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Altered interleukin 12 and nitric oxide levels in recurrrent miscarriage
European Journal of Obstetrics & Gynecology and Reproductive Biology 75 (1997) 211–214
Altered interleukin 12 and nitric oxide levels in recurrrent miscarriage a, a a a b Rhoda Wilson *, Iain McInnes , Bernard Leung , James H. McKillop , James J. Walker a
Department of Medicine, Glasgow Royal Infirmary, Glasgow G31 2 ER, UK Department of Obstetrics and Gynaecology, St James Hospital, Leeds, UK
b
Received 16 January 1997; received in revised form 15 April 1997; accepted 2 June 1997
Abstract The causes of recurrent miscarriage are not fully understood. Recent studies have suggested that whilst a T H 2 type immune response may be associated with a healthy pregnancy, miscarriage may be associated with a T H 1 type response. Serum levels of nitric oxide (NO) and Interleukin 12 (IL 12) were measured in; healthy non-pregnant women; healthy pregnant women; women suffering spontaneous abortion; pregnant women with a history of recurrent miscarriage; non-pregnant women with a history of recurrent miscarriage. Normal pregnancy was associated with a significant decrease in serum levels of nitrite (13.0 vs. 22.0 P,0.0001). In women admitted with spontaneous abortion there was a significant increase in the levels of nitrite (16.0 vs. 13.0 P,0.05), but no change in IL 12 compared to normal pregnant women. In pregnant women with a history of recurrent miscarriage levels of nitrite (16.0 vs. 13.0 P,0.05) and IL 12 (10.0 vs. 6.0 P,0.0006) were significantly elevated compared to normal pregnancy. When these women were sampled prior to becoming pregnant the levels of NO were found to be significantly lower than those in the non-pregnant control group (13.1 vs. 22.0 P,0.05) although levels of IL 12 were unchanged. No correlation was found between serum nitrite and IL 12 levels. This report further supports the idea that polarisation of the immune response during pregnancy may predispose to recurrent miscarriage. 1997 Elsevier Science Ireland Ltd. Keywords: Interleukin 12; Nitric oxide; Miscarriage; Immune response
1. Introduction Ten–fifteen percent of all pregnancies end in miscarriage. Although most women will suffer no further problems in subsequent pregnancies, approximately 1% will suffer recurrent miscarriage, defined as 3 or more miscarriages before 24 weeks gestation [1]. The aetiology of miscarriage is often multifactorial with some cases due to infection and others to chromosomal abnormalities [2]. However in many patients, the cause remains unknown. Cytokine and soluble cytokine receptor levels are elevated in the serum and decidua of recurrent miscarriage patients, indicating that immunological mechanisms may be important [3]. The effector T cell response may be divided functionally *Corresponding author. Tel: 144-141-2115418; fax: 144-1415522953.
into two subsets. T H 1 cells are characterised by the production of IFNg and IL2 with subsequent development of cell mediated responses, whereas T H 2 cells selectively produce IL 4, IL 5 and IL 13 and predispose to a humoral response. Recent studies have shown that trophoblast stimulated peripheral blood cells from recurrent miscarriage patients generate an excess of T H 1 derived cytokine in contrast to controls, indicating that T H 2 responses may be the predominant situation during pregnancy and that alteration of this state may lead to pathology [4]. We have recently observed increased numbers of the antibody producing B cell subset CD5 / 20 these patients, together with other tissue specific autoantibodies [5]. Moreover elevated serum levels of lupus anticoagulant have been reported [6], indicating a role for humoral mechanisms. Interleukin 12 (IL 12) is a macrophage and B cell derived cytokine which is crucial for the maturation of the Th1 phenotype in vivo and in vitro. Exogenous administra-
0301-2115 / 97 / $17.00 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S0301-2115( 97 )00124-3
212
R. Wilson et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 75 (1997) 211 – 214
tion of IL 12 will enhance autoimmune responses and inhibition of IL 12 effects may prejudice T cell responses to the Th2 phenotype [7]. We have recently shown that IL 12 production is closely related to the generation of NO in the MRL murine autoimmunity model [8]. As NO has been implicated in the events surrounding early abortion [9] we therefore hypothesised that parallel production of IL 12 and nitric oxide (NO) might occur in recurrent miscarriage. We now report the presence of elevated levels of NO and IL 12 in spontaneous abortion and recurrent miscarriage in contrast to those measured in normal pregnancy.
