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T-helper 2-cytokine levels in women with threatened abortion
European Journal of Obstetrics & Gynecology and Reproductive Biology 111 (2003) 43–49
T-helper 2-cytokine levels in women with threatened abortion Roberto Paradisia,*, Mario Maldini-Casadeib, Paola Bonib, Paolo Busacchia, Eleonora Porcua, Stefano Venturolia a
Department of Obstetrics and Gynecology and Reproductive Biology, University Alma Mater Studiorum of Bologna, S. Orsola Hospital, Massarenti 13, Bologna 40138, Italy b Central Laboratory, S. Orsola Hospital, Bologna, Italy Received 30 April 2002; received in revised form 7 January 2003; accepted 29 January 2003
Abstract Objective: Considering that successful embryo development has been immunologically attributed to a T-helper 2 phenomenon and that threatened abortion is a very frequent but pathogenetically not well-defined clinical entity, our purpose was to investigate serum levels of the main T-helper 2-type cytokines during the evolution of this condition. Study design: Three T-helper 2-type cytokines (interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-13 (IL-13)) were measured by an enzyme-linked immunosorbent assay (ELISA) in serum of 12 women with threatened abortion both at hospital admission and discharge time. Fourteen women with missed abortion, 14 normal pregnant women and 14 normal non-pregnant women represent study control groups. Results: Serum concentrations of the selected T-helper 2-type cytokines showed no significant differences in women with threatened abortion with those of normal pregnant and non-pregnant women, whereas showed significantly lower values in women with missed abortion. Conclusion: Our data (a) confirm the concept that first-trimester normal pregnancy is a T-helper 2 phenomenon, (b) show that threatened abortion, when T-helper 2-biased, may tend to a positive evolution of the condition, (c) display that interleukin-10, particularly, may represent a useful diagnostic and prognostic marker for predicting the normal continuance of the pregnancy in threatened abortion, (d) confirm the existence of a T-helper 2-type pattern deficiency in missed abortion, and finally (e) may open the way to new T-helper 2-biased immune therapies in case of difficult first-trimester pregnancies. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cytokines; Interleukin-6; Interleukin-10; Interleukin-13; Threatened abortion
1. Introduction In spite of the world population explosion in the last century the process leading human pregnancy is fairly inefficient because 70% of conceptions fail to achieve viability. Spontaneous abortion is the most common complication of pregnancy and is classically defined as a clinically recognized pregnancy loss before 20 weeks’ gestation and occurs at a frequency of 15% [1]. While recurrent spontaneous abortion has been extensively studied [1,2], sporadic spontaneous abortion and, in particular, the first step of this pathological condition, the threatened abortion, has been characterized to a lesser extent. Threatened abortion refers to an intrauterine viable clinical pregnancy accompanied by an intrauterine source of painless vaginal bleeding. A subchorionic hematoma caused by partial placental abruption is often the source of bleeding but not necessarily associated with ultimate pregnancy loss [3]. * Corresponding author. Tel.: þ39-051-6363494; fax: þ39-051-301994. E-mail address: [email protected] (R. Paradisi).
Threatened abortion, in fact, may settle with normal continuation of the pregnancy or progress and be accompanied by pain to become an inevitable abortion. To day, do not exist specific markers that can help to predict if pregnancy continues developing until delivery or terminates in abortion. Signs and symptoms of pregnancy, such as nausea, vomiting, tiredness, breast tenderness may be present in both conditions, so the clinical assessment of threatened abortion is unreliable in most cases and should be superseded by ready access to sonographic assessment [4]. Even though pelvic examination and sonographic scan may usually lead to a proper diagnosis, the use of transvaginal color Doppler ultrasound is not helpful for predicting positive pregnancy outcome in threatened abortion because no apparent circulatory alteration occurs [5], excluding a vascular pathogenesis. Doubling of b-hCG subunit levels every 48–72 h is often but not always diagnostically useful [6], limiting the importance of endocrine factors in threatened abortion. No specific infection has been identified as primary cause of abortion, moreover infections have occurred in women with successful pregnancy at the same rate as those experiencing
0301-2115/$ – see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0301-2115(03)00119-2
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R. Paradisi et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 111 (2003) 43–49
pregnancy loss [7], minimizing the role of microbiologic factors in threatened abortion. Considering that the fetal allograft host reaction in maternal tissue is considered as a paradox by immunologists and the positive or negative outcome of feto-placental unit implant in maternal uterus remains not well understood, an immunological pathogenesis has been advanced in recurrent spontaneous abortion of unknown origin [1]. Immunity is regulated by CD4 (þ) T cells which can be divided into T-helper 0–2 cells based on their prevalent profile of cytokine production [8–10]. T-helper 0 cells may be considered as progenitor cells producing both T-helper 1 and 2 cells. T-helper 1 cells primarily secrete interferon-g but also interleukin-2, interleukin-12, interleukin-15 and tumor necrosis factor-b. T-helper 2 cells primarily secrete interleukin-10 (IL-10), but also interleukin-4, interleukin-5, interleukin-6 (IL-6), interleukin-9 and interleukin-13 (IL-13) [8–10]. T-helper 1-type cytokines mediate delayed hypersensitivity and drive cellular immunity, while T-helper 2-type cytokines activate B cells, causing their differentiation into antibody producing cells [11]. Cytokine network has been suggested to be involved with positive or negative evolution of the ongoing pregnancies [12]. A T-helper 2-type cytokine profile may be associated with successful pregnancy, whereas the presence of a dominant T-helper 1-type cytokine profile may be indicative of a pathological pregnancy both in experimental animals [12] and in human beings [13,14]. In humans, however, all the studies reported on the effects of the cytokine network in pregnancy have been performed in normal pregnancy or in manifested spontaneous abortion (recurrent or sporadic) [13,15–23]. If immunological factors may contribute to the pathogenesis of many, if not all, cases of threatened abortion also need to be elucidated. There are no reports on cytokine detection in women with threatened abortion and more simply diagnostic and eventual prognostic immune-derived markers of a semi-allograft rejection are needed for the management of threatened abortion and for predicting pregnancy outcome. Here we evaluate the role of interleukin-6, interleukin-10 and interleukin-13, some of the most representative cytokines of the T-helper 2-type cytokine network [10] during threatened abortion with the attempt to evaluate the existence of immunological factors in the pathogenesis of this pathological condition and identify eventually predictors of its evolution.
2. Materials and methods This study was approved by our local ethical committee and all the patients gave informed consent. Twelve in-patient pregnant women for a threatened abortion during the first-trimester of pregnancy (mean S:D:, 9:9 1:6 weeks gestation) entered the study as a study group (threatened abortion group). These women presented no previous history of abortion, ectopic pregnancy or pre-term delivery.
