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Preimplantation genetic diagnosis for the treatment of failed in vitro fertilization–embryo transfer and habitual abortion
FERTILITY AND STERILITY威 VOL. 81, NO. 5, MAY 2004 Copyright ©2004 American Society for Reproductive Medicine Published by Elsevier Inc. Printed on acid-free paper in U.S.A.
Preimplantation genetic diagnosis for the treatment of failed in vitro fertilization– embryo transfer and habitual abortion Martin Wilding, Ph.D.,a Robert Forman, M.D.,b George Hogewind, M.D.,b Loredana Di Matteo, Ph.D.,c Fulvio Zullo, M.D.,d Fulvio Cappiello, M.D.,a and Brian Dale, Ph.D.a Center for Reproductive Biology, Clinica Villa del Sole, Naples, Italy; CRM London, London, United Kingdom; Il Universita` degli Studi di Napoli, Naples, Italy; and Institute of Gynecological and Pediatric Sciences, University of Catanzaro “Magna Grecia,” Catanzaro, Italy
Received December 23, 2002; revised and accepted October 6, 2003. Supported in part by a grant from Organon, Milan, Italy. Reprint requests: Dr. Martin Wilding, Center for Reproductive Biology, Clinica Villa del Sole, Via Manzoni, 15, 80126 Naples, Italy (FAX: 00-39081-5479251; E-mail: [email protected]). a Center for Reproductive Biology, Clinica Villa del Sole, Naples, Italy. b CRM London, Park Lorne, London, United Kingdom. c Facolta di Medicina e Chirurgia, II Universita` degli Studi di Napoli, Naples, Italy. d Institute of Gynecological and Pediatric Sciences, University of Catanzaro “Magna Grecia,” Catanzaro, Italy. 0015-0282/04/$30.00 doi:10.1016/j.fertnstert.2003. 10.028
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Objective: To apply preimplantation genetic diagnosis (PGD) for the treatment of patients with a history of failed IVF-ET or habitual aborters. Design: Prospective clinical study. Setting: Tertiary center for assisted reproduction. Patient(s): Ninety-four couples with failed IVF-ET after ⬎2 IVF cycles and 64 couples with ⬎2 spontaneous abortions. Intervention(s): Patients were prepared for oocyte retrieval using standard controlled ovarian hyperstimulation protocols after standard laboratory techniques. Blastomeres from 6- to 8-cell embryos were analysed using fluorescence in situ hybridization with commercial chromosomal probes, and normoploid embryos were transferred on day 3 after fertilization. Main Outcome Measure(s): Pregnancy and implantation rates and live births. Result(s): Both 3- and 5-probe PGD resulted in a significantly higher outcome than controls for failed IVF-ET. Five-probe PGD appeared to be more suitable for habitual aborters. Conclusion(s): This pilot study suggests that 3-probe PGD is a valid option for failed IVF-ET patients. The use of five or more probes is indicated for habitual aborters. (Fertil Steril威 2004;81:1302⫺4. ©2004 by American Society for Reproductive Medicine.) Key Words: In vitro fertilization, fluorescence in situ hybridization, human embryo, spontaneous pregnancy loss
Chromosomal aneuploidy is one of the major factors that can negatively affect the establishment of a viable pregnancy through the failure of embryos to implant or the spontaneous loss of the fetus in the first trimester (1–7). The analysis of human preimplantation embryos developing in vitro has demonstrated that embryos with both normal and abnormal morphology can be affected by aneuploidy (7–10). This suggests that the transfer of embryos on the basis of morphology alone is a poor indicator of implantation potential, and this factor may be especially critical in patients with a history of failure to conceive (11). Preimplantation genetic diagnosis (PGD) is a technique in which the chromosomal constituent
of human embryos developing in vitro can be analyzed and chromosomally abnormal embryos can be deselected for transfer (12–16). Preimplantation genetic diagnosis is indicated for the treatment of pathologies such as mosaic karyotype or advanced maternal age in the couple undergoing IVF (17, 18). In this study, we test whether PGD is a valid technique for patients in whom multiple cycles of IVF-ET have failed or for fertile patients in whom the developing fetus is habitually lost in the first trimester of pregnancy. We show that, whereas the analysis of three chromosomes using fluorescence in situ hybridization (FISH) is successful for the treatment of failed IVF-ET, 5-color FISH using chromosomes 13, 16, 18, 21, and 22 is indicated for the treatment of habitual aborters.
MATERIALS AND METHODS Study Design This study is a prospective pilot study of the use of PGD in patients with a history of at least two cycles of failed implantation after IVF with either IVF or intracytoplasmic sperm injection (failed IVF-ET) and fertile patients affected by at least two incidences of spontaneous recurrent pregnancy loss (habitual aborters). Patients seeking assisted reproduction between 1999 and 2001 agreed to a cycle of PGD and provided written consent. Institutional Review Board approval was obtained because of the absence of such a board, however, the experimental protocols adhered to the Helsinki declaration of 1975 and successive modifications. All couples went through a gynecological and andrological workup to exclude known causes of infertility. Couples were included in the trial if the female menstrual cycle ranged from 24 to 35 days (intra-individual variability, ⫾3 days), hysteroscopy revealed a normal uterine cavity, the karyotype of both subjects of the couple was normal, and biochemical assessments demonstrated the absence of metabolic, autoimmune, and infectious disorders. Patients were excluded from the trial if the female basal FSH was ⬎10 IU/L, body mass index [weight (kg)/height (m)2 ⫻ 100] was ⬎29, biochemical and/or ultrasound evidence suggested polycystic ovarian syndrome, the female partner had stage III–IV endometriosis, autoimmune, thyroid, or chromosomal abnormalities were present, only one ovary was present, hysteroscopy showed an abnormal uterine cavity, or the male partner had obstructive or nonobstructive azoospermia. No age criteria were imposed.
IVF and ET Techniques Patients were prepared by standard ovarian hyperstimulation regimes including down-regulation of the pituitary gland with a GnRH agonist followed by ovarian stimulation with exogenous recombinant FSH. Starting doses of FSH were in the range of 225–300 IU and were followed by adjustment of the stimulation protocol after day 5 depending on the ovarian response as determined by the levels of serum E2 and ultrasound analysis of folliculogenesis. Oocyte retrieval was performed at the Center for Reproductive Biology, Clinica Villa del Sole, in Naples, Italy, 36 hours after the administration of 10,000 IU -hCG when at least three follicles 18 –20 mm in diameter were observed by ultrasound examination. IVF techniques followed standard protocols for oocyte and sperm preparation. Commercially prepared culture media were used for all laboratory protocols (Medicult, Copenhagen, Denmark). Fertilization was checked 16 –20 hours after insemination to determine the presence of two clear pronuclei. A morphological and developmental check was performed at 40 – 41 hours after fertilization and 64 – 65 hours after fertilization. ET was performed using a soft ET catheter (Edwards-Wallace, SIMS Portex, Hyde, Kent, UK). FERTILITY & STERILITY威
Pregnancies were reported when blood hCG was positive 15 days after ET and subsequent ultrasound examination of the uterus 7 weeks after ET demonstrated a clear gestational sac(s) with fetal heart beat(s) present. In addition, the number of live, term births was recorded.
