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The RsaI Polymorphism in the Estrogen Receptor-ß Gene Is Associated with Male Infertility

Department of Gynecology and Obstetrics (E.L.A., T.B.H.), Rikshospitalet University Hospital, 0027 Oslo, Norway; Faculty of Health Sciences (E.L.A., T.B.H.), Oslo University College, and Cancer Registry of Norway (T.G.), N-0130 Oslo, Norway; Institution of Clinical Sciences, Fertility Center, and Department of Urology (A.G., Y.L.G.), Malmö University Hospital, Lund University, 221 00 Malmö, Sweden; Department of Oncology (O.S., J.E., M.C.), Lund University Hospital, 221 85 Lund, Sweden; and Department of Molecular Medicine (A.N.), Karolinska Institutet, SE-171 77 Stockholm, Sweden

Address all correspondence and requests for reprints to: Elin L. Aschim, Faculty of Health Sciences, Oslo University College, P.O. Box 4, St. Olavs Plass, N-0130 Oslo, Norway. E-mail: elin.aschim{at}hf.hio.no; or Yvonne Giwercman, Malmö University Hospital, Entrance 46, Floor 4, SE 205 02 Malmö, Sweden.

	Abstract

Top Abstract Introduction Patients and Methods Results Discussion References

 
Context: Hypospadias, cryptorchidism, testicular cancer, and low semen quality have been proposed as being parts of the testicular dysgenesis syndrome (TDS) hypothetically due to changes in the androgen-estrogen balance in utero. Estrogens and estrogen receptors (ERs) play a role in regulating testicular function. ERß contains two silent polymorphisms, RsaI (G1082A) and AluI (G1730A).

Objective: We investigated the significance of these polymorphisms in the etiology of disorders being part of TDS.

Setting: The patients were recruited consecutively through university hospital clinics.

Participants: Four groups of Caucasian patients were included: 106 men from infertile couples with a sperm concentration less than 5 x 10⁶ spermatozoa/ml, 86 testicular cancer patients, 51 boys with hypospadias, and 23 cases with cryptorchidism. Military conscripts (n = 186) with sperm concentration higher than 5 x 10⁶ spermatozoa/ml served as controls.

Main Outcome Measures: ERß polymorphisms RsaI and AluI were determined by allele-specific PCR. In addition, reproductive hormone analyses were performed in controls and infertile men.

Results: Compared with the controls, the frequency of the heterozygous RsaI AG-genotype was three times higher in infertile men (13.2 vs. 4.3%; P = 0.01). The heterozygous RsaI AG genotype was associated with an approximately 20% reduction in LH concentration, compared with the wild-type RsaI GG genotype in both controls and infertile men. Subjects with testicular cancer, hypospadias, or cryptorchidism did not differ from controls regarding the frequency of any of the polymorphisms.

Conclusions: Polymorphisms in ERß may have modulating effects on human spermatogenesis. The phenotype of TDS seems to be, at least partly, determined by the genotype.

	Introduction

Top Abstract Introduction Patients and Methods Results Discussion References

 
IN RECENT YEARS there have been reports on increasing prevalence of hypospadias, cryptorchidism (1), and testicular germ cell cancer (TGCC) (2, 3) as well as declining semen quality (4, 5), recently referred to as the testicular dysgenesis syndrome (TDS) (6). It has been hypothesized that changes in the androgen-estrogen balance in utero could lead to impairment of embryonic programming including the development of male reproductive organs during fetal life, resulting in one or more of the above-mentioned conditions. The hormonal balance could, for example, be disturbed by increased exposure to estrogenic or antiandrogenic endocrine disruptors (6, 7). Alterations in genes involved in androgen or estrogen action or metabolism could predispose for the development of some TDS conditions, by rendering some individuals more sensitive to an exogenously or endogenously derived disruption of the androgen-estrogen balance.

