Preterm delivery is the leading cause of infant mortality and morbidity as well as medical expenditure for infants. However, despite the dramatic improvement in obstetric management and decades of public health intervention toward reducing adverse pregnancy outcomes, the incidence of preterm delivery remains unchanged.1 The strong resistance of preterm delivery to social and medical interventions not only indicates the failure to identify major etiological factor(s) but also suggests that the underlying causes for preterm delivery may be strongly related to host factors as opposed to environmental factors.
In recent years, important progress has been made in understanding the important role that immunology and human leukocyte antigen (HLA) alleles play in reproduction.2–5 Immunologists have long been interested in maternal tolerance of the fetus because the fetus is essentially an allograft.3,4,6 In recent decades, research into this area has revealed that immunological mechanisms are a crucial part of a successful pregnancy. One of the discoveries was that parental sharing of HLA alleles may lead to adverse pregnancy outcomes, including infertility, recurrent miscarriages, low birth weight, preeclampsia, birth defects, and sudden infant death syndrome, although the findings have not been consistent.4,7–21
We hypothesized that it is not simply the parental sharing of HLA alleles; rather, it is the transmission of parentally shared HLA alleles that increases the risk of preterm delivery and possibly other adverse pregnancy outcomes. The transmission of parentally shared HLA alleles to offspring results in maternal–fetal sharing of parentally shared HLA alleles. This 3-way sharing of HLA alleles (mother–father and mother–fetus) could potentially alter normal immunological responses of mother to fetus that is required to establish normal maternal–fetal immune tolerance and to sustain a healthy pregnancy.
Given our hypothesis, the term “maternal–fetal sharing” of HLA allele(s) implies maternal–fetal sharing of HLA allele(s) that are shared by parents. It is a conditional sharing, not a sharing of any HLA allele(s) between a mother and her fetus. A fetus will always receive 1 HLA allele from its mother at any given HLA locus. If this maternal HLA allele is not carried (shared) by the father, the HLA allele will not be considered shared by the mother and the fetus even although the mother and her fetus have the same HLA allele (Fig. 1). Therefore, we propose that maternal–fetal sharing of HLA alleles in relation to adverse pregnancy outcomes involves 2 crucial conditions: 1) parental sharing of the same HLA allele(s) and 2) transmission of such parentally shared allele(s) to the fetus. This hypothesis is different from a traditional hypothesis of HLA–disease association in which specific HLA alleles are correlated with diseases across families. We are not concerned with any specific HLA allele. Rather, we are concerned with alleles that are carried (shared) by both parents. Although parental sharing of HLA alleles has been associated with adverse pregnancy outcomes, no reports have directly examined the effect of transmission of parentally shared HLA alleles to the fetus because, in the past, most studies in this area have been done with recurrent miscarriages from which obtaining fetal tissues for HLA typing has rarely been done. The current study was conducted to test our hypothesis that transmission of parentally shared HLA alleles to offspring (not parental sharing itself) is associated with an increased risk of preterm delivery. The study also was initiated based on our previous findings that recurrent risk of preterm delivery will be reduced if a woman with a history of preterm delivery changes partners.22
MATERIALS AND METHODS
The Institutional Review Boards of Kaiser Permanente and Children's Hospital Oakland Research Institute both approved the protocol and conduct of the study. Informed consent was obtained from all participants. We conducted a population-based family study among members of the Kaiser Permanente Medical Care Program, a group-model integrated healthcare delivery system, in the Bay Area Region of Northern California. Kaiser Permanente Medical Care Program members are representative of the underlying population in the service areas. We selected case families that met the following eligibility criteria: 1) had at least 1 early preterm singleton birth (35 weeks of gestation or less) from the same parents; 2) at least 1 of the early preterm births was the first-born singleton for the mother; 3) preterm delivery was not caused by known factors, such as incompetent cervix or congenital abnormality of the uterus; and 4) both parents spoke English. We oversampled multiplex families (2 or more preterm delivery at 35 weeks of gestation or less from the same parents) because multiplex families are generally more likely to have underlying host (familial) etiology. A total of 48 multiplex and 25 simplex families with samples from 219 participants (3 members in each family) were included in the final analysis. Because our analysis did not reveal any noticeable difference in the transmission pattern of HLA alleles associated with preterm delivery between multiplex and simplex families, to increase the power of the analysis, we presented our results with multiplex and simplex families combined. Our focus on first-born preterm birth was based on the hypothesized underlying immunological mechanisms and the findings from studies of recurrent miscarriages indicating that the association between immunological factors and adverse pregnancy outcomes is different between women with and without previous live birth.23,24
Buccal cells were collected from the first-born children and their parents from all participating families to determine parental HLA types and the transmission of parental alleles to the offspring. Mothers were interviewed for potential risk factors for preterm delivery and complete reproductive history. Fifty-seven percent of eligible families participated in the study. In addition to refusals, approximately 20% of nonparticipation was the result of an inability to locate the biological father. Our HLA typing results indicated that all participating fathers were biological fathers of the participating children.