Embryo Biopsy and FISH Procedures Preimplantation genetic diagnosis using 3-probe analysis by FISH was used in a series of 42 patients attending for assisted reproduction. This group of patients included 26 failed IVF-ET couples and 16 habitual aborters. A further 12 failed IVF-ET couples that declined PGD technology gave consent for their data to be used as controls. No control data could be obtained for habitual aborters that declined PGD technology because of the lack of need for these couples to undergo assisted reproduction. Preimplantation genetic diagnosis using 5-probe FISH was applied to 35 failed IVF-ET couples and 48 habitual aborters. Again, 21 failed IVF-ET couples that declined PGD technology gave consent for their data to be used as controls. As for 3-probe PGD, no control data were available for habitual aborters. Embryos with over five cells on day 3 after fertilization (64 – 65 hours) were biopsied. Embryos were placed in a biopsy medium deficient in both calcium and magnesium to loosen attachments between blastomeres (embryo biopsy medium; Medicult). A hole was drilled in the zona pellucida overlying the blastomeres to be removed using acidified Tyrodes solution (Medicult) expelled from a 2–3 m diameter assisted hatch pipette (Research Instruments, Cornwall, UK). After rupture of the zona pellucida, a 30 m diameter blastomere biopsy pipette (Research Instruments) was used to remove the blastomeres for analysis. Biopsied blastomeres were subjected to a brief hypotonic shock in 1% sodium citrate followed by attachment to a slide and fixation in Carnoys fixative. Fixed blastomeres were dehydrated in an ethanol series before hybridization. The probes for 3-probe FISH were as follows: LSI 13 (RB-1, 13q14 green, Vysis, Downers Grove, IL), CEP 18 (Alpha Satellite DYZ3 orange/green, Vysis), and LSI 21 (orange, region 21q22.13– q22.2, Vysis). Five-probe FISH was performed with the following probes: LSI 13 (RB-1, 13q14 orange, Vysis), CEP 16 (Satellite II DNA/D16Z3, aqua, Vysis), CEP 18 (Alpha Satellite DYZ3 orange/aqua, Vysis), LSI 21 (region 21q22.13– q22.2, orange/green, Vysis), and LSI 22q (bcr region 22q11.2, green, Vysis). Slides were codenatured on a programmable slide warmer (Vysis Hybrite). Excess probe was removed by washing in 0.4⫻ SSC solution containing 0.3% IPEGAL CA-630 (Sigma, Milan, Italy) at 73°C followed by a wash in 2⫻ SSC ⫹ 0.1% IPEGAL CA-630 (Sigma) at room temperature. Slides were then washed in phosphate-buffered saline, dehydrated, and air-dried. A counter-stain containing 125 ng/mL 4⬘,6-diamidino-2-phenylindole, dihydrochloride was used to visualize nuclei. 1303
TABLE 1 Three-color PGD cycles for failed IVF-ET.
No. of patients Mean age (years) No. of previous cycles/pregnancy losses No. of oocyte retrievals No. of mature oocytes retrieved (mean ⫾ SD/cycle) No. of fertilized embryos (fertilization rate) No. of embryos biopsied No. of normoploid embryos (% of cohort) Monosomies (% of cohort)b Trisomies (% of cohort)c Otherd (% of cohort)e No. of ETs (mean ⫾ SD) No. of clinical pregnancies (% pregnancies/transfer) No. of fetal heartbeats (implantation rate) No. of live births
Controls
PGD
Pa
12 35.5 ⫾ 4.0 31 15 169 (11.3 ⫾ 6.3) 138 (81.6) NA NA NA NA NA 55 (3.7 ⫾ 0.7) 2 (13.0) 3 (5.4) 3
26 36.3 ⫾ 3.8 84 34 286 (9.1 ⫾ 3.2) 223 (78) 187 119 (64) 12 (6.4)a 6 (3.3) 50 (26.7) 116 (3.3 ⫾ 0.6) 18 (53.0) 26 (22.4) 24
NA .9 NA NA .7 .5 NA NA NA NA NA .7 .02 .01 NA
Note: Data are presented as actual numbers. NA ⫽ not applicable. a P ⬍ .05 is considered statistically significant. b Includes 10 mono-21, 1 mono-13, and 1 mono-18. c Includes 2 trisomy 21 and 4 trisomy 18. e Includes 3 mono-13, 6 mono-16, 4 mono-18, 4 mono-21, 2 mono-22, and 10 combined monosomies. d Includes 3 tri-13, 2 tri-16, 3 tri-21, 2 tri-22, and 6 combined trisomies. d Includes haploid, triploid, combined monosomies, trisomies, and chaotic mosaics because of the difficulty in distinguishing among these karyotypes under the biopsy conditions. Wilding. Three- and 5-color PGD. Fertil Steril 2004.
FISH signals were observed and recorded using a Nikon Genikon karyotype system based on a Nikon Eclipse microscope (Nikon, Florence, Italy). Embryos diagnosed as normoploid after FISH contained two distinct signals for each of the chromosomes tested. Embryos were diagnosed with simple aneuploidies when the tested chromosomes had a single signal either more or less than diploid. Embryos were diagnosed as chaotic mosaics when multiple aneuploidies were present, suggesting random chromosome segregation. Other forms of embryo abnormality (haploidy, triploidy, etc.) were diagnosed as observed.
Statistical Analyses
All data are presented as means ⫾ SD except where stated otherwise. All statistics were performed using the Sigma Stat package (SPSS, Ekrath, Germany). The Mann-Whitney rank sum test was used to adjust the t-test for small populations of data. The z-test with Yates correction was used to determine the significance of proportions.
RESULTS Preimplantation Genetic Diagnosis for Failed IVF-ET In total, 34 3-probe PGD cycles and 15 control IVF cycles were performed with the 38 couples (Table 1). The maternal age for controls was 35.5 ⫾ 4.0 years, and for PGD patients, 36.3 ⫾ 3.8 years. Clinical IVF parameters were not significantly different between the two groups (Table 1). After biopsy, a total of 119 embryos (64%) were diagnosed as 1304 Wilding et al.
