- Email: [email protected]
Cytogenetic analysis of early nonviable pregnancies after assisted reproduction treatment
Cytogenetic analysis of early nonviable pregnancies after assisted reproduction treatment In our series, a very high prevalence of chromosomal abnormalities was observed in spontaneous miscarriages, irrespective of how pregnancy was achieved, by assisted reproductive technology (ART) or natural conception. This high prevalence was equally represented in both groups, with no statistical difference in type of chromosomal abnormalities or in total frequency; however, the incidence of monosomy X is increased and the polyploidies are decreased in abortus after intracytoplasmic sperm injection (ICSI) when it is compared with miscarriages after spontaneous gestations. (Fertil Steril 2010;93:289–92. 2010 by American Society for Reproductive Medicine.)
Sporadic early pregnancy loss occurs in 10%–20% of clinically recognized gestations (1). Fetoplacental chromosomal abnormalities account for 50% of sporadic first trimester miscarriages (2– 4), and the other 50% are usually classified as idiopathic. After assisted reproductive technology (ART) the miscarriage rate might increase to 12%–40%, depending on the technique used (5–8), the number of embryos transferred (9), and maternal age (10– 12). Although recent developments in ART, such as intracytoplasmic sperm injection (ICSI), have been associated with a higher incidence of aneuploid and, in particular, of sex chromosome de novo aberrations in prenatal or child born testing (13, 14), no data from large series have been reported on chromosomal abnormalities in miscarriage after ART (15–20). To our knowledge, this is the largest series. The aim of the present work was to evaluate the type and frequency of chromosomal abnormalities occurring in early pregnancy loss after different ART treatments. Furthermore, the results allow us to determine whether different ART treatments might increase the risk of chromosomal abnormal conceptus. A cohort of 676 spontaneous miscarriages from the Instituto Valenciano de Infertilidad was processed by our Cytogenetic Laboratory between September 1996 and December 2007. Of these, 587 were successfully karyotyped. Most women (76.83%; 451/587) M. Carmen Martınez, M.Sc.a Carmen Mendez, B.Sc.a Jaime Ferro, M.D.b Maria Nicolas, M.D.a Vicente Serra, M.D., Ph.D.b Jose Landeras, M.D.a a Instituto Valenciano de Infertilidad in Murcia (IVI-Murcia), Murcia b Instituto Valenciano de Infertilidad, Valencia, Spain Received February 10, 2009; revised June 26, 2009; accepted July 14, 2009; published online September 11, 2009. M.C.M. has nothing to disclose. C.M. has nothing to disclose. J.F. has nothing to disclose. M.N. has nothing to disclose. V.S. has nothing to disclose. J.L. has nothing to disclose. Reprint requests: M. Carmen Martınez, M.Sc., IVI-Murcia, C/Navegante Macıas del Poyo no. 5, 30007, Murcia, Spain (FAX: 34-968-23-40-63; E-mail: [email protected]).
0015-0282/10/$36.00 doi:10.1016/j.fertnstert.2009.07.989
had conceived with the use of ART. Nonviable spontaneous pregnancies from couples who had not undergone ART treatment (136/587) acted as the control group. The mean age of patients was 35.41 4.94 years in the donor cycle group and 32.16 8.13 years in the control group. The mean gestational age was 8.19 2.42 weeks in the ART group versus 8.30 2.99 weeks in the control group. In 98.28% of cases, the pregnancy terminated before the 12th week of gestation. A precurettage hysteroembryoscopy was performed to obtain selective chorionic and embryo samples in 413 woman and a total of 449 gestational sacs were biopsied, using a previously developed