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Original Studies |
Department of Obstetrics and Gynecology, National University of Singapore, National University Hospital, Singapore 119074
Address all correspondence and requests for reprints to: Associate Prof. Ashim C. Roy, Department of Obstetrics and Gynecology, National University of Singapore, National University Hospital, Lower Kent Ridge Road, Singapore 119074. E-mail: obgroyac{at}nus.edu.sg
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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and ERß, are known. ERß
messenger ribonucleic acid occurs maximally in the ovaries and
granulosa cells; thus, ERß may be essential for normal ovulation. In
a recent gene knockout study, it has been shown that ERß gene null
female mice develop normal reproductive tract and ovaries during pre-
and neonatal periods, but have an abnormal frequency of spontaneous
ovulation in adulthood. In the present case-control study, we explored
the association of two recently described ERß gene polymorphisms,
RsaI and AluI, with ovulatory
dysfunctions. The respective frequencies of these polymorphisms were
significantly higher in patients than in controls
(P = 0.009 and P = 0.059). The
polymorphisms were significantly associated with ovulatory
dysfunctions, especially in patients homozygous for the polymorphisms
(P = 0.016 and P = 0.038,
respectively). The compound homozygosity of the polymorphisms was seen
only in patients (n = 5) and not controls (P =
0.009). The serum levels of LH, FSH, and progesterone were lower in the
homozygous and compound homozygous than in the respective
nonpolymorphic patients. All five compound homozygous patients had
ovulatory dysfunctions with no etiological pathology. Our results
suggest that ERß gene RsaI and AluI
polymorphisms may be associated with ovulatory defects in some
patients, especially those with unknown causes. |
Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Estrogen triggers a broad array of tissue- and organ-specific
physiological responses by binding to a nuclear receptor protein, the
estrogen receptor (ER). The ER is classified as a class 1 member of the
superfamily of nuclear hormone receptors, defined as ligand-inducible
transcription factors (4). ERs have been identified in the
human oocyte (5), granulosa cells (6), and
ovarian epithelial cells (7). Two subtypes of ER are now
known, ER and ERß (8). Both ER and ERß genes are
expressed in the human ovary (7, 9).
Recent findings strongly suggest that ER polymorphisms may be
associated with the outcome of in vitro fertilization and
pregnancy rates (10, 11). They may cause a phenotypic
disadvantage to patients undergoing ovarian hyperstimulation.
Previously, Hill et al. (12) reported an
association between ER polymorphisms and ER expression in human breast
cancer. Moreover, it has recently been observed that a polymorphism in
the CYP17 gene involved in estrogen synthesis could create an
additional promoter site in the gene and influence production of the
hormone (13).
Although polymorphisms may not be directly linked to a certain disease, they may be useful tools in the study of multifactorial disorders (14, 15). Moreover, association studies of the polymorphisms may be meaningful when applied to genes with a clear biological significance in the disorder (16). Single nucleotide polymorphisms (SNPs) are the most frequently found DNA sequence variations in the human genome (17). There is now considerable interest in the discovery and characterization of SNPs to enable the analysis of potential relationships between genotypes and phenotypes (18).
As ERß messenger ribonucleic acid (mRNA) occurs maximally in ovaries and granulosa cells (8), the ERß gene is likely to have a role in ovulatory function. A recent ERß knockout study in female mice has confirmed that ERß is essential for normal ovulation efficiency, but not for sexual differentiation, fertility, or lactation (19).
Recently, several genetic variants of ERß gene were described, with evidence of the highest incidence of RsaI and AluI polymorphisms in the probands of different weight extremes in the German population (20). In this study the significance of these common ERß polymorphisms, RsaI and AluI, in ovulatory dysfunction was evaluated.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Ninety-eight local Chinese women with ovulation defects and menstrual disorders attending the National University Hospital, Singapore, were recruited for this study. Their age ranged from 14–39 yr (mean ± SD, 25.6 ± 6.71 yr). Serum progesterone (P) values less than 31.8 nmol/L confirmed the deranged ovulatory status in all the patients. Of 98 patients, 33 showed ovulation disturbances of unknown causes (idiopathic), 30 had associated polycystic ovary syndrome (PCOS), 13 had ovulatory defects because of reproductive causes (premature ovarian failure, gonadal dysgenesis, resistant ovarian syndrome, fibroids, and ovarian endometrial cyst), and 22 showed ovulatory defects of nonreproductive causes (hyperthyroidism, hypothyroidism, diabetes, malnutrition, exercise, stress, and obesity).