DNA was extracted from the buccal cells by vortexing 3 brushes in 50 mM of NaOH, lysing at 95°C, and then neutralizing with 1 M of Tris-HCl, pH 8. Molecular HLA typing requires the use of polymerase chain reaction for DNA amplification and immobilized sequence-specific oligonucleotide probe array methods for intermediate resolution HLA typing to define alleles or allelic groups at better-than-serological equivalence (Dynal Reli HLA DRB Typing Kit; Dynal, Oslo, Norway). In addition, higher-resolution probe arrays (Roche Molecular Systems, Alameda, CA) and allelic-group-specific amplifications were used to define ambiguous allelic combinations and to determine alleles and genotypes for class I HLA-B and class II DRB1 loci in family cases. HLA-B and DRB1 loci were chosen because both are involved in antigen presentation and recognition and both were most frequently associated, in previous studies, with adverse pregnancy outcomes, such as recurrent miscarriages and preeclampsia.5,11,25–27 HLA typing was performed without knowledge of the case–control status of the specimens, and HLA typing of children was blinded to their parental HLA types. Parental sharing was determined at both a molecular and serological-equivalent level.
We examined the association between transmission of parentally shared HLA alleles and the risk of preterm delivery based on case-parental trios. This study design is similar to the transmission disequilibrium test/case-parental control study28–30 type design. However, when we used this transmission disequilibrium test/case-parental control–type analysis, the emphasis was not any specific “disease” allele across the studied families; rather, we were interested in any alleles that were shared by both parents that could be different from family to family. The advantage of the transmission disequilibrium test/case-parental control design is that it eliminates concerns related to selection bias, confounding resulting from population stratification, and any other potential biases resulting from environmental factors. Based on Mendel's laws of genetic segregation and independent assortment, for heterozygous parents who share 1 HLA allele, offspring should have a 50% chance of receiving the parentally shared HLA allele from 1 parent and a 25% chance of receiving the shared allele from both parents. Any significantly greater probability of transmission of the parentally shared allele compared with the unshared allele to preterm cases will provide etiological evidence between the transmission of parentally shared HLA alleles and the risk of preterm delivery.
This transmission disequilibrium test/case-parental control type of analysis is robust against any biases caused by environmental factors and population stratification. However, segregation distortion, although rare, could potentially produce a spurious association. Although studies of HLA regions have not provided any evidence of segregation distortion at those loci, to be conservative, we examined the potential existence of segregation in randomly selected controls families that met the following criteria: 1) had no preterm delivery in the woman's reproductive history; 2) had at least 2 singleton births with the same father; and 3) had no history of recurrent miscarriages (3 or more) or preeclampsia that have been reported to be associated with parental sharing of HLA alleles. Cases and controls were comparable in demographic characteristics, including maternal age, education, parental race/ethnicity, and parity, as well as sex of offspring. If population controls demonstrate no segregation distortion at those loci, an association based on this case–parental control study will provide powerful support for a genuine link. At the beginning of the study, we also collected samples from normal full-term offspring who were siblings of preterm cases. Those 15 full-term siblings could also serve as “sib-matched controls” to determine the potential existence of segregation distortion. Because the transmission pattern between the population controls and the sib-matched controls was very similar as expected according to Mendel's laws, we combined both controls to increase the sample size in the final analysis.