technique (21, 22). One of the advantages of this technique is that it permitted us to analyze twin pregnancies separately; there were 32 sets of diamniotic twins and 3 triple gestations. The criteria for inclusion in the study group were the finding of a first trimester nonviable pregnancy and no or minimal bleeding. The selected tissues were subjected to cytogenetic analysis (adapted protocol) (23). Distributions were compared using the c2 test; mean maternal and gestational ages were compared using an analysis of variance (ANOVA) test. Statistical calculations were performed using the SPSS software package program, version 12.0 for windows (SPSS Inc., Chicago, IL). For all test thresholds, a value of P<.05 was used for significance. The data of both study groups are summarized in Table 1. Overall cytogenetic results revealed that 52.64% (309/587) of the miscarriages showed an abnormal karyotype. The frequency was 55.14% (75/136) in the case of spontaneous gestation and 51.88% (234/451) after ART, 70.66% (53/75) after artificial insemination (P<.027), and 47.82% (11/ 23) when the treatment was IVF. The rate was 51.82% (85/164) in abortus after ICSI and 60.86% (28/46) after an IVF/ICSI cycle. The lowest prevalence was found in pregnancy losses after IVF treatment in patients in an oocyte donation program at 23.80% (5/21; P<.008), after ICSI treatment in patients in an oocyte donation program at 41.66% (25/60; P<.082), and in embryos that had previously been screened for aneuploidies during preimplantation genetic diagnosis at 43.54% (27/62; P<.13, not significant [NS]). The most prevalent anomalies observed in early pregnancy loss after ART were autosomal trisomies (67.09%; 157/234), which affected chromosomes 16 (23.56%), 22 (17.19%), 15 (9.55%), 21 (8.91%), 13 (3.82%), and 14 (3.18%). There was a double aneuploidy in 6.41% of cases compared with a 58.66% (44/75) trisomy rate in spontaneous gestation, in which chromosomes 16
Fertility and Sterility Vol. 93, No. 1, January 2010 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.
289
290 Martınez et al. Correspondence
TABLE 1 Comparison of distribution of miscarriages after spontaneous and ART gestation according to cytogenetic categories. Gestation group Spontaneous Total ART Insemination IVF ICSI IVF/ICSI Oocyte donor IVF Oocyte donor ICSI PGS
Total
Abnormal case (%)
Trisomies (%)
Double trisomies (%)
45,X (%)
47.XXY (%)
Monosomy 21 (%)
Polyploidy (%)
Structural anomalies (%)
Embryonic mosaicism
136 451 75 23 164 46 21 60 62
75 (55.14) 234 (51.88) 53 (70.66)a 11 (47.82) 85 (51.82) 28 (60,86) 5 (23.80)b 25 (41.66)c 27 (43.54)
44 (58.66) 157 (67.09) 33 (62.26) 5 (45.45) 63 (74.11) 23 (82.14) 3 (60.00) 11 (44.00) 19 (70.37)
4 (5.33) 15 (6.41) 4 (7.54) 2 (18.18) 4 (4.70) 2 (7.14) 0 0 3 (11.11)
4 (5.33) 24 (10.25) 3 (5.66) 2 (18.18) 6 (7.05) 0 0 10 (40.00)d 3 (11.11)
0 1 (0.42) 0 0 1 (1.17) 0 0 0 0
2 (2.66) 0 0 0 0 0 0 0 0
16 (21.23) 23 (9.82) 12 (22.64) 2 (18.18) 5 (5.88)e 1 (3.57) 0 2 (8.00) 1 (3.70)
5 (6.66) 14 (5.98) 1 (1.88) 0 6 (7.05) 2 (7.14) 2 (40.0) 2 (8.00) 1 (3.70)
8 (5.88) 21 (4.65) 6 (8.00) 0 6 (3.65) 2 (4.34) 1 (4.76) 1 (1.66) 3 (11.11)
ICSI ¼ intracytoplasmic sperm injection; PGS ¼ preimplantation genetic screening. a P< .027, statistical significance compared with the spontaneous gestation group. b P< .008, statistical significance compared with the spontaneous gestation group. c P< .001, statistical significance compared with the spontaneous gestation group. d P<0.001, statistical significance compared with the spontaneous gestation group. e P<0.061, statistical significance compared with the spontaneous gestation group. Martınez. Correspondence. Fertil Steril 2010.