Three- and 5-color PGD
suitable for transfer in patients undergoing PGD, whereas 55 embryos were transferred in the control group (Table 1). A total of 18 pregnancies (53% pregnancy rate/transfer) with an implantation rate of 22.4% were achieved in the 34 PGD procedures performed (Table 1). In contrast, controls were characterized by a pregnancy rate of 13% (two pregnancies in 15 cycles) and an implantation rate of 5.4% (three embryos implanting/55 transferred, Table 1). Sixty-eight out of the 187 embryos (36%) obtained from group A patients undergoing PGD were diagnosed with chromosomal abnormalities after PGD (Table 1). Eighteen embryos had simple or multiple aneuploidies, while 50 embryos had more complex chromosomal abnormalities (Table 1). These abnormalities could not be separated into chaotic mosaics or complex aneuploidies because the removal of a single blastomere does not permit the accurate diagnosis of the embryo karyotype. A second series of failed IVF-ET patients were treated with 5-probe PGD using fluorescence probes directed to chromosomes 13, 16, 18, 21, and 22. In total, a series of 56 patients were treated with this protocol. This patient group included 35 patients in which the PGD protocol was applied and 21 controls. Again, maternal age, fertilization rates, the rate of embryo development, and number of embryos transferred were not significantly different between the two groups (Table 2). After 5-probe PGD, 62.1% of embryos were diagnosed as normoploid, 29 embryos had single or complex monosomies, 16 embryos were affected by trisomies, and 54 embryos had complex chromosomal abnorVol. 81, No. 5, May 2004
TABLE 2
TABLE 3
Five-color FISH for failed IVF-ET. Controls
PGD-FISH for habitual aborters. PGD
Pa
No. of patients 21 35 NA Mean age (years) 35.1 ⫾ 3.3 34.3 ⫾ 3.7 .9 No. of previous cycles/pregnancy 77 102 NA losses No. of oocyte retrievals 24 41 NA No. of mature oocytes retrieved 198 (8.2 ⫾ 5.0) 399 (8.5 ⫾ 4.4) .9 (mean ⫾ SD/cycle) No. of fertilized embryos 158 (80) 303 (76) .32 (fertilization rate) No. of embryos biopsied NA 262 NA Normoploid NA 163 (62.1) NA Monosomiesb NA 29 (11.1)c NA Trisomiesc NA 16 (6.1)d NA Otherd NA 54 (20.6) NA No. of normoploid embryos (% NA 163 (62) .92 of cohort) No. of ETs (mean ⫾ SD) 90 (3.8 ⫾ 1.2) 163 (3.9 ⫾ 1.0) .95 No. of clinical pregnancies (% 6 (25.0) 16 (39.0) .38 pregnancies/transfer) No. of fetal heartbeats 9 (10.0) 34 (21.0) .04 (implantation rate) No. of live births 7 33 NA Note: Data are presented as actual numbers. NA ⫽ not applicable. a P ⬍.05 is considered statistically significant. b Includes 3 mono-13, 6 mono-16, 4 mono-18, 4 mono-21, 2 mono-22, and 10 combined monosomies. c Includes 3 tri-13, 2 tri-16, 3 tri-21, 2 tri-22, and 6 combined trisomies. d Includes haploid, triploid, combined monosomies, trisomies, and chaotic mosaics because of the difficulty in distinguishing among these karyotypes under the biopsy conditions. Wilding. Three- and 5-color PGD. Fertil Steril 2004.
malities (Table 2). Six of the control (25% pregnancy rate/ transfer) and 16 PGD patients (39% pregnancy rate/transfer) achieved pregnancy after a total of 65 PGD cycles; Table 2). Although the pregnancy rate was not significantly higher than in the control cycles, the implantation rate after PGD was significantly higher than in controls (Table 2).
Preimplantation Genetic Diagnosis for Habitual Aborters Habitual aborters are otherwise fertile couples that have experienced more than two episodes of spontaneous pregnancy termination within the first trimester. Where no diagnosis could be made of this problem, 3-probe PGD was offered to these couples. In total, 16 couples accepted 3-probe PGD technology (Table 3). One hundred fifty-five mature oocytes were retrieved in the 26 cycles performed by these couples. Of these, 127 fertilized normally after IVF (82% fertilization rate; Table 3). One hundred twelve embryos were considered to be of sufficient quality 64 – 65 hours after fertilization for the biopsy procedure, and of these, 83 embryos were considered normoploid after 3-probe FISH (Table 3). A total of three clinical pregnancies were FERTILITY & STERILITY威
Three-color PGD No. of patients Mean age (years) No. of previous cycles/pregnancy losses No. of oocyte retrievals No. of mature oocytes retrieved (mean ⫾ SD/ cycle) No. of fertilized embryos (fertilization rate) No. of embryos biopsied Normoploid Monosomies Trisomies Othere No. of normoploid embryos (% of cohort) No. of ETs (mean ⫾ SD) No. of clinical pregnancies (% pregnancies/transfer) No. of fetal heartbeats (implantation rate) No. of live births
Five-color PGD
16 37.6 ⫾ 4.1 44
48 35.4 ⫾ 3.3 177
26 155 (5.6 ⫾ 4.4)
62 700 (10.3 ⫾ 5.0)
127 (82)
553 (79)
112 83 (74.1%) 4 (3.6%)a 3 (2.7%)c 22 (19.6%) 83 (74)
501 311 (62.1%) 71 (14.2%)b 11 (2.1%)d 108 (21.6%) 311 (62)
83 (2.9 ⫾ 1.3) 3 (11.5)
276 (3.8 ⫾ 1.0) 22 (35.5)
4 (4.8) 4
58 (21.1) 54
Note: Data are presented as actual numbers. NA ⫽ not applicable. a Includes 1 mono-18 and 3 mono-13. c Includes 1 trisomy 21, 1 trisomy 18 and 1 trisomy 13. b Includes 6 mono-13, 14 mono-16, 11 mono-18, 3 mono-21, 12 mono-22, and 25 combined monosomies. d Includes 1 tri-13, 3 tri-16, 1 tri-21, 3 tri-22, and 3 combined trisomies. e Includes haploid, triploid, combined monosomies, trisomies, and chaotic mosaics because of the difficulty of distinguishing among these karyotypes under the biopsy conditions. Statistical analysis was not performed on the groups because the two patient groups were selected in diverse periods and therefore are not derived from the same distribution. Wilding. Three- and 5-color PGD. Fertil Steril 2004.
obtained out of the 26 cycles performed (11.5% pregnancy rate/transfer), and a total of four gestational sacs with fetal heart beats were observed (implantation rate, 4.8%; Table 3). All four sacs gave rise to healthy births (Table 3). The low success rate using three-chromosome FISH for patients affected by this type of infertility suggests that an extension of the FISH protocol to cover chromosomes 16 and 22 could help this group of patients. We therefore offered 5-probe PGD-FISH, using fluorescent probes for chromosomes 13, 16, 18, 21, and 22 on a second series of patients. In total, a series of 48 patients were treated with this protocol (Table 3). After 62 oocyte retrievals, 22 patients achieved pregnancy with 5-probe FISH (35.5% pregnancy rate/transfer; Table 3). A total of 58 gestational sacs with fetal heart beats were obtained after the transfer of 276 embryos (implantation rate, 21.1%; Table 3). Of these, 54 resulted in healthy live births (Table 3). 1305
The above data suggest that the analysis of five chromosomes by FISH during PGD cycles is more reliable for IVF outcome than the detection of three chromosomes in habitual aborters. We examined whether this increase in reliability was due to an increase in the detection efficiency of a particular type of aneuploidy or karyotype. The use of fivecolor FISH for patients attending the PGD program for multiple spontaneous pregnancy loss was correlated with an increase in the detection of monosomic embryos (Table 3). Monosomies detected included a proportion of both single and combined monosomies for chromosomes 16 and 22 (Table 3). Although the comparison between 3- and 5-probe FISH for habitual aborters is tentative in the present work, the data suggest a correlation between the presence of aneuploidies not detected by three-chromosome FISH and the increase in outcome observed.