During hospitalization all the patients were maintained at complete rest in bed and routine examinations with ultrasound and endocrine (b-hCG) evaluation were accomplished. The viability of the fetus and its normal cardiac activity were observed in all the subjects. No progesterone therapy was performed. Blood specimens were collected twice, the former at admission time and the latter at discharge time about after 2–3 days. None patient exited in spontaneous abortion during and after hospitalization and all were subsequently followed successfully to term. Fourteen in-patient age-, weight- and height-matched pregnant women for a diagnosed missed abortion during the firsttrimester of pregnancy (mean S:D:, 10:0 1:6 weeks gestation) entered the study as the first control group (missed abortion control group). Blood specimens were taken before miscarriage by D&C. Each abortive tissue was kariotyped and all resulted not chromosomically abnormal. Fourteen out-patient age-, weight- and height-matched pregnant women undergoing a normal, non-pathologic pregnancy during the first-trimester of pregnancy (mean S:D:, 9:8 0:7 weeks gestation) entered the study as the second control group (pregnant control group). They were attending routine prenatal visit and ultrasound examination to ascertain the exact week of pregnancy and document normal fetal cardiac activity and the viability of the fetus. Blood specimens for routine haemodynamic and biochemical examinations were taken. Fourteen healthy age-, weight- and heightmatched non-pregnant women with normal ovulatory menstrual cycles chosen among hospital and faculty employees entered the study as the third control group (non-pregnant control group). Blood specimens were collected in early follicular phase (days 2–5 of the cycle). Blood was collected by venipuncture and serum was isolated shortly thereafter. Aliquots of serum were stored frozen at 70 8C without interruption until assay. Concentrations of the cytokines interleukin-6, interleukin-10 and interleukin-13 were measured by using commercially available enzyme-linked immunosorbent assay (ELISA) kits. These included a kit for interleukin-6 (Diagnostic Products Corporation, Los Angeles, CA), interleukin-10 (PerSeptive Biosystems, Framingham, MA), and interleukin-13 (Immunotech, Marseille, France). The assays are based on a solid-phase enzyme immunoassay employing the dual-antibody sandwich principle and were performed according to the manifacturer’s procedures, and recombinant reference cytokine samples served as positive controls for calibration. Sensitivities of the methods relative to the study conditions were as follows: interleukin-6, 5 pg/ml; interleukin-10, 1 pg/ml; and interleukin-13, 1.5 pg/ml. Inter- and intra-assay variabilities were 9.6, 5.5 and 9.5% and 8, 13.1 and 3.8% for interleukin-6, interleukin-10, and interleukin-13, respectively. No cross-reactions against other standard cytokines were observed. Each sample was tested in duplicate and in blinded fashion so that the technician conducting the assay was not aware of the source of samples under test.
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Data analysis was performed by analysis of variance (ANOVA) and non-parametric Mann–Whitney U-test and Spearman rank correlation, as appropriate. A P-value of <0.05 was accepted as statistically significant. Results are expressed as mean S:D.
3. Results Blood leukocyte count in normal non-pregnant control group was significantly lower with respect to all pregnant
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groups (P < 0:05 versus threatened abortion group; P < 0:005 versus missed abortion group; P < 0:05 versus normal pregnant control group) (Table 1). Serum interleukin-6 concentrations in women with threatened abortion showed no significant differences with those of normal pregnant and non-pregnant women (P ¼ NS). No significant differences were also found between the two evaluations within the threatened abortion group. On the contrary, interleukin-6 was measured in serum of women with missed abortion with significantly lower levels than that of women with threatened abortion (P < 0:005 and
Table 1 Numbers of white blood cells (WBC) and serum concentrations of interleukin-6 (IL-6), interleukin-10 (IL-10) and interleukin-13 (IL-13) in threatened abortion, missed abortion, pregnant and non-pregnant groups Threatened abortion group
7.8 1.9 14.9 6.1***,z 5.3 1.7y,z 3.9 2.1*,z
Pregnant control group
Non-pregnant control group
Second sample (discharge time)
First sample (admission time) WBC (103/ml) Interleukin-6 (pg/ml) Interleukin-10 (pg/ml) Interleukin-13 (pg/ml)
Missed abortion control group
13.8 5.1** 4.9 1.7y 4.2 2.5*
8.6 9.1 2.0 2.4
2.6 2.9 1.1 0.5
7.7 13.5 4.4 5.7
2.1 2.3 1.4 4.6
6.3 12.6 5.6 3.8
1.1 2.1 4.3 2.1
*
P < 0:05 vs. missed abortion group. P < 0:01 vs. missed abortion group. *** P < 0:005 vs. missed abortion group. y P < 0:001 vs. missed abortion group. z P ¼ NS vs. same group at discharge time and pregnant and non-pregnant control groups. **
Fig. 1. Distribution of interleukin-6 levels (pg/ml) in serum of the threatened abortion group at admission (A) and discharge (B) time, missed abortion control group (C), pregnant control group (D) and non-pregnant control group (E). Number (2) represents the number of cases with overlapping values. ( ) mean S:D.
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R. Paradisi et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 111 (2003) 43–49
Fig. 2. Distribution of interleukin-10 levels (pg/ml) in serum of the threatened abortion group at admission (A) and discharge (B) time, missed abortion control group (C), pregnant control group (D) and non-pregnant control group (E). Numbers (2 or 3) represent the number of cases with overlapping values. ( ) mean S:D.
Fig. 3. Distribution of interleukin-13 levels (pg/ml) in serum of the threatened abortion group at admission (A) and discharge (B) time, missed abortion control group (C), pregnant control group (D) and non-pregnant control group (E). Numbers (2 or 3) represent the number of cases with overlapping values. ( ) mean S:D.
