BRIEF REPORT |
Sperm and Blastomere Aneuploidy Detection in Reproductive Genetics and Medicine
Società Italiana Studi di Medicina della Riproduzione (S.I.S.Me.R.), Reproductive Medicine Unit, Bologna, Italy
Correspondence to: Luca Gianaroli, S.I.S.Me.R., Reproductive Medicine Unit, Via Mazzini, 123 40138 Bologna, Italy. E-mail: sismer{at}sismer.it
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Summary |
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(J Histochem Cytochem 53:261267, 2005)
Key Words: aneuploidy assisted reproduction embryo culture embryo development fluorescence in situ hybridization implantation preimplantation genetic diagnosis
EVALUATION OF EMBRYO morphology and developmental rate is the current means of formulating a prognosis for implantation. Although good correlation exists between the morphological aspect of developing embryos and viability, this approach is not highly efficient because more than two thirds of the transferred embryos do not result in a pregnancy that is carried to term. In patients with poor prognosis, the rate of embryo loss is even more severe and seems to be related to a high incidence of chromosomally abnormal embryos that, in some categories, exceeds 60%. In these cases, regular embryo growth and even development to blastocyst are not reliable tools for selection against numerical or structural alterations in chromosomes.
Preimplantation genetic diagnosis (PGD) for aneuploidy provides information for embryonic chromosomal assessment. This information is of great importance in some groups of patients and contributes an additional criterion for embryo selection. Data reported on the clinical outcome after PGD for aneuploidy show the main results are an increased implantation rate, a reduced incidence of spontaneous abortion, and a minimized risk of trisomic conception (Gianaroli et al. 1999,2003
; Munné et al. 1999
; Munné 2002
). Thus, this approach is aimed both at improving the chances of healthy implantation and at increasing the efficiency of assisted reproductive techniques by refining the methods of embryo selection when morphological criteria alone are not sufficient to identify viable embryos.
The data obtained by the chromosomal analysis of preimplantation embryos have confirmed the exposure to nondisjunction events during oogenesis, providing the molecular basis for the known decline in the reproductive outcome of women in advanced age. These findings suggest that chromosomal abnormalities could be the reason for poor reproductive performance in other groups of infertile patients and encourage an attempt to identify categories of patients for whom PGD for aneuploidy might be of benefit.
Conventional karyotype analysis is not possible in preimplantation embryos because of the low yield of metaphase chromosomes after anti-mitotic treatment. Therefore, alternative approaches have been developed, and PGD for aneuploidy is mostly based on fluorescence in situ hybridization (FISH) in interphase nuclei (Figure 1A).
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The aim of this study was to evaluate the results generated by the analysis of preimplantation embryos for the chromosomes that are mainly involved in numerical defects in the human. Special attention was dedicated to estimating the paternal contribution to aneuploidy by directly analyzing chromosomes on spermatozoa and by evaluating the incidence of aneuploidy in embryos in relation to the sperm quality.
Advanced maternal age has been the first indication for PGD of aneuploidy based on the consideration that the selection of euploid embryos could reverse the age effect. Other categories of younger patients with a poor prognosis for pregnancy were also included in the study: (a) multiple unexplained failures in three or more consecutive cycles of in vitro fertilization (IVF); (b) an altered karyotype caused by gonosomal mosaicism or by structural abnormalities such as translocations or inversions; (c) the occurrence of repeated spontaneous abortions in couples with a normal karyotype; and (d) a condition of gonadal failure in women (poor responders) and men (azoospermia).
Between September 1996 and December 2003, PGD for aneuploidy was performed in 1029 conception cycles at S.I.S.ME.R. Reproductive Medicine Unit (Table 1). A diagnosis was obtained in 5115 embryos (99% of 5152 biopsied embryos); of these, 1680 had a normal chromosomal complement (33%) and made the transfer possible for 699 cycles (68%) with an average of 1.7 ± 0.7 euploid embryos replaced. Two hundred eight clinical pregnancies were generated (30%) with an implantation rate of 21.3%.
