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Chromosomal aberrations in couples undergoing intracytoplasmic sperm injection: influence on implantation and ongoing pregnancy rates
FERTILITY AND STERILITYt VOL. 70, NO. 5, NOVEMBER 1998 Copyright ©1998 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
Chromosomal aberrations in couples undergoing intracytoplasmic sperm injection: influence on implantation and ongoing pregnancy rates Michael C. W. Scholtes, M.D.,* Claudia Behrend, M.D.,† Jutta Dietzel-Dahmen, Ph.D.,† Dagmar G. van Hoogstraten, M.D.,* Kersten Marx, M.D.,* Suzanne Wohlers, M.D.,* Hugo Verhoeven, M.D.,* and Gerard H. Zeilmaker, Ph.D.‡ Centre for Reproductive Medicine and Genetics, Du¨sseldorf, Germany
Objective: To determine the incidence of chromosomal aberrations in couples undergoing intracytoplasmic sperm injection (ICSI) and their influence on subsequent implantation and ongoing pregnancy rates. Design: Prospective study. Setting: Fertility center. Patient(s): Candidates for ICSI. Intervention(s): Chromosomes were trypsin-banded in 2,280 patients. In all cases, 10 metaphases were karyotyped. Sex chromosome analysis was performed in 10 additional metaphases. When apparent chromosomal aberrations were detected, 100 metaphases were analyzed. Main Outcome Measure(s): Implantation and ongoing pregnancy rates in couples with a chromosomal disorder. Results: A chromosomal abnormality was demonstrated in 7.2% of all couples. Among the male partners, 4.48% had aberrations. Autosomal aberrations were present in 2.96%, and numerical or structural sex chromosome abnormalities were found in 1.52%. Among the female partners, numerical or structural abnormalities were documented in 9.79%. Only 2.32% of the female partners had autosomal structural abnormalities. Numerical or structural anomalies involving sex chromosomes were found in 7.47%. Implantation rates of 9.4% and 16.3% per embryo were observed in female partners with sex chromosome mosaicism and autosomal aberrations, respectively. In male partners, the respective rates were 3.8% and 23.1%.
Received December 26, 1997; revised and accepted June 15, 1998. Reprint requests: Gerard H. Zeilmaker, Ph.D., Department of Endocrinology and Reproduction, Erasmus University, Postbox 1738, 3000 DR Rotterdam, the Netherlands (FAX: 31-104366832). * In Vitro Fertilization Department. † Department of Genetics. ‡ Department of Endocrinology and Reproduction, Erasmus University, Rotterdam, the Netherlands. 0015-0282/98/$19.00 PII S0015-0282(98)00310-0
Conclusion(s): The incidence of chromosomal disorders in couples seeking ICSI treatment is considerable, especially minor mosaicism (,10%) of sex chromosomes in the female partners. Preliminary data indicate a low implantation rate in couples with minor mosaicism of sex chromosomes. (Fertil Sterilt 1998;70:933–7. ©1998 by American Society for Reproductive Medicine.) Key Words: Intracytoplasmic sperm injection, genetic aberration, minor mosaicism, implantation rate, karyotype
Because chromosomally abnormal sperm are used in intracytoplasmic sperm injection (ICSI), this procedure theoretically increases the risk of propagating disorders. This risk seems to be especially high when the male partner has a very low sperm count and impaired sperm morphology (1– 4). However, reports in the literature indicate only a slightly higher percentage of chromosomal disorders in offspring conceived with the use of ICSI and no difference in the rate of major malformations (5). Regulatory mechanisms appear to be involved in the (partial) elimination of chromosomally aberrant embryos (1, 6). Analysis of
the somatic karyotype of all potential candidates for ICSI may reduce the risk of producing offspring with chromosomal abnormalities. Guidelines produced by the German Association for Reproductive Medicine and Endocrinology, dictate that both male and female partners be karyotyped and counseled before undergoing treatment with ICSI, regardless of the cause of their infertility. In the present study, we report the results of cytogenetic analyses of couples entering our ICSI program. We also discuss preliminary data concerning implantation and ongoing 933
TABLE 1 Cytogenetic analysis of 1,116 male and 1,164 female patients undergoing ICSI: autosomal aberrations. Male autosomes
Aberration Numerical Marker chromosomes
Structural Translocations
Inversions
Female autosomes
Karyotype
No. of aberrations
47,XY,1mar (unknown origin) 47,XY,1dic (15)(q11) 47,XY,1i (13)(pter-.p10)
1 (0.09) 1 (0.09) 1 (0.09)
Robertsonian translocations -der(13;14) (q10;q10) -der(14;21) (q10;q10) -der(14;15) (q10;q10) Reciprocal translocations (see Table 3) Paracentric (see Table 3) Pericentric Inv(9)qh
Duplications
Aberration
No. of aberrations
Numerical Marker chromosomes
47,XX,1mar
0
Structural Translocations
Robertsonian translocations
0
8 (0.72) 2 (0.18) 1 (0.09) 9 (0.80) Inversions 2 (0.18) 0 8 (0.72) 0
Karyotype
Duplications
Reciprocal translocations (see Table 3)
8 (0.69)
Paracentric (see Table 3) Pericentric (see Table 3) Inv(9)qh 46,XX,dup(9)(q21.1q21.2) 46,XX,dup(9)(p12p12)
2 (0.17) 3 (0.26) 12 (1.03) 1 (0.08) 1 (0.08)
Note: Values in parentheses are percentages.
pregnancy rates in patients with chromosomal disorders.
MATERIALS AND METHODS Over a period of 26 months (May 1995 to June 1997), cytogenetic studies were performed in 2,280 patients who were referred for treatment with ICSI at our center for reproductive medicine and genetics. Of the 2,280 patients investigated, 1,164 were female and 1,116 were male.
poor fertilization in the past, in 9%. Male factor infertility was diagnosed only after multiple successive (.2) sperm analyses with an interval of at least 6 weeks. Statistical analysis was performed by x2 analysis and Fisher’s two-tailed exact test.
RESULTS
For each patient, an initial cytogenetic study of peripheral blood cells was undertaken according to the standard protocol of Moorhead et al. (7). Chromosomes were trypsinbanded according to the method of Klinger (8). In cases of structural aberrations, additional banding techniques were performed, including C-banding (9) and nucleolar organizing-staining with the use of the technique developed by Bloom and Goodpasture (10).
