The effect of CGG repeat number on ovarian response among fragile X premutation carriers undergoing preimplantation genetic diagnosis

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1 GENETICS The effect of repeat number on ovarian response among fragile X premutation carriers undergoing preimplantation genetic diagnosis Guy Bibi, M.D., a Mira Malcov, Ph.D., a Yaron Yuval, M.D., b Adi Reches, M.D., b Dalit Ben-Yosef, Ph.D., a Beni Almog, M.D., a Ami Amit, M.D., a and Foad Azem, M.D. a a Racine IVF Unit and b Prenatal Diagnosis Unit, Genetic Institute, Lis Maternity Hospital, Tel Aviv Sourasky Medical Center and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Objective: To assess ovarian response among carriers of FMR1 premutation who undergo preimplantation genetic diagnosis (PGD). Design: Retrospective study. Setting: Academic IVF unit. Patient(s): Of 18 carriers of FMR1 premutation referred to PGD, eight had <1 repeats and ten had R1 repeats. Intervention(s): Controlled ovarian stimulation (COH) and PGD. Main Outcome Measure(s): Correlation between the number of repeats and the level of E 2 at day of hcg administration, number of retrieved oocytes, number of two-pronuclear (2PN) zygotes, and dose of recombinant FSH. Result(s): There was a positive correlation between repeats and the level of E 2 at day of hcg administration, number of retrieved oocytes, and number of 2PN zygotes. There was a negative correlation between number of repeats and the total dose of gonadotropins. The E 2 level and the number of retrieved oocytes and 2PN zygotes were significantly higher and the dose of gonadotropins significantly lower for premutation patients with R1 repeats compared with <1 repeats. Conclusion(s): There is a positive correlation between E 2 level, retrieved oocytes, 2PN zygotes, and number of repeats. Premutation carriers with <1 repeats suffer from impaired ovarian response and decreased fertilization rate. (Fertil Steril Ò 21;94: Ó21 by American Society for Reproductive Medicine.) Key Words: FMR1 premutation, ovarian response, PGD, COH Fragile X syndrome is the most common cause of inherited mental retardation as well as the most common known genetic cause of autism (1). This disorder is associated with a dynamic triple repeat sequence mutation in the X-linked gene known as fragile X mental retardation 1 (FMR1) (2) and characterized by chromosomal fragility at Xq27.3 (3). The mutation in the FMR1 gene was found to be due to an expanded sequence of trinucleotide repeats in the untranslated region, which was hypermethylated in affected individuals (4, ). The number of repeats present in normal alleles varies from 6 to 2 (2). The consequence of Received January 29, 29; revised April 13, 29; accepted April 14, 29; published online May 29, 29. G.B. has nothing to disclose. M.M. has nothing to disclose. Y.Y. has nothing to disclose. A.R. has nothing to disclose. D.B.-Y. has nothing to disclose. B.A. has nothing to disclose. A.A. has nothing to disclose. F.A. has nothing to disclose. Reprint requests: Foad Azem, M.D., Sara Racine IVF Unit, Lis Maternity Hospital, Tel Aviv Sourasky Medical Center, 6 Weizman Street, Tel Aviv 64239, Israel (FAX: þ ; Azemf@tasmc. health.gov.il). full mutation, i.e., >2 repeats, is the absence of the fragile X mental retardation protein (FMRP), an RNA-binding protein that leads to the features of fragile X syndrome (6). The mutation tends to expand in size as it is passed from mother to offspring. Premutation alleles range from to 199 trinucleotide repeats. Carriers do not have fragile X related mental retardation, but they are at risk for expansion from premutation to full mutation over several generations (7, 8). The intermediate range consists of alleles of 4 4 repeats which may be unstable when transmitted from parent to child over several generations (9). Premature ovarian failure (POF) is a condition in which women develop infertility and cessation of menses associated with high levels of gonadotropins and low levels of estrogen before the age of 4 years (1). Earlier studies demonstrated that the prevalence of POF among fragile X premutation carriers ranges from 13% 26%, compared with.4% 14% in the full mutation carriers (11 13). Premutation carriers have an elevation in the serum FSH level (14 16) and a decrease in the serum inhibin B level, 1-282/$36. Fertility and Sterility â Vol. 94, No. 3, August doi:1.116/j.fertnstert Copyright ª21 American Society for Reproductive Medicine, Published by Elsevier Inc.

