{"paper_id":"2f64675c-9b3c-4d07-9c3d-bf55b39eb195","body_text":"ORIGINAL ARTICLE\nGranulocyte-Colony Stimulating Factor as Treatment\nOption in Patients with Recurrent Miscarriage\nClaudia Santjohanser • Catherine Knieper •\nCordula Franz • Kaino Hirv • Osama Meri •\nManfred Schleyer • Wolfgang Wu¨rfel • Bettina Toth\nReceived: 11 May 2012 / Accepted: 20 December 2012 / Published online: 24 January 2013\n/C211L. Hirszfeld Institute of Immunology and Experimental Therapy, Wroclaw, Poland 2013\nAbstract In 1–5 % of patients during childbearing years\nrecurrent miscarriages (RM) occur. There are established\nrisk factors like anatomical, endocrine and hemostatic\ndisorders as well as immunological changes in the maternal\nimmune system. Nevertheless, further elucidation of the\npathogenesis remains a matter of debate. In addition, there\nare no standardized immunological treatment strategies.\nRecent studies indicate possible effects of tumor necrosis\nfactor a blocker and granulocyte-colony stimulating factor\n(G-CSF) concerning live birth rate (LBR) in RM patients.\nTherefore, we performed a retrospective cohort study in\npatients undergoing assisted reproductive treatment (ART)\nwith known RM analysing the possible benefits of G-CSF\napplication. From January 2002 to December 2010, 127\npatients (199 cylces) with RM (at least 2 early miscar-\nriages) 49 (72 cycles) receiving G-CSF and 78 (127 cycles)\ncontrols receiving either no medication (subgroup 1) or\nCortisone, intravenous immunoglobulins or low molecular\nweight heparin (subgroup 2) undergoing ART for in vitro\nfertilisation/intracytoplasmic sperm injection were ana-\nlysed. G-CSF was administered weekly once (34 Mill) in\n11 patients, 38 patients received 2 9 13 Mill G-CSF per\nweek until the 12th week of gestation. Statistical analysis\nwas performed with SPSS for Windows (19.0), p\\ 0.05\nsignificant. The mean age of the study population was\n37.3 ± 4.4 years (mean ± standard deviation) and dif-\nfered not significantly between patients and subgroups.\nHowever, the number of early miscarriages was signifi-\ncantly higher in the G-CSF group as compared to the\nsubgroups (G-CSF 2.67 ± 1.27, subgroup 1 0.85 ± 0.91,\nsubgroup 2 0.64 ± 0.74) and RM patients receiving G-CSF\nhad significantly more often a late embryo transfer (day 5)\n(G-CSF 36.7 %, subgroup 1 12.1 %, subgroup 2 8.9 %).\nThe LBR of patients and the subgroups differed signifi-\ncantly (G-CSF 32 %, subgroup 1 13 %, subgroup 2 14 %).\nSide effects were present in less than 10 % of patients,\nconsisting of irritation at the injection side, slight leuko-\ncytosis, rise of the temperature ( \\38 /C176C), mild bone pain\nand hyperemesis gravidarum. None of the newborn showed\nany kind of malformations. According to our data, G-CSF\nseems to be a safe and promising immunological treatment\noption for RM patients. However, with regard to the ret-\nrospective setting and the possible bias of a higher rate of\nlate embryo transfers in the G-CSF group additional studies\nare needed to further strengthen our results.\nKeywords Recurrent miscarriage /C1\nAssisted reproduction /C1Immunological treatment /C1\nG-CSF\nIntroduction\nRecurrent miscarriage (RM) is defined as more than three\nmiscarriages by the World Health Organization (WHO)\n(Stirrat 1990). The American Society for Reproductive\nMedicine defines it as two or more consecutive pregnancy\nW. Wu¨rfel and B. Toth contributed equally.