Mannan-binding lectin concentration during normal human pregnancy

David C. Kilpatrick

Academic Unit, Department of Transfusion Medicine, 2 Forrest Road, Edinburgh EH1 2QN, UK


    Abstract
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 Abstract
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 Materials and methods
 Results
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Relative deficiency of mannan-binding lectin (MBL) is associated with recurrent miscarriage. To investigate whether MBL concentration alters as a result of pregnancy, serial measurements were made in 14 patients before and during normal early pregnancy. Additionally, a longitudinal study was made of one individual over several years and including two normal pregnancies. Three (20%) subjects experienced a significant increase in MBL concentration during pregnancy. It was concluded that a modest but significant increase in MBL concentration can result from pregnancy, but it is a rare occurrence in the first trimester.

Key words: mannan-binding lectin/recurrent miscarriage/recurrent spontaneous abortion


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mannan-binding lectin (MBL) is an opsonic C-type lectin and an important component of the innate immune system (reviewed by Turner, 1996, 1998). Individuals with low MBL concentrations seem to be more susceptible to infectious disease generally (Garred et al., 1995Go; Summerfield et al., 1997Go; Kakkanaiah et al., 1998Go), and are over-represented in cohorts of patients with specific bacterial, viral or protozoal infections (Luty et al., 1998Go; Hibberd et al., 1999Go; Yuen et al., 1999Go).

Individuals with low concentrations of MBL are also over-represented among patients with unexplained recurrent miscarriage (Kilpatrick et al., 1995Go, 1999Go; Christiansen et al., 1999Go). An MBL concentration of <=0.1 µg/ml (found in ~10% of the general population) in either parent is a risk factor for spontaneous abortion (Kilpatrick et al., 1999Go), but with an odds ratio of ~2.2, this has limited predictive value for individual couples. MBL deficiency was not evident in women with explained recurrent miscarriage or with endometriosis (Kilpatrick et al., 1996Go), or in couples with pre-eclampsia (Kilpatrick, 1996Go).

Although circulating MBL concentrations are largely genetically determined (Madsen et al., 1995Go), MBL is an acute phase reactant (Thiel et al., 1992Go; Arai et al., 1993Go), and its concentration can vary with physiological conditions. It is not known if MBL concentration alters in response to pregnancy, and therefore whether that consideration could influence the classification of MBL measurements made during pregnancy, or alter the status of an MBL deficient individual as a result of pregnancy. In this study, longitudinal measurements were made before and during normal pregnancy in the same individual subjects. It appears that there is generally little or no increase in MBL concentration during the critical first trimester of pregnancy.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
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MBL was assayed as previously described (Kilpatrick et al., 1995Go). Briefly, diluted sera were incubated in mannan-coated wells of an enzyme-linked immunosorbent assay (ELISA) plate in the presence of calcium, and, after washing, a murine monoclonal antibody to MBL was added. Bound immune complexes were visualized with anti-mouse immunoglobulin conjugated to alkaline phosphatase followed by p-nitrophenyl phosphate as substrate. This method has intra-assay and inter-assay coefficients of variation of 3.5 and 7% respectively, and results obtained in a quality assurance exercise showed excellent (r > 0.99) correlation with values obtained by Dr S.Thiel (University of Aarhus, Aarhus, Denmark) using an internationally distributed MBL standard (Thiel et al., 1992Go).

It is virtually impossible in practice to study randomly selected, obstetrically normal women of proven fertility. Therefore, most of the sera were obtained from women originally presenting with idiopathic recurrent miscarriage described elsewhere (Kilpatrick and Liston, 1994Go). Patients who had experienced three or more consecutive miscarriages were considered to be idiopathic aborters if they had normal platelets, thyroid hormone and glucose concentrations, and if both they and their partners had normal karyotypes. Patients suspected of uterine abnormalities were investigated by hysterosalpingography and/or tests of cervical resistance. Furthermore, patients considered for immunotherapy did not have anti-nuclear antibodies, lupus anti-coagulants, cardiolipin antibodies, or anti-paternal (anti-partner) T or B lymphocyte antibodies at presentation. Stored serum samples for this investigation were selected from patients who had been randomized to receive either autologous lymphocytes (no specific treatment) or injections of their partners' lymphocytes (immunotherapy), and who subsequently had normal pregnancies. The former group (age range: 23–40 years; mean number of abortions: 3.3) were obviously the most suitable, but adequate sets of sera were available from only eight such patients. In order to increase the number of patients in the study and in particular to include more subjects with low MBL concentrations, sera from a further six patients (age range: 30–34 years; mean number of abortions: 3.7) who had received injections of their partners' cells were selected.

A pregnancy was considered normal if it was of more than 38 weeks duration, resulted in the birth of a healthy baby >2.5 kg in birthweight, and was characterized by the absence of maternal disease or neonatal complications. At least two blood samples (typically taken several months apart) were obtained from each patient before pregnancy was achieved.

Statistical analyses were performed using Prism for Windows software from Graph Pad (San Diego, CA, USA); significance of the difference between means was tested using parametric (unpaired t-tests) computations after testing the data for normality.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Serum samples were tested from eight patients with a history of recurrent miscarriage who received no specific treatment and had normal pregnancies terminating in live births. In each case two pre-pregnancy samples were obtained (typically 6–8 months prior to conception). At least one further sample was obtained during early pregnancy. There was no consistent pattern of change associated with pregnancy, and only one patient had an appreciable (66%) increase in MBL concentration (Figure 1Go).



