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Placental transfer of rubella-specific IgG in fullterm and preterm newborns
International Journal of Gynecology and Obstetrics 81 (2003) 157–162
Article
Placental transfer of rubella-specific IgG in fullterm and preterm newborns M. Doroudchia, A. Samsami Dehaghanib, K. Emadc, A. Ghaderia,* a
Department of Immunology, Medical School, Shiraz University of Medical Sciences, Shiraz, Iran b Department of Obstetrics and Gynecology, Shiraz University of Medical Sciences, Shiraz, Iran c Department of Pediatrics, Shiraz Hospital, Shiraz, Iran
Received 16 September 2002; received in revised form 9 December 2002; accepted 10 December 2002
Abstract Objectives: This study was undertaken to investigate placental transfer of anti-rubella IgG immunoglobulins in Iranian mothers. Methods: In total, 231 pregnant women and their paired infants enrolled in this study of which, 197 gave birth to fullterm and 26 gave birth to preterm infants. Rubella specific antibodies were detected by an in-house whole-virus ELISA assay in maternal and cord sera of 188 fullterm and 26 preterm infants. Results: A highly significant correlation was observed between anti-rubella IgG in newborns in total, in preterm and fullterm neonates with their paired mothers (P-valuess0.0001, 0.002, 0.0001, respectively). A borderline significant difference was observed between mean anti-rubella IgG in fullterm and preterm neonates (Ps0.04). Mean cordymaternal ratio of anti-rubella IgG was 0.83 which was surprisingly low. A significant lower anti-rubella IgG was observed in newborns born from mothers with blood group Bq than those born from mothers with blood groups Aq (Ps0.04) and Oq (Ps0.02), respectively. The same difference was observed between mean maternal anti-rubella IgG in those with blood groups Bq and Aq (Ps0.04) and those with blood groups Bq and Oq (Ps0.05). In addition, a low frequency of Bq blood group in high positive sera and a high frequency of this blood group among low positive and negative sera was detected. Conclusions: Our data suggest that the main factors that influence the infants’ rubellaspecific IgG concentration are maternal concentration of this immunoglobulin, maternal blood group, and neonatal gestational age. 䊚 2003 International Federation of Gynecology and Obstetrics. Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Iranian; Placental transfer; Preterm; Rubella
1. Introduction Placental transfer of maternal immunoglobulins is the major route of protection of infants during *Corresponding author. Tel.: q98-711-233-4589; fax: q98711-233-4589. E-mail address: [email protected] (A. Ghaderi).
and after gestation. This process has an active nature and starts during the first trimester of gestation, however, the concentration of neonatal IgG does not seem to exceed one-third of maternal IgG at this time w1,2x. It has been suggested that prematurity, low birthweight and maternal immunoglobulin concentration may interfere with the
0020-7292/03/$30.00 䊚 2003 International Federation of Gynecology and Obstetrics. Published by Elsevier Science Ireland Ltd. All rights reserved. doi: 10.1016/S0020-7292Ž02.00442-3
158
M. Doroudchi et al. / International Journal of Gynecology and Obstetrics 81 (2003) 157–162
materno-fetal transport of antibodies w3,4x. In this regard, preterm infants may be more vulnerable to viral and bacterial infections. Rubella is an endemic disease in Iran; however, the rubella vaccine is not currently part of routine immunization schedule of this country. Therefore, young mothers and their infants are solely dependent on mothers’ history of previous rubella infection. To assess the degree of rubella immunity in mothers and transfer of this immunity to newborns, anti-rubella IgG titer was determined in 219 mothers and their fullterm and preterm newborns. 2. Subjects and methods 2.1. Study population In total, 231 pregnant women who attended to the hospitals of Shiraz University of Medical Sciences in 1999 were included in this study. For 227 individuals gestational age was known of which, 26 gave birth to preterm (-37 weeks), 197 gave birth to fullterm ()37 weeks) infants and four were aborted. Sera were separated from blood samples on the same day of sampling, were aliquoted in 0.5 ml volumes and stored at y20 8C until use. Data including gestational age, previous abortions, parity, mothers’ blood group and newborns’ weights were recorded at the time of sampling.