During 1995–1997, systematic cytogenetic testing was performed in 2,280 patients undergoing ICSI in our clinic. Of the 2,280 patients investigated, 1,164 were female and 1,116 were male.
In all cases, 10 metaphases of peripheral lymphocytes were karyotyped. An additional 10 metaphases were evaluated for their number of gonosomes. If there was any sign of mosaicism, 100 metaphases from two independent cell cultures were analyzed.
The frequency of aberrations was 4.48% in the male partners. Autosomal aberrations were observed in 33 men (2.96%) (Table 1), and numerical or structural sex chromosome abnormalities were found in 17 (1.52%) (Table 2).
The median age of the male partners was 34.5 years (range, 22– 66 years). The median age of the female partners was 32.5 years (range, 19 – 49 years). The selection criteria for ICSI treatment were extreme oligospermia, asthenospermia, or teratozoospermia, defined according to World Health Organization criteria (,5 3 106/mL, ,25% progressive motility, and ,15% normal forms) in 91% of couples and other indications, such as total fertilization failure in previous IVF attempts or successive 934
Scholtes et al.
Chromosomal aberrations in ICSI
A chromosomal abnormality was found in 7.2% of the couples. Single-cell aberrations were not included. Inversions of chromosome 9 are not considered to be pathologic but were included in Table 1 for the sake of completeness.
Numerical or structural abnormalities were documented in 9.79% of the female partners. Autosomal structural abnormalities were found in 27 women (2.32%) (Table 1), and numerical anomalies involving sex chromosomes were found in 87 (7.47%) (Table 2). There did not appear to be an age-related distribution of gonosomal aberrations. It is of interest that we found a high incidence of minor sex chromosome mosaicism in the female partners. This is a surprising finding because male factor infertility is the primary indication for ICSI. Vol. 70, No. 5, November 1998
TABLE 2 Cytogenetic analysis of 1,116 male and 1,164 female patients undergoing ICSI: sex chromosome aberrations. Male sex chromosomes
Karyotype
No. of aberrations
47,XXY 47,XYY 47,XXY/46,XY 47,XYY/46,XY 47,XYY/49,XYYYY/46,XY 45,X/47,XYY 45,X/46,XY
3 (0.27) 3 (0.27) 2 (0.18) 3 (0.27) 1 (0.09) 1 (0.09) 2 (1.18)
46,X,inv(y)(q11.2q12) 46,X,inv(y)(p11.2q12)
1 (0.09) 1 (0.09) 0
Aberration Numerical Complete Mosaicism
Structural Complete
Female sex chromosomes
Mosaicism
Karyotype
No. of aberrations
47,XXX 45,X/46,XX 47,XXX/46,XX 45,X/47,XXX/46,XX 45,X/48,XXXX/46,XX 45,X/47,XXX/48,XXXX/46,XX 45,X/47,XXX/48,XXXX/49,XXXXX/46,XX
1 (0.08) 48 (4.12) 7 (0.60) 22 (1.89) 4 (0.34) 2 (0.17) 1 (0.08)
Aberration Numerical Complete Mosaicism
Structural Complete
46,X,del(X)(pter-.q22:)
1 (0.08)
Mosaicism
46,Xr(X)/46,XX
1 (0.08)
Note: Values in parentheses are percentages.
In only two couples entering the ICSI program were both partners affected. In one couple, the male partner had Klinefelter’s syndrome and his partner had a 45,X/47,XXX/ 46,XX mosaic (2%/2%/96%). In the other couple, both partners had a minor sex chromosome mosaic: 47,XYY/46,XY (4%/96%) in the man and 45,X/46,XX (5%/95%) in the woman. Data on the nature of the autosomal aberrations found in the male and female partners and further details regarding the anomalies involved are provided in Table 3.
An embryo implantation rate of 9.4% and an ongoing pregnancy rate per transfer of 12.5% were observed in the female partners with sex chromosome mosaicism and were not statistically different from the rates in the female partners with autosomal aberrations. The implantation rate was significantly lower (P ,.05) in couples with a male gonosomal aberration than in couples with a male autosomal aberration (Table 4).
DISCUSSION TABLE 3 Detailed list of all autosomal aberrations in 2,280 patients undergoing ICSI. Female aberrations Reciprocal translocations 46,XX,t(12;16)(q24.3;p11.2) 46,XX,t(2;20)(q14.3;p13) 46,XX,t(12;19) 46,XX,t(1;2)(q42;p11.2) 46,XX,t(1;8)(p10;q10) 46,XX,t(7;9)(p10;p10) 46,XX,t(3;13)(q21;q22) 46,XX,t(2;7)(p25;q22) Paracentric inversions 46,XX,inv(7)(p15.1p22) 46,XX,inv(12)(q21q23) Pericentric inversions 46,XX,inv(10)(p11.2q21.2) 46,XX,inv(10)(p11q22) 46,XX,inv(10)(p11q22) Duplications 46,XX,dup(9)(q21.1q21.2) 46,XX,dup(9)(p12p12)
FERTILITY & STERILITYt
Male aberrations
46,XY,t(1;9)(q44;p11.2) 46,XY,t(4;11;18)(p12;p15.1;q23) 46,XY,t(3;10)(p25;q26) 46,XY,t(1;3)(p36.5;q21) 46,XY,t(8;13)(q24.1;q22) 46,XY,t(9;14)(p22;q22) 46,Y,t(X;3)(q26;q23) 46,XY,t(4;19)(q34;q13.3) 46,XY,t(6;18)(q26;q23) 46,XY,inv(3)(p21p25) 46,XY,inv(2)(q21q22)
According to federal guidelines in Germany, all couples undergoing ICSI must be karyotyped before treatment, regardless of the cause of their infertility. The high percentage of chromosomal aberrations found in the female partners of couples undergoing ICSI in the present study raises the question of whether the incidence is increased in infertile couples in general, as has been demonstrated in couples undergoing regular treatment with IVF (11). Studies of the prevalence of chromosomal disorders in patients undergoing ICSI generally lack normal control groups. Prospective studies that include control groups should be carried out because minor mosaicism has been dismissed as a cultural artifact or considered to be of no relevance. The results of our study confirm that there is a high rate of chromosomal anomalies in both the male and female partners of infertile couples undergoing ICSI. Infertility has been correlated with chromosomal abnormalities in different IVF patient groups (11, 12). Conflicting data have been published on the frequency of gonosomal aberrations. The number of cytogenetic analyses in IVF is limited. Moreover, the incidence of gonosomal anomalies may be related to the indication for IVF. 935
TABLE 4 Subdivision of chromosomal aberrations according to male/female sex chromosome or autosomal anomalies: influence on implantation and ongoing pregnancy rates.
