Molecular Human Reproduction, Vol. 7, No. 2, 201-203,
February 2001
© 2001 European Society of Human Reproduction and Embryology
Implantation and pregnancy |
The functionally important IL-10 promoter polymorphism (–1082G
A) is not a major genetic regulator in recurrent spontaneous abortions
1 Department of Medical Microbiology, University of Oulu, P.O.Box 5000, 90014 OULU, and 2 The Finnish Red Cross Blood Transfusion Service, Tissue Typing Laboratory, Helsinki, Finland
Abstract
Enhanced secretion of anti-inflammatory Th2 cytokines is a characteristic feature in normal physiological pregnancy. In recurrent spontaneous abortions (RSA), however, defective production of interleukin-10 (IL-10) and other Th2 cytokines has been shown in humans. Association studies have shown that a base exchange polymorphism (guanine
adenine) at position –1082 of the IL-10 promoter is associated with differential IL-10 production. Since factors contributing to IL-10 production appear to be important in RSA, we studied the IL-10 genotypes of 38 Finnish women with a history of three or more consecutive abortions and 131 ethnically matched healthy controls. No significant differences in the –1082 allele or genotype frequencies were found between the controls and the RSA women. The present study suggests that the IL-10 –1082 (GA) polymorphism is not a major genetic regulator in RSA.
abortion/IL-10/polymorphism/pregnancy
Introduction
Spontaneous abortion is one of the most common complications of pregnancy. The aetiology of recurrent spontaneous abortions (RSA) remains unresolved in ~50% of the cases. The possible immunological aetiologies of pregnancy failure have been researched intensively (for review, see Clark et al., 1999). In normal pregnancy, a shift to Th2 cytokine production with abundant interleukin-10 (IL-10) is considered important. In RSA, on the contrary, maternal decidual T-cells (Piccini et al., 1998
) and mitogen-activated (PBMC) blood mononuclear cells (Marzi et al., 1996) show decreased expression of IL-10. There is also evidence of a diminished Th2 type immune response to placental and trophoblastic antigens in RSA (Hill et al., 1995; Raghupathy et al., 1999). Serum IL-10 concentrations are also low in pre-eclampsia, which is another common disorder of pregnancy (Hennessy et al., 1999). Thus, IL-10 could be an important anti-inflammatory cytokine contributing to the outcome of pregnancy (Hanna et al., 2000).
During pregnancy, IL-10 is produced locally in the feto–placental unit by cytotrophoblasts, and it up-regulates the human leukocyte antigen (HLA)-G expression of cytotrophoblasts at the feto–maternal barrier. It has been suggested that this protects the fetus from rejection (Moreau et al., 1999). IL-10 has important modulatory effects against pro-inflammatory cytokines, especially interferon-
(IFN-) and tumour necrosis factor-
(TNF-), both of which have been shown to be detrimental to the feto–placental unit in a murine model (Chaouat et al., 1995), while high serum TNF- values have been reported in women undergoing RSA (Mallman et al., 1991; Szekeres-Bartho et al., 1996; Deneys and De Bruyere, 1997). However, contradictory results have also been published (Schust and Hill, 1996).
IL-10 production is under genetic control in humans. It has been shown that adenine (A) at the site –1082 in the promoter region of the IL-10 gene is associated with low and guanine (G) with high production of IL-10 (Turner et al., 1997). Therefore, we were interested to see whether this polymorphism is a genetic regulator in RSA.
Materials and methods
Patient samples
DNA samples were available from 38 women belonging to the original study group of 59 Finnish women (mean age 33 years, range 26–46) with a history of at least three consecutive spontaneous abortions in early pregnancy (mean number of abortions three, range 3–8). All women had regular menstrual cycles and were healthy. Parental karyotypes were analysed. The women underwent careful clinical examinations, including hysteroscopy and serial endometrial biopsies, as well as analyses of tissue antibodies and autoantibodies. Plasma glucose and thyroid-stimulating hormone concentrations were also measured. In addition, a possible infectious aetiology was studied by assessing the immunoglobulin (Ig)G and IgM antibodies to cytomegalovirus and Toxoplasma gondii using enzyme immunoassay. Moreover, microbiological samples were obtained from the cervix and uterine cavity during hysteroscopy, and Chlamydia trachomatis, Neisseria gonorrhoea, Listeria monocytogenes and herpes simplex viruses were cultured as described earlier (Tulppala et al., 1993). No obvious cause for RSA was found in our study group, and the abortions were thus classified as `unexplained'. A total of 27 women had no previous children and 11 had had one to two children before the episode of consecutive abortions.