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(27.27%), 22 (20.45%), 15 (6.81%), 21 (4.54%), 13 (9.09%), and 14 (6.81%) were affected. A double aneuploidy occurred in 5.33% of the cases. It has been observed that trisomies for all autosomes can be implied in early embryonic loss with the exception of chromosome 1. In the group receiving ART, monosomy X was observed in 10.25% of cases (24/234) and it was observed in 5.33% (4/75) of the spontaneous group. Polyploidy was observed in 9.82% of ART cases (23/234) versus 21.23% of cases (16/75) in the control group (spontaneous pregnancies) and 5.98% of abnormalities (14/234) were structural in the first group versus 6.66% (5/ 75) in the second one. We considered the oocyte donor cycle treatments in a different group because it was expected that the lower mean age of the donor women would not increase the incidence of autosomal trisomies or monosomies X (24). Although when the oocyte donor cycles were separated into IVF and ICSI groups, the frequency of embryonic anomalies was, curiously, significantly (statistically significant) higher for monosomy X in the ICSI group (P<.001), with most of the monosomy X cases happening after an ICSI procedure (79.16%;19/24). A higher incidence of de novo aneuploidy has been previously reported in sex chromosomes in prenatal diagnosis or children born after ICSI treatment (14, 25). The present study demonstrates these same results in abortus from women who had undergone ICSI, mainly with regard to monosomy X. This anomaly was the most frequent sex chromosomal anomaly observed in our series, compared with the other sex chromosomal anomalies like 47,XXY or 47,XYY reported in the prenatal or neonatal studies mentioned previously. This event may be due to the lower viability in utero of monosomy X (99% end in spontaneous loss). An increased incidence of monosomy X in ICSI abortus compared with IVF abortus has also been recently reported (19). The high rate of monosomy X in our donation/ICSI group might indicate that ICSI facilitates the transmission of a sex chromosomal abnormality from sperm to conceptus and this might be related to the higher rate of sex chromosomal aneuploidy found in sperm of subfertile men (26, 27) or by mitotic errors related to damage in the cytoskeleton caused by the injection (17). Other investigators have proposed a preferential location of the X chromosome in the subacrosomal region of the sperm nucleus, which leads to reduced DNA decondensation (28) of this region or propensity for inactivation and loss rate for ICSI embryos (29). According our results, in an oocyte donation program, the risk of embryos with chromosomopathies is significantly lower when the clinician decides on IVF versus an ICSI treatment. Furthermore, if we consider that age is not relevant to the final rate of aneuploidy in this group, then ICSI could be the reason. This last event would lead us to believe that the clinician should choose IVF with donor treatment whenever possible. In this sense, as other investigators have noted, this finding would lead to us to further research the risk of sex chromosomal anomalies in offspring and to advise undergoing prenatal cytogenetic diagnosis in pregnancies after ICSI.
On the other hand, a lower frequency (not statistically significant) of polyploidy was observed in the ICSI group (P<.061)—possibly polyploidy decreased after ICSI due to double sperm fecundation being avoided with this treatment. Our mosaicism rate from embryos in spontaneous gestation was 5.88% (8/136) versus 3.65% (3/164) in the ICSI group. The hypothesized higher risk of postzygotic events as a consequence of the ICSI procedure leading to a higher proportion of chromosomal mosaicism (30), a phenomenon observed by other investigators, was not confirmed in this study. We also considered, in a different ICSI group, a total of 62 cases in which embryos were screened for the most frequent aneuploidy type by preimplantation diagnosis treatments (chromosomes 13, 18, 21, 22, 16, X, and Y). We then identified 19 embryos with trisomies (70.37%), finding the most prevalent chromosomes to be numbers 3 and 15; double trisomies for these chromosomes were not analyzed using fluorescent in situ hibridization (FISH). Trisomies for chromosomes 13, 18 and 21 have not been observed in this group, although some chromosomes previously analyzed went undetected by FISH, probably due to mosaicism (5/62). These mosaics could be identified in three cases during the fetal loss analysis; in the other two cases the low proportion of normal cells could have been hidden by the overgrowth of the abnormal cells. Another interesting observation is that all chromosomes contribute to early pregnancy failure but chromosomes 13, 14, 15, 16, 18, 21, and 22 appear to be the more common culpits, which must be taken into consideration in embryo chromosomal screening programs to improve implantation rates or in women of advanced age or who suffer recurrent miscarriages (31). This option increases the interest in classifying a miscarriage as chromosomally normal or abnormal. This classification could help to determine when to use this treatment to reduce the miscarriage rate (32) and it is also being developed as an alternative to prenatal diagnosis in IVF (33). Although preimplantation genetic screening (PGS) was introduced into clinical practice to screen for aneuploid embryos to improve implantation rates, prevent abortus in women of advanced age, or women who suffered recurrent miscarriages (31), PGS has recently been criticized by several investigators (34–37) regarding its efficiency and safety. Some investigators prefer an oocyte donation program or even surrogacy when the maternal environment needs to be substituted, instead of PGS (38). The debate on the usefulness of PGS is ongoing, therefore more randomized clinical trials are needed. At present, the number of cases analyzed in this study is not large enough to establish separate subgroups according to different infertility etiologies. Acknowledgments: The authors thank all Instituto Valenciano de Infertilidad medical staff for the collection of samples and the technical personnel for their technical assistance. We gratefully acknowledge Dr. J. Lopez for his assistance with the data analysis.
