| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
ORIGINAL RESEARCH |
From the Department of Obstetrics and Gynecology, University of Utah Health Sciences Center, Salt Lake City, Utah; emerGen Laboratories, Salt Lake City, Utah; Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, British Columbia, Canada; Department of Obstetrics and Gynecology, Washington University, St. Louis, Missouri; and Department of Human Genetics, University of Chicago, Chicago, Illinois.
Address reprint requests to: Amy E. Sullivan, MD, University of Utah Health Sciences Center, Room 2B200, 50 North Medical Drive, Salt Lake City, Utah 84132; E-mail: amy.sullivan{at}hsc.utah.edu.
 |
ABSTRACT |
|---|
TOP
 ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
METHODS: A multicenter study was performed. A power calculation determined that 101 patients were needed to detect a difference in skewed X inactivation between patients and controls. Patients were entered into a prospective trial of mononuclear-cell immunotherapy and subsequently tested for skewed X inactivation. Age-matched controls had one live birth and no prior miscarriages. Results from our X inactivation assay were compared with those from an independent genetics laboratory.
RESULTS: Greater than 75% skewing was seen in 22.6% of patients and 26.5% controls (P = .52). Greater than 90% skewing was seen in 6.6% of patients and 3.9% of controls (P = .77). There were 19.8% of primary aborters and 32% of secondary aborters with greater than 75% skewed X inactivation (P = .38). There were 4.9% of primary aborters and 12.0% of secondary aborters with greater than 90% skewed X inactivation (P = .27) Neither greater than 75% nor greater than 90% skewed X inactivation impacted next pregnancy outcomes (odds ratios = 0.87 [95% confidence interval (CI) 0.34, 2.3] and 1.4 [95% CI 0.27, 7.5], respectively). Results of the exchange of samples with an independent laboratory were highly correlated (
= 0.987, P < .001, coefficient of variation = 5.5%).
CONCLUSION: Skewed X chromosome inactivation is not associated with recurrent miscarriage. A patient’s X chromosome inactivation status does not predict next pregnancy outcome. Our assay correlates with another experienced laboratory.
Recurrent miscarriage, defined as three or more losses before 20 weeks’ gestation, is a common clinical problem that affects approximately 1% of all women of reproductive age.1 Numerous etiologic factors have been implicated in recurrent miscarriage, including genetic abnormalities, endocrine disorders, infection, uterine anomalies, and autoimmune disorders. Despite this extensive list, factors that may be associated with recurrent miscarriage are identified in only 20–60% of patients.2,3 This diagnostic uncertainty results in significant emotional duress for both patients and their families. It is also difficult for physicians to counsel or treat patients when the cause of recurrent miscarriage remains unknown. Recently, a relationship has been identified between the molecular phenotype of skewed X chromosome inactivation and recurrent early pregnancy loss.4–7
X chromosome inactivation is a naturally occurring process, wherein one of two X chromosomes present in every female cell line is inactivated during the late blastocyst stage of embryogenesis.8 The process of X chromosome inactivation assures appropriate "dosage compensation" so there is equal X chromosome expression in both females and males. Typically, X chromosome inactivation is random, so that maternally and paternally derived X chromosomes are inactivated with approximately the same frequency. As a result, X chromosomes inherited from either parent are expressed in an approximately 1-to-1 ratio. A patient is considered to have skewed X chromosome inactivation if there is preferential inactivation of either the maternally or the paternally derived X chromosome, deviating from the 1-to-1 ratio. To date there is no consensus in the literature as to what percentage of X chromosome inactivation should be used to define skewed inactivation. However, prior studies have used greater than 70% inactivation of an X chromosome from a particular parent as a cutoff value for skewed X chromosome inactivation9; some studies use greater than 90%,4,5,7 whereas still others use greater than 95%.6