Reference population
The control group, included to allow statistical comparisons, consisted of 131 healthy Finnish adults of whom 80 were female (mean age of the female subjects 46 years, range 24–64; mean age of the male subjects 48 years, range 32–69).
Determination of the –1082 IL-10 polymorphism
Total genomic DNA from peripheral blood leukocytes was extracted by the salting out procedure (Miller et al., 1988). IL-10 –1082GA polymorphism detection was done with allele refractory mutation system (ARMS)–polymerase chain reaction (PCR). The target sequences for the primers were obtained from GenBank (accession number X78437). An additional mismatch (underlined) was inserted into the 3' penultimate nucleotide of allele-specific forward primers to prevent non-specific amplification of the opposite allele
The A allele was amplified with the forward primer, fpena (5'-AAC ACT ACT AAG GCT TCT TTG GGT A-3'), and the G allele was amplified with the primer, fpeng (5'-AAC ACT ACT AAG GCT TCT TTG GGT G-3'). The primer, grev2 (5'-GTA AGC TTC TGT GGC TGG AGT C-3'), was used as a reverse primer in both reactions. These reactions amplify allele-specific fragments of 161 bases of the promoter of the IL-10 gene. An additional upstream sense primer, afor3 (5'-TTT CCA GAT ATC TGA AGA AGT CCT G-3'), was included with the reverse primer grev2 in both PCR reactions to amplify an internal control band (313 bp). PCR was performed in a total volume of 10 µl (100 ng of genomic DNA, 1x PCR buffer with 1.5 mmol/l MgCl2, 200 µmol/l of each nucleotide, 0.8 IU Taq polymerase and 0.5 µmol/l of each primer). The cycling conditions were as follows: an initial denaturation at 95°C for 5 min, followed by 30 cycles at 95°C for 30 s and 63°C for 30 s. The final extension step was at 72°C for 5 min. The PCR products were visualized under UV light with ethidium bromide stain after agarose gel electrophoresis. The ARMS–PCR method was validated by determining the IL-10–1082 alleles of 36 samples by sequencing (ABI Prism; Perkin Elmer, Foster City, CA, USA). The genotypes obtained with both methods were identical.
Statistical analysis
Fisher's exact test was used to compare the allele and genotype frequencies. Additionally, we performed simulations to assess the power of the sample to detect an allele association. We defined the relative risk (RR) for the –1082A substitution as 1.0 when the allele frequencies in the affected subjects did not differ from those recorded in our control group (0.58). This locus attains a maximum value of RR 1.7 when all the affected chromosomes have –1082A substitution. By simulation, we created 10 000 artificial data sets comparable to the size of our study group of affected chromosomes (n = 38) and comparable to the relative risks of 1.2 (–1082A allele frequency 1.2x0.58 = 0.70), 1.4 (–1082A allele frequency 1.4x0.58 = 0.81) and 1.6 (–1082A allele frequency 1.6x0.58 = 0.93). The strengths of the detected associations in the 
2 test at the probability level of 95% in three different data sets are reported.
Results
The IL-10 polymorphism was analysed in 38 women with RSA and 131 ethnically matched healthy control subjects. There were no significant differences in the IL-10 (–1082GA) genotype or allele frequencies between the RSA women and the controls (Table I). All the samples in our series could be genotyped with the ARMS–PCR method described above. The allelic frequencies of IL-10 –1082 in our control population closely resembled those published earlier in Finland (Helminen et al., 1999). To study the power of our study design, e.g. how small a genetic effect we would be able to exclude from our data set, we used a permutation test. Based on our results, our power to detect an allele association was good enough to exclude a RR of >1.4 for the locus in RSA. If the locus had had a RR of 1.6, we would have found a significant association at P < 0.00001, and if the locus had had a RR of 1.4, we would still have found the association at P < 0.02. Very small effects, however, could not be excluded.