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tions collected in Japan. J Obstet Res 2004;30: 237–41. 5. Wennerholm UB, Hamberger L, Nilsson L, Wennergren M, Wikl M, Bergh C. Obstetrics and perinatal outcome of children conceived from cryopreserved embryos. Hum Reprod 1997;12:1819–25.
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6. Aytoz A, Van den Abbeel E, Bonduelle M, Camus M, Joris H, Van Steirteghem A, et al. Obstetric outcome of pregnancies after the transfer of cryopreserved and fresh embryos obtained by conventional in-vitro fertilization and intracytoplasmic sperm injection. Hum Reprod 1999;10:2619–24. 7. Palmero GD, Neri QV, Hariprashad JJ, Davis OK, Veeck LL, Rosenwaks Z. ICSI and its outcome. Semin Reprod Med 2000;18:161–9. 8. Westergaard HB, Johansen AM, Erb K, Andersen AN. Danish National IVF Registry 1994 and 1995. Treatment, pregnancy outcome and complications during pregnancy. Acta Obstet Gynecol Scand 2000;79:384–9. 9. Balen AH, McDougall J, Tan SL. The influence of the number of embryos transferred in 1060 in-vitro fertilization pregnancies on miscarriage rates and pregnancy outcome. Hum Reprod 1993;8:1324–8. 10. Lass A, Croucher C, Duffy S, Dawson K, Margara R, Winston RM. One thousand initiated cycles of in vitro fertilization in women > or ¼ 40 years of age. Fertil Steril 1998;70:1030–4. 11. Pantos K, Athanasiou V, Stefanidis K, Stavrou D, Vaxevanoglou T, Chronopoulou M. Influence of advanced age on the blastocyst development rate and pregnancy rate in assisted reproductive technology. Fertil Steril 1999;71:1144–6. 12. Nikolettos N, Kupker W, Al-Hasani S, Demirel LC, Schopper B, Sturm R, et al. ICSI outcome in patients of 40 years age and over: a retrospective analysis. Eur J Obstet Gynecol Reprod Biol 2000;91:177–82. 13. Tarlatzis BC, Grimbizis G. Pregnancy and child outcome after assisted reproduction techniques. Hum Reprod 1999;14(Suppl 1):231–42. 14. Bonduelle M, Van Assche E, Joris H, Keymolen K, Devroey P, Van Steirteghem A, et al. Prenatal testing in ICSI pregnancies: incidence of chromosomal anomalies in 1586 karyotypes and relation to sperm parameters. Hum Reprod 2002;17:2600–14. 15. Plachot M. Chromosome analysis of spontaneous abortions after IVF. A European survey. Hum Reprod 1989;4:425–9. 16. Shields LE, Serafın PC, Schenken RS, Moore CM. Chromosomal analysis of pregnancy losses in patients undergoing assisted reproduction. J Assist Reprod Genet 1992;9:57–60.
