말초혈액 자연살해세포 분획 및 세포용해 활성도 분석을 통한 습관성 유산 위험군의 진단적 유용성에 관한 연구 = Increased Peripheral NK Cell Fraction and Their Cytolytic activity in Patients with History of Recurrent Spontaneous Abortion

국문 초록 (Abstract)

목 적: 임신 전 $CD3^-/CD56^+/CD16^+$ 말초혈액 자연살해세포 (pbNK cell)의 분획과 세포용해 활성도를 정상군과 습관성 유산의 기왕력을 가진 환자군으로 나누어 비교, 분석하고 습관성 유산의 위험도를 제시할 수 있는 각각의 cut-off value를 설정하고자 하였다. 연구방법: 전향적 연구로서 습관성 유산의 기왕력이 있는 여성을 환자군 (n=35)으로 하였으며, 대조군으로 불임이나 습관성 유산의 기왕력이 없으며 정상아의 출산 경험이 있는 여성을 대조군 (n=15)으로 설정하였다. 유세포분석기를 이용하여 pbNK cell 분획 및 세포용해 활성도를 측정 후 그 결과를 비교 분석하였다. 결 과: pbNK cells의 분획은 습관성 유산 환자군에서 대조군에 비해 통계적으로 유의하게 높은 결과를 보였다($14.2{\pm}5.2$ vs. $9.4{\pm}3.7%$, p=0.002, 95% confidence interval [CI] 1.8~7.8). Receiver operating characteristic curve (ROC) 곡선을 이용하여 pbNK cell의 분획에 대한 cut-off values을 12.1%로 정하였을 때 습관성 유산의 위험도는 8.4배 증가하였다. pbNK cell의 K562 세포용해 활성도를 3가지 다른 Effector to Target (E:T) 비율 (50:1, 25:1, 12.5:1)을 사용하여 측정한 결과 각각의 경우에 있어 습관성 유산 환자군에서 대조군에 비해 통계적으로 유의하게 증가된 결과를 보였다 ($48.3{\pm}19.0$ vs. $31.3{\pm}11.9%$ in 50:1 ratio, p=0.002; $37.0{\pm}18.1$ vs. $20.2{\pm}9.2%$ in 25:1 ratio, p<0.001; $23.5{\pm}12.7$ vs. $12.4{\pm}7.3%$ in 12.5:1 ratio, p=0.001). ROC 곡선을 이용하여 각각 E:T 비율에서 세포용해 활성도의 cut-off values (43.1% in 50:1, 26.9% in 25:1, and 17.4% in 12.5:1)을 설정하여 분석한 결과 습관성 유산의 위험도는 각각 10.0배, 11.4배, 그리고 15.0배 증가된 결과를 보였다. 결 론: 원인이 분명하지 않은 습관성 유산 환자에서 pbNK cell의 분획과 세포용해 활성도를 측정하는 것은 면역학적 원인, 특히 동종면역 요인에 의한 습관성 유산의 유용한 진단 지표로 이용될 수 있을 것으로 사료된다. 향후 동종 면역반응에 의한 습관성 유산 환자에서 면역학적 원인의 치료 전, 후 pbNK cell의 분획과 세포용해 활성도를 측정, 비교하여 그 효과를 증명하는 연구가 필요할 것으로 생각된다.

다국어 초록 (Multilingual Abstract)