\nC. Santjohanser /C1O. Meri /C1M. Schleyer /C1W. Wu¨rfel\nKinderwunsch Centrum Mu¨nchen, Munich, Germany\nC. Knieper /C1C. Franz /C1B. Toth ( &)\nDepartment of Gynecological Endocrinology\nand Fertility Disorders, University of Heidelberg,\nVossstrasse 9, 69115 Heidelberg, Germany\ne-mail: bettina.toth@med.uni-heidelberg.de\nK. Hirv\nCenter for Human Genetics and Laboratory Medicine,\nMartinsried, Germany\nArch. Immunol. Ther. Exp. (2013) 61:159–164\nDOI 10.1007/s00005-012-0212-z\n123\n\nlosses documented by ultrasound or histopathologic\nexamination ( 2013). It affects approximately 1–5 % of\ncouples trying to conceive and remains unexplained in\nabout 50 % (Faridi and Agrawal 2011; Toth et al. 2010b).\nImmunologic processes play a main part during implanta-\ntion and embryo as well as fetal development (Yang et al.\n2010). Most recently, several authors published data on\nalterations in the humoral and adaptive immune system in\nRM patients and controls (Hiby et al. 2010; Jin et al. 2011;\nToth et al. 2010a; Wang et al. 2010; Yang et al. 2010).\nSince the discovery of regulatory T cells (Tregs), the\nTh1/Th2 paradigm (Jin et al. 2011; Mosmann and Coffman\n1989) was challenged towards a more complex model\n(Kumar et al. 2011; Wang et al. 2010) also including\ndendritic cells. Dendritic cells present affiliated antigens\nand are able to induce a switch in the immune answer.\nBeing in the immuntolerant ‘‘steady state’’, they support\nthe physiologic immunoarchitecture of the placenta. When\nthey are exposed to inflammatory stress, they promote\neffector cells like cytotoxic T cells to expand and therefore\nhighly contribute to a cytotoxic reaction (Scholz et al.\n2008; Segerer et al. 2012).\nFocussing on the Tregs and Th17 interaction, some\naberrations seem to be present in RM patients. Tregs are\nimmunmodulating cells suppressing lymphocytes and are\nable to produce transforming growth factor b and inter-\nleukin 10. There is evidence that Treg levels are reduced in\nRM patients and that a high level of Tregs in the peripheral\nblood correlates with implantation success after in vitro\nfertilisation (IVF) (Lee et al. 2012; Zhou et al. 2012). In\naddition, low circulating CD4\n?CD25?Foxp3? Treg cells\nseem to predict miscarriage in pregnant women with a\nhistory of failure (Winger et al. 2009). T cell subsets like\nTh17 promote a pro-inflammatory immune reaction and are\nnegatively controlled by Tregs (D’Addio et al. 2011). In\nthe peripheral blood of patients with idiopathic RM, the\nexpression of Th17-positive cells seems to be significantly\nincreased when compared to women with healthy preg-\nnancies (Wang et al. 2010). In addition, the suppressive\nactivity of Tregs towards Th17-positive cells was\ndecreased in RM patients (Wang et al. 2010).\nHowever, the ‘‘bench to bedside’’ process is not finished\nas there are no standardized procedures to detect immu-\nnological disorders in RM patients and accordingly no\nstandardized treatment options to rule out and treat RM\npatients with immunologic disorders. Even though altera-\ntions in natural killer (NK) cells, lymphocytes, T cells or\nintereleukins can be detected in detail, no gold standards\nfor medical interventions do exist.\nSo far, immunmodulatory therapies in RM patients\ninclude paternal immunization, intravenous immunoglob-\nulins (ivIgG) as well as Cortisone administration. Recent\nstudies indicate a possible role of tumor necrosis factor\n(TNF)-a blockers and granulocyte-colony stimulating fac-\ntor (G-CSF) in RM patients (Scarpellini and Sbracia 2009;\nWinger et al. 2009).