Aberration Male gonosomes Male autosomes Female gonosomes Female autosomes
No. of patients
No. of cycles (no. of ETs)
9 27 54 15
14 (12) 40 (33) 94 (72) 27 (20)
No. of pregnancies (percentage of ETs) 1 (8.3) 11 (33) 13 (18) 7 (40)
Implantation rate per embryo (%)
No. of ongoing pregnancies/no. of births (%)
Prenatal anomaly*
3.8† 23.1† 9.4 16.3
0/1 (8.3) 0/9 (27.2) 2/7 (12.5) 0/5 (25)
— 2‡ — —
* Reported chromosomal aberration. † Values with the same superscripts were significantly different (P , .05). ‡ One selective fetocide (trisomy 9), one balanced rob (13;14).
Numerous surveys have been conducted to determine the correlation between autosomal and sex chromosome abnormalities and male infertility. Most studies confirm a genetic basis, especially in patients with impaired spermatogenesis (1– 4, 13–15). Fluorescence in situ hybridization (FISH) has made it possible to perform comprehensive studies on numerical chromosome abnormalities in human spermatozoa (16 –20). Fluorescence in situ hybridization overcomes the major disadvantage of karyotyping a limited number of spermatozoa after penetration of zona-free hamster oocytes and subsequent decondensation. However, FISH does not discriminate between dead and vital, motile and immotile, or normal and grossly abnormal cells. In practice, this means that the frequency of abnormal chromosomes is not automatically the same in the gametes used for fertilization. In contrast with FISH, karyotyping shows all chromosomes and covers all types of structural rearrangements. Therapeutic possibilities offered by the introduction of ICSI have increased our awareness of the potential for propagating chromosomal aberrations in the next generation (21). Numerical and structural chromosomal anomalies, even those involved in spermiogenesis, are compatible with fertilization in ICSI (22, 23). However, complex structural rearrangements seem to be incompatible with fertilization in ICSI (24). According to the recent publication of in ‘t Veld et al. (25), infertile men with idiopathic oligozoospermia or azoospermia should be offered genetic testing and counseling before ICSI treatment is considered. The study on which this conclusion was based involved a subgroup of oligospermic or azoospermic male patients. It was not clear whether these men were actually taking part in the ICSI program. This group, therefore, may not be representative of male patients undergoing treatment with ICSI because no patients with cryptorchidism were included. Meiotic studies in men indicate that the sex chromosomes, along with chromosome 21, may be more susceptible 936
Scholtes et al.
Chromosomal aberrations in ICSI
to nondisjunction than are the other chromosomes (18, 26). Even more remarkable is the fact that a high incidence of sex chromosome aneuploidy occurs in female patients undergoing ICSI, with 45,X metaphases being more common than 47,XXX cells (27). The predominant loss of an X chromosome may be related to age and caused by prematurely separating centromeres, which do not attach to the spindle fibers and hence cause nondisjunction (28, 29) in the oocyte and somatic cells. However, in our ICSI studies, we could not find a relation between patient age and 46,XX/45,X mosaicism, possibly because of the relatively narrow age range of the female partners. It is still unknown whether the high incidence of minor gonosomal mosaicism can cause infertility as well as problems in implantation and further progress of a subsequent pregnancy. Although our observations are limited, the preliminary data indicate that there may be comparatively low implantation and ongoing pregnancy rates in couples with gonosomal aberrations. We were not able to find a statistically significant difference between female sex chromosome anomalies and female autosomal aberrations, as we were with male abnormalities. We initially intended not to label the data statistically significant because we wanted to stress the questionable clinical significance of any differences found. The number of aberrations found in conjunction with pregnancy probably is too small for us to analyze. Future studies are necessary. On average, after meiosis, 50% of translocations are unbalanced. The frequency depends on which chromosomes are involved. For these men, the risk of having an offspring with unbalanced translocation is theoretically high. However, the available data from newborns conceived with the use of ICSI do not indicate a higher rate of autosomal abnormalities in this group. The incidence of sex chromosome aneuploidies is increased to 1.2% compared with 0.83% in an unselected population (30, 31). The reduced transmission of unbalanced translocations probably is due to natural selection of embryos (6). Vol. 70, No. 5, November 1998
To date, few data are available concerning the influence of minor (,10%) mosaicism of sex chromosomes in peripheral lymphocytes on fertility and subsequent pregnancy in female patients. Data on this subject should be interpreted cautiously; for example, the karyotype of peripheral lymphocytes cannot be extrapolated automatically to the germ cell line. There is evidence that a maternal predisposition to chromosome aneuploidy may exist in patients undergoing IVF and may be associated with failed IVF cycles (32). Decreased implantation and ongoing pregnancy rates might be in line with these observations. Further, a recent publication from an Australian IVF group (33) points out possible negative aspects of ICSI therapy in a follow-up study of children performed 1 year after birth. In addition, the use of immature gametes further increases the risk that unexpected negative side effects will occur. Karyotyping may help in elucidating the long-term effects of ICSI therapy and excluding possible parental factors until more reassuring data from large prospective (blinded) follow-up studies are available. We therefore conclude that genetic testing and counseling of both male and female partners is recommended before treatment with ICSI. Our findings indicate that cytogenetic analysis should be considered seriously in all infertile couples before methods of assisted conception are used. References 1. Chandley AC, Edmond P, Christie S, Gowans L, Fletcher J, Frackiewicz A, et al. Cytogenetics and infertility in man. I. Karyotype and seminal analysis: results of a five-year survey of men attending a subfertility clinic. Ann Hum Genet 1975;39:231–54. 2. Chandley AC. The chromosomal basis of human infertility. Br Med Bull 1979;35:181– 6. 3. Ferguson-Smith MAB, Lennox B, Mack WS, Steward JSS. Klinefelter’s syndrome: frequency and testicular morphology in relation to nuclear sex. Lancet 1957;2:167–9. 4. Retief AE, Van Zyl JA, Menkveld R, Fox MF, Kotze GM, Brusnicky J. Chromosome studies in 496 infertile males with a sperm count below 10 million/ml. Hum Genet 1984;66:162– 4. 5. Bonduelle M, Wilikens A, Buysse A, Van Assche E, Wisanto A, Devroey P, et al. Prospective follow-up study of 877 children born after intracytoplasmic sperm injection (ICSI), with ejaculated epididymal and testicular spermatozoa and after replacement of cryopreserved embryos obtained after ICSI. Hum Reprod 1996;11(Suppl 4):131–55. 6. Plachot M, Veiga A, Montagut J, de Grouchy J, Calderon G, Lepretre S, et al. Are clinical and biological IVF parameters correlated with chromosomal disorders in early life: a multicentric study. Hum Reprod 1988;3:627–35. 7. Moorhead PS, Nowell PC, Mellmann WJ, Battips DM, Hungerford DA. Chromosome preparations of leucocyte cultures from human peripheral blood. Exp Cell Res 1960;20:613– 6. 8. Klinger HP. Rapid processing of primary embryonic tissues for chromosome banding pattern analysis. Cytogenetics 1972;11:424 –35. 9. Sumner AT. A simple technique for demonstrating centromeric heterochromatin. Exp Cell Res 1972;75:304 – 6. 10. Bloom SE, Goodpasture C. An improved technique for selective silver staining of nucleolar organizer regions in human chromosomes. Hum Genet 1976;34:199 –206.
