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Practical considerations of embryo manipulation: preimplantation genetic typing
ELSEVIER
PRACTICAL CONSIDERATIONS OF EMBRYO MANIPULATION: PREIMPLANTATION GENETIC TYPING J. F. Garcia Department of Aniil
Health and Production, %o Paul0 State University, UNESP Aracatuba, Brazil ABSTRACT
In the past years, research in embryo technologies is moving to the establishment of preimplantation genetic typing or also denominated preimplantation genetic diagnosis (PGD). The objectives of these tests are the prevention of genetic diseases transmission and the prediction of phenotypic characteristics, as well as sex determination, genetic disorders and productive and reproductive profiles, prior to the embryo transfer or freezing, during early stages of development. This paper points out the state-of-the-art of PGD, mainly in cattle and discuss the perspectives of multiloci genetic analysis of embryos. Q2001 by Elsevier Science Inc. Key words: embryo sexing, PEP-PCR, PGD, bovine, cattle
INTRODUCTION Routine use of artificial insemination in cattle during the last decades enables development and application of new biotechniques for animal production improvement, in special embryo transfer related to procedures which allowed the development of parallel techniques as in vitro fertilization (IVF) and embryo cloning. Advances in DNA technologies have opened several perspectives for genetic selection of domestic species, aiming the improvement of productivity. In the last years, research in embryo technologies is moving to the establishment of preimplantation genetic typing or also denominated preimplantation genetic diagnosis (PGD). The objectives of these tests are the prevention of genetic diseases transmission and the prediction of phenotypic characteristics, as well as sex determination, genetic disorders and productive and reproductive profiles, prior to the embryo transfer or heezing, during early stages of development (12).
THE BEGINNING: EMBRYO SEXING Embryo sexing was the first, most widely spread and so far, the most important application of PGD in domestic animals. The idea of predetermining the sex of livestock has been the aim of several groups in the last decades. It is possible to reach this goal by the separation of Y Acknowledgments: Prof. Dr. J. A. Visintin and DVM P. Hassun Filho, S. Zapata, M. Nogueira, F. Pupim, B. Barros, A. Castro and S. Uvo. Theriogenology 56: 1393-t 399, 2001 Q 2001 Elsevier Science Inc.
0093-691WOl/$-see front matter PII: SOOSS-691X(01)00642-2
Theriogenology
chromosome carrying spermatozoa or by embryo analysis (17). Embryo sexing procedures can be divided into invasive and noninvasive protocols, based on the zona pellucida penetration by glass needle, biopsy pipette or microblade during embryo manipulation for biopsy collection. Enzymatic determination (27) and immunological approach (1) in spite of their noninvasive characteristics, has not yet proven effective in embryo sex determination for commercial purposes. On the other hand, invasive methods have become more common (28). Besides this, the development of polymerase chain reaction - PCR (21) made DNA amplification from small amount of template possible (sometimes a single cell genome). Several papers were published on this matter, using different bovine Y chromosome derived DNA sequences (23, 18, 16, 10, 7, 5, 19, 26, 20, 13, 15). Recently, embryo sexing based on PCR methodology is becoming more common for commercial purposes (25,4,8,24). Acceptable when analyzed all same paper the cryopreservation, freezing (25).
pregnancy rates (453%) in PCR sexed embryos (n = 833) were described biopsy systems in conjunction (microblade, needle and glass pipette). In the authors compare blastomere aspiration and microblade biopsy before suggesting the feasibility of using blastomere aspiration prior to embryo
Trying to make the application of bovine embryo PCR sexing in field conditions easier, a simplified the protocol was developed by performing the biopsy procedures without micromanipulators (but using microblade) and by detecting the Y chromosome DNA amplification directly from the test tube containing ethidium bromide, eliminating the electrophoresis step (4). This paper presents the standardization results of this technique using experimental embryos (not transferred to recipients), only suggesting the possible application of this new protocol in field conditions. Recent papers from the same group are reinforcing the use of this protocol under field conditions (2, 3.) Massive work was conducted between 1992 and 1997, when 4183 bovine embryos were biopsied using aspiration, microblade and needle methods. Several analyses were performed involving comparisons related to year, season, month, number of flushes per cow, number of corpora lutea, bull, donor, embryo stage, grade,‘freezing procedure and biopsy method (24). The most relevant data on this paper are the pregnancy rates after the freezing of aspirated or needle biopsied embryos (33 and 41% respectively), and the high rate of correct sex determination (93%). These values were considered good enough for the commercial application of this technique. Basically, it is possible to extract from the literature, and also from the commercial initiatives in this area, that the results are not good enough when the freezing of microblade biopsied embryos is necessary, even using different freezing protocols. Embryo freezing prior to transfer is obligatory in the majority of the ET systems around the world, since the costs for maintenance of a high number of recipients is prohibitive. Previous reports from our group (8) described pregnancy rate of 45.8% after the transfer of biopsied embryos frozen using one-step protocol (ethylene glycol) and also 91% of correct sex determination.