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In the patients with repeated IVF failures, the most frequent defects were complex abnormalities, haploidy, and polyploidy, which accounted for 64% of total abnormalities. This frequency was significantly different from that in patients of advanced maternal age (57%, p<0.001), suggesting the possibility of alterations in the mechanisms of cell division, such as asynchrony between karyokinesis and cytokinesis, or centriolar defects. The male gamete could have a role in determining this condition, because the first oocyte divisions are controlled by the sperm-derived centriole and corresponding microtubule organizing regions (Schatten 1994). The sperm centrosome also determines the plane of the first cleavage, with critical consequences for the polarity and ploidy of the resulting embryo (Edwards and Beard 1997
). In the present study, the proportion of couples with male-factor infertility was 71% in the group with repeated IVF failures vs 44% in the group of patients with advanced maternal age (p<0.001). This difference in the frequency of male-factor infertility could explain the results of the chromosomal analysis in the resulting embryos for which complex abnormalities were the prevailing defect.
Besides demonstrating the clinical relevance of PGD for aneuploidy, PGD also provides information on early embryology. Two factors are especially relevant: (a) the association between chromosomal status and embryo morphology and (b) the frequency with which specific chromosomes undergo aneuploid events.
As shown in Figure 2, embryos with seven or eight cells at 62 hr after insemination, with less than 10% fragmentation, absence of multinucleation, or cytoplasmic irregularities, have the highest chances of being chromosomally normal (Magli et al. 2001). At this stage, more than half of the abnormalities (825 abnormal embryos, representing 51% of the 1617 analyzed) are monosomies and trisomies (30% of total abnormalities), whereas complex abnormalities, haploidy, and polyploidy prevail in embryos cleaving at an abnormal rate, either too slowly (four to six cells early on day 3) or too fast (more than eight cells early on day 3). The presence of an aneuploid complement does not preclude development of a morphologically normal blastocyst, confirming that morphological criteria alone are of limited value in poor-prognosis patients (Magli et al. 2000
).
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The widespread use of ICSI for the treatment of extremely severe male-factor infertility in reproductive medicine and the data from the follow-up of the children born have raised concern about the safety of the procedure (Bonduelle et al. 2002). The resulting increased incidence of de novo chromosomal abnormalities suggested the possibility that in selected groups of patients the paternal contribution to aneuploidy could be relevant. This subject was indirectly investigated in a study in which a group of 136 patients underwent PGD for aneuploidy (Gianaroli et al. 2000b
). Only couples with a female partner younger than 36 years were admitted to evaluate the effect of sperm indices on the chromosomal constitution of preimplantation embryos. The results were analyzed by dividing the patients in four groups according to the sperm parameters: (a) normospermic patients requiring ICSI for previous fertilization failures, (b) moderately oligoastenoteratospermic (OAT) patients, (c) severely OAT patients, and (d) azoospermic patients with sperm retrieved by microsurgical interventions in the epididymus using microsurgical epididymal sperm aspiration (MESA) or in the testis using testicular sperm extraction (TESE). The data were compared with data obtained from normospermic patients undergoing conventional insemination.
There were no differences in the overall percentage of chromosomally abnormal embryos in the different groups. A higher incidence of monosomies and trisomies, however, was found in embryos generated by MESA and TESE sperm. In addition, the rate of aneuploidy for sex chromosomes increased proportionally with the severity of the male-factor condition. The extension of this study to other cases allowed the evaluation of differences between MESA and TESE embryos and, in the TESE embryos, between those generated by patients with obstructive and non-obstructive azoospermia. In agreement with other reports (Silber et al. 2003), complex abnormalities were the prevailing defect in embryos generated by TESE patients with non-obstructive azoospermia. In addition, this group also presented a significant increase in gonosomal aneuploidy (unpublished data).