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Figure 1. Mannan-binding lectin (MBL) concentrations during the first trimester of normal pregnancy. Each symbol represents an individual patient. Two samples were obtained from each patient over a variable time-span before pregnancy occurred, giving the pre-pregnancy (PRE) values as the mean and range of those two values. Gestational age on the abscissa is given in weeks of pregnancy (i.e. time from last menstrual period).

 
Another group of sera from six recurrent miscarriage patients who had been treated with leukocyte immunotherapy was tested; three had had blood samples collected until halfway through their pregnancies, and all had normal pregnancies resulting in live births. In principle, this investigation could have been complicated by a direct influence of the leukocyte injections, but in no instance was a significant change apparent in samples obtained 1 month after immunization (data not shown). There was essentially no change in the three patients with low pre-pregnancy MBL concentrations (nos 4, 5 and 6); little change in two patients with above-average levels (nos 2 and 3); and a modest (53%) rise in one patient (no. 1; Table IGo).


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Table I. MBL concentrations (µg/ml) in leukocyte immunotherapy patients
 
Finally, samples were obtained from one normal (no adverse obstetric history) individual before, during, between, and after two uncomplicated pregnancies (Figure 2Go). No dramatic changes were evident, but the MBL concentration during both pregnancies was higher than the non-pregnant mean (from 15 samples collected over several years), and in both pregnancies the highest values occurred during the third trimester. The mean (±SD) concentration during the pregnancies (5.31 ± 0.52) was significantly higher than the mean of the non-pregnancy samples (3.84 ± 0.46), when the data were analysed statistically (P < 0.0001).



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Figure 2. Individual MBL concentration variation throughout two normal pregnancies. Samples were obtained from a healthy individual before the first pregnancy, after the second pregnancy, during and between the pregnancies. Each gestational period (continuous line) is denoted from the estimated times of conception (C1 and C2) until term (T1 and T2), although conventional weeks of pregnancy (time from last menstrual period) is given along the abscissa. Values obtained outside pregnancy are joined by a broken line. The dotted line indicates the mean MBL concentration of the 15 samples obtained during the non-pregnant periods over several years.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ideally, it would be desirable to investigate randomly selected, obstetrically normal women of proven fertility, but such an aim would be unrealistic. In practice, it is extremely difficult to obtain blood samples before pregnancy or from early in the first trimester unless the subjects were previously referred to hospital for some complaint, probably related to fertility. In this instance, the bulk of the subjects studied first presented with a history of miscarriage. However, all of the pregnancies included in this investigation were normal in the accepted sense. Moreover, it has been estimated that about half of couples who have had only three consecutive pregnancy losses are just unlucky (discussed by Christiansen, 1997).

This study is the first to examine MBL concentration as a function of pregnancy and includes a unique case report of an obstetrically normal individual before, during and after two successful pregnancies. The latter provides a unique opportunity to observe normal variation in MBL over time during several periods of the non-pregnant state, against which to compare the values obtained during periods of pregnancy. The selection of samples from untreated recurrent miscarriage patients depended on the availability of samples, while the treated patients' samples were chosen to provide a suitable range of initial MBL concentrations. It must be emphasized that this work was designed to look for changes induced by pregnancy and is not an extension of previous work relating MBL deficiency to risk of miscarriage.

It is clear that only a minority of normal pregnancies is characterized by a small increase in maternal MBL concentration, at least during the early stages of pregnancy when most miscarriages occur. This unremarkable but useful finding is consistent with the putative Th-2 bias in normal pregnancy (Raghupathy, 1997), and the rather weak MBL response to surgery or infection (Thiel et al., 1992Go). It is noteworthy that in individuals with low MBL concentrations (typical of homozygotes or heterozygotes for structural gene mutations) there was never any change greater than that which could be accounted for by intra-assay variation. It is also clear, however, that a modest increase in MBL concentration can be induced as a function of pregnancy, and the possibility is raised by the single example that this increase might peak in the third trimester.

It is concluded that subjects whose relative MBL deficiency might be considered clinically significant in this context (Kilpatrick et al., 1999Go) are unlikely to be misclassified for risk of pregnancy failure if tested during early pregnancy. (Women with MBL <=0.1 µg/ml are twice as likely to miscarry as women with median levels.) Nevertheless, recurrent miscarriage patients should ideally be tested for MBL when not pregnant, as indeed were those previously described (Kilpatrick et al., 1995Go, 1999Go; Christiansen et al., 1999Go).


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Arai, T., Tabona, P. and Summerfield, J.A. (1993) Human mannose-binding protein gene is regulated by interleukins, dexamethasone and heat shock. Q. J. Med., 86, 575–582.[Medline]

Christiansen, O.B. (1997) Epidemiological, immunogenetic and immunotherapeutic aspects of unexplained recurrent miscarriage. Danish Med. Bull., 44, 396–424.[ISI][Medline]

Christiansen, O.B., Kilpatrick, D.C., Souter, V. et al. (1999) Mannan-binding lectin deficiency is associated with unexplained recurrent miscarriage. Scand. J. Immunol., 49, 193–196.[ISI][Medline]

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Kilpatrick, D.C., Bevan, B.H., Liston, W.A. et al. (1996) Lectins in the feto–placental unit: a review. In Van Driessche, E. et al. (eds), Lectins – Biology, Biochemistry, Clinical Chemistry, vol. 11. Textop, Hellerup, pp. 161–167.

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Submitted on August 20, 1999; accepted on January 6, 2000.