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Hum. Reprod. Advance Access originally published online on January 8, 2008
Human Reproduction 2008 23(3):688-692; doi:10.1093/humrep/dem415
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© The Author 2008. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

X chromosome inactivation patterns in patients with idiopathic premature ovarian failure

Sang Ho Yoon1, Young Min Choi2,3,5, Min A. Hong2, Byung Moon Kang4, Jin Ju Kim2, Eung Gi Min1, Jung Gu Kim2 and Shin Yong Moon2,3

1 Department of Obstetrics and Gynecology, Dongguk University International Hospital, Goyang, South Korea 2 Department of Obstetrics and Gynecology, Seoul National University College of Medicine, 28 Yungun-dong, Chongno-ku, Seoul 110-744, South Korea 3 The Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University College of Medicine, Seoul, South Korea 4 Department of Obstetrics and Gynecology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, South Korea

5 Correspondence address. Fax: +82-2-762-3599; E-mail: ymchoi{at}snu.ac.kr


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Funding
 References
 
BACKGROUND: X chromosome aberrations have been reported as the cause of extremely skewed X chromosome inactivation (XCI). The purpose of this study was to investigate whether skewed XCI is associated with idiopathic premature ovarian failure (POF).

METHODS: The XCI status was evaluated in Korean women by the methylation assay of androgen receptor locus in 126 idiopathic POF patients (35.3 ± 13.9 years old, mean ± SD) and 126 age-matched controls (35.2 ± 13.9 years). The incidence of skewed XCI in POF group was compared with that of control. The correlation between age and skewed XCI was also evaluated within both groups.

RESULTS: The incidence of extremely skewed XCI (≥90%) was 3.9 versus 2.7% (P = 0.710) in POF and control group, respectively. No significant differences were found in the incidence of skewed XCI on all three levels (≥90, ≥80 and ≥70%) compared between these two groups. The calculation of correlation coefficients showed that, in both POF and control group, there were no significant correlations between age and XCI ratio. Neither was there increasing tendency of skewed XCI according to the increase of age in both groups. Furthermore, there were no significant differences when the XCI ratios were analysed according to the age of onset of ovarian failure.

CONCLUSIONS: The incidence of skewed XCI in Korean POF population was not significantly different from control, implying that skewed XCI may not be associated with idiopathic POF. There were also no significant correlations between age and skewed X-inactivation patterns in both groups.

Key words: androgen receptor gene/methylation-specific PCR/premature ovarian failure/skewed X chromosome inactivation


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Funding
 References
 
Premature ovarian failure (POF) is generally defined as the cessation of ovarian function with elevated gonadotropins and low estrogen levels before or at the age of 40 (Coulam, 1982Go), and the incidence is estimated to occur in 1% of women of reproductive age (Coulam et al., 1986Go). The fertility of affected women is severely compromised and they need long-term hormone therapy to relieve symptoms of estrogen deficiency and to maintain bone density (Devi et al., 1998Go). Although known etiologies of POF include genetic, autoimmune and metabolic causes, as well as viral infections, cytotoxic drugs and radiation (Rebar, 1982Go; Coulam et al., 1983Go; Conway, 1997Go; Anasti, 1998Go), the etiology remains unknown in a large proportion of cases and they are classified as idiopathic POF. POF is, however, often associated with abnormalities of X chromosome (Mumm et al., 2001Go; Goswami and Conway, 2005Go). X monosomy, as in Turner syndrome, or X-deletions and translocations are known to be responsible for POF (Therman et al., 1990Go; Davis et al., 2000Go). Disruption of genes involved in maintaining ovarian function, due to structural aberrations of X chromosome, might directly cause ovarian dysfunction.