DISCUSSION In this study, we tested whether the genetic diagnosis of aneuploidy in human preimplantation embryos improves the prognosis after IVF in couples suffering from two specific infertility problems: repetitive failed IVF-ET and habitual abortion. Preimplantation genetic diagnosis protocols used either 3-probe analysis examining chromosomes 13, 18, and 21 or 5-probe analysis for chromosomes 13, 16, 18, 21, and 22. Chromosomes 13, 18, 21 were chosen because they are the cause of the three known aneuploidies that can result in live births (1, 8 –10, 15), and chromosomes 16 and 22 were chosen because they are the cause of the aneuploidies that are known to be common in early pregnancy loss (19, 20). Both protocols resulted in the detection of chromosomal abnormalities in approximately 38% of embryos that would have been considered suitable for transfer into the patient’s uterus. The outcome of IVF in failed IVF-ET patients was significantly improved after both 3- and 5-probe PGD with respect to controls. However, 5-probe FISH did not appear to offer advantages over 3-probe FISH in this limited trial. These data suggest that PGD can offer advantages over simply repeating the IVF procedure in failed IVF-ET patients by simply deselecting chromosomally abnormal embryos. Further, the trial suggests that 3-probe PGD may be sufficient in eliminating chromosomally abnormal embryos in these cases. Although the analysis of as few as three chromosomes appears to aid in the treatment of patients suffering from multiple cycles of failed IVF-ET, little was gained with 3-probe PGD for patients suffering from habitual abortion. The use of 3-probe PGD resulted in three pregnancies in a total of 26 oocyte retrievals. Aneuploidies of chromosomes 16 and 22 are often implicated in spontaneous pregnancy loss (19, 20). In these patients, 5-probe FISH appeared to assist in a positive outcome. Of the 48 couples included in the trial, 22 achieved pregnancy after 62 oocyte retrievals 1306 Wilding et al.
Three- and 5-color PGD
(35.5% pregnancy rate/oocyte retrieval). Even more pertinent to the study was the observation that 54 of these embryos resulted in the birth of a healthy baby. Although no control data were available for this group of patients in the present study, the data suggest that PGD can improve the prognosis for this group of patients. We examined the types of chromosomal abnormality detected by 5-color FISH in this group of patients. Although the level of complex chromosomal aneuploidies was similar to that of three-chromosome analysis, we noted a significant increase in the detection of monosomies for the chromosomes tested (Table 3). Of the 71 monosomic embryos characterized, 26 (36.6%) were single monosomies for chromosomes 16 or 22 and a further 25 (35.2%) were combined monosomies. Although these data do not demonstrate that monosomies for chromosomes 16 and 22 specifically caused the previously low outcome in this group of patients, the data suggest that high levels of specific monosomies are correlated with the low outcome in this group of patients. This pilot study therefore suggests that the use of 3-probe PGD is often sufficient to enable the selection of chromosomally normal embryos for transfer in patients characterized by previous IVF failure. The data further suggest that 5-probe PGD is more useful for habitual aborters. Although the data are interesting, it is not clear from the current limited sample of patients whether the current protocol can be applied to all patient groups (for example, patients of advanced age). A larger, prospective, randomized, controlled, multicenter trial comparing PGD with other IVF protocols (such as changing the ovarian stimulation protocol or embryo selection criteria) is required to determine the accuracy of the current data, and could produce a definitive protocol for these patients. References 1. Hassold T, Chiu D. Maternal age-specific rates of numerical chromosome abnormalities with special reference to trisomy. Hum Genet 1985;70:11–17. 2. Hassold T, Jacobs P. Trisomy in man. Ann Rev Genet 1984;18:69 –97. 3. Yoon P, Freeman S, Sherman S, Taft LF, Gu Y, Pettay D, et al. Advanced maternal age and the risk of Downs syndrome characterized by the meiotic stage of the chromosomal error: a population-based study. Am J Hum Genet 1996;58:628 –33. 4. Dailey T, Dale B, Cohen J, Munne S. Association between nondisjunction and maternal age in meiosis-II human oocytes. Am J Hum Genet 1996;59:176 –84. 5. Antonarakis S, Petersen M, McInnis M, Adelsberger PA, Schinzel AA, Binkert F, et al. The meiotic stage of non-disjunction in trisomy 21: determination by using DNA polymorphisms. Am J Hum Genet 1992; 50:544 –50. 6. Mikkelsen M, Halberg A, Poulsen H. Epidemiological study of Downs syndrome in Denmark, including family studies of chromosomes and DNA markers. Dev Brain Dysfunct 1995;8:4 –12. 7. Munne S, Alikani M, Tomkin G, Grifo J, Cohen J. Embryo morphology, developmental rates and maternal age are correlated with chromosome abnormalities. Fertil Steril 1995;64:382–91. 8. Munne S, Grifo J, Cohen J. Chromosome abnormalities in human arrested preimplantation embryos: a multiple-probe FISH study. Am J Hum Genet 1994;55:150 –9. 9. Harper JC, Coonen E, Handyside AH, Winston RM, Hopman AH, Delhanty JD. Mosaicism of autosomes and sex chromosomes in morphologically normal, monospermic human embryos. Prenat Diagn 1995;15:41–50. 10. Delhanty JDA, Harper JC, Ao A, Handyside AH, Winston RM. Multicolor FISH detects frequent chromosomal mosaicism and chaotic
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11. 12. 13. 14. 15.
division in normal preimplantation embryos from fertile patients. Hum Genet 1997;99:755–60. Nass TE, Escudero T, Marello EC, Velilla E, Sandalinas M, Werlin MB, et al. Dissociation between embryo morphology and euploidy in IVF patients with poor prognosis. Fertil Steril 2002;77(Suppl 3):S11. Delhanty JDA. Chromosome analysis by FISH in human preimplantation genetics. Hum Reprod 1997;12:153–5. Handyside AH, Kontogianni EH, Hardy K, Winston RML. Pregnancies from biopsied human preimplantation embryos sexed by Y specific DNA amplification. Nature 1990;344:768 –70. Handyside AH, Lesko LG, Tarin JJ, Winston RML, Hughes MR. Birth of a normal girl after in-vitro fertilization and preimplantation diagnostic testing for cystic fibrosis. N Engl J Med 1992;327:905–8. Munne S, Lee A, Rosenwaks Z, Grifo J, Cohen J. Diagnosis of major chromosome abnormalities in human preimplantation embryos. Hum Reprod 1993;8:2185–91.
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16. Grifo JA, Tang YX, Munn S, Alikani M, Cohen J, Rosenwaks Z. Healthy deliveries from biopsied human embryos. Hum Reprod 1994; 9:912–6. 17. Gianaroli L, Magli MC, Munne S, Fiorentino A, Montanaro N, Ferraretti AP. Will preimplantation genetic diagnosis assist patients with a poor prognosis to achieve pregnancy? Hum Reprod 1997;12: 1762–7. 18. Munne S, Magli MC, Cohen J, Morton P, Sadowy S, Gianaroli L, et al. Positive outcome after preimplantation diagnosis of aneuploidy in human embryos. Hum Reprod 1999;14:2191–9. 19. Carp H, Toder V, Aviram A, Daniely M, Mashiach S, Barkai G. Karyotype of the abortus in recurrent miscarriage. Fertil Steril 2001; 75:678 –82. 20. Munne S, Magli MC, Bahce M, Fung J, Legator M, Morrison L, et al. Preimplantation diagnosis for the aneuploidies most commonly found in spontaneous abortions and live births: XY,13,14,15,16,18 and 22. Prenat Diagn 1998;18:1459 –66.