2. Patients and methods Local ethical committee approval was obtained prior to collection of samples. Clotted venous blood samples obtained from 108 Caucasian women attending Glasgow Royal Maternity hospital were immediately spun at 500 g for 10 min and serum was stored at 2708C until assay in single batches. 5 distinct patient groups were identified. Group 1 comprised 31 healthy non-pregnant females. Group 2 comprised 18 healthy primigravidae of 7.261.5 weeks gestation. All pregnancies continued successfully to term. Group 3 comprised 10 women of 7.561.9 weeks gestation, admitted during spontaneous abortion, none of whom were recurrent aborters. Group 4 comprised 29 women of 6.661.8 weeks gestation all with a previous history of recurrent miscarriage. Of these 13 later aborted (Group 4a) and 16 continued successfully to term (Group 4b). Group 5 comprised 20 non-pregnant females with a previous history of recurrent miscarriage, who were attending a pre-pregnancy clinic because of this significant past history. Serum nitrate levels were measured by chemiluminescence as previously described [10]. Briefly, after reduction of nitrate using nitrate reductase (Sigma, Poole, UK) samples were injected into boiling glacial acetic acid (Sigma), 6% sodium iodide (Sigma) then NO produced in this reaction was carried in gaseous nitrogen into an NO analyser (Dasibi, Japan) adapted for the purpose. Sodium nitrate standards were prepared and provided a standard curve for comparison. Lower limit of detection was 2 mM. IL 12 levels were measured using a commercially available sandwich ELISA purchased from R&D Systems (Oxon, UK). The assay which was used according to manufacturers instructions had a sensitivity of 5 pg / ml.
3. Statistics Results are expressed as medians. Results were tested for statistical significance using a Mann Whitney Test.
4. Results Results of serum nitrate levels are given in Fig. 1. Compared to non-pregnant controls serum nitrate levels were significantly reduced in healthy first trimester pregnant women. In contrast, in first trimester pregnant women with previous history of recurrent miscarriage but no current active clinical pathology, serum nitrate levels were significantly higher than those predicted by the normal pregnancy group. Moreover, nitrate levels in pregnant women with a history of recurrent miscarriage were equivalent to those found in patients undergoing spontaneous abortion. When pregnant women with previous recurrent miscarriage were further divided into subsequent failed or successful outcome, serum nitrate levels in those whose pregnancies subsequently failed were significantly higher than in normal pregnancy (16.0 vs. 13.0 P,0.05). In contrast, nitrate levels were not significantly different from normal pregnancy in those women who progressed to a successful delivery (12.4 vs. 13.0 N.S). Normal pregnancy was not associated with significantly increased levels of serum IL 12 (Fig. 2). Serum IL-12 was normal during spontaneous abortion compared to the control group. In contrast, levels of IL 12 were significantly increased in first trimester pregnant women with a history of recurrent miscarriage compared to normal pregnancy. When this group were again subdivided by outcome, those whose pregnancy subsequently failed exhibited higher serum IL-12 levels than pregnant controls (12.4 vs. 8.0 P,0.05), whereas, those with successful outcome did not significantly differ (10.8 vs. 8.0 N.S.). No correlation was found between IL 12 and serum nitrate levels (r50.23 N.S.).