The association between transmission of parentally shared HLA alleles and the risk of preterm delivery is presented in Table 1. Among 73 families with at least 1 preterm delivery at 35 weeks of gestation or greater and whose members were typed for HLA-B and DRBI loci, 34 cases were eligible for further analysis because the parents shared HLA alleles at either HLA-B or DRB1 loci. Next, we excluded cases and controls whose parents (either or both) could not provide information for determining preferential transmission because either 1) they were homozygous at the shared locus, 2) they shared both B and DRB1 alleles that may be on different chromosomes, in which case offspring would always receive one parentally shared allele regardless of which chromosome was transmitted, or 3) they shared both alleles at B locus, in which case offspring would always receive 1 parentally shared B allele regardless of which B allele was transmitted. Therefore, our analysis was conducted based on families in which both parents shared only 1 HLA allele (B or DRB1 locus) and were heterozygous at the shared locus, allowing a more focused and straightforward analysis to test our hypothesis.
Among the families that were eligible for analyzing preferential transmission of parentally shared HLA alleles, we compared our observed transmission distributions from the mother, the father, and the combination of both parents with the expected corresponding distributions using the expected number based on Mendel's Laws of segregation.
A significantly higher proportion of preterm cases (83%) received parentally shared HLA allele from their mothers than expected (50%): odds ratio (OR) 4.8; 95% confidence interval (CI) 1.6–19.2 (Table 1). The proportion of preterm cases who received parentally shared HLA allele from their father (57%) was not statistically different from the expected (50%): OR 1.3; 95% CI 0.5–3.3 (Table 1). A significantly higher proportion (48%) of preterm cases received parentally shared HLA allele from both parents than expected (25%): OR 5.5; 95% CI 1.2–51.1. In contrast, significantly fewer cases (9%) receive no parentally shared HLA allele from either parent compared with 25% expected (Table 1).
Transmission patterns in our full-term controls confirmed that there was no segregation distortion in transmission of HLA-B and DR alleles to offspring in this study population. The transmission distribution pattern was almost identical to the expected pattern based on Mendel's laws. Therefore, the observed preferential transmission of parentally shared HLA alleles to preterm cases is not confounded by segregation distortion, nor is it likely to be explained by other known genetic and environmental factors.
Because of the small number of parents who shared B allele (5 heterozygous parents), we were unable to evaluate the effect of transmission of parentally shared HLA allele separately for B and DR loci. Also, the preferential transmission of parentally shared alleles did not seem to be related to any specific HLA alleles.
In this population-based family study, we observed that parentally shared HLA alleles were preferentially transmitted to offspring who were delivered prematurely. The preferential transmission of parentally shared HLA alleles was more pronounced for transmission from both parents or from mothers to offspring (Table 1). The above-observed preferential transmission of parentally shared HLA alleles was statistically significant, taking into account the sample size. These findings provide the evidence that transmission of parentally shared HLA alleles may increase the risk of preterm delivery. Based on Mendel's laws of segregation and independent assortment, our analysis of transmission of parentally shared HLA alleles using families with heterozygous parents provided an efficient and unbiased assessment of the relationship between the transmission of parentally shared HLA alleles and the risk of preterm delivery. If transmission of parentally shared HLA alleles were not related to the risk of preterm delivery, both shared and unshared alleles should have had an equal chance (50%) of being transmitted to preterm offspring. No external factors are likely to influence transmission. Because our HLA typing of offspring was blinded to parental sharing status, our findings are also unlikely the result of technical biases. Therefore, our findings of an association between transmission of parentally shared HLA alleles and the risk of preterm delivery suggest a potential etiological connection between transmission of parentally shared HLA alleles and the risk of preterm delivery.
In contrast to the preferential transmission of parentally shared HLA alleles in preterm births, the transmission pattern from our population-based and sib-matched full-term controls was almost identical to the transmission pattern predicted by the Mendel's Laws. This observation further supports the etiological link between preferential transmission of parentally shared HLA alleles and the risk of preterm delivery and ruled out the possibility of segregation distortion as the reason for the observed preferential transmission.
