise mechanisms of maternal fetal tolerance are complex and numerous; however, they are out of the scope of this review. Maternal immunology in recurrent miscarriage Several immunological mechanisms have been associated with recurrent miscarriage [1,13]. Most studies indicate higher levels of activated immune cells and associated cytokines, and lower levels of regulatory cells and associated cytokines in women with recurrent miscarriage. Differences have been found both locally as well as in the peripheral blood. Tissue recovered after both spontaneous as well as induced abortions showed lower levels of
Treg cells in spontaneous miscarriages [14,15]. Other studies showed that women with recurrent miscarriage have lower levels of Treg cells in their peripheral blood and also reduced suppressive function of Treg cells compared to a control group [14,16]. These Treg cells could also be less stable, as gene expression of certain genes related to Treg cell stability was less in women with recurrent miscarriage [17]. Other evidence implying Treg cells in the pathophysiology of recurrent miscarriage are altered Treg/ Th17 balances found in recurrent miscarriage. Higher levels of Th17 cells and Th17 associated cytokines have been found in decidual tissue and peripheral blood of women with recurrent miscarriage [18,19], indicating an imbalance between Treg and Th17 cells in these women [20]. Most likely the regulation of this balance is dysfunctional, as it has been shown that peripheral blood mononuclear cells from women with recurrent miscarriage show greater proliferation of Th17 and fewer Treg cells once cultured in the presence of sperm antigens from their male partner [17]. An interesting question would be whether this inadequate skewing of the maternal immune system is also influenced by paternal factors, and is caused by differences in semen constituents. It is known that seminal fluid induces Treg cell generation, and that the duration of sexual relationship is related to pregnancy outcome [21,22]. Inadequate immune modulating factors in seminal fluid could lead to inadequate adaptation of the maternal immune system, and make a couple prone for recurrent miscarriage. More studies analyzing the role of semen constituents in couples with recurrent miscarriage are needed. Another mechanism which is also implied in the immune regulation in recurrent miscarriage is altered Interleukin 6 (IL6) signaling. IL6 is known to influence the differentiation of naı¨ve T cells away from Treg cells toward Th17 cells [23]. Studies have found lower levels of IL6 in endometrial tissue in the mid-secretory phase of the cycle, together with increased serum levels of IL6 in women with recurrent miscarriage compared to controls [24–27]. The higher levels of IL6 could explain the finding that sera from women with recurrent miscarriage inhibit the expansion of the Treg cell populations [24]. Other immune cells associated with recurrent miscarriage are NK cells and their cytokines [28–30]. Although studies differ in their methods, most studies found higher levels of NK cells in the peripheral blood of women with recurrent miscarriage [30–32], whereas also an altered balance in inhibitory and activating killer immunoglobulin-like receptors (KIR) was found [24,33–35]. KIRs are cell proteins on the surface of NK cells. Whereas inhibitory KIRs prevent activation, activating KIRs lead to cytotoxic activity [36]. Altered balances in these KIRs could lead to an improper activation of the NK cells. More evidence for NK cell dysfunction in recurrent miscarriage comes from a finding that NK cells were partially impaired in women with recurrent miscarriage, and with that were less successful in inhibiting Th17 cells [12]. Immune modulators Since the maternal immune system is implied in the etiology of part of the unexplained recurrent miscarriages, several immune modulating therapies have been investigated, including aspirin, paternal leukocyte immunization, progesterone, and steroids [37]. As the causes of recurrent miscarriage are multi factorial, selection of couples by immunological analysis could improve therapeutic outcomes and therefore influence the results of the different studies. However, in only a few studies pre-immune screening of the subjects was performed. Most studies used either paternal white cell immunization (12 trials, 641 women, Peto OR 1.23, 95% confidence interval (CI) 0.89 to 1.70) or intravenous administration of immunoglobulins (eight trials, 303 women, Peto OR 0.98, 95% CI 0.61 to 1.58) as therapy [37]. Although a Cochrane review of 2006
Please cite this article in press as: Prins JR, et al. Immunomodulators to treat recurrent miscarriage. Eur J Obstet Gynecol (2014), http:// dx.doi.org/10.1016/j.ejogrb.2014.07.038
