Objective
The investigators tested the hypothesis that maternal-fetal immune interactions could be important in initiating spontaneous labor onset by examining if labor was delayed when fetuses share maternal HLA antigen types.
Study Design
HLA antigen types A, B, and DR in 200 Danish mother-infant pairs delivering in 42–44 weeks (postterm) were compared with 195 mother-infant pairs delivering in 37–40 weeks (term).
Results
Sharing of HLA A and B antigens was more common than expected in postterm deliveries. Odds ratios were 1.54 (95% confidence interval [CI], 1.01–2.35) and 1.75 (95% CI, 0.87–3.52), respectively (risk per shared antigen: 1.40 [95% CI, 1.04–1.90] per unit increase). Adding stringent birth-length criteria for postmaturity (92 cases; 168 controls) strengthened risks associated with antigen sharing to 1.57 (95% CI, 0.90–2.74) and 2.60 (95% CI, 1.15–5.88), respectively (risk per shared antigen: 1.60 (95% CI, 1.10–2.32).
Conclusion
Postterm-delivered infants had more HLA A and B antigens in common with their mothers, suggesting that recognition of HLA antigen differences by adaptive immunity may have a role in triggering labor onset.
The factors precipitating spontaneous labor onset are poorly understood. Premature deliveries may occur for reasons related to factors either in the mother or infant, complicating their understanding. However, few factors are known to affect the risk of postterm delivery. Typically, mothers delivering postterm are healthy and infants are normal, albeit with the potential complications and increased mortality of having outgrown their placental life support. We reasoned that immunologic factors might be important in triggering spontaneous labor and that these factors would be best studied by comparing postterm to term infants.
Paternal-contributed HLA antigens should be recognized and rejected by the mother when they are foreign to her immune system. The fetus escapes immunologic detection by down-regulating expression of the heterogeneous class Ia HLA A and B antigens and up-regulating expression of the highly conserved HLA class Ib G antigen. These HLA antigen changes occur soon after conception and persist throughout pregnancy. We speculated that subtle up-regulation of class 1a HLA antigens in the last days of pregnancy could be a trigger for provoking an immune response and cytokines that initiate labor. If immune interaction between mother and placenta were important, then mothers who shared HLA antigen identity with their child might be delayed in recognizing the “foreignness” of the placenta. To examine the role of immunologic challenge in labor onset, we proposed that infants sharing HLA antigen types with their mothers would more likely deliver postterm than those with only minimal HLA antigen sharing.
Materials and Methods
Study participants were obtained from the Danish National Birth Cohort (DNBC), a nationwide study that initially enrolled 101,045 pregnant women from 1996–2002 and sought follow-up of liveborn offspring (n = 96,842). A detailed description of this cohort study has been previously published. We acknowledge that there is variation in the usage of “term” and “postterm” pregnancy duration. For convenience, we defined “term” as 37–40 weeks’ estimated gestation, as in common usage, and “postterm” as ≥42 weeks’ duration. Gestations estimated at 41 weeks were excluded to enhance the contrast between the duration of the 2 pregnancy groups. The current study compares the frequency of HLA antigen sharing between mothers and infants for infants delivered at term and postterm by these definitions. This study was approved by the Scientific Ethics Committee, Copenhagen.
Table 1 presents a flowchart by which subjects were selected. We required that both parents be born in Denmark and that complete information for essential parameters be known. Only a woman’s first DNBC pregnancy resulting in a live-born singleton was considered. Mothers were ineligible if they self-reported at enrollment that they were in poor health or if they reported hypertension, whether or not pregnancy related. Because induction and cesarean delivery were commonly done on postterm infants, we could not use mode of delivery as a criterion for selection. Among term births, 5% were induced and 12% taken operatively. Many mothers delivering at term would likely have started labor prior to the intervention, but this information was not available. Although a few of these infants might have gone on to become postterm pregnancies in the absence of intervention, the resulting small misclassification bias is conservative, in that it would favor the null hypothesis. Because obstetricians generally intervene when pregnancies reach 41 or 42 weeks’ gestation, few women were reported to have had 43 or 44 weeks’ gestations and they were grouped with 42-week pregnancies. Gestation estimates of ≥45 weeks were considered erroneous, and these pregnancies were excluded. All infants were without recorded malformations of any kind. We also required that samples from the mother (venous blood) and infant (umbilical cord blood) be available in the DNBC bio-bank. We had a surplus of eligible cases and controls, and the selection for inclusion was random (computer generated), the objective being to obtain data on at least 200 term and 200 postterm mother-infant pairs, allowing for about 15% losses. This study was powered to have an 80% chance of finding as statistically significant ( P < .05) a 2-fold increase in association with HLA antigen sharing.