Skewed X chromosome inactivation has been detected in females who are carriers of X-linked mutations, including hemophilia A and Duchenne muscular dystrophy.10–13 In these cases, the abnormal X chromosome is silenced and the normal X chromosome is preferentially expressed. Lanasa et al hypothesized that skewed X inactivation is a marker for women who carry X-linked embryo-lethal mutations on one of their X chromosomes and who may therefore be at risk for recurrent pregnancy loss.4,5,14 Alternatively, the association between skewed X chromosome inactivation and recurrent miscarriage may be multifactorial and not necessarily closely related to the inheritance of an embryo-lethal X-linked mutation.15 Although associations between skewed X inactivation and recurrent miscarriage have been reported, some studies found no relationship between skewed X inactivation and recurrent miscarriage (Kristiansen M, Knudsen GP, Hagen CB, Erstavik KH. X inactivation patterns in females with recurrent spontaneous abortions [abstract]. Presented at the International Symposium on X-chromosome Inactivation in Mammals. Novosibirsk, Russia, September 1999). Regardless, it has yet to be determined whether a patient’s X chromosome inactivation status is useful for determining the outcome of subsequent pregnancies in women with recurrent miscarriage. The purpose of this study was to estimate whether 1) there is an association between skewed X chromosome inactivation and unexplained recurrent early pregnancy loss in a well-defined sample of women with recurrent miscarriage, and 2) whether a woman’s skewed X inactivation status is predictive of a successful outcome in a subsequent, prospectively followed pregnancy.
|
MATERIALS AND METHODS |
|---|
|
TOP
ABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Previous studies have reported a 13–32% difference in the skewed X inactivation status between patients and controls. Based on these studies, we assumed a rate of 5% skewed X inactivation in controls and 18% skewed X inactivation in patients and performed a power calculation. We determined that using an of 0.05, ß of 0.20, and a power of 0.80, 101 patients were needed in this study to detect a 13% difference in skewed X inactivation between patients and controls. Of the 171 couples in the Recurrent Miscarriage trial, 118 were included in this study; 53 couples were excluded because deoxyribonucleic acid (DNA) was not obtained from both partners. Among the 118 couples, 110 were white, 2 were black, 3 were Hispanic, 1 was East Asian, and 2 were Asian Indian. Mean maternal age was 32.6 years (standard deviation [SD] 4.8; range 22–41 years) and the mean number of previous miscarriages was 4.3 (SD 1.7; range 3–13). Twenty-five (21%) of the couples had a previous live-born child. Sixty-six received treatment with their partner’s mononuclear cells, and 52 received saline as a placebo.
There are no known differences in skewed X chromosome inactivation between women of different racial backgrounds, and therefore controls were 117 healthy white women with at least one live birth and no history of spontaneous abortions. Controls were age matched to patients on a one-to-one basis and were recruited from the Obstetrics and Gynecology clinic at the University of Utah.
All patients had a sample of peripheral blood drawn, and DNA was extracted from the leukocytes. The X chromosome inactivation assay is based on the fact that the androgen receptor locus is methylated on the inactive X chromosome and unmethylated on the active X chromosome.17 In addition, there is a highly polymorphic CAG repeat just adjacent to the 5’ coding region of the androgen receptor gene. In patients who are heterozygous for the polymorphism, a distinction can be made between the two X chromosomes, and the degree of inactivation of each X chromosome can subsequently be determined. The X chromosome inactivation assay will therefore be informative only in cases that are heterozygous.
Deoxyribonucleic acid samples from all centers were coded and analyzed as a single group. Deoxyribonucleic acid was digested with HpaII, a methylation-sensitive restriction enzyme that cleaves only the unmethylated (active) X chromosome at the androgen receptor site. The products were then amplified by polymerase chain reaction (PCR) with fluorescent-labeled primers. Only the androgen receptor gene from the undigested (inactive) X chromosome would be amplified because the unmethylated active X chromosome would be cleaved by HpaII. The products were then electrophoresed on an ABI 3700 sequencer (Applied Biosystems, Foster City, CA). Peak heights were analyzed using GeneScan software (Applied Biosystems), and the percentage of X chromosome inactivation was determined as described elsewhere.18 To estimate the accuracy of our X chromosome inactivation protocol, we performed the assay four times on 31 randomly selected samples. The interassay coefficient of variation in our laboratory was 2.5%.
