Obstetrics & Gynecology:
Progesterone Receptor Genotype, Family History, and Spontaneous Preterm Birth
Manuck, Tracy A. MD; Major, Heather D. MD; Varner, Michael W. MD; Chettier, Rakesh MS; Nelson, Lesa BS; Esplin, M. Sean MD
From the University of Utah Department of Obstetrics and Gynecology; and Taueret Laboratories, Salt Lake City, Utah.
Supported by the University of Utah Department of Obstetrics and Gynecology.
Presented in part at the Society for Gynecologic Investigation Annual Meeting in Glasgow, Scotland, March 17-21, 2009.
Corresponding author: Tracy A. Manuck, MD, 30 North 1900 East, Room 2B200, Salt Lake City, UT 84132; e-mail: firstname.lastname@example.org.
Financial Disclosure Lesa Nelson and Rakesh Chettier are affiliated with Taueret Laboratories, LLC, which received payment for genotyping costs. The other authors did not report any potential conflicts of interest.
OBJECTIVE: To examine whether women with a personal or family history of preterm birth are more likely to have genetic variation in the human progesterone receptor (hPR).
METHODS: Women with a singleton preterm birth at less than 37 weeks of gestation between 2002 and 2006 were identified from a prospectively collected clinical and biologic obstetric database (cases). Women in the control group were those with only term deliveries at or above 38 weeks of gestation. The Utah Population Database was queried for family history (first- or second-degree relative) of preterm birth. DNA was extracted from stored buffy coats and genotyped for six single nucleotide polymorphisms in the hPR.
RESULTS: One hundred fifty-four patients (92 women in the preterm case group, 62 women in the term control group) were included. All were white or Hispanic. There were no statistical differences between white and Hispanic allele frequencies. Women in the preterm case group were more likely to carry the minor allele, G (minor allele frequency 0.29 compared with 0.18, P=.035) for rs471767, and were more likely to carry the GT haplotype across rs471767 and rs578029 compared with women in the term control group. Similar haplotype block variation was seen among women with preterm birth plus a family history of preterm birth.
CONCLUSION: Allele and haplotype frequencies in the hPR are significantly different among women with preterm birth and women with preterm birth plus a family history of preterm birth. This suggests the hPR gene may be a candidate for association with preterm birth or familial preterm birth.
LEVEL OF EVIDENCE: III
More than 12% of all pregnancies in the United States are delivered preterm, and rates continue to rise.1 Prematurity is the leading cause of perinatal morbidity and mortality; premature newborns have a 40-fold increase in mortality compared with their term counterparts.2,3
Mounting evidence suggests that a genetic component may play a role in the development of spontaneous preterm birth in many women. The strongest predictor of spontaneous preterm birth is a previous history of spontaneous preterm birth.4,5 Spontaneous preterm birth recurs in 35–40% of women, and a tendency for repeat preterm birth at a similar gestational age has been observed.4,6 Ward and colleagues7 studied 236 women with at least one spontaneous preterm birth and their families and found that the coefficient of kinship for familial preterm delivery was more than 50 standard deviations higher than randomly selected population controls, suggesting preterm birth is associated with heritable genes.
Progesterone is essential to the maintenance of pregnancy, and several studies using supplemental progesterone in varying forms have shown reductions in spontaneous preterm birth among women at particularly high risk of prematurity.8–10 The human progesterone receptor (hPR) is a member of the steroid and thyroid receptor superfamily. Single nucleotide polymorphisms (SNPs) within the hPR gene have been identified and have been found to be associated with several reproductive disorders.11–13 Limited studies have examined the relationship between hPR gene polymorphisms and preterm birth, with conflicting results.14–16
The objective of our study was to examine whether women with spontaneous preterm birth are more likely to have genetic variation in the hPR compared with women with only term deliveries. We further hypothesized that women with both a family and personal history of preterm birth are more likely to have variation in the hPR compared with women with only term deliveries and no family history of preterm birth.
Women with a DNA sample available and a singleton spontaneous preterm birth at less than 37 weeks of gestation occurring between November 2002 and September 2006 were identified from a prospectively collected obstetric database at the University of Utah (Salt Lake City, UT). Women delivering a singleton nonanomalous fetus at greater than or equal to 38 weeks of gestation at the time of enrollment and with no prior preterm deliveries were also identified from the same prospective database. Women had been recruited at the time of their delivery hospitalization and had previously provided consent for future biologic tissue analyses, including genetic studies.