Although the underlying mechanisms for our findings are not known at this point, our findings suggest that a successful pregnancy may require a 3-way immunological interaction between mothers and fathers and between mothers and their fetuses. This observation is consistent with emerging knowledge in reproductive immunology. Although the exact details are still being studied, it is generally established that immunological disparity between mother and father and between mother and the fetus is healthy for a successful pregnancy.4,5 The underlying mechanisms are likely to involve the establishment of maternal–fetal immunological tolerance, which may require appropriate fetal stimulation and maternal immunological reaction.4,5,8,21,31 Maternal–fetal tolerance is established likely through 1) production of blocking factors that prevent maternal recognition of fetal alloantigens; 2) activation of suppressor T cells that down-regulate the maternal immunoresponse; and/or 3) suppression of fetal expression of antigens.2,4,32 This immunological interaction involves antigen presentation and recognition between mother and father and between a mother and her fetus. The HLA loci play an essential role in antigen presentation and recognition. Thus, one of the potential mechanisms could be that maternal–fetal sharing of HLA alleles caused by the transmission of parentally shared HLA alleles can significantly reduce maternal–fetal disparity, thus altering the normal maternal-fetal interaction. Without appropriate fetal stimulation and normal maternal immunological reaction, maternal ability to establish healthy immune tolerance that is needed to sustain a successful pregnancy may be adversely impacted. Indeed, recent studies have shown that maternal level of HLA antigens is associated with preterm delivery and other adverse pregnancy outcomes.20,33 With increasing understanding of the role of reproductive immunology in determining pregnancy outcomes, new hypotheses and mechanisms are likely to emerge to elucidate the mechanisms for our findings.
Our findings that maternal transmission of parentally shared HLA alleles was more pronounced than paternal transmission also are consistent with those from large-scale epidemiological studies that suggested that although both parents contribute to the risk of preterm delivery, mothers play a far more important role in determining the risk of preterm delivery in future pregnancies than fathers do.22,34,35 Although the mother, like the father, contributes to the HLA identity, she must also interact with the fetus to establish immune tolerance. Therefore, it is conceivable that transmission of parentally shared HLA allele from a mother to the fetus may be more likely to interfere with the normal immunological interactions between a mother and the fetus, resulting in abnormal pregnancy outcomes including preterm delivery.
Although the exact mechanistic pathways of the relationship between transmission of parentally shared HLA alleles and preterm delivery are not currently well understood, one of the potential culprits is likely the resulting pathological changes in the placenta which is both crucial for sustaining a healthy pregnancy and for providing the interface for maternal–fetal immunological exchange.6,36–38 The placenta also is likely involved in labor initiation.7
Our findings that it is the transmission of parentally shared alleles, not parental sharing itself, that increases the risk of preterm delivery may also provide an explanation for previous inconsistent results for the relationship between HLA sharing and recurrent miscarriage. Parental sharing of HLA alleles is a precondition for transmission of parentally shared HLA alleles. However, parental sharing itself does not necessarily mean transmission of parentally shared alleles. In our study, parental sharing by itself was not associated with an increased risk of preterm delivery, but transmission of parentally shared HLA alleles was (Table 1).
One important limitation of the study was the relatively small sample size in the final analysis. Although we started with a reasonable number of participants, because of the restriction of requiring parental sharing of HLA alleles and both heterozygous parents at the shared HLA locus, the number of informative families was reduced significantly. Nonetheless, our finding remains statistically significant. The small number of informative families also limited our ability to conduct subgroup analysis, such as examination of different associations between multiplex and simplex families of preterm delivery.
If our findings that transmission of parentally shared HLA allele increases the risk of preterm delivery are corroborated in future studies, the result will represent a significant advance in our understanding of the etiology of preterm delivery and other adverse pregnancy outcomes. It also will change the conceptualization of the causes for preterm delivery and possibly other adverse pregnancy outcomes. These findings may then have important implications for obstetric practice and improvement of pregnancy outcomes as well as infant morbidity and mortality. In addition, the findings will also have important implications for basic research into understanding of reproductive immunology.
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