5. Discussion Successful outcome of pregnancy requires active adaptation of the maternal immune system. T H 2 type cytokines released by the trophoblast, early in the course of a normal pregnancy may suppress the cellular immune responses that could endanger the foetus [7]. Recurrent miscarriage has been associated with T H 1 type immune response directed at the trophoblast. Recent studies in mice have shown there may be a role for NO either as an effector molecule mediating early murine embryo loss or in the in situ activation of decidual macrophages which was observed as an early event preceding spontaneous abortion [9]. These studies used tissue obtained from the site of implantation. The current study examined peripheral blood and found serum levels of IL12 and NO to be significantly elevated in pregnant women with a history of recurrent miscarriage compared to normal pregnant women. The increases which we observed occurred early in the first trimester, before there was any active sign of miscarriage. These changes were possibly initiated in the uterus. Although neither the NO or IL 12 were of any predictive
R. Wilson et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 75 (1997) 211 – 214
213
Fig. 1. Definition of groups. Group 1: non-pregnant; Group 2: pregnant; Group 3: spontaneous abortion; Group 4: recurrent miscarriage; Group 5: recurrent miscarriage–pre-pregnancy. Levels of serum nitrate were measured as described previously. 1 P,0.05; 11 P,0.0001 vs. Group 1.
Fig. 2. Definition of groups. Group 1: non-pregnant; Group 2: pregnant; Group 3: spontaneous abortion; Group 4: recurrent miscarriage; Group 5: recurrent miscarriage–pre-pregnancy. Levels of IL 12 were measured as described previously. 111 P,0.001 vs. Group 1; ***P,0.0006 vs. Group 2; 8P,0.05 vs Group 4.
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R. Wilson et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 75 (1997) 211 – 214
value, levels of both were significantly higher in those women with a history of recurrent miscarriage whose pregnancies ultimately failed compared to those with a history of recurrent miscarriage whose pregnancies on this occasion continued to term. Moreover patterns seen in women with a history of recurrent miscarriage resembled those found in women admitted during spontaneous abortion. Our results implicate IL 12 and NO in pregnancy loss. It is possible that the T H 2 response seen in continuing pregnancy helps prevent generation of IL 12 and NO. However in recurrent miscarriage, events occur which facilitate production of NO and IL 12 indicative of a T H 1 type response. Previous studies have found NO to be detectable early on in placental development. In addition increased serum and urine levels of NO have been found in women suffering pre term labour. Whether the elevated levels of NO and IL 12 found in this study are the result of increased immunological activity within the peripheral circulation or whether they are the result of events in the uterus is not clear. What has emerged from the present study is that early in the course of a normal pregnancy NO production falls and IL 12 production becomes detectable. These changes do not occur in women with a history of recurrent miscarriage regardless of the outcome of their pregnancy. This study confirms the idea that T H cells (or cytokine producing T H cells) are involved in the aetiology of recurrent miscarriage. The increased immunological activity seen previously in these women and the increased number of antibody producing B cells may be responsible for the increased levels of NO and IL 12.
Acknowledgements We are grateful to Professor F.Y. Liew, Department of Immunology, Western Infirmary, Glasgow for his help in this project.
References [1] Stirred GM. Lancet 1990;336:673–5. [2] Clark DA, Bonnar J, editors. Recent advances in Obstetrics and Gynaecology. Churchill Livingstone. Longman Group 1989:25–35. [3] Clark DA, Lea RG, Podor T, Daya S, Banwatt, Hailey C. Cytokines Determining the Success or failure of Pregnancy. Frontiers in Human Reproduction. 1991;626:524–36. [4] Hill JA, Katalin P, Anderson DJ. T helper 1 type immunity to trophoblasts in women with recurrent spontaneous abortion. JAMA 1995;273:1933–6. [5] Roberts J, Jenkins C, Wilson R, Maclean MA, McKillop JH, Walker JJ. Eur J Endo 1966;134:84–6. [6] Ware-Branch D. Autoimmunity and Pregnancy Loss. J Am Med Assoc 1990;264:1453–4. [7] Lieblau RS, Singer S, McDevit HO. Th 1 and ThH2 CD41 T cells in the pathogenesis of organspecific diseases. Immunol Today 1995;16:34–8. [8] Huang FP, Feng GJ, Lindop G, Stott D, Liew FY. The role of IL 12 and nitric oxide in the development of spontaneous autoimmune diseas in MRL / MP-lpr / lpr mice. J Exp Med 1996;183:1447–59. [9] Haddad EK, Dudos AJ, Barnes MG. Early embryo loss is associated with local production of nitric oxide by decidual mononuclear cells. J Exp Med 1995;182:1143–52. [10] Aoki T. Continous flow determination of nitrite with membrane separation chemiluminesence detection. Biomed Chromatog 1994; 128–130.