Theriogenology
1395
In the past years we have been working in answering the crescent claim for embryo sexing by PCR in Brazil. Comparing the advantages and disadvantages of each biopsy method available (microblade or aspiration) and different PCR systems, we decided to work with blastomere aspiration and amplification of a repetitive Y chromosome DNA sequence followed by electrophoresis. Using this approach we are able to determine the male embryos or conclude for a “presumptive female” diagnosis. The reason for that is the nonutilization of a control autossomal region, which could discriminate the male and female biopsies with higher accuracy. In contrast with other available tests (for example the YCD from the US company AB Technology) that use an autosomal control, the system we are applying does not use this strategy. The results from the last 24 months show us that the methodology we chose can be applied under field conditions (Table 1 and 2). The strategies for implanting this technology in the field conditions are: i) the f&zing of all embryos (selected as Grade 1 or 2 - IETS) after biopsy procedure (which explains the option for aspiration), ii) do not use autossomal control in order to make the PCR reaction easy (we are accepting a 14% misdiagnosis in “presumptive ternales” and a 7% misdiagnosis in male), iii) work in large programs for bull production or dairy herd (at least 300 embryo transfers per year), iv) reach a feasible cost for the test (about US% 20.00 per test), v) create operational facilities to the practitioners that are performing the biopsy and embryo freezing (shipment of biopsies, PCR testing and results mailing), and vi) recommend micromanipulation system presenting a reasonable cost/benefit relation. With the rapid global changes in cattle market we consider the implantation of embryo sexing systems absolutely necessary in two circumstances: i) intensive dairy production where ET is applied routinely and ii) elite beef herds where the cows have progressively lower value meanwhile the bulls market is always growing.
Table 1. Pregnancy rates and PCR sexing results obtained from April 1999 to March 2001, Total .._.I, _I. ___1-_Aspiration biopsied embryos prior fieezing Nonbiopsied embryos prior freezing ..---
512
No. Pregnancies 234
560
250
Pregnancy Rates ‘.
45.70% 44.64%
Table 2. General data of PCR sexing obtained from April 1999 to March 200 1, for the embryos already transferred and with ultrasound (US) sexing or calving.