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11. Hens L, Bonduelle M, Liebaers I, Devroey P, Van Steirteghem AC. Chromosome aberrations in 500 couples referred for in-vitro fertilization or related fertility treatment. Hum Reprod 1988;3:451–7. 12. Lange R, Johannson G, Engel W. Chromosome studies in in-vitro fertilization patients. Hum Reprod 1993;8:572– 4. 13. Pandiyan N, Jequier AM. Mitotic chromosomal anomalies among 1210 infertile men. Hum Reprod 1996;11:2604 – 8. 14. Bourrouillou G, Dastugue N, Colombies P. Chromosome studies in 952 infertile males with a sperm count below 10 million/ml. Hum Genet 1985;71:366 –7. 15. Bourrouillou G, Calvas P, Bujan L, Mieusset R, Mansat A, Pontonnier F. Mitotic chromosomal anomalies among infertile men (letter). Hum Reprod 1997;12:2337– 8. 16. Martin RH, Ko E, Chan K. Detection of aneuploidy in human interphase spermatozoa by fluorescence in situ hybridization (FISH). Cytogenet Cell Genet 1993;64:23– 6. 17. Robbins WA, Segraves R, Pinkel D, Wyrobek AJ. Detection of aneuploid human sperm by fluorescence in situ hybridization: evidence for a donor difference in frequency of sperm disomic for chromosomes 1 and Y. Am J Hum Genet 1993;52:799 – 807. 18. Spriggs EL, Rademaker AW, Martin RH. Aneuploidy in human sperm: the use of multicolor FISH to test various theories of nondisjunction. Am J Hum Genet 1996;58:356 – 62. 19. Martin RH, Spriggs E, Rademaker AW. Multicolor fluorescence in situ hybridization analysis of aneuploidy and diploidy frequencies in 225,846 sperm from 10 normal men. Biol Reprod 1996;54:394 – 8. 20. Downie SE, Flaherty SP, Matthews CD. Detection of chromosomes and estimation of aneuploidy in human spermatozoa using fluorescence in-situ hybridization. Mol Hum Reprod 1997;3:585–98. 21. Engel W, Murphy D, Schmid M. Are there genetic risks associated with microassisted reproduction? Hum Reprod 1996;11:2359 –70. 22. Testart J, Gautier E, Brami C, Rolet F, Sedbon E, Thebault A. Intracytoplasmic sperm injection in infertile patients with structural chromosome abnormalities. Hum Reprod 1996;11:2609 –12. 23. Tournaye H, Staessen C, Libaers I, van Assche E, Deveoey P, Bonduelle M, et al. Testicular sperm recovery in nine 47,XXY Klinefelter patients. Hum Reprod 1996;11:1644 –9. 24. Siffroi JP, Benzacken B, Straub B, le Bourhis C, North MO, Curotti G, et al. Assisted reproductive technology and complex chromosomal rearrangements: the limits of ICSI. Mol Human Reprod 1997;3:847–51. 25. in ‘t Veld PA, Halley DJJ, van Hemel JO, Niermeijer MF, Dohle G, Weber RAF. Genetic counselling before intracytoplasmic sperm injection. Lancet 1997;350:490. 26. Spriggs EL, Rademaker AW, Martin RH. Aneuploidy in human sperm: results of two- and three-color fluorescence in situ hybridization using centromeric probes for chromosomes 1, 12, 15, 18, X, and Y. Cytogenet Cell Genet 1995;71:47–53. 27. Galloway SM, Buckton KE. Aneuploidy and ageing: chromosome studies on a random sample of the population using G-banding. Cytogenet Cell Genet 1978;20:78 –95. 28. Vig BK. Sequence of centromere separation: orderly separation of multicentric chromosomes in mouse L cells. Chromosoma 1984;90:39 – 45. 29. Fitzgerald PH, Pickering AF, Mercer JM, Miethke PM. Premature centromere division: a mechanism of non-disjunction causing X chromosome aneuploidy in somatic cells of man. Ann Hum Genet 1975; 38:417–28. 30. Hook EB. Chromosome abnormalities: prevalence, risks and recurrence. In: Brock DJH, Rodeck CH, Ferguson-Smith MAB, editors. Prenatal diagnosis and screening. Edinburgh: Churchill Livingstone, 1992:351–92. 31. Jacobs PA, Browne C, Gregson N, Joyce C, White H. Estimates of the frequency of chromosome abnormalities detectable in unselected newborns using moderate levels of banding. J Med Genet 1992;29:103– 8. 32. Zenzes MT, Wang P, Casper RF. Evidence for maternal predisposition to chromosome aneuploidy in multiple oocytes of some in vitro fertilization patients. Fertil Steril 1992;57:143–9. 33. Bowen JR, Gibson FL, Leslie GI, Saunders DM. Medical and developmental outcome at 1 year for children conceived by intracytoplasmic sperm injection. Lancet 1998;351:1529 –34.