These results showed the importance of including the chromosomal analysis of sperm cells in the preliminary tests given infertile couples, especially in cases of repeated failures in cycles of assisted reproduction. Several reports have documented the greater frequency of chromosomal abnormalities in spermatozoa from infertile males as compared with the normospermic population (Egozcue et al. 1997; In't Veld et al. 1997
; Pang et al. 1999
; Bernardini et al. 2000
).
A FISH test with probes specific for chromosomes 13, 15, 16, 17, 18, 21, 22, X, and Y was developed and applied to 96 sperm samples (15 normospermic, 59 OAT, 14 severely OAT, and 8 MESA and TESE spermatozoa). When available, 5000 spermatozoa were analyzed by combining the three probes in three different sets. In cases of extremely severe oligospermia, each spermatozoon was analyzed with a five-probe mixture while its position on the slide was recorded. A second-round hybridization followed with the four remaining probes on the same slide, allowing analysis of nine chromosomes for each sperm cell (unpublished data). Results were analyzed using a statistical analysis based on chi-square test by applying the binomial distribution of Poissons. The results were interpreted by assigning clinical relevance of the detected aneuploidy at p values lower than 0.001. In these cases, patients were sent to the andrologist to evaluate the necessity of a therapy aimed at improving sperm indices, including the proportion of aneuploid cells. The highest incidence of aneuploidy was detected in samples belonging to severely OAT and MESA-TESE patients (Table 2). The difference was significantly relevant when compared with the normospermic population.
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In our center, for patients of poor prognosis, the first treatment option is generally FISH on embryos. After a failed cycle of PGD for aneuploidy on blastomeres with no transfer caused by FISH results or no pregnancy after repeated PGD cycles, FISH on sperm is recommended to the male partner. If the results are within the normal range, a cycle with polar body biopsy is the following step. Conversely, if the results are significantly abnormal, the patient is referred to the andrologist; if therapy is recommended, the FISH test is repeated after completion of the treatment (Magli et al. 2004). The aim of this approach is to determine whether the aneuploidy originates in the gametes or in the embryos. In some cases, comparison with the results obtained by FISH analysis is informative and permits correlations between aneuploidy in gametes and embryos to be established. Table 3 shows the data from 27 couples that underwent FISH on sperm and embryos. Although a higher number of cases will be needed to draw valid conclusions and to control for the dominant effect of female age, this approach seems to be promising in indicating correlations between sperm aneuploidy and the resulting embryos. According to these very preliminary data, when sperm aneuploidy exceeds 10% in patients younger than 36 years, mosaicism seems to be the prevailing defect in the generated embryos.
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Footnotes |
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Received for publication June 4, 2004; accepted December 16, 2004
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Literature Cited |
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Bernardini L, Gianaroli L, Fortini D, Conte N, Magli MC, Cavani S, Gaggero G, et al. (2000) Frequency of hyper-, hypohaploid and diploidy in ejaculate, epididymal and testicular germ cells of infertile patients. Hum Reprod 15:21652172
Bonduelle M, Van Assche E, Joris H, Keymolen K, Devroey P, Van Steirteghem A, Liebaers I (2002) Prenatal testing in ICSI pregnancies: incidence of chromosomal anomalies in 1586 karyotypes and relation to sperm parameters. Hum Reprod 17:26002614