solubilized rubella antigen in carbonate-bicarbonate buffer (pH 9.6) was coated onto ELISA plate in proper concentration. After washing with PBS containing 0.05% Tween 20, plates were blocked with PBS containing 1% gelatin for 1 h at 37 8C. Specific IgG was detected using a horseradish peroxidase (HRP)-conjugated goat anti-human IgG (Calbiochem, USA) and enzyme substrate, orthophenylene diamine dihydrochloride (DAKO, Denmark) plus H2O2 in citrate buffer (pH 5.5). The reaction was stopped by adding 50 ml of 12.5% H2SO4 solution after 10 min. The absorbances were measured at 490 nm using an ELISA spectrophotometer (Titertecplus-MS2 reader, Finland). Concentration of RV-specific IgG (IUyml) was determined from logarithmic regression plots as described previously w6x. 2.4. Statistical methods Student’s t-tests for unpaired and paired samples were used to compare the mean concentration of anti-rubella IgG between study groups. The chisquare test was used to evaluate the effect of different factors on seropositivity rate. One-way ANOVA and Levene’s test were used to compare the variances of the groups. Statistical analyses were performed using SPSS for Windows software version 6.0. 3. Results
2.2. Virus culture and antigen preparation The Takahashi strain of rubella virus was cultured in monolayers of Vero cell line grown in RPMI-1640 containing 3% fetal bovine serum at 37 8C in a 5% CO2-containing atmosphere. Fortyeight hours postinfection, the medium was replaced with RPMI-1640 medium without serum. Detergent-solubilized whole virus preparations were prepared by polyethylene glycol-6000 precipitation according to the method described by Mitchell et al. w5x. 2.3. Whole virus ELISA An in-house ELISA assay was performed as previously described w6x. Briefly, Triton X-100-
Of 231, 219 paired cordymother sera were available for measurement of anti-rubella IgG. The mean"S.D. age of mothers was 23.5"5.3 years and median age of mothers was 22 years. The mean"S.D. of newborn weights in total, among fullterm newborns and preterm neonates were 3175"432 g, 3223"418 g and 2833"382 g, respectively. Table 1 summarizes the main characteristics of the studied cases. The mean"S.D. of anti-rubella IgG in mothers sera was determined to be 104.8"46.8 IUyml and mean"S.D. of antirubella IgG in infants sera was determined to be 83.9"45.9 IUyml. The Mean cordymaternal ratio of anti-rubella IgG was 0.83"0.38 (range 0.24– 2.68) among which 165 pairs had ratios less than 1 and 54 pairs had ratios more than 1. Twelve out
M. Doroudchi et al. / International Journal of Gynecology and Obstetrics 81 (2003) 157–162 Table 1 Characteristics of patients
159
Table 2 Concentration of anti-rubella IgG in neonates of mothers according to the history of previous abortion(s) No. of cases
Gestational age )37 weeks -37 weeks Unknowna Abortionb No abortion One abortion Two abortions Three abortions Unknown Delivery Normal vaginal Cesarean section Unknown Blood group Aq Bq ABq Oq Ay By ABy Oy Unknown Parity 1 2 3 4 5 6 7 8 9 Unknown Total
Abortion 197 26 8 206 18 4 2 1 216 10 5 70 46 16 76 7 6 0 4 6 105 59 24 17 11 7 3 3 1 1 231
a
Data were not available. All cases are considered here whether the paired maternaly cord anti-rubella IgG was available or not. b
of 219 (5.5%) infants were negative for antirubella IgG and three out of 219 (1%) mothers were negative for anti-rubella IgG. Mean and median maternal IgG for the 12 negative infants were 45"24 IU and 45 IU, respectively. A highly significant correlation was observed between anti-rubella IgG in newborns and their paired mothers (Ps0.0001). There was also a significant correlation between anti-rubella IgG in newborns and their paired mothers in preterm (Ps
0
1
2
Mean"S.D. of anti90.7"53.1 61.9"38.9* 85.5"35.8 rubella IgG (IUyml) No. of cases 193 18 4 *P-0.05.
0.002) and fullterm (Ps0.0001) neonates. The mean"S.D. of anti-rubella IgG in fullterm and preterm neonates were 87.5"47.8 and 70.8"32.5 IUyml, respectively. Levene’s test for equality of variances revealed a borderline difference between the two groups (Ps0.054) and the t-test for twosamples with unequal variances revealed a significant difference between fullterm and preterm neonates (Ps0.04). No correlation was observed between anti-rubella IgG in neonates and type of delivery, parity, and neonates’ weights. However, the concentration of anti-rubella IgG in neonates of mothers with one abortion was lower than those without abortion and this difference was found to be significant (Ps0.03) (Table 2). Analysis of variance revealed a significant difference between mean anti-rubella IgG in newborns of mothers with blood group Bq in comparison to newborns born from mothers with blood groups Aq and Oq (P-0.05). Table 3 illustrates the mean"S.D. of anti-rubella IgG in newborns born from mothers with different blood groups. Performing the t-test between these groups confirmed a significant lower anti-rubella IgG concentration in newborns born from mothers with Table 3 Concentration of anti-rubella IgG in neonates of mothers according to maternal blood group Blood group
Mean"S.D. of antirubella IgG (IU) No. of cases *P-0.05.