In men, estrogens are synthesized from testosterone via the action of aromatase cytochrome P450. Estrogens seem to play an important role for male fertility, which was demonstrated by the finding that aromatase deficiency caused progressive infertility in adult mice (8) and reduced sperm production and sperm motility in humans (9, 10). In contrast, increased levels of estrogens in utero have, at least in some studies, been shown to lead to TDS-like conditions in both mice (11, 12, 13, 14) and men (15, 16). It is important to emphasize, however, that the hypothesized relationship between estrogens and TDS development is still a matter of controversy.

Estrogen signaling in the cell is mediated by estrogen receptors (ERs), of which at least two subtypes exist, ER and ERß. Two silent polymorphisms in ER have been associated with azoospermia or severe oligozoospermia (17, 18), whereas no associations between ER polymorphisms and cryptorchidism or TGCC have been found (19). Recently several sequence variants of the ERß gene have been described (20), including two silent GA polymorphisms, RsaI and AluI. Both polymorphisms have been overrepresented in ovulatory dysfunctions (21). However, studies on genetic variants of ERß with respect to TDS are still lacking. Such information might add to our knowledge regarding the role of estrogens in the physiology and pathophysiology of male reproductive systems.

Accordingly, our aim was to investigate the two ERß polymorphisms with respect to male infertility, TGCC, hypospadias, and cryptorchidism. Furthermore, by comparing reproductive hormone concentrations in subjects with different ERß genotypes, we wished to evaluate whether these polymorphic forms might play a role for ERß function in vivo.

	Patients and Methods

Top Abstract Introduction Patients and Methods Results Discussion References

 
Infertile men

One hundred six consecutive Caucasian men from infertile couples were included in the study. All men presented with sperm concentrations lower than 5 x 10⁶/ml in all (at least two) ejaculates, delivered for examination. Although the fertility of their female partners was not explored, these men are for the sake of simplicity referred to as infertile throughout this paper. Men with known genetic causes of infertility, e.g. Klinefelter syndrome or Y-chromosome microdeletions, as well as those with a history of cryptorchidism were excluded.

TGCC patients

During the period March 2001 to August 2002, all patients under the age of 50 yr with the diagnosis of TGCC passing through the outpatient clinic of the Department of Oncology, Lund University Hospital (Lund, Sweden) were asked to participate in a study of reproductive function. Seventy-nine percent of the patients accepted to participate. Blood samples for DNA analysis were drawn from 86 of them, all being Caucasians. Among these, 28 presented with a diagnosis of seminoma and 58 were nonseminomas, the latter group including 14 that had tumors with a seminoma component. The distribution of histological tumor types and clinical stages in those who denied taking part in the study, was the same as among the participants (data not shown).

Boys with hypospadias

Fifty-one consecutive Caucasian boys with normal male 46, XY karyotype, referred for surgery due to isolated hypospadias, were included in the study. The study group included glanular (n = 21), penile (n = 13), and penoscrotal (n = 17) hypospadias cases.

Men with a history of cryptorchidism

Twenty-three men with a history of cryptorchidism were included in the study. Of these, 15 were originally excluded from the group of infertile men, whereas eight were from the military conscripts group (see below).

Controls

Normal controls served a group of 186 Swedish military conscripts without genital abnormalities and with sperm concentration higher than 5 x 10⁶ spermatozoa/ml. All underwent scrotal ultrasound, which did not reveal testicular microlithiasis indicative of carcinoma-in situ of the testis (22). This cohort was derived from an original group of 203 conscripts with Swedish parents. Subsequently, eight men were excluded due to cryptorchidism (see above) and eight due to sperm concentration lower than 5 x 10⁶ spermatozoa/ml.

Informed consent was obtained from all subjects or their parents, according to protocols approved by the ethical review boards of Karolinska Institutet and Lund University.