2 which reflects a decreased follicle number (16). Furthermore, premutation carriers entered menopause approximately years earlier than noncarrier women (13) and typically do not respond well to exogenous gonadotropin stimulation (17). The underlying molecular mechanism of ovarian failure is not clear, but the full mutation females who have reduced FMR1 protein are not affected with POF. The relationship between the number of repeats and the age of last menstrual cycle is nonlinear (13, 18, 19). The risk of POF appears to increase with increasing premutation repeat size between 9 and 99, after which the risk plateaus or even decreases for women with repeat sizes >1 (19). Carriers of fragile X premutation are currently offered preimplantation genetic diagnosis (PGD) as a diagnostic tool for prevention of the subsequent birth of an affected baby (17, 2). Similar to other IVF procedures, the PGD is based upon controlled ovarian hyperstimulaion (COH) and ovum pickup (2). The fact that some of these patients may suffer from POF or altered ovarian reserve creates an obstacle for the success of the procedure. Some centers stopped offering PGD for fragile X carriers, and some have suggested other alternatives, such as egg donation (21). Platteau et al. (17) reported that fragile X patients needed significantly more ampules of FSH to yield the same number of oocytes compared with overall intracytoplasmic sperm injection (ICSI) patients. The purpose of the present study was to examine the ovarian response in relation to number of trinucleotide repeats in premutation carriers undergoing COH for PGD. The primary end points included: level of E 2 at the day of hcg administration, number of retrieved oocytes, number of two-pronuclei pre-embryos (2PN), and gonadotropin dose. The secondary end point was to compare the ovarian response to the administered gonadotrophin dose between two subgroups of premutation carriers using 1 repeats as a cutoff point. MATERIALS AND METHODS Patients Candidates for PGD of fragile X syndrome were recruited from the fragile X screening program or following the birth of an affected individual in the immediate family. Because the risk for expansion to a full mutation is directly related to the number of repeats in the carrier mother (22, 23), only patients with >7 repeats or those with an affected offspring diagnosed before birth or already born were enrolled in the PGD program. Between January 26 and July 28, 18 couples were referred to our IVF unit seeking PGD for fragile X syndrome after genetic consultation. All of the the women completed a reproductive history questionnaire. The study was approved by the local ethics committee. Ovarian stimulation was achieved using a gonadotropinreleasing hormone analogue (Decapeptyl; Ferring, Keil, Germany) from the first day of the cycle followed by recombinant FSH (rfsh; Gonal F [Serono, Geneva, Switzerland] or Puregon [Organon, Oss, The Netherlands]) from the third day of the cycle. Human chorionic gonadotropin (Ovitrelle 2 mg; Serono) was given when at least three R17-mm follicles were demonstrated. Oocyte retrieval was scheduled 3 36 hours after administration of hcg. Oocytes were denuded of cumulus cells using hyaluronidase and a fine hand-drawn glass Pasteur pipette. Fertilization was performed by ICSI using a Nikon inverted microscope (Diaphot 3) with Narishige micromanipulators. Oocytes were examined 18 2 hours after ICSI under a dissecting microscope for the presence of pronuclei. Embryo cleavage rate and morphology were evaluated on day 3, before biopsy. The PGD procedure and multiplex nested polymerase chain reaction were as described elsewhere (2). The luteal phase support was supplemented by daily 6 mg micronized progesterone (Utrogestan; Pipette, Brussels, Belgium). Statistics Correlations between E 2 levels, age, number of retrieved oocytes, number of 2PN zygotes, total dose of gonadotropins, and number of repeats were evaluated using Pearson or Spearman correlations as appropriate. The two-sample t test was applied for testing differences between the two study subgroups of premutation carriers. Multiple logistic regressions were applied for testing the difference between the study groups and correlations between the studied parameters adjusted for confounders. Results are expressed as mean SD unless otherwise indicated. All tests applied were two tailed, and a P value of %% was considered to be significant. The data were analyzed using the SAS software (SAS Institute, Cary, NC) (24). RESULTS The study