Objective
The purpose of this study was to evaluate the association between unexplained stillbirth (SB) and single-nucleotide polymorphisms (SNPs) in genes involved in placental function using a well-characterized cohort.
Study Design
Placentas were obtained from 50 unexplained SB and 46 live birth controls. Classification of stillbirth was by Wigglesworth criteria. SBs were stratified by weight: appropriate (AGA-SB) and small for gestational age (SGA-SB, less than the 10th percentile) and gestational age: before 32 and after 32 weeks. Placental DNA was extracted and various SNPs in the endothelial nitric oxide synthase (eNOS), Klotho, hypoxic inducible factor-1α, and and tumor necrosis factor-α genes were evaluated.
Results
None of the SNPs were associated with SB overall. Significantly different genotype distribution emerged for eNOS-SNP rs1800783 when comparing AGA-SB with SGA-SB and control ( P = .004). Its allele-A was more frequent in AGA-SB compared with both controls ( P = .03) and SGA-SB ( P = .001). No differences were seen accordingly to gestational age.
Conclusion
Unexplained stillbirth in the setting of adequate growth is associated with carrier of allele A of rs1800783 eNOS gene in the placenta.
Stillbirth (SB) is a relatively frequent complication of pregnancy, occurring in 3-6 of 1000 pregnancies in developed countries. Over the past several decades, improvements in maternity care have resulted in reduction in stillbirth in high-income countries, but more recently, this decline has halted, and SB now accounts for half of perinatal mortality. Although several conditions including fetal malformation, chromosomal aberration, and maternal and/or fetal infections are known to predispose to this condition, up to 50-70% of SB remain unexplained. Furthermore, because effective preventative strategies have not yet been identified, few obstetric complications are as emotionally devastating as stillbirth for both patients and clinicians.
A substantial proportion of unexplained SBs in high-income countries are suspected to be due to abnormalities in placental function and development, whether or not associated with impaired fetal growth. Placental disorders may cause stillbirth through several mechanisms including vascular dysfunction, angiogenic imbalance, inflammation, and oxidative stress. However, to date the specific underlying pathogenetic pathway has not been clearly identified.
Single-gene mutations may cause abnormal placental function. These mutations can lead to fetal death at midgestation because of dysfunction in pathways involved in placental growth, development, or angiogenesis. Endothelial nitric oxide synthase (eNOS), Klotho, hypoxic inducible factor-1α (HIF-1α), and tumor necrosis factor (TNF-α) genes are critical for a number of such pathways. No reports to date have specifically addressed their association with SB using placental tissues from well-characterized cases and controls.
Endothelial nitric oxide synthase is the rate-limiting enzyme in nitric oxide (NO) production, the most potent smooth muscle vasodilator and the molecule responsible for the vascular adaptation occurring in normal pregnancy. Transgenic mice deficient in eNOS have abnormal prenatal development, fetal growth restriction, and late fetal death. Studies have shown that NO concentrations are significantly reduced in amniotic fluid of pregnancies complicated by SB compared with live birth controls.
The Klotho gene encodes for a single-pass transmembrane protein. The extracellular domain is subject to ectodomain shedding, and as a result, the Klotho protein exists in 2 forms: membrane and secreted Klotho. The latter acts as a humoral factor with pleiotropic activities, including suppression of growth factor signaling and oxidative stress, maintenance of vascular integrity, and inflammation. In mice, a defect in Klotho expression leads to a syndrome resembling the aging process, whereas its overexpression leads to an extended life span. Moreover, reduced Klotho levels have been associated with endothelial dysfunction, altered angiogenesis, and vasculogenesis. To date, Klotho-related pathways have not been investigated in obstetrics.
Hypoxia-inducible factors are essential key regulators of placental vascularization and trophoblast differentiation. They mediate the response to hypoxia by inducing genes involved in glycolysis, red blood cell production, and angiogenesis. They are also involved in placental development through interaction with hormones, growth factors, and cytokines. Hypoxia-inducible factor-deficient embryo mice exhibit defective placental vascularization and cell differentiation, leading to fetal death. In humans, hypoxia-inducible factors were found to be overexpressed in placentas obtained from pregnancies complicated by preeclampsia and intrauterine growth restriction.