kale diagnosed embryos “Presumptive female” diagnosed embryos Total analyzed embryos
No. Tested Embryos 79 54 133
Confirmed Results by US or Calving 74 41 115
Rate of Correct Sexing
93.7% 75.9% 86.5%
Theriogenology
1396
THE FUTURE: MULTILOCUS GENETIC TYPING The large amount of data provided by the almost finished “genome projects” (human, mouse, rat) will lead us to several molecular markers to be applied in livestock industry, in special coupled to the embryo technology. A limiting factor to conventional PCR strategy for PGD is the possibility of only one round of diagnosis per biopsy since all biopsy material is consumed in each test. The improvement of new molecular biology techniques is demonstrating the feasibility of analyzing more than one locus through the DNA amplification of a single cell (9). The feasibility of analysis of different loci from a single human embryonic cell was reported, coupling PCR procedure to a technique denominated primer extension preamplification (PEP), that consists of the exponential DNA amplification using 1Smer random oligonucleotides, prior to the gene specific PCR (30). Figure 1 illustrates the potential of PEP-PCR for PGD. Limphoblasts from Duchenne’s muscular distrophy patient were individually placed into tubes and submitted to PEP-PCR. After preamplification step the solution was divided in 6 aliquots, and each of them submitted to a specific PCR. Five exons of muscular distrophy gene and ZFX/ZFY loci were analyzed. The same procedure was tested with amniocytes, chorionic villus cells and blastomeres, reaching 93% of correct results and indicating perspectives for the use of PEP-PCR for multiple PGD in embryos (14). It was considered that the amount of DNA present in an embryo biopsy could be used for a limited number of loci analysis using a single multiplex PCR and that several repetitions should be conducted in order to avoid the random mismatches that occurs in conventional PCR. To increase the number of loci analyzed per embryo it was employed PEP-PCR in one fourth of each bovine embryo, showing the feasibility of multiple analysis of kappa casein, bovine leucocyte adhesion deficiency (BLAD) and microsatellite D9S1, and sex-determining loci BRY. 1, Bov97M and ZFWZFY. The conclusion was that PEP allows the analysis of approximately 40 genomic regions per quarter embryo and that the combined use of Bov97M and ZFX/ZFY offers high fidelity in bovine embryo sexing routine (11). PEP-PCR followed by nested-PCR for human amelogenin (AMGX and AMGY) and cystic fibrosis (AF508) was reported, presenting 93% of sensibility and 100% of correct diagnosis for amelogenin and 95% correct diagnosis for cystic fibrosis, showing the importance of this PGD in X chromosome-linked recessive genetic disorders (22). SRY, HPRT e DNXds3 loci were tested in mice leucocyte DNA and embryo biopsies, revealing a lo-fold increase in sensitivity of PCR afler previous PEP procedure, indicating this technique as a good alternative for multiloci diagnosis (29). A recent paper described a procedure for multiple genotype analysis (determination of sex and three genetic markers) from a single cell derived Iroombovine preimplantation embryo, using PEP-PCR. Efficiency of genotyping by standard PCR for kappa casein, growth hormone (GH) and prolactin (PRL) polymorphic alleles was 91, 88 and 89%, respectively. Sexing
1397
Theriogenology
efficiency in the multiplex PCR was 91%, based on the amplification of Y-specific locus using kappa casein internal standard (6). In the near fitture, the results of large-scale genome projects will provide new molecular markers to be applied for genotyping either in animal breeding programs or early stage embryo PGD. Better understanding and development of the techniques for multiloci PGD will be essential when new markers become available.
PCR => LHR
GnRHR
BRY.4a
’
”
Mcmsate6les
Electrophwetogram in automated DNA snakier
Electrophoresis
I
144
2b2
1
