|Year : 2016 | Volume
| Issue : 2 | Page : 85-89
Comparison of salivary immunoglobulin A levels in children delivered by cesarean section with those delivered via vaginal delivery
Priya Subramaniam, Shilpy Dwivedi, KL Girish Babu
Department of Pedodontics and Preventive Dentistry, The Oxford Dental College, Hospital and Research Center, Bengaluru, Karnataka, India
|Date of Web Publication||9-Aug-2016|
Department of Pedodontics and Preventive Dentistry, The Oxford Dental College, Hospital and Research Center, Bengaluru - 560 068, Karnataka
Source of Support: None, Conflict of Interest: None
Background: Salivary immunoglobulin A (SIgA) is an important factor in the oral defense mechanism. SIgA levels in infants have shown to vary widely. Mode of delivery may influence SIgA levels. The present study was conducted to evaluate levels of SIgA in infants at different ages, and to compare it with the mode of delivery.
Methodology: The study group consisted of 279 healthy neonates and infants up to the age of 9 months. They were divided according to age: Group I: 6–10 days; Group II: 6–8 weeks; Group III: 6 months, and Group IV: 9 months. Unstimulated whole saliva was collected from the floor of the mouth and buccal sulcii by means of sterile polyethylene pipettes. Saliva was collected 1 h after feeding. All salivary samples were collected in disposable sterile vials and transferred suitably to a laboratory for estimation of SIgA level using enzyme-linked immunosorbent assay. Data were subjected to statistical analysis.
Results: There were 146 infants that were delivered by cesarean section and 133 vaginally delivered infants. There was a significant difference in the SIgA levels between vaginal delivery and cesarean section infants (P < 0.001).
Conclusion: Mode of delivery appears to have influence on the levels of SIgA in saliva of neonates and infants.
Keywords: Infants, mode of delivery, saliva, salivary immunoglobulin A
|How to cite this article:|
Subramaniam P, Dwivedi S, Girish Babu K L. Comparison of salivary immunoglobulin A levels in children delivered by cesarean section with those delivered via vaginal delivery. Saudi J Oral Sci 2016;3:85-9
|How to cite this URL:|
Subramaniam P, Dwivedi S, Girish Babu K L. Comparison of salivary immunoglobulin A levels in children delivered by cesarean section with those delivered via vaginal delivery. Saudi J Oral Sci [serial online] 2016 [cited 2019 Jul 24];3:85-9. Available from: http://www.saudijos.org/text.asp?2016/3/2/85/188074
| Introduction|| |
Saliva plays an important role in oral defense mechanisms because it contains several factors that protect both hard and soft tissues. The development of the immune system in infants is characterized by the induction of an antigen-specific immune response and maintenance of immunological tolerance against commonly found compounds in the environment of the infant. Immunoglobulins in saliva consist predominantly of secretory immunoglobulin A (sIgA), with smaller amounts of serum immunoglobulins IgG, IgA, and IgM.
Salivary Immunoglobulin A (SIgA) is a type of sIgA. Secretory IgA is the dominant immunoglobulin isotype on all mucosal surfaces and in external secretions including saliva. Secretory IgA has several effector functions, i.e., agglutination, precipitation, opsonization, suppression of inflammation, plasmid curing, inhibition of colonization, and neutralization of toxins, viruses, and enzymes.
SIgA has shown to be present in saliva and other secretions at birth., It is considered as an indicator of maturation of mucosal immune system in children. SIgA levels in infants have shown to vary widely. SIgA levels are low in saliva during the first few months, and in longitudinal studies involving infants, stabilization of SIgA levels have seldom reached before 12–24 months. Mode of delivery can influence SIgA production in infancy. Mode of delivery may possibly have, via gut microbiota development, significant effects on immunological functions in an infant.
At birth, there is incomplete development of the immune system. Lack of antigenic experience and prevalence of suppression during fetal life are responsible for “physiological” immaturity of immune function in newborn infants. During and shortly after birth, the oral epithelial surfaces of infants become colonized by various bacterial species, which provide an antigenic stimulus for development of the immune system in infants.
The rate of cesarean deliveries has increased 10-fold worldwide during recent times. Infants born by cesarean section showed higher levels of immunoglobulin-producing cells in their peripheral blood compared to those born by vaginal delivery. There is a possibility that mode of delivery may influence the immunological functions in an infant. SIgA antibodies neutralize antigenic components involved in microbial virulence and might block surface adhesins important for colonization of the mucosa.
The present study was conducted to evaluate and compare SIgA levels in children delivered by cesarean section with those delivered via vaginal delivery.
| Methodology|| |
The study protocol was approved by the Ethical Committee of the Institution. Written informed consent was taken from the parents prior to collection of salivary sample. Prior to conducting the study, nature of the study was explained and permission was obtained from the authorities of various hospitals in Bangalore.
