A successful pregnancy requires an optimal interplay between the maternal immune system and the semi-allogeneic fetus. Breakdown of the maternal immune tolerance may result in fetal rejection. Thus, the maternal tolerance toward the fetus has to be maintained both locally at the fetal-maternal interface and systemically, since bidirectional trafficking of cells and soluble HLA between the mother and the fetus takes place (1–3). As early as 4 weeks of gestation, semi-allogeneic fetal DNA can be detected in the maternal circulation (2) and the presence of this fetal microchimerism can persist for decades after delivery (4).

Inherited paternal HLA antigens (IPA) of fetal origin are able to prime maternal immune responses at the fetal-maternal interface as well as in the maternal circulation (5, 6). These immune responses may lead to the production of child-specific HLA antibodies (7–9). The maternal production child-specific HLA antibodies of the IgG isotype requires interaction between activated B-cells and primed T-helper cells. First, B-cell activation occurs upon antigenic uptake of IPA by the B-cell receptor (10). Subsequently, upon T-cell recognition of degraded IPA presented on HLA class-II molecules, T-helper cells provide co-stimulation via CD40–CD40L interaction and secrete cytokines (10, 11). These signals drive proliferation and differentiation of naive B cells into memory cells and plasma cells and induce IgM to IgG isotype switching (10, 11). Thus, the maternal production of child-specific IgG HLA antibodies requires the activation of B cells by T-helper cells where both B cells and T-helper cells respond to the same antigen, a phenomenon called linked recognition (12).

Despite abundant allogeneic fetal contact, only 10–40% of the mothers develop child-specific HLA antibodies (8, 9). The exact mechanism behind HLA antibody formation is currently unclear. Increasing gravidity (8, 13) and the fetal and maternal HLA phenotype combination (14) may be important determinants in the immunogenicity toward IPA. We previously showed that HLA antibody formation during a successful pregnancy without prior miscarriages is related to the number of predicted HLA-derived T-helper epitopes as determined by the PIRCHE-II model (Predicted Indirectly ReCognizable HLA Epitopes) (15). This model identifies the number of mismatched HLA-derived peptides that can be presented by HLA class-II molecules, designated as PIRCHE-II (16).

HLA antibodies play an important role in organ transplantation; the presence of pre-transplantation donor-specific HLA antibodies is associated with antibody-mediated rejection and an impaired graft survival (17–20). Therefore, more insight into the immunogenicity of mismatched HLA after pregnancy may have has implications in the transplantation field. In contrast to transplantation, the effect of IPA-specific HLA antibodies on the fetus is presumably rather harmless, as the prevalence of IPA-specific HLA antibodies is relatively high in normal pregnancies. However, both beneficial and harmful effects of HLA antibodies on pregnancy outcome have been described, indicating that the role of IPA-specific HLA antibodies on pregnancy outcome is debatable (21). Most of these studies focused on HLA antibody formation in (recurrent) miscarriage(s), whereas studies about the effect of a prior miscarriage on HLA antibody formation during a subsequent successful pregnancy are limited. In the present study, we investigate for the first time the effect of a single previous miscarriage on HLA antibody formation during a subsequent first successful pregnancy.

Maternal immune responses can be formed against IPA of the fetus during pregnancy, leading to IPA-specific antibodies and T cells (5, 7). Despite the clinical relevance of HLA-specific antibodies in transplantation outcome, the clinical relevance of paternal HLA-specific antibodies in pregnancy outcome is currently unclear (21). The present study was initiated to investigate the effect of a first pregnancy and a first miscarriage on HLA antibody formation during a subsequent first successful pregnancy.

In our cohort of 287 mother–child pairs, we investigated HLA immunization against mismatched IPA of the most recent child in secundigravidae with or without a single prior miscarriage. The percentage of immunogenic HLA was significantly lower in secundigravidae with a prior miscarriage compared to secundigravidae without a prior miscarriage. Several studies have shown that the prevalence of HLA antibodies increases with the number of successful pregnancies (9, 13). Our data show that the relation between increasing gravidity and the prevalence of HLA antibody formation is absent in secundigravidae with a prior miscarriage, indicating that a previous miscarriage behaves differently when compared to a previous successful pregnancy.