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Chromosomal aberrations in couples undergoing intracytoplasmic sperm injection: influence on implantation and ongoing pregnancy rates Michael C. W. Scholtes, M.D.,* Claudia Behrend, M.D.,† Jutta Dietzel-Dahmen, Ph.D.,† Dagmar G. van Hoogstraten, M.D.,* Kersten Marx, M.D.,* Suzanne Wohlers, M.D.,* Hugo Verhoeven, M.D.,* and Gerard H. Zeilmaker, Ph.D.‡ Centre for Reproductive Medicine and Genetics, Du¨sseldorf, Germany
Objective: To determine the incidence of chromosomal aberrations in couples undergoing intracytoplasmic sperm injection (ICSI) and their influence on subsequent implantation and ongoing pregnancy rates. Design: Prospective study. Setting: Fertility center. Patient(s): Candidates for ICSI. Intervention(s): Chromosomes were trypsin-banded in 2,280 patients. In all cases, 10 metaphases were karyotyped. Sex chromosome analysis was performed in 10 additional metaphases. When apparent chromosomal aberrations were detected, 100 metaphases were analyzed. Main Outcome Measure(s): Implantation and ongoing pregnancy rates in couples with a chromosomal disorder. Results: A chromosomal abnormality was demonstrated in 7.2% of all couples. Among the male partners, 4.48% had aberrations. Autosomal aberrations were present in 2.96%, and numerical or structural sex chromosome abnormalities were found in 1.52%. Among the female partners, numerical or structural abnormalities were documented in 9.79%. Only 2.32% of the female partners had autosomal structural abnormalities. Numerical or structural anomalies involving sex chromosomes were found in 7.47%. Implantation rates of 9.4% and 16.3% per embryo were observed in female partners with sex chromosome mosaicism and autosomal aberrations, respectively. In male partners, the respective rates were 3.8% and 23.1%.
Received December 26, 1997; revised and accepted June 15, 1998. Reprint requests: Gerard H. Zeilmaker, Ph.D., Department of Endocrinology and Reproduction, Erasmus University, Postbox 1738, 3000 DR Rotterdam, the Netherlands (FAX: 31-104366832). * In Vitro Fertilization Department. † Department of Genetics. ‡ Department of Endocrinology and Reproduction, Erasmus University, Rotterdam, the Netherlands. 0015-0282/98/$19.00 PII S0015-0282(98)00310-0
Conclusion(s): The incidence of chromosomal disorders in couples seeking ICSI treatment is considerable, especially minor mosaicism (,10%) of sex chromosomes in the female partners. Preliminary data indicate a low implantation rate in couples with minor mosaicism of sex chromosomes. (Fertil Sterilt 1998;70:933–7. ©1998 by American Society for Reproductive Medicine.) Key Words: Intracytoplasmic sperm injection, genetic aberration, minor mosaicism, implantation rate, karyotype
Because chromosomally abnormal sperm are used in intracytoplasmic sperm injection (ICSI), this procedure theoretically increases the risk of propagating disorders. This risk seems to be especially high when the male partner has a very low sperm count and impaired sperm morphology (1– 4). However, reports in the literature indicate only a slightly higher percentage of chromosomal disorders in offspring conceived with the use of ICSI and no difference in the rate of major malformations (5). Regulatory mechanisms appear to be involved in the (partial) elimination of chromosomally aberrant embryos (1, 6). Analysis of
the somatic karyotype of all potential candidates for ICSI may reduce the risk of producing offspring with chromosomal abnormalities. Guidelines produced by the German Association for Reproductive Medicine and Endocrinology, dictate that both male and female partners be karyotyped and counseled before undergoing treatment with ICSI, regardless of the cause of their infertility. In the present study, we report the results of cytogenetic analyses of couples entering our ICSI program. We also discuss preliminary data concerning implantation and ongoing 933
TABLE 1 Cytogenetic analysis of 1,116 male and 1,164 female patients undergoing ICSI: autosomal aberrations. Male autosomes
Aberration Numerical Marker chromosomes
Structural Translocations
Inversions
Female autosomes
Karyotype
No. of aberrations
47,XY,1mar (unknown origin) 47,XY,1dic (15)(q11) 47,XY,1i (13)(pter-.p10)
1 (0.09) 1 (0.09) 1 (0.09)
Robertsonian translocations -der(13;14) (q10;q10) -der(14;21) (q10;q10) -der(14;15) (q10;q10) Reciprocal translocations (see Table 3) Paracentric (see Table 3) Pericentric Inv(9)qh
Duplications
Aberration
No. of aberrations
Numerical Marker chromosomes
47,XX,1mar
0
Structural Translocations
Robertsonian translocations
0
8 (0.72) 2 (0.18) 1 (0.09) 9 (0.80) Inversions 2 (0.18) 0 8 (0.72) 0
Karyotype
Duplications
Reciprocal translocations (see Table 3)
8 (0.69)
Paracentric (see Table 3) Pericentric (see Table 3) Inv(9)qh 46,XX,dup(9)(q21.1q21.2) 46,XX,dup(9)(p12p12)
2 (0.17) 3 (0.26) 12 (1.03) 1 (0.08) 1 (0.08)
Note: Values in parentheses are percentages.
pregnancy rates in patients with chromosomal disorders.
MATERIALS AND METHODS Over a period of 26 months (May 1995 to June 1997), cytogenetic studies were performed in 2,280 patients who were referred for treatment with ICSI at our center for reproductive medicine and genetics. Of the 2,280 patients investigated, 1,164 were female and 1,116 were male.
poor fertilization in the past, in 9%. Male factor infertility was diagnosed only after multiple successive (.2) sperm analyses with an interval of at least 6 weeks. Statistical analysis was performed by x2 analysis and Fisher’s two-tailed exact test.
RESULTS
For each patient, an initial cytogenetic study of peripheral blood cells was undertaken according to the standard protocol of Moorhead et al. (7). Chromosomes were trypsinbanded according to the method of Klinger (8). In cases of structural aberrations, additional banding techniques were performed, including C-banding (9) and nucleolar organizing-staining with the use of the technique developed by Bloom and Goodpasture (10).