Altered interleukin 12 and nitric oxide levels in recurrrent miscarriage a, a a a b Rhoda Wilson *, Iain McInnes , Bernard Leung , James H. McKillop , James J. Walker a
Department of Medicine, Glasgow Royal Infirmary, Glasgow G31 2 ER, UK Department of Obstetrics and Gynaecology, St James Hospital, Leeds, UK
b
Received 16 January 1997; received in revised form 15 April 1997; accepted 2 June 1997
Abstract The causes of recurrent miscarriage are not fully understood. Recent studies have suggested that whilst a T H 2 type immune response may be associated with a healthy pregnancy, miscarriage may be associated with a T H 1 type response. Serum levels of nitric oxide (NO) and Interleukin 12 (IL 12) were measured in; healthy non-pregnant women; healthy pregnant women; women suffering spontaneous abortion; pregnant women with a history of recurrent miscarriage; non-pregnant women with a history of recurrent miscarriage. Normal pregnancy was associated with a significant decrease in serum levels of nitrite (13.0 vs. 22.0 P,0.0001). In women admitted with spontaneous abortion there was a significant increase in the levels of nitrite (16.0 vs. 13.0 P,0.05), but no change in IL 12 compared to normal pregnant women. In pregnant women with a history of recurrent miscarriage levels of nitrite (16.0 vs. 13.0 P,0.05) and IL 12 (10.0 vs. 6.0 P,0.0006) were significantly elevated compared to normal pregnancy. When these women were sampled prior to becoming pregnant the levels of NO were found to be significantly lower than those in the non-pregnant control group (13.1 vs. 22.0 P,0.05) although levels of IL 12 were unchanged. No correlation was found between serum nitrite and IL 12 levels. This report further supports the idea that polarisation of the immune response during pregnancy may predispose to recurrent miscarriage. 1997 Elsevier Science Ireland Ltd. Keywords: Interleukin 12; Nitric oxide; Miscarriage; Immune response
1. Introduction Ten–fifteen percent of all pregnancies end in miscarriage. Although most women will suffer no further problems in subsequent pregnancies, approximately 1% will suffer recurrent miscarriage, defined as 3 or more miscarriages before 24 weeks gestation [1]. The aetiology of miscarriage is often multifactorial with some cases due to infection and others to chromosomal abnormalities [2]. However in many patients, the cause remains unknown. Cytokine and soluble cytokine receptor levels are elevated in the serum and decidua of recurrent miscarriage patients, indicating that immunological mechanisms may be important [3]. The effector T cell response may be divided functionally *Corresponding author. Tel: 144-141-2115418; fax: 144-1415522953.
into two subsets. T H 1 cells are characterised by the production of IFNg and IL2 with subsequent development of cell mediated responses, whereas T H 2 cells selectively produce IL 4, IL 5 and IL 13 and predispose to a humoral response. Recent studies have shown that trophoblast stimulated peripheral blood cells from recurrent miscarriage patients generate an excess of T H 1 derived cytokine in contrast to controls, indicating that T H 2 responses may be the predominant situation during pregnancy and that alteration of this state may lead to pathology [4]. We have recently observed increased numbers of the antibody producing B cell subset CD5 / 20 these patients, together with other tissue specific autoantibodies [5]. Moreover elevated serum levels of lupus anticoagulant have been reported [6], indicating a role for humoral mechanisms. Interleukin 12 (IL 12) is a macrophage and B cell derived cytokine which is crucial for the maturation of the Th1 phenotype in vivo and in vitro. Exogenous administra-
0301-2115 / 97 / $17.00 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S0301-2115( 97 )00124-3
212
R. Wilson et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 75 (1997) 211 – 214
tion of IL 12 will enhance autoimmune responses and inhibition of IL 12 effects may prejudice T cell responses to the Th2 phenotype [7]. We have recently shown that IL 12 production is closely related to the generation of NO in the MRL murine autoimmunity model [8]. As NO has been implicated in the events surrounding early abortion [9] we therefore hypothesised that parallel production of IL 12 and nitric oxide (NO) might occur in recurrent miscarriage. We now report the presence of elevated levels of NO and IL 12 in spontaneous abortion and recurrent miscarriage in contrast to those measured in normal pregnancy.
