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Preembryonic diagnosis for sickle cell disease
Molecular and Cellular Endocrinology 183 (2001) S19– S22 www.elsevier.com/locate/mce
Preembryonic diagnosis for sickle cell disease Anver Kuliev *, Svetlana Rechitsky, Oleg Verlinsky, Charles Strom, Yury Verlinsky Reproducti6e Genetics Institute, Chicago, IL 60657, USA
Abstract Embryos found to be abnormal during preimplantation genetic diagnosis are discarded or analyzed to confirm the diagnosis. The destruction of affected embryos is ethically unacceptable to some couples. We developed a preembryonic genetic diagnosis, that uses sequential first and second polar body removal, followed by oocyte freezing at the pronuclear stage. This was applied in a patient at risk of having a child with sickle cell disease, who suffered hyper-stimulation syndrome. Fourteen oocytes were obtained and tested for the maternal sickle cell allele by PCR analysis of the first and second polar body. Immediately after procedure of polar body removal, the pronuclear-stage oocytes were frozen. Six mutation-free oocytes detected by polar body analysis were then thawed, allowed to cleave, and transferred in the two consecutive clinical cycles, both resulting in clinical pregnancies, one of which resulted in birth of a healthy child. The oocytes predicted to contain abnormal b-globin gene were not further cultured, to avoid formation and discard of the affected embryos. The results demonstrate feasibility of preembryonic diagnosis for single gene disorders, avoiding the establishment and destruction of mutant embryos. © 2001 Published by Elsevier Science Ireland Ltd. Keywords: First and second polar body; Multiplex nested PCR; Preembryonic diagnosis; Preimplantation genetic diagnosis; Sickle cell disease
1. Introduction Preimplantation genetic diagnosis (PGD) has been used for several hundred patients at risk of having children with genetic and chromosomal disorders. Two different approaches have been used; including cleavage-stage embryo biopsy and polar body (PB) sampling, both of which have resulted in the births of unaffected children (IWGPG, 1998). In cleavage-stage embryo biopsy, unaffected embryos are selected for transfer or frozen for subsequent transfer. Affected embryos may be discarded, frozen indefinitely, or destroyed during diagnostic confirmation. In the standard sequential PB approach, the first PB (PB1) is removed prior to fertilization, the second PB (PB2) is removed after fertilization and all fertilized embryos are cultured pending the availability of the genotype results. Therefore, both affected and unaffected oocytes are fertilized and developed to a cleavage-stage, so the same situation occurs as in blastomere biopsy, where embryos have been established that are later discovered to be affected. * Corresponding author.
The destruction or indefinite frozen storage of affected embryos is ethically unacceptable to some groups and couples on ethical and/or religious grounds. For these couples, there is a need for an ethically more palatable technique. We developed a method to complete the PB1 and PB2 removal prior to the fusion of the male and female pronucleus, followed by freezing all oocytes in the pronuclear phase. After analysis, in a subsequent menstrual cycle, only the oocytes predicted to have inherited the normal maternal allele are thawed and cultured to allow fusion of the pronuclei, embryo development, and transfer. Since zygotes prior to pronuclear fusion are not yet considered embryos and no abnormal oocytes are thawed and cultured, no affected embryos are established, causing this technique to be ethically more acceptable to many couples. Therefore, this technique creates a new class of genetic diagnosis, preembryonic genetic diagnosis, which pushes the frontier of genotyping to an even earlier stage. The present paper describes the case of preembryonic diagnosis performed for sickle cell disease, resulting in two unaffected singleton pregnancies and the birth of a healthy child.