Bq
Aq
Oq
65.3"51.6*
89.1"45.8
81.8"38.8
42
65
68
M. Doroudchi et al. / International Journal of Gynecology and Obstetrics 81 (2003) 157–162
160
Table 4 Blood groups frequencies in high positive ()100 IUyml), low positive ()20 IUyml, -50 IUyml) and negative (-20 IUyml) infants’ sera Blood group
High positivea ()100 IUyml) Low positive ()20, -50 IUyml) Negative (-20 IUyml) a
Aq (%)
Bq (%)
ABq (%)
Oq (%)
Ay (%)
By (%)
Oy (%)
23y76 (30.26%) 10y48 (20.83%) 3y13 (23.08%)
9y76 (11.84%) 16y48 (33.33%) 4y13 (30.77%)
4y76 (5.26%) 3y48 (6.25%) 1y13 (7.69%)
28y76 (36.85%) 15y48 (31.25%) 4y13 (30.77%)
2y76 (2.63%) 2y48 (4.17%) 1y13 (7.69%)
3y76 (3.95%) 2y48 (4.17%) 0 (0%)
3y76 (3.95%) 0 (0%) 0 (0%)
Four cases were missing.
blood group Bq than those born from mothers with blood groups Aq (Ps0.04) and Oq (Ps 0.02), respectively. The same difference was observed between mean maternal anti-rubella IgG in those with blood groups Bq and Aq (Ps 0.04) and those with blood groups Bq and Oq (Ps0.05). Comparison of blood groups frequencies between high positive ()100 IUyml), low positive ()20 IUyml, -50 IUyml) and negative (-20 IUyml) infants’ sera (Table 4) revealed a low frequency of Bq blood group in high positive sera and a high frequency of this blood group among low positive and negative sera. In this regard, the ratio of high positive Aq sera to high positive Bq sera was 23y9 (2.6) whereas this ratio in low positive and negative sera was found to be 10y16 (0.63) and 3y4 (0.75), respectively. The ratio of high positive Oq sera to high positive Bq sera was 28y9 (3.1), whereas this ratio in low positive and negative sera was found to be 15y16 (0.94) and 4y4 (1.0), respectively. 4. Discussion Materno-fetal transfer of antibodies has always been a subject of controversy. Cordymaternal ratio of specific antibodies for some infective agents has been reported to be greater than 1 w7–9x. However, for some other infections and in some ethnic groups this ratio is less than or equal to 1 w10,11x. Moreover, a lower transfer of maternal specific IgG has been reported in preterm infants in different studies w10,12,13x. We detected a highly significant correlation between anti-rubella
IgG in newborns and their mothers (Ps0.001). This correlation was stronger in fullterm (Ps 0.0001) than preterm (Ps0.002) infants. In a study on placental transfer of maternal rubella antibodies, Linder et al. detected a significant difference in geometric mean titers of neutralizing antibodies between full-term and preterm infants w13x. Compared to the Linder group, we detected a less significant (Ps0.04) difference between rubella antibodies in preterm and full-term infants and this might be related to the higher birthweight of preterm infants in our study compared to their study. A significant difference between mean antirubella IgG in neonates of mothers with one abortion compared to those without abortion was observed. Mean age, parity, blood group distribution and maternal IgG in mothers without abortion and those with one and two abortions were compared. There was no major difference in the age, parity or blood group, however, the maternal rubella IgG correlated well with the infant rubella IgG in the three groups. Therefore, it seems more likely that the observed difference was related to the maternal rubella IgG concentration. In the present study mean cordymaternal ratio of rubella antibodies was found to be 0.83 which is less than 1. This observation is in agreement with the reported low level of antibody transfer in African women which has been suggested to be related to the rate of endemicity of infectious diseases w11x. In a study in a West African population, Okoko et al. found an association between placental malarial infection and reduction in the
M. Doroudchi et al. / International Journal of Gynecology and Obstetrics 81 (2003) 157–162
transfer of measles antibodies w12x. The malarial infection of placenta in our study population was not investigated, however, the rate of malaria infection in Fars, Iran is much lower than Africa. The incidence of malaria infection in Fars province in the sampling period has been 25 per 100 000 w14x. This rate is not comparable with West Africa in which the average predicted prevalence for more than 50% of its population is 30–70% w15x. Therefore, the basis of this low cordymaternal ratio of rubella antibodies in our cases remains to be elucidated. A significant lower anti-rubella IgG concentration was observed in newborns of mothers with blood group Bq compared with those born from mothers with blood groups Aq (Ps0.04) and