These potentially eligible patients were then linked to the Utah Population Database. The Utah Population Database is a unique resource of linked records, including birth certificates, death certificates, pedigrees, and other vital data for more than 6 million individuals. This database has previously been used to assess for familial disposition to pregnancy complications, including preterm delivery, preeclampsia, and operative delivery.4,17–19 Each individual's pedigree was queried for the number of first- and second-degree relatives with a documented prior delivery less than 37 weeks of gestation.
Patients with multiple gestations, those carrying a fetus with suspected major anomalies, and those who could not be linked to the Utah Population Database were excluded. This study was approved by the institutional review boards at the University of Utah and the Resource for Genetic Epidemiology (Utah Population Database).
Clinical data were abstracted from the patient's chart at the time of delivery and biologic sample collection. Data were verified by the review of medical records at the time of inclusion in this analysis.
DNA was extracted from stored buffy coats using standard methods. Patients were subsequently genotyped for six previously identified SNPs in the region of the hPR, including rs471767, rs578029, rs503362, rs582691, rs10985068, and rs10501973, using TaqMan chemistry (Applied Biosystems, Foster City, CA).
Clinical characteristics were compared using χ2 and Fisher exact text as appropriate. Minor allele frequencies were calculated for each SNP and were compared using χ2 and Fishers exact text. Genotypes for each SNP were compared using the “dominant” model of inheritance. Odds ratios of preterm birth were calculated using women homozygous for the major allele as the referent group. Deviation from Hardy-Weinberg equilibrium was assessed. Linkage disequilibrium and haplotype frequencies were estimated, and PHASE version 2.1 (University of Chicago, Chicago, IL) was used to account for haplotype phase uncertainty.20 Stepwise multivariable logistic regression was used to correct for possible confounders. Population attributable risk was calculated from the logistic model as described by Greenland.21 Data were analyzed using STATA version 10.0 (College Station, TX). Significance was set at P<.05.
One hundred fifty-four patients were genotyped, including 92 women in the preterm case group and 62 women in the term control group. All patients were self-identified as either white (85.7%) or Hispanic (14.3%); no statistical differences were seen in white allele frequencies when compared with Hispanic allele frequencies (data not shown); therefore, the two population groups were examined together. All SNPs were in Hardy-Weinberg equilibrium. Maternal demographic information and delivery characteristics are displayed in Table 1. Of the 92 women in the preterm case group, 35 (38%) delivered very preterm, less than 34 weeks of gestation.
Minor allele frequencies were compared between cases and controls for each SNP (Table 2). Genotypes were compared using the dominant model of inheritance (assumes that one copy of the minor allele is sufficient for disease; compares those homozygous for the major allele with the others) (Table 2). Women in the preterm birth case group were more likely to carry the minor allele, G, for rs471767, when compared with the women in the term control group (minor allele frequency 0.29 compared with 0.18, respectively, P=.035). Carriage of the minor allele, G, for rs471767 conferred an almost twofold increased odds of preterm birth (odds ratio [OR] 1.85, 95% confidence interval [CI] 1.04–3.26). There were no significant differences in the genotypes or minor allele frequencies between women in the case group and women in the control group for the other five SNPs studied.
Haplotype analysis results are shown in Table 3. Of note, women in the case and control groups had significantly different haplotypes across rs471767 and rs578029. The incidence of the uncommon GT haplotype was 0.105 among women in the preterm case group compared with 0.0096 among women in the term control broup (P=.003).
We next examined the role of family history. A similar percentage of women in the preterm case group and in the term control group had first- and/or second-degree relatives with documented preterm deliveries (Table 4). There were 25 women who delivered preterm and had a family history of preterm birth (preterm and positive family history). When compared with 44 women who delivered at term and had no family history of preterm birth, although those delivering preterm and with a positive family history did not have significantly different minor allele frequencies for the individual SNP studied, these women had significantly different haplotype distributions (Table 5).