R. Paradisi et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 111 (2003) 43–49
P < 0:01 versus threatened abortion group at admission and discharge time, respectively; Table 1). Interleukin-6 concentrations in missed abortion group were also lower than those in pregnant and non-pregnant control groups (P < 0:001 and P < 0:005, respectively). By single distribution, interleukin-6 concentrations in serum of women with missed abortion showed almost always lower values when compared to the mean values of the other groups demonstrating only a relative overlap (Fig. 1). Correlation between interleukin-6 levels and blood leukocyte count was not significant (r ¼ 0:09, P ¼ NS). Serum interleukin-10 concentrations in women with threatened abortion showed no significant differences with those of normal pregnant and non-pregnant women (P ¼ NS). No significant differences were also found between the two evaluations within the threatened abortion group. On the contrary, interleukin-10 was measured in serum of women with missed abortion with highly significant lower levels than that of women with threatened abortion (P < 0:001 versus threatened abortion group at both admission and discharge time; Table 1). Interleukin-10 concentrations in missed abortion group were also markedly lower than those in pregnant and non-pregnant control groups (P < 0:001). By single distribution, interleukin-10 concentrations in serum of women with missed abortion showed lower values when compared to the mean values of the other groups demonstrating practically almost no overlap (Fig. 2). Correlation between interleukin-10 levels and blood leukocyte count was not significant (r ¼ 0:10, P ¼ NS). Serum interleukin-13 concentrations in women with threatened abortion showed no significant differences with those of normal pregnant and non-pregnant women (P ¼ NS). No significant differences were also found between the two evaluations within the threatened abortion group. On the contrary, interleukin-13 was measured in serum of women with missed abortion with sufficiently significant lower levels than that of women with threatened abortion (P < 0:05 versus threatened abortion group at both admission and discharge time; Table 1). Interleukin-13 concentrations in missed abortion group were also lower than those in pregnant and non-pregnant control groups (P < 0:01). By single distribution, interleukin-13 concentrations in serum of women with missed abortion showed values at the lower limits of the range of the values of the other groups (Fig. 3). Correlation between interleukin-13 levels and blood leukocyte count was not significant (r ¼ 0:20, P ¼ NS).
4. Discussion Fetus is partly allogenic to the mother, but is not commonly rejected by the maternal immune system. In the last decade has emerged the concept that fetal comfort may be preserved by the action of the 2-type and not the 1-type cytokines, or successful pregnancy is a T-helper 2 phenomenon [12,24], in
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particular, in the mid-late first-trimester pregnancy during placental formation [10]. Briefly, exist in experimental animals beneficial cytokines (anti-inflammatory cytokines secreted by T-helper 2-type cells) which can enhance fetal growth and survival and deleterious cytokines (pro-inflammatory cytokines secreted by T-helper 1-type cells) which can compromise pregnancy causing the death of the fetoplacental unit [12,24]. The positive emerging role, particularly, of interleukin-10 associated with normal pregnancy and the negative deleterious effects of interferon-g that may compromise pregnancy have been documented [12,24]. Similar results have been proposed also in human beings. A lot of studies demonstrating a role of T-helper 1-type immunity in reproductive failure and of T-helper 2-type immunity in successful pregnancy have been reported, but always in normal and/or in clearly compromised pregnancies [13,15–23,25]. Here, we analyzed for the first time T-helper 2-type cytokine profile in threatened abortion, the first step towards claimed spontaneous abortion or remission of symptoms. On the other hand, threatened abortion is a dramatic clinical problem by a socio-economical point of view, because is the most frequent condition among the patients who attend the out-patient emergency unit. The rationale for investigating the presence of selected cytokines directly in the serum of women with threatened abortion is derived from the fact that cytokines concentrations have already been measured in the sera of pregnant and aborting women as well as in several other situations, infections disease particularly, in which cytokines are detectable in the circulation [21]. All the three selected T-helper 2-type cytokines presented similar concentrations in women with threatened abortion and in women with regular pregnancy suggesting the existence of an immune T-helper 2-type pattern also in threatened abortion condition. Moreover all the patients of the threatened abortion group showed similar results in their cytokine profiles in both investigations, at admission and discharge time, confirming a maintenance of the immune Thelper 2-type pattern during hospitalization in concomitance with the remission of symptoms. On the other hand, none patient of this group tended to abortion, to verify, in the case, a switch to an immune T-helper 1-type pattern. Furthermore all the T-helper 2-type cytokines studied, interleukin-10 particularly, showed significantly lower concentrations in missed abortion group, strengthening the hypothesis that a T-helper 2-type pattern deficiency may help a shift towards a T-helper 1-type pattern typical of the pregnancy loss. Taken together these data sustain the existence of an active T-helper 2-type response in threatened abortion with positive evolution, contributing to create an immunological environment that is conductive to a resolution of this condition and to a regular continuance of the pregnancy. Lacking data in the literature for comparison with threatened abortion, our data agree, however, with the majority of the studies reported in normal pregnancy and recurrent or sporadic spontaneous abortions. All ‘‘in vitro’’ studies
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[13–15,18,25], grossly agree in the detection of higher levels of T-helper 2-type cytokines in normal pregnancy compared to recurrent or sporadic spontaneous abortions and higher levels of T-helper 1-type cytokines in recurrent or sporadic spontaneous abortions compared to normal pregnancy. ‘‘In vivo’’ studies on T-helper 1–2-biased in terms of circulating levels of cytokines in the serum also indicate a prevalently T-helper 2-biased in normal pregnancy and T-helper 1biased in recurrent or sporadic spontaneous abortions [17,19–22]. Only Schust and Hill [16] found no detectable serum levels of cytokines in any patient sample regardless of pregnancy status or pregnancy outcome. On the contrary, Vassiliadis et al. [17] detected very high serum cytokine levels with values of a different order of magnitude (ng/ml versus pg/ml). Several mechanisms may be involved in threatened abortion to influence positively or negatively the T-helper 1/T-helper 2 balance in favor of one or other direction. In normal pregnancy interleukin-6, interleukin-10 and interleukin-13 are spontaneously secreted by human trophoblast and play crucial T-helper 2-type roles in implantation and continuation of early pregnancy [10]. Hill et al. [13] suggested that in cases with unexplained recurrent abortion may exist a primary defect that can lead to an inability to activate this T-helper 2-response. It is likely, however, that the primary protective role for the T-helper 2-type cytokines in the maintenance of normal pregnancy may not be a direct one, but indirect. In