\nTNF-a is a pro-inflammatory Th1 cytokine and the ratio\nof TNF-a producing T cells seems to be significantly higher\nin patients with recurrent implantation failure (RIF) and RM\npatients than in fertile controls (Kwak-Kim et al. 2003;N g\net al. 2002) making it a new treatment target for RM\npatients. Winger et al. ( 2009) administered TNF- a inhibi-\ntors and ivIgGs in combination with low molecular weight\nheparin (LMWH) in RM patients, which elevated the live\nbirth rate (LBR).\nG-CSF is synthesized by immune and endometrial cells\n(Makinoda et al. 2008). Recently, a few studies indicated\npossible benefits of G-CSF administration in RIF and RM\npatients (Scarpellini and Sbracia 2009; Wurfel et al. 2010).\nScarpellini and Sbracia ( 2009) investigated G-CSF\nadministration in patients with idiopathic RM and reached\na LBR of 82.8 % in the treatment group compared to\n48.5 % in the control group.\nWithin our study, we investigated G-CSF application in\nRM patients undergoing assisted reproductive therapy\n(ART) and compared the LBR with RM patients receiving\nno medication or LMWH, Cortisone or ivIgG. The study\npopulation consisted of 127 patients and 78 controls\nundergoing 199 and 127 in vitro fertilisation/intracyto-\nplasmic sperm injection (IVF/ICSI) cycles, respectively.\nMaterials and Methods\nSince 2002, RM patients who underwent ART at the Kin-\nderwunsch Centrum Munich (KCM, Germany) were offered\nG-CSF treatment. In total, 7,410 were treated at the KCM\nduring this period, including 649 RM patients. All patients\nunderwent IVF/ICSI. The data were evaluated according to\nthe following inclusion and exclusion criteria: at least two\nearly miscarriages (excluding RM patients with biochemical\npregnancies), no maternal or paternal chromosomal aberra-\ntions, no ART with heterologous oocytes or sperms, no\nimmunmodulatory treatments like TNF-a blocker or donor\nleukocytes. In addition, patients were all negative for rele-\nvant uterine anomalies, infections and endocrine\ndysfunctions (like congenital adrenal hyperplasia or Base-\ndow disease). The thyroid-stimulating hormone (TSH) level\nof patients with Hashimoto thyroiditis was adjusted by thy-\nroid hormones to values \\2.5 IU/ml. In cases with\ncoagulation disorders, like mutations in the factor V Leiden\nor prothrombin gene, LMWH was administered daily,\npatients with anti-phospholipid antibodies were treated with\nLMWH and acetylsalicylic acid (Aspirin).\nAccording to the guidelines of the German Society for\nGynecology and Obstetrics the following investigations\n160 Arch. Immunol. Ther. Exp. (2013) 61:159–164\n123\n\nwere performed: anti-phospholipid antibodies, such as anti-car-\ndiolipin antibodies, lupus-anticoagulants andb2-glycoproteins.\nIn patients strongly suspected of having anti-phospholipid\nsyndrome with a negative test result non-established anti-\nphospholipid-antibodies such as phophatidylserin as well\nas inositol and annexin V levels were tested. To exclude\nautoimmune thyroid disease we measured basal TSH and\nanti-TPO antibodies in all patients. In case of suspected\nintestinal diseases, such as celiac disease, we examined\ntransglutaminase antibodies. In addition, we investigated\nNK cell levels and the Th1/Th2 ratio in the peripheral\nblood of all patients. Furthermore, we performed a cross-\nmatch-test and HLA typing of both partners. Since 2006,\nkiller-cell immunglobulin-like receptor (KIR)-typing, and\nmore recently, regulatory T cells were additionally part of\nour routine set up.