included 18 fertile women (mean age years, range 27 4 years) with a history of at least one termination of pregnancy due to prenatal diagnosis of a full mutation fragile X embryo in 14 of them. The other four patients were diagnosed as premutation carriers after screening tests. The patients underwent a total of 71 PGD cycles for fragile X syndrome. Their demographic and premutation molecular data are presented in Table 1. All fragile X premutation carriers had an ovulatory menstrual cycle during the study. The mean basal FSH level was miu/ml (range miu/ml). The mean of cycles/couple was , and the mean number of repeats was The correlation between the number of repeats and milestones of ovarian response is presented in Fig 1. We found a positive and significant correlation between the mean number of repeats and the mean number of retrieved oocytes (P¼.29), the mean number of 2PN zygotes (P¼.34), and the mean E 2 serum level at the day of hcg administration (P<.1). 87 Bibi et al. repeat, ovarian stimulation and PGD Vol. 94, No. 3, August 21

3 TABLE 1 Demographic, reproductive, and molecular characteristics of study population. Patient no. Age (y) Basal FSH (miu/l) FMR1 repeats a Reproductive history b G1PA1, 1 TOP G3P1A2, AC, 2 TOP G1PA1, 1 TOP G1P1, AB, 1 TOP G1P1A1, AC, 1 TOP GP, G1PA1, 1 TOP G1PA1, 1 TOP G2PA2, 2 TOP G1PA1, 1 TOP G1PA1, 1 TOP G1PA1, 1 TOP G1PA1, 1 TOP GP, G1P, 1 CM G1P1, 1 TOP G1P1, 1 SA GP1, 3 SA, 2 TOP a Maternal premutated allele. b A ¼ abortions; AB ¼ affected brother; AC ¼ affected child.; CM ¼ chemical pregnancy; G ¼ gravity; P ¼ parity; SM ¼ spontaneous abortion; TOP ¼ termination of pregnancy. Bibi. repeat, ovarian stimulation and PGD. Fertil Steril 21. We also found a negative significant correlation between the repeats and the mean dose of rfsh (P<.1). After adjustments for age, the positive correlation between the number of repeats and the E 2 level at the day of hcg administration remained significant (P¼.3), as did the correlation between the number of repeats and the number of retrieved oocytes (P¼.74) and 2PN zygotes (P¼.92; Fig. 1). After adjustments for age, the negative correlation between the number of repeats and the mean dose of administered rfsh remained significant (P<.1). Eight premutation carriers with <1 repeats (group A) underwent 22 PGD cycles, and ten premutation carriers with R1 repeats (group B) had 49 PGD cycles. Group A included three women with 7 repeats, three with 8 repeats, and two with 9 repeats, and Group B included one woman with 12 repeats, five with 1 repeats, and four with 2 repeats. Southern blot analysis was carried out to be sure of the allele size and to exclude full mutation carriers. The mean basal FSH level in group A was compared with in group B. Table 2 presents a comparison of the ovarian response between premutation carrier groups. The E 2 levels at the day of hcg administration, the number of retrieved oocytes, and the number of 2PN zygotes were significantly lower in group A compared with group B. Furthermore, the dose of rfsh given during the COH was significantly higher in group A compared with group B. DISCUSSION This study is the first to concentrate on the correlation between repeats and ovarian response after COH in patients who underwent PGD. This study differs from earlier ones which looked into the association between fragile X premutation and POF. Ennis et al. (18) and Sullivan et al. (13) demonstrated that the highest risk for ovarian dysfunction, defined by the age at menopause and prevalence of POF, occurred among carriers with a middle range of repeats (8 1). Allen et al. (2) confirmed the nonlinear association of repeat size and ovarian insufficiency: Carriers with 8 99 repeats had increased rates of menstrual dysfunction, infertility, and dizygotic twinning compared with noncarriers. Carriers with 8 99 repeats also had a 7-year reduction in mean age at menopause and consequently an increased prevalence of POF (32% vs. 1%) and increased risk of osteoporosis (2). Finally, carriers of both smaller and larger premutation repeat sizes also suffered from ovarian insufficiency, but not to as great an extent as those with 8 99 repeats. The association between repeats and ovarian response during COH, however, is less documented. Preimplantation genetic diagnosis has opened a new avenue of looking into the ovarian response. Patients with premutation who undergo PGD need to recruit a sufficient number of follicles to perform the genetic analysis related to difficulties of replication of the mutant gene. In a study Fertility and Sterility â 871