| Criterion | Case pairs, n | Control pairs, n |
|---|---|---|
| Gestational age at birth, wk | ≥42 | 37–40 |
| Case/control selection process | ||
| Initial pool of mother-child pairs | 7514 | 46,180 |
| Exclusion of pregnancies with maternal gestational hypertension | 766 | 5026 |
| Exclusion of pairs without blood samples for mother and child | 2927 | 17,801 |
| Exclusion of pairs without maternal smoking information | 7 | 135 |
| Exclusion of infants with ≥1 parent born outside Denmark | 316 | 1933 |
| Final pool of potential mother-child pairs | 3498 | 21,285 |
| Random selection of 233 pairs | 233 | 233 |
| Exclusions during analytic phase | ||
| Technical failure of PCR amplification | 23 | 27 |
| Failure to genotype all 3 loci | 6 | 9 |
| Mother-child mismatch at ≥1 locus | 4 | 2 |
| Final no. of mother-child pairs included in analysis | 200 | 195 |
The gestational age is critical to establishing a postterm delivery. In the DNBC, estimates of gestational age show close agreement whether using last normal menstrual period or ultrasound. We used established DNBC criteria, in which the earliest available reliable data were given priority, as follows: (1) the enrollment visit (usually the initial doctor visit in the first trimester)—gestation was based on the last normal menstrual period, but strict guidelines were used to assure the last menses was entirely normal and precisely dated (42% in postterm infants; 46% in controls); (2) the 30-week interview—(typically the second interview)—gestation was primarily based on ultrasound results (52% and 51%, respectively); (3) the postpartum interview—the infant’s physical assessment examination contributed to the estimation (4% and 2%, respectively); and (4) if no other data were available, the physician/midwife assessment in birth records maintained by the Danish National Birth Registry was used (2% and 1%, respectively).
Samples were prepared as dried blood spots on filter paper and stored at –20°C until testing. DNA was extracted from punch samples using Qiagen Maxi-kit (VWR, Stockholm, Sweden) and arranged in 96-well trays, each with quality control and blank samples. The DNA samples were amplified by polymerase chain reaction, and the HLA antigen genotype was determined by using the bead/reverse transcriptase sequence-specific oligonucleotide method (One Lambda LABType SSO, Canoga Park, CA). HLA antigen type was determined at an intermediate (antigen) level of resolution for HLA A, B, and DR(B1) antigen, according to the protocol in the One Lambda LABType SSO kit. Six samples with ambiguous typing results in initial tests were resolved by further tests. Samples were placed randomly on test plates, and the laboratory was masked as to the identity of the mother-infant pairs and their case-control status.
Half of the HLA antigens must be acquired from the mother and cannot be recognized as foreign to her immune system. By chance, some paternally contributed HLA antigens may be the same as those in the mother. A priori, we predicted cell-mediated immunity would be most important. HLA antigen A and B interactions are critical to cellular immunity. If so, infants with more HLA A and B antigens in common with their mothers would be most likely to be born postterm. In contrast, DR antigens are involved in humoral immune reactions, and we expected little influence from sharing of DR antigens.
We initially identified 233 eligible postterm and 233 term control pairs. However, a technical problem prevented polymerase chain reaction-amplification in 1 plate, resulting in the loss of data from 23 case and 27 control pairs. We regard this amplification failure as a random loss. Subjects in whom only 1 targeted antigen was identified were considered homozygous for the allele. Six case and 9 control pairs were excluded, because we failed to identify all 3 targeted antigens in both mother and infant. Four case and 2 control pairs were excluded, because HLA antigen types of the infants did not match that of their mothers at 1 antigen (usually) or more, due to sample mislabeling or laboratory error. The final analysis included only mother-infant pairs with complete and compatible HLA antigen types.