\nOnly 127 RM patients (199 treatment cycles) fulfilled the\ninclusion criteria. In total, n = 49 patients received G-CSF,\n11 patients 1 9 34 Mill IU per week and 38 patients\n2 9 13 Mill IU G-CSF/week starting at the day of embryo\ntransfer until the 12th week of pregnancy. The subgroup 1,\nn = 33 patients (n = 46 cycles), did not receive any med-\nication, the subgroup 2 consisted of n = 45 patients\n(n = 81 cycles) with other medicaments like LMWH en-\noxaparin-natrium 40 mg subcutaneously once daily,\nacetylsalicylic acid (100 mg), folic acid (5 mg) or Predni-\nsone/Dexamethasone (2.5–5.0 mg/0.5 mg) starting in the\nmiddle of the previous cycle until the evidence of an\nembryonic heart beat and Doxycycline (1 9 100 mg for\n5 days) beginning at the day of the embryo transfer. All\nstudy patients received folic acid (0.5 mg) and progesterone\nvaginally (3 9 200 mg in the luteal phase until 12th week\nof pregnancy).\nAll patients underwent ART, 80 % with a long stimu-\nlation protocol and the others with an antagonist protocol.\nPatients with regular cycles started with gonadotropin-\nreleasing hormone agonist on day 24 of the preceding\ncycle. Additionally, daily recombinant follicle-stimulating\nhormone (FSH) and/or human menopausal gonadotropin\n(HMG; 125–200 IU) injection were administered from the\nthird day of the menstrual cycle. Patients were closely\nmonitored (vaginal ultrasound, taking into account the\nnumber and medium size of follicles of both ovaries and\nthe endometrial thickness as well as concentrations of\nestradiol, progesterone and luteinizing hormone). The FSH/\nHMG dose was adjusted according to the individual\novarian response. Ovulation was induced with 10,000 IU\nhCG when at least three follicles\n[17 mm were present\n36–38 h before oocyte pick up. Depending on the sperm\nparameters according to the WHO guidelines, an IVF/ICSI\ntreatment was performed. The embryonal development was\nclosely monitored and an ultrasound-guided embryo\ntransfer was performed on day 1–3 (75.6 %), day 4 (3.9 %)\nor day 5 (20.5 %) after oocyte pick up.\nData were analyzed with SPSS for Windows, release\n19.0 (SPSS; Chicago, IL, USA). Results are given as\nmean ± standard deviation (minimum–maximum), unless\nstated otherwise. Differences between the groups were\nanalysed by the non-parametric Mann–Whitney test and the\nv\n2 test, p\\ 0.05 were regarded as statistically significant.\nResults\nStudy Population\nMean age was 37.3 ± 4.4 years (26–45). Patients receiving\nG-CSF had undergone significantly more early ( p\\ 0.001)\nand late miscarriages ( p\\ 0.001) as compared to the sub-\ngroup 1 and 2. In general, 6.02± 3.8 (0–17) ART cycles were\nperformed before G-CSF treatment administration. 8.9± 5.1\n(1–23) oocytes were reached during oocyte pick up of which\n5.2 ± 3.5 (1–13) could be fertilized. Most embryo transfers\nwere performed with 2 ± 0.6 (1–3) embryos and embryo\ntransfer took place at day 5 in 37 % of the patients receiving\nG-CSF compared to 12 % in the controls without any\nTable 1 Study population\nG-CSF (N = 49) No medication (subgroup 1)\n(N = 33)\nOther medication (subgroup 2)\n(N = 45)\nAge (years) 37.63 ± 3.97 (27–44) 37.61 ± 4.41 (26–44), p = 0.73 37.61 ± 4.41 (26–44), p = 0.48\nNumber of cycles undergone before treatment 6.53 ± 3.99 (1–16) 5.27 ± 2.98 (0–12), p = 0.22 6.02 ± 4.1 (0–17), p = 0.56\nNumber of oocytes 9.41 ± 5.59 (1–23) 9.58 ± 5.04 (1–23), p = 0.65 7.84 ± 4.47 (1–18), p = 0.17\nNumber of fertilized oocytes 5.69 ± 3.92 (1–13) 4.97 ± 3.04 (1–12), p = 0.78 4.91 ± 3.43 (1–13), p = 0.38\nNumber of transferred embryos 2.14 ± 0.68 (1–3) 2.45 ± 0.67 (1–3), p = 0.04 2.36 ± 0.57 (1–3), p = 0.13\nNumber of early miscarriages (anamnestic) 2.67 ± 1.27 (1–6) 0.85 ± 0.91 (0–3), p\\ 0.001 0.64 ± 0.74 (0–3), p\\ 0.001\nNumber of late miscarriages (anamnestic) 0.2 ± 0 (0–1) 1.82 ± 1.04 (0–4), p\\ 0.001 1.78 ± 1.02 (0–4), p\\ 0.001\nValues are given as mean ± SD (minimum–maximum), patients receiving G-CSF or no medication at all (subgroup 1) or other medications like\nLMWH, ASS 100, folic acid, Prednisone/Dexamethasone or Doxycycline (subgroup 2)\nArch. Immunol. Ther. Exp. (2013) 61:159–164 161\n123\n\nmedication ( p = 0.05) and 9 % in the controls receiving\nadditional medications (p = 0.005) (Table 1).