fact, an important, if not major, role for Thelper 2-type cells is downregulate T-helper 1-immunity, suppressing interferon-g and other related T-helper 1-type cytokine production [26], because interleukin-6 and interleukin-10 are potent inhibitors of T-helper 1 effector function [11]. T-helper 1-type cytokines, on the other hand, can be harmful to pregnancy either by direct cytotoxic action on throphoblast cells, by inhibition of throphoblast growth, by enhancement of natural killer activity, or even via activated macrophages [27]. Lim et al. [2] suggested that the T-helper 2-response allows the production of blocking antibodies to mask fetal trophoblast antigens from immunological recognition by maternal T-helper 1 cell-mediated/cytotoxic response which is responsible for rejection of semi-allografts. Natural killer cell-mediated immunity against trophoblast has also been evidenced. A correlation between fetal resorption and an increased appearance of natural killer cells at the feto-maternal interface [28] as well as elevated natural killer cell levels in the blood in women aborting kariotypically normal embryos [29] have been observed. Considering that T-helper 2-type cytokines counter natural killer cell activation [12], an inhibition of this activity may be a credible explanation for immunological protection against abortion [29]. Moreover, interleukin-10 reduces expression of HLA classes I and II molecules, whereas interferon-g augment this expression [30]. The T-helper 1–2-type shift of cytokine profile in normal pregnancy and eventually threatened abortion may contribute to modify the expression of HLA molecules in the developing fetal tissues, reducing potentially the
risk of rejection. On the other hand, it will be helpful during threatened abortion to avoid infection, autoimmune disease, stress, that may well result in a strong enough T-helper 1biased immunity to compromise pregnancy, because T-helper 1-type cells induced by infection may traverse the fetal interface or may product cytokines that affect the trophoblast [26]. Other mechanisms hypothetically involved in threatened abortion to maintain a pro-T-helper 2 and an anti-T-helper 1-biased can be the hormones, in particular the progesterone, that in mice eliciting from lymphocytes the production of progesterone-induced blocking factor which suppresses natural killer activity, may act as immunomodulator by altering the T-helper 1/T-helper 2 balance in favor of the latter [31]. Piccinni et al. [9] observed in humans also that progesterone promotes T-helper 2-type cytokine production and may contribute to the regulation of the immune homeostasis during pregnancy, whereas progesterone analogues as well as 17b-estradiol have no effect on cytokine production. These effects, however, have been obtained in ‘‘in vitro’’ experiments by administering progesterone at levels comparable to those present at the feto-maternal interface during pregnancy, that are about 100 times the physiological progesterone concentrations found in serum during pregnancy. At these doses therefore progesterone may function as a potent inducer of T-helper 2-type cytokine production [9] and/or blocker of T-helper 1-type cytokine production [32]. By a clinical point of view reassurance that most pregnancy with threatened abortion go to term is the mainstay of management, even though attempts to maintain pregnancy are reasonable only in those cases in which the fetus is not chromosomically and/or anatomically seriously affected. In such case, abortion may be considered a part of the quality control process in human reproduction [33]. The historic administration of hCG and progesterone (at the traditional progestative doses) is no longer practised. On the other hand, no trials, at our knowledge, are in progress in human threatened abortion concerning the ‘‘in vivo’’ administration of pure micronized progesterone at the above mentioned immunomodulatory doses, to sustain the concept of successful pregnancy as progesterone-induced T-helper 2 phenomenon. This study is not able to demonstrate a direct cause/effect relationship between circulating T-helper 2-type cytokines and the evolution of threatened abortion, but support, however, the concept that (a) first-trimester normal pregnancy is T-helper 2-biased, (b) threatened abortion also, when T-helper 2-biased, may tend to a positive evolution of the condition and to a normal continuance of the pregnancy and (c) a T-helper 2-type pattern deficiency is joined with missed abortion. Moreover, serum interleukin-10, particularly, when assayed by highly sensitive ELISA kits, may represent an acceptable peripheral marker of immunity potentially occurring locally at the feto-maternal interface and predictor of pregnancy outcome in cases of unexplained threatened abortion.
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Acknowledgements The work was supported in part by grants from Ministero dell’Istruzione, dell’Universita` e della Ricerca, Rome, Italy (funds ex-60%, 1997, 1998 and 1999). References [1] Hill JA. Sporadic and recurrent spontaneous abortion. Curr Probl Obstet Gynecol Fertil 1994;17:114–62. [2] Lim KIH, Odukoya OA, Li TC, Cooke ID. Cytokines and immunoendocrine factors in recurrent miscarriage. Human Reprod Update 1996;2:469–81. [3] Dickey RP, Olar TT, Curole DW, Taylor SN, Matulich EM. Relationship of first-trimester subchorionic bleeding detected by color Doppler ultrasound to subchorionic fluid, clinical bleeding and pregnancy outcome. Obstet Gynecol 1992;80:415–20. [4] Chung TK, Sahota DS, Lau TK, Mongelli JM, Spencer JA, Haines CJ. Threatened abortion: prediction of viability based on signs and symptoms. Aust N Z J Obstet Gynecol 1999;39:443–7. [5] Alcazar JL, Ruiz-Perez ML. Uteroplacental circulation in patients with first-trimester threatened abortion. Fertil Steril 2000;73:130–5. [6] Lagrew D, Wilson EA, Jawad J. Determination of gestational age by serum concentration human chorionic gonadotropin. Obstet Gynecol 1983;62:37–40. [7] Simpson JL, Mills JL, Kim H, Holmes LB, Lee J, Metzer B, et al. Infectious process: an infrequent cause of first trimester spontaneous abortion. Human Reprod 1996;11:668–72. [8] Mosmann TR, Cherwinski HM, Bond MW, Giedlin MA, Coffman RL. Two types of murine helper T cell clones. I. Definition according to profiles of lymphokine activities and secreted proteins. J Immunol 1986;136:2348–57. [9] Piccinni M-P, Giudizi M-G, Biagiotti R, Beloni L, Giannarini L, Sampognaro S, et al. Progesterone favors the development of human T helper cells producing Th2-type cytokines and promotes both IL-4 production and membrane CD30 expression in established Th1 cell clones. J Immunol 1995;155:128–33. [10] Dealtry GB, O’Farrell MK, Fernandez N. The Th2 cytokine environment of the placenta. Int Arch Allergy Immunol 2000;123: 107–19. [11] Fiorentino DF, Bond MW, Mosmann TR. Two types of mouse T helper cell. IV. Th2 clones secrete a factor that inhibits cytokine production by Th1 clones. J Exp Med 1989;170:2081–95. [12] Wegmann TG, Lin H, Guilbert L, Mosmann TR. Bidirectional cytokine interactions in the maternal–fetal relationship: is successful pregnancy a Th2 phenomenon? Immunol Today 1993;14:353–6. [13] Hill JA, Polgar K, Anderson DJ. T-helper 1-type immunity to trophoblast in women with recurrent spontaneous abortion. JAMA 1995;273:1933–6. [14] Raghupathy R, Makhseed M, Azizieh F, Hassan N, Al-Azemi M, AlShamali E. Maternal Th1- and Th2-type reactivity to placental antigens in normal human pregnancy and unexplained recurrent spontaneous abortion. Cell Immunol 1999;196:122–30. [15] Marzi M, Vigano A, Trabattoni D, Villa ML, Salvaggio A, Clerici E, et al. Characterization of type 1 and type 2 cytokine production profile in physiologic and pathologic human pregnancy. Clin Exp Immunol 1996;106:127–33.