Allele-specific PCR

In all subjects, allele-specific PCR was performed to detect the RsaI and AluI variants of ERß. For each polymorphism two reactions per subject were run, using a specific primer for either the polymorphic A variant or for the wild-type G variant, together with an upstream and a downstream primer. PCR conditions were established to generate both a control fragment and a shorter, allele-specific band in the presence of the variant and only the control fragment in its absence.

Allele-specific PCR of the RsaI polymorphism was performed in a total volume of 25 µl containing 25 ng genomic DNA, 45 mmol/liter KCl, 10 mmol/liter Tris HCl (pH 9.1), 0.1% Tween 20, 0.2 mmol/liter deoxynucleotide triphosphate, 1.5 mmol/liter MgCl₂, 1 U Dynazyme Taq polymerase (Finnzymes Oy, Espoo, Finland), and 0.5 µmol/liter of each of the primers RsaI forward (Fw), RsaI reverse (Rev), and either RsaI RevA or RsaI RevG. Primer sequences are presented in Table 1. Amplification was performed for 35 cycles; each cycle including denaturation for 1 min at 96 C, primer annealing for 30 sec at 58 C, and primer extension for 3 min at 72 C, with an initial denaturation step for 3 min at 96 C, and a final extension step for 7 min at 72 C. For the AluI polymorphism, an annealing temperature of 54 C for 30 sec was used. Other conditions were the same as for the RsaI reaction. The sequences of the primers are presented in Table 1.

Analysis of restriction fragment length polymorphisms

Both the RsaI and AluI polymorphisms are restriction fragment length polymorphisms, and digestion with the respective restriction enzymes was performed according to the manufacturer (Fermentas, Helsingborg, Sweden) to verify the results from the allele-specific PCR. In the RsaI polymorphism, a G to A nucleotide exchange at nucleotide 1082 in exon 5 created a recognition site for RsaI, and in the AluI polymorphism an exchange of G to A at nucleotide 1730 in the noncoding end of exon 8 introduced a recognition site for AluI (nucleotide numbering according to GenBank accession no. AB006590). In both positions a G nucleotide was considered the wild-type sequence and was not digestible by RsaI or AluI.

RsaI digestion produced one uncleaved band of 409 bp in subjects with the homozygous wild-type GG genotype, two bands of 110 and 299 bp in homozygous polymorphic AA subjects, and all three bands in heterozygous AG carriers. AluI digestion yielded one band of 405 bp in the uncleaved homozygous wild-type GG polymorphism, two bands of 163 and 242 bp in the homozygous polymorphic AA polymorphism, and all three bands in heterozygous AG subjects.

Hormone analysis

Inhibin B levels were assessed using a specific immunometric assay, as previously described (23), with a detection limit of 15 ng/liter and total assay variation coefficients less than 7%. In military conscripts, circulating levels of FSH, LH, SHBG, testosterone, and estradiol were measured by an automated fluorescence detection system (Autodelfia, Wallac Oy, Turku, Finland) at the routine clinical chemistry laboratory, Uppsala University Hospital. Intraassay and total assay variation was below the level of 4 and 7.5%, respectively.

In infertile men, analyses were performed at Malmö University Hospital (Malmö, Sweden) using immunometric ELISAs with a commercially available kit (Oxford Bio-Innovation Ltd., Oxfordshire, UK). Laboratory total assay variation was 12.4% at 25 ng/liter and 12.8% at 305 ng/liter. Testosterone levels were measured using an immunoassay (Access; Beckman Coulter Inc., Fullerton, CA). Laboratory total assay variation was 2.8% at 2.9 nmol/liter and 3.2% at 8.1 nmol/liter. Plasma FSH and LH concentrations were measured by means of immunoassays (Immuno 1; Bayer Diagnostics Division, Tarrytown, NJ). Laboratory total assay variation for FSH was 2.5% at 2.9 IU/liter and 1.4% at 15 IU/liter, and for LH it was 2.6% at 3.0 IU/liter and 1.7% at 15 IU/liter. Serum SHBG was measured using an immunoassay (Immulite 2000; Diagnostic Products Corp., Los Angeles, CA). Total assay variation was 3.7% at 29 nmol/liter and 6.7% at 85 nmol/liter. Because the methods of hormonal measurement applied to samples from controls and infertile men were not the same, we did not compare the hormone levels between the two groups but instead compared the hormone concentrations between the genotypes within each group.

