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1 大韓不妊學會誌 : 第 32 卷第 3 號 2005 Kor. J. Fertil. Steril., Vol. 32, No. 3, 2005, 9 습관성유산환자에서저용량면역글로불린치료와말초혈액내 Natural Killer (NK) 세포의임계치에관한연구 성균관대학교의과대학, 삼성제일병원산부인과불임및생식내분비 차선화 김해숙 김혜옥 송인옥 유근재 궁미경 강인수 양광문 The Preconceptional Level of Peripheral Natural Killer Cells which was Expected to Bring Successful Treatment Outcome using Low-dose Intravenous Gamma Immunoglobulin (IVIg) Infusion in Patients with Recurrent Spontaneous Abortion Sun Hwa Cha, Hae Suk Kim, Hye Ok Kim, In Ok Song, Keun Jai Yoo, Mi Kyung Koong, Inn Soo Kang, Kwang Moon Yang Department of Obstetrics and Gynecology, Samsung Cheil Hospital & Women's Healthcare Center Sungkyunkwan University School of Medicine, Seoul, Korea Objectives: We aimed to investigate the clinical effect of low-dose intravenous immunoglobulin treatment in unexplained recurrent spontaneous aborters (RSA) with elevated peripheral CD56+ natural killer (NK) cell levels and to determine the pre-conceptional NK cell percentage predictive of subsequent successful pregnancy outcome. Materials and Methods: Sixty four cases of unexplained recurrent miscarriage with elevated peripheral NK cells (>15%) were received low dose IVIg infusion at the dosage of 400 mg/kg/month after confirmation of gestational sac and continued until 20 weeks. The patients were divided into two groups according to the pregnancy outcome: Group I was success of treatment defined as live birth at or after 25 gestational weeks and Group II was failure of treatment. The preconceptional levels of the peripheral blood NK cells were compared between two groups. Results: Fifty-three pregnancies resulted in live births after 25 weeks and 11 resulted in abortion (Overall success rate of IVIG treatment was 82.8%). Preconceptional CD56+ NK cell percentage in group II (27.4±1.9%) was higher than those in group I (22.3±0.8%). By using ROC curve, optimal discrimination between success and failure of treatment was achieved with 27% of preconceptional NK cell percentage. Conclusion: In RSA patients with elevated NK cells, we suggest that preconceptional peripheral blood CD56+ NK cell level could be a useful marker for predicting successful treatment outcome of low-dose IVIg infusion. Key Words: Recurrent spontaneous abortion, Natural killer cell, Intravenous immunoglobulin 주관책임자 : 양광문, 우 ) 서울특별시중구묵정동 1-19번지, 삼성제일병원의학연구소산부인과불임및생식내분비분과. Tel: (02) , Fax: (02) , ykm2955@yahoo.co.kr