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[16] Schust DJ, Hill JA. Correlation of serum cytokine and adhesion molecule determinations with pregnancy outcome. J Soc Gynecol Invest 1996;3:259–61. [17] Vassiliadis S, Ranella A, Papadimitriou L, Makrygiannakis A, Athanassakis I. Serum levels of pro- and anti-inflammatory cytokines in non-pregnant women, during pregnancy, labour and abortion. Mediat Inflamm 1998;7:69–72. [18] Makhseed M, Raghupathy R, Azizieh F, Al-Azemi MM, Hassan NA, Bandar A. Mitogen-induced cytokine responses of maternal lymphocytes suggest a differential Th-type bias in normal pregnancy and pregnancy failure. Am J Reprod Immunol 1999;42:273–81. [19] Lim KJH, Odukova OA, Ajjan RA, Li T-C, Weetman AP, Cooke ID. The role of T-helper cytokines in human reproduction. Fertil Steril 2000;73:136–42. [20] Jenkins C, Roberts J, Wilson R, MacLean MA, Shilito J, Walker JJ. Evidence of a TH 1 type response associated with recurrent miscarriage. Fertil Steril 2000;73:1206–8. [21] Makhseed M, Raghupathy R, Azizieh F, Farhat R, Hassan N, Bandar A. Circulating cytokines and CD30 in normal human pregnancy and recurrent spontaneous abortions. Human Reprod 2000;15:2011–7. [22] Zenclussen AC, Kortebani G, Mazzolli A, Margni R, Malan Borel Y. Interleukin-6 and solubile interleukin-6 receptor serum levels in recurrent spontaneous abortion women immunized with paternal white cells. Am J Reprod Immunol 2000;44:22–9. [23] Koumantaki Y, Matalliotakis I, Sifakis S, Kyriakou D, Neonaki M, Goymenou A, et al. Detection of interleukin-6, interleukin-8, and interleukin-11 in plasma from women with spontaneous abortion. Eur J Obstet Gynecol Reprod Biol 2001;98:66–71. [24] Chaouat G, Meliani AA, Martal J, Raghupathy R, Ellior J, Mosmann T, et al. IL-10 prevents naturally occurring fetal loss in the CBA DBA/2 mating combination, and local defect in IL-10 production in this abortion-prone combination is corrected by in vivo injection of INF-g. J Immunol 1995;154:4261–8. [25] Piccinni M-P, Beloni L, Livi C, Maggi E, Scarselli GF, Romagnani S. Detective production of both leucemia inhibitory factor and type 2 Thelpre cytokines by decidual T cells in unexplained recurrent abortions. Nat Med 1998;4:1020–4. [26] Raghupathy R. Th1-type immunity is incompatible with successful pregnancy. Immunol Today 1997;18:478–82. [27] Duclos AJ, Haddad EK, Baines GG. Presence of activated macrophages in a murine model of early embryo loss. Am J Reprod Immunol 1995;33:354–66. [28] Gendron RL, Baines M. Immunohistological analyses of decidual natural killer cells during spontaneous abortion in mice. Cell Immunol 1988;113:261–7. [29] Clark DA, Arck PC, Jalali R, Merali FS, Manuel J, Chaouat G, et al. Psycho-neuro-cytokine/endocrine pathways in immunoregulation during pregnancy. Am J Reprod Immunol 1996;35:330–7. [30] Moore KW, O’Garra A, de W Malefyt P, Vieira P, Mosmann TR. Interleukin-10. Annu Rev Immunol 1993;11:165–99. [31] Szekeres-Bartho J, Wegmann TG. A progesterone-dependent immunomodulatory protein alters the Th1/Th2 balance. J Reprod Immunol 1996;31:81–95. [32] Choi BC, Polgar K, Xiao L, Hill JA. Progesterone inhibits in vitro embryotoxic Th1 cytokine production to trophoblast in women with recurrent pregnancy loss. Human Reprod 2000;15(Suppl 1):46–59. [33] Szabo I, Szilagyi A. Management of threatened abortion. Early Pregnancy 1996;2:233–40.
T-helper 2-cytokine levels in women with threatened abortion Roberto Paradisia,*, Mario Maldini-Casadeib, Paola Bonib, Paolo Busacchia, Eleonora Porcua, Stefano Venturolia a
Department of Obstetrics and Gynecology and Reproductive Biology, University Alma Mater Studiorum of Bologna, S. Orsola Hospital, Massarenti 13, Bologna 40138, Italy b Central Laboratory, S. Orsola Hospital, Bologna, Italy Received 30 April 2002; received in revised form 7 January 2003; accepted 29 January 2003
Abstract Objective: Considering that successful embryo development has been immunologically attributed to a T-helper 2 phenomenon and that threatened abortion is a very frequent but pathogenetically not well-defined clinical entity, our purpose was to investigate serum levels of the main T-helper 2-type cytokines during the evolution of this condition. Study design: Three T-helper 2-type cytokines (interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-13 (IL-13)) were measured by an enzyme-linked immunosorbent assay (ELISA) in serum of 12 women with threatened abortion both at hospital admission and discharge time. Fourteen women with missed abortion, 14 normal pregnant women and 14 normal non-pregnant women represent study control groups. Results: Serum concentrations of the selected T-helper 2-type cytokines showed no significant differences in women with threatened abortion with those of normal pregnant and non-pregnant women, whereas showed significantly lower values in women with missed abortion. Conclusion: Our data (a) confirm the concept that first-trimester normal pregnancy is a T-helper 2 phenomenon, (b) show that threatened abortion, when T-helper 2-biased, may tend to a positive evolution of the condition, (c) display that interleukin-10, particularly, may represent a useful diagnostic and prognostic marker for predicting the normal continuance of the pregnancy in threatened abortion, (d) confirm the existence of a T-helper 2-type pattern deficiency in missed abortion, and finally (e) may open the way to new T-helper 2-biased immune therapies in case of difficult first-trimester pregnancies. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cytokines; Interleukin-6; Interleukin-10; Interleukin-13; Threatened abortion
1. Introduction In spite of the world population explosion in the last century the process leading human pregnancy is fairly inefficient because 70% of conceptions fail to achieve viability. Spontaneous abortion is the most common complication of pregnancy and is classically defined as a clinically recognized pregnancy loss before 20 weeks’ gestation and occurs at a frequency of 15% [1]. While recurrent spontaneous abortion has been extensively studied [1,2], sporadic spontaneous abortion and, in particular, the first step of this pathological condition, the threatened abortion, has been characterized to a lesser extent. Threatened abortion refers to an intrauterine viable clinical pregnancy accompanied by an intrauterine source of painless vaginal bleeding. A subchorionic hematoma caused by partial placental abruption is often the source of bleeding but not necessarily associated with ultimate pregnancy loss [3]. * Corresponding author. Tel.: þ39-051-6363494; fax: þ39-051-301994. E-mail address: [email protected] (R. Paradisi).