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17. Causio F, Fischetto R, Sarcina E, Geusa S, Tartagni M. Chromosome analysis of spontaneous abortions after in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). A European survey. Eur J Obstet Gynecol Reprod Biol 2002;105:44–8. 18. Lathi RB, Milki AA. Rate of aneuploidy in miscarriages following in vitro fertilization and intracytoplasmic sperm injection. Fertil Steril 2004;84: 1270. 19. Ma S, Philipp T, Zhao Y, Stetten G, Robinson WP, Kalousek D. Frequency of chromosomal abnormalities in spontaneous abortions derived from intracytoplasmic sperm injection compared with those from in vitro fertilization. Fertil Steril 2006;85: 236–9. 20. Bettio D, Venci A, Levi Setti PE. Chromosomal abnormalities in miscarriages after different assisted reproduction procedures. Placenta 2008;29:126–8. 21. Ferro J, Serra V, Pellicer A, Remohı J. Histeroembrioscopıa: tecnicas y aplicaciones. Cuad Med Reprod 2001;7:139–54. 22. Filly RA. Ultrasound evaluation during the first trimester. In: Callen PW, ed. Ultrasonography in obstetrics and gynecology. Philadelphia: WB Saunders, 1994:63–85. 23. Barch MJ, Knutsen T, Spurbeck JL. The AGT cytogenetics laboratory manual. Philadelphia: Lippincott-Raven, 1997. 187–90. 24. Warburton D. Biological aging and the etiology of aneuploidy. Cytogenet Genome Res 2005;111: 266–72. 25. Lam R, Ma S, Robinson WP, Chan T, Yuen B. Cytogenetic investigation of fetuses and infants conceived through intracytoplasmic sperm injection. Fertil Steril 2001;76:1272–5. 26. Macas E, Imthrn B, Keller P. Increased incidence of numerical chromosome abnormalities in spermatozoa injected into human oocytes by ICSI. Hum Reprod 2001;16:115–20. 27. Tang SS, Grao H, Robinson WP, Ho Yuen B, Ma S. An association between sex chromosomal aneuploidy in sperm and an abortus with 45, X of paternal origin: possible transmission of chromosomal abnormalities through ICSI. Hum Reprod 2004;19:147–51.
28. Sbracia M, Baldi M, Cao D, Sandrelli A, Chiandetti A, Poverin R, et al. Preferential location of sex chromosomes, their aneuploidy in human sperm, and their role in determining sex chromosome aneuploidy in embryos after ICSI. Hum Reprod 2002;17:320–4. 29. Greaves IK, Rens W, Ferguson-Smith MA, Griffin D, Marshall Graves JA. Conservation of chromosome arrangement and position of the X in mammalian sperm suggest functional significance. Chromosome Res 2003;11:503–12. 30. In´t Veld P, Brandenburg H, Verhoeff A, Dhondt M, Los F. Sex chromosomal anomalies and intracytoplasmic sperm injection. Lancet 1995;345:773. 31. Donoso P, Staessen C, Fauser BC, Devroey P. Current value of preimplantation genetic aneuploidy screening in IVF. Hum Reprod Update 2007;13: 15–25. 32. Vidal F, Rubio C, Simon C. Is there a place for preimplantation genetic diagnosis screening in recurrent miscarriage patients? J Reprod Fertil Suppl 2000;55:143–6. 33. Delhanty JD, Harper JC. Pre-implantation genetic diagnosis. Baillieres Best Pract Res Clin Obstet Gynaecol 2000;14:691–708. 34. Cohen J, Grifo J. Multicentre trial of preimplantation genetic screening reported in the New England Journal of Medicine: an in-depth look at the findings. Reprod Biomed Online 2007;15: 365–6. 35. Munne S, Cohen J, Simpson JL. In vitro fertilization with preimplantation genetic screening. N Engl J Med 2007;357:1769–70. 36. Munne S, Gianaroli L, Tur-Kaspa I, Magli C, Sandalinas M, Grifo J, et al. Substandard application of preimplantation genetic screening may interfere with its clinical success. Fertil Steril 2007;88:781–4. 37. Kulie A, Verlynsky Y. Impact of preimplantation genetic diagnosis for chromosomal disorders on reproductive outcome. Reprod Biomed Online 2008;16:9–10. 38. Carp HJ, Dirnfeld M, Dor J, Grudzinskas JG. ART in recurrent miscarriage: preimplantation genetic diagnosis/screening or surrogacy? Hum Reprod 2004;19:1502–5.