Semen analysis

The ejaculate was obtained by masturbation after a minimum 48 h of sexual abstinence. The assessment of concentration was performed as recommended by the World Health Organization’s recommendations by use of a modified Neubauer chamber and positive displacement pipettes for proper dilution of the ejaculate (24). Three laboratory technicians performed the analyses of the ejaculates. The interobserver coefficient of variation was found to be 8.5% for concentration assessment. The laboratory participates in an external Quality Control Program, organized by Nordic Association for Andrology and European Society of Human Reproduction and Embryology.

Statistical analysis

The distributions of ERß polymorphisms were compared between the patient groups and controls using Fisher’s exact test. Median hormone levels for the different genotypes, in controls and infertile men, separately, were compared by Mann-Whitney U test applying the SPSS statistical software (version 11.0; SPSS, Inc., Chicago, IL). All statistical tests were two sided. P < 0.05 was considered statistically significant.

	Results

Top Abstract Introduction Patients and Methods Results Discussion References

 
Infertile men

The RsaI AA genotype was found in one control subject only. When analyzing the distribution of the RsaI polymorphism, we found that the infertile men had approximately three times higher frequency of the heterozygous RsaI AG genotype than controls (P = 0.01; Table 2 and Fig. 1). The infertile men did not differ from the controls regarding the AluI polymorphism.

View larger version (11K): [in this window] [in a new window]   FIG. 1. Proportion of RsaI AG genotype in the different study groups. Significant difference from controls is marked with an asterisk.

The total group of TGCC patients did not differ from the controls with regard to any of the two ERß polymorphisms (Table 2). Patients with nonseminomas with a seminoma component had five times higher frequency of the RsaI AG genotype (P = 0.03) and a 68% higher frequency of the heterozygous AluI AG genotype (P = 0.05), compared with controls.

Boys with hypospadias

No significant differences between controls and patients with hypospadias were found with respect to the two ERß polymorphisms (Table 2).

Men with cryptorchidism

The RsaI AG genotype was three times more frequent among the men with a history of cryptorchidism, compared with the controls, although this difference did not reach statistical significance (P = 0.11). This group of men with cryptorchidism did not differ from the controls regarding the frequency of AluI genotypes.

Hormone analysis

In both controls and infertile men, the heterozygous polymorphic RsaI AG genotype was associated with an approximately 20% reduction in the median concentration of LH (P = 0.05 and P = 0.04, respectively), compared with the homozygous wild-type RsaI GG genotype (Table 3 and Fig. 2). No such differences were found for FSH, inhibin B, testosterone, estradiol, or SHBG.

View larger version (11K): [in this window] [in a new window]   FIG. 2. Box plots showing median and interquartile range of the LH levels according to RsaI genotype in controls and infertile men. The median LH level was significantly lower in RsaI AG, compared with RsaI GG in both groups.

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

The finding of decreased LH levels in men with the RsaI AG genotype, despite unchanged testosterone and estradiol concentration, might indicate that this genotype implies an increased ERß activity, leading to increased estrogen sensitivity. The finding that the RsaI AG group presented with lower LH values in controls as well as in the infertile men makes it more probable that this is a biologically relevant observation and not just a result of multiple testing. Correspondingly, decreased LH levels were reported in Chinese patients with ovulatory dysfunction presenting with combined RsaI and AluI AA genotypes (21).