2. Patients and methods Local ethical committee approval was obtained prior to collection of samples. Clotted venous blood samples obtained from 108 Caucasian women attending Glasgow Royal Maternity hospital were immediately spun at 500 g for 10 min and serum was stored at 2708C until assay in single batches. 5 distinct patient groups were identified. Group 1 comprised 31 healthy non-pregnant females. Group 2 comprised 18 healthy primigravidae of 7.261.5 weeks gestation. All pregnancies continued successfully to term. Group 3 comprised 10 women of 7.561.9 weeks gestation, admitted during spontaneous abortion, none of whom were recurrent aborters. Group 4 comprised 29 women of 6.661.8 weeks gestation all with a previous history of recurrent miscarriage. Of these 13 later aborted (Group 4a) and 16 continued successfully to term (Group 4b). Group 5 comprised 20 non-pregnant females with a previous history of recurrent miscarriage, who were attending a pre-pregnancy clinic because of this significant past history. Serum nitrate levels were measured by chemiluminescence as previously described [10]. Briefly, after reduction of nitrate using nitrate reductase (Sigma, Poole, UK) samples were injected into boiling glacial acetic acid (Sigma), 6% sodium iodide (Sigma) then NO produced in this reaction was carried in gaseous nitrogen into an NO analyser (Dasibi, Japan) adapted for the purpose. Sodium nitrate standards were prepared and provided a standard curve for comparison. Lower limit of detection was 2 mM. IL 12 levels were measured using a commercially available sandwich ELISA purchased from R&D Systems (Oxon, UK). The assay which was used according to manufacturers instructions had a sensitivity of 5 pg / ml.
3. Statistics Results are expressed as medians. Results were tested for statistical significance using a Mann Whitney Test.
4. Results Results of serum nitrate levels are given in Fig. 1. Compared to non-pregnant controls serum nitrate levels were significantly reduced in healthy first trimester pregnant women. In contrast, in first trimester pregnant women with previous history of recurrent miscarriage but no current active clinical pathology, serum nitrate levels were significantly higher than those predicted by the normal pregnancy group. Moreover, nitrate levels in pregnant women with a history of recurrent miscarriage were equivalent to those found in patients undergoing spontaneous abortion. When pregnant women with previous recurrent miscarriage were further divided into subsequent failed or successful outcome, serum nitrate levels in those whose pregnancies subsequently failed were significantly higher than in normal pregnancy (16.0 vs. 13.0 P,0.05). In contrast, nitrate levels were not significantly different from normal pregnancy in those women who progressed to a successful delivery (12.4 vs. 13.0 N.S). Normal pregnancy was not associated with significantly increased levels of serum IL 12 (Fig. 2). Serum IL-12 was normal during spontaneous abortion compared to the control group. In contrast, levels of IL 12 were significantly increased in first trimester pregnant women with a history of recurrent miscarriage compared to normal pregnancy. When this group were again subdivided by outcome, those whose pregnancy subsequently failed exhibited higher serum IL-12 levels than pregnant controls (12.4 vs. 8.0 P,0.05), whereas, those with successful outcome did not significantly differ (10.8 vs. 8.0 N.S.). No correlation was found between IL 12 and serum nitrate levels (r50.23 N.S.).
5. Discussion Successful outcome of pregnancy requires active adaptation of the maternal immune system. T H 2 type cytokines released by the trophoblast, early in the course of a normal pregnancy may suppress the cellular immune responses that could endanger the foetus [7]. Recurrent miscarriage has been associated with T H 1 type immune response directed at the trophoblast. Recent studies in mice have shown there may be a role for NO either as an effector molecule mediating early murine embryo loss or in the in situ activation of decidual macrophages which was observed as an early event preceding spontaneous abortion [9]. These studies used tissue obtained from the site of implantation. The current study examined peripheral blood and found serum levels of IL12 and NO to be significantly elevated in pregnant women with a history of recurrent miscarriage compared to normal pregnant women. The increases which we observed occurred early in the first trimester, before there was any active sign of miscarriage. These changes were possibly initiated in the uterus. Although neither the NO or IL 12 were of any predictive
R. Wilson et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 75 (1997) 211 – 214
213
Fig. 1. Definition of groups. Group 1: non-pregnant; Group 2: pregnant; Group 3: spontaneous abortion; Group 4: recurrent miscarriage; Group 5: recurrent miscarriage–pre-pregnancy. Levels of serum nitrate were measured as described previously. 1 P,0.05; 11 P,0.0001 vs. Group 1.