X chromosome inactivation (XCI) is a phenomenon whereby one of the two X chromosomes in each somatic cell of healthy human female becomes inactivated to compensate for the differences in the X-linked gene dosage between males and females (Lyon, 1989Go). This inactivation occurs randomly on both the maternally and the paternally derived X chromosome very early in embryonic development (Lyon, 1961Go, 1972Go). The incidence of skewed XCI in normal women is controversial. It has been suggested that the incidence of skewed XCI may vary with age (Busque et al., 1996Go) and estimated incidence of extremely skewed XCI in the general female population is 1.5–23% (Busque et al., 1996Go; Lanasa et al., 1999Go; Sharp et al., 2000Go). Many X-linked syndromes have been reported in which carrier females exhibit extremely skewed X-inactivation (Wengler et al., 1995Go; Orstavik et al., 1996Go; Devriendt et al., 1997Go). It is well documented that structural abnormalities of X chromosome, such as large deletions and unbalanced X/autosome translocation, result in skewed patterns of XCI, with the abnormal X chromosome being inactive in most cells for the better genetic balance (Therman and Patau, 1974Go; Mcmahon and Monk, 1983Go; Willard, 1995Go; Mumm et al., 2001Go).

Recently, there was a report that skewed XCI was significantly increased in a Japanese group of women with POF (Sato et al., 2004Go). They suggested that cryptic abnormalities of X chromosome or gene mutations may lead to skewed XCI and result in POF. However, conflicting results that skewed XCI was not associated with POF were reported by Italian group with larger cohort (Bione et al., 2006Go). They pointed out that small sample size and the broad age range of POF patients in the Japanese study could be a reason for the discrepancy with their data. They also suggested that small deletions or mutations in X-linked genes do not appear to be a common feature of POF patients and X-linked genes involved in POF may be too few or not able to interfere with XCI. We therefore investigated whether skewed XCI is associated with idiopathic POF that may have underlying chromosomal aberrations in our Korean population. For this, we analysed XCI patterns by the methylation assay of androgen receptor (AR) (CAG)n polymorphism and the incidence of skewed XCI in POF group was compared with that of control. The correlation between the age and skewed XCI was also evaluated within both groups.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Funding
 References
 
Subjects
A total of 126 Korean women affected by idiopathic POF were recruited between July 1999 and March 2004. For the purpose of this study, the following criteria for diagnosing POF were used (Anasti, 1998Go): ≥4 months of amenorrhea and two serum FSH levels of ≥40 mIU/ml obtained ≥1 month apart in a women aged ≤40 years. All of the patients were given a protocol for a check of their medical and gynecologic history. Subjects underwent gynecologic examination, pelvic ultrasound evaluation, serum hormone level analysis [i.e. LH, FSH, estradiol (E2), TSH and prolactin], autoimmune disease work up and karyotyping. After that, patients with known cause of POF were excluded in advance: cytogenetic abnormalities, previous chemo- or radiotherapy, previous bilateral oophorectomy. Control group included 126 healthy Korean women who had normal menstrual cycles (21–35 days) and normal ovarian feature by ultrasonography. The women recruited as control were age-matched. The review board for human research of Seoul National University Hospital approved this project, and written informed consent was obtained from each woman.

XCI assay
X-inactivation patterns were determined in blood DNA by using a modification of the methylation analysis of the AR locus as described previously (Allen et al., 1992Go). To assess differential methylation, samples were first digested with methylation-sensitive restriction enzymes, HpaII and HhaI, and then amplified by PCR. When the DNA is digested initially with restriction enzyme, PCR amplification will only occur if the restriction sites are methylated (and hence DNA not digested); if any of the sites are unmethylated, digestion will occur and amplication will not be possible. DNA from male control was used as a digestion control because male X chromosome is always active and unmethylated.