0303-7207/01/$ - see front matter © 2001 Published by Elsevier Science Ireland Ltd. PII: S 0 3 0 3 - 7 2 0 7 ( 0 1 ) 0 0 5 6 9 - X
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A. Kulie6 et al. / Molecular and Cellular Endocrinology 183 (2001) S19–S22
2. Material and methods A 33-year old women and spouse at risk for the birth of a child with sickle cell disease were referred for PGD to avoid a possible termination of a pregnancy following prenatal diagnosis. A standard IVF protocol was initiated but the patient suffered hyper-stimulation syndrome, which precluded transfer of embryos in the current cycle. Twenty-eight mature oocytes were aspirated and placed in culture medium. Of the 28 aspirated oocytes, 14 extruded PB1 that were removed by micromanipulation as described elsewhere (Verlinsky and Kuliev, 1993). The oocytes were then fertilized by intracytoplasmic sperm injection. As soon as the PB2 was removed, and prior to the fusion of the male and female pronuclei, all embryos were frozen. The PB1 and PB2 were analyzed by multiplex nested PCR as described elsewhere (Rechitsky et al., 1998). Genotyping was performed using multiplex PCR because the most important source of diagnostic error in PGD occurs when only one allele of a heterozygous cell amplifies in a process known as allele-drop out (ADO). This occurs in approximately 5– 10% of PB analyses in the beta globin gene (Rechitsky et al., 1998). We developed a technique to detect ADO to prevent misdiagnosis. This involves nested, multiplex PCR with completely separate primer sets for the sickle cell mutation and two linked short tandem repeats (STR) markers; one located at the 5% end of the b-globin gene (5% STR) and the other in the human thyrosin hydroxylase gene (THO-STR). The mother was heterozygous for both linked markers and the phase of the linkage is known by family analysis using her affected child. To detect potential contamination with extraneous DNA and identify the embryo that implanted and established pregnancy, three additional non-linked STRs were amplified, including STR at the 5% untranslated region of human coagulation factor A subunit gene (HUMF13A01), STR for Von Willibrands Disease (vWF), and an STR for chromosome 21 (D21S11). Primers were synthesized on an Applied Biosystems 381 A Synthesizer. The list of primer sequences, reaction conditions and details of the nested PCR have been described earlier (Rechitsky et al., 1998; Kuliev et al., 1998). In brief, the first round PCR (25 cycles) was performed with a mixture of outside primers for bglobin gene and STRs, and then the resulting PCR products were aliquotted and amplified separately with inside primers for each individual locus in the second PCR round. For sickle cell mutation analysis, the resulting PCR products were restriction digested for 3 h overnight with DdeI (Promega) under the conditions recommended by the manufacturer, followed by polyacrylamide gel electrophoresis (PAGE) as described elsewhere (Rechitsky et al., 1998). For STR fragment
analyses, the PCR products were analyzed by PAGE alone. The pronuclear-stage oocytes predicted to be normal were thawed, cultured to develop into the cleaving embryos of 6–8 cell stage and transferred back to the patient in the two subsequent clinical cycles. The oocytes predicted to contain the mutant maternal gene were not thawed, but analyzed directly at the pronuclear-stage for the confirmation of PB diagnosis.
3. Results and discussion Of 28 aspirated oocytes, 14 extruded their PB1 and were studied for the presence of sickle cell mutation. Following intracytoplasmic sperm injection, PB2s were extruded from 13 of them and studied. Results of both PB1 and PB2 were available in 12 of these 13 oocytes (Fig. 1). Overall, six oocytes were predicted to contain a normal allele (Fig. 1, oocyte number 3, 5, 9, 10, 13 and 14) based on heterozygous status of PB1 and hemizygous mutant status of PB2. As seen from Fig. 1, a probable case of ADO was detected in oocyte number 6. Although the sickle cell analysis of PB1 showed only the normal allele and the 5%-STR showed only six repeats, it was heterozygous for the THO-STR, suggesting that this is a case of ADO. Therefore, without simultaneous amplification of linked STRs, a misdiagnosis of the heterozygous oocyte as homozygous due to ADO would have occurred leading to a misdiagnosis of the maternal contribution to the zygote. In this particular instance, the error would have caused an unaffected zygote to be misdiagnosed as affected, but the reverse could also occur. Subsequently, the patient was prepared for a frozen embryo transfer. In the first frozen cycle, four zygotes determined to have the maternal unaffected allele were thawed and cultured. Three developed into cleaving embryos of acceptable quality and were transferred (Fig. 1, embryo number 3, 10, 14), resulting in a singleton pregnancy, which was spontaneously aborted (Verlinsky et al., 1999). In the second frozen cycle, two unaffected embryos were transferred (Fig. 1, embryo number 5 and 9) and resulted in a singleton pregnancy and birth of an unaffected child, following confirmation of PB diagnosis by chorionic villus sampling (CVS). The results of the application of non-linked markers allowed not only the exclusion of a possible DNA contamination but also the identification of the embryo (number 9) that implanted yielding a clinical pregnancy. All the remaining oocytes predicted to contain an abnormal gene were not further cultured, but exposed directly to PCR analysis at the pronuclear stage for confirmation of diagnosis as frozen samples, and were shown to be abnormal as predicted by PB analysis.