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Sporadic early pregnancy loss occurs in 10%–20% of clinically recognized gestations (1). Fetoplacental chromosomal abnormalities account for 50% of sporadic first trimester miscarriages (2– 4), and the other 50% are usually classified as idiopathic. After assisted reproductive technology (ART) the miscarriage rate might increase to 12%–40%, depending on the technique used (5–8), the number of embryos transferred (9), and maternal age (10– 12). Although recent developments in ART, such as intracytoplasmic sperm injection (ICSI), have been associated with a higher incidence of aneuploid and, in particular, of sex chromosome de novo aberrations in prenatal or child born testing (13, 14), no data from large series have been reported on chromosomal abnormalities in miscarriage after ART (15–20). To our knowledge, this is the largest series. The aim of the present work was to evaluate the type and frequency of chromosomal abnormalities occurring in early pregnancy loss after different ART treatments. Furthermore, the results allow us to determine whether different ART treatments might increase the risk of chromosomal abnormal conceptus. A cohort of 676 spontaneous miscarriages from the Instituto Valenciano de Infertilidad was processed by our Cytogenetic Laboratory between September 1996 and December 2007. Of these, 587 were successfully karyotyped. Most women (76.83%; 451/587) M. Carmen Martınez, M.Sc.a Carmen Mendez, B.Sc.a Jaime Ferro, M.D.b Maria Nicolas, M.D.a Vicente Serra, M.D., Ph.D.b Jose Landeras, M.D.a a Instituto Valenciano de Infertilidad in Murcia (IVI-Murcia), Murcia b Instituto Valenciano de Infertilidad, Valencia, Spain Received February 10, 2009; revised June 26, 2009; accepted July 14, 2009; published online September 11, 2009. M.C.M. has nothing to disclose. C.M. has nothing to disclose. J.F. has nothing to disclose. M.N. has nothing to disclose. V.S. has nothing to disclose. J.L. has nothing to disclose. Reprint requests: M. Carmen Martınez, M.Sc., IVI-Murcia, C/Navegante Macıas del Poyo no. 5, 30007, Murcia, Spain (FAX: 34-968-23-40-63; E-mail: [email protected]).
0015-0282/10/$36.00 doi:10.1016/j.fertnstert.2009.07.989
had conceived with the use of ART. Nonviable spontaneous pregnancies from couples who had not undergone ART treatment (136/587) acted as the control group. The mean age of patients was 35.41 4.94 years in the donor cycle group and 32.16 8.13 years in the control group. The mean gestational age was 8.19 2.42 weeks in the ART group versus 8.30 2.99 weeks in the control group. In 98.28% of cases, the pregnancy terminated before the 12th week of gestation. A precurettage hysteroembryoscopy was performed to obtain selective chorionic and embryo samples in 413 woman and a total of 449 gestational sacs were biopsied, using a previously developed technique (21, 22). One of the advantages of this technique is that it permitted us to analyze twin pregnancies separately; there were 32 sets of diamniotic twins and 3 triple gestations. The criteria for inclusion in the study group were the finding of a first trimester nonviable pregnancy and no or minimal bleeding. The selected tissues were subjected to cytogenetic analysis (adapted protocol) (23). Distributions were compared using the c2 test; mean maternal and gestational ages were compared using an analysis of variance (ANOVA) test. Statistical calculations were performed using the SPSS software package program, version 12.0 for windows (SPSS Inc., Chicago, IL). For all test thresholds, a value of P<.05 was used for significance. The data of both study groups are summarized in Table 1. Overall cytogenetic results revealed that 52.64% (309/587) of the miscarriages showed an abnormal karyotype. The frequency was 55.14% (75/136) in the case of spontaneous gestation and 51.88% (234/451) after ART, 70.66% (53/75) after artificial insemination (P<.027), and 47.82% (11/ 23) when the treatment was IVF. The rate was 51.82% (85/164) in abortus after ICSI and 60.86% (28/46) after an IVF/ICSI cycle. The lowest prevalence was found in pregnancy losses after IVF treatment in patients in an oocyte donation program at 23.80% (5/21; P<.008), after ICSI treatment in patients in an oocyte donation program at 41.66% (25/60; P<.082), and in embryos that had previously been screened for aneuploidies during preimplantation genetic diagnosis at 43.54% (27/62; P<.13, not significant [NS]). The most prevalent anomalies observed in early pregnancy loss after ART were autosomal trisomies (67.09%; 157/234), which affected chromosomes 16 (23.56%), 22 (17.19%), 15 (9.55%), 21 (8.91%), 13 (3.82%), and 14 (3.18%). There was a double aneuploidy in 6.41% of cases compared with a 58.66% (44/75) trisomy rate in spontaneous gestation, in which chromosomes 16
Fertility and Sterility Vol. 93, No. 1, January 2010 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.