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Preimplantation genetic diagnosis for the treatment of failed in vitro fertilization– embryo transfer and habitual abortion Martin Wilding, Ph.D.,a Robert Forman, M.D.,b George Hogewind, M.D.,b Loredana Di Matteo, Ph.D.,c Fulvio Zullo, M.D.,d Fulvio Cappiello, M.D.,a and Brian Dale, Ph.D.a Center for Reproductive Biology, Clinica Villa del Sole, Naples, Italy; CRM London, London, United Kingdom; Il Universita` degli Studi di Napoli, Naples, Italy; and Institute of Gynecological and Pediatric Sciences, University of Catanzaro “Magna Grecia,” Catanzaro, Italy
Received December 23, 2002; revised and accepted October 6, 2003. Supported in part by a grant from Organon, Milan, Italy. Reprint requests: Dr. Martin Wilding, Center for Reproductive Biology, Clinica Villa del Sole, Via Manzoni, 15, 80126 Naples, Italy (FAX: 00-39081-5479251; E-mail: [email protected]). a Center for Reproductive Biology, Clinica Villa del Sole, Naples, Italy. b CRM London, Park Lorne, London, United Kingdom. c Facolta di Medicina e Chirurgia, II Universita` degli Studi di Napoli, Naples, Italy. d Institute of Gynecological and Pediatric Sciences, University of Catanzaro “Magna Grecia,” Catanzaro, Italy. 0015-0282/04/$30.00 doi:10.1016/j.fertnstert.2003. 10.028
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Objective: To apply preimplantation genetic diagnosis (PGD) for the treatment of patients with a history of failed IVF-ET or habitual aborters. Design: Prospective clinical study. Setting: Tertiary center for assisted reproduction. Patient(s): Ninety-four couples with failed IVF-ET after ⬎2 IVF cycles and 64 couples with ⬎2 spontaneous abortions. Intervention(s): Patients were prepared for oocyte retrieval using standard controlled ovarian hyperstimulation protocols after standard laboratory techniques. Blastomeres from 6- to 8-cell embryos were analysed using fluorescence in situ hybridization with commercial chromosomal probes, and normoploid embryos were transferred on day 3 after fertilization. Main Outcome Measure(s): Pregnancy and implantation rates and live births. Result(s): Both 3- and 5-probe PGD resulted in a significantly higher outcome than controls for failed IVF-ET. Five-probe PGD appeared to be more suitable for habitual aborters. Conclusion(s): This pilot study suggests that 3-probe PGD is a valid option for failed IVF-ET patients. The use of five or more probes is indicated for habitual aborters. (Fertil Steril威 2004;81:1302⫺4. ©2004 by American Society for Reproductive Medicine.) Key Words: In vitro fertilization, fluorescence in situ hybridization, human embryo, spontaneous pregnancy loss
Chromosomal aneuploidy is one of the major factors that can negatively affect the establishment of a viable pregnancy through the failure of embryos to implant or the spontaneous loss of the fetus in the first trimester (1–7). The analysis of human preimplantation embryos developing in vitro has demonstrated that embryos with both normal and abnormal morphology can be affected by aneuploidy (7–10). This suggests that the transfer of embryos on the basis of morphology alone is a poor indicator of implantation potential, and this factor may be especially critical in patients with a history of failure to conceive (11). Preimplantation genetic diagnosis (PGD) is a technique in which the chromosomal constituent
of human embryos developing in vitro can be analyzed and chromosomally abnormal embryos can be deselected for transfer (12–16). Preimplantation genetic diagnosis is indicated for the treatment of pathologies such as mosaic karyotype or advanced maternal age in the couple undergoing IVF (17, 18). In this study, we test whether PGD is a valid technique for patients in whom multiple cycles of IVF-ET have failed or for fertile patients in whom the developing fetus is habitually lost in the first trimester of pregnancy. We show that, whereas the analysis of three chromosomes using fluorescence in situ hybridization (FISH) is successful for the treatment of failed IVF-ET, 5-color FISH using chromosomes 13, 16, 18, 21, and 22 is indicated for the treatment of habitual aborters.
MATERIALS AND METHODS Study Design This study is a prospective pilot study of the use of PGD in patients with a history of at least two cycles of failed implantation after IVF with either IVF or intracytoplasmic sperm injection (failed IVF-ET) and fertile patients affected by at least two incidences of spontaneous recurrent pregnancy loss (habitual aborters). Patients seeking assisted reproduction between 1999 and 2001 agreed to a cycle of PGD and provided written consent. Institutional Review Board approval was obtained because of the absence of such a board, however, the experimental protocols adhered to the Helsinki declaration of 1975 and successive modifications. All couples went through a gynecological and andrological workup to exclude known causes of infertility. Couples were included in the trial if the female menstrual cycle ranged from 24 to 35 days (intra-individual variability, ⫾3 days), hysteroscopy revealed a normal uterine cavity, the karyotype of both subjects of the couple was normal, and biochemical assessments demonstrated the absence of metabolic, autoimmune, and infectious disorders. Patients were excluded from the trial if the female basal FSH was ⬎10 IU/L, body mass index [weight (kg)/height (m)2 ⫻ 100] was ⬎29, biochemical and/or ultrasound evidence suggested polycystic ovarian syndrome, the female partner had stage III–IV endometriosis, autoimmune, thyroid, or chromosomal abnormalities were present, only one ovary was present, hysteroscopy showed an abnormal uterine cavity, or the male partner had obstructive or nonobstructive azoospermia. No age criteria were imposed.
IVF and ET Techniques Patients were prepared by standard ovarian hyperstimulation regimes including down-regulation of the pituitary gland with a GnRH agonist followed by ovarian stimulation with exogenous recombinant FSH. Starting doses of FSH were in the range of 225–300 IU and were followed by adjustment of the stimulation protocol after day 5 depending on the ovarian response as determined by the levels of serum E2 and ultrasound analysis of folliculogenesis. Oocyte retrieval was performed at the Center for Reproductive Biology, Clinica Villa del Sole, in Naples, Italy, 36 hours after the administration of 10,000 IU -hCG when at least three follicles 18 –20 mm in diameter were observed by ultrasound examination. IVF techniques followed standard protocols for oocyte and sperm preparation. Commercially prepared culture media were used for all laboratory protocols (Medicult, Copenhagen, Denmark). Fertilization was checked 16 –20 hours after insemination to determine the presence of two clear pronuclei. A morphological and developmental check was performed at 40 – 41 hours after fertilization and 64 – 65 hours after fertilization. ET was performed using a soft ET catheter (Edwards-Wallace, SIMS Portex, Hyde, Kent, UK). FERTILITY & STERILITY威
Pregnancies were reported when blood hCG was positive 15 days after ET and subsequent ultrasound examination of the uterus 7 weeks after ET demonstrated a clear gestational sac(s) with fetal heart beat(s) present. In addition, the number of live, term births was recorded.