Genomic DNA was isolated from peripheral blood by Wizard DNA extraction kit (Promega, Madison, WI, USA). A 100 ng portion of DNA from each sample was digested at 37°C for 2 h with 10 U HpaII and 10 U HhaI in a total volume of 20 µl 1x reaction buffer and a no-enzyme control digest was also setup for each sample. After digestion, restriction enzymes were heat-inactivated by incubation at 65°C for 10 h. PCR was performed in a total 50 µl reaction volumes containing genomic DNA 1 µg, 5 µl 10x PCR buffer (final concentrations: 10 mM Tris–HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2), 2.5 mM dNTPs, 1.25 U AmpliTaq DNA polymerase (Perkin-Elmer, USA), 10% dimethylsulphoxide, 20 pmol/l of each forward and reverse primers. Digested and undigested DNA was then amplified in duplicate PCRs with PCR primers, both of which amplify the highly polymorphic CAG repeat region in Exon 1 of the AR gene at Xq12. The sequences of the primers used in the PCR were as follows: forward; 5’-GCT GTG AAG GTT GCT GTT CCT CAT-3’ labeled with 5’-phosphoamidite dye, reverse; 5’-TCC AGA ATC TGT TCC AGA GCG TGC-3’. The samples were amplified for 30 cycles comprising of 15 s at 95°C, 30 s at 60°C and 30 s at 72°C with an initial denaturation at 95°C for 5 min. Electrophoresis was carried out through 4% polyacrylamide gel in an ABI 377 DNA sequencer. Gels were stained with ethidium bromide and photographed under ultra-violet light. The size of PCR product from each allele was analysed by Genescan software for the quantification by peak height.

Differences in the size ratio of the heterozygous two-peak patterns suggested skewed XCI. XCI ratio was calculated as described previously (Sharp et al., 2000Go). XCI status was classified as random (XCI < 70% skewing), or skewed (XCI ≥ 70%). When the ratio was ≥90%, the XCI pattern was considered to be extremely skewed.

Statistical analysis
Data were analysed by {chi}2-test, Fisher’s exact test and one-way analysis of variance. In all tests, significance was accepted for P-value <0.05. Pearson’s correlation coefficient was also used to measure the level of correlation between age and XCI ratio in both groups. All data analyses were performed using the Statistical Package for the Social Sciences (SPSS) (version 12.0, SPSS Inc., Chicago, IL, USA).


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Funding
 References
 
We have examined XCI patterns from 126 women with idiopathic POF and 126 age-matched control women. The mean age of onset of ovarian failure was 27.5 ± 8.95 years but the mean age at the time of blood withdrawal was 35.3 ± 13.9 years therefore, the mean age of control group was 35.2 ± 13.9 years. In addition, the mean values of FSH, LH and E2 in POF group were 75.1 ± 31.0mIU/ml, 33.5 ± 20.7mIU/ml and 24.7 ± 23.5pg/ml, respectively.

The 102/126 (81.0%) versus 113/126 (89.7%) cases in POF and control group were informative heterozygous at the AR (CAG)n polymorphism. The incidence of extremely skewed XCI (≥90%) was 4 (3.9%) versus 3 (2.7%) cases (P = 0.710) in POF and control group, respectively. No significant differences were found in the incidence of skewed XCI on all three levels (≥90, ≥80 and ≥70%) compared between both groups (Table I). Also, the incidence of extremely skewing in normal control group was 2.7%. The distribution of XCI patterns in POF and control group is shown in Fig. 1. The incidence of XCI showed random pattern in both groups. The mean XCI ratios in POF and control group were 66.2 ± 11.3 and 66.6 ± 10.9%, respectively. Fig. 1 also showed that both groups have similar X-inactivation patterns irrespective of the presence of POF.


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  		Table I. Incidence of skewed XCI in Korean women with and without POF

 

Figure 1
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  		Figure 1: Distribution of XCI ratios in POF and control group

X-inactivation values are expressed as the percentage ratio of the predominantly inactive allele to the predominantly active allele. The mean XCI ratio in POF and control group was 66.2 ± 11.3% and 66.6 ± 10.9% (mean ± SD), respectively

 
The X-inactivation ratio was also analysed according to the age of onset of ovarian failure and no significant differences in X-inactivation ratios were found (Table II). The calculation of correlation coefficients showed that, in both POF and control group, there were no significant correlations between age and XCI ratio (R = 0.144, P = 0.150 in POF; R = –0.009, P = 0.927 in control group). Neither was there an increasing tendency for skewed XCI ratio with increase of age in both groups (Fig. 2).