2 근래에습관성유산의원인과관련하여 natural killer (NK) 세포에대한많은연구가이루어지고있다. 정상적으로임신초기에는말초혈액내 NK 세포의활성도와비율은감소하는반면습관성유산환자에서는증가되어있고, 임신전에측정한경우에도역시습관성유산의경력이있는여성에서 NK 세포의비율은더높았다. 1~4 정상염색체를갖는태아를유산한여성에서혈액내 NK 세포가증가한소견을보이며, 5,6 비임신여성에서 NK 세포가증가한경우다음임신에서유산될가능성이높다고보고되었다. 7,8 요컨대 NK 세포를조절함으로써성공적인임신의예후를기대할수있고, 면역글로불린은이러한면역학적인원인의습관성유산환자에서착상부위의 NK 세포의활성도를낮추어모체내동종면역체인태아를공격하는것을막음으로써유산을방지하는효과를기대한다. 9,10 그러나아직까지면역학적원인의습관성유산환자에서면역글로불린치료의효과에대해서논란이되고있는데, 1995년 Coulam 등과 Christiansen 등은면역글로불린과위약의임신성공률을비교한연구에서각각 62.1% vs. 34.4% (odds ratio, 3.12), 52.9% vs. 29.4% (odds ratio, 2.70) 으로면역글로불린의효용성을입증하였으나, 11, 년 German RSA/ IVIG group과 1998년 Stephenson 등은 60.6% vs. 67.7% (odds ratio, 0.73), 50.0% vs. 52.6% (odds ratio, 0.90) 으로의미가없다고보고하였다. 13,14 한편국내에서김등은 3회이상의반복적자연유산을경험한습관성유산여성에서습관성유산의기왕력이없는여성에비해유의하게임신전말초혈액림프구의 CD56+ NK 세포의백분율이높음 (8.9% vs. 24.5%) 을보고하였고, 15 저자들은이전의연구에서 CD56+ NK 세포가 15% 이상증가한여성에서저용량의면역글로불린을주사함으로써치료하지않은군에비해통계적으로유의하게유산을방지할수있음을보고하였다. 16 이에저자들은면역학적원인에의한습관성유산환자중, 면역글로불린치료후 25주이상임신유지에성공한군과실패한군사이의임신전말초혈액내 CD56+ NK 세포의백분율을비교하고, 저용량면역글로불린치료의성공을예측할수있는임신전말초혈액내 CD56+ NK 세포의백분율 의임계치에대해알아보고자하였다. 연구대상및방법 1. 연구대상 2001년 12월 1일부터 2002년 9월 30일까지삼성제일병원습관성유산클리닉을내원한여성중임신 20주이전에 2번이상의반복적자연유산을경험한원인불명의습관성유산환자를포함하였다. 즉, 임신전에자궁난관조영술, 복강경, 자궁내시경, 부부염색체검사, 자가항체검사 (anticardiolipin antibody, antinuclear antibody, antimicrosome antibody, antithyroglobulin antibody, lupus anticoagulant), 배란 7일후황체호르몬검사, 자궁내막검사등을시행하여염색체이상, 자궁의해부학적이상, 내분비이상, 감염에의한습관성유산을제외하였다. 이후임신된 64명에서이전의저자들의연구에근거하여, 15,16 임신전말초혈액임파구에서 CD56+ NK cell 이차지하는백분율을측정하여 15% 이상증가된환자를대상으로질초음파검사를통해임신낭을확인한직후부터임신 20주정도까지 4주간격으로환자몸무게의 Kg당 400 mg 용량의면역글로불린을정맥내주사하였다. 면역글로불린치료후재태연령 25주이상임신유지에성공한환자군 (Group I) 과재태연령 25주미만에임신이종결된대조군 (Group II) 에서양군의임신전 CD16+ 및 CD56+ NK cell 백분율을비교하였다. 2. CD16+ 및 CD56+ NK cell 백분율의측정각각의환자로부터말초혈액 10 ml를채취한후 F-H gradient solution을이용하여림프구나큰단핵세포를 2000 µ 정도로세척하여분리하였다. CD16+ 및 CD56+ NK cell에간접형광염색을하여 FAC- Scan (Becton-Dickinson Co, mauntainview, U.S.A) 기기를이용하여 CD16+ 및 CD56+ NK cell을분석하여절대수와백분율을구하였다. 3. 통계분석통계적유의성검정은 χ 2 test로하였고, p<0.05일때통계적으로유의한차이가있다고정의하였다. ROC (Receiver operating characteristics) curve를통해

3 Table 1. Clinical characteristics of the treatment success (Group I) and failure group (Group II) Group I (n=53) Group II (n=11) P-value Age (year) 30.4± ±1.1 NS No. of SAB * 2.5± ±0.5 NS Weeks of SAB 8.98± ±1.1 NS Primary SAB (%) 44/53 (83) 8/11 (72.7) NS Autoantibody positivity (%) 30/53 (56.6) 8/11 (72.7) NS CD 56+NK cell percentage (%) 22.3± ± CD 16+NK cell percentage (%) 13.6± ±2.2 NS Values were mean ± SE. Group I: pregnancy sustained beyond 25 gestational weeks Group II: pregnancy terminated below 25 gestational weeks *SAB: spontaneous abortion Antithyroglobin Ab, antimicrosome Ab, antinuclear Ab, antiphospholipid Ab, lupus anticoagulant, rheumatic Ab. CD56+, CD16+ NK cell percentage in peripheral blood lymphocyte 면역글로불린치료의효용성을증명할수있는임계치를구하였다. 결 과 연구대상 64명중임신초기에저용량면역글로불린을투여받고 25주이상임신이성공적으로유지된군은 53명으로 82.8% 의치료성공률을나타냈다. 이들치료성공군 (Group I, 53명 ) 과 25주미만에임신이종결된치료실패군 (Group II, 11명 ) 간의평균나이, 평균자연유산횟수, 일차성혹은이차성습관성유산의분포, 자가항체양성률에유의한차이는없었다 (Table 1). 이번임신에서초기에자연유산된치료실패군 (group II) 11명은 6주에서 20주사이에임신이종결되었으며, 이들중 5예에서수태산물에대한염색체검사를시행하였고, 4예에서는정상소견을보였고, 다른 1예에서는삼염색체 (21 trisomy) 가진단되었다. 치료실패군 (Group II) 11명의 CD56+ NK 세포백분율은평균 27.4% 로치료성공군 (Group I, 53명, 22.3%) 에비해서는유의하게높은수치를나타냈다 (p=0.008) (Figure 1). 그러나말초혈액림프구의 CD16+ NK 세포백분율은양군간의통계학적으로유의한차이는없었다 (Table 1). Figure 1. Pre-conceptional CD56+ natural killer (NK) cell percentage in each groups. 저용량면역글로불린의치료성공을예측할수있는임계치로는임신전혈액내 CD56+ NK 세포백분율이 27% 이하일때 (OR 4.58, 95% CI 1.18~ 17.8) 로산정되었다 (Table 2). 고 찰 임신초기에프로제스테론, 임신단백인 TJ6, 그외단핵구나영양막세포 (trophoblast) 에서분비하는사이토카인에의해유도되는세포자멸사 (apo