Fig. 2. Definition of groups. Group 1: non-pregnant; Group 2: pregnant; Group 3: spontaneous abortion; Group 4: recurrent miscarriage; Group 5: recurrent miscarriage–pre-pregnancy. Levels of IL 12 were measured as described previously. 111 P,0.001 vs. Group 1; ***P,0.0006 vs. Group 2; 8P,0.05 vs Group 4.
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R. Wilson et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 75 (1997) 211 – 214
value, levels of both were significantly higher in those women with a history of recurrent miscarriage whose pregnancies ultimately failed compared to those with a history of recurrent miscarriage whose pregnancies on this occasion continued to term. Moreover patterns seen in women with a history of recurrent miscarriage resembled those found in women admitted during spontaneous abortion. Our results implicate IL 12 and NO in pregnancy loss. It is possible that the T H 2 response seen in continuing pregnancy helps prevent generation of IL 12 and NO. However in recurrent miscarriage, events occur which facilitate production of NO and IL 12 indicative of a T H 1 type response. Previous studies have found NO to be detectable early on in placental development. In addition increased serum and urine levels of NO have been found in women suffering pre term labour. Whether the elevated levels of NO and IL 12 found in this study are the result of increased immunological activity within the peripheral circulation or whether they are the result of events in the uterus is not clear. What has emerged from the present study is that early in the course of a normal pregnancy NO production falls and IL 12 production becomes detectable. These changes do not occur in women with a history of recurrent miscarriage regardless of the outcome of their pregnancy. This study confirms the idea that T H cells (or cytokine producing T H cells) are involved in the aetiology of recurrent miscarriage. The increased immunological activity seen previously in these women and the increased number of antibody producing B cells may be responsible for the increased levels of NO and IL 12.
Acknowledgements We are grateful to Professor F.Y. Liew, Department of Immunology, Western Infirmary, Glasgow for his help in this project.
References [1] Stirred GM. Lancet 1990;336:673–5. [2] Clark DA, Bonnar J, editors. Recent advances in Obstetrics and Gynaecology. Churchill Livingstone. Longman Group 1989:25–35. [3] Clark DA, Lea RG, Podor T, Daya S, Banwatt, Hailey C. Cytokines Determining the Success or failure of Pregnancy. Frontiers in Human Reproduction. 1991;626:524–36. [4] Hill JA, Katalin P, Anderson DJ. T helper 1 type immunity to trophoblasts in women with recurrent spontaneous abortion. JAMA 1995;273:1933–6. [5] Roberts J, Jenkins C, Wilson R, Maclean MA, McKillop JH, Walker JJ. Eur J Endo 1966;134:84–6. [6] Ware-Branch D. Autoimmunity and Pregnancy Loss. J Am Med Assoc 1990;264:1453–4. [7] Lieblau RS, Singer S, McDevit HO. Th 1 and ThH2 CD41 T cells in the pathogenesis of organspecific diseases. Immunol Today 1995;16:34–8. [8] Huang FP, Feng GJ, Lindop G, Stott D, Liew FY. The role of IL 12 and nitric oxide in the development of spontaneous autoimmune diseas in MRL / MP-lpr / lpr mice. J Exp Med 1996;183:1447–59. [9] Haddad EK, Dudos AJ, Barnes MG. Early embryo loss is associated with local production of nitric oxide by decidual mononuclear cells. J Exp Med 1995;182:1143–52. [10] Aoki T. Continous flow determination of nitrite with membrane separation chemiluminesence detection. Biomed Chromatog 1994; 128–130.