Sample size estimation
The size of the study group was estimated using the following formula:
With the risk for type I error, set at α = 0.05 and power (1-β) = 0.8. This would allow a detection of a 35% difference between the groups. The minimum sample size obtained was 128.
Three hundred subjects for the purpose of this cross-sectional study were selected from the Gynecological/Obstetrics and Pediatric Departments of government and private hospitals of Bangalore. Information regarding the age of child, mode of delivery, term of pregnancy, health of mother during pregnancy, and health of infant at birth was obtained from the mothers and hospital records using a data recording sheet. Exclusion criteria followed was complicated deliveries, incubated infants, antibiotic treatment of mother or infant during first 30 days of life, serious metabolic or infectious complications during the first 30 days of life and congenital anomalies. Neonates and infants that were delivered vaginally and cesarean section were selected. Those born at term (37–41 weeks of gestation, appropriate for gestation age) and infants of normal birth weight (2.5–3.5 Kg) and without any underlying disease were included in the study.
Twenty-one subjects could not be included and therefore 279 healthy neonates and infants, up to the age of 9 months, formed the study group. They were divided according to age as follows: Group I: 6–10 days; Group II: 6–8 weeks; Group III: 6 months, and Group IV: 9 months.
The feeding practice in Groups I and II was exclusively breastfeeding and in Groups III and IV, it was a combination of breast milk and a semisolid diet.
Estimation of salivary immunoglobulin A levels
Unstimulated whole saliva was collected from the floor of the mouth and buccal sulcii by means of sterile polyethylene pipettes. Care was taken not to cause mucosal trauma so as to prevent contamination with blood. Saliva was collected late morning and at least 1 h after feeding to avoid potential contamination of the saliva by milk. All samples were collected in disposable sterile vials and transferred to a laboratory in carbon dioxide snow (dry ice) and ice packs at -50°C. The samples were kept at -85°C in order to prevent degradation of protein  that might affect the estimation of IgA levels in the samples. Analysis was done within 1 week of collection, and each sample was analyzed in duplicate.
The samples were placed in Eppendorf tubes, clarified by centrifugation at ×10,000 g for 15 min at 4°C and frozen at -20°C for examination using enzyme-linked immunosorbent assay (ELISA) (BHAT BIO-SCAN ® Human IgA ELISA). To avoid serum contamination of the saliva samples, an ELISA kit specific for transferrin was used. The transferrin concentration in mg/dl for each saliva sample tested was determined by converting its optical density (OD) value using the formula: OD = ¼ cx b
The amount of total IgA was determined by ELISA. The microplates, having 96 wells each, were coated with goat anti-human α-chain purified antibodies, diluted at 1/500 in buffer I (sodium carbonate pH 9.6 with 0.2% sodium azide) and incubated. Plates were then blocked with 0.1% bovine albumin in buffer II (phosphate-buffered saline with 0.05% Tween 20 and 0.02%NaN3) for 30 min and incubated with diluted saliva samples (1/1600 in buffer II with 0.1% bovine albumin). The plates were incubated with the primary antibody (rabbit anti-human α-chain) diluted in buffer II with 0.1% albumin (1/1600) for 2 h and bound primary antibody was detected by reaction with goat antirabbit IgG conjugated to alkaline phosphatase diluted in buffer II (1/20,000). Substrate (p-nitrophenyl phosphate (1 mg/ml); diluted in buffer IV (0.05 M sodium carbonate, pH 9.8, withMgCl2) was added to each well, and after 30 min, the reaction was stopped by the addition of 1N sodium hydroxide. Conversion of substrate was determined at OD using an ELISA reader. The plates were washed 3 times with 0.9% NaCl, 0.05%Tween 20 following each step. Unless otherwise stated, all incubation times were 2 h and all reactions took place on a rocking platform at room temperature. Salivary antibody concentrations were calculated by reference to pooled standard saliva obtained from 10 control patients with high levels of antibody activity. A standard dilution curve ranging from 1: 200 to 1: 6400 was assayed with every microtiter plate. To calculate the concentration of IgA in the pooled saliva in lg/ml, a commercial kit specific for SIgA secretory was used. The IgA concentration in l µg/ml for each saliva tested was determined by comparing its OD value to the linear portion of the standard curve and using the formula: (OD = c (LognX) + b).