Oq (Ps0.02). Moreover, a low frequency of Bq blood group in high positive sera and a high frequency of this blood group among low positive and negative sera were observed. To our best knowledge, there is no report on the association of ABO blood group antigens and rubella virus infection or immunity. However, many investigators have reported the association of different diseases and blood group antigens w16–18x. This association in some cases has been related to the existence of bacterial, viral or parasite receptors w19–23x, however, in some instances it is not associated with the binding of microorganisms w24x, or there is no direct rational for the reported associations w25–27x. Our data need to be confirmed in other investigations and the involved mechanisms remains to be clarified. In this study we investigated the placental transfer of rubella specific antibodies in Iranian mothers to find out how much our infants can rely on maternal antibodies for immunity to this virus. It was found that the mean cordymaternal ratio is less than 1 and the factors that affect infants rubella-specific IgG are maternal concentration of this immunoglobulin, maternal blood group, and neonatal gestational age. Acknowledgments This work was financially supported by the Department of Research and Technology, Ministry of Health and Medical Education, Tehran, Iran and
161
the Shiraz University of Medical Sciences, Shiraz, Iran. References w1x Kohler PF, Farr RS. Elevation of cord over maternal IgG immunoglobulin: evidence for an active placental IgG transport. Nature 1966;210:1070 –1071. w2x Jauniaux E, Jurkovic D, Gulbis B, Liesnard C, Lees C, Campbell S. Materno-fetal immunoglobulin transfer and passive immunity during the first trimester of human pregnancy. Hum Reprod 1995;10:3293 –3297. w3x Wesumperuma HL, Perera AJ, Pharoah PO, Hart CA. The influence of prematurity and low birthweight on transplacental antibody transfer in Sri Lanka. Ann Trop Med Parasitol 1999;93:169 –177. w4x Hood N, Chan MC, Maxwell SM, Familusi JB, Hart CA. Placental transfer of tetanus toxoid antibodies in Nigerian mothers. Ann Trop Pediatr 1994;14:179 –182. w5x Mitchell LA, Zhang T, Ho M, Decarie D, Tingle AJ, Zrein M, et al. Characterization of rubella virus-specific antibody responses by using a new synthetic-peptidebased enzyme linked immunosorbent assay. J Clin Microbiol 1992;30:1841 –1847. w6x Doroudchi M, Samsami Dehaghani A, Emad K, Ghaderi A. Seroepidemiological survey of rubella immunity among three populations in Shiraz, Islamic Republic of Iran. East Mediterr Health J 2001;7:128 –138. w7x Toivanen P, Mantyjarvi R, Hirvonen T. Maternal antibodies in human foetal sera at different stages of gestation. Immunology 1968;15:395 –403. w8x Goncalves G, Cutts FT, Hills M, Rebelo-Andrade H, Trigo FA, Barros H. Transplacental transfer of measles and total IgG. Epidemiol Infect 1999;122:273 –279. w9x Sato H, Albrecht P, Reynolds DW, Stagno S, Ennis FA. Transfer of measles, mumps, and rubella antibodies from mother to infant. Its effect on measles, mumps, and rubella immunization. Am J Dis Child 1979;133:1240 –1243. w10x Nagao AT, Costa-Carvalho BT, Arslanian C, Sole D, Naspitz C, Carneiro-Sampaio MM. Placental transfer of IgG antibodies against Haemophilus influenzae type b capsular polysaccharide in Brazilian term and preterm newborns. J Trop Pediatr 1999;45:171 –173. w11x Gendrel D, Richard-Lenoble D, Blot P, Fribourg-Blanc A. Transfer of measles immunoglobulins and antibodies from mother to child in Africa and Europe. Presse Med 1988;17:1633 –1636. w12x Okoko BJ, Wesuperuma LH, Ota MO, Banya WA, Pinder M, Gomez FS, et al. Influence of placental malaria infection and maternal hypergammaglobulinaemia on materno-foetal transfer of measles and tetanus antibodies in a rural west African population. J Health Popul Nutr 2001;19:59 –65. w13x Linder N, Sirota L, Aboudy Y, German B, Lifshits T, Barnea BS, et al. Placental transfer of maternal rubella