Edwards RG, Beard HK (1997) Oocyte polarity and cell determination in early mammalian embryos. Mol Hum Reprod 3:863905[Abstract]
Egozcue J, Blanco J, Vidal F (1997) Chromosome studies in human sperm nuclei using fluorescence in situ hybridization (FISH). Hum Reprod Update 3:441452
Ferraretti AP, Magli MC, Kopcow L, Gianaroli L (2004) Prognostic role of preimplantation genetic diagnosis for aneuploidy in assisted reproductive technology outcome. Hum Reprod 19:694699
Gianaroli L, Magli MC, Ferraretti AP, Munné S (1999) Preimplantation diagnosis for aneuploidies in patients undergoing in vitro fertilization with a poor prognosis: identification of the categories for which it should be proposed. Fertil Steril 72:837844[CrossRef][Medline]
Gianaroli L, Magli MC, Ferraretti AP, Fortini D, Tabanelli C, Gergolet M (2000a) Gonadal activity and chromosomal constitution of in vitro generated embryos. Mol Cell Endocrinol 161:111116[CrossRef][Medline]
Gianaroli L, Magli MC, Ferraretti AP, Iammarrone E (2000b) Preimplantation diagnosis after assisted reproduction techniques for genetically-determined male infertility. J Endocrinol Invest 23:711716[Medline]
Gianaroli L, Magli MC, Ferraretti AP, Tabanelli C, Trombetta C, Boudjema E (2002) The role of preimplantation diagnosis for aneuploidies. Reprod BioMed Online 4:3136[Medline]
Gianaroli L, Magli MC, Fiorentino F, Baldi M, Ferraretti AP (2003) Clinical value of preimplantation genetic diagnosis. Placenta 24:7783[CrossRef][Medline]
In't Veld PA, Broekmans FJ, de France HF, Pearson PL, Pieters MH, van Kooij RJ (1997) Intracytoplasmic sperm injection (ICSI) and chromosomally abnormal spermatozoa. Hum Reprod 12:752754[Abstract]
Koehler KE, Hawley RS, Sherman S, Hassold T (1996) Recombination and non-disjunction in humans and flies. Hum Mol Genet 5:14951504[Abstract]
Magli MC, Jones GM, Gras L, Gianaroli L, Korman L, Trounson AO (2000) Chromosome mosaicism in day 3 aneuploid embryos that develop to morphologically normal blastocysts in vitro. Hum Reprod 15:17811786
Magli MC, Gianaroli L, Ferraretti AP (2001) Chromosomal abnormalities in embryos. Mol Cell Endocrinol 183:2934[CrossRef][Medline]
Magli MC, Gianaroli L, Ferraretti AP, Toschi M, Esposito F, Fasolino MC (2004) The combination of polar body and embryo biopsy does not affect embryo viability. Hum Reprod 19:11631169
Munné S, Magli C, Cohen J, Morton P, Sadowy S, Gianaroli L, Tucker M, et al. (1999) Positive outcome after preimplantation diagnosis of human embryos. Hum Reprod 14:21912199
Munné S (2002) Preimplantation genetic diagnosis of numerical and structural chromosomal abnormalities. Reprod Biomed Online 4:183196[Medline]
Munné S, Bahçe M, Sandalinas M, Escudero T, Márquez C, Velilla E, Colls P, et al. (2004) Differences in chromosome susceptibility to aneuploidy and survival to first trimester. Reprod Biomed Online 8:8190[Medline]
Palermo GP, Joris H, Devroey P, Van Steirteghem AC (1992) Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 340:1718[CrossRef][Medline]
Pang MG, Hoegerman SF, Cuticchia AJ, Moony SY, Doncel GF, Acosta AA, Kearns WG (1999) Detection of aneuploidy for chromosomes 4, 6, 7, 8, 9, 10, 11, 12, 13, 17, 18, 21, X and Y by fluorescence in-situ hybridization in spermatozoa from nine patients with oligoasthenoteratozoospermia undergoing intracytoplasmic sperm injection. Hum Reprod 14:12661273
Schatten G (1994) The centrosome and its mode of inheritance: the reduction of the centrosome during gametogenesis and its restoration during fertilization. Dev Biol 165:299335[CrossRef][Medline]
Silber S, Escudero T, Lenahan K, Abdelhadi I, Kilani Z, Munne S (2003) Chromosomal abnormalities in embryos derived from testicular sperm extraction. Fertil Steril 79:3038[CrossRef][Medline]