To test the reliability of our X chromosome inactivation assay, 20 samples of DNA that were tested at our institution using the protocol described above were randomly selected and also tested in the laboratory of Dr. Wendy Robinson at the University of British Columbia in Vancouver. Researchers from the University of British Columbia were blinded to the X chromosome inactivation results from our laboratory. There were minor differences in both the X chromosome inactivation assay and the quantification techniques between the two laboratories. Researchers at the Medical Genetics laboratory at the University of British Columbia recommend using a secondary cutter, the PCR conditions vary between the University of Utah and the University of British Columbia protocols, and the University of British Columbia laboratory calculates the percentage of skewing with respect to area under the curves, whereas the University of Utah laboratory uses peak heights (Beever CL, Jiang RH, Brown CJ, Robinson WP. Variability in DNA methylation assays of X chromosome inactivation (XCI) [abstract]. Presented at the American Society of Human Genetics meeting; October 2001; San Diego, California). 19 To estimate whether the difference in the quantification technique would independently contribute to significant variations in results, the data from the 20 comparison samples tested at the University of Utah were quantified using both peak heights and area under the curves, and the results were compared. To compare the skewed X inactivation assay techniques independently, results from our assay and results from the University of British Columbia’s assay were both quantified using peak heights and then compared.
The ages of the patients and controls were compared using the t test. 
2 analysis was used to compare the skewed X chromosome inactivation status in patients and controls. There were only a small number of patients that demonstrated greater than 95% skewed X chromosome inactivation, and therefore we used both greater than 75% and greater than 90% inactivation of a particular X chromosome to define skewed X inactivation. Patients who were primary aborters and those who were secondary aborters were separately compared with controls. A logistic regression model was used to determine the effect of greater than 75% skewed X inactivation, greater than 90% skewed X inactivation, maternal age, the number of previous miscarriages, and a history of a previous live birth on the next pregnancy outcome. Correlation of the results of the X chromosome inactivation assay techniques and quantification methods performed in our institution and in the University of British Columbia laboratory was assessed by both the coefficient of variation and the reliability coefficient (). The interassay coefficient of variation was also determined for our laboratory.
|
RESULTS |
|---|
|
TOP
ABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The average skewing was 66.4% in patients and 67.2% in controls (P = .65). Greater than 75% skewing was noted in 24 of 106 patients (22.6%) and 27 of 102 controls (26.5%) (P = .52) (Table 1
). Extremely skewed X chromosome inactivation (greater than 90%) was detected in 7 of 106 patients (6.6%) and 4 of 102 controls (3.9%) (P = .77). Results were informative in 81 of 93 patients with recurrent primary abortion (patients who never had a live birth) and 25 of 25 patients with recurrent secondary abortion (patients who had at least one previous successful pregnancy). Recurrent secondary abortion patients demonstrated an increase in skewed X chromosome inactivation; however, the difference was not statistically significant when compared with controls. Sixteen primary aborters (19.8%) and 8 secondary aborters (32%) demonstrated greater than 75% skewed X chromosome inactivation (P = .38). Four primary aborters (4.9%) and three secondary aborters (12%) displayed greater than 90% skewed X chromosome inactivation (P = .27). When only white patients were included in the analysis, the results were similar.
