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10.3109/14767058.2011.636108

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The Journal of Maternal-Fetal and Neonatal Medicine, 2012; 25(5): 531–534© 2012 Informa UK, Ltd.ISSN 1476-7058 print/ISSN 1476-4954 onlineDOI: 10.3109/14767058.2011.636108

08 September 2011

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20 October 2011

Objective: To prospectively investigate the potential role of the lectin pathway of complement in intrauterine-growth-restriction (IUGR, associated with impaired immunocompetence and increased risk for neonatal infections), by determining cord blood concentrations of mannose-binding lectin (MBL), H-ficolin and L-ficolin (important mediators of neonatal innate immunity) in IUGR and appropriate for gestational age (AGA) pregnancies. Furthermore, we aimed to describe correlations among cord blood MBL, H- and L-ficolin concentrations and with several demo-graphic parameters of the infants at birth. Methods: Serum MBL, H- and L-ficolin concentrations were determined by ELISA in 154 mixed arteriovenous cord blood samples from IUGR (n = 50) and AGA (n = 104) singleton full-term infants. Results: Cord blood MBL concentrations were significantly lower in IUGR cases than AGA controls (p = 0.029). No differences in cord blood H- and L-ficolin concentrations were observed between groups. In the IUGR group, cord blood MBL concentrations negatively correlated with respec-tive L-ficolin ones (r = −0.442, p = 0.001). Conclusions: The relatively decreased MBL expression in IUGR fetuses at term could possibly contribute to IUGR-associated neonatal immunodeficiency, predisposing to increased susceptibility to infections. The negative correlation between MBL and L-ficolin concentrations in the IUGR group might suggest an underlying immune variation and needs to be further investigated.

Keywords: H-ficolin, L-ficolin, mannose-binding lectin, neonate, umbilical cord blood

IntroductionEarly infancy is a vulnerable period of transition from the passive, transplacentally acquired immunity to the extrauterine, adaptive one. The latter is severely impaired during the neonatal period, due to immature B and T cell function, resulting to high suscep-tibility to infections [1,2]. This propensity is further enhanced in prematurity and intrauterine-growth restriction (IUGR [3]).

In this respect, several lines of evidence from both human and animal studies suggest that IUGR subjects present with compro-mised immunocompetence and show persistent immunological abnormalities [4,5], including a decrease in IgG concentrations, phagocytic index and lysozymes, as well as neutropenia, especially in cases of IUGR due to preeclampsia [6]. As a result, culture-proven sepsis is more common in IUGR infants [7].

Since neonates experience both humoral and cellular immu-nodeficiency, they rely on genetically determined innate defence mechanisms [1,8]. Innate immunity is mainly mediated by host proteins that recognize conserved pathogen-associated molecular patterns, such as repetitive sugar arrays present on many micro-organisms but not on mammalian cells [9]. The lectin pathway of complement represents an important component of innate immu-nity and consists of mannose-binding lectin (MBL), H-ficolin and L-ficolin, which recognize various microorganisms [9]. Both MBL and ficolins rely on MBL-associated serine protease-2 to activate the complement system [9].

MBL is produced by the liver and plays an important role in the neonatal immune system, acting as a first line of defence at the time of primary contact with a pathogen, mediating its elimi-nation through the activation of complement in an antibody-independent manner [10]. MBL deficiency has been investigated in various settings [11] and an increased susceptibility to sepsis has been demonstrated in MBL deficient neonates [12–14]. More interestingly, low cord blood MBL concentrations have been asso-ciated with an increased frequency of infections in early child-hood [15]. On the contrary, in spite of the close structural and functional similarities, the role of ficolins in sepsis remains largely unknown [16]. However, the presence of ficolins in cord blood has been confirmed [17].

Although the role of the lectin pathway of complement activa-tion is crucial early postpartum [1,8], the current knowledge on lectin pathway proteins in IUGR neonates is very limited [18].

Therefore, the aim of this study was to investigate, for the first time to our knowledge, the possible role of the proteins of the entire lectin pathway in the host defense of IUGR neonates, by determining and comparing cord blood MBL, H- and L-ficolin concentrations in well-characterized IUGR and appropriate for gestational age (AGA) pregnancies. Furthermore, the potential association of the above concentrations with several maternal and fetal anthropometric/clinical variables was explored.