Figure 1. General overview of PEP-PCR protocol in preimplantation genetic diagnosis. The strategy allows the determination of several genetic characteristics from biopsies containmg small amount of cells.
1398
Theriogenology
REFERENCES 1. ANDERSON, G. B. Identification of embryonic sex by detection of H-Y antigen. Theriogenology, 1987,21:12-28. 2. BREDBACKA, P. Recent developments in embryo sexing and its field application. Reprod. Nutr. Dev. 1998; 38:605-13 3. .BREDBACKA, P.; PEIPPO. J.; JAAKMA. U. A simplified protocol for PCR-sexing of bovine embryos: A field trial. Theriogenology, 1996; 45218. .. .. 4. BREDBACKA, P., KANKAANPAA, A.; PEIPPO, J. PCR-sexing of bovine embryos: A simplified protocol. Theriogenology, 1995; 44:167-76. 5. BREDBACKA, P; BREDBACKA, K, PEIPPO, .I. Experiences of using PCR for sexing bovine embryos. Reproduction of Domestic Animals, 1991; 26;76-7. 6. CHRENEK, P, BOULANGER, L, HEYMAN, Y, UHRIN, P, LAURINCIK, J, BULLA, J, RENARD, J.P. Sexing and multiple genotype analysis from a single cell of bovine embryo. Theriogenology 2001; 55:1071-81. 7. COTINOT, C.; KIRSZENBAUM, M.; LEONARD, M.; GIANQUINTO, L. VAIMAN, M. Isolation of bovine Y-derived sequence: potential use in embryo sexing. Genomics, 1991; 10:646-53. 8. GARCIA, J. F.; NOGUEIRA, M. F. G.; PUPIM, F. P. V.; PANTANO, T.; VISINTIN, J. A, Pregnancy rates of blastomere biopsied bovine embryos hozen in ethylene glycol. Theriogenology, 1997; 47:268. 9. HASSUN, P. A.; MELLO, M. R. B.; PORTO, L. P. C.; GARCIA, J. F. Bovine embryo sexing by primer extension preamplification - polymerase chain reaction (PEP-PCR). Theriogenology 1999; 51:398. 10. HERR, C. M.; HOLT, N. A.; MATTHAEI, K. I.; REED, K. C. Sex of progeny horn bovine embryos sexed with a rapid Y-chromosome detection assay. Theriogenology, 1990; 33:247. 11. HOCHMAN, D.; ZARON, Y.; DEKEL, I.; FELDMESSER, E.; MEDRANO, J. F.; SHANI, M.; RON, M. Multiple genotype analysis and sexing of IVF bovine embryos. Theriogenology, 1995; 7:1063-75. 12. JARRELL, V. L. Featured article: predicting genotypes of oocytes, zygotes and preimplantation embryos. Embryo Transfer Newsletter 1994; 12:2. 13. KIRKPATRICK, B. W.; MONSON, R. L. Sensitive sex determination assay applicable to bovine embryos derived from IVM and IVF. Journal of Reproduction and Fertility 1993; 98:225-40. 14. KRISTJANSSON, K.; CHONG, S. S.; VEYVER., V.; SUBRAMANIAN, S.; SNABES, M. C.; HUGHES, M. R Preimplantation single cell analyses of distrophm gene deletions using whole genome amplification. Nature Genetics 1994; 6: 19-23. 15. MACHATY, Z. PALDI, A.; CSkI, T.; VARGA, Z.; KISS, I.; BzkRANDI, Z.; VAJTA, G. Biopsy and sex determination by PCR of IVF bovine embryos. Journal of Reproduction and Fertility 1993; 98:467-70. 16. MATTHEWS, M. E Repeated DNA sequences from the bovine Y-chromosome. Australia 1990. Thesis (Ph.D.) - Australian National University. 17. McEVOY, J. D. Alteration of sex ratio. Animal Breeding Abstracts, 1992; 60:97- 111. 18. MILLER, J. R.; KOOPMAN, M. Isolation and characterization of two male-specific DNA tiagments from the bovine gene. Animal Genetics 1990; 21:77-82.
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19. PEURA, T.; HYTTINEN, J. M.; TURUNEN, M.; JANNE, J. A reliable sex determination assay for bovine preimplantation embryos by the polymerase chain reaction. Theriogenology 1991,35:547-55. 20. POLLEVICK, G. D.; GIAMBIAGI, S.; MANCARDI, S.; DE LUCA, L.; BURRONE, 0.; FRASCH, A. C. C.; UGALDE, R. Sex determination of bovine embryo by restriction fragment polymorphism of PCR amplified ZEWZFY loci. Bio/Technology 1992; 10:805-7. 21. SAIKI, R. K.; GELFAND, D. H.; STOFFEL, S.; HIGUCHI, R.; HORN, G. T.; MULLIS, K. B; ERLICH, H. A. Primer-directed enzymatic amplification of DNA with a termostable DNA polymerase. Science 1988; 239:487-91. 22. SCHAAFF, F.; WEDEMANN, H.; SCHWINGER, E. Analysis of sex and AF508 in single amniocytes using primer extension preamplification. Human Genetic 1996; 98: 158-6 1. 