Embryo Biopsy and FISH Procedures Preimplantation genetic diagnosis using 3-probe analysis by FISH was used in a series of 42 patients attending for assisted reproduction. This group of patients included 26 failed IVF-ET couples and 16 habitual aborters. A further 12 failed IVF-ET couples that declined PGD technology gave consent for their data to be used as controls. No control data could be obtained for habitual aborters that declined PGD technology because of the lack of need for these couples to undergo assisted reproduction. Preimplantation genetic diagnosis using 5-probe FISH was applied to 35 failed IVF-ET couples and 48 habitual aborters. Again, 21 failed IVF-ET couples that declined PGD technology gave consent for their data to be used as controls. As for 3-probe PGD, no control data were available for habitual aborters. Embryos with over five cells on day 3 after fertilization (64 – 65 hours) were biopsied. Embryos were placed in a biopsy medium deficient in both calcium and magnesium to loosen attachments between blastomeres (embryo biopsy medium; Medicult). A hole was drilled in the zona pellucida overlying the blastomeres to be removed using acidified Tyrodes solution (Medicult) expelled from a 2–3 m diameter assisted hatch pipette (Research Instruments, Cornwall, UK). After rupture of the zona pellucida, a 30 m diameter blastomere biopsy pipette (Research Instruments) was used to remove the blastomeres for analysis. Biopsied blastomeres were subjected to a brief hypotonic shock in 1% sodium citrate followed by attachment to a slide and fixation in Carnoys fixative. Fixed blastomeres were dehydrated in an ethanol series before hybridization. The probes for 3-probe FISH were as follows: LSI 13 (RB-1, 13q14 green, Vysis, Downers Grove, IL), CEP 18 (Alpha Satellite DYZ3 orange/green, Vysis), and LSI 21 (orange, region 21q22.13– q22.2, Vysis). Five-probe FISH was performed with the following probes: LSI 13 (RB-1, 13q14 orange, Vysis), CEP 16 (Satellite II DNA/D16Z3, aqua, Vysis), CEP 18 (Alpha Satellite DYZ3 orange/aqua, Vysis), LSI 21 (region 21q22.13– q22.2, orange/green, Vysis), and LSI 22q (bcr region 22q11.2, green, Vysis). Slides were codenatured on a programmable slide warmer (Vysis Hybrite). Excess probe was removed by washing in 0.4⫻ SSC solution containing 0.3% IPEGAL CA-630 (Sigma, Milan, Italy) at 73°C followed by a wash in 2⫻ SSC ⫹ 0.1% IPEGAL CA-630 (Sigma) at room temperature. Slides were then washed in phosphate-buffered saline, dehydrated, and air-dried. A counter-stain containing 125 ng/mL 4⬘,6-diamidino-2-phenylindole, dihydrochloride was used to visualize nuclei. 1303
TABLE 1 Three-color PGD cycles for failed IVF-ET.
No. of patients Mean age (years) No. of previous cycles/pregnancy losses No. of oocyte retrievals No. of mature oocytes retrieved (mean ⫾ SD/cycle) No. of fertilized embryos (fertilization rate) No. of embryos biopsied No. of normoploid embryos (% of cohort) Monosomies (% of cohort)b Trisomies (% of cohort)c Otherd (% of cohort)e No. of ETs (mean ⫾ SD) No. of clinical pregnancies (% pregnancies/transfer) No. of fetal heartbeats (implantation rate) No. of live births
Controls
PGD
Pa
12 35.5 ⫾ 4.0 31 15 169 (11.3 ⫾ 6.3) 138 (81.6) NA NA NA NA NA 55 (3.7 ⫾ 0.7) 2 (13.0) 3 (5.4) 3
26 36.3 ⫾ 3.8 84 34 286 (9.1 ⫾ 3.2) 223 (78) 187 119 (64) 12 (6.4)a 6 (3.3) 50 (26.7) 116 (3.3 ⫾ 0.6) 18 (53.0) 26 (22.4) 24
NA .9 NA NA .7 .5 NA NA NA NA NA .7 .02 .01 NA
Note: Data are presented as actual numbers. NA ⫽ not applicable. a P ⬍ .05 is considered statistically significant. b Includes 10 mono-21, 1 mono-13, and 1 mono-18. c Includes 2 trisomy 21 and 4 trisomy 18. e Includes 3 mono-13, 6 mono-16, 4 mono-18, 4 mono-21, 2 mono-22, and 10 combined monosomies. d Includes 3 tri-13, 2 tri-16, 3 tri-21, 2 tri-22, and 6 combined trisomies. d Includes haploid, triploid, combined monosomies, trisomies, and chaotic mosaics because of the difficulty in distinguishing among these karyotypes under the biopsy conditions. Wilding. Three- and 5-color PGD. Fertil Steril 2004.
FISH signals were observed and recorded using a Nikon Genikon karyotype system based on a Nikon Eclipse microscope (Nikon, Florence, Italy). Embryos diagnosed as normoploid after FISH contained two distinct signals for each of the chromosomes tested. Embryos were diagnosed with simple aneuploidies when the tested chromosomes had a single signal either more or less than diploid. Embryos were diagnosed as chaotic mosaics when multiple aneuploidies were present, suggesting random chromosome segregation. Other forms of embryo abnormality (haploidy, triploidy, etc.) were diagnosed as observed.
Statistical Analyses
All data are presented as means ⫾ SD except where stated otherwise. All statistics were performed using the Sigma Stat package (SPSS, Ekrath, Germany). The Mann-Whitney rank sum test was used to adjust the t-test for small populations of data. The z-test with Yates correction was used to determine the significance of proportions.
RESULTS Preimplantation Genetic Diagnosis for Failed IVF-ET In total, 34 3-probe PGD cycles and 15 control IVF cycles were performed with the 38 couples (Table 1). The maternal age for controls was 35.5 ⫾ 4.0 years, and for PGD patients, 36.3 ⫾ 3.8 years. Clinical IVF parameters were not significantly different between the two groups (Table 1). After biopsy, a total of 119 embryos (64%) were diagnosed as 1304 Wilding et al.