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concluded that overall these therapies do not improve live birth rate in women with recurrent miscarriage [37], some studies did find normalization of immune parameters in women with recurrent miscarriage. Methods between the studies vary and it can be argued that immunizing the women by injecting paternal cells intravenously is immunologically not an optimal method as most of the immune reaction will be cytotoxic driven. Immunizing subcutaneously could improve results, as this shows similarities to the ‘natural’ immunizing method (coitus), and dendrictic cells and macrophages will present the antigens [22]. Interestingly, a few studies have shown that once immunizing recurrent miscarriage patients with mononuclear cells from their husbands, levels of IL6 decrease to normal levels [24,25,38]. More interestingly, immunization of women with recurrent miscarriage with paternal cells led to an increase in the Treg cell frequency comparable to levels of fertile women [24]. In mice models it has been shown that after increasing the levels of Treg cells abortion prone mice are protected against fetal loss [28,39]. Although these latter results hold potential, there is no evidence yet that immunizing with paternal cells does improve pregnancy outcome. Future studies should consider using the subcutaneously route to immunize women. Because of the role of Treg cells in recurrent miscarriage, several other strategies have been used to improve their levels. The use of granulocyte colony stimulating factor (G-CSF) has been evaluated for the treatment of recurrent miscarriage. G-CSF and GM-CSF are cytokines known to recruit and activate tolerogeneic dendritic cells which are implied in the generation of Treg cells [10,39,40]. A small study showed a reduction in the incidence of miscarriage in a following pregnancy in women treated with G-CSF (odds ratio 5.1; 95% CI 1.5–18.4), together with an increase in levels of HCG [41]. As these results were only obtained in small studies, these results have to be confirmed in a larger randomized trial. The use of progesterone is of recent interest. Progesterone suppresses Th17 cell responses and enhances development and possible function of Treg cells [42,43]. A recent Cochrane review stated that there is no evidence that progesterone could prevent miscarriage [44]. Interestingly, a subgroup analysis, only analyzing women with recurrent miscarriage, within this Cochrane review of four trials analyzing the effect of progesterone in women with recurrent miscarriage showed a significant decrease in miscarriage (total of 225 women, Peto OR 0.39; 95% CI 0.21 to 0.72) [7,44–47]. However, most of this data is over 50 years old and these results should be confirmed in larger randomized clinical trials. Other strategies include therapies to modulate NK cell numbers. In patients with recurrent miscarriage several abnormalities were demonstrated in cellular immunity and increased numbers of NK cells were found in peripheral blood. Since prednisolone decreases numbers of NK cells in women with recurrent miscarriage [48], a recent pilot feasibility study administered prednisolone or a placebo to women with recurrent miscarriage, in this study no difference was found in live birth rate (95% CI 0.79–2.86) [49]. There is also no evidence for a beneficial treatment effect of intravenous immunoglobulins in recurrent miscarriage patients [37]. One study analyzed the effects of intravenous immunoglobulin on levels of Treg and Th17 cells in women with recurrent miscarriage with prescreened immune abnormalities [50]. Levels of Treg cells and Th17 cells did not change in the whole patient group, however, when subjects were divided in groups based on their immune cell levels, intravenous immunoglobulins led to higher levels of Treg cells in the lowest Treg cell number group and lower levels of Th17 cells in the higher Th17 cell number groups [50]. Although this study shows that administration of immunoglobulins could restore part of an immune balance in part of recurrent miscarriage there was no effect seen on pregnancy rate
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[50]. However, this was not a double blind placebo controlled trial, therefore further evaluation is needed. Being able to influence both immune and haemostatic characteristics, the effect of aspirin on recurrent miscarriage has been evaluated. No improvement in live birth rate was however observed after administration of aspirin with or without a low molecular weight heparin [51]. To summarize the above, there is no clear evidence yet that immune modulation benefit couples with recurrent miscarriage. Since the causes of recurrent miscarriage are multi factorial, inclusion and treatment of patients who are likely to benefit from immune modulating therapy is essential. Adequate screening of both maternal immune cell subsets and their function and possibly also immune skewing factors in semen could define couples in which the immune system might be the cause of recurrent miscarriage. In these cases, specific immunotherapy might benefit pregnancy outcome. However, similar to