Objective: To testify whether the increased peripheral blood natural killer (pbNK) cells fraction and their cytolytic activity could coincide with patient's history of recurrent spontaneous abortion (RSA) and to evaluate these factors are can be valuable diagnostic markers in RSA. Methods: Women with a history of RSA comprised the patient group (n=35). Normal fertile women, who were experienced at least one healthy term birth without history of infertility or recurrent miscarriage, were included as the healthy control group (n=15). The pbNK cells of $CD3^-/CD56^+/CD16^+$ and their cytolytic activities against K562 cells were measured by flow cytometry and the values were compared between study and control groups. Results: Proportions of pbNK cells among peripheral blood monocytes (PBMC) ($14.2{\pm}5.2$ vs. $9.4{\pm}3.7%$, p=0.002, 95% confidence interval [CI], 1.8 to 7.8) was significantly higher in the patient group. The odds ratio of having RSA history was increased as 8.4 folds (59% of sensitivity, 80% of specificity, and 95% CI: 2.0 to 35.8) in patients who showed pbNK cells fraction above 12.1% which was determined as cut-off value by using ROC curve analysis. The cytolytic activities of pbNK cells which measured by three different ratio of effecter pbNK cells to target K562 cells and calculated by the percent of cytolytic K562 cells, were significantly higher in study group than that of control group (in 50:1 ratio, $48.3{\pm}19.0$ vs. $31.3{\pm}11.9%$, p=0.002; in 25:1 ratio, $37.0{\pm}18.1$ vs. $20.2{\pm}9.2%$, p<0.001; in 12.5:1 ratio, $23.5{\pm}12.7$ vs. $12.4{\pm}7.3%$, p=0.001). With the cut-off values of cytolytic activity of pbNK cells as 43.1% (50:1), 26.9% (25:1), and 17.4% (12.5:1) each, the risk of having RSA history was increased by 10.0, 11.4, and 15.0 folds in patients who had increased in each effector of pbNK to target of K562 cells ratio. Conclusion: The analysis of pbNK cells fraction and their cytolytic activity can be valuable diagnostic markers for RSA. We are going to planning the large scaled studies which include the data of obstetric outcomes in subsequent pregnancies to clarify our results of this study.

참고문헌 (Reference)

1 Kwak JY, "Up-regulated expression of CD56+, CD56+/ CD16+, and CD19+ cells in peripheral blood lymphocytes in pregnant women with recurrent pregnancy losses" 34 : 93-99, 1995

2 Cha SH, "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" 32 : 217-222, 2005

3 Hogge WA, "The clinical use of karyotyping spontaneous abortions" 189 : 397-400, 2003

4 Cooper MA, "The biology of human natural killer-cell subsets" 22 : 633-640, 2001

5 Higuchi K, "Suppression of natural killer cell activity by monocytes following immunotherapy for recurrent spontaneous aborters" 33 : 221-227, 1995

6 Hannes M, "Recurrent spontaneous miscarriage" 13 : 103-106, 1992

7 Yamada H, "Pre-conceptional natural killer cell activity and percentage as predictors of biochemical pregnancy and spontaneous abortion with normal chromosome karyotype" 50 : 351-534, 2003

8 Park DW, "Peripheral blood NK cells reflect changes in decidual NK cells in women with recurrent miscarriages" 63 : 173-180, 2010

9 Dwyer JM, "Manipulating the immune system with immune globulin" 326 : 107-116, 1992

10 Daya S, "Intravenous immunoglobulin therapy for recurrent spontaneous abortion: a meta-analysis" 39 : 69-76, 1998

11 Ruiz JE, "Intravenous immunoglobulin inhibits natural killer cell activity in vivo in women with recurrent spontaneous abortion" 35 : 370-375, 1996

12 Beer AE, "Immunophenotypic profiles of peripheral blood lymphocytes in women with recurrent pregnancy losses and in infertile women with multiple failed in vitro fertilization cycles" 35 : 376-382, 1996

13 Trundley A, "Human uterine leukocytes and pregnancy" 63 : 1-12, 2004

14 Choudhury SR, "Human reproductive failure I: immunological factors" 7 : 113-134, 2001

15 Cooper MA, "Human natural killer cells: a unique innate immunoregulatory role for the CD56(bright) subset" 97 : 3146-3151, 2001

16 Stray-Pedersen B, "Etiologic factors and subsequent reproductive performance in 195 couples with a prior history of habitual abortion" 148 : 140-146, 1984

17 Loke YW, "Decidua in human implantation" 10 (10): 14-21, 1995

18 Yokoyama M, "Cytotoxic cells directed against placental cells detected in human habitual abortions by an in vitro terminal labeling assay" 31 : 197-204, 1994

19 Daya S, "Critical analysis of intravenous immunoglobulin therapy for recurrent miscarriage" 5 : 475-482, 1999

20 Coulam CB, "Correlation of NK cell activation and inhibition markers with NK cytoxicity among women experiencing immunologic implantation failure after in vitro fertilization and embryo transfer" 20 : 58-62, 2003

21 Kwak-Kim J, "Clinical implication of natural killer cells and reproduction" 59 : 388-400, 2008