23. SCHROEDER A.; MILLER, J. R.; THOMSEN, P. D.; ROSCHLAUD, K.; AVERY, B.; POULSEN, P. H.; SCHMIDT, M.; SCHWERIN, M. Sex determination of bovine embryos using the polymerase chain reaction. Animal Biotechnology, v.1, n. 1-2, p.121-33, 1990. 24. SHEA, B. F. Determinin g the sex of bovine embryos using polymerase chain reaction results: A six-year retrospective study. Theriogenology 1999; 5 1:841-54. 25. THIBIER, M.; NIBART, M. The sexing of bovine embryos in the field. Theriogenology 1995; 43:71-80. 26. UTSUMI, K.; KAWAMOTO, T.; KIM, J. H.; TAKEDA, H.; IRITANI, A. Sexing of bovine embryos by polymerase chain reaction using Y-specific DNA as primer. In: International Congress on Animal Reproduction, 12, The Hague. Proceedings, 1992, p.757-9. 27. WILLIAMS. T. J. A technique for sexing mouse embryos by a visual calorimetric assay of the X-linked enzyme glucose 6 phosphate dehydrogenase. Theriogenology 1986; 25:733-9. 28. YANG, X.; ANDERSON, G. B. Micromanipulation of mammalian embryos: principles, progress and future possibilities. Theriogenology 1992; 38:3 15-35. 29. ZAPATA, S. T. Micromanipula@o e diagnostic0 genetico pre-implantacional em embrioes murinos pela tecnica de PEP-PCR (“Primer extension preamplitication- polymerase chain reaction”). Sb Paulo, 1998. (Mestrado) - Faculdade de Medicina Veterinkia e Zootecnia, Universidade de Sao Paulo. 30. ZHANG, L.; CUI, X.; SCHIMITT, K.; HUBERT, R.; NAVIDI, W.; ARNHEIM, N. Whole genome amplification from a single cell: implication for genetic analysis. Proceedings of the National Academy of Science of the United States of America 1992; 89:5847-51.
PRACTICAL CONSIDERATIONS OF EMBRYO MANIPULATION: PREIMPLANTATION GENETIC TYPING J. F. Garcia Department of Aniil
Health and Production, %o Paul0 State University, UNESP Aracatuba, Brazil ABSTRACT
In the past years, research in embryo technologies is moving to the establishment of preimplantation genetic typing or also denominated preimplantation genetic diagnosis (PGD). The objectives of these tests are the prevention of genetic diseases transmission and the prediction of phenotypic characteristics, as well as sex determination, genetic disorders and productive and reproductive profiles, prior to the embryo transfer or freezing, during early stages of development. This paper points out the state-of-the-art of PGD, mainly in cattle and discuss the perspectives of multiloci genetic analysis of embryos. Q2001 by Elsevier Science Inc. Key words: embryo sexing, PEP-PCR, PGD, bovine, cattle
INTRODUCTION Routine use of artificial insemination in cattle during the last decades enables development and application of new biotechniques for animal production improvement, in special embryo transfer related to procedures which allowed the development of parallel techniques as in vitro fertilization (IVF) and embryo cloning. Advances in DNA technologies have opened several perspectives for genetic selection of domestic species, aiming the improvement of productivity. In the last years, research in embryo technologies is moving to the establishment of preimplantation genetic typing or also denominated preimplantation genetic diagnosis (PGD). The objectives of these tests are the prevention of genetic diseases transmission and the prediction of phenotypic characteristics, as well as sex determination, genetic disorders and productive and reproductive profiles, prior to the embryo transfer or heezing, during early stages of development (12).
THE BEGINNING: EMBRYO SEXING Embryo sexing was the first, most widely spread and so far, the most important application of PGD in domestic animals. The idea of predetermining the sex of livestock has been the aim of several groups in the last decades. It is possible to reach this goal by the separation of Y Acknowledgments: Prof. Dr. J. A. Visintin and DVM P. Hassun Filho, S. Zapata, M. Nogueira, F. Pupim, B. Barros, A. Castro and S. Uvo. Theriogenology 56: 1393-t 399, 2001 Q 2001 Elsevier Science Inc.