the therapeutic options, no guidelines toward specific immune screening have been made yet. Possible immune therapies have to be proven safe for both mother and child. During pregnancy the fetal immune system is shaped, and most likely the fetal immune system is influenced by maternal immune factors. There is evidence that prenatal maternal immune factors could influence the fetal immune system, and with that leading to fetal immune programming [5]. As modulating the fetal immune system via maternal modulation could lead to for example allergies, cardiovascular diseases in later life, and could also have an influence on neurodevelopment, great care is needed before administering modulators of the maternal immune system to women suffering from recurrent miscarriage. Conclusion Although there is a scientific rationale to modulate the maternal immune system as a therapy for recurrent miscarriage, caution is very much needed. Since the causes of recurrent miscarriage are multi factorial, a strategy toward selection of patients, who might benefit from maternal immune modulation, has to be developed. For most of the above mentioned modulators, the exact working mechanisms are not known, and the possible effects of such a (immune) modulator on fetal and neonatal immune function are also unknown. Although some immune modulators do normalize immune abnormalities found in women with recurrent miscarriage, there is no proven effect on pregnancy outcome yet. Therefore, more specific and demonstrably safe maternal immune modulators have to be developed. Possible promising immune modulators should first be tested in randomized clinical trials before being used in daily clinical practice. Although it can be tempting to give a promising therapy to these sometimes desperate couples, only therapies with a scientifically proven beneficial and safe effect should be administered to prevent providing false hope to couples experiencing recurrent miscarriage. Conflict of interest statement None of the authors report any potential conflict of interest. References [1] Larsen EC, Christiansen OB, Kolte AM, Macklon N. New insights into mechanisms behind miscarriage. BMC Med 2013;11:154. [2] Regan L, Rai R. Epidemiology and the medical causes of miscarriage. Baillieres Best Pract Res 2000;14:839–54. [3] Hsu P, Nanan RK. Innate and adaptive immune interactions at the fetal– maternal interface in healthy human pregnancy and pre-eclampsia. Front Immunol 2014;5:125. [4] Robertson SA. Immune regulation of conception and embryo implantation-all about quality control. J Reprod Immunol 2010;85:51–7.
Please cite this article in press as: Prins JR, et al. Immunomodulators to treat recurrent miscarriage. Eur J Obstet Gynecol (2014), http:// dx.doi.org/10.1016/j.ejogrb.2014.07.038
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An imbalance in interleukin-17-producing T and Foxp3(+) regulatory T cells in women with idiopathic recurrent pregnancy loss. Hum Reprod (Oxford England) 2011;26:2964–71. [21] Kho EM, McCowan LM, North RA, et al. Duration of sexual relationship and its effect on preeclampsia and small for gestational age perinatal outcome. J Reprod Immunol 2009;82:66–73. [22] Robertson SA, Prins JR, Sharkey DJ, Moldenhauer LM. Seminal fluid and the generation of regulatory T cells for embryo implantation. Am J Reprod Immunol 2013;69:315–30. [23] Bettelli E, Carrier Y, Gao W, et al. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 2006; 441:235–8. [24] Arruvito L, Billordo A, Capucchio M, Prada ME, Fainboim L. IL-6 trans-signaling and the frequency of CD4+FOXP3+ cells in women with reproductive failure. J Reprod Immunol 2009;82:158–65. [25] Zenclussen AC, Kortebani G, Mazzolli A, Margni R, Malan Borel I. Interleukin-6 and soluble interleukin-6 receptor serum levels in recurrent spontaneous abortion women immunized with paternal white cells. Am J Reprod Immunol 2000;44:22–9. [26] Jasper MJ, Tremellen KP, Robertson SA. Reduced expression of IL-6 and IL1alpha mRNAs in secretory phase endometrium of women with recurrent miscarriage. J Reprod Immunol 2007;73:74–84. [27] von Wolff M, Thaler CJ, Strowitzki T, Broome J, Stolz W, Tabibzadeh S. Regulated expression of cytokines in human endometrium throughout the menstrual cycle: dysregulation in habitual abortion. Mol Hum Reprod 2000; 6:627–34. [28] Zenclussen AC, Gerlof K, Zenclussen ML, et al. Abnormal T-cell reactivity against paternal antigens in spontaneous abortion: adoptive transfer of pregnancy-induced CD4+ CD25+ T regulatory cells prevents fetal rejection in a murine abortion model. Am J Pathol 2005;166:811–22. [29] Tang AW, Alfirevic Z, Quenby S. Natural killer cells and pregnancy outcomes in women with recurrent miscarriage and infertility: a systematic review. Hum Reprod (Oxford England) 2011;26:1971–80. [30] Seshadri S, Sunkara SK. Natural killer cells in female infertility and recurrent miscarriage: a systematic review and meta-analysis. Hum Reprod Update 2014;20:429–38.