4 FIGURE 1 After adjustments for age, positive correlation was found between the mean number of repeats and (A) the E 2 level at the day of hcg administration (P¼.3), (B) the number of retrieved oocytes (P¼.7), and (C) the number of two-pronuclear zygotes 2PN (P¼.9). (D) Negative correlation between the number of repeats and the mean dose of administered recombinant FSH (P<.1). 9 8 A (r=.42, p=.3) 4 B (r=.32, p=.7) 7 4 E2 (pg/ml )* Retrived oocytes C (r=.31, p=.9) 7 D (r=-.46, p=.1) PN rfsh (IU/L )* Bibi. repeat, ovarian stimulation and PGD. Fertil Steril 21. by Platteau et al. (17) in which premutation carriers underwent PGD, the authors reported that fragile X patients needed significantly more ampules of FSH to yield the same number of oocytes compared with ICSI patients overall. Furthermore, some of the cycles were canceled due to low response to COH. Platteau et al., however, did not correlate the ovarian response with number of repeats (17). TABLE 2 Comparison of ovarian response between premutation carrier groups (mean ± SD). <1 repeats (group A) R1 repeats (group B) P value E 2 (pg/ml) 1,391 1,78 3,6 2,32. No. of retrieved oocytes No. of 2PN No. of cycles Dose of FSH (IU/L) 3,8 1,21 2, <.1 Age (y) Note: 2PN ¼ two-pronuclear zygote. Bibi. repeat, ovarian stimulation and PGD. Fertil Steril Bibi et al. repeat, ovarian stimulation and PGD Vol. 94, No. 3, August 21

5 Some centers have stopped offering PGD for fragile X carriers, and some have suggested other alternatives, such as egg donation (21). In the present study, we found a positive and significant correlation between the mean number of repeats and the milestones of ovarian response: E 2 levels, retrieved oocytes, and 2PN zygotes. Furthermore, these milestones were significantly higher among patients with R1 repeats than among patients with <1 repeats. The findings of a linear association with repeats in the present study are in accordance with those of Allen et al. (2), who found a nonlinear association of menopause age with premutation size, indicating that the age at menopause decreases with increasing repeat number until about 8 repeats, after which increasing repeat number is associated with an increasing age at menopause. The linear correlation in the present study is mainly attributed to the fact that the lowest number of repeats of the fragile X mutations in our patients were in the transition zone of 7 8 repeats, from which the correlation is linear even in the Allen et al. model (2). It is worth noting that the earlier studies cited were concerned with POF rather than ovarian response during COH. We also found a negative and significant correlation between the number of repeats and the dose of rfsh. These findings are in agreement with those of Platteau et al. (17), who reported that premutation carriers who underwent PGD needed significantly more ampules of FSH to yield the same number of oocytes compared with ICSI patients overall. The present findings show that although the ovarian response was associated with the number of repeats, patients with <1 repeats had a lower ovarian response compared with those with R1 repeats. This difference in ovarian response was, however, not associated with basal FSH levels, which were within the normal range. The present study cohort included fertile women with a history of termination of pregnancies or birth of affected babies, whereas patients in most of the cited studies were infertile. Nonetheless, our observations are in accordance with those of Gleicher et al. (26), who recently reported a strong statistical correlation between the number of triple repeats in FMR1 alleles and ovarian function, as represented by FSH and antimullerian hormone levels. In clinical terms, this means that the number of repeats correlates, in increasing order, with a clinical diagnosis of normal ovarian function, premature ovarian ageing, and POF. Patients with <1 repeats should be considered to be potentially low responders, and, as such, the COH protocol should be adjusted to recruit as many oocytes as possible to enable PGD performance. Furthermore, these patients should be counseled about the risk of POF despite the presence of normal levels of basal FSH. In summary, the present study demonstrates a positive correlation between the number of repeats and the parameters of ovarian response among patients who undergo COH for PGD. Furthermore, premutation carriers with <1 repeats suffer from impaired ovarian response and a decreased