\nIn total, 71.36 % patients received good quality\nembryos, 73.61 % with G-CSF treatment, 62.22 % in\nsubgroup 1 and 76.25 % in subgroup 2. Good quality of an\nembryo was defined as an adequate morphology on the\nspecific day of transfer according to the most recently\npublished Istanbul criteria ( 2011).\nG-CSF Treatment\nSignificant differences were present between RM patients\nreceiving G-CSF and subgroups without G-CSF consider-\ning pregnancy rate (PR) and LBR. A PR of 47 % and a\nLBR of 32 % were achieved after G-CSF administration.\nIn comparison to the G-CSF group, the subgroup of RM\npatients with some other medication (subgroup 2; Fig. 1)\nshowed a PR of 27 %, p = 0.016, and a LBR of 14 %,\np = 0.006, while the subgroup with no medication reached\naP Ro f2 4% , p = 0.016, and a LBR of 13 %, p = 0.016\n(subgroup 1; Figs. 1 and 2).\nFocussing on the subgroup of RM patients with G-CSF\ntreatment, more patients receiving 1 9 34 Mill IU G-CSF\n(72 %) delivered a healthy baby compared to 21 % patients\nreceiving 2 9 13 Mill IU G-CSF, p = 0.001.\nConsidering the occurrence of twin pregnancies, 4 %\n(2/49) of patients receiving G-CSF delivered a multiple\npregnancy, compared to 0 % of patients without G-CSF\n(subgroup 2) and 6 % (2/33) of patients without G-CSF or\nany other medication (subgroup 1).\nSide effects were present in less than 10 % of patients,\nconsisting of irritation at the injection side, slight leuko-\ncytosis, rise in the temperature ( \\38 /C176C), mild bone pain\nand hyperemesis gravidarum. None of the newborn showed\nany kind of malformations.\nDiscussion\nWithin our retrospective cohort study, PR and LBR of RM\npatients with G-CSF treatment were compared to patients\nreceiving other relevant medications like LMWH and\nAspirin, folic acid, Cortisone or Doxycyclin and a sub-\ngroup of patients without any medication. There were\nsignificant differences, concerning PR and LBR between\nthe subgroups: a PR of 47 % was achieved after G-CSF\nadministration, of 27 % in RM patients with alternative\nmedications and 24 % in cycles without medication.\nHowever, we had some bias in our study: First of all,\npatients receiving G-CSF had significantly more often a\nday 5 embryo transfer as compared to the other patients. Of\ncourse, this ‘‘benefit’’ was counteracted due to the fact that\nG-CSF patients have undergone more early and late mis-\ncarriages, thus being a more severely affected RM\nsubgroup. Another bias might be that the subgroup 2,\nwhich received a wide range of medical treatments for\ndifferent indications, was a heterogeneous population.\nThus, it is complicated to reconcile and compare these\npatients. This could be one of the reasons for the low rates\nof pregnancy success.\nSo far, there are no treatment options in patients with\nunexplained RM which are established and distinctly jus-\ntified. According to a recent literature review, even the\napplication of LMWH could not be proven to be com-\npletely safe and generally be of beneficial impact in\npatients undergoing ART (Bohlmann 2011).