4 Table 2. Prediction of successful pregnancy stratified by preconceptional level of CD56+ NK cell in RSA patients treated with low-dose IVIg Cut-off values of CD56+ cell 27% 28% Sensitivity (%) Specificity (%) Positive predictive value (%) Negative predictive value (%) Relative risk % CI 1.18~ ~8.82 ptosis) 등의기전에의해정상에서는 CD56+ NK 세포가감소하는것으로알려져있다. 17~21 그러나면역학적원인의습관성유산여성의면역표현형 (immunophenotype) 을조사하면많은수에서말초혈액내 CD56+, CD56+/16+ NK 세포와 CD19+ B 세포가현저하게증가되어있다. 4 이러한 NK 세포는임신초기에영양막세포를인식하는데실제습관성유산여성의말초혈액에서얻은 NK 세포가자가태반세포에대한세포독성을나타냄을증명할수있었다. 22 이런환자에서임신초기에 CD56+ NK 세포를감소시킴으로써반복적인유산을막고성공적인임신결과를얻을수있다. 그러나면역글로불린의치료를받고도자연유산이된여성에서는 CD56+ NK 세포의감소가관찰되지않았다. 10 Yamada 등은원인불명의습관성유산여성에서임신전 NK 세포활성도가 46% 이상일때상대위험도가 3.6, 말초혈액림프구중 CD56+ 세포백분율이 16.4% 이상일때상대위험도가 4.9로향후임신에서생화학적임신과정상염색체를갖는태아의자연유산의위험률이더높았음을증명하였고, 따라서 CD56+ NK 세포가향후임신의결과를예측하고치료방침을세우는데있어효율적인방법이라고주장하였다. 6 본연구에서도임신전반기에저용량면역글로불린치료를받은습관성유산여성중 25주미만에유산된군의임신전말초혈액내 CD56+ NK 세포의백분율는 27.4% 로 25주이상임신유지에성공 한군에비해통계학적으로유의하게높은수치를나타내었다. 이는앞서언급한바와같이 NK 세포의비율이높을수록유산의위험이높고, 이를적절하게조절하는것이향후성공적인임신유지에중요하다는것을설명할수있다. CD56+ NK 세포가증가된습관성유산여성에서면역글로불린치료후 in vivo와 in vitro 모두에서말초혈액내 CD56+, CD56+/16+ NK 세포가효과적으로억제되었다. 이는면역글로불린이 Th-2 cytokine 면역반응이우세한방향으로면역체계를유도하여 Th-1 cytokine에의한항체의존세포매개세포독성 (antibody-dependent cell-mediated cytotoxicity) 을약화시킴으로써 NK 세포의활성화를저하시키는것으로가정하였다. 10,23,24 Ruiz 등은습관성유산여성에서면역글로불린치료이전과치료후 7~11일에측정한 NK 세포의활성도를비교한 in vivo 연구에서다음임신시임신유지에성공하여출산한예에서는 NK 세포의활성도가현저히감소하거나변화가없었던반면, 유산이되었던예는유산당시그활성도가현저히증가되어있었고, 임신이되지않은 1예에서는매우높은 NK 세포독성을나타낸것으로보아 NK 세포활성도가임신의예후및면역글로불린치료의효용성을예측하는데도움이될수있다고보고하였다. 10 본연구에서사용한저용량의면역글로불린치료는태아의생존가능성이있는 25주를기준으로하여그이상임신이지속되었을때를치료의성공으로간주한다면약 82.8% 의치료성공률을보였다. 이는기존의 randomized, double-blind controlled study에대한 meta-analysis에서면역글로불린치료의 overall success rate를 62.4% 로보고한것에비해높게나타났다. 이는아마도기존의연구들과달리본연구에서는임신전말초혈액내 CD56+ NK 세포가증가한경우, 즉면역학적원인에기인한습관성유산여성에만선택적으로면역글로불린을주입함으로써치료의효용성을높여주었다고할수있겠다. 비슷하게 Kwak 등의연구에서임신전 NK 세포가 12% 미만으로면역글로불린을주입하지않은군과 12% 이상이면서저용량면역글로불린을주입한군, 면역글로불린주입을하지않은군간의

5 출산율 (live birth rate) 은각각 83.3% (15/18), 100% (9/9), 33.3% (2/6) 로유의한차이를보였다. 특히이들은 CD56+ NK 세포의활성도를미리측정하여조절함으로써유산율뿐만아니라조산및자궁내태아성장지연역시감소하였다고보고하였다. 24 한편아직까지습관성유산여성에서사용하는면역글로불린치료의적절한용량에대한연구는정립된바없다. 이전에발표되었던연구들에서사용한면역글로불린치료의용량과주입시기는매우다양하고결과역시다양하게나타났다. 다만 Ruiz은습관성유산여성에서채취한혈액을이용한 in vitro 실험에서면역글로불린을처리하였을때 93% 에서용량에비례하여 (dose-dependent) NK 세포활성도가감소하는것을증명하고이는 effector cell 수준에서 immunoglobulin의 antigen binding portion에의해매개되기때문이라고설명하였다. 