One hundred and fifty apparently healthy local Chinese women, aged 18–44 yr (mean ± SD[SCAP], 32.7 ± 4.56 yr) with spontaneous regular menstrual cycles (intervals between 23 and 39 days) were used as control subjects. They had normal ovulatory cycles, as indicated by serum P levels (>10 ng/mL) measured during the ovulatory and midluteal phases. Informed consent of the patients and approval of the study by the ethical committee were obtained.
PCR
Nuclear DNA was extracted from the peripheral leukocytes by a standard procedure. The ligand binding domain of exon 5 and the 3'-untranslated region of exon 8 of the ERß gene were amplified using the following primers: A, 5'-TCTTGCTTTCCCCAGGCTTT-3'; B, 5'-ACCTGTCCAGAACAAGATCT-3'; C, 5'-TTTTTGTCCCCATAGTAACA-3'; and D, 5'-AATGAGGGACCACAGCA-3', respectively. Amplification using the PCR technique was performed for 40 cycles at an annealing temperature of 52 C as described previously (21). Amplification of exons 5 and 8 yielded the desired products of 156 and 307 bp, respectively.
Restriction fragment length polymorphism (RFLP)
The PCR products were analyzed for RFLP using RsaI and AluI restriction enzymes as described previously (22). Nucleotide exchange G-A at nucleotide 1082 in exon 5 created a recognition site for RsaI, and exchange at nucleotide 1730 in exon 8 introduced a recognition site for AluI. G at nucleotide 1730 was considered the wild-type sequence, because it was detected predominantly in the studied population.
Serum levels of estradiol (E2), P, testosterone (T), LH, and FSH were measured by their specific RIAs using reagents provided by WHO under the Matched Reagent Program (23).
Statistical analysis
The Hardy-Weinberg equilibrium was estimated by the
2 test, and all serum values were subjected to
one-way ANOVA to achieve homogeneity of variance. Statistical tests of
significance and 2 analysis were carried out
using SPSS for Windows 8.0 (SPSS, Inc., Chicago, IL).
P < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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).
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).
The respective incidence of RsaI and AluI
genotypes in 98 patients and 150 control subjects is shown in Table 1. The respective frequency of
RsaI and AluI polymorphisms was significantly
higher in patients than controls (P = 0.009;
2 = 9.517 and P = 0.059;
2 = 5.671, respectively). The occurrence of
respective homozygous RsaI and AluI polymorphisms
was also significantly higher in patients (8.2% and 11.2%) than in
controls (1.3% and 4%; P = 0.016 and
P = 0.038; Table 1). Interestingly, compound
homozygosity of the polymorphisms was seen only in the patients (n
= 5) and not in the controls (P = 0.009). Furthermore,
the respective frequency of RsaI and AluI
polymorphisms was significantly higher in idiopathic patients than
controls (P < 0.001; 2 =
23.19 and P < 0.001; 2 =
17.160, respectively). The respective occurrence of homozygous
RsaI and AluI polymorphisms was also
significantly higher in idiopathic patients than controls
(P < 0.001 and P = 0.001; Table 1).
All five compound homozygous patients presented ovulatory dysfunctions
of unknown causes (P < 0.001). The frequency of
RsaI polymorphism, but not of AluI polymorphism,
was significantly higher in PCOS patients than controls
(P = 0.019 and P = 0.568,
respectively).
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. Serum levels of
E2 and T did not show any significant differences
in patients among the three genotypes of RsaI polymorphism
(0.828 
P 1.000), AluI
polymorphism (0.151 P 0.886), and RR-AA and
rr-aa genotypes (0.456 P 0.714).
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Serum levels of P in the RsaI homozygous (1.72 nmol/L), AluI homozygous (2.45 nmol/L), and compound homozygous for both polymorphisms (0.80 nmol/L) were significantly lower than those in the respective nonpolymorphic patients (rr, 7.85 nmol/L; aa, 8.90 nmol/L; rr-aa, 7.73 nmol/L; P < 0.001).
Serum levels of FSH were slightly lower in the RsaI
homozygous (2.89 IU/L) than in the corresponding nonpolymorphic
patients (3.76 IU/L; P = 0.822). They were also lower
in the AluI homozygous (2.53 IU/L) than in the corresponding
nonpolymorphic (4.75 IU/L) patients (P = 0.093). For
the patients homozygous for both RsaI and AluI
polymorphisms (2.06 IU/L), FSH levels were lower than those in the
patients without these polymorphisms (4.19 IU/L), but the difference
was not statistically significant (P = 0.221). Clinical
and endocrinological profiles of the patients compound homozygous for
both polymorphisms are shown in Table 3.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Thus, normal ovarian function depends on a multitude of auto- and paracrine effects of estrogen. It acts in concert with the gonadotropins secreted from the anterior pituitary and provides successful folliculogenesis and an intrafollicular steroid environment, which are essential for the development and maturation of oocytes. Alternations in the steroid profile during maturation induce changes in the oocyte that affect the fertilization process (32, 33).