During 1995–1997, systematic cytogenetic testing was performed in 2,280 patients undergoing ICSI in our clinic. Of the 2,280 patients investigated, 1,164 were female and 1,116 were male.
In all cases, 10 metaphases of peripheral lymphocytes were karyotyped. An additional 10 metaphases were evaluated for their number of gonosomes. If there was any sign of mosaicism, 100 metaphases from two independent cell cultures were analyzed.
The frequency of aberrations was 4.48% in the male partners. Autosomal aberrations were observed in 33 men (2.96%) (Table 1), and numerical or structural sex chromosome abnormalities were found in 17 (1.52%) (Table 2).
The median age of the male partners was 34.5 years (range, 22– 66 years). The median age of the female partners was 32.5 years (range, 19 – 49 years). The selection criteria for ICSI treatment were extreme oligospermia, asthenospermia, or teratozoospermia, defined according to World Health Organization criteria (,5 3 106/mL, ,25% progressive motility, and ,15% normal forms) in 91% of couples and other indications, such as total fertilization failure in previous IVF attempts or successive 934
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A chromosomal abnormality was found in 7.2% of the couples. Single-cell aberrations were not included. Inversions of chromosome 9 are not considered to be pathologic but were included in Table 1 for the sake of completeness.
Numerical or structural abnormalities were documented in 9.79% of the female partners. Autosomal structural abnormalities were found in 27 women (2.32%) (Table 1), and numerical anomalies involving sex chromosomes were found in 87 (7.47%) (Table 2). There did not appear to be an age-related distribution of gonosomal aberrations. It is of interest that we found a high incidence of minor sex chromosome mosaicism in the female partners. This is a surprising finding because male factor infertility is the primary indication for ICSI. Vol. 70, No. 5, November 1998
TABLE 2 Cytogenetic analysis of 1,116 male and 1,164 female patients undergoing ICSI: sex chromosome aberrations. Male sex chromosomes
Karyotype
No. of aberrations
47,XXY 47,XYY 47,XXY/46,XY 47,XYY/46,XY 47,XYY/49,XYYYY/46,XY 45,X/47,XYY 45,X/46,XY
3 (0.27) 3 (0.27) 2 (0.18) 3 (0.27) 1 (0.09) 1 (0.09) 2 (1.18)
46,X,inv(y)(q11.2q12) 46,X,inv(y)(p11.2q12)
1 (0.09) 1 (0.09) 0
Aberration Numerical Complete Mosaicism
Structural Complete
Female sex chromosomes
Mosaicism
Karyotype
No. of aberrations
47,XXX 45,X/46,XX 47,XXX/46,XX 45,X/47,XXX/46,XX 45,X/48,XXXX/46,XX 45,X/47,XXX/48,XXXX/46,XX 45,X/47,XXX/48,XXXX/49,XXXXX/46,XX
1 (0.08) 48 (4.12) 7 (0.60) 22 (1.89) 4 (0.34) 2 (0.17) 1 (0.08)
Aberration Numerical Complete Mosaicism
Structural Complete
46,X,del(X)(pter-.q22:)
1 (0.08)
Mosaicism
46,Xr(X)/46,XX
1 (0.08)
Note: Values in parentheses are percentages.
In only two couples entering the ICSI program were both partners affected. In one couple, the male partner had Klinefelter’s syndrome and his partner had a 45,X/47,XXX/ 46,XX mosaic (2%/2%/96%). In the other couple, both partners had a minor sex chromosome mosaic: 47,XYY/46,XY (4%/96%) in the man and 45,X/46,XX (5%/95%) in the woman. Data on the nature of the autosomal aberrations found in the male and female partners and further details regarding the anomalies involved are provided in Table 3.
An embryo implantation rate of 9.4% and an ongoing pregnancy rate per transfer of 12.5% were observed in the female partners with sex chromosome mosaicism and were not statistically different from the rates in the female partners with autosomal aberrations. The implantation rate was significantly lower (P ,.05) in couples with a male gonosomal aberration than in couples with a male autosomal aberration (Table 4).
DISCUSSION TABLE 3 Detailed list of all autosomal aberrations in 2,280 patients undergoing ICSI. Female aberrations Reciprocal translocations 46,XX,t(12;16)(q24.3;p11.2) 46,XX,t(2;20)(q14.3;p13) 46,XX,t(12;19) 46,XX,t(1;2)(q42;p11.2) 46,XX,t(1;8)(p10;q10) 46,XX,t(7;9)(p10;p10) 46,XX,t(3;13)(q21;q22) 46,XX,t(2;7)(p25;q22) Paracentric inversions 46,XX,inv(7)(p15.1p22) 46,XX,inv(12)(q21q23) Pericentric inversions 46,XX,inv(10)(p11.2q21.2) 46,XX,inv(10)(p11q22) 46,XX,inv(10)(p11q22) Duplications 46,XX,dup(9)(q21.1q21.2) 46,XX,dup(9)(p12p12)
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Male aberrations
46,XY,t(1;9)(q44;p11.2) 46,XY,t(4;11;18)(p12;p15.1;q23) 46,XY,t(3;10)(p25;q26) 46,XY,t(1;3)(p36.5;q21) 46,XY,t(8;13)(q24.1;q22) 46,XY,t(9;14)(p22;q22) 46,Y,t(X;3)(q26;q23) 46,XY,t(4;19)(q34;q13.3) 46,XY,t(6;18)(q26;q23) 46,XY,inv(3)(p21p25) 46,XY,inv(2)(q21q22)
According to federal guidelines in Germany, all couples undergoing ICSI must be karyotyped before treatment, regardless of the cause of their infertility. The high percentage of chromosomal aberrations found in the female partners of couples undergoing ICSI in the present study raises the question of whether the incidence is increased in infertile couples in general, as has been demonstrated in couples undergoing regular treatment with IVF (11). Studies of the prevalence of chromosomal disorders in patients undergoing ICSI generally lack normal control groups. Prospective studies that include control groups should be carried out because minor mosaicism has been dismissed as a cultural artifact or considered to be of no relevance. The results of our study confirm that there is a high rate of chromosomal anomalies in both the male and female partners of infertile couples undergoing ICSI. Infertility has been correlated with chromosomal abnormalities in different IVF patient groups (11, 12). Conflicting data have been published on the frequency of gonosomal aberrations. The number of cytogenetic analyses in IVF is limited. Moreover, the incidence of gonosomal anomalies may be related to the indication for IVF. 935
TABLE 4 Subdivision of chromosomal aberrations according to male/female sex chromosome or autosomal anomalies: influence on implantation and ongoing pregnancy rates.