fertilization rate. REFERENCES 1. Hagerman RJ. Lessons from fragile X regarding neurobiology, autism, and neurodegeneration. J Dev Behav Pediatr 26;27: Broadie K, Pan L. Translational complexity of the fragile X mental retardation protein: insights from the fly. Mol Cell 2;17: Sutherland GR, Ashforth PL. X-linked mental retardation with macroorchidism and the fragile site at Xq 27 or 28. Hum Genet 1979;48: Oberle I, Rousseau F, Heitz D, Kretz C, Devys D, Hanauer A, et al. Instability of a base pair DNA segment and abnormal methylation in fragile X syndrome. Science 1991;22: Verkerk AJ, Pieretti M, Sutcliffe JS, Fu YH, Kuhl DP, Pizzuti A, et al. Identification of a gene (FMR-1) containing a repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome. Cell 1991;6: Pieretti M, Zhang FP, Fu YH, Warren ST, Oostra BA, Caskey CT, et al. Absence of expression of the FMR-1 gene in fragile X syndrome. Cell 1991;66: Nolin SL, Lewis FA 3rd, Ye LL, Houck GE Jr, Glicksman AE, Limprasert P, et al. Familial transmission of the FMR1 repeat. Am J Hum Genet 1996;9: Murray A, Macpherson JN, Pound MC, Sharrock A, Youings SA, Dennis NR, et al. The role of size, sequence and haplotype in the stability of FRAXA and FRAXE alleles during transmission. Hum Mol Genet 1997;6: Nolin SL, Brown WT, Glicksman A, Houck GE Jr, Gargano AD, Sullivan A, et al. Expansion of the fragile X repeat in females with premutation or intermediate alleles. Am J Hum Genet 23;72: Goswami D, Conway GS. Premature ovarian failure. Hum Reprod Update 2;11: Uzielli ML, Guarducci S, Lapi E, Cecconi A, Ricci U, Ricotti G, et al. Premature ovarian failure (POF) and fragile X premutation females: from POF to fragile X carrier identification, from fragile X carrier diagnosis to POF association data. Am J Med Genet 1999;84: Allingham-Hawkins DJ, Babul-Hirji R, Chitayat D, Holden JJ, Yang KT, Lee C, et al. Fragile X premutation is a significant risk factor for premature ovarian failure: the International Collaborative POF in Fragile X study preliminary data. Am J Med Genet 1999;83: Sullivan AK, Marcus M, Epstein MP, Allen EG, Anido AE, Paquin JJ, et al. Association of FMR1 repeat size with ovarian dysfunction. Hum Reprod 2;2: Murray A, Webb J, MacSwiney F, Shipley EL, Morton NE, Conway GS. Serum concentrations of follicle stimulating hormone may predict premature ovarian failure in FRAXA premutation women. Hum Reprod 1999;14: Hundscheid RD, Braat DD, Kiemeney LA, Smits AP, Thomas CM. Increased serum FSH in female fragile X premutation carriers with either regular menstrual cycles or on oral contraceptives. Hum Reprod 21;16: Welt CK, Smith PC, Taylor AE. Evidence of early ovarian aging in fragile X premutation carriers. J Clin Endocrinol Metab 24;89: Platteau P, Sermon K, Seneca S, Van Steirteghem A, Devroey P, Liebaers I. Preimplantation genetic diagnosis for fragile Xa syndrome:- difficult but not impossible. Hum Reprod 22;17: Ennis S, Ward D, Murray A. Nonlinear association between repeat number and age of menopause in FMR1 premutation carriers. Eur J Hum Genet 26;14: Wittenberger MD, Hagerman RJ, Sherman SL, McConkie-Rosell A, Welt CK, Rebar RW, et al. The FMR1 premutation and reproduction. Fertil Steril 27;87:46 6. Fertility and Sterility â 873

6 2. Malcov M, Naiman T, Yosef DB, Carmon A, Mey-Raz N, Amit A, et al. Preimplantation genetic diagnosis for fragile X syndrome using multiplex nested PCR. Reprod Biomed Online 27;14: Apessos A, Abou-Sleiman PM, Harper JC. Delhanty JDA Preimplantation genetic diagnosis of the fragile X syndrome by use of linked polymorphic markers. Prenat Diagn 21;21: Fu YH, Kuhl DP, Pizzuti A, Pieretti M, Sutcliffe JS, Richards S, et al. Variation of the repeat at the fragile X site results in genetic instability: resolution of the Sherman paradox. Cell 1991;67: Geva E, Yaron Y, Shomrat, Ben-Yehuda A, Zabari S, eretz H, et al. The risk of fragile X premutation expansion is lower in carriers detected by general prenatal screening than in carriers from known fragile X families. Genetic Testing 2;4: SAS procedures guide. Cary (NC): SAS Institute. 2. Allen EG, Sullivan AK, Marcus M, Small C, Dominguez C, Epstein MP, et al. Examination of reproductive aging milestones among women who carry the FMR1 premutation. Hum Reprod 27;22: Gleicher N, Weghofer A, Barad DH. A pilot study of premature ovarian senescence: I. Correlation of triple repeats on the FMR1 gene to ovarian reserve parameters FSH and antim ullerian hormone. Fertil Steril 29;91: Bibi et al. repeat, ovarian stimulation and PGD Vol. 94, No. 3, August 21