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M. Doroudchi et al. / International Journal of Gynecology and Obstetrics 81 (2003) 157–162 antibodies to full-term and preterm infants. Infection 1999;27:203 –207. Kadivar MR, Gharachahi AM, Kazerooni PA, Ghahramani I, Keshtgari M, Bakhtiari H, et al. A guide for prevention of communicable diseases. Fifth annual report of communicable diseases. Disease Control Unit. Shiraz University Press, 2000. Kleinschmidt I, Omumbo J, Briet O, van de Giesen N, Sogoba N, Mensah NK, et al. An empirical malaria distribution map for West Africa. Trop Med Int Health 2001;6:779 –786. Ali S, Niang MA, N’doye I, Critchlow CW, Hawes SE, Hill AV, et al. Secretor polymorphism and human immunodeficiency virus infection in Senegalese women. J Infect Dis 2000;181:737 –739. Balajee SA, Menon T, Ranganathan S. ABO blood groups in relation to the infection rate of dermatophytosis. Mycoses 1996;39:475 –478. Kauffmann F, Frette C, Pham QT, Nafissi S, Bertrand JP, Oriol R. Associations of blood group-related antigens to FEV1, wheezing, and asthma. Am J Respir Crit Care Med 1996;153:76 –82. Barnwell JW, Nichols ME, Rubinstein P. In vitro evaluation of the role of the Duffy blood group in erythrocyte invasion by Plasmodium vivax. J Exp Med 1989;169:1795 –1802. Brown KE, Hibbs JR, Gallinella G, Anderson SM, Lehman ED, McCarthy P, et al. Resistance to parvovirus
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B19 infection due to lack of virus receptor (erythrocyte P antigen). N Engl J Med 1994;330:1192 –1196. Nishimura H, Sugawara K, Kitame F, Nakamura K. Attachment of influenza C virus to human erythrocytes. J Gen Virol 1988;69:2545 –2553. Boren T, Falk P, Roth KA, Larson G, Normark S. Attachment of Helicobacter pylori to human gastric epithelium mediated by blood group antigens. Science 1993;262:1892 –1895. Appelmelk BJ, Monteiro MA, Martin SL, Moran AP, Vandenbroucke-Grauls CM. Why Helicobacter pylori has Lewis antigens. Trends Microbiol 2000;8:565 –570. Blackwell CC, Dundas S, James VS, Mackenzie DA, Braun JM, Alkout AM, et al. Blood group and susceptibility to disease caused by Escherichia coli O157. J Infect Dis 2002;185:393 –396. Glass RI, Holmgren J, Haley CE, Khan MR, Svennerholm AM, Stoll BJ, et al. Predisposition for cholera of individuals with O blood group; possible evolutionary significance. Am J Epidemiol 1985;121:791 –796. Prata A. Influence of the host related factors in the development of the hepatosplenic form of Schistosomiasis mansoni. Mem Inst Oswaldo Cruz 1992;87:39 –44. Darbinian VZh, Nersisian VM, Musazlian NO, Maretirosian IG, Akopian LP. Blood group factor and HLArelated epilepsy among the Armenian population. Zh Nevropatol Psikhiatr S S Korsakova 1990;90:85 –87.
Article
Placental transfer of rubella-specific IgG in fullterm and preterm newborns M. Doroudchia, A. Samsami Dehaghanib, K. Emadc, A. Ghaderia,* a
Department of Immunology, Medical School, Shiraz University of Medical Sciences, Shiraz, Iran b Department of Obstetrics and Gynecology, Shiraz University of Medical Sciences, Shiraz, Iran c Department of Pediatrics, Shiraz Hospital, Shiraz, Iran
Received 16 September 2002; received in revised form 9 December 2002; accepted 10 December 2002
Abstract Objectives: This study was undertaken to investigate placental transfer of anti-rubella IgG immunoglobulins in Iranian mothers. Methods: In total, 231 pregnant women and their paired infants enrolled in this study of which, 197 gave birth to fullterm and 26 gave birth to preterm infants. Rubella specific antibodies were detected by an in-house whole-virus ELISA assay in maternal and cord sera of 188 fullterm and 26 preterm infants. Results: A highly significant correlation was observed between anti-rubella IgG in newborns in total, in preterm and fullterm neonates with their paired mothers (P-valuess0.0001, 0.002, 0.0001, respectively). A borderline significant difference was observed between mean anti-rubella IgG in fullterm and preterm neonates (Ps0.04). Mean cordymaternal ratio of anti-rubella IgG was 0.83 which was surprisingly low. A significant lower anti-rubella IgG was observed in newborns born from mothers with blood group Bq than those born from mothers with blood groups Aq (Ps0.04) and Oq (Ps0.02), respectively. The same difference was observed between mean maternal anti-rubella IgG in those with blood groups Bq and Aq (Ps0.04) and those with blood groups Bq and Oq (Ps0.05). In addition, a low frequency of Bq blood group in high positive sera and a high frequency of this blood group among low positive and negative sera was detected. Conclusions: Our data suggest that the main factors that influence the infants’ rubellaspecific IgG concentration are maternal concentration of this immunoglobulin, maternal blood group, and neonatal gestational age. 䊚 2003 International Federation of Gynecology and Obstetrics. Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Iranian; Placental transfer; Preterm; Rubella
1. Introduction Placental transfer of maternal immunoglobulins is the major route of protection of infants during *Corresponding author. Tel.: q98-711-233-4589; fax: q98711-233-4589. E-mail address: [email protected] (A. Ghaderi).