The study of ERß null female mice (19) suggested that reduced ovulation efficiency could be primarily due to a defect within ovarian tissue, more precisely an inability of E2 to exert its effect on the granulosa cells of maturing follicles. An impaired ability to produce an adequate gonadotropin surge was also considered to be a contributing factor to the decreased ovulation rate (34).
In this case-control study, the incidence of homozygous RsaI polymorphism and AluI polymorphism was higher in patients than controls. Moreover, the coexistence of RsaI and AluI homozygosity was seen only in the patients (n = 5) and not in the controls. In addition, 6 of 8 patients homozygous for RsaI, 8 of 11 patients homozygous for AluI, and all 5 patients homozygous for both RsaI and AluI polymorphisms were found to have ovulatory defects of unknown causes. These results suggest that homozygous RsaI and AluI polymorphisms may be associated with ovulatory dysfunctions, especially in patients with no known causes of the dysfunctions. Interestingly, the frequency of RsaI polymorphism was significantly higher in PCOS patients than controls. However, this finding in the PCOS patients needs to be confirmed in a separate study involving a large number of patients.
In this study gradient patterns of LH and P levels were observed in the patients carrying homozygous and compound homozygous polymorphisms. Serum levels of these hormones were apparently lower in homozygous and compound homozygous patients than in their respective nonpolymorphic counterparts; these were lower in the compound homozygous than in the homozygous patients. It is possible that in ovarian tissue where the hormones and their respective receptors coexist, some loss of ERß action occurs in the homozygous patients, which is greater in the compound homozygous subjects. This, in turn, results in an alteration in the generation and/or function of other hormones, such as LH and P (34). Serum FSH levels showed an apparent gradient pattern without statistical significance.
Although serum E2 levels were low in all patients, these were within the normal range of the hormone. The female mice lacking ERß also showed normal serum E2 levels (19), indicating that the biosynthesis of estrogen was within normal limits.
Like ER, ERß is also present in various regions of the
hypothalamus (35). E2 was shown to
induce GnRH release from the hypothalamus as well as increase the level
of GnRH receptors in the anterior pituitary (26). It is
likely that a lack of hypothalamic ERß function reduces the positive
regulatory effect of E2 on the
hypothalamic-pituitary axis, causing a reduction in the frequency
and/or amplitude of the preovulatory gonadotropin surge and, hence,
defective ovulation (34). The reduction in the
preovulatory gonadotropin surge may also account for the decreased
levels of LH and, to some extent, of FSH in the polymorphic homozygous
patients.
Although RsaI and AluI polymorphisms in the ERß gene do not lead to amino acid changes in the ERß protein, it is possible that these polymorphisms are in linkage disequilibrium with other regulatory sequence variations that may affect gene expression or function (36). It is now becoming increasingly clear that SNPs, the most frequently found sequence variations in DNA in the human genome, may be useful as markers for the identification of genetic factors associated with complex disease traits (17). Furthermore, it has been recently reported that genes containing SNPs can cause different structural folds of mRNA (37). These mRNA variants may possess different biological functions that interact with other cellular components.
In conclusion, in this case-control study significant associations of the homozygous RsaI and AluI polymorphisms with ovulatory dysfunctions were seen. Serum levels of LH, P, and, to a lesser extent, FSH showed gradient patterns in the patients homozygous and particularly in those compound homozygous for RsaI and AluI, polymorphisms. Interaction of environmental factors with a small number of major causative genes including those involved in paracrine and autocrine modifications of ovulatory functions may contribute to these associations. Thus, our study, the first of its kind in this area, postulates that genetic variability of the ERß gene may be associated with ovulatory dysfunction, especially that of unknown cause.
Received November 22, 1999.
Revised March 28, 2000.
Revised June 19, 2000.
Accepted September 19, 2000.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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and ß in cultured human
ovarian surface epithelial cells. Mol Hum Reprod. 4:811–815. (ER-) and
(ER-ß) ß mRNA in the midgestational human fetus. J Clin
Endocrinol Metab. 82:3509–3512. immunoreactivity in neurons of the rat forebrain. Endocrinology. 139:5267–5270.This article has been cited by other articles:
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