Threatened abortion, in fact, may settle with normal continuation of the pregnancy or progress and be accompanied by pain to become an inevitable abortion. To day, do not exist specific markers that can help to predict if pregnancy continues developing until delivery or terminates in abortion. Signs and symptoms of pregnancy, such as nausea, vomiting, tiredness, breast tenderness may be present in both conditions, so the clinical assessment of threatened abortion is unreliable in most cases and should be superseded by ready access to sonographic assessment [4]. Even though pelvic examination and sonographic scan may usually lead to a proper diagnosis, the use of transvaginal color Doppler ultrasound is not helpful for predicting positive pregnancy outcome in threatened abortion because no apparent circulatory alteration occurs [5], excluding a vascular pathogenesis. Doubling of b-hCG subunit levels every 48–72 h is often but not always diagnostically useful [6], limiting the importance of endocrine factors in threatened abortion. No specific infection has been identified as primary cause of abortion, moreover infections have occurred in women with successful pregnancy at the same rate as those experiencing
0301-2115/$ – see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0301-2115(03)00119-2
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pregnancy loss [7], minimizing the role of microbiologic factors in threatened abortion. Considering that the fetal allograft host reaction in maternal tissue is considered as a paradox by immunologists and the positive or negative outcome of feto-placental unit implant in maternal uterus remains not well understood, an immunological pathogenesis has been advanced in recurrent spontaneous abortion of unknown origin [1]. Immunity is regulated by CD4 (þ) T cells which can be divided into T-helper 0–2 cells based on their prevalent profile of cytokine production [8–10]. T-helper 0 cells may be considered as progenitor cells producing both T-helper 1 and 2 cells. T-helper 1 cells primarily secrete interferon-g but also interleukin-2, interleukin-12, interleukin-15 and tumor necrosis factor-b. T-helper 2 cells primarily secrete interleukin-10 (IL-10), but also interleukin-4, interleukin-5, interleukin-6 (IL-6), interleukin-9 and interleukin-13 (IL-13) [8–10]. T-helper 1-type cytokines mediate delayed hypersensitivity and drive cellular immunity, while T-helper 2-type cytokines activate B cells, causing their differentiation into antibody producing cells [11]. Cytokine network has been suggested to be involved with positive or negative evolution of the ongoing pregnancies [12]. A T-helper 2-type cytokine profile may be associated with successful pregnancy, whereas the presence of a dominant T-helper 1-type cytokine profile may be indicative of a pathological pregnancy both in experimental animals [12] and in human beings [13,14]. In humans, however, all the studies reported on the effects of the cytokine network in pregnancy have been performed in normal pregnancy or in manifested spontaneous abortion (recurrent or sporadic) [13,15–23]. If immunological factors may contribute to the pathogenesis of many, if not all, cases of threatened abortion also need to be elucidated. There are no reports on cytokine detection in women with threatened abortion and more simply diagnostic and eventual prognostic immune-derived markers of a semi-allograft rejection are needed for the management of threatened abortion and for predicting pregnancy outcome. Here we evaluate the role of interleukin-6, interleukin-10 and interleukin-13, some of the most representative cytokines of the T-helper 2-type cytokine network [10] during threatened abortion with the attempt to evaluate the existence of immunological factors in the pathogenesis of this pathological condition and identify eventually predictors of its evolution.
2. Materials and methods This study was approved by our local ethical committee and all the patients gave informed consent. Twelve in-patient pregnant women for a threatened abortion during the first-trimester of pregnancy (mean S:D:, 9:9 1:6 weeks gestation) entered the study as a study group (threatened abortion group). These women presented no previous history of abortion, ectopic pregnancy or pre-term delivery.
During hospitalization all the patients were maintained at complete rest in bed and routine examinations with ultrasound and endocrine (b-hCG) evaluation were accomplished. The viability of the fetus and its normal cardiac activity were observed in all the subjects. No progesterone therapy was performed. Blood specimens were collected twice, the former at admission time and the latter at discharge time about after 2–3 days. None patient exited in spontaneous abortion during and after hospitalization and all were subsequently followed successfully to term. Fourteen in-patient age-, weight- and height-matched pregnant women for a diagnosed missed abortion during the firsttrimester of pregnancy (mean S:D:, 10:0 1:6 weeks gestation) entered the study as the first control group (missed abortion control group). Blood specimens were taken before miscarriage by D&C. Each abortive tissue was kariotyped and all resulted not chromosomically abnormal. Fourteen out-patient age-, weight- and height-matched pregnant women undergoing a normal, non-pathologic pregnancy during the first-trimester of pregnancy (mean S:D:, 9:8 0:7 weeks gestation) entered the study as the second control group (pregnant control group). They were attending routine prenatal visit and ultrasound examination to ascertain the exact week of pregnancy and document normal fetal cardiac activity and the viability of the fetus. Blood specimens for routine haemodynamic and biochemical examinations were taken. Fourteen healthy age-, weight- and heightmatched non-pregnant women with normal ovulatory menstrual cycles chosen among hospital and faculty employees entered the study as the third control group (non-pregnant control group). Blood specimens were collected in early follicular phase (days 2–5 of the cycle). Blood was collected by venipuncture and serum was isolated shortly thereafter. Aliquots of serum were stored frozen at 70 8C without interruption until assay. Concentrations of the cytokines interleukin-6, interleukin-10 and interleukin-13 were measured by using commercially available enzyme-linked immunosorbent assay (ELISA) kits. These included a kit for interleukin-6 (Diagnostic Products Corporation, Los Angeles, CA), interleukin-10 (PerSeptive Biosystems, Framingham, MA), and interleukin-13 (Immunotech, Marseille, France). The assays are based on a solid-phase enzyme immunoassay employing the dual-antibody sandwich principle and were performed according to the manifacturer’s procedures, and recombinant reference cytokine samples served as positive controls for calibration. Sensitivities of the methods relative to the study conditions were as follows: interleukin-6, 5 pg/ml; interleukin-10, 1 pg/ml; and interleukin-13, 1.5 pg/ml. Inter- and intra-assay variabilities were 9.6, 5.5 and 9.5% and 8, 13.1 and 3.8% for interleukin-6, interleukin-10, and interleukin-13, respectively. No cross-reactions against other standard cytokines were observed. Each sample was tested in duplicate and in blinded fashion so that the technician conducting the assay was not aware of the source of samples under test.