Materials and methodsThe Ethics Committee of our teaching hospital approved the study protocol and written informed consent was acquired from all recruited mothers before cord blood sampling. From July 2009 to December 2009, one hundred and fifty four Caucasian women were included in a prospective study. During this time period,

The potential role of the lectin pathway of complement in the host defence of full-term intrauterine growth restricted neonates at birth

Despina D. Briana1, Sofia Liosi1, Dimitrios Gourgiotis2, Maria Boutsikou1, Stavroula Baka1, Antonios Marmarinos2, Dimitrios Hassiakos1 & Ariadne Malamitsi-Puchner1

1Neonatal Division, 2nd Department of Obstetrics and Gynecology and 2Research Laboratories, 2nd Department of Pediatrics, Athens University Medical School, Athens, Greece

Correspondence: Prof. Ariadne Malamitsi-Puchner, MD, Neonatal Division, 2nd Department of Obstetrics and Gynecology, 19 Soultani Street, 10682, Athens, Greece. Tel: +30 6944443815. Fax: +30 2107233330. E-mail: amalpu@aretaieio.uoa.gr; amalpu@tellas.gr

(Received 08 September 2011; accepted 20 October 2011)

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50 asymmetric IUGR singleton, full-term infants (birth weight ≤5th customized centile) were consecutively born and the AGA singleton, full-term baby, born simultaneously or immediately before/afterwards was enrolled as control (totally n = 104).

The Gestation Related Optimal Weight computer-generated program was used to calculate the customized centile for each pregnancy, taking into consideration significant determinants of birth weight, such as maternal height and booking weight, ethnic group, parity, gestational age and gender [19]. Gestational age was determined, according to the date of the last menstrual period and was confirmed by early antenatal ultrasound. Birth weight was measured with an electronic scale.

IUGR fetuses were closely observed by Doppler studies every 10–15 days from the 32nd gestational week and onwards. Three consecutive measurements of the pulsatility index (PI) of the uterine, umbilical and cerebral arteries were performed and the mean value was recorded. Mean PI values of the uterine and umbilical arteries [20,21] were progressively found to be in the upper physiological limits for the corresponding gestational age in 30 cases (ranging between the 90th and the 95th percentile). In eight cases, respective information was lacking. In the remaining 12 cases, PI values showed increased impedance to flow, being above the 95th percentile for gestational age.

Six of the 50 mothers with IUGR offspring presented with preec-lampsia, five presented with pregnancy-induced hypertension, 15 suffered from various diseases, such as iron-deficient anemia (three cases), severe type type I diabetes mellitus (five cases) and hypo-thyroidism (seven cases). The remaining 24 women were smoking >10 cigarettes/day during the whole duration of pregnancy.

Amniotic fluid was diminished in all IUGR cases. For the evaluation of the amniotic fluid, the largest fluid column on the vertical plane was assessed and was defined as diminished if <2 cm. Placental weights were reduced [22] ranging from 310 to 450 g.

In the AGA group, mothers were healthy and were either non-smokers or abstained from smoking during pregnancy. Placentas were normal in appearance and weight [22], ranging from 480 to 621 g.

Tests for congenital infections were negative in all women and their offspring had no symptoms of intrauterine infection or signs of genetic syndromes. One- and five-minute Apgar scores were in all infants ≥8. All neonates were breastfed and they all adapted well to extrauterine life. Demographic data of included IUGR and AGA infants and their mothers are presented in Table I.

After double clamping of the umbilical cord, mixed arterio-venous cord blood (reflecting the fetal state) was collected in pyrogen-free tubes. Serum was separated by centrifugation and was kept frozen at −80°C until assay. The determination of serum MBL, H- and L-ficolin concentrations was performed by ELISA (Hycult Biotechnology b.v., Uden, The Netherlands).

The minimum detectable concentration, inter- and intra-assay coefficients of variation for MBL, H-ficolin and L-ficolin were: 0.41 ng/ml, 8.7% and 4.3%, respectively, 0.008 µg/ml, 7.9% and 8%, respectively, and 16 ng/ml, 5.6% and 5.7%, respectively.