Three- and 5-color PGD
suitable for transfer in patients undergoing PGD, whereas 55 embryos were transferred in the control group (Table 1). A total of 18 pregnancies (53% pregnancy rate/transfer) with an implantation rate of 22.4% were achieved in the 34 PGD procedures performed (Table 1). In contrast, controls were characterized by a pregnancy rate of 13% (two pregnancies in 15 cycles) and an implantation rate of 5.4% (three embryos implanting/55 transferred, Table 1). Sixty-eight out of the 187 embryos (36%) obtained from group A patients undergoing PGD were diagnosed with chromosomal abnormalities after PGD (Table 1). Eighteen embryos had simple or multiple aneuploidies, while 50 embryos had more complex chromosomal abnormalities (Table 1). These abnormalities could not be separated into chaotic mosaics or complex aneuploidies because the removal of a single blastomere does not permit the accurate diagnosis of the embryo karyotype. A second series of failed IVF-ET patients were treated with 5-probe PGD using fluorescence probes directed to chromosomes 13, 16, 18, 21, and 22. In total, a series of 56 patients were treated with this protocol. This patient group included 35 patients in which the PGD protocol was applied and 21 controls. Again, maternal age, fertilization rates, the rate of embryo development, and number of embryos transferred were not significantly different between the two groups (Table 2). After 5-probe PGD, 62.1% of embryos were diagnosed as normoploid, 29 embryos had single or complex monosomies, 16 embryos were affected by trisomies, and 54 embryos had complex chromosomal abnorVol. 81, No. 5, May 2004
TABLE 2
TABLE 3
Five-color FISH for failed IVF-ET. Controls
PGD-FISH for habitual aborters. PGD
Pa
No. of patients 21 35 NA Mean age (years) 35.1 ⫾ 3.3 34.3 ⫾ 3.7 .9 No. of previous cycles/pregnancy 77 102 NA losses No. of oocyte retrievals 24 41 NA No. of mature oocytes retrieved 198 (8.2 ⫾ 5.0) 399 (8.5 ⫾ 4.4) .9 (mean ⫾ SD/cycle) No. of fertilized embryos 158 (80) 303 (76) .32 (fertilization rate) No. of embryos biopsied NA 262 NA Normoploid NA 163 (62.1) NA Monosomiesb NA 29 (11.1)c NA Trisomiesc NA 16 (6.1)d NA Otherd NA 54 (20.6) NA No. of normoploid embryos (% NA 163 (62) .92 of cohort) No. of ETs (mean ⫾ SD) 90 (3.8 ⫾ 1.2) 163 (3.9 ⫾ 1.0) .95 No. of clinical pregnancies (% 6 (25.0) 16 (39.0) .38 pregnancies/transfer) No. of fetal heartbeats 9 (10.0) 34 (21.0) .04 (implantation rate) No. of live births 7 33 NA Note: Data are presented as actual numbers. NA ⫽ not applicable. a P ⬍.05 is considered statistically significant. b Includes 3 mono-13, 6 mono-16, 4 mono-18, 4 mono-21, 2 mono-22, and 10 combined monosomies. c Includes 3 tri-13, 2 tri-16, 3 tri-21, 2 tri-22, and 6 combined trisomies. d Includes haploid, triploid, combined monosomies, trisomies, and chaotic mosaics because of the difficulty in distinguishing among these karyotypes under the biopsy conditions. Wilding. Three- and 5-color PGD. Fertil Steril 2004.
malities (Table 2). Six of the control (25% pregnancy rate/ transfer) and 16 PGD patients (39% pregnancy rate/transfer) achieved pregnancy after a total of 65 PGD cycles; Table 2). Although the pregnancy rate was not significantly higher than in the control cycles, the implantation rate after PGD was significantly higher than in controls (Table 2).
Preimplantation Genetic Diagnosis for Habitual Aborters Habitual aborters are otherwise fertile couples that have experienced more than two episodes of spontaneous pregnancy termination within the first trimester. Where no diagnosis could be made of this problem, 3-probe PGD was offered to these couples. In total, 16 couples accepted 3-probe PGD technology (Table 3). One hundred fifty-five mature oocytes were retrieved in the 26 cycles performed by these couples. Of these, 127 fertilized normally after IVF (82% fertilization rate; Table 3). One hundred twelve embryos were considered to be of sufficient quality 64 – 65 hours after fertilization for the biopsy procedure, and of these, 83 embryos were considered normoploid after 3-probe FISH (Table 3). A total of three clinical pregnancies were FERTILITY & STERILITY威
Three-color PGD No. of patients Mean age (years) No. of previous cycles/pregnancy losses No. of oocyte retrievals No. of mature oocytes retrieved (mean ⫾ SD/ cycle) No. of fertilized embryos (fertilization rate) No. of embryos biopsied Normoploid Monosomies Trisomies Othere No. of normoploid embryos (% of cohort) No. of ETs (mean ⫾ SD) No. of clinical pregnancies (% pregnancies/transfer) No. of fetal heartbeats (implantation rate) No. of live births
Five-color PGD
16 37.6 ⫾ 4.1 44
48 35.4 ⫾ 3.3 177
26 155 (5.6 ⫾ 4.4)
62 700 (10.3 ⫾ 5.0)
127 (82)
553 (79)
112 83 (74.1%) 4 (3.6%)a 3 (2.7%)c 22 (19.6%) 83 (74)
501 311 (62.1%) 71 (14.2%)b 11 (2.1%)d 108 (21.6%) 311 (62)
83 (2.9 ⫾ 1.3) 3 (11.5)
276 (3.8 ⫾ 1.0) 22 (35.5)
4 (4.8) 4
58 (21.1) 54
Note: Data are presented as actual numbers. NA ⫽ not applicable. a Includes 1 mono-18 and 3 mono-13. c Includes 1 trisomy 21, 1 trisomy 18 and 1 trisomy 13. b Includes 6 mono-13, 14 mono-16, 11 mono-18, 3 mono-21, 12 mono-22, and 25 combined monosomies. d Includes 1 tri-13, 3 tri-16, 1 tri-21, 3 tri-22, and 3 combined trisomies. e Includes haploid, triploid, combined monosomies, trisomies, and chaotic mosaics because of the difficulty of distinguishing among these karyotypes under the biopsy conditions. Statistical analysis was not performed on the groups because the two patient groups were selected in diverse periods and therefore are not derived from the same distribution. Wilding. Three- and 5-color PGD. Fertil Steril 2004.
obtained out of the 26 cycles performed (11.5% pregnancy rate/transfer), and a total of four gestational sacs with fetal heart beats were observed (implantation rate, 4.8%; Table 3). All four sacs gave rise to healthy births (Table 3). The low success rate using three-chromosome FISH for patients affected by this type of infertility suggests that an extension of the FISH protocol to cover chromosomes 16 and 22 could help this group of patients. We therefore offered 5-probe PGD-FISH, using fluorescent probes for chromosomes 13, 16, 18, 21, and 22 on a second series of patients. In total, a series of 48 patients were treated with this protocol (Table 3). After 62 oocyte retrievals, 22 patients achieved pregnancy with 5-probe FISH (35.5% pregnancy rate/transfer; Table 3). A total of 58 gestational sacs with fetal heart beats were obtained after the transfer of 276 embryos (implantation rate, 21.1%; Table 3). Of these, 54 resulted in healthy live births (Table 3). 1305
The above data suggest that the analysis of five chromosomes by FISH during PGD cycles is more reliable for IVF outcome than the detection of three chromosomes in habitual aborters. We examined whether this increase in reliability was due to an increase in the detection efficiency of a particular type of aneuploidy or karyotype. The use of fivecolor FISH for patients attending the PGD program for multiple spontaneous pregnancy loss was correlated with an increase in the detection of monosomic embryos (Table 3). Monosomies detected included a proportion of both single and combined monosomies for chromosomes 16 and 22 (Table 3). Although the comparison between 3- and 5-probe FISH for habitual aborters is tentative in the present work, the data suggest a correlation between the presence of aneuploidies not detected by three-chromosome FISH and the increase in outcome observed.