Tumor necrosis factor-α is a well-known potent proinflammatory cytokine, produced by trophoblast, leucocytes, and macrophages. It plays a role in insulin resistance and in the pathogenesis of the metabolic syndrome such as endothelial activation, obesity, and dyslipidemia. Pregnancies complicated by preeclampsia have increased levels of TNF-α in plasma and amniotic fluid and gene overexpression in their placenta. It also appears to play a role in preterm labor and preterm premature rupture of the membranes.
Based on their biological functions, eNOS, HIF-1, Klotho, and TNF-α are plausible candidate genes to be associated with SB, and polymorphisms within these genes could potentially be important as genetic markers of susceptibility to SB. Therefore, our objective was to compare the distribution of placental eNOS, Klotho, HIF-1α, and TNF-α gene single-nucleotide polymorphisms (SNPs) in a well-characterized cohort of unexplained SB and control live births.
Materials and Methods
Study populations
Placenta samples were collected from stillbirths and control live births between January 2005 and January 2009 at the Policlinico Hospital of Modena, Italy. The diagnosis of SB was based on the World Health Organization criteria defined as fetal death at longer than 22 weeks of gestation or with birthweight of 500 g or greater if the gestational age was unknown. Controls were obtained from women having an uneventful pregnancy at term. The institutional review board for the diagnostic evaluation and sample collection of stillborn infants was approved by the Italian Ministry of Education, University, and Research; subsequent institutional review board approval was obtained for genetics and deoxyribonucleic acid (DNA)-based research.
All stillbirths underwent evaluation according to a specific standardized clinical protocol. Those SBs with a negative work up were considered unexplained and eligible for inclusion in the study. On the contrary, SBs with positive work-up because of congenital abnormality, twin-to-twin transfusion syndrome, infection, and intrapartum asphyxia were considered explained and were excluded.
Standardized clinical protocol
Maternal serology, microbiology, and placental and fetal karyotypes were obtained. Maternal serologic testing included: toxoplasmosis, rubella, cytomegalovirus serology, and human immunodeficiency virus; serum parvovirus titers; thyroid function; indirect Coombs; and inherited and acquired thrombophilias. All tests were systematically performed following enrollment. Microbiology included the following: gram staining; aerobic and anaerobic cultures on vaginal, placental, and fetal swabs (auricular and nasal-pharyngeal cavity); and fetal blood drawn by an intracardiac fetal puncture prior to autopsy. In addition, a complete fetal autopsy was performed by a perinatal pathologist, and a detailed placental gross and histological examination was also undertaken.
At delivery, 4 placental biopsies were taken. Central chorionic tissue areas were dissected and the maternal decidual and amniotic membrane were removed. Tissues were stored at −80°C until use.
All information were recorded and, once the diagnostic data were available, were evaluated in a systematic and unbiased manner by a panel of 2 obstetricians, 1 neonatologist, and 1 pathologist, who reviewed the cases and classified them based on the Wigglesworth stillbirth classification systems. Subsequently, SB cases were stratified according to birthweight: appropriate for gestational age (AGA-SB; 10th percentile or greater) and small for gestational age (SGA-SB, less than the 10th percentile); and gestational age (SBs occurring before 32 weeks [<32 weeks-SB] and after 32 weeks [>32 weeks-SB]).
Genotyping
DNA extraction and amplification were performed at the Mother-Infant Research Laboratory at the Policlinico Hospital of Modena, Italy. Genotyping assay was done at the University of Texas Medical Branch Laboratory (Galveston, TX).
Researchers and laboratory personnel were blinded to the case/control status of the biologic samples. DNA was extracted from frozen placental tissues using a QIAmp DNA microkit by QIAGEN (Valencia, CA) in accordance with the manufacturer’s protocol. We excluded placentas the samples of which either failed to genotype or had greater than 30% of their genotype results missing.
We selected 5 SNPs for eNOS (rs1007311, rs891512, rs1800779, rs1800783, and rs1800780), 2 SNPs for Klotho (rs9536314 and rs564481), 1 for HIF-1α (rs11549465), and 1 for TNF-α (rs1800629).
SNP analysis was performed using the Taqman SNP allele discrimination assay for sample genotyping (Applied Biosystems, Inc, Foster City, CA). Predesigned Taqman assays were used and genotypes were determined using Applied Biosystems automated Taqman genotyping software, SDS version 2.1.