and after gestation. This process has an active nature and starts during the first trimester of gestation, however, the concentration of neonatal IgG does not seem to exceed one-third of maternal IgG at this time w1,2x. It has been suggested that prematurity, low birthweight and maternal immunoglobulin concentration may interfere with the
0020-7292/03/$30.00 䊚 2003 International Federation of Gynecology and Obstetrics. Published by Elsevier Science Ireland Ltd. All rights reserved. doi: 10.1016/S0020-7292Ž02.00442-3
158
M. Doroudchi et al. / International Journal of Gynecology and Obstetrics 81 (2003) 157–162
materno-fetal transport of antibodies w3,4x. In this regard, preterm infants may be more vulnerable to viral and bacterial infections. Rubella is an endemic disease in Iran; however, the rubella vaccine is not currently part of routine immunization schedule of this country. Therefore, young mothers and their infants are solely dependent on mothers’ history of previous rubella infection. To assess the degree of rubella immunity in mothers and transfer of this immunity to newborns, anti-rubella IgG titer was determined in 219 mothers and their fullterm and preterm newborns. 2. Subjects and methods 2.1. Study population In total, 231 pregnant women who attended to the hospitals of Shiraz University of Medical Sciences in 1999 were included in this study. For 227 individuals gestational age was known of which, 26 gave birth to preterm (-37 weeks), 197 gave birth to fullterm ()37 weeks) infants and four were aborted. Sera were separated from blood samples on the same day of sampling, were aliquoted in 0.5 ml volumes and stored at y20 8C until use. Data including gestational age, previous abortions, parity, mothers’ blood group and newborns’ weights were recorded at the time of sampling.
solubilized rubella antigen in carbonate-bicarbonate buffer (pH 9.6) was coated onto ELISA plate in proper concentration. After washing with PBS containing 0.05% Tween 20, plates were blocked with PBS containing 1% gelatin for 1 h at 37 8C. Specific IgG was detected using a horseradish peroxidase (HRP)-conjugated goat anti-human IgG (Calbiochem, USA) and enzyme substrate, orthophenylene diamine dihydrochloride (DAKO, Denmark) plus H2O2 in citrate buffer (pH 5.5). The reaction was stopped by adding 50 ml of 12.5% H2SO4 solution after 10 min. The absorbances were measured at 490 nm using an ELISA spectrophotometer (Titertecplus-MS2 reader, Finland). Concentration of RV-specific IgG (IUyml) was determined from logarithmic regression plots as described previously w6x. 2.4. Statistical methods Student’s t-tests for unpaired and paired samples were used to compare the mean concentration of anti-rubella IgG between study groups. The chisquare test was used to evaluate the effect of different factors on seropositivity rate. One-way ANOVA and Levene’s test were used to compare the variances of the groups. Statistical analyses were performed using SPSS for Windows software version 6.0. 3. Results
2.2. Virus culture and antigen preparation The Takahashi strain of rubella virus was cultured in monolayers of Vero cell line grown in RPMI-1640 containing 3% fetal bovine serum at 37 8C in a 5% CO2-containing atmosphere. Fortyeight hours postinfection, the medium was replaced with RPMI-1640 medium without serum. Detergent-solubilized whole virus preparations were prepared by polyethylene glycol-6000 precipitation according to the method described by Mitchell et al. w5x. 2.3. Whole virus ELISA An in-house ELISA assay was performed as previously described w6x. Briefly, Triton X-100-