0093-691WOl/$-see front matter PII: SOOSS-691X(01)00642-2
Theriogenology
chromosome carrying spermatozoa or by embryo analysis (17). Embryo sexing procedures can be divided into invasive and noninvasive protocols, based on the zona pellucida penetration by glass needle, biopsy pipette or microblade during embryo manipulation for biopsy collection. Enzymatic determination (27) and immunological approach (1) in spite of their noninvasive characteristics, has not yet proven effective in embryo sex determination for commercial purposes. On the other hand, invasive methods have become more common (28). Besides this, the development of polymerase chain reaction - PCR (21) made DNA amplification from small amount of template possible (sometimes a single cell genome). Several papers were published on this matter, using different bovine Y chromosome derived DNA sequences (23, 18, 16, 10, 7, 5, 19, 26, 20, 13, 15). Recently, embryo sexing based on PCR methodology is becoming more common for commercial purposes (25,4,8,24). Acceptable when analyzed all same paper the cryopreservation, freezing (25).
pregnancy rates (453%) in PCR sexed embryos (n = 833) were described biopsy systems in conjunction (microblade, needle and glass pipette). In the authors compare blastomere aspiration and microblade biopsy before suggesting the feasibility of using blastomere aspiration prior to embryo
Trying to make the application of bovine embryo PCR sexing in field conditions easier, a simplified the protocol was developed by performing the biopsy procedures without micromanipulators (but using microblade) and by detecting the Y chromosome DNA amplification directly from the test tube containing ethidium bromide, eliminating the electrophoresis step (4). This paper presents the standardization results of this technique using experimental embryos (not transferred to recipients), only suggesting the possible application of this new protocol in field conditions. Recent papers from the same group are reinforcing the use of this protocol under field conditions (2, 3.) Massive work was conducted between 1992 and 1997, when 4183 bovine embryos were biopsied using aspiration, microblade and needle methods. Several analyses were performed involving comparisons related to year, season, month, number of flushes per cow, number of corpora lutea, bull, donor, embryo stage, grade,‘freezing procedure and biopsy method (24). The most relevant data on this paper are the pregnancy rates after the freezing of aspirated or needle biopsied embryos (33 and 41% respectively), and the high rate of correct sex determination (93%). These values were considered good enough for the commercial application of this technique. Basically, it is possible to extract from the literature, and also from the commercial initiatives in this area, that the results are not good enough when the freezing of microblade biopsied embryos is necessary, even using different freezing protocols. Embryo freezing prior to transfer is obligatory in the majority of the ET systems around the world, since the costs for maintenance of a high number of recipients is prohibitive. Previous reports from our group (8) described pregnancy rate of 45.8% after the transfer of biopsied embryos frozen using one-step protocol (ethylene glycol) and also 91% of correct sex determination.
Theriogenology
1395
In the past years we have been working in answering the crescent claim for embryo sexing by PCR in Brazil. Comparing the advantages and disadvantages of each biopsy method available (microblade or aspiration) and different PCR systems, we decided to work with blastomere aspiration and amplification of a repetitive Y chromosome DNA sequence followed by electrophoresis. Using this approach we are able to determine the male embryos or conclude for a “presumptive female” diagnosis. The reason for that is the nonutilization of a control autossomal region, which could discriminate the male and female biopsies with higher accuracy. In contrast with other available tests (for example the YCD from the US company AB Technology) that use an autosomal control, the system we are applying does not use this strategy. The results from the last 24 months show us that the methodology we chose can be applied under field conditions (Table 1 and 2). The strategies for implanting this technology in the field conditions are: i) the f&zing of all embryos (selected as Grade 1 or 2 - IETS) after biopsy procedure (which explains the option for aspiration), ii) do not use autossomal control in order to make the PCR reaction easy (we are accepting a 14% misdiagnosis in “presumptive ternales” and a 7% misdiagnosis in male), iii) work in large programs for bull production or dairy herd (at least 300 embryo transfers per year), iv) reach a feasible cost for the test (about US% 20.00 per test), v) create operational facilities to the practitioners that are performing the biopsy and embryo freezing (shipment of biopsies, PCR testing and results mailing), and vi) recommend micromanipulation system presenting a reasonable cost/benefit relation. With the rapid global changes in cattle market we consider the implantation of embryo sexing systems absolutely necessary in two circumstances: i) intensive dairy production where ET is applied routinely and ii) elite beef herds where the cows have progressively lower value meanwhile the bulls market is always growing.
Table 1. Pregnancy rates and PCR sexing results obtained from April 1999 to March 2001, Total .._.I, _I. ___1-_Aspiration biopsied embryos prior fieezing Nonbiopsied embryos prior freezing ..---
512
No. Pregnancies 234
560
250
Pregnancy Rates ‘.