The test for normality using Kolmogorov–Smirnov test proved that the data do not follow normal distribution. Hence, the nonparametric test, namely Mann–Whitney U-test was used to test the equality of two groups.
| Results|| |
There were 146 infants that were delivered by cesarean section and 133 vaginally delivered infants [Table 1]. There was a significant difference in the SIgA levels between vaginal delivery and cesarean section full-term infants (P < 0.001) [Table 2]. In Groups I, II, and IV, a significant difference was observed in SIgA levels between cesarean section infants and vaginally delivered infants (P < 0.001). Regardless of the mode of delivery, SIgA levels were lowest in the 6–10 day age group and were highest in the 9-month-old age group [Table 3].
|Table 2: Comparison of salivary immunoglobulin A levels according to mode of delivery|
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|Table 3: Comparison of salivary immunoglobulin A levels according to age and mode of delivery|
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| Discussion|| |
Controversy exists with regard to detectable levels of SIgA at birth. Levels of SIgA in saliva during the first 10 days after birth have been reported to be difficult to measure in some studies;, however, few investigators have reported measurable concentrations of SIgA in the saliva of newborn infants.,, An association between age and feeding has been reported with SIgA production in infancy, but the available data on SIgA levels in breast- and formula-fed infants are conflicting.,
Immunization is given at birth and during early infancy. At 6 and 9 months, there is an eruption of primary teeth along with initiation of weaning and change in diet. It is also the time for immunization. To study the possible variations in SIgA levels, different age groups were selected for the present study.
Several factors might influence the development of an effective mucosal immune response including gestational age, maternal antibodies, feeding practices, exposure to antigens, and nutritional status. Since immunization may alter estimation of SIgA levels, salivary samples were collected prior to the scheduled date of immunization.
Levels of SIgA observed can be effected by the timing of saliva collection after feeding.,,,
Unlike adults, the mucosa of young children tends to be particularly responsive to microbial stimuli. Infants can be exposed to all types of antigens during and soon after delivery. In new-born infants, the cell-mediated immunological response can mature with the help of certain stimuli.
It was thought that vaginally delivered babies are exposed to more bacterial species and strains from the birth canal earlier than the relatively aseptically delivered cesarean section infants. However, Streptococcus mutans colonization within the mouth was detected significantly later in vaginally delivered than in cesarean section-born infants. Infants born by cesarean delivery have a higher level of immunoglobulin-producing cells in their peripheral blood compared to those born by vaginal delivery. The oral cavity of cesarean section infants was seen to be colonized 11.7 months earlier than vaginally delivered infants.
Vaginally delivered infants showed a different microbial composition in the intestines compared with infants delivered by cesarean section (C-section) or with the aid of instruments. By the age of 1, infants delivered by C-section have a lower ratio of anaerobic bacteria in the gut than vaginally delivered infants. A significantly higher prevalence of oral streptococci and lactobacilli was found in vaginally delivered infants when compared to those delivered by C-section.
Within the parameters of the low power (0.8) in this exploratory study, all infants delivered vaginally had significantly higher SIgA levels than those delivered by C-section. This could be due to the immune response to antigenic stimuli as a result of bacterial colonization. This difference was significant in early life (both 6–10 days and 6–8 weeks) and then later again at 9 months. However, by 6 months of age, the immune response was not significantly different from that of C-section. It may be attributed to the introduction of new foods around this age for all infants and further colonization of both gut and oral cavity. Infants delivered by cesarean section may have an impaired leukocyte function because of the lack of the pressure normally experienced at birth.
SIgA concentration has also been shown to be influenced by the presence of teeth. In our study, teeth had not yet erupted in 95% of the 6-month-old infants. However, all the 9-month-old infants had, at least, two erupted primary incisors and these infants also showed significantly higher SIgA levels.
Regardless of the mode of delivery, SIgA levels were seen to increase with age. Since this study was of a cross-sectional design, investigations with longitudinal follow-up of infants are necessary to validate these findings. SIgA plays an important role in defense against mucosal pathogens. It is essential to establish a relationship between feeding practices and SIgA levels in infants so that children have a well-developed mucosal immune system early in life which would probably aid in the future resistance to dental caries and periodontal diseases.
| Conclusion|| |
Mode of delivery appears to have an influence on levels of SIgA in saliva of neonates and infants. Infants who were vaginally delivered had significantly higher SIgA levels than that of cesarean section delivered infants.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
M'Rabet L, Vos AP, Boehm G, Garssen J. Breast-feeding and its role in early development of the immune system in infants: Consequences for health later in life. J Nutr 2008;138:1782S-90S.
Seidel BM, Schulze B, Kiess W, Vogtmann C, Borte M. Determination of secretory IgA and albumin in saliva of newborn infants. Biol Neonate 2000;78:186-90.
Kawabata S, Terao Y, Fujiwara T, Nakagawa I, Hamada S. Targeted salivary gland immunization with plasmid DNA elicits specific salivary immunoglobulin A and G antibodies and serum immunoglobulin G antibodies in mice. Infect Immun 1999;67:5863-8.
Wan AK, Seow WK, Purdie DM, Bird PS, Walsh LJ, Tudehope DI. Immunoglobulins in saliva of preterm and full-term infants. Oral Microbiol Immunol 2003;18:72-8.