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Discussion
Normal human pregnancy is characterized by a Th2 shift (Wegman et al., 1993; Raghupathy et al., 1999), which involves a decrease in the production of IL-2 and IFN- and an increase in the production of IL-4 and IL-10 (Marzi et al., 1996). It has recently been suggested that HLA class II associations with RSA might be explained by a predisposition to hypersecretion of TNF-, as the encoding gene TNFA is located in linkage disequilibrium with the HLA region on 6p21 (Christiansen, 1999). Like IL-10, TNF- production is under genetic control. The TNFA promoter region polymorphism (–308GA) regulating TNF- transcription was, however, not associated with preterm deliveries (Dizon-Townson et al., 1997). Although the TNFA promoter region has not been studied in RSA, the NcoI restriction site in the TNFB gene, which is in strong linkage disequilibrium with the –308GA substitution of TNFA, has been studied previously (Laitinen et al., 1992); this study showed no evidence of an association between the NcoI polymorphism of the TNFB gene and RSA.
To our knowledge, IL-10 polymorphisms have not been previously characterized in RSA. We became interested in IL-10 as an important antagonist for TNF- and a major anti-inflammatory cytokine at the feto–placental interface. However, no association between RSA and the IL-10 –1082 promoter region polymorphism could be found. Since the –1082A substitution is common in the general population (allele frequency 0.58), the maximal genetic effect that the allele can have in the development of the disease is not more than 1.7-fold compared with the risk of the disease in the population at large. However, even minor genetic regulators, when they are common in the population, can have a significant role in the molecular genetic aetiology of the disease. While the demonstration or exclusion of the possible effect of a rare allele requires large association studies, the evaluation of the genetic effects of common alleles needs less statistical power. Even though our study group was rather small, we had adequate power to detect genetic effects that increase the risk of the disease to more than 1.4-fold. However, to exclude effects smaller than that (RR <1.4), a large association study would have been needed.
Our results suggest that a –1082A substitution in the regulation of IL-10 production is unlikely to be the major cause of the diminished IL-10 production in the women with RSA, as interpreted within the limitation of a small study group. Thus, other mechanisms regulating the Th1/Th2 balance during pregnancy are more important. Progesterone-induced blocking factor (PIBF) and leukaemia inhibitory factor (LIF) contribute to the Th2 shift during pregnancy (Szekeres-Bartho et al., 1996; Piccini et al., 1998). However, even more general factors, possibly genetic ones, have been suggested to explain the tendency of RSA women to secrete Th1 type cytokines instead of Th2 type molecules. An enhanced Th1 type immune reaction against trophoblast antigens has been demonstrated in non-pregnant women with previous RSA compared with women with successful pregnancies (Hill et al., 1995). This is highly interesting, as no pregnancy-related regulatory mechanisms were influencing the PBMC cytokine secretion in the study setting of Hill et al. Based on their findings, it is reasonable to speculate that the diminished IL-10 response in women with RSA is not exclusively due to pregnancy-related factors. This hypothesis naturally highlights the significance of genetic factors in IL-10 regulation among RSA patients. The present study shows that the IL-10 –1082 (GA) polymorphism is not a feasible candidate in this respect.
Acknowledgments
The authors would like to thank Dr Saija Koskimies and Dr Maija Tulppala for providing the sample set, Dr Vesa Ollikainen for the simulation studies and Mrs Eeva-Liisa Heikkinen for her skilful technical assistance. This study was supported by a grant from the University of Oulu Foundation (to J.K.).
Notes
3 To whom correspondence should be addressed. E-mail: jari.karhukorpi{at}oulu.fi 
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Submitted on August 17, 2000; accepted on December 5, 2000.
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