Our results suggest that a prior miscarriage has a different immunological impact on a subsequent successful pregnancy than a prior successful pregnancy. The lower immunogenicity observed in secundigravidae with a prior miscarriage cannot be explained by altered numbers of mismatched eplets and PIRCHE-II (Figure 4). Alternatively, the lower percentage of immunogenic antigens among secundigravidae with prior miscarriage in our population may result from tolerizing effects of a first short allogeneic interaction during the prior miscarriage. These tolerizing effects may be caused by fetal microchimerism, as the increased occurrence and long-term persistence of fetal microchimerism in the maternal system after or during fetal loss has been described previously (24, 25). Alternatively, the low percentage of immunogenic antigens among secundigravidae with a single prior miscarriage might also be explained by natural selection of a particular HLA genotype during a subsequent pregnancy. The chance of inheriting an alternative paternal haplotype during a subsequent successful pregnancy compared to the previous miscarriage is 50%. However, a previous miscarriage may further stimulate HLA genotype diversity by putting additive selective pressure on a subsequent pregnancy. Either directly or via modulating the maternal immune system, the HLA genotype of the miscarried fetus may discriminate against that particular HLA genotype during or shortly after conception (26). If this hypothesis is correct, a previous miscarried fetus facilitates the selection of the HLA genotype of a subsequent child. Such a selection may be achieved via a maternal immune response directed against the HLA genotype that is similar to the HLA genotype of the miscarriage itself, resulting in either selective abortion of the fetus or via a female alloimmune response against certain HLA genotypes present in seminal fluid, as seminal plasma contains soluble HLA (27) and spermatozoa also express both HLA class-I and class-II (28). However, currently no data are available to support such a natural selection of a particular HLA genotype. To challenge this hypothesis, the HLA typing of the current child should be compared with the HLA typing of the previous miscarried fetus. HLA typing of the miscarried fetus is not available for the current cohort and is in general hard to obtain. Alternatively, inclusion of paternal HLA typing may provide a better insight in this mechanism.

The duration of maternal exposure to allo-epitopes is significantly shorter during a miscarriage compared to a full-term pregnancy. Therefore, one might argue that alloimmunization is negligible in pregnancies that end in a miscarriage and that the alloimmunization pattern of secundigravidae with a prior miscarriage is more comparable to the alloimmunization pattern of primigravidae. In this study, we showed that the percentage of immunogenic antigens for secundigravidae with a prior miscarriage was also lower than the percentage of immunogenic antigens observed for primigravidae (Figure 1), demonstrating that the immunization pattern in secundigravidae with a prior miscarriage differs from the immunization pattern that was observed in primigravidae. Thus, despite a shorter duration of maternal allo-exposure during pregnancy loss, the effect of a prior miscarriage on a subsequent pregnancy cannot be neglected in terms of HLA antibody formation.

Although our investigation on the differential effect of a first pregnancy and a first miscarriage on a subsequent successful pregnancy are unprecedented, our observation might be supported by previous reports. For example, Triulzi et al. showed that women with a single pregnancy that ended in a miscarriage had a diminished HLA alloimmunization compared to women with a single pregnancy that ended in a successful delivery (13). Furthermore, Masson et al. reported that the HLA immunization incidence was diminished in women with a single successful pregnancy that was preceded by one or more miscarriages compared to women with a single successful pregnancy that was not preceded by one or more miscarriages (29). However, the latter study did not take the number of prior miscarriages into account. In our population, we observed that women with a single successful pregnancy that was preceded by two or more miscarriages had a higher percentage of immunogenic antigens than women with a single successful pregnancy that was preceded by a single miscarriage (Figure 2). This observation indicates that the number of prior miscarriages may have impact on HLA sensitization during a subsequent successful pregnancy.

The probability of HLA antibody formation increases with the number of PIRCHE-II in pregnancies that were not preceded by one or more miscarriages, including secundigravidae without a prior miscarriage (15). In our cohort of secundigravidae with a prior miscarriage, the single immunogenic HLA had a number of PIRCHE-II that was comparable to the other non-immunogenic HLA (Figure 4), indicating that the PIRCHE-II effect is absent in pregnancies that were preceded by miscarriages.

Our study has limited details about the miscarried fetus itself, as the paternity, HLA typing, and cause of the miscarriage were not documented. Furthermore, the miscarriages in our cohort were self-reported. Since a majority of the miscarriages are unnoticed (30), it may well be that the number of prior miscarriages is underestimated. Therefore, also in the secundigravidae without prior miscarriage group and in the primigravidae group some women might have previous miscarriages, which may led to underestimation of immunization toward IPA in normal pregnancies. Moreover, serum samples for HLA antibody analysis after the miscarriage are lacking for our cohort. These latter serum samples may answer the question whether the mother had developed HLA antibodies against the miscarried fetus or not and would provide more insight in the possible mechanisms behind our observations.

In summary, we showed that a previous miscarriage and a previous successful pregnancy have a different impact on HLA antibody formation during a subsequent successful pregnancy. In contrast to successful pregnancies, increasing gravidity is not related to increased child-specific HLA antibody formation in secundigravidae with a prior miscarriage. Further details about the miscarried fetus itself or paternal HLA typing will be required to explain the observed different impact of a previous miscarriage and a previous successful pregnancy on child-specific HLA antibody formation during a subsequent successful pregnancy. These data may help to understand the mechanism of child-specific HLA antibody formation during a successful pregnancy that was preceded by a miscarriage and therefore will have implications in the transplantation field.

All the authors met the authorship criteria as described by Frontiers in Immunology. KG, GH, SS, and ES were involved in design of the work and interpretation of the data. HD and IH were involved in acquisition of the data. All the authors were involved in drafting or revising the manuscript and approved the final version. All the authors agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

The authors of this manuscript have conflicts of interests to disclose. The UMCU has filed a patent application on the prediction of an alloimmune response against mismatched HLA. ES is listed as inventor on this patent. The other authors have no conflicts of interest to disclose as described by the Frontiers in Immunology.