23 이에저자들은저용량면역글로불린치료의성공을예측할수있는 CD56+ NK 세포의임계치를알아보고자하였고, CD56+ NK 세포가 27% 이하일때에는저용량만으로도유의하게 (OR 4.58, 95% CI 1.18~17.8) 효과를기대할수있으나 28% 이상에서는통계적으로유의한차이는없었다. 최근 Morikawa 등은 4회이상반복자연유산의경력이있는원인불명의습관성유산여성에서임신확인직후대용량의면역글로불린 (over the course of 5 days at a dose of 20 g/d, for a total dose of 100 g) 을투여한결과, 태아의염색체이상으로인한자연유산을제외하면 100% 에서임신유지에성공하였고, 말초혈액내 CD56+ NK 세포의비율도치료이전에비해유의하게감소하였다고보고하였다. 25 본연구에서저용량면역글로불린치료로실패한군에서특히임신전 CD56+ NK 세포가현저하게상승된여성에서는이처럼면역글로불린의용량이나주입시기를달리함으로써임신유지의성공을기대해볼수있겠다. 이상의연구결과로원인불명의습관성유산여성에서면역글로불린치료를시행하는데있어말초혈액내 CD56+ NK 세포의활성도를측정하는것은유용한지표로이용될수있고, 한편 NK 세포의비율이현저하게상승된경우에는저용량의면역글로불린치료만으로는잘조절되지않아효과 적으로유산을방지하지못함을알수있었다. 따라서향후면역글로불린용량과반응에관련된체계적인연구가필요할것으로사료된다. 참고문헌 1. Wegmann TG, Lin H, Guilbert L, Mosmann TR. Bidirectional cytokine interactions in the maternalfetal relationship: Is successful pregnancy a TH2 phenomenon? Immunol Today 1993; 14: Higuchi K, Aoki K, Kimbara T, Hosoi N, Yamamoto T, Okada H. Suppression of natural killer cell activity by monocytes following immunotherapy for recurrent spontaneous aborters. Am J Reprod Immunol 1995; 33: Kwak JY, Beaman KD, Gilman-Sachs A, Ruiz JE, Schewitz D, Beer AE. Up-regulated expression of CD56+, CD56+/CD16+, and CD19+ cells in peripheral blood lymphocytes in pregnant women with recurrent pregnancy losses. Am J Reprod Immunol 1995; 34: Beer AE, Kwak JY, Ruiz JE. Immunophenotypic profiles of peripheral blood lymphocytes in women with recurrent pregnancy losses and in infertile women with multiple failed in vitro fertilization cycles. Am J Reprod Immunol 1996; 35: Clark DA, Coulam CB. Is there an immunological cause of repeated pregnancy wastage? Adv Obstet Gynecol 1995; 3: Yamada H, Morikawa M, Kato EH, Shimada S, Kobashi G, Minakami H. Pre-conceptional naturalkiller-cell activity and percentage as predictors of biochemical pregnancy and spontaneous abortion with normal chromosome karyotype. Am J Reprod Immunol 2003; 50: Aoki K, Kajiura S, Matsumoto Y, Ogasawara M, Okada S, Yagami Y, et al. Preconceptional naturalkiller-cell activity as a predictor of miscarriage. Lancet 1995; 345: Emmer PM, Veerhoek M, Nelen WL, Steegers EA, Joosten I. Natural killer cell reactivity and HLA-G

6 in recurrent spontaneous abortion. Transplant Proc 1999; 31: Kwak JY, Kwak FM, Ainbinder SW, Ruiz AM, Beer AE. Elevated peripheral blood natural killer cells are effectively downregulated by immunoglobulin G infusion in women with recurrent spontaneous abortions. Am J Reprod Immunol 1996; 35: Ruiz JE, Kwak JY, Baum L, Gilman-Sachs A, Beaman KD, Kim YB, et al. Intravenous immunoglobulin inhibits natural killer cell activity in vivo in women with recurrent spontaneous abortion. Am J Reprod Immunol 1996; 35: Coulam CB, Krysa L, Sterm J, Bustillo M. Intravenous immunoglobulin for treatment of recurrent pregnancy loss. Am J Reprod Immunol 1995; 34: Christiansen OB, Mathiesen O, Husth