With regard to increased estrogen sensitivity in RsaI AG subjects, our findings are in accordance with the observation of increased risk of reduced sperm quality in men exposed to the potent estrogen diethylstilbestrol in utero (25). The latter finding, however, might be a chance observation because it was not confirmed in a later study (26). Furthermore, it is well known that spermatogenic arrest occurs when men are on a long-term estrogen replacement therapy before sex change surgery (27, 28), and hence, it seems plausible to assume that increased exposure to estrogens hampers spermatogenesis. This effect might be indirect, mediated through lower gonadotropin secretion and as one of the consequences of a decreased testosterone synthesis by the Leydig cells. In our study, however, RsaI AG and RsaI GG subjects had similar circulating testosterone levels, suggesting that this was not the mechanism behind the low sperm concentration in our infertile subjects. An additional direct role of estrogen in spermatogenesis is indicated by the detection of ERß in human testis in the Sertoli cells as well as germ cells, including round spermatids (29) and the fact that the splicing of this gene seems to be cell-dependent within the human testis (28).

The mechanisms behind altered ERß function in subjects with RsaI polymorphisms remain to be elucidated. The G to A change does not lead to amino acid changes in the protein. It can be speculated, however, that this polymorphism is in linkage disequilibrium with other genetic variations that could affect gene expression or function. A recent study showed that the RsaI polymorphism was in complete linkage disequilibrium with a polymorphism located at the splice acceptor site just before exon 8 in ERß (30). This may potentially affect the splicing of this exon, leading to proteins with different properties than the wild-type ERß (31, 32). The RsaI polymorphism could also have a direct effect through changing the nucleotide sequence and thereby the secondary structure of the ERß mRNA, possibly leading to changes in mRNA syntheses, splicing, maturation, transport, translation, or degradation (33, 34).

We found no clear association between ERß polymorphisms and hypospadias or cryptorchidism, although the latter group presented with equally high prevalence of the RsaI AG polymorphism as the infertile men. This finding was possibly linked to the infertility status of the majority of these men rather than to cryptorchidism per se.

Nonseminomatous TGCC with seminoma components was associated with an increased frequency of both RsaI and AluI AG genotypes. However, because no such association was found in patients with pure nonseminomas or seminomas, this might be a chance finding.

As controls we included military conscripts, who can be considered as representative for the general population of Swedish adolescents (35). None of them presented with genital abnormalities, and men with a history of cryptorchidism were excluded from this group. Although their age was below the peak incidence of TGCC, scrotal examination gave no indication of carcinoma in situ of the testis in any of these men. However, although we selected the controls based on sperm concentration above 5 x 10⁶ spermatozoa/ml, it cannot be excluded that some of these men in the future might experience infertility problems. On the other hand, we believe that any misclassification would introduce a nondifferential bias and result in an underestimation of the association between genotype and infertility or TGCC rather than produce false-positive results. Furthermore, because we used consecutive cases, we believe that the results of this study can be generalized to the respective groups of patients.

The distribution of RsaI and AluI genotypes in our controls was similar to that in previous studies in Caucasians (20, 36, 37, 38). This indicates that the comparison of Swedish controls with case groups including a fraction of non-Swedish Caucasians in our study is justifiable.

In conclusion, we found an association between the RsaI genotype of the ERß gene and male infertility, which may be related to effects on LH secretion. Our findings demonstrate that genetic polymorphisms in the ERß gene might modify the phenotypic outcome in TDS.

	Footnotes

 
This work was supported by grants from Swedish Governmental Funding for Clinical Research, Swedish Research Council (Grants 521-2004-6072 and K2003-732KX-14506-01A), Swedish Cancer Society, the Gunnar Nilssons Cancer Foundation, the Foundation for Urological Research, and Research Council of Norway (Grant 142439/310).

First Published Online July 5, 2005

Abbreviations: ER, Estrogen receptor; Fw, forward; Rev, reverse; TDS, testicular dysgenesis syndrome; TGCC, testicular germ cell cancer.

Received February 7, 2005.

Accepted June 23, 2005.

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Top Abstract Introduction Patients and Methods Results Discussion References

 

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