Analysis
The clinical characteristics of the study populations were compared using a χ 2 or Fisher exact test. SNPs were tested for Hardy-Weinberg equilibrium. Genotype distribution and allelic frequencies between groups were compared using a χ 2 or Fisher exact test. Analysis was done first between SBs and controls and then stratified by birthweight (SGA-SB and AGA-SB) and gestational age (<32 weeks-SB and >32 weeks-SB). Because this was an exploratory study, no power calculation was conducted, no adjustments were made for multiple comparisons, and all comparisons are reported. All statistical analyses were performed with SPSS version 15.0 (SPSS Inc, Chicago, IL).
Results
We collected a total of 67 consecutive SB cases, and 52 were considered unexplained and enrolled in the study. These were matched with 52 controls. After excluding placental samples that failed to genotype or had greater than 30% of their genotype missing (2 SBs and 6 controls), we had 96 placentas (50 SB cases and 46 controls) available for analysis. Based on birthweight, 25 SBs were grouped as AGA and 25 as SGA. When stratified by gestational age, 24 stillbirths were less than 32 weeks and 26 were longer than 32 weeks.
Of the 9 selected SNPs, the assay for 1 SNP (eNOS SNP rs1800780) failed to genotype in greater than 30% of the samples and was excluded from further analysis. The distribution of the genotypes in the overall cohort was in Hardy-Weinberg equilibrium.
The clinical characteristics of women with SBs and controls are listed in Table 1 . No significant differences emerged between the 2 groups, other than gestational ages and birthweights.
| Characteristic | SB (n = 50) | Controls (n = 46) |
|---|---|---|
| Maternal age, y a | 30.9 ± 5.2 (19–40) | 29.5 ± 4.9 (23–39) |
| Gestational age at delivery, wks a | 33.8 ± 5.1 (24–41) | 37.8 ± 6.8 (29–41) |
| Nulliparity, n (%) b | 28 (56) | 30 (65.2) |
| Birthweight, g a | 1952 ± 1049 (325–3900) | 2748 ± 789 (1165–4030) |
| Male sex, n (%) b | 21 (42) | 23 (50) |
The histopathological examination was performed in 45 SB cases (90%), and evidence of abnormal placental growth and vascular development was shown in a significant proportion of SB cases (40 SBs, 80% of total cases). Placental karyotyping failed in 16 of 50 SB cases (32%). In the remnant, normal karyotype was found.
Genotypic distributions and allelic frequencies of cases and controls for all the investigated SNPs are described in Tables 2 and 3 , respectively. When the analysis was performed for overall SB, there were no significant differences in genotype distribution or allelic frequencies between SB and control placentas for any of the analyzed SNPs. However, when the analysis was stratified by birthweight, different genotype distribution was found for eNOS rs1800783 when comparing AGA-SB vs control ( P = .02) and AGA-SB vs SGA-SB ( P = .002). Most importantly, carriers of the allele A of the eNOS rs1800783 were more frequent in AGA-SB compared with both controls ( P = .03; odds ratio, 2.36; 95% confidence interval, 1.1–5.3) and SGA-SB ( P = .001; odds ratio, 0.24; 95% confidence interval, 0.09–0.60) as shown in the Figure . None of the other analyzed SNPs were significantly different between the studied subgroups.
| Variable | SB (n = 50) | SGA SB (n = 25) | AGA SB (n = 25) | CTR (n = 46) | <32 wks (SB = 24) | >32 wks (SB = 26) |