Statistical analysis

MBL data were not normally distributed, whereas data regarding H- and L-ficolin presented with normal distribution. Furthermore, variables regarding birth weight, gestational and maternal age were normally distributed (Kolmogorov–Smirnov test). Independent samples T-test was used to detect differences in normally distributed variables between IUGR cases and AGA controls. Otherwise, non-parametric Mann–Whitney U-Test was applied. Pearson’s χ-square test was used to detect differences concerning gender, delivery mode and parity between groups.

Linear regression analysis was applied to examine the possible effect of different independent variables such as group, maternal age, parity, gestational age, mode of delivery, birth weight, customized centile and gender on H- and L-ficolin concentra-tions. Spearman’s or Pearson’s correlation coefficient was used, as appropriate, to detect any positive or negative correlations. A p < 0.05 was considered statistically significant. Statistical analysis was performed, by using SPSS 11.5 (Chicago IL, USA).

ResultsSerum MBL, H- and L-ficolin values are shown in Figures 1, 2 and 3 [mean, (95% confidence intervals)]. Cord blood

Table I. Demographic data for appropriate for gestational age (AGA) and intrauterine growth restricted (IUGR) neonates and their mothers.

Demographic data

AGA cases IUGR cases

p valueMean ± SD/

median (range)Mean ± SD/

median (range)Birth weight (g) 3280 ± 290.28 2507 ± 264.5 <0.001Birth weight centile 43.5 (20–89) 3.0 (0–5) <0.001Gestational age (weeks) 38.98 ± 1.07 38.36 ± 1.27 NSMaternal age (years) 30.10 ± 5.0 32.06 ± 4.53 0.02Gender n (%) 0.038 Male 63 (60.6) 21 (42) Female 41 (39.4) 29 (58) Mode of delivery n (%) 0.001 Vaginal 76 (73.1) 22 (44) Caesarean section 28 (26.9) 28 (56) Parity n (%) NS Primigravida 72 (69.2) 31 (62) Other 32 (30.8) 19 (38) NS, non significant.

Figure 1. Box and whiskers plots of cord blood mannose-binding lectin (MBL) concentrations in appropriate for gestational age (AGA) and intrauterine growth restricted (IUGR) groups. Each box represents the median concentration with the interquartile range (25th and 75th percentiles). The upper and lower whiskers represent the range.

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Mannose-binding protein MBL concentrations were significantly lower in IUGR cases, compared to AGA controls [median (range): 310.09 (1.24–3525.24) ng/ml and 1117.19 (8.84–4989.37) ng/ml, respectively, p = 0.029].

By contrast, no significant differences in H- and L-ficolin concentrations were observed between IUGR and AGA groups (mean values ± SD: 16.40 ± 6.2 µg/ml versus 16.02 ± 5.86 µg/ml and 4141.07 ± 1474.7 ng/ml versus 4395.47 ± 1684.22 ng/ml, respectively). In the IUGR group, cord blood MBP concentrations negatively correlated with L-ficolin ones (r = −0.442, p = 0.001).

In both groups, the effect of maternal age, parity, gestational age, delivery mode, customized centile, birth weight and gender on MBL, H- and L-ficolin concentrations was not significant.

DiscussionThe results of this study indicate that cord blood MBL concen-trations are significantly lower in IUGR cases, compared to AGA controls. Furthermore, the IUGR neonates of our cohort present with a mean cord blood MBL value of 0.31 µg/ml, which may be regarded as deficient, since cut-off plasma MBL concentrations of 0.4 µg/ml in preterm neonates and 0.7 µg/ml in term neonates have previously been chosen to define MBL deficiency [23,24].

MBL levels are genetically determined, although a large interin-dividual variability exists, in part due to its behaviour as a reactant phase protein [25]. Three major mutant alleles in exon 1, as well as mutations in the promoter region of the MBL2 gene, have been associated with MBL deficiency [26]. However, there is a high rate of haplotype variation between ethnic groups and within these groups, MBL concentrations vary considerably [27]. Thus, studies in adults usually define MBL deficiency on MBL2 genotype [24]. Nevertheless, neonates can have low MBL concentrations despite wild-type haplotypes [28]. Therefore, MBL deficiency at birth has been proposed to be defined by decreased MBL concentrations and not by MBL2 genotype [24].