[31] Lachapelle MH, Miron P, Hemmings R, Roy DC. Endometrial T, B, and NK cells in patients with recurrent spontaneous abortion. Altered profile and pregnancy outcome. J Immunol 1996;156:4027–34. [32] Quenby S, Bates M, Doig T, et al. Pre-implantation endometrial leukocytes in women with recurrent miscarriage. Hum Reprod (Oxford England) 1999; 14:2386–91. [33] Vargas RG, Bompeixe EP, Franca PP, Marques de Moraes M, da Graca Bicalho M. Activating killer cell immunoglobulin-like receptor genes’ association with recurrent miscarriage. Am J Reprod Immunol 2009;62:34–43. [34] Varla-Leftherioti M, Keramitsoglou T, Parapanissiou E, et al. HLA-DQA1*0505 sharing and killer immunoglobulin-like receptors in sub fertile couples: report from the 15th international histocompatibility workshop. Tissue Antigens 2010;75:668–72. [35] Varla-Leftherioti M, Spyropoulou-Vlachou M, Niokou D, et al. Natural killer (NK) cell receptors’ repertoire in couples with recurrent spontaneous abortions. Am J Reprod Immunol 2003;49:183–91. [36] Bryceson YT, Chiang SC, Darmanin S, et al. Molecular mechanisms of natural killer cell activation. J Innate Immu 2011;3:216–26. [37] Porter TF, LaCoursiere Y, Scott JR. Immunotherapy for recurrent miscarriage. In: The Cochrane database of systematic reviews; 2006 (CD000112). [38] Gafter U, Sredni B, Segal J, Kalechman Y. Suppressed cell-mediated immunity and monocyte and natural killer cell activity following allogeneic immunization of women with spontaneous recurrent abortion. J Clin Immunol 1997;17:408–19. [39] Clark DA, Fernandes J, Banwatt D. Prevention of spontaneous abortion in the CBA DBA/2 mouse model by intravaginal TGF-beta and local recruitment of CD4+ 8+ FOXP3+ cells. Am J Reprod Immunol 2008;59:525–34. [40] Sato K, Yamashita N, Baba M, Matsuyama T. Modified myeloid dendritic cells act as regulatory dendritic cells to induce anergic and regulatory T cells. Blood 2003;101:3581–9. [41] Scarpellini F, Sbracia M. Use of granulocyte colony-stimulating factor for the treatment of unexplained recurrent miscarriage: a randomised controlled trial. Hum Reprod (Oxford England) 2009;24:2703–8. [42] Lee JH, Lydon JP, Kim CH. Progesterone suppresses the mTOR pathway and promotes generation of induced regulatory T cells with increased stability. Eur J Immunol 2012;42:2683–96. [43] Xu L, Dong B, Wang H, et al. Progesterone suppresses Th17 cell responses, and enhances the development of regulatory T cells, through thymic stromal lymphopoietin-dependent mechanisms in experimental gonococcal genital tract infection. Microbes Infect/Inst Pasteur 2013;15:796–805. [44] Haas DM, Ramsey PS. Progestogen for preventing miscarriage. In: The Cochrane database of systematic reviews; 2013 (10:CD003511). [45] Goldzieher JW. Double-blind trial of a progestin in habitual abortion. JAMA 1964;188:651–4. [46] Levine L. Habitual abortion: a controlled study of progestational therapy. West J Surg Obstet Gynecol 1964;72:30–6. [47] Swyer GI, Daley D. Progesterone implantation in habitual abortion. Br Med J 1953;1:1073–7. [48] Quenby S, Kalumbi C, Bates M, Farquharson R, Vince G. Prednisolone reduces preconceptual endometrial natural killer cells in women with recurrent miscarriage. Fertil Steril 2005;84:980–4. [49] Tang AW, Alfirevic Z, Turner MA, Drury JA, Small R, Quenby S. 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 (Oxford England) 2013;28:1743–52. [50] Kim DJ, Lee SK, Kim JY, et al. Intravenous immunoglobulin g modulates peripheral blood th17 and foxp3(+) regulatory T cells in pregnant women with recurrent pregnancy loss. Am J Reprod Immunol 2014;71:441–50. [51] Kaandorp SP, Goddijn M, van der Post JA, et al. Aspirin plus heparin or aspirin alone in women with recurrent miscarriage. N Engl J Med 2010;362:1586–96.