Maruyama K, Hida M, Kohgo T, Fukunaga Y. Changes in salivary and fecal secretory IgA in infants under different feeding regimens. Pediatr Int 2009;51:342-5.
Gleeson M, Dobson AJ, Firman DW, Cripps AW, Clancy RL, Wlodarczyk JH, et al.
The variability of immunoglobulins and albumin in salivary secretions of children. Scand J Immunol 1991;33:533-41.
Alaluusua S. Longitudinal study of salivary IgA in children from 1 to 4 years old with reference to dental caries. Scand J Dent Res 1983;91:163-8.
Avanzini MA, Plebani A, Monafo V, Pasinetti G, Teani M, Colombo A, et al.
A comparison of secretory antibodies in breast-fed and formula-fed infants over the first six months of life. Acta Paediatr 1992;81:296-301.
Huurre A, Kalliomäki M, Rautava S, Rinne M, Salminen S, Isolauri E. Mode of delivery – Effects on gut microbiota and humoral immunity. Neonatology 2008;93:236-40.
Murray PR, Rosenthal KS, Kobayashi GS, Pfaller MA. Commensal and pathogenic microbial flora in humans. Medical Microbiology. St. Louis: Mosby Inc.; 2002. p. 78-87.
Marsh PD. Oral ecology and its impact on oral microbial diversity. In: Kuramitsu HK, Ellen RP, editors. Oral Bacterial Ecology: The Molecular Basis. Wymondham: Horizon Scientific Press; 2000. p. 11-65.
Brandtzaeg P. Mucosal immunity: Induction, dissemination, and effector functions. Scand J Immunol 2009;70:505-15.
Fitzsimmons SP, Evans MK, Pearce CL, Sheridan MJ, Wientzen R, Cole MF. Immunoglobulin A subclasses in infants' saliva and in saliva and milk from their mothers. J Pediatr 1994;124:566-73.
Castro GF, Souza IP, Lopes S, Stashenko P, Teles RP. Salivary IgA to cariogenic bacteria in HIV-positive children and its correlation with caries prevalence and levels of cariogenic microorganisms. Oral Microbiol Immunol 2004;19:281-8.
Gleeson M, Cripps AW, Clancy RL, Husband AJ, Hensley MJ, Leeder SR. Ontogeny of the secretory immune system in man. Aust N
Z J Med 1982;12:255-8.
Selner JC, Merrill DA, Claman HN. Salivary immunoglobulin and albumin: Development during the newborn period. J Pediatr 1968;72:685-9.
Weemaes C, Klasen I, Göertz J, Beldhuis-Valkis M, Olafsson O, Haraldsson A. Development of immunoglobulin A in infancy and childhood. Scand J Immunol 2003;58:642-8.
Piirainen L, Pesola J, Pesola I, Komulainen J, Vaarala O. Breastfeeding stimulates total and cow's milk-specific salivary IgA in infants. Pediatr Allergy Immunol 2009;20:295-8.
Choo S, Zhang Q, Seymour L, Akhtar S, Finn A. Primary and booster salivary antibody responses to a 7-valent pneumococcal conjugate vaccine in infants. J Infect Dis 2000;182:1260-3.
Tappuni AR, Challacombe SJ. A comparison of salivary immunoglobulin A (IgA) and IgA subclass concentrations in predentate and dentate children and adults. Oral Microbiol Immunol 1994;9:142-5.
Stephens S. Development of secretory immunity in breast fed and bottle fed infants. Arch Dis Child 1986;61:263-9.
Gross SJ, Buckley RH. IgA in saliva of breast-fed and bottle-fed infants. Lancet 1980;2:543.
Wu CY, Demeure C, Kiniwa M, Gately M, Delespesse G. IL-12 induces the production of IFN-gamma by neonatal human CD4 T cells. J Immunol 1993;151:1938-49.
Li Y, Caufield PW, Dasanayake AP, Wiener HW, Vermund SH. Mode of delivery and other maternal factors influence the acquisition of Streptococcus mutans
in infants. J Dent Res 2005;84:806-11.
Nelun Barfod M, Magnusson K, Lexner MO, Blomqvist S, Dahlén G, Twetman S. Oral microflora in infants delivered vaginally and by caesarean section. Int J Paediatr Dent 2011;21:401-6.
Schlinzig T, Johansson S, Gunnar A, Ekström TJ, Norman M. Epigenetic modulation at birth – Altered DNA-methylation in white blood cells after caesarean section. Acta Paediatr 2009;98:1096-9.
Favier CF, Vaughan EE, De Vos WM, Akkermans AD. Molecular monitoring of succession of bacterial communities in human neonates. Appl Environ Microbiol 2002;68:219-26.
[Table 1], [Table 2], [Table 3]