Several lines of evidence suggest a fetal origin of cord blood MBL [28,29]. In this respect, lower cord blood MBL levels than maternal ones and a lack of correlation between cord blood and maternal MBL levels were reported [28]. Additionally, cord blood MBL concentrations correlate with the individual MBL levels later in life [30]. Relatively, the differences between umbilical cord and neonatal plasma MBL concentrations within 24 h after birth were reported to be negligible [24,29], and, thus, it has been proposed that neonatal MBL concentrations can reliably be measured in umbilical cord blood [24].

There is considerable evidence that MBL insufficiency is associated with an opsonization defect and can increase susceptibility to various infections in both adults and neonates [11–14]. Furthermore, a high prevalence of MBL deficiency has been demonstrated in preterm neonates [13,24], probably due to a maturational defect, possibly involving the liver secretory capacity [13]. Thus, the relatively decreased MBL expression in IUGR fetuses of our cohort may represent an additional mecha-nism, by which IUGR predisposes to increased susceptibility to infections.

On the contrary, the findings of the present study suggest a lack of significant differences in cord blood H- and L-ficolin concentra-tions between IUGR cases and AGA controls. However, a striking association between cord blood L-ficolin deficiency and low birth weight, independently of gestational age, has been recently docu-mented [18]. In comparison to MBL, much less has been reported on the role of ficolins in the perinatal period. Furthermore, disease-association studies involving ficolins are currently in their infancy. In this respect, a higher frequency of L-ficolin defi-ciency has previously been reported in children with recurrent respiratory infections [31]. Additionally, a recent preliminary study indicated a strong relationship between decreased H-ficolin concentrations and a high susceptibility to neonatal infections [32]. On the other hand, ficolins and the classical complement pathway activities are not influenced in a similar way during preg-nancy and postpartum, as MBL [33]. Therefore, the data of this study may suggest that, as compared to MBL, ficolins are probably differentially regulated during the perinatal period. Alternatively,

Figure 2. Mean (95% confidence intervals) cord blood H-ficolin concentrations in and intrauterine growth restricted (IUGR) groups.

Figure 3. Mean (95% confidence intervals) cord blood L-ficolin concentrations in and intrauterine growth restricted (IUGR) groups.

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ficolins may not be involved in the pathogenesis of the enhanced neonatal immunodeficiency associated with IUGR.

Additionally, in the narrow age interval studied, no significant effect of fetal maturation on MBL, H- or L-ficolin concentrations was observed in the present report. On the contrary, several groups have reported a correlation between cord blood MBL concentrations and gestational age [12,13,34]; however others found no such relation-ship [18,29]. As far as ficolins are concerned, a positive association between cord blood levels and gestational age has been recorded, although existing only until term, in line with our results [17,18].

Furthermore, in accordance with previous reports [18,32,34], our findings further confirm that the lectin pathway of comple-ment activation may not be fully functional at birth, as concen-trations of the lectin pathway proteins measured were lower, compared with children and adults [35].

Finally, our data suggest the presence of a negative correlation between cord blood MBL and L-ficolin concentrations only in the IUGR group. The domain organizations of MBL and ficolins are very similar in that both, consist of a collagen-like domain and a carbohydrate-binding domain [16]. Furthermore, serum MBL and ficolins recognize specific pathogens and eliminate them, by acting as opsonins and by activating the lectin complement pathway [16]. This indicates that both MBL and ficolins play roles in innate immunity in a similar way [16]. Thus, it may be speculated that the negative correlation between cord blood MBL and L-ficolin concentrations in our IUGR cohort probably suggests the pres-ence of an underlying immune variation associated with IUGR.

In conclusion, our findings provide evidence that full-term IUGR neonates present with relative MBL deficiency at birth. The possible relation of this finding with the high susceptibility of IUGR subjects to infections may have widespread clinical and therapeutic implications. On the other hand, whether ficolins are implicated in the pathogenesis of neonatal immunodeficiency associated with IUGR needs to be further investigated by larger studies.

Declaration of interest: The authors report no conflicts of interest.

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