Variables considered for inclusion in the logistic regression model included maternal age, history of a previous preterm delivery, history of cervical loop electrosurgical excision procedure, family history of preterm birth, male fetus, marital status, tobacco use during pregnancy, and carriage of the GT haplotype across rs471767 and rs578029. Only carriage of the GT haplotype (OR 14.3, 95% CI 1.81–112.9, P=.012), history of a previous preterm delivery (OR 7.7, 95% CI 2.5–24.2, P<.001), and maternal age (OR 0.96, 95% CI 0.90–1.02, P=.15) remained in the final model. The population attributable risk was calculated from the final regression model, and for the GT haplotype, was found to be 15.2% (95% CI 8.7–21.2%).
In this patient population of white and Hispanic women, variation in some areas of the hPR occurs more frequently in women with spontaneous preterm birth less than 37 weeks of gestation. These data suggest that the hPR may be a candidate gene involved in the etiology of preterm birth.
Nuclear hPRs primarily exist in two different distinct isoforms, PR-A and PR-B, and have been found in gestational tissues including the amion and chorion.22 Both are encoded from a single gene by transcription from two different promoters, and they have differing roles. PR-A is smaller and lacks the 164 N-terminal amino acids that form an activation domain on the receptor. It inhibits the transcription of progesterone receptive genes. In contrast, PR-B increases transcription of progesterone-responsive genes and has an overall quiescent effect on the myometrium.11,23
Given the differing roles of these receptors, it has been proposed that the responsiveness of target tissues to progesterone depends not only on the level of progesterone but also on the ratio of PR isoforms.24,25 It has also been hypothesized that a relative increase in the ratio of PR-A to PR-B may contribute to a functional withdrawal of progesterone and lead to the initiation of labor.26,27 Importantly, rs471767 is a SNP located just upstream of the hPR promotor. Thus, it is plausible that genetic variation in this region could alter transcription of PR-A in relation to PR-B.
We found that the hPR haplotype block encompassing SNPs rs471767 and rs578029 was strongly associated with preterm birth; in our regression model, carriage of the GT haplotype conferred a 14-fold increased risk of preterm birth. This suggests that some of the markers studied in this region may be in linkage disequilibrium with other functional genes associated with prematurity. The haplotype associations were present in all women with preterm birth as well as those delivering preterm with a positive family history. These data suggest that women at the highest risk of prematurity are also at highest risk of genetic differences in their hPR. The population attributable risk of carriage of the GT haplotype was 15.2%. This is similar to a previous genetics of preterm birth study, which found the population attributable risk of a single gene among African-American women with preterm premature rupture of membranes to be 12%.28 Given the multifactorial etiology of spontaneous preterm birth, this relatively low population attributable risk is not unexpected.
There have been few previous studies of the hPR gene in women with preterm birth. Diaz-Cueto et al14 studied a Hispanic population of 64 women in a preterm case group and 54 women in a term control group and concluded that polymorphisms in the hPR gene are unlikely to be associated with preterm birth. Luo and colleagues16 genotyped 78 primarily Hispanic patients with preterm birth for three PR SNP and found an association between PR genotype and preterm birth in two of these SNP but only for women with a body mass index less than 18.5 kg/m2. In 2007, Ehn et al15 examined 18 SNPs in 415 maternal–fetal infant pairs and found a relationship between prematurity and SNP rs503362 (P=.008). In contrast to these findings, we did not find an association between rs503362 and prematurity.
We recognize the limited power of this study to definitively exclude the role of individual polymorphisms in preterm birth due to a small sample size. We do not have consistent prepregnancy body mass index data on this cohort and were thus unable to compare our findings with those of Lou et al.16 Additionally, more than half of our preterm cases were delivered during the “late preterm” period, between 34 and 36 weeks of gestation. Although late preterm newborns do not generally have the same high rates of morbidity and mortality as very premature newborns, they do constitute the largest group of preterm newborns and have generally been understudied. Our study had several additional strengths; our population consisted of only white and Hispanic patients; prior studies have shown a negligible risk of confounding due to admixture of these populations.29 Clinical data and biologic samples were collected prospectively. Additionally, we have unique, objective family history data from the Utah Population Database.
Further study is needed to confirm these results and to examine the region upstream of the hPR promoter in depth. Confirmation of these results could lead to further study of the specific gene product from this region and refinement of therapeutic modalities for the treatment and/or prevention of prematurity.
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