Aberration Male gonosomes Male autosomes Female gonosomes Female autosomes
No. of patients
No. of cycles (no. of ETs)
9 27 54 15
14 (12) 40 (33) 94 (72) 27 (20)
No. of pregnancies (percentage of ETs) 1 (8.3) 11 (33) 13 (18) 7 (40)
Implantation rate per embryo (%)
No. of ongoing pregnancies/no. of births (%)
Prenatal anomaly*
3.8† 23.1† 9.4 16.3
0/1 (8.3) 0/9 (27.2) 2/7 (12.5) 0/5 (25)
— 2‡ — —
* Reported chromosomal aberration. † Values with the same superscripts were significantly different (P , .05). ‡ One selective fetocide (trisomy 9), one balanced rob (13;14).
Numerous surveys have been conducted to determine the correlation between autosomal and sex chromosome abnormalities and male infertility. Most studies confirm a genetic basis, especially in patients with impaired spermatogenesis (1– 4, 13–15). Fluorescence in situ hybridization (FISH) has made it possible to perform comprehensive studies on numerical chromosome abnormalities in human spermatozoa (16 –20). Fluorescence in situ hybridization overcomes the major disadvantage of karyotyping a limited number of spermatozoa after penetration of zona-free hamster oocytes and subsequent decondensation. However, FISH does not discriminate between dead and vital, motile and immotile, or normal and grossly abnormal cells. In practice, this means that the frequency of abnormal chromosomes is not automatically the same in the gametes used for fertilization. In contrast with FISH, karyotyping shows all chromosomes and covers all types of structural rearrangements. Therapeutic possibilities offered by the introduction of ICSI have increased our awareness of the potential for propagating chromosomal aberrations in the next generation (21). Numerical and structural chromosomal anomalies, even those involved in spermiogenesis, are compatible with fertilization in ICSI (22, 23). However, complex structural rearrangements seem to be incompatible with fertilization in ICSI (24). According to the recent publication of in ‘t Veld et al. (25), infertile men with idiopathic oligozoospermia or azoospermia should be offered genetic testing and counseling before ICSI treatment is considered. The study on which this conclusion was based involved a subgroup of oligospermic or azoospermic male patients. It was not clear whether these men were actually taking part in the ICSI program. This group, therefore, may not be representative of male patients undergoing treatment with ICSI because no patients with cryptorchidism were included. Meiotic studies in men indicate that the sex chromosomes, along with chromosome 21, may be more susceptible 936
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to nondisjunction than are the other chromosomes (18, 26). Even more remarkable is the fact that a high incidence of sex chromosome aneuploidy occurs in female patients undergoing ICSI, with 45,X metaphases being more common than 47,XXX cells (27). The predominant loss of an X chromosome may be related to age and caused by prematurely separating centromeres, which do not attach to the spindle fibers and hence cause nondisjunction (28, 29) in the oocyte and somatic cells. However, in our ICSI studies, we could not find a relation between patient age and 46,XX/45,X mosaicism, possibly because of the relatively narrow age range of the female partners. It is still unknown whether the high incidence of minor gonosomal mosaicism can cause infertility as well as problems in implantation and further progress of a subsequent pregnancy. Although our observations are limited, the preliminary data indicate that there may be comparatively low implantation and ongoing pregnancy rates in couples with gonosomal aberrations. We were not able to find a statistically significant difference between female sex chromosome anomalies and female autosomal aberrations, as we were with male abnormalities. We initially intended not to label the data statistically significant because we wanted to stress the questionable clinical significance of any differences found. The number of aberrations found in conjunction with pregnancy probably is too small for us to analyze. Future studies are necessary. On average, after meiosis, 50% of translocations are unbalanced. The frequency depends on which chromosomes are involved. For these men, the risk of having an offspring with unbalanced translocation is theoretically high. However, the available data from newborns conceived with the use of ICSI do not indicate a higher rate of autosomal abnormalities in this group. The incidence of sex chromosome aneuploidies is increased to 1.2% compared with 0.83% in an unselected population (30, 31). The reduced transmission of unbalanced translocations probably is due to natural selection of embryos (6). Vol. 70, No. 5, November 1998
To date, few data are available concerning the influence of minor (,10%) mosaicism of sex chromosomes in peripheral lymphocytes on fertility and subsequent pregnancy in female patients. Data on this subject should be interpreted cautiously; for example, the karyotype of peripheral lymphocytes cannot be extrapolated automatically to the germ cell line. There is evidence that a maternal predisposition to chromosome aneuploidy may exist in patients undergoing IVF and may be associated with failed IVF cycles (32). Decreased implantation and ongoing pregnancy rates might be in line with these observations. Further, a recent publication from an Australian IVF group (33) points out possible negative aspects of ICSI therapy in a follow-up study of children performed 1 year after birth. In addition, the use of immature gametes further increases the risk that unexpected negative side effects will occur. Karyotyping may help in elucidating the long-term effects of ICSI therapy and excluding possible parental factors until more reassuring data from large prospective (blinded) follow-up studies are available. We therefore conclude that genetic testing and counseling of both male and female partners is recommended before treatment with ICSI. Our findings indicate that cytogenetic analysis should be considered seriously in all infertile couples before methods of assisted conception are used. References 1. Chandley AC, Edmond P, Christie S, Gowans L, Fletcher J, Frackiewicz A, et al. Cytogenetics and infertility in man. I. Karyotype and seminal analysis: results of a five-year survey of men attending a subfertility clinic. Ann Hum Genet 1975;39:231–54. 2. Chandley AC. The chromosomal basis of human infertility. Br Med Bull 1979;35:181– 6. 3. Ferguson-Smith MAB, Lennox B, Mack WS, Steward JSS. Klinefelter’s syndrome: frequency and testicular morphology in relation to nuclear sex. Lancet 1957;2:167–9. 4. Retief AE, Van Zyl JA, Menkveld R, Fox MF, Kotze GM, Brusnicky J. Chromosome studies in 496 infertile males with a sperm count below 10 million/ml. Hum Genet 1984;66:162– 4. 5. Bonduelle M, Wilikens A, Buysse A, Van Assche E, Wisanto A, Devroey P, et al. Prospective follow-up study of 877 children born after intracytoplasmic sperm injection (ICSI), with ejaculated epididymal and testicular spermatozoa and after replacement of cryopreserved embryos obtained after ICSI. Hum Reprod 1996;11(Suppl 4):131–55. 6. Plachot M, Veiga A, Montagut J, de Grouchy J, Calderon G, Lepretre S, et al. Are clinical and biological IVF parameters correlated with chromosomal disorders in early life: a multicentric study. Hum Reprod 1988;3:627–35. 7. Moorhead PS, Nowell PC, Mellmann WJ, Battips DM, Hungerford DA. Chromosome preparations of leucocyte cultures from human peripheral blood. Exp Cell Res 1960;20:613– 6. 8. Klinger HP. Rapid processing of primary embryonic tissues for chromosome banding pattern analysis. Cytogenetics 1972;11:424 –35. 9. Sumner AT. A simple technique for demonstrating centromeric heterochromatin. Exp Cell Res 1972;75:304 – 6. 10. Bloom SE, Goodpasture C. An improved technique for selective silver staining of nucleolar organizer regions in human chromosomes. Hum Genet 1976;34:199 –206.