|---|---|---|---|---|---|---|
| eNOS rs1007311 | ||||||
| AA | 16 (32) | 7 (28) | 9 (36) | 12 (26) | 9 (37) | 7 (27) |
| AG | 22 (44) | 11 (44) | 11 (44) | 28 (61) | 10 (42) | 12 (46) |
| GG | 12 (24) | 7 (28) | 5 (20) | 6 (13) | 5 (21) | 7 (27) |
| eNOS rs891512 | ||||||
| AA | 4 (8) | 1 (4) | 3 (12) | 4 (9) | 2 (8) | 2 (8) |
| AG | 21 (42) | 11 (44) | 10 (40) | 17 (37) | 11 (46) | 10 (38) |
| GG | 25 (50) | 13 (52) | 12 (48) | 25 (54) | 11 (46) | 14 (54) |
| eNOS rs1800779 | ||||||
| AA | 17 (34) | 10 (40) | 7 (28) | 12 (26) | 7 (29) | 10 (38) |
| AG | 21 (42) | 12 (48) | 9 (36) | 23 (50) | 10 (42) | 11 (42) |
| GG | 12 (24) | 3 (12) | 9 (36) | 11 (24) | 7 (29) | 5 (19) |
| eNOS rs1800783 a | ||||||
| AA | 17 (34) | 3 (12) | 15 (60) | 12 (26) | 9 (37) | 8 (31) |
| AT | 20 (40) | 13 (52) | 6 (24) | 24 (52) | 9 (38) | 11 (42) |
| TT | 13 (26) | 9 (36) | 4 (16) | 10 (22) | 6 (25) | 7 (27) |
| Klotho rs564481 | ||||||
| CC | 23 (46) | 12 (48) | 11 (44) | 22 (48) | 11 (46) | 12 (46) |
| CT | 21 (42) | 11 (44) | 10 (40) | 19 (41) | 10 (42) | 11 (42) |
| TT | 6 (12) | 2 (8) | 4 (16) | 5 (11) | 3 (12) | 3 (12) |
| Klotho rs9536314 | ||||||
| GG | 1 (2) | 1 (4) | 0 (0) | 2 (4) | 1 (4) | 0 (0) |
| GT | 11 (22) | 4 (16) | 7 (28) | 11 (24) | 6 (25) | 5 (19) |
| TT | 38 (76) | 20 (80) | 18 (72) | 33 (72) | 17 (71) | 21 (81) |
| HIF-1α rs11549465 | ||||||
| CC | 41 (82) | 20 (80) | 21 (84) | 40 (87) | 18 (75) | 23 (88) |
| CT | 8 (16) | 5 (20) | 3 (12) | 5 (11) | 5 (21) | 3 (12) |
| TT | 1 (2) | 0 (0) | 1 (4) | 1 (2) | 1 (4) | 0 (0) |
| TNF-α rs1800629 | ||||||
| AA | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| AG | 5 (10) | 2 (8) | 3 (12) | 8 (17) | 2 (8) | 3 (12) |
| GG | 45 (90) | 23 (92) | 22 (88) | 38 (83) | 22 (92) | 23 (88) |
a CTR vs AGA-SB vs SGA-SB: P = .004; CTR vs AGA-SB: P = .02; AGA-SB vs SGA-SB: P = .002.
| Variable | SB (n = 50) | SGA-SB (n = 25) | AGA-SB (n = 25) | CTR (n = 46) | <32 wks SB (n = 48) | >32 wks SB (n = 52) |
|---|---|---|---|---|---|---|
| eNOS rs1007311 | ||||||
| A | 54 (54) | 25 (50) | 29 (58) | 52 (57) | 28 (58) | 26 (50) |
| G | 46 (46) | 25 (50) | 21 (42) | 40 (43) | 20 (42) | 26 (50) |
| eNOS rs891512 | ||||||
| A | 29 (29) | 13 (26) | 16 (32) | 25 (27) | 15 (31) | 14 (27) |
| G | 71 (71) | 37 (74) | 34 (68) | 67 (73) | 33 (69) | 38 (73) |
| eNOS rs1800779 | ||||||
| A | 55 (55) | 32 (64) | 23 (46) | 47 (51) | 24 (50) | 31 (60) |
| G | 45 (45) | 18 (36) | 27 (54) | 45 (49) | 24 (50) | 21 (40) |
| eNOS rs1800783 a | ||||||
| A | 54 (54) | 19 (38) | 36 (72) | 48 (52) | 27 (56) | 27 (52) |
| T | 46 (46) | 31 (62) | 14 (28) | 44 (48) | 21 (44) | 25 (48) |
| Klotho rs564481 | ||||||
| C | 67 (67) | 35 (70) | 32 (64) | 63 (68) | 32 (67) | 35 (67) |
| T | 33 (33) | 15 (30) | 18 (36) | 29 (32) | 16 (33) | 17 (33) |
| Klotho rs9536314 | ||||||
| G | 13 (13) | 6 (12) | 7 (14) | 15 (16) | 8 (17) | 5 (10) |
| T | 87 (87) | 44 (88) | 43 (86) | 77 (84) | 40 (83) | 47 (90) |
| HIF-1α rs11549465 | ||||||
| C | 90 (90) | 45 (90) | 45 (90) | 85 (92) | 41 (85) | 49 (94) |
| T | 10 (10) | 5 (10) | 5 (10) | 7 (8) | 7 (15) | 3 (6) |
| TNF-α rs1800629 | ||||||
| A | 5 (5) | 2 (4) | 3 (6) | 8 (9) | 2 (4) | 3 (6) |
| G | 95 (95) | 48 (96) | 47 (94) | 84 (91) | 46 (96) | 49 (94) |
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