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ORIGINAL RESEARCH |
From the Departments of Gynecologic Endocrinology and Reproductive Medicine and Obstetrics and Gynecology, University of Vienna School of Medicine, Vienna, Austria.
Address reprint requests to: Fritz Nagele, MD, Department of Gynecologic Endocrinology and Reproductive Medicine, Waehringer Guertel 18–20, Vienna A-1090, Austria; E-mail: fritz.nagele{at}akh-wien.ac.at.
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ABSTRACT |
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METHODS: In a case control study, we studied 125 women with a history of three or more consecutive pregnancy losses before 20 weeks’ gestation and 137 healthy controls with at least two live births and no history of pregnancy loss. Peripheral venous puncture, DNA extraction, and polymerase chain reaction followed by restriction fragment length polymorphism analysis were used to genotype women for the presence of the A218C polymorphism in intron 7 of the tryptophan hydroxylase gene.
RESULTS: Allele frequencies among women with idiopathic recurrent miscarriage and controls were 32.4% and 38.7%, respectively, for allele A (wild type) and 67.6% and 61.3%, respectively, for allele C (mutant). No association between the presence of allele C and idiopathic recurrent miscarriage was found (P = .3; odds ratio 1.31; 95% confidence interval 0.93, 1.87). Genotype frequencies also were not significantly different between the study group (C/C: 44.8%; A/C: 45.6%; A/A: 9.6%) and the control group (C/C: 37.2%; A/C: 48.2%; A/A: 14.6%; P = .2). Between women with primary and women with secondary idiopathic recurrent miscarriage, no statistically significant differences with respect to allele frequencies were observed (63% vs 62% for allele C and 31% vs 38% for allele A; P = .3).
CONCLUSION: The A218C polymorphism in intron 7 of the tryptophan hydroxylase gene is not associated with idiopathic recurrent miscarriage.
Immunologic factors are believed to play a major etiologic role in spontaneous miscarriage.1 Physiologically, the maternal immune response is suppressed selectively during pregnancy, as demonstrated by the reduced production of proinflammatory cytokines,2 the increased production of asymmetrically glycosylated blocking antibodies,3 and lymphatic expression of the progesterone-induced blocking factor.4
Another mechanism to protect the fetus from maternal alloimmune rejection involves local catabolism of tryptophan at the placental level.5 Tryptophan deficiency leads to inhibition of maternal T cell proliferation via a tryptophan-sensitive cell cycle arrest.6 This is in accordance with the finding of decreased systemic tryptophan plasma concentrations during physiologic pregnancy.7 Also, the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase is expressed by the syncytiotrophoblast and mediates a local placental milieu of low tryptophan concentrations. Munn et al5 showed that expression of indoleamine 2,3-dioxygenase is required to prevent rejection of the fetus in murine pregnancy. In vitro studies involving peripheral blood mononuclear cells suggest that in humans, macrophages suppress T cell proliferation through an indoleamine 2,3-dioxygenase–mediated tryptophan catabolism.8 Given the crucial role of tryptophan in maternal-fetal immune homeostasis, the tryptophan-degrading enzyme tryptophan hydroxylase might be involved in the pathophysiology of fetal rejection and subsequent miscarriage.
Tryptophan hydroxylase catalyzes the biopterin-dependent mono-oxygenation of tryptophan to 5-hydroxytryptophan, the decarboxylated form of the neurotransmitter serotonin.9 Expression of tryptophan hydroxylase is limited to specific cells—eg, mast cells, mononuclear leukocytes, beta cells of the islets of Langerhans, raphe neurons, and intestinal and pancreatic enterochromaffin cells.10–14 Tryptophan hydroxylase is the rate-limiting enzyme in the synthesis of serotonin.15
The human tryptophan hydroxylase gene is located on the short arm of chromosome 11 at p15.4–p15.5. A polymorphism consisting of an A
C substitution at position 218 in intron 7 of the gene has been described.16 This polymorphism is associated with increased local tryptophan concentrations and reduced serotonin concentrations, a history of suicide attempts among alcoholic individuals, suicidal behavior among depressed patients, bipolar disorder, and impulsive-aggressive behavior.17–20
In the present study, we attempted to establish an association between the A218C polymorphism of the tryptophan hydroxylase gene and the occurrence of idiopathic recurrent miscarriage. We hypothesized that the incidence of this polymorphism, associated with changes in local tryptophan concentrations, would be increased among women with idiopathic recurrent miscarriage.
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MATERIALS AND METHODS |
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The diagnosis of idiopathic recurrent miscarriage was made on the basis of a documented history of at least three spontaneous, consecutive miscarriages before 20 weeks’ gestation (each time with the same partner). Each woman underwent a diagnostic work-up to rule out a verifiable cause of the recurrent miscarriages. Diagnostic procedures included hysteroscopy; paternal and maternal karyotyping; cervical cultures for chlamydia, Ureaplasma, and mycoplasma; a comprehensive hormonal status evaluation; and evaluation of antiphospholipid syndrome with immunoglobulin (Ig) M and IgG anticardiolipin antibody assessment and lupus anticoagulant testing. Among these women, primary recurrent miscarriage was defined as no history of a pregnancy lasting more than 20 weeks. Secondary recurrent miscarriage was defined as a history of at least one pregnancy lasting more than 20 weeks.