\nBy now, the only randomised controlled trial concerning\nG-CSF treatment in RM patients was the one by Scarpellini\net al. ( 2009). Altogether, 68 women with primary unex-\nplained RM, all with at least four consecutive miscarriages,\nwere divided in a study ( n = 35) and a placebo ( n =\n33)\ngroup. In contrast to our study, the patients were all\nFig. 1 Pregnancy rates of patients receiving either G-CSF, no\nmedication at all (subgroup 1) or other medications like LMWH, ASS\n100, folic acid, Prednisone/Dexamethasone or Doxycycline (subgroup\n2). *p\\ 0.05\nLive birth rate\n0%\n5%\n10%\n15%\n20%\n25%\n30%\n35%\n40%\n45%\n50%\nG-CSF no medication other medication\n* **\nFig. 2 Live birth rate of patients receiving either G-CSF, no\nmedication at all (subgroup 1) or other medications like LMWH, ASS\n100, folic acid, Prednisone/Dexamethasone or Doxycycline (subgroup\n2). *p\\ 0.05; **p\\ 0.01\n162 Arch. Immunol. Ther. Exp. (2013) 61:159–164\n123\n\nyounger than 39 years, did not undergo ART and received\na G-CSF dosage which was 1 lg (100,000 IU)/kg/day\nstarting at the sixth day after ovulation. In addition, we did\nnot adjust our G-CSF dosage due to patients weight and did\nnot administer G-CSF daily. Taking an example of a 70 kg\npatient, the dosage per week of Scarpellini would be much\nhigher than ours (49 Mill IU vs. 26, respectively, 34 Mill IU).\nAlthough our study population was slightly larger, we did\nnot have the opportunity to form a placebo group consid-\nering the strict laws of the German Ethical committees.\nScarpellini and Sbracia (2009) did not report on significant\nside effects of G-CSF application except a mild skin rash\nand slight leukocyte elevation which corresponds to our\ndata.\nG-CSF has multiple effects on the immune system like\ninduction of the Th2 answer (Pan et al. 1995) and inhibi-\ntion of NK cells (Schlahsa et al. 2011) and blood\nmononuclear cells (Kitabayashi et al. 1995; Sugita et al.\n2003). Moreover, experiments on animals and cell lines\nhave proven a protective impact on fetuses (Novales et al.\n1993) and trophoblast cells (Marino and Roguin 2008).\nPresuming that an immunologic disorder might be one\nof the main reasons for RM, these patients could benefit\nconcerning the immunmodulatory functions of G-CSF.\nFurthermore, in vitro G-CSF application seems to be\nfavourable on the decidualization process of endometrial\nstromal cells (Tanaka et al. 2000). However, its specific\neffects on the endometrium as well as the feto-maternal\ninterphase are not fully understood yet.\nAs RM is a very heterogeneous pathology, the main\nproblem which we are facing is to specify the subgroup of\nRM patients, which benefits from G-CSF treatment. This\ncould be a surrogate immunological marker which we were\nnot able to identify yet. Scarpellini and Sbracia (2009) only\nselected patients with idiopathic RM. Winger et al. ( 2009),\nwho administered Humira, chose patients with a significant\nTh1/Th2 bias.\nOur data suggest that RM patients might benefit from\nG-CSF treatment, but a placebo-controlled clinical trial\nincluding more RM patients is needed as well as the\nongoing analysis of relevant immunological markers in\norder to further specify which RM patient will benefit from\nimmunmodulatory treatments.\nReferences\nAlpha Scientists in Reproductive Medicine and ESHRE Special\nInterest Group of Embryology (2011) The Istanbul consensus\nworkshop on embryo assessment: proceedings of an expert\nmeeting. Hum Reprod 26:1270–1283\nBohlmann MK (2011) Effects and effectiveness of heparin in assisted\nreproduction. J Reprod Immunol 90:82–90\nD’Addio F, Riella LV, Mfarrej BG et al (2011) The Link between the\nPDL1 costimulatory pathway and Th17 in fetomaternal toler-\nance. 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