Mol. Hum. Reprod. Advance Access originally published online on June 6, 2007
Molecular Human Reproduction 2007 13(8):537-540; doi:10.1093/molehr/gam041
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Estrogen receptor alpha polymorphisms and fertility in populations with different reproductive patterns
1 Department of Genetics and Molecular Biology, University ‘La Sapienza’, P.le A. Moro 5, 00185 Rome, Italy 2 CNR Institute of Molecular Biology and Pathology, c/o Department of Genetics and Molecular Biology, University ‘La Sapienza’, P.le A. Moro 5, 00185 Rome, Italy 3 Department of Animal and Human Biology, University ‘La Sapienza’, P.le A. Moro 5, 00185 Rome, Italy 4 Department of Biology, University ‘Tor Vergata’, Via della Ricerca Scientifica 1, Rome, Italy
5 Correspondence address. Tel: +390 64 9912825; Fax: +39 44 56866; E-mail: rosamaria.corbo{at}uniroma1.it
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Abstract |
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The estrogen receptor (ER) plays an important role in mediating estrogen action on target tissues. ER-alpha, the most abundant, is found in all human reproductive tissues and studies on alpha-ER knockout mice have highlighted its role in reproduction. ER-alpha gene (ESR1) polymorphisms have been associated with a variety of disorders including human infertility. In this study, we examined the association of ESR1 PvuII and XbaI polymorphisms with fertility in two populations with different reproductive patterns and precisely in a sample of healthy Italian men and women (n = 178) and in a sample of healthy African-Ecuadorian women (n = 57). ESR1 xx and ppxx genotypes among the Italian men were found to be associated with an above-median number of children (P = 0.01 and P = 0.004, respectively). ESR1 pp genotype among the Italian women showed a tendency to be associated with a lower number of abortions (P = 0.04), whereas ESR1 pp and ppxx genotypes among African-Ecuadorian women were associated with a higher number of children (P = 0.02 and P = 0.03, respectively). These results are consistent with previous observations indicating a role of ESR1 genotypes in human infertility and give insight into the complex interactions between genotypes and reproductive behaviours in human populations.
Key words: African-Ecuadorian women/ESR1 gene polymorphisms/fertility/Italian men and women
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Introduction |
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The estrogen receptor (ER) plays an important role in mediating estrogen action on target tissues. Two subtypes of ER are known, ER-alpha encoded by the ESR1 gene on chromosome 6 (Menasce et al., 1993) and ER-beta encoded by the ESR2 gene on chromosome 14 (Enmark et al., 1997). ER-alpha, the first identified and the most abundant, is found in all human reproductive tissues. Its part in reproduction has been elucidated by studies on male and female alpha-ER knockout (ERKO) mice that showed complete infertility (Matthews and Gustafsson, 2003). The female mice were infertile because they were anovulatory, had altered pituitary gonadotrophin concentrations and impaired uterine response to estrogen (Hewitt and Korach, 2003), whereas the absence of ER-alpha in the alpha-ERKO male mice resulted in severely impaired spermatogenesis and sperm production (Couse et al., 2001). ESR1 gene common polymorphisms in humans have been associated with breast cancer, spontaneous abortion, osteoporosis, age of menarche (Leher et al., 1990, 1993; Andersen et al., 1994; Curran et al., 2001; Gennari et al., 2005; Stavrou et al., 2006) and more recently with female and male infertility. ESR1 PvuII polymorphism (rs2234693) in women was found to affect pregnancy rate following in vitro fertilization (IVF) (Georgiou et al., 1997; Sundarrajan et al., 1999), whereas in males ESR1 XbaI polymorhism (rs9340799) was suggested to have an effect on azoospermia or idiopathic severe oligospermia (Kukuvitis et al., 2002).
Human fertility is a complex character determined by interactions between genetic and environmental factors. A recent series of twin studies provided evidence that genetic factors represent a significant component of human fertility measured as waiting time to pregnancy, completed family size and age at first conception (Rodgers et al., 2001; Christensen et al., 2003; Kohler et al., 2006). In addition, behavioural genetic studies documented genetic influences on fertility precursors such as onset of puberty, sexual behaviour and desire for children.
Molecular genetic investigations on specific ‘fertility genes’ are quite scarce (Gerdes et al., 1996; Kohler et al., 2006). Recently, we studied the role of the apolipoprotein E gene (APOE) in human reproduction and confirmed its effect on fertility in developed and pre-modern populations (Corbo et al., 2004a,b). With the present study, we extended our analysis to ESR1 as a potential human fertility candidate gene. ESR1 PvuII and XbaI polymorphisms were examined in a sample of Italian men and women and a sample of African-Ecuadorian women. The Italian sample comprised subjects born before 1930 and living in a society where the demographic transition (increase in life expectancy and declining fertility) had started (Ulizzi et al, 1979). In Italian women born in the same geographic area and in the same period (1900–1930), the fertility rate decreased from 4.8 to 2.4 and the mean age at marriage was 22.4 years (Istituto Centrale di Statistica, 1974). The African-Ecuadorian sample was collected from a pre-industrial population which at the time of sample collection did not practice contraception, and so can be considered as a population with natural fertility. Demographic variables on the African-Ecuadorian women's fertility (fertility rate: 6.5; age at first birth: 18.2 years) and newborn viability are reported elsewhere (Corbo et al., 2004b). The study of populations living under different environmental conditions, including reproductive patterns, could help to identify possible genetic–environmental interactions in the determination of a multifactorial trait like fertility.