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11. Hens L, Bonduelle M, Liebaers I, Devroey P, Van Steirteghem AC. Chromosome aberrations in 500 couples referred for in-vitro fertilization or related fertility treatment. Hum Reprod 1988;3:451–7. 12. Lange R, Johannson G, Engel W. Chromosome studies in in-vitro fertilization patients. Hum Reprod 1993;8:572– 4. 13. Pandiyan N, Jequier AM. Mitotic chromosomal anomalies among 1210 infertile men. Hum Reprod 1996;11:2604 – 8. 14. Bourrouillou G, Dastugue N, Colombies P. Chromosome studies in 952 infertile males with a sperm count below 10 million/ml. Hum Genet 1985;71:366 –7. 15. Bourrouillou G, Calvas P, Bujan L, Mieusset R, Mansat A, Pontonnier F. Mitotic chromosomal anomalies among infertile men (letter). Hum Reprod 1997;12:2337– 8. 16. Martin RH, Ko E, Chan K. Detection of aneuploidy in human interphase spermatozoa by fluorescence in situ hybridization (FISH). Cytogenet Cell Genet 1993;64:23– 6. 17. Robbins WA, Segraves R, Pinkel D, Wyrobek AJ. Detection of aneuploid human sperm by fluorescence in situ hybridization: evidence for a donor difference in frequency of sperm disomic for chromosomes 1 and Y. Am J Hum Genet 1993;52:799 – 807. 18. Spriggs EL, Rademaker AW, Martin RH. Aneuploidy in human sperm: the use of multicolor FISH to test various theories of nondisjunction. Am J Hum Genet 1996;58:356 – 62. 19. Martin RH, Spriggs E, Rademaker AW. Multicolor fluorescence in situ hybridization analysis of aneuploidy and diploidy frequencies in 225,846 sperm from 10 normal men. Biol Reprod 1996;54:394 – 8. 20. Downie SE, Flaherty SP, Matthews CD. Detection of chromosomes and estimation of aneuploidy in human spermatozoa using fluorescence in-situ hybridization. Mol Hum Reprod 1997;3:585–98. 21. Engel W, Murphy D, Schmid M. Are there genetic risks associated with microassisted reproduction? Hum Reprod 1996;11:2359 –70. 22. Testart J, Gautier E, Brami C, Rolet F, Sedbon E, Thebault A. Intracytoplasmic sperm injection in infertile patients with structural chromosome abnormalities. Hum Reprod 1996;11:2609 –12. 23. Tournaye H, Staessen C, Libaers I, van Assche E, Deveoey P, Bonduelle M, et al. Testicular sperm recovery in nine 47,XXY Klinefelter patients. Hum Reprod 1996;11:1644 –9. 24. Siffroi JP, Benzacken B, Straub B, le Bourhis C, North MO, Curotti G, et al. Assisted reproductive technology and complex chromosomal rearrangements: the limits of ICSI. Mol Human Reprod 1997;3:847–51. 25. in ‘t Veld PA, Halley DJJ, van Hemel JO, Niermeijer MF, Dohle G, Weber RAF. Genetic counselling before intracytoplasmic sperm injection. Lancet 1997;350:490. 26. Spriggs EL, Rademaker AW, Martin RH. Aneuploidy in human sperm: results of two- and three-color fluorescence in situ hybridization using centromeric probes for chromosomes 1, 12, 15, 18, X, and Y. Cytogenet Cell Genet 1995;71:47–53. 27. Galloway SM, Buckton KE. Aneuploidy and ageing: chromosome studies on a random sample of the population using G-banding. Cytogenet Cell Genet 1978;20:78 –95. 28. Vig BK. Sequence of centromere separation: orderly separation of multicentric chromosomes in mouse L cells. Chromosoma 1984;90:39 – 45. 29. Fitzgerald PH, Pickering AF, Mercer JM, Miethke PM. Premature centromere division: a mechanism of non-disjunction causing X chromosome aneuploidy in somatic cells of man. Ann Hum Genet 1975; 38:417–28. 30. Hook EB. Chromosome abnormalities: prevalence, risks and recurrence. In: Brock DJH, Rodeck CH, Ferguson-Smith MAB, editors. Prenatal diagnosis and screening. Edinburgh: Churchill Livingstone, 1992:351–92. 31. Jacobs PA, Browne C, Gregson N, Joyce C, White H. Estimates of the frequency of chromosome abnormalities detectable in unselected newborns using moderate levels of banding. J Med Genet 1992;29:103– 8. 32. Zenzes MT, Wang P, Casper RF. Evidence for maternal predisposition to chromosome aneuploidy in multiple oocytes of some in vitro fertilization patients. Fertil Steril 1992;57:143–9. 33. Bowen JR, Gibson FL, Leslie GI, Saunders DM. Medical and developmental outcome at 1 year for children conceived by intracytoplasmic sperm injection. Lancet 1998;351:1529 –34.
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