|
). Neither greater than 75% skewed X chromosome inactivation nor greater than 90% skewed X chromosome inactivation predicted the outcome of the next pregnancy. Additionally, neither maternal age nor a history of a prior live birth was useful in predicting the next pregnancy outcome. Only the number of previous miscarriages was associated with a subsequent spontaneous abortion (odds ratio = 1.3, 95% confidence interval 1.003, 1.6), similar to previous reports in this sample.16,20
|
= 0.987, P < .001, coefficient of variation = 5.5%) (Figure 2). As expected, a comparison of quantification methods showed that the results were also highly correlated when the skewed X inactivation data run from a single laboratory were quantified using both area under the curve and peak height (mean difference = 2.1%, r2 = .969, coefficient of variation = 2.0%, P < .001). A specific comparison of the assay technique at the University of Utah to the assay technique employed by the University of British Columbia also demonstrated highly correlated results, with a mean difference of 2.4% (r2 = .969, coefficient of variation = 3.6%, P < .001).
|
|
DISCUSSION |
|---|
|
TOP
ABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Our patients were a well-characterized population experiencing idiopathic recurrent miscarriage who were recruited into a multicenter, randomized clinical trial.16 We found that only 6.6% of patients had greater than 90% skewed X inactivation, a figure much lower than the 13–32% rate reported in prior studies,5–7 whereas the 3.9% rate of skewed X inactivation in our control population is similar to other published control data.4,5,7 We have also shown that neither greater than 75% skewed X chromosome inactivation nor greater than 90% skewed X chromosome inactivation was predictive of a successful outcome in the next pregnancy.
Our findings may differ from those of others for several reasons. First, the selection of women with recurrent miscarriage in our study differs from the selection by others. Our study included patients with three or more pregnancy losses. Patients with structural chromosome abnormalities, uterine anomalies, antiphospholipid antibodies, luteal phase defects, and thyroid disorders were excluded. In contrast, Lanasa et al included patients experiencing only two or more pregnancy losses and did not exclude patients on the basis of luteal phase or thyroid abnormalities.4 Sangha et al included patients with three or more losses, excluding only those with documented parental structural chromosomal abnormalities.7 Our strict inclusion criteria assured that the population we evaluated for skewed X inactivation consisted of patients with true idiopathic recurrent miscarriage. Second, our study demonstrated an increase in skewed X chromosome inactivation between secondary recurrent abortion patients and controls; however, this difference did not reach statistical significance. This may be because of the small number of patients with secondary abortion in our study group. Other groups have not made a distinction between primary and secondary abortion patients. It is possible that their studies included a greater number of secondary abortion patients, which could account for the increase in skewed X chromosome inactivation that they report in recurrent miscarriage cases. Finally, our study included age-matched controls, whereas in other studies the patients were significantly older than their controls.4,5 Because skewed X chromosome inactivation increases with age,18 the age discrepancy in other studies could have resulted in an apparent increase in skewed X chromosome inactivation in recurrent miscarriage cases when compared with their controls.
This multicenter study included 110 white women and 10 women of different ethnicities. All of the age-matched controls were white. There have been no reports of differences in skewed X chromosome inactivation in women of different ethnicities; however, this has not been directly studied. To ensure that our homogeneous control population did not skew our results, the data were analyzed excluding the eight non-white women in whom the assay was informative. The results of this secondary analysis were similar to the primary analysis.
It does not appear that interlaboratory variation in either assay technique or data calculation affected these results. In our study, X chromosome inactivation results correlated highly with those from the laboratory at the University of British Columbia, although minor variations in both assay technique and quantification methods exist. Furthermore, we demonstrated that the interassay coefficient of variation in our laboratory is 2.5% (data not shown). Both the low inter- and intralaboratory coefficient of variation suggest that our X chromosome inactivation assay is highly reproducible.
Skewed X chromosome inactivation may be detected in many women merely because of the limited precursor cell pool size present during the late blastocyst stage when the "choice" of which X chromosome will be inactivated is made.21,22 In this stage of fetal development, there are approximately 10–20 cells that will subsequently form the embryo; the other cells differentiate into extraembryonic tissue.23 Thus it is possible that some women may demonstrate skewed X inactivation by chance alone, rather than because they carry X-linked mutations. If this were the case, then a relationship between skewed X chromosome inactivation and recurrent miscarriage would not be evident, as in our study.