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Materials and Methods |
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Subjects
The Italian sample (86 healthy unrelated men and 92 healthy unrelated women in the post-reproductive age) was collected for a multidisciplinary study (LONCILE study) which investigated anthropological and biological characteristics of the elderly population of the Cilento area in the district of Salerno, southern Italy (Cresta and Gregorio, 2001). All the subjects were collected consecutively without selection criteria. A questionnaire asking about the number of children, pregnancies and spontaneous abortions was administered to the women; the men were asked only about the number of children. In addition to verify whether the study sample constituted of elderly people was representative of the fertile population, a control sample drawn from the present population of reproductive age (<40 years) was examined as well for ESR1 allele frequencies.
The African-Ecuadorian sample analysed in this paper (57 healthy women apparently unrelated and of all age classes) is part of a larger sampling, randomly collected, constituted of African-Ecuadorian women (n = 124) living along the Cayapas River and its tributaries in the province of Esmeraldas, northwestern Ecuador. Data on fertility were collected through interviews with the women. Demographic data concerning women's fertility were reported elsewhere (Corbo et al., 2004b). Blood samples could only be collected from part of the total sample.
Collection of biological material for the scientific studies was approved by the institutional ethics committees. Informed consent was obtained from all subjects.
Laboratory methods
Venous blood was drawn in ethylenediaminetetraacetic acid (EDTA) from all subjects after overnight fasting. High molecular weight DNA was extracted from whole blood according to the procedure described by Miller et al. (Miller et al., 1988). The two ESR1 intronic polymorphisms were examined according to the technique of Yaich et al. (Yaich et al., 1992) with slight modifications. In particular, a different primer couple was designed: 5'-AGGGTTATGTGGCAATGACG-3' as forward primer and 5'-CCTGCACCAGAATATGTTACCT-3' as reverse primer. The PCR product was digested overnight by PvuII and XbaI, and then visualized on agarose gel. ESR1*P and *X alleles indicate the absence of a cutting site for the PvuII and XbaI restriction enzymes, respectively, whereas ESR1*p and *x alleles indicate the presence of a cutting site.
Statistical analysis
Allele frequencies were determined by the gene counting method; the agreement of the genotype distribution with Hardy–Weinberg expectations was verified by an exact test. Both samples were in Hardy–Weinberg equilibrium. The linkage disequilibrium was calculated with the EH program by Xie and Ott (Xie and Ott, 1993), available at http://linkage.rockefeller.edu/soft/.
Comparisons among the mean number of children associated with different ESR1 genotypes were performed by parametric (ANOVA) and non-parametric tests (Kruskal–Wallis). As the age range of the African-Ecuadorian women was 15–82 years, the number of children was adjusted for mother's age by linear regression analysis prior to the analyses. The chi-square test for trend was used to verify the presence of a linear trend between the presence of 0, 1, 2 ESR1* p or x alleles or px haplotypes and the proportion of subjects with an above-median number of children.
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Results |
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The characteristics of the samples are reported in Table 1. The observed fertility rate for the Italian women was an intermediate value with respect to the range of 4.8–2.4 reported for cohorts of women born in the same geographic area and in the same period (1900–1930) (Istituto Centrale di Statistica, 1974), i.e. in the period when family planning began to be accepted (Ulizzi et al., 1979). The fertility rate observed in the African-Ecuadorian sample was similar to the value reported for African-Ecuadorian women (6.5) (Corbo et al., 2004b).
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ESR1*P and *X allele frequencies observed in the Italian sample of elderly subjects were 0.494 and 0.508, respectively. They were very similar to those found in the sample of young controls (ESR1*P = 0.522; ESR1*X = 0.504, P > 0.05 for both polymorphisms) and therefore they can be considered representative of the population of reproductive age. ESR1*P and *X allele frequencies were 0.404 and 0.281, respectively, in the African-Ecuadorian sample. Because of the proximity of the two ESR1 restriction fragment length polymorphisms, the linkage disequilibrium between them was analysed. They were in strong linkage disequilibrium (P < 0.0001), with a tendency of the ESR1*P allele to be associated with ESR1*X and ESR1*p to be associated with ESR1*x. PX and px were the most frequent haplotypes in both the Italian (estimated frequencies = 0.490 and 0.453, respectively) and the African-Ecuadorian sample (estimated frequencies = 0.270 and 0.586, respectively). For this reason, all the analyses were performed for the combined PvuII and XbaI genotypes as well.