45.70% 44.64%
Table 2. General data of PCR sexing obtained from April 1999 to March 200 1, for the embryos already transferred and with ultrasound (US) sexing or calving.
kale diagnosed embryos “Presumptive female” diagnosed embryos Total analyzed embryos
No. Tested Embryos 79 54 133
Confirmed Results by US or Calving 74 41 115
Rate of Correct Sexing
93.7% 75.9% 86.5%
Theriogenology
1396
THE FUTURE: MULTILOCUS GENETIC TYPING The large amount of data provided by the almost finished “genome projects” (human, mouse, rat) will lead us to several molecular markers to be applied in livestock industry, in special coupled to the embryo technology. A limiting factor to conventional PCR strategy for PGD is the possibility of only one round of diagnosis per biopsy since all biopsy material is consumed in each test. The improvement of new molecular biology techniques is demonstrating the feasibility of analyzing more than one locus through the DNA amplification of a single cell (9). The feasibility of analysis of different loci from a single human embryonic cell was reported, coupling PCR procedure to a technique denominated primer extension preamplification (PEP), that consists of the exponential DNA amplification using 1Smer random oligonucleotides, prior to the gene specific PCR (30). Figure 1 illustrates the potential of PEP-PCR for PGD. Limphoblasts from Duchenne’s muscular distrophy patient were individually placed into tubes and submitted to PEP-PCR. After preamplification step the solution was divided in 6 aliquots, and each of them submitted to a specific PCR. Five exons of muscular distrophy gene and ZFX/ZFY loci were analyzed. The same procedure was tested with amniocytes, chorionic villus cells and blastomeres, reaching 93% of correct results and indicating perspectives for the use of PEP-PCR for multiple PGD in embryos (14). It was considered that the amount of DNA present in an embryo biopsy could be used for a limited number of loci analysis using a single multiplex PCR and that several repetitions should be conducted in order to avoid the random mismatches that occurs in conventional PCR. To increase the number of loci analyzed per embryo it was employed PEP-PCR in one fourth of each bovine embryo, showing the feasibility of multiple analysis of kappa casein, bovine leucocyte adhesion deficiency (BLAD) and microsatellite D9S1, and sex-determining loci BRY. 1, Bov97M and ZFWZFY. The conclusion was that PEP allows the analysis of approximately 40 genomic regions per quarter embryo and that the combined use of Bov97M and ZFX/ZFY offers high fidelity in bovine embryo sexing routine (11). PEP-PCR followed by nested-PCR for human amelogenin (AMGX and AMGY) and cystic fibrosis (AF508) was reported, presenting 93% of sensibility and 100% of correct diagnosis for amelogenin and 95% correct diagnosis for cystic fibrosis, showing the importance of this PGD in X chromosome-linked recessive genetic disorders (22). SRY, HPRT e DNXds3 loci were tested in mice leucocyte DNA and embryo biopsies, revealing a lo-fold increase in sensitivity of PCR afler previous PEP procedure, indicating this technique as a good alternative for multiloci diagnosis (29). A recent paper described a procedure for multiple genotype analysis (determination of sex and three genetic markers) from a single cell derived Iroombovine preimplantation embryo, using PEP-PCR. Efficiency of genotyping by standard PCR for kappa casein, growth hormone (GH) and prolactin (PRL) polymorphic alleles was 91, 88 and 89%, respectively. Sexing
1397
Theriogenology
efficiency in the multiplex PCR was 91%, based on the amplification of Y-specific locus using kappa casein internal standard (6). In the near fitture, the results of large-scale genome projects will provide new molecular markers to be applied for genotyping either in animal breeding programs or early stage embryo PGD. Better understanding and development of the techniques for multiloci PGD will be essential when new markers become available.
PCR => LHR
GnRHR
BRY.4a
’
”
Mcmsate6les
Electrophwetogram in automated DNA snakier
Electrophoresis
I
144
2b2
1
Figure 1. General overview of PEP-PCR protocol in preimplantation genetic diagnosis. The strategy allows the determination of several genetic characteristics from biopsies containmg small amount of cells.
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