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290 Martınez et al. Correspondence
TABLE 1 Comparison of distribution of miscarriages after spontaneous and ART gestation according to cytogenetic categories. Gestation group Spontaneous Total ART Insemination IVF ICSI IVF/ICSI Oocyte donor IVF Oocyte donor ICSI PGS
Total
Abnormal case (%)
Trisomies (%)
Double trisomies (%)
45,X (%)
47.XXY (%)
Monosomy 21 (%)
Polyploidy (%)
Structural anomalies (%)
Embryonic mosaicism
136 451 75 23 164 46 21 60 62
75 (55.14) 234 (51.88) 53 (70.66)a 11 (47.82) 85 (51.82) 28 (60,86) 5 (23.80)b 25 (41.66)c 27 (43.54)
44 (58.66) 157 (67.09) 33 (62.26) 5 (45.45) 63 (74.11) 23 (82.14) 3 (60.00) 11 (44.00) 19 (70.37)
4 (5.33) 15 (6.41) 4 (7.54) 2 (18.18) 4 (4.70) 2 (7.14) 0 0 3 (11.11)
4 (5.33) 24 (10.25) 3 (5.66) 2 (18.18) 6 (7.05) 0 0 10 (40.00)d 3 (11.11)
0 1 (0.42) 0 0 1 (1.17) 0 0 0 0
2 (2.66) 0 0 0 0 0 0 0 0
16 (21.23) 23 (9.82) 12 (22.64) 2 (18.18) 5 (5.88)e 1 (3.57) 0 2 (8.00) 1 (3.70)
5 (6.66) 14 (5.98) 1 (1.88) 0 6 (7.05) 2 (7.14) 2 (40.0) 2 (8.00) 1 (3.70)
8 (5.88) 21 (4.65) 6 (8.00) 0 6 (3.65) 2 (4.34) 1 (4.76) 1 (1.66) 3 (11.11)
ICSI ¼ intracytoplasmic sperm injection; PGS ¼ preimplantation genetic screening. a P< .027, statistical significance compared with the spontaneous gestation group. b P< .008, statistical significance compared with the spontaneous gestation group. c P< .001, statistical significance compared with the spontaneous gestation group. d P<0.001, statistical significance compared with the spontaneous gestation group. e P<0.061, statistical significance compared with the spontaneous gestation group. Martınez. Correspondence. Fertil Steril 2010.
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(27.27%), 22 (20.45%), 15 (6.81%), 21 (4.54%), 13 (9.09%), and 14 (6.81%) were affected. A double aneuploidy occurred in 5.33% of the cases. It has been observed that trisomies for all autosomes can be implied in early embryonic loss with the exception of chromosome 1. In the group receiving ART, monosomy X was observed in 10.25% of cases (24/234) and it was observed in 5.33% (4/75) of the spontaneous group. Polyploidy was observed in 9.82% of ART cases (23/234) versus 21.23% of cases (16/75) in the control group (spontaneous pregnancies) and 5.98% of abnormalities (14/234) were structural in the first group versus 6.66% (5/ 75) in the second one. We considered the oocyte donor cycle treatments in a different group because it was expected that the lower mean age of the donor women would not increase the incidence of autosomal trisomies or monosomies X (24). Although when the oocyte donor cycles were separated into IVF and ICSI groups, the frequency of embryonic anomalies was, curiously, significantly (statistically significant) higher for monosomy X in the ICSI group (P<.001), with most of the monosomy X cases happening after an ICSI procedure (79.16%;19/24). A higher incidence of de novo aneuploidy has been previously reported in sex chromosomes in prenatal diagnosis or children born after ICSI treatment (14, 25). The present study demonstrates these same results in abortus from women who had undergone ICSI, mainly with regard to monosomy X. This anomaly was the most frequent sex chromosomal anomaly observed in our series, compared with the other sex chromosomal anomalies like 47,XXY or 47,XYY reported in the prenatal or neonatal studies mentioned previously. This event may be due to the lower viability in utero of monosomy X (99% end in spontaneous loss). An increased incidence of monosomy X in ICSI abortus compared with IVF abortus has also been recently reported (19). The high rate of monosomy X in our donation/ICSI group might indicate that ICSI facilitates the transmission of a sex chromosomal abnormality from sperm to conceptus and this might be related to the higher rate of sex chromosomal aneuploidy found in sperm of subfertile men (26, 27) or by mitotic errors related to damage in the cytoskeleton caused by the injection (17). Other investigators