For each antigen, a score was developed according to how many antigens the infant had in common with his or her mother. The score could be 1 (maternally acquired antigen only) or 2 (paternally acquired antigen matched maternally acquired antigen). The possible composite scores for A and B types in a mother-infant pair ranged from 2 (maternally contributed antigens only) to 4 (all antigens matched maternally acquired antigens). Similarly, scores for HLA A, B, and DR antigens ranged from 3–6. Statistically, associations for having antigens in common were examined by odds ratios (ORs) estimates with 2-sided 95% confidence intervals produced using logistic regression (SAS, version 9.2; SAS Institute, Cary, NC). Although not hypothesized, specific HLA antigen type might have been a factor in either the mother or the child only. Therefore, we also examined univariate P values and ORs for individual antigens. Analysis of individual HLA antigen types is presented when types were found in at least 10 cases or controls.
As a sensitivity test, we adopted more stringent criteria for postterm delivery. Of infant birthweight, body mass index, and length, birth length most clearly differed between term and postterm infants ( Figure ). We therefore modified the criteria for postterm-delivered infants to require both an estimated gestational age of 42–44 weeks and a birth length of ≥54 cm. Similarly, term newborns were required to have an estimated gestational age of 37–40 weeks and a birth length of <54 cm. The analysis was then repeated in this subset, with the a priori expectation that the associations would be stronger than in the main study, whether significant or otherwise.
Results
The analysis was done on 200 case and 195 control mother-infant pairs. Their characteristics are given in Table 2 . Postterm-delivering mothers tended to be primiparous and to smoke less. Postterm infants tended to be firstborns and were longer and had a larger head circumference than term infants.
| Characteristics | Case pairs (n = 200) | Control pairs (n = 195) | |||||||
|---|---|---|---|---|---|---|---|---|---|
| Mean | (SD) | n | (%) | Mean | (SD) | n | (%) | P value | |
| Maternal characteristics | |||||||||
| Age at delivery, y | 29.7 | (4.4) | 30.1 | (4.0) | .34 | ||||
| Prepregnancy weight, kg | 67.6 | (12.6) | 66.6 | (11.6) | .44 | ||||
| Prepregnancy body mass index | 23.6 | (4.2) | 23.5 | (4.1) | .83 | ||||
| Cesarean delivery (emergency) | 9 | (5) | 14 | (7) | .26 | ||||
| Delivery after induction | 70 | (35) | 9 | (5) | < .0001 | ||||
| Parents born in Denmark | |||||||||
| 2 | 185 | (93) | 181 | (93) | |||||
| 1 a | 15 | (7) | 14 | (7) | .90 | ||||
| Any smoking during pregnancy | 48 | (24) | 59 | (30) | .16 | ||||
| Smoking >30 wk | 25 b | (16) | 24 b | (12) | .95 | ||||
| Infant characteristics | |||||||||
| Apgar score, 5 min | 9.9 | (0.5) | 9.9 | (0.5) | .99 | ||||
| Birthweight, g | 3837 | (551) | 3599 | (453) | < .001 | ||||
| Birth length, cm | 53.2 | (4.4) | 52.1 | (2.1) | .003 | ||||
| Gestational age at birth, wk | 42 c | 39 c | < .001 | ||||||
| Head circumference, cm | 35.4 | (5.3) | 34.7 | (4.6) | .14 | ||||
| Birth order | |||||||||
| 1 | 107 | (54) | 86 | (44) | |||||
| 2 | 54 | (27) | 66 | (34) | |||||
| 3 | 24 | (12) | 28 | (14) | |||||
| ≥4 | 3 | (2) | 4 | (2) | .42 | ||||
| Unknown | 12 | (6) | 11 | (6) | |||||
| Grandparents born in Denmark | |||||||||
| All 4 | 174 | (87) | 169 | (87) | |||||
| ≥1 born outside Denmark | 14 | (7) | 14 | (7) | |||||
| ≥1 missing birth place d | 12 | (7) | 12 | (6) | .99 | ||||
| Male sex | 101 | (51) | 103 | (53) | .64 | ||||
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