Alternatively, it is possible that skewed X chromosome inactivation in humans is a heritable genetic trait that can be identified in family members in multiple generations. For example, there is a locus in mice on the X chromosome, known as the X controlling element, that influences which X chromosome will be inactivated.24 More recently, Percec et al reported that there are autosomal factors located on chromosome 15 in the mouse that also affect X chromosome inactivation.25 Whether similar loci exist in humans is not clear. Skewed X chromosome inactivation appears to be inherited in some families, but most of these cases have been identified as a result of a proband female expressing an X-linked disease.6,26 One group has mapped skewed X chromosome inactivation in humans to loci on the X chromosome.27 Therefore, skewed X inactivation may be a heritable genetic variant not necessarily related to recurrent miscarriage.
Recurrent pregnancy loss is a difficult situation for both patients and clinicians. A large proportion of women have no identifiable cause, which results in speculative counseling about subsequent pregnancy outcomes. The prospect of a new and plausible cause of recurrent miscarriage would be very attractive to clinicians, patients, and researchers. However, in contrast to other recent reports, we find no association between skewed X chromosome inactivation and recurrent miscarriage in a well-characterized population of women experiencing idiopathic recurrent pregnancy loss. Skewed X chromosome inactivation did not correlate with the outcome of a subsequent pregnancy. Therefore, testing women with idiopathic recurrent miscarriage for skewed X chromosome inactivation is not indicated.
|
Footnotes |
|---|
doi:10.1016/S0029-7844(03)00345-4
Received September 23, 2002. Received in revised form December 30, 2002. Accepted January 2, 2003.
|
REFERENCES |
|---|
|
TOP
ABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
2. Hatasaka HH, Varner MW. Recurrent pregnancy loss. Curr Opin Obstet Gynecol 1994;6:503–9.[Medline]
3. Branch DW. Management of recurrent early pregnancy loss. ACOG practice bulletin no. 24. Washington: American College of Obstetricians and Gynecologists, 2001.
4. Lanasa MC, Hogge WA, Kubik C, Blancato J, Hoffman EP. Highly skewed X-chromosome inactivation is associated with idiopathic recurrent spontaneous abortion. Am J Hum Genet 1999;65:252–4.[Medline]
5. Lanasa MC, Hogge WA, Kubik CJ, Ness RB, Harger J, Nagel T, et al. A novel X chromosome-linked genetic cause of recurrent spontaneous abortion. Am J Obstet Gynecol 2001;185:563–8.[Medline]
6. Pegoraro E, Whitaker J, Mowery-Rushton P, Surti U, Lanasa M, Hoffman EP. Familial skewed X inactivation: A molecular trait associated with high spontaneous-abortion rate maps to Xq28. Am J Hum Genet 1997;61: 160–70.[Medline]
7. Sangha KK, Stephenson MD, Brown CJ, Robinson WP. Extremely skewed X-chromosome inactivation is increased in women with recurrent spontaneous abortion. Am J Hum Genet 1999;65:913–7.[Medline]
8. Lyon MF. Gene action in the X-chromosome of the mouse (Mus musculus L). Nature 1961;190:372–3.[Medline]
9. Uehara S, Hashiyada M, Sato K, Sato Y, Fujimori K, Okamura K. Preferential X-chromosome inactivation in women with idiopathic recurrent pregnancy loss. Fertil Steril 2001;76:908–14.[Medline]
10. Tanner SM, Orstavik KH, Kristiansen M, Lev D, Lerman-Sagie T, Sadeh M, et al. Skewed X-inactivation in a manifesting carrier of X-linked myotubular myopathy and in her non-manifesting carrier mother. Hum Genet 1999; 104:249–53.[Medline]
11. Tenenhouse HS. X-linked hypophosphataemia: A homologous disorder in humans and mice. Nephrol Dial Transplant 1999;14:333–41.