Jelmer R. Prins* Tom E.C. Kieffer Sicco A. Scherjon Department of Obstetrics and Gynecology, University of Groningen, University Medical Center Groningen, PO Box 30001, Hanzeplein 1, 9700 RB Groningen, The Netherlands *Corresponding
author. Tel.: +31 50 3613020; fax: +31 50 3611806 E-mail address: [email protected] (J.R. Prins). 25 June 2014 Accepted 29 July 2014
Please cite this article in press as: Prins JR, et al. Immunomodulators to treat recurrent miscarriage. Eur J Obstet Gynecol (2014), http:// dx.doi.org/10.1016/j.ejogrb.2014.07.038
EURO-8674; No. of Pages 4 European Journal of Obstetrics & Gynecology and Reproductive Biology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Invited editorial
Immunomodulators to treat recurrent miscarriage
A B S T R A C T
Keywords: Recurrent miscarriage Immune system Therapy Review
Recurrent miscarriage is a reproductive disorder affecting many couples. Although several factors are associated with recurrent miscarriage, in more than 50% of the cases the cause is unknown. Maladaptation of the maternal immune system is associated with recurrent miscarriage and could explain part of its pathophysiology. Modulating the maternal immune system toward pregnancy tolerance could benefit pregnancy outcome. Although there is a clear scientific rationale that modulating the maternal immune system could benefit recurrent miscarriage, only a few studies suggest possible beneficial effects of immune modulators as a therapy for recurrent miscarriage. Therapies skewing the maternal immune response to a tolerating regulatory T cell rich environment seem especially promising; however, more research is needed to find effective and safe maternal immune modulators for reproductive pathologies as recurrent miscarriage. Moreover, the possible side effects on maternal, fetal, and neonatal immune function are essentially unknown, and its elucidation is crucial before any possible therapeutic strategies could be clinically implemented. ß 2014 Elsevier Ireland Ltd. All rights reserved.
Introduction Recurrent miscarriage is a reproductive disorder distressing many couples. Depending on the definition used, it is estimated that about 1–3% of couples seeking to reproduce are affected by it [1,2]. In contrast to a sporadic miscarriage which is often caused by a conceptus having structural malformations or chromosomal abnormalities, the causes of recurrent miscarriage are far from completely understood [1]. Although some factors are clearly associated with recurrent miscarriage, most cases are idiopathic, and no possible cause is evident. The maternal immune system plays an important role in the success of pregnancy. Optimal regulation of the maternal immune system is essential to tolerate the allogeneic fetus. Improper regulation leads to several reproductive disorders, such as recurrent miscarriage [1]. The complex mechanisms behind the tolerance toward the fetus are not fully elucidated [3]. The important regulating role of the maternal immune system gives the female reproductive system the possibility to accommodate or reject a conceptus depending on maternal, environmental, and fetal factors [4]. Since the maternal immune system is implicated in the pathogenesis of recurrent miscarriage, modulation of the maternal immune system holds potential, and gives hope for treatment and prevention of recurrent miscarriage. Immune modulators being
able to skew the maternal immune system toward fetal tolerance could benefit pregnancy outcome. However, skewing the maternal immune system toward tolerance could also result in tolerance of fetuses which would have been rejected rightfully by the maternal quality control system. Moreover, modulation of a pregnancy immune environment could very well influence fetal and neonatal development and have a role in the priming of the fetal immune system [5]. Furthermore, modulation of the maternal immune system could cause long term effects, causing the mother to be vulnerable for immune associated diseases in later live. Although no evidence of long term maternal or fetal effects has been found yet, caution is essential before implying immune modulating therapies in clinical settings without a proven and clear beneficial effect, and without evidence of safety. This short review aims to examine and highlight some of the possibilities of modulation of the maternal immune system as a treatment option for recurrent miscarriage. Methods For this review, papers were included using combinations of the following search terms in Pubmed: ‘recurrent miscarriage, recurrent abortions, deciduas, immune, placenta, pregnancy, abortion, regulatory T cells, Treg cells, paternal, NK cells, cytokines, Th17 cells, Th1 cells, progesterone, G-CSF, GM-CSF, (immune)
http://dx.doi.org/10.1016/j.ejogrb.2014.07.038 0301-2115/ß 2014 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: Prins JR, et al. Immunomodulators to treat recurrent miscarriage. Eur J Obstet Gynecol (2014), http:// dx.doi.org/10.1016/j.ejogrb.2014.07.038
G Model