have proposed a preferential location of the X chromosome in the subacrosomal region of the sperm nucleus, which leads to reduced DNA decondensation (28) of this region or propensity for inactivation and loss rate for ICSI embryos (29). According our results, in an oocyte donation program, the risk of embryos with chromosomopathies is significantly lower when the clinician decides on IVF versus an ICSI treatment. Furthermore, if we consider that age is not relevant to the final rate of aneuploidy in this group, then ICSI could be the reason. This last event would lead us to believe that the clinician should choose IVF with donor treatment whenever possible. In this sense, as other investigators have noted, this finding would lead to us to further research the risk of sex chromosomal anomalies in offspring and to advise undergoing prenatal cytogenetic diagnosis in pregnancies after ICSI.
On the other hand, a lower frequency (not statistically significant) of polyploidy was observed in the ICSI group (P<.061)—possibly polyploidy decreased after ICSI due to double sperm fecundation being avoided with this treatment. Our mosaicism rate from embryos in spontaneous gestation was 5.88% (8/136) versus 3.65% (3/164) in the ICSI group. The hypothesized higher risk of postzygotic events as a consequence of the ICSI procedure leading to a higher proportion of chromosomal mosaicism (30), a phenomenon observed by other investigators, was not confirmed in this study. We also considered, in a different ICSI group, a total of 62 cases in which embryos were screened for the most frequent aneuploidy type by preimplantation diagnosis treatments (chromosomes 13, 18, 21, 22, 16, X, and Y). We then identified 19 embryos with trisomies (70.37%), finding the most prevalent chromosomes to be numbers 3 and 15; double trisomies for these chromosomes were not analyzed using fluorescent in situ hibridization (FISH). Trisomies for chromosomes 13, 18 and 21 have not been observed in this group, although some chromosomes previously analyzed went undetected by FISH, probably due to mosaicism (5/62). These mosaics could be identified in three cases during the fetal loss analysis; in the other two cases the low proportion of normal cells could have been hidden by the overgrowth of the abnormal cells. Another interesting observation is that all chromosomes contribute to early pregnancy failure but chromosomes 13, 14, 15, 16, 18, 21, and 22 appear to be the more common culpits, which must be taken into consideration in embryo chromosomal screening programs to improve implantation rates or in women of advanced age or who suffer recurrent miscarriages (31). This option increases the interest in classifying a miscarriage as chromosomally normal or abnormal. This classification could help to determine when to use this treatment to reduce the miscarriage rate (32) and it is also being developed as an alternative to prenatal diagnosis in IVF (33). Although preimplantation genetic screening (PGS) was introduced into clinical practice to screen for aneuploid embryos to improve implantation rates, prevent abortus in women of advanced age, or women who suffered recurrent miscarriages (31), PGS has recently been criticized by several investigators (34–37) regarding its efficiency and safety. Some investigators prefer an oocyte donation program or even surrogacy when the maternal environment needs to be substituted, instead of PGS (38). The debate on the usefulness of PGS is ongoing, therefore more randomized clinical trials are needed. At present, the number of cases analyzed in this study is not large enough to establish separate subgroups according to different infertility etiologies. Acknowledgments: The authors thank all Instituto Valenciano de Infertilidad medical staff for the collection of samples and the technical personnel for their technical assistance. We gratefully acknowledge Dr. J. Lopez for his assistance with the data analysis.
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