Table 2 shows the mean number of children associated with ESR1 genotypes in Italian men. Subjects carrying pp or xx genotypes or both had a higher number of children, and the difference among the genotypes was not significant only for the PvuI polymorphism. The chi-square test for trend (Table 3) revealed that the proportion of subjects with an above-median number of children (number of children = 3) tended to significantly increase with the presence in the genotypes of 0, 1 or 2 ESR1*p or *x alleles or the px haplotype.
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In the sample of Italian women (Table 4), the ESR1 genotypes were not found to be associated with a different number of children. However, there was a trend of a lower number of abortions among women homozygous for the pp genotype than those carrying the PP or Pp genotypes (P = 0.04). Specifically, 30% of women carrying the ESR1*pp genotype had at least one spontaneous abortion, whereas 51% of women carrying the ESR1*PP or Pp genotype had at least one spontaneous abortion (Fisher's exact test; P = 0.15).
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Among the African-Ecuadorian women (Table 5), the ESR1 pp genotype was found associated with the highest number of children (P = 0.02), about twice that associated with the PP genotype. The chi-square test for trend showed a linear trend between the proportion of subjects with an above-median number of children (number of children = 8) and the presence of 0, 1 or 2 ESR1*p allele and px haplotype (Table 6). A similar although not significant trend was observed for the XbaI polymorphism.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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With this study, we investigated the possible impact of ESR1 gene variation on human fertility. On the whole, the results point to an increased reproductive efficiency associated with ESR1 xx and pp genotypes. In the sample of healthy Italian males, ESR1 xx and ppxx genotypes were found to be associated with a higher number of children. This result seems to parallel observations by Kukuvitis et al. (Kukuvitis et al., 2002), who found in a sample of infertile Greek men a significantly lower frequency of ESR1 xx genotypes among subjects with azoospermia or idiopathic severe oligospermia. The ESR1 pp genotype in our sample of Italian women showed a tendency to be associated with a lower number of abortions, whereas in the African-Ecuadorian women, it was associated with a higher number of children. The same result was found when the combined ESR1 ppxx genotypes were examined. These findings are consistent with previous data (Georgiou et al., 1997; Sundarrajan et al., 1999) showing a higher pregnancy rate in infertile women undergoing IVF and carrying the ESR1 pp genotype. The observation of comparable results in populations with different reproductive patterns (number of children, age at first birth), such as Italians and African-Ecuadorians in this study (Number of children 3.7 versus 7.6; estimated age at first birth for Italian women sample 22.4 versus 18.2 years for African-Ecuadorian women), suggests that the effect of the ESR1 genotype on fertility is independent of the environmental context.
In vitro studies have demonstrated that enhancer activity differs, although not significantly, among ESR1 haplotypes, the highest being associated with ESR1 xp haplotype and ESR1*x allele (Maruyama et al., 2000). This difference suggested that the expression of ESR1 could be regulated depending on the ESR1 genotype. According to present data, it could be hypothesized that the presence of ESR1 xx and pp genotypes, increasing the ESR1 function, may also affect estrogen biological action and its role on reproductive efficiency.
It is interesting to observe how the phenotypic effects of ESR1 genotype vary according to reproductive environments: in women from populations at natural fertility (African-Ecuadorian women), ESR1 genotype influences the fertility level and its effect emerges only when elevated reproductive efficiency (above-median number of children) is considered. In populations where family planning has started (Italian women), the phenotypic effect begins to shift towards anomalous conditions such as recurrent abortion; finally, the effects of ESR1 genotype manifest in modern societies as successful outcome in women undergoing IVF (Georgiou et al., 1997; Sundarrajan et al., 1999). Similarly, in healthy Italian males, whose reproductive life took place at the beginning of the demographic transition, the ESR1 genotype seems to act by increasing reproductive efficiency, whereas in males living in modern societies, it appears to exert a protective effect against azoospermia or idiopathic oligospermia (Kukuvitis et al., 2002). In other words, within the context of modern reproductive behaviours (postponement and reduction of fertility), the phenotype associated with ESR1 xx and pp genotypes tends to be more apparent because, rather than influence the number of children, it seems to be associated with having children or not instead. This picture could represent an example of the emerging relevance of genetic factors in determining human fertility in Western low-fertility societies, as shown by Kohler et al. (Kohler et al., 2002), and reveals the complex interactions between genetic factors and reproductive environments in the determination of human fertility.
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Acknowledgements |
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We are grateful to K.A. Britsch for reviewing the manuscript. Financial support from University ‘la Sapienza’.
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Submitted on March 22, 2007; resubmitted on April 26, 2007; accepted on May 1, 2007.
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