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therapy, and prevention’. Systematic reviews were used for additional search through their reference list. Most (immune) therapies for recurrent miscarriage have been reviewed separately recently in meta-analyses, these Cochrane meta-analyses were also used in this review. Results Recurrent miscarriage Recurrent miscarriage is a disorder which is not consistently defined within existing literature. Most studies define recurrent miscarriage as three or more consecutive miscarriages before 20 weeks of gestation [1,6]. Depending on the definition it is estimated that 1–3% of couples seeking to reproduce are affected by it [1,2]. Although several causes are associated with recurrent miscarriage including anatomical anomalies, genetic abnormalities, thrombophilia, lifestyle, and hormonal dysfunction, in more than 50% of the couples suffering from recurrent miscarriage no such associations are found [1,7,8]. Most likely, a part of these unexplained miscarriages is caused by malfunctioning of maternal immune regulation. Immunology during pregnancy It is known that regulation of the maternal immune system is essential to prevent rejection of the fetus. Several mechanisms are involved in the induction of maternal tolerance, and as adequate regulation is important, most likely redundant mechanisms prevent rejection of the fetus. Besides the immune changes seen locally at the fetal maternal interface, peripheral immune responses are also altered during pregnancy [9]. Regulatory T (Treg) cells are a key player within the regulation of maternal immune tolerance [10]. Lower numbers and altered function of Treg cells have been associated with adverse pregnancy outcomes in both human and animal studies, and it has been shown that adequate numbers and function of Treg cells are essential for pregnancy success [10]. During pregnancy the balance of T cell subsets is essential for preventing fetal rejection, for example increased levels of Th1 and Th17 related cells and cytokines are associated with adverse pregnancy outcomes. Depending on the cytokine environment, naı¨ve T cells will differentiate to a T cell subset. Other immune cells which are important during pregnancy are NK cells and macrophages [3]. NK cells are regarded as important in the regulation of vascularization and invasion processes during pregnancy [11]. All immune cells work together to ensure fetal tolerance. A good example of this is the recent finding that NK cells are able to inhibit the Th17 cells, and with that skew the maternal immune system toward tolerance [12]. The mechanisms causing the shift of the maternal immune system toward fetal tolerance are not fully known, but most likely endogenous and exogenous factors, including exposure to semen, hormones, and lifestyle, modulate the maternal immune system toward tolerance [10]. The precise mechanisms of maternal fetal tolerance are complex and numerous; however, they are out of the scope of this review. Maternal immunology in recurrent miscarriage Several immunological mechanisms have been associated with recurrent miscarriage [1,13]. Most studies indicate higher levels of activated immune cells and associated cytokines, and lower levels of regulatory cells and associated cytokines in women with recurrent miscarriage. Differences have been found both locally as well as in the peripheral blood. Tissue recovered after both spontaneous as well as induced abortions showed lower levels of
Treg cells in spontaneous miscarriages [14,15]. Other studies showed that women with recurrent miscarriage have lower levels of Treg cells in their peripheral blood and also reduced suppressive function of Treg cells compared to a control group [14,16]. These Treg cells could also be less stable, as gene expression of certain genes related to Treg cell stability was less in women with recurrent miscarriage [17]. Other evidence implying Treg cells in the pathophysiology of recurrent miscarriage are altered Treg/ Th17 balances found in recurrent miscarriage. Higher levels of Th17 cells and Th17 associated cytokines have been found in decidual tissue and peripheral blood of women with recurrent miscarriage [18,19], indicating an imbalance between Treg and Th17 cells in these women [20]. Most likely the regulation of this balance is dysfunctional, as it has been shown that peripheral blood mononuclear cells from women with recurrent miscarriage show greater proliferation of Th17 and fewer Treg cells once cultured in the presence of sperm antigens from their male partner [17]. An interesting question would be whether this inadequate skewing of the maternal immune system is also influenced by paternal factors, and is caused by differences in semen constituents. It is known that seminal fluid induces Treg cell generation, and that the duration of sexual relationship is related to pregnancy outcome [21,22]. Inadequate immune modulating factors in seminal fluid could lead to inadequate adaptation of the maternal immune system, and make a couple prone for recurrent miscarriage. More studies analyzing the role of semen constituents in couples with recurrent miscarriage are needed. Another mechanism which is also implied in the immune regulation in recurrent miscarriage is altered Interleukin 6 (IL6) signaling. IL6 is known to influence the differentiation of naı¨ve T cells away from Treg cells toward Th17 cells [23]. Studies have found lower levels of IL6 in endometrial tissue in the mid-secretory