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Data analysis was performed by analysis of variance (ANOVA) and non-parametric Mann–Whitney U-test and Spearman rank correlation, as appropriate. A P-value of <0.05 was accepted as statistically significant. Results are expressed as mean S:D.
3. Results Blood leukocyte count in normal non-pregnant control group was significantly lower with respect to all pregnant
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groups (P < 0:05 versus threatened abortion group; P < 0:005 versus missed abortion group; P < 0:05 versus normal pregnant control group) (Table 1). Serum interleukin-6 concentrations in women with threatened abortion showed no significant differences with those of normal pregnant and non-pregnant women (P ¼ NS). No significant differences were also found between the two evaluations within the threatened abortion group. On the contrary, interleukin-6 was measured in serum of women with missed abortion with significantly lower levels than that of women with threatened abortion (P < 0:005 and
Table 1 Numbers of white blood cells (WBC) and serum concentrations of interleukin-6 (IL-6), interleukin-10 (IL-10) and interleukin-13 (IL-13) in threatened abortion, missed abortion, pregnant and non-pregnant groups Threatened abortion group
7.8 1.9 14.9 6.1***,z 5.3 1.7y,z 3.9 2.1*,z
Pregnant control group
Non-pregnant control group
Second sample (discharge time)
First sample (admission time) WBC (103/ml) Interleukin-6 (pg/ml) Interleukin-10 (pg/ml) Interleukin-13 (pg/ml)
Missed abortion control group
13.8 5.1** 4.9 1.7y 4.2 2.5*
8.6 9.1 2.0 2.4
2.6 2.9 1.1 0.5
7.7 13.5 4.4 5.7
2.1 2.3 1.4 4.6
6.3 12.6 5.6 3.8
1.1 2.1 4.3 2.1
*
P < 0:05 vs. missed abortion group. P < 0:01 vs. missed abortion group. *** P < 0:005 vs. missed abortion group. y P < 0:001 vs. missed abortion group. z P ¼ NS vs. same group at discharge time and pregnant and non-pregnant control groups. **
Fig. 1. Distribution of interleukin-6 levels (pg/ml) in serum of the threatened abortion group at admission (A) and discharge (B) time, missed abortion control group (C), pregnant control group (D) and non-pregnant control group (E). Number (2) represents the number of cases with overlapping values. ( ) mean S:D.
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Fig. 2. Distribution of interleukin-10 levels (pg/ml) in serum of the threatened abortion group at admission (A) and discharge (B) time, missed abortion control group (C), pregnant control group (D) and non-pregnant control group (E). Numbers (2 or 3) represent the number of cases with overlapping values. ( ) mean S:D.
Fig. 3. Distribution of interleukin-13 levels (pg/ml) in serum of the threatened abortion group at admission (A) and discharge (B) time, missed abortion control group (C), pregnant control group (D) and non-pregnant control group (E). Numbers (2 or 3) represent the number of cases with overlapping values. ( ) mean S:D.
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P < 0:01 versus threatened abortion group at admission and discharge time, respectively; Table 1). Interleukin-6 concentrations in missed abortion group were also lower than those in pregnant and non-pregnant control groups (P < 0:001 and P < 0:005, respectively). By single distribution, interleukin-6 concentrations in serum of women with missed abortion showed almost always lower values when compared to the mean values of the other groups demonstrating only a relative overlap (Fig. 1). Correlation between interleukin-6 levels and blood leukocyte count was not significant (r ¼ 0:09, P ¼ NS). Serum interleukin-10 concentrations in women with threatened abortion showed no significant differences with those of normal pregnant and non-pregnant women (P ¼ NS). No significant differences were also found between the two evaluations within the threatened abortion group. On the contrary, interleukin-10 was measured in serum of women with missed abortion with highly significant lower levels than that of women with threatened abortion (P < 0:001 versus threatened abortion group at both admission and discharge time; Table 1). Interleukin-10 concentrations in missed abortion group were also markedly lower than those in pregnant and non-pregnant control groups (P < 0:001). By single distribution, interleukin-10 concentrations in serum of women with missed abortion showed lower values when compared to the mean values of the other groups demonstrating practically almost no overlap (Fig. 2). Correlation between interleukin-10 levels and blood leukocyte count was not significant (r ¼ 0:10, P ¼ NS). Serum interleukin-13 concentrations in women with threatened abortion showed no significant differences with those of normal pregnant and non-pregnant women (P ¼ NS). No significant differences were also found between the two evaluations within the threatened abortion group. On the contrary, interleukin-13 was measured in serum of women with missed abortion with sufficiently significant lower levels than that of women with threatened abortion (P < 0:05 versus threatened abortion group at both admission and discharge time; Table 1). Interleukin-13 concentrations in missed abortion group were also lower than those in pregnant and non-pregnant control groups (P < 0:01). By single distribution, interleukin-13 concentrations in serum of women with missed abortion showed values at the lower limits of the range of the values of the other groups (Fig. 3). Correlation between interleukin-13 levels and blood leukocyte count was not significant (r ¼ 0:20, P ¼ NS).