The control group consisted of 137 women with at least two live births and no history of miscarriage. Patients were recruited consecutively at our outpatient clinic for postmenopausal disorders between January 1996 and September 1999. All control women were postmenopausal, to rule out possible future miscarriages after inclusion in the study. Written informed consent was obtained from participating women. To avoid confounding by ethnicity, only white women were included in the study and control groups. To avoid confounding by genetic admixture, only women whose parents were of the same ethnicity were enrolled.
Blood was drawn from the antecubital vein, and DNA was extracted using the QIAGEN System (QIAamp DNA Blood Midi Kit; QIAGEN GmbH, Hilden, Germany). DNA was stored at 4C until analyzed. Following the polymerase chain reaction (PCR) strategy described by Nielsen et al,16 we amplified DNA with primers from intron 7 of the tryptophan hydroxylase gene. The PCR conditions comprised an initial denaturation step at 94C for 4 minutes; followed by 45 cycles at 94C for 1 minute, 55C for 1 minute, and 72C for 2 minutes; and a final extension step at 72C for 4 minutes. Oligonucleotide primers (forward: 5'-TTCCATCCGTCCTGTGGCT-GGTTA-3'; reverse: 5'-TTTGAACAGCCTCCTCTG-AAGCGC-3') were used to generate an 880 base-pair product. With the use of a restriction fragment length polymorphism strategy, the PCR product was digested by BfaI (New England Biolabs, Beverly, MA), separated on a 2% agarose gel and stained with SYBR Green I (FMC, Bio Products Europe, Denmark) (Figure 1
). The polymorphic allele containing the BfaI restriction site was designated allele C. The wild-type allele without the BfaI restriction site was designated allele A.
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2 test. The odds ratio (OR) was used as a measure of the strength of the association between allele and genotype frequencies and idiopathic recurrent miscarriage. All P values were two-tailed, and 95% confidence intervals (CIs) were calculated. P < .05 was considered statistically significant. We calculated the power to detect a difference between women with idiopathic recurrent miscarriage and control women for the tryptophan hydroxylase polymorphism. Data from Table 1 were used. For the comparison, we used a total of 262 women (524 alleles). A power of 27% was achieved. We calculated the necessary sample to achieve a power of 80% and an 
error of .05 using the Yates correction factor.
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RESULTS |
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Allele frequencies among women with idiopathic recurrent miscarriage and controls were 32.4% and 38.7%, respectively, for allele A (wild type) and 67.6% and 61.3%, respectively, for allele C (mutant). No association between the presence of allele C and idiopathic recurrent miscarriage was observed (P = .3; OR 1.31; 95% CI 0.93, 1.87). Genotype frequencies also were not significantly different between the study group (C/C: 44.8%; A/C: 45.6%; A/A: 9.6%) and the control group (C/C: 37.2%; A/C: 48.2%; A/A: 14.6%; P = .2) (Table 1
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Between women with primary and women with secondary idiopathic recurrent miscarriage, no statistically significant differences with respect to allele frequencies were observed (63% vs 62% for allele C and 31% vs 38% for allele A; P = .3).
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DISCUSSION |
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The A218C polymorphism of the tryptophan hydroxylase gene is an intronic mutation and therefore unlikely to change the amino acid sequence of any viable exon directly. However, the A218C site is located in a potential GATA transcription factor binding site, and this might influence tryptophan hydroxylase gene expression.21 Alternatively, this polymorphism might be in linkage disequilibrium with other mutations of the tryptophan hydroxylase gene coding sequence, might be in a regulatory region, or might be in a neighboring gene. Although the exact mechanism linking the A218C polymorphism to reduced expression of the gene product remains unknown, the functional relevance of this polymorphism has been demonstrated for a series of clinical syndromes—eg, bipolar disorder, suicide attempts, and suicidal behavior.17–20
A search of PubMed for the period from January 1966 to December 2000 using the search terms "tryptophan," "tryptophan hydroxylase," "abortion," "recurrent abortion," "miscarriage," "recurrent miscarriage," "mutation," and "polymorphism" did not reveal any reports of a genetic variant of the tryptophan hydroxylase gene among women with idiopathic recurrent miscarriage. However, our study fell short of determining a significant effect of the tryptophan hydroxylase gene on idiopathic recurrent miscarriage. We found no association between the tryptophan hydroxylase genotype and allele frequencies and idiopathic recurrent miscarriage in a representative white population. The possibility that alternative mutations or polymorphisms of the tryptophan hydroxylase gene might segregate with idiopathic recurrent miscarriage in this population or in a population with a different ethnic background cannot be ruled out by our study.
In summary, we have demonstrated that the tryptophan hydroxylase polymorphism in intron 7 of the tryptophan hydroxylase gene is not associated with idiopathic recurrent miscarriage in a white population. These data indicate that the tryptophan hydroxylase gene is not a candidate gene for this condition.
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Footnotes |
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Received January 23, 2001. Received in revised form May 23, 2001. Accepted June 7, 2001.
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REFERENCES |
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