phase of the cycle, together with increased serum levels of IL6 in women with recurrent miscarriage compared to controls [24–27]. The higher levels of IL6 could explain the finding that sera from women with recurrent miscarriage inhibit the expansion of the Treg cell populations [24]. Other immune cells associated with recurrent miscarriage are NK cells and their cytokines [28–30]. Although studies differ in their methods, most studies found higher levels of NK cells in the peripheral blood of women with recurrent miscarriage [30–32], whereas also an altered balance in inhibitory and activating killer immunoglobulin-like receptors (KIR) was found [24,33–35]. KIRs are cell proteins on the surface of NK cells. Whereas inhibitory KIRs prevent activation, activating KIRs lead to cytotoxic activity [36]. Altered balances in these KIRs could lead to an improper activation of the NK cells. More evidence for NK cell dysfunction in recurrent miscarriage comes from a finding that NK cells were partially impaired in women with recurrent miscarriage, and with that were less successful in inhibiting Th17 cells [12]. Immune modulators Since the maternal immune system is implied in the etiology of part of the unexplained recurrent miscarriages, several immune modulating therapies have been investigated, including aspirin, paternal leukocyte immunization, progesterone, and steroids [37]. As the causes of recurrent miscarriage are multi factorial, selection of couples by immunological analysis could improve therapeutic outcomes and therefore influence the results of the different studies. However, in only a few studies pre-immune screening of the subjects was performed. Most studies used either paternal white cell immunization (12 trials, 641 women, Peto OR 1.23, 95% confidence interval (CI) 0.89 to 1.70) or intravenous administration of immunoglobulins (eight trials, 303 women, Peto OR 0.98, 95% CI 0.61 to 1.58) as therapy [37]. Although a Cochrane review of 2006
Please cite this article in press as: Prins JR, et al. Immunomodulators to treat recurrent miscarriage. Eur J Obstet Gynecol (2014), http:// dx.doi.org/10.1016/j.ejogrb.2014.07.038
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concluded that overall these therapies do not improve live birth rate in women with recurrent miscarriage [37], some studies did find normalization of immune parameters in women with recurrent miscarriage. Methods between the studies vary and it can be argued that immunizing the women by injecting paternal cells intravenously is immunologically not an optimal method as most of the immune reaction will be cytotoxic driven. Immunizing subcutaneously could improve results, as this shows similarities to the ‘natural’ immunizing method (coitus), and dendrictic cells and macrophages will present the antigens [22]. Interestingly, a few studies have shown that once immunizing recurrent miscarriage patients with mononuclear cells from their husbands, levels of IL6 decrease to normal levels [24,25,38]. More interestingly, immunization of women with recurrent miscarriage with paternal cells led to an increase in the Treg cell frequency comparable to levels of fertile women [24]. In mice models it has been shown that after increasing the levels of Treg cells abortion prone mice are protected against fetal loss [28,39]. Although these latter results hold potential, there is no evidence yet that immunizing with paternal cells does improve pregnancy outcome. Future studies should consider using the subcutaneously route to immunize women. Because of the role of Treg cells in recurrent miscarriage, several other strategies have been used to improve their levels. The use of granulocyte colony stimulating factor (G-CSF) has been evaluated for the treatment of recurrent miscarriage. G-CSF and GM-CSF are cytokines known to recruit and activate tolerogeneic dendritic cells which are implied in the generation of Treg cells [10,39,40]. A small study showed a reduction in the incidence of miscarriage in a following pregnancy in women treated with G-CSF (odds ratio 5.1; 95% CI 1.5–18.4), together with an increase in levels of HCG [41]. As these results were only obtained in small studies, these results have to be confirmed in a larger randomized trial. The use of progesterone is of recent interest. Progesterone suppresses Th17 cell responses and enhances development and possible function of Treg cells [42,43]. A recent Cochrane review stated that there is no evidence that progesterone could prevent miscarriage [44]. Interestingly, a subgroup analysis, only analyzing women with recurrent miscarriage, within this Cochrane review of four trials analyzing the effect of progesterone in women with recurrent miscarriage showed a significant decrease in miscarriage (total of 225 women, Peto OR 0.39; 95% CI 0.21 to 0.72) [7,44–47]. However, most of this data is over 50 years old and these results should be confirmed in larger randomized clinical trials. Other strategies include therapies to modulate NK cell numbers. In patients with recurrent miscarriage several abnormalities were demonstrated in cellular immunity and increased numbers of NK cells were found in peripheral blood. Since prednisolone decreases numbers of NK cells in women with