12. Van den Veyver IB. Skewed X inactivation in X-linked disorders. Semin Reprod Med 2001;19:183–91.[Medline]
13. Villard L, Levy N, Xiang F, Kpebe A, Labelle V, Chevillard C, et al. Segregation of a totally skewed pattern of X chromosome inactivation in four familial cases of Rett syndrome without MECP2 mutation: Implications for the disease. J Med Genet 2001;38:435–42.
14. Lanasa MC, Hogge WA. X chromosome defects as an etiology of recurrent spontaneous abortion. Semin Reprod Med 2000;18:97–103.[Medline]
15. Robinson WP, Beever C, Brown CJ, Stephenson MD. Skewed X inactivation and recurrent spontaneous abortion. Semin Reprod Med 2001;19:175–81.[Medline]
16. Ober C, Karrison T, Odem RR, Barnes RB, Branch DW, Stephenson MD, et al. Mononuclear-cell immunisation in prevention of recurrent miscarriages: A randomised trial. Lancet 1999;354:365–9.[Medline]
17. Allen RC, Zoghbi HY, Moseley AB, Rosenblatt HM, Belmont JW. Methylation of HpaII and HhaI sites near the polymorphic CAG repeat in the human androgen-receptor gene correlates with X chromosome inactivation. Am J Hum Genet 1992;51:1229–39.[Medline]
18. Sharp A, Robinson D, Jacobs P. Age- and tissue-specific variation of X chromosome inactivation ratios in normal women. Hum Genet 2000;107:343–9.[Medline]
19. Lau AW, Brown CJ, Penaherrera M, Langlois S, Kalousek DK, Robinson WP. Skewed X-chromosome inactivation is common in fetuses or newborns associated with confined placental mosaicism. Am J Hum Genet 1997;61: 1353–61.[Medline]
20. Aldrich C, Verp MS, Walker MA, Ober C. A null mutation in HLA-G is not associated with preeclampsia or intrauterine growth retardation. J Reprod Immunol 2000; 47:41–8.[Medline]
21. Belmont JW. Genetic control of X inactivation and processes leading to X-inactivation skewing. Am J Hum Genet 1996;58:1101–8.[Medline]
22. Plenge R. A promoter mutation in the XIST gene in two unrelated families with skewed X-chromosome inactivation. Nat Genet 1997;17:353–6.[Medline]
23. Lyon MF. Possible mechanisms of X chromosome inactivation. Nat New Biol 1971;232:229–32.[Medline]
24. Cattanach BM. Control of chromosome inactivation. Annu Rev Genet 1975;9:1–18.[Medline]
25. Percec I, Plenge RM, Nadeau JH, Bartolomei MS, Willard HF. Autosomal dominant mutations affecting X inactivation choice in the mouse. Science 2002;296:1136–9.
26. Orstavik KH, Orstavik RE, Eiklid K, Tranebjaerg L. Inheritance of skewed X chromosome inactivation in a large family with an X-linked recessive deafness syndrome. Am J Med Genet 1996;64:31–4.[Medline]
27. Naumova AK, Olien L, Bird LM, Smith M, Verner AE, Leppert M, et al. Genetic mapping of X-linked loci involved in skewing of X chromosome inactivation in the human. Eur J Hum Genet 1998;6:552–62.[Medline]
This article has been cited by other articles:
|
|
 |
|
  E. Pasquier, C. Bohec, L. De Saint Martin, C. Le Marechal, M.T. Le Martelot, S. Roche, Y. Laurent, C. Ferec, M. Collet, and D. Mottier Strong evidence that skewed X-chromosome inactivation is not associated with recurrent pregnancy loss: an incident paired case control study Hum. Reprod., November 1, 2007; 22(11): 2829 - 2833. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