DISCUSSION In this study, we tested whether the genetic diagnosis of aneuploidy in human preimplantation embryos improves the prognosis after IVF in couples suffering from two specific infertility problems: repetitive failed IVF-ET and habitual abortion. Preimplantation genetic diagnosis protocols used either 3-probe analysis examining chromosomes 13, 18, and 21 or 5-probe analysis for chromosomes 13, 16, 18, 21, and 22. Chromosomes 13, 18, 21 were chosen because they are the cause of the three known aneuploidies that can result in live births (1, 8 –10, 15), and chromosomes 16 and 22 were chosen because they are the cause of the aneuploidies that are known to be common in early pregnancy loss (19, 20). Both protocols resulted in the detection of chromosomal abnormalities in approximately 38% of embryos that would have been considered suitable for transfer into the patient’s uterus. The outcome of IVF in failed IVF-ET patients was significantly improved after both 3- and 5-probe PGD with respect to controls. However, 5-probe FISH did not appear to offer advantages over 3-probe FISH in this limited trial. These data suggest that PGD can offer advantages over simply repeating the IVF procedure in failed IVF-ET patients by simply deselecting chromosomally abnormal embryos. Further, the trial suggests that 3-probe PGD may be sufficient in eliminating chromosomally abnormal embryos in these cases. Although the analysis of as few as three chromosomes appears to aid in the treatment of patients suffering from multiple cycles of failed IVF-ET, little was gained with 3-probe PGD for patients suffering from habitual abortion. The use of 3-probe PGD resulted in three pregnancies in a total of 26 oocyte retrievals. Aneuploidies of chromosomes 16 and 22 are often implicated in spontaneous pregnancy loss (19, 20). In these patients, 5-probe FISH appeared to assist in a positive outcome. Of the 48 couples included in the trial, 22 achieved pregnancy after 62 oocyte retrievals 1306 Wilding et al.
Three- and 5-color PGD
(35.5% pregnancy rate/oocyte retrieval). Even more pertinent to the study was the observation that 54 of these embryos resulted in the birth of a healthy baby. Although no control data were available for this group of patients in the present study, the data suggest that PGD can improve the prognosis for this group of patients. We examined the types of chromosomal abnormality detected by 5-color FISH in this group of patients. Although the level of complex chromosomal aneuploidies was similar to that of three-chromosome analysis, we noted a significant increase in the detection of monosomies for the chromosomes tested (Table 3). Of the 71 monosomic embryos characterized, 26 (36.6%) were single monosomies for chromosomes 16 or 22 and a further 25 (35.2%) were combined monosomies. Although these data do not demonstrate that monosomies for chromosomes 16 and 22 specifically caused the previously low outcome in this group of patients, the data suggest that high levels of specific monosomies are correlated with the low outcome in this group of patients. This pilot study therefore suggests that the use of 3-probe PGD is often sufficient to enable the selection of chromosomally normal embryos for transfer in patients characterized by previous IVF failure. The data further suggest that 5-probe PGD is more useful for habitual aborters. Although the data are interesting, it is not clear from the current limited sample of patients whether the current protocol can be applied to all patient groups (for example, patients of advanced age). A larger, prospective, randomized, controlled, multicenter trial comparing PGD with other IVF protocols (such as changing the ovarian stimulation protocol or embryo selection criteria) is required to determine the accuracy of the current data, and could produce a definitive protocol for these patients. References 1. Hassold T, Chiu D. Maternal age-specific rates of numerical chromosome abnormalities with special reference to trisomy. Hum Genet 1985;70:11–17. 2. Hassold T, Jacobs P. Trisomy in man. Ann Rev Genet 1984;18:69 –97. 3. Yoon P, Freeman S, Sherman S, Taft LF, Gu Y, Pettay D, et al. Advanced maternal age and the risk of Downs syndrome characterized by the meiotic stage of the chromosomal error: a population-based study. Am J Hum Genet 1996;58:628 –33. 4. Dailey T, Dale B, Cohen J, Munne S. Association between nondisjunction and maternal age in meiosis-II human oocytes. Am J Hum Genet 1996;59:176 –84. 5. Antonarakis S, Petersen M, McInnis M, Adelsberger PA, Schinzel AA, Binkert F, et al. The meiotic stage of non-disjunction in trisomy 21: determination by using DNA polymorphisms. Am J Hum Genet 1992; 50:544 –50. 6. Mikkelsen M, Halberg A, Poulsen H. Epidemiological study of Downs syndrome in Denmark, including family studies of chromosomes and DNA markers. Dev Brain Dysfunct 1995;8:4 –12. 7. Munne S, Alikani M, Tomkin G, Grifo J, Cohen J. Embryo morphology, developmental rates and maternal age are correlated with chromosome abnormalities. Fertil Steril 1995;64:382–91. 8. Munne S, Grifo J, Cohen J. Chromosome abnormalities in human arrested preimplantation embryos: a multiple-probe FISH study. Am J Hum Genet 1994;55:150 –9. 9. Harper JC, Coonen E, Handyside AH, Winston RM, Hopman AH, Delhanty JD. Mosaicism of autosomes and sex chromosomes in morphologically normal, monospermic human embryos. Prenat Diagn 1995;15:41–50. 10. Delhanty JDA, Harper JC, Ao A, Handyside AH, Winston RM. Multicolor FISH detects frequent chromosomal mosaicism and chaotic
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11. 12. 13. 14. 15.
division in normal preimplantation embryos from fertile patients. Hum Genet 1997;99:755–60. Nass TE, Escudero T, Marello EC, Velilla E, Sandalinas M, Werlin MB, et al. Dissociation between embryo morphology and euploidy in IVF patients with poor prognosis. Fertil Steril 2002;77(Suppl 3):S11. Delhanty JDA. Chromosome analysis by FISH in human preimplantation genetics. Hum Reprod 1997;12:153–5. Handyside AH, Kontogianni EH, Hardy K, Winston RML. Pregnancies from biopsied human preimplantation embryos sexed by Y specific DNA amplification. Nature 1990;344:768 –70. Handyside AH, Lesko LG, Tarin JJ, Winston RML, Hughes MR. Birth of a normal girl after in-vitro fertilization and preimplantation diagnostic testing for cystic fibrosis. N Engl J Med 1992;327:905–8. Munne S, Lee A, Rosenwaks Z, Grifo J, Cohen J. Diagnosis of major chromosome abnormalities in human preimplantation embryos. Hum Reprod 1993;8:2185–91.
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16. Grifo JA, Tang YX, Munn S, Alikani M, Cohen J, Rosenwaks Z. Healthy deliveries from biopsied human embryos. Hum Reprod 1994; 9:912–6. 17. Gianaroli L, Magli MC, Munne S, Fiorentino A, Montanaro N, Ferraretti AP. Will preimplantation genetic diagnosis assist patients with a poor prognosis to achieve pregnancy? Hum Reprod 1997;12: 1762–7. 18. Munne S, Magli MC, Cohen J, Morton P, Sadowy S, Gianaroli L, et al. Positive outcome after preimplantation diagnosis of aneuploidy in human embryos. Hum Reprod 1999;14:2191–9. 19. Carp H, Toder V, Aviram A, Daniely M, Mashiach S, Barkai G. Karyotype of the abortus in recurrent miscarriage. Fertil Steril 2001; 75:678 –82. 20. Munne S, Magli MC, Bahce M, Fung J, Legator M, Morrison L, et al. Preimplantation diagnosis for the aneuploidies most commonly found in spontaneous abortions and live births: XY,13,14,15,16,18 and 22. Prenat Diagn 1998;18:1459 –66.
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