recurrent miscarriage [48], a recent pilot feasibility study administered prednisolone or a placebo to women with recurrent miscarriage, in this study no difference was found in live birth rate (95% CI 0.79–2.86) [49]. There is also no evidence for a beneficial treatment effect of intravenous immunoglobulins in recurrent miscarriage patients [37]. One study analyzed the effects of intravenous immunoglobulin on levels of Treg and Th17 cells in women with recurrent miscarriage with prescreened immune abnormalities [50]. Levels of Treg cells and Th17 cells did not change in the whole patient group, however, when subjects were divided in groups based on their immune cell levels, intravenous immunoglobulins led to higher levels of Treg cells in the lowest Treg cell number group and lower levels of Th17 cells in the higher Th17 cell number groups [50]. Although this study shows that administration of immunoglobulins could restore part of an immune balance in part of recurrent miscarriage there was no effect seen on pregnancy rate
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[50]. However, this was not a double blind placebo controlled trial, therefore further evaluation is needed. Being able to influence both immune and haemostatic characteristics, the effect of aspirin on recurrent miscarriage has been evaluated. No improvement in live birth rate was however observed after administration of aspirin with or without a low molecular weight heparin [51]. To summarize the above, there is no clear evidence yet that immune modulation benefit couples with recurrent miscarriage. Since the causes of recurrent miscarriage are multi factorial, inclusion and treatment of patients who are likely to benefit from immune modulating therapy is essential. Adequate screening of both maternal immune cell subsets and their function and possibly also immune skewing factors in semen could define couples in which the immune system might be the cause of recurrent miscarriage. In these cases, specific immunotherapy might benefit pregnancy outcome. However, similar to the therapeutic options, no guidelines toward specific immune screening have been made yet. Possible immune therapies have to be proven safe for both mother and child. During pregnancy the fetal immune system is shaped, and most likely the fetal immune system is influenced by maternal immune factors. There is evidence that prenatal maternal immune factors could influence the fetal immune system, and with that leading to fetal immune programming [5]. As modulating the fetal immune system via maternal modulation could lead to for example allergies, cardiovascular diseases in later life, and could also have an influence on neurodevelopment, great care is needed before administering modulators of the maternal immune system to women suffering from recurrent miscarriage. Conclusion Although there is a scientific rationale to modulate the maternal immune system as a therapy for recurrent miscarriage, caution is very much needed. Since the causes of recurrent miscarriage are multi factorial, a strategy toward selection of patients, who might benefit from maternal immune modulation, has to be developed. For most of the above mentioned modulators, the exact working mechanisms are not known, and the possible effects of such a (immune) modulator on fetal and neonatal immune function are also unknown. Although some immune modulators do normalize immune abnormalities found in women with recurrent miscarriage, there is no proven effect on pregnancy outcome yet. Therefore, more specific and demonstrably safe maternal immune modulators have to be developed. Possible promising immune modulators should first be tested in randomized clinical trials before being used in daily clinical practice. Although it can be tempting to give a promising therapy to these sometimes desperate couples, only therapies with a scientifically proven beneficial and safe effect should be administered to prevent providing false hope to couples experiencing recurrent miscarriage. Conflict of interest statement None of the authors report any potential conflict of interest. References [1] Larsen EC, Christiansen OB, Kolte AM, Macklon N. New insights into mechanisms behind miscarriage. BMC Med 2013;11:154. [2] Regan L, Rai R. Epidemiology and the medical causes of miscarriage. Baillieres Best Pract Res 2000;14:839–54. [3] Hsu P, Nanan RK. Innate and adaptive immune interactions at the fetal– maternal interface in healthy human pregnancy and pre-eclampsia. Front Immunol 2014;5:125. [4] Robertson SA. Immune regulation of conception and embryo implantation-all about quality control. J Reprod Immunol 2010;85:51–7.
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Jelmer R. Prins* Tom E.C. Kieffer Sicco A. Scherjon Department of Obstetrics and Gynecology, University of Groningen, University Medical Center Groningen, PO Box 30001, Hanzeplein 1, 9700 RB Groningen, The Netherlands *Corresponding
author. Tel.: +31 50 3613020; fax: +31 50 3611806 E-mail address: [email protected] (J.R. Prins). 25 June 2014 Accepted 29 July 2014
Please cite this article in press as: Prins JR, et al. Immunomodulators to treat recurrent miscarriage. Eur J Obstet Gynecol (2014), http:// dx.doi.org/10.1016/j.ejogrb.2014.07.038