Of 231, 219 paired cordymother sera were available for measurement of anti-rubella IgG. The mean"S.D. age of mothers was 23.5"5.3 years and median age of mothers was 22 years. The mean"S.D. of newborn weights in total, among fullterm newborns and preterm neonates were 3175"432 g, 3223"418 g and 2833"382 g, respectively. Table 1 summarizes the main characteristics of the studied cases. The mean"S.D. of anti-rubella IgG in mothers sera was determined to be 104.8"46.8 IUyml and mean"S.D. of antirubella IgG in infants sera was determined to be 83.9"45.9 IUyml. The Mean cordymaternal ratio of anti-rubella IgG was 0.83"0.38 (range 0.24– 2.68) among which 165 pairs had ratios less than 1 and 54 pairs had ratios more than 1. Twelve out
M. Doroudchi et al. / International Journal of Gynecology and Obstetrics 81 (2003) 157–162 Table 1 Characteristics of patients
159
Table 2 Concentration of anti-rubella IgG in neonates of mothers according to the history of previous abortion(s) No. of cases
Gestational age )37 weeks -37 weeks Unknowna Abortionb No abortion One abortion Two abortions Three abortions Unknown Delivery Normal vaginal Cesarean section Unknown Blood group Aq Bq ABq Oq Ay By ABy Oy Unknown Parity 1 2 3 4 5 6 7 8 9 Unknown Total
Abortion 197 26 8 206 18 4 2 1 216 10 5 70 46 16 76 7 6 0 4 6 105 59 24 17 11 7 3 3 1 1 231
a
Data were not available. All cases are considered here whether the paired maternaly cord anti-rubella IgG was available or not. b
of 219 (5.5%) infants were negative for antirubella IgG and three out of 219 (1%) mothers were negative for anti-rubella IgG. Mean and median maternal IgG for the 12 negative infants were 45"24 IU and 45 IU, respectively. A highly significant correlation was observed between anti-rubella IgG in newborns and their paired mothers (Ps0.0001). There was also a significant correlation between anti-rubella IgG in newborns and their paired mothers in preterm (Ps
0
1
2
Mean"S.D. of anti90.7"53.1 61.9"38.9* 85.5"35.8 rubella IgG (IUyml) No. of cases 193 18 4 *P-0.05.
0.002) and fullterm (Ps0.0001) neonates. The mean"S.D. of anti-rubella IgG in fullterm and preterm neonates were 87.5"47.8 and 70.8"32.5 IUyml, respectively. Levene’s test for equality of variances revealed a borderline difference between the two groups (Ps0.054) and the t-test for twosamples with unequal variances revealed a significant difference between fullterm and preterm neonates (Ps0.04). No correlation was observed between anti-rubella IgG in neonates and type of delivery, parity, and neonates’ weights. However, the concentration of anti-rubella IgG in neonates of mothers with one abortion was lower than those without abortion and this difference was found to be significant (Ps0.03) (Table 2). Analysis of variance revealed a significant difference between mean anti-rubella IgG in newborns of mothers with blood group Bq in comparison to newborns born from mothers with blood groups Aq and Oq (P-0.05). Table 3 illustrates the mean"S.D. of anti-rubella IgG in newborns born from mothers with different blood groups. Performing the t-test between these groups confirmed a significant lower anti-rubella IgG concentration in newborns born from mothers with Table 3 Concentration of anti-rubella IgG in neonates of mothers according to maternal blood group Blood group
Mean"S.D. of antirubella IgG (IU) No. of cases *P-0.05.
Bq
Aq
Oq
65.3"51.6*
89.1"45.8
81.8"38.8
42
65
68
M. Doroudchi et al. / International Journal of Gynecology and Obstetrics 81 (2003) 157–162
160
Table 4 Blood groups frequencies in high positive ()100 IUyml), low positive ()20 IUyml, -50 IUyml) and negative (-20 IUyml) infants’ sera Blood group
High positivea ()100 IUyml) Low positive ()20, -50 IUyml) Negative (-20 IUyml) a
Aq (%)
Bq (%)
ABq (%)
Oq (%)
Ay (%)
By (%)
Oy (%)
23y76 (30.26%) 10y48 (20.83%) 3y13 (23.08%)
9y76 (11.84%) 16y48 (33.33%) 4y13 (30.77%)
4y76 (5.26%) 3y48 (6.25%) 1y13 (7.69%)
28y76 (36.85%) 15y48 (31.25%) 4y13 (30.77%)
2y76 (2.63%) 2y48 (4.17%) 1y13 (7.69%)
3y76 (3.95%) 2y48 (4.17%) 0 (0%)
3y76 (3.95%) 0 (0%) 0 (0%)
Four cases were missing.