M, Ramsussen KL, Ingersler HJ, Lauristen JG, et al. Placebocontrolled trial of treatment of unexplained secondary recurrent spontaneous abortion with i.v. immunoglobulin. Hum Reprod 1995; 10: Intravenous immunoglobulin in the prevention of recurrent miscarriage. The German RSA/IVIG Group [editorial]. Br J Obstet Gynaecol 1994; 101: Stephenson MD, Dreher K, Houlihan E, Wu V. Prevention of unexplained recurrent spontaneous abortion using intravenous immunoglobulin: a prospective, randomized, double, blinded, placebo controlled trial. Am J Reprod Immunol 1998; 39: 김문영, 양광문, 김해숙, 차선화, 궁미경, 강인수등. 말초혈액 CD56+ Natural Killer cell의증가에기인하여면역글로불린치료후분만한습관성유산환자에서향후계획임신전시행한말초혈액 NK cell의변화에대한연구. 대한산부회지 2004; 47: 차선화, 양광문, 박찬우, 김해숙, 조동희, 강인수, 궁미경등. 말초혈액 CD56+ Natural Killer Cell 증가에기인한습관성유산환자에서정맥내면역글로블린치료의효과에관한연구. 대한불임학회지 2005; 32: Szekeres-Bartko J, Faust Z, Varga P, Szereday L, Kelemen K. The immunological pregnancy protective effect of progesterone is manifested via controlling cytokene production. Am J Reprod Immunol 1996; 35: Ribbing SL, Hoverland RC, Beaman KD. T-cell suppressor factor play an integral role in preventing fetal rejection. J Reprod Immunol 1988; 14: Nichols TC, Kang J, Angkachatchai V, Beer AE, Beaman KD. Expression of a membrane form of the pregnancy associated protein TJ6 on lymphocytes. Cell Immunol 1994; 155: Beaman KD, Angkahatchai V, Gilman-Sacks A. TJ6: The pregnancy-associated cytokine. Am J Reprod Immunol 1996; 35: Clark DA, Vince G, Flanders KC, Hirte H, Starkey P. CD56+ lymphoid cells in human first trimester pregnancy decidua as a source of novel TGF-β 2-related immunosuppressive factors. Hum Reprod 1994; 9: Yokoyama M, Sano M, Sonoda K, Nozaki M, Nakamura GI, Nakano H. Cytotoxic cells directed against placental cells detected in human habitual abortions by an in vitro terminal labeling assay. Am J Reprod Immunol 1994; 31: Ruiz JE. Effect of intravenous immunoglobulin on natural killer cell activity in women with recurrent spontaneous abortion. Master thesis, Finch university of health sciences/the chicagomedical school, United states; Kwak JY, Kwak FM, Gilman-Sachs A, Beaman KD, Cho DD, Beer AE. Immunoglobulin G infusion treatment for women with recurrent spontaneous abortions and elevated CD56+ Natural killer cells. Early pregnancy 2000; 4: Morikawa M, Yamada H, Kato EH, Shimada S, Kishi T, Yamada T, et al. Massive intravenous immunoglobulin treatment in women with four or more recurrent spontaneous abortions of unexplained etiology: down-regulation of NK cell activity and subsets. Am J Reprod Immunol 2001; 46:

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대한진단검사의학회지제 26 권제 3 호 2006 Korean J Lab Med 2006;26:198-203 원저 진단면역학 습관성유산환자단핵구에서의 CD56/CD16 항원표현과혈청 TNF-alpha 정량 김혜련 1 박애자 1 이미경 1 조동희 2 중앙대학교의과대학진단검사의학과교실 1, 성균관의대삼성제일병원진단검사의학과 2 CD56/CD16 Expression