4. Discussion Fetus is partly allogenic to the mother, but is not commonly rejected by the maternal immune system. In the last decade has emerged the concept that fetal comfort may be preserved by the action of the 2-type and not the 1-type cytokines, or successful pregnancy is a T-helper 2 phenomenon [12,24], in
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particular, in the mid-late first-trimester pregnancy during placental formation [10]. Briefly, exist in experimental animals beneficial cytokines (anti-inflammatory cytokines secreted by T-helper 2-type cells) which can enhance fetal growth and survival and deleterious cytokines (pro-inflammatory cytokines secreted by T-helper 1-type cells) which can compromise pregnancy causing the death of the fetoplacental unit [12,24]. The positive emerging role, particularly, of interleukin-10 associated with normal pregnancy and the negative deleterious effects of interferon-g that may compromise pregnancy have been documented [12,24]. Similar results have been proposed also in human beings. A lot of studies demonstrating a role of T-helper 1-type immunity in reproductive failure and of T-helper 2-type immunity in successful pregnancy have been reported, but always in normal and/or in clearly compromised pregnancies [13,15–23,25]. Here, we analyzed for the first time T-helper 2-type cytokine profile in threatened abortion, the first step towards claimed spontaneous abortion or remission of symptoms. On the other hand, threatened abortion is a dramatic clinical problem by a socio-economical point of view, because is the most frequent condition among the patients who attend the out-patient emergency unit. The rationale for investigating the presence of selected cytokines directly in the serum of women with threatened abortion is derived from the fact that cytokines concentrations have already been measured in the sera of pregnant and aborting women as well as in several other situations, infections disease particularly, in which cytokines are detectable in the circulation [21]. All the three selected T-helper 2-type cytokines presented similar concentrations in women with threatened abortion and in women with regular pregnancy suggesting the existence of an immune T-helper 2-type pattern also in threatened abortion condition. Moreover all the patients of the threatened abortion group showed similar results in their cytokine profiles in both investigations, at admission and discharge time, confirming a maintenance of the immune Thelper 2-type pattern during hospitalization in concomitance with the remission of symptoms. On the other hand, none patient of this group tended to abortion, to verify, in the case, a switch to an immune T-helper 1-type pattern. Furthermore all the T-helper 2-type cytokines studied, interleukin-10 particularly, showed significantly lower concentrations in missed abortion group, strengthening the hypothesis that a T-helper 2-type pattern deficiency may help a shift towards a T-helper 1-type pattern typical of the pregnancy loss. Taken together these data sustain the existence of an active T-helper 2-type response in threatened abortion with positive evolution, contributing to create an immunological environment that is conductive to a resolution of this condition and to a regular continuance of the pregnancy. Lacking data in the literature for comparison with threatened abortion, our data agree, however, with the majority of the studies reported in normal pregnancy and recurrent or sporadic spontaneous abortions. All ‘‘in vitro’’ studies
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[13–15,18,25], grossly agree in the detection of higher levels of T-helper 2-type cytokines in normal pregnancy compared to recurrent or sporadic spontaneous abortions and higher levels of T-helper 1-type cytokines in recurrent or sporadic spontaneous abortions compared to normal pregnancy. ‘‘In vivo’’ studies on T-helper 1–2-biased in terms of circulating levels of cytokines in the serum also indicate a prevalently T-helper 2-biased in normal pregnancy and T-helper 1biased in recurrent or sporadic spontaneous abortions [17,19–22]. Only Schust and Hill [16] found no detectable serum levels of cytokines in any patient sample regardless of pregnancy status or pregnancy outcome. On the contrary, Vassiliadis et al. [17] detected very high serum cytokine levels with values of a different order of magnitude (ng/ml versus pg/ml). Several mechanisms may be involved in threatened abortion to influence positively or negatively the T-helper 1/T-helper 2 balance in favor of one or other direction. In normal pregnancy interleukin-6, interleukin-10 and interleukin-13 are spontaneously secreted by human trophoblast and play crucial T-helper 2-type roles in implantation and continuation of early pregnancy [10]. Hill et al. [13] suggested that in cases with unexplained recurrent abortion may exist a primary defect that can lead to an inability to activate this T-helper 2-response. It is likely, however, that the primary protective role for the T-helper 2-type cytokines in the maintenance of normal pregnancy may not be a direct one, but indirect. In fact, an important, if not major, role for Thelper 2-type cells is downregulate T-helper 1-immunity, suppressing interferon-g and other related T-helper 1-type cytokine production [26], because interleukin-6 and interleukin-10 are potent inhibitors of T-helper 1 effector function [11]. T-helper 1-type cytokines, on the other hand, can be harmful to pregnancy either by direct cytotoxic action on throphoblast cells, by inhibition of throphoblast growth, by enhancement of natural killer activity, or even via activated macrophages [27]. Lim et al. [2] suggested that the T-helper 2-response allows the production of blocking antibodies to mask fetal trophoblast antigens from immunological recognition by maternal T-helper 1 cell-mediated/cytotoxic response which is responsible for rejection of semi-allografts. Natural killer cell-mediated immunity against trophoblast has also been evidenced. A correlation between fetal resorption and an increased appearance of natural killer cells at the feto-maternal interface [28] as well as elevated natural killer cell levels in the blood in women aborting kariotypically normal embryos [29] have been observed. Considering that T-helper 2-type cytokines counter natural killer cell activation [12], an inhibition of this activity may be a credible explanation for immunological protection against abortion [29]. Moreover, interleukin-10 reduces expression of HLA classes I and II molecules, whereas interferon-g augment this expression [30]. The T-helper 1–2-type shift of cytokine profile in normal pregnancy and eventually threatened abortion may contribute to modify the expression of HLA molecules in the developing fetal tissues, reducing potentially the
risk of rejection. On the other hand, it will be helpful during threatened abortion to avoid infection, autoimmune disease, stress, that may well result in a strong enough T-helper 1biased immunity to compromise pregnancy, because T-helper 1-type cells induced by infection may traverse the fetal interface or may product cytokines that affect the trophoblast [26]. Other mechanisms hypothetically involved in threatened abortion to maintain a pro-T-helper 2 and an anti-T-helper 1-biased can be the hormones, in particular the progesterone, that in mice eliciting from lymphocytes the production of progesterone-induced blocking factor which suppresses natural killer activity, may act as immunomodulator by altering the T-helper 1/T-helper 2 balance in favor of the latter [31]. Piccinni et al. [9] observed in humans also that progesterone promotes T-helper 2-type cytokine production and may contribute to the regulation of the immune homeostasis during pregnancy, whereas progesterone analogues as well as 17b-estradiol have no effect on cytokine production. These effects, however, have been obtained in ‘‘in vitro’’ experiments by administering progesterone at levels comparable to those present at the feto-maternal interface during pregnancy, that are about 100 times the physiological progesterone concentrations found in serum during pregnancy. At these doses therefore progesterone may function as a potent inducer of T-helper 2-type cytokine production [9] and/or blocker of T-helper 1-type cytokine production [32]. By a clinical point of view reassurance that most pregnancy with threatened abortion go to term is the mainstay of management, even though attempts to maintain pregnancy are reasonable only in those cases in which the fetus is not chromosomically and/or anatomically seriously affected. In such case, abortion may be considered a part of the quality control process in human reproduction [33]. The historic administration of hCG and progesterone (at the traditional progestative doses) is no longer practised. On the other hand, no trials, at our knowledge, are in progress in human threatened abortion concerning the ‘‘in vivo’’ administration of pure micronized progesterone at the above mentioned immunomodulatory doses, to sustain the concept of successful pregnancy as progesterone-induced T-helper 2 phenomenon. This study is not able to demonstrate a direct cause/effect relationship between circulating T-helper 2-type cytokines and the evolution of threatened abortion, but support, however, the concept that (a) first-trimester normal pregnancy is T-helper 2-biased, (b) threatened abortion also, when T-helper 2-biased, may tend to a positive evolution of the condition and to a normal continuance of the pregnancy and (c) a T-helper 2-type pattern deficiency is joined with missed abortion. Moreover, serum interleukin-10, particularly, when assayed by highly sensitive ELISA kits, may represent an acceptable peripheral marker of immunity potentially occurring locally at the feto-maternal interface and predictor of pregnancy outcome in cases of unexplained threatened abortion.
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