blood group Bq than those born from mothers with blood groups Aq (Ps0.04) and Oq (Ps 0.02), respectively. The same difference was observed between mean maternal anti-rubella IgG in those with blood groups Bq and Aq (Ps 0.04) and those with blood groups Bq and Oq (Ps0.05). Comparison of blood groups frequencies between high positive ()100 IUyml), low positive ()20 IUyml, -50 IUyml) and negative (-20 IUyml) infants’ sera (Table 4) revealed a low frequency of Bq blood group in high positive sera and a high frequency of this blood group among low positive and negative sera. In this regard, the ratio of high positive Aq sera to high positive Bq sera was 23y9 (2.6) whereas this ratio in low positive and negative sera was found to be 10y16 (0.63) and 3y4 (0.75), respectively. The ratio of high positive Oq sera to high positive Bq sera was 28y9 (3.1), whereas this ratio in low positive and negative sera was found to be 15y16 (0.94) and 4y4 (1.0), respectively. 4. Discussion Materno-fetal transfer of antibodies has always been a subject of controversy. Cordymaternal ratio of specific antibodies for some infective agents has been reported to be greater than 1 w7–9x. However, for some other infections and in some ethnic groups this ratio is less than or equal to 1 w10,11x. Moreover, a lower transfer of maternal specific IgG has been reported in preterm infants in different studies w10,12,13x. We detected a highly significant correlation between anti-rubella
IgG in newborns and their mothers (Ps0.001). This correlation was stronger in fullterm (Ps 0.0001) than preterm (Ps0.002) infants. In a study on placental transfer of maternal rubella antibodies, Linder et al. detected a significant difference in geometric mean titers of neutralizing antibodies between full-term and preterm infants w13x. Compared to the Linder group, we detected a less significant (Ps0.04) difference between rubella antibodies in preterm and full-term infants and this might be related to the higher birthweight of preterm infants in our study compared to their study. A significant difference between mean antirubella IgG in neonates of mothers with one abortion compared to those without abortion was observed. Mean age, parity, blood group distribution and maternal IgG in mothers without abortion and those with one and two abortions were compared. There was no major difference in the age, parity or blood group, however, the maternal rubella IgG correlated well with the infant rubella IgG in the three groups. Therefore, it seems more likely that the observed difference was related to the maternal rubella IgG concentration. In the present study mean cordymaternal ratio of rubella antibodies was found to be 0.83 which is less than 1. This observation is in agreement with the reported low level of antibody transfer in African women which has been suggested to be related to the rate of endemicity of infectious diseases w11x. In a study in a West African population, Okoko et al. found an association between placental malarial infection and reduction in the
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transfer of measles antibodies w12x. The malarial infection of placenta in our study population was not investigated, however, the rate of malaria infection in Fars, Iran is much lower than Africa. The incidence of malaria infection in Fars province in the sampling period has been 25 per 100 000 w14x. This rate is not comparable with West Africa in which the average predicted prevalence for more than 50% of its population is 30–70% w15x. Therefore, the basis of this low cordymaternal ratio of rubella antibodies in our cases remains to be elucidated. A significant lower anti-rubella IgG concentration was observed in newborns of mothers with blood group Bq compared with those born from mothers with blood groups Aq (Ps0.04) and Oq (Ps0.02). Moreover, a low frequency of Bq blood group in high positive sera and a high frequency of this blood group among low positive and negative sera were observed. To our best knowledge, there is no report on the association of ABO blood group antigens and rubella virus infection or immunity. However, many investigators have reported the association of different diseases and blood group antigens w16–18x. This association in some cases has been related to the existence of bacterial, viral or parasite receptors w19–23x, however, in some instances it is not associated with the binding of microorganisms w24x, or there is no direct rational for the reported associations w25–27x. Our data need to be confirmed in other investigations and the involved mechanisms remains to be clarified. In this study we investigated the placental transfer of rubella specific antibodies in Iranian mothers to find out how much our infants can rely on maternal antibodies for immunity to this virus. It was found that the mean cordymaternal ratio is less than 1 and the factors that affect infants rubella-specific IgG are maternal concentration of this immunoglobulin, maternal blood group, and neonatal gestational age. Acknowledgments This work was financially supported by the Department of Research and Technology, Ministry of Health and Medical Education, Tehran, Iran and
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