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  		Table II. The comparison of XCI ratio according to the age of onset of ovarian failure

 

Figure 2
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  		Figure 2: Correlation analysis between age and X-inactivation ratio in POF and control group

(A) POF group. (B) control group. The expressed age is the age of POF and control at the time of blood withdrawal

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Funding
 References
 
This study was performed to determine whether skewed XCI is associated with idiopathic POF. To do this, we excluded POF patients who were found to have chromosomal abnormalities (i.e. X-monosomy, X-translocations) and then, investigated XCI status. There was an age gap of 8 years from the point of POF to the time of blood withdrawal. As there were previous reports that the skewing of XCI could be acquired with age (Busque et al., 1996Go; Sharp et al., 2000Go), it is possible that the discrepancy between the age of POF and the entry time to the study may have an effect on the variations of skewing pattern. Therefore, control women were age-matched not according to the age of onset of ovarian failure but to the age at the time of blood withdrawal for DNA preparation, and this may be more scientifically accurate. Moreover, as the ages of POF with extreme skewing were 22, 33, 38 and 59 years and those in control were 20, 36 and 41 years, we concluded that extreme skewing in our cohort was not influenced by age.

The present study did not show a significant difference in the incidence of skewed XCI between POF and control group. Both groups showed similar distribution patterns of XCI. Thus, our data for the Korean population support the previous results that the distribution of skewed XCI was not different from control in the Italian POF population (Bione et al., 2006Go). There was also a report that cryptic rearrangements in the Xq critical region are unlikely to be a common cause of POF (Portnoi et al., 2006Go). However, it is still possible that underlying X chromosome aberrations may be associated with idiopathic POF. There is a possibility that cryptic rearrangements might not alter the skewing state of XCI and small rearrangements are less likely to cause skewing.

We also assumed that as the age of onset of ovarian failure is young, including primary POF, this could result in increased patterns of skewed XCI. In our cohort, 11 women had a primary amenorrhea and the informative XCI ratios in this group were 54, 56, 57, 59, 64, 70, 76 and 93%. In contrast to the previous results (Bretherick et al., 2007Go), the patterns of skewed XCI were not significantly different between primary and secondary POF. It has been also suggested that severe skewing occurs more frequently in older females of general population (Busque et al., 1996Go). In contrast to the expectation, in our cohort, we could not find any correlation between age and XCI ratio in POF, nor was the correlation significant in control group. Taking everything into consideration, these results suggest that age may not be the contributing factor to the skewing of XCI. However, it is possible that the relatively small size of our cohort may be responsible for this discrepancy.

To evaluate the XCI status, a methylation sensitive PCR assay was used. It is well documented that the methylation of HpaII and HhaI sites in the AR gene correlates with X inactivation (Allen et al., 1992Go). To avoid underestimation of the true degree of skewing, we also utilized double digestion with both HpaII and HhaI enzymes (Sharp et al., 2000Go). Nevertheless, in our study, there was a relatively low incidence of extreme skewing (≥90%) of 2.7% in normal control compared to the previous reports of 1.5–23% in general population (Busque et al., 1996Go; Lanasa et al., 1999Go; Sharp et al., 2000Go; Bione et al., 2006Go). The incidence of skewed XCI in normal women is controversial; however, this result suggests that extremely skewed XCI may not be a relatively common phenomenon at least in Korean women.

In summary, the incidence of skewed XCI in Korean POF population was not significantly different from control, implying that skewed XCI may not be associated with idiopathic POF. Skewing state of XCI did not change with age in this study and extremely skewed XCI was not a relatively common phenomenon, at least in Korean women. Since subtle chromosomal aberrations may not alter the pattern of skewed XCI, X-inactivation assay cannot be currently recommended as a diagnostic tool for idiopathic POF. Further studies using the method of array comparative genomic hybridization will be helpful to explore whether there are underlying small mutations of X chromosome in the women with POF who showed extremely skewed XCI.


   Funding
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Funding
References
 
This study was supported by a grant (01-PJ10-PG6-01GN13-0002) from the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Funding
 References
 
Allen RC, Zoghbi HY, Moseley AB, Rosenblatt HM, Belmont JW. Methylation of Hpa II and Hha I sites near the polymorphic CAG repeat in the human androgen-receptor gene correlates with X-chromosome inactivation. Am J Hum Genet (1992) 51:1229–1239.[Web of Science][Medline]

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Submitted on August 16, 2007; resubmitted on November 25, 2007; accepted on December 9, 2007.


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