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H. Schonewille et al.
paternal HLA antigens (NIPAs) significantly more often than against NIMAs.8 However, a protective effect of HLA NIMAs exposure on later renal and stem cell transplant outcome was not confirmed in all further studies.9-11
Studies in mice models showed that a maximum immune tolerance to NIMA is obtained when in utero expo- sure to NIMA is followed by breastfeeding (BF).12,13 One study in humans showed a superior graft survival of mater- nal and sibling renal transplants when the recipient was breastfed.14 Other studies in humans also showed that the duration of BF was associated with autoimmune diseases later in life.15,16
Therefore, the controversial results on the role of expo- sure to NIMAs on later immunity when challenged by pregnancy, transfusion or transplantation, may - among other factors - be due to different BF habits. Breast milk con- tains soluble molecules such as HLA, immunoglobulins and extracellular vesicles, as well as viable cells, the latter already observed by Antoni van Leeuwenhoek in the 17th century.17–19
Despite ante- and postnatal anti-D immunoprophylaxis since 1998, Rhesus D antibodies are still the most frequent cause of severe HDFN. We previously showed that, yearly, about 15 pregnancies complicated by anti-D, four by anti-K and one by anti-c required intra-uterine transfusions (IUT).20 RhD immunoprophylaxis however hampers investigation of the effect of D NIMA exposure in utero and by BF on the anti-D response towards a D-positive child.
Severe HDFN is nowadays successfully treated with IUT. Unfortunately, such IUTs expose the mother to RBC anti- gens of the fetus and IUT donors, often leading to the induction of additional RBC antibodies.21
In the present study we investigated the hypothesis, that BF may affect immunity against non-inherited maternal red blood cell antigens, when encountered later in life through pregnancy or by transfusion, in a cohort of mothers whose fetuses were treated with IUT because of HDFN.
Methods
Study design
A cohort study of 125 grandmother-mother-child combinations, participating in the LOTUS (LOng Term follow Up after intrauter- ine transfusionS) study. In short, all women with children who were treated with IUT for HDFN from 1987-2008 were eligible. Details of the population and the methods adopted have been published previously22 (see Online Supplementary Appendix for details). All participating women were asked to invite their moth- ers to participate. Grandmothers were asked to complete a ques- tionnaire on duration of breastfeeding (regardless of exclusivity). The study was approved by the ethics committee of the Leiden University Medical Center (P08.080).
Data collection and intra-uterine transfusion policy
All participants and IUT donors were typed for relevant RBC antigens (see Online Supplementary Appendix for details). The mothers were screened for RBC antibodies as previously described.23 Maternal transfusion history including date, number and donor of each IUT and number of pregnancies were collected. Over the years the transfusion policy changed with increasing degree of extended RBC antigen matching between mother and IUT donor and also procedural technique (see Online Supplementary Appendix for details).20,24
The following was determined:
1. Identification of non-D RBC antigens (C, c, E, e, K, Fya, Fyb, Jka, Jkb, M, S and s) expressed by the child or IUT donor(s) but not by the mother i.e., mismatched antigens.
2. The presence or absence of maternal antibodies against each of these mismatched antigens.
3. For each mismatched antigen, whether the grandmother car- ried the antigen as a NIMA.
Statistical analyses
Univariate logistic regression was used to calculate odds ratio (OR) and 95% confidence intervals (CIs). The presence of antibod- ies was used as the dependent variable. BF duration was analysed categorized as 0, 1, 2, 3, 4-6 and >6 months and in a sensitivity analysis dichotomized (≤ or > 0, 1, 2, 3, 4 and 6 months). Adjusted odds ratio (aOR) was calculated in the final multivariate logistic regression model. The following variables were considered poten- tial confounders for RBC antibodies: ABO compatibility between mother and child, maternal HLA-DRB1*15 genotype,25 number of IUTs (categorized as 1, 2, 3, 4 and >4), number of pregnancies (cat- egorized as ≤2, 3 and >3), year of IUT (categorized in 5-year- blocks; 1988-93, 1994-98, 1999-03 and 2004-08) and RBC antigen immunogenicity (high: C, c, E, e and K and low: Fya, Fyb, Jka, Jkb, M, S and s antigens).
The associations between the duration of BF and the induction of antibodies were adjusted for potential confounders (i.e., P-val- ues <0.2 in univariate analyses). To test for effect modification, two interaction terms (NIMA by months of BF and NIMA by anti- gen immunogenicity) were added to the model. The variables NIMA, months of BF, and the interaction term (NIMA by months of BF) were forced into the model.
All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS Inc, Chicago, IL, USA). A 95% CI not overlapping the null value 1.00 for OR was regarded statistically significant.
Results
Study population
A total of 125 grandmother-mother-child pairs partici- pated in the study. The mothers (median age at follow up, 40 years; range 24-52) gave birth to 399 children of which 143 were treated for HDFN with a total of 405 IUTs. The median birth order of the first IUT-treated child was three; there were three cases with an affected first child. The antibodies primarily responsible for HDFN were anti-D (n=93), anti-K (n=19), anti-c (n=11) and anti-Cw and -Kpa, one each (see Table 1 for additional characteristics).
A total of 549 RBC antigens - other than D - were not expressed by the mothers. For 171 of these antigens, there was no exposure by child or donor, and due to incomplete child and/or donor RBC typing, exposure was not known for 48 antigens. Consequently, analyses were restricted to the remaining 330 antigen exposures (median 3; range 1-5 per mother), of which 123 were NIMA re-exposures and 207 not previously exposed to as NIMA. These resulted in 158 antibodies, 54 (44%) against NIMA of which six antibodies caused the HDFN, and 104 (50%) against antigens not exposed to as NIMA, of which 26 caused the HDFN (Figure 2).
Breastfeeding duration and antibody responses
The median period that the 125 mothers were breastfed for was two months (range 0-12); 45 mothers were not
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