A. Kulie6 et al. / Molecular and Cellular Endocrinology 183 (2001) S19–S22
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Fig. 1. Diagnostic scheme and sequential polar body analysis for sickle cell disease. Upper panel, schematic representation of beta globin locus and linked markers on chromosome 11p15.5 used for this study. STR, short tandem repeat in 5% untranslated region of b-globin gene. SCA-Dde 1, Dde 1 restriction site at the sickle cell mutation (A T missense mutation). THO-STR, short tandem repeat in thyrosin hydroxylase gene on chromosome 11p15.5. i, intron. Arrows represent primer sets for nested PCR. The lower panels represent series which all include the first polar body (PB1) followed by the second polar body (PB2) in the lane to its immediate right for single zygotes. The oocyte number that appears at the bottom of the figure signifies the oocyte number for all analyses shown. The upper panel represents the Dde1 restriction digestion for the sickle cell mutation genotyping. The middle panel represents the 5% STR analysis. The lower panel represents the THO-STR analysis. ET1, embryos that were thawed and cultured for transfer in the first frozen cycle. ET2, embryos that were thawed and cultured for transfer in the second frozen cycle. Chorionic villus sampling (CVS), genotype of embryo resulting in pregnancy, prenatal diagnosis and birth of unaffected child. ADO, notes analysis in which ADO was detected by linked marker analysis.
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The presented data demonstrate the feasibility of performing preembryonic diagnosis for single gene disorders, which has resulted in obtaining two unaffected pregnancies and the birth of a healthy, unaffected child. PGD by PB1 testing has also been described for the maternally derived translocations (Munne et al., 1998). In our earlier work, we have attempted to perform PGD for single gene disorders by the use of PB1 analysis alone (Verlinsky et al., 1992). Although this allowed pre-selection of a few mutation-free oocytes inferred from the homozygous abnormal status of PB1, the majority of oocytes were heterozygous after the first meiotic division, so the genotype of the resulting embryos could not be predicted, thus limiting the number of normal embryos for transfer. To overcome this problem, a two-step oocyte testing process by sequential analysis of both PB1 and PB2 has been introduced, resulting in pregnancies and births of a number of children free of genetic disorders, including the clinical pregnancy in the case of sickle cell disease (Verlinsky et al., 1997). However, in most of these cases, the oocytes tested by PB analysis were kept in culture, so that they developed into a cleavage stage embryo by the time the genotyping results were obtained. To avoid discarding these preimplantation embryos, we have introduced freezing of oocytes immediately following fertilization and extrusion of PB2 and prior to fusion of the male and female pronuclei, which is considered the beginning of the embryonic period (Larsen, 1994). In fact, freezing may be omitted in the near future as recent developments in PCR analysis would allow com-
pleting the genetic diagnosis before pronuclei fusion. This opens a possibility for application of PGD for those couples who cannot accept any intervention or discarding of the human embryos.
References International Working Group on Preimplantation Genetics, 1998. Preimplantation diagnosis: an alternative to prenatal diagnosis of genetic and chromosomal disorders. Report of the Eight Annual Meeting in Association with International Conference on Prenatal Diagnosis and Therapy, Los Angeles, June 1998. J. Assist. Reprod. Genet. 16 161 – 164. Kuliev, A., Rechitsky, S., Verlinsky, O., et al., 1998. Preimplantation diagnosis of thalassemias. J. Assist. Reprod. Genet. 15, 219 – 225. Larsen, W.J., 1994. Human Embryology, Churchill Livingstone. Munne, S., Morrison, L., Fung, J., et al., 1998. Spontaneous abortions are reduced after preconception diagnosis of translocations. Assist. Reprod. Genet. 15, 290 – 296. Rechitsky, S., Strom, C., Verlinsky, O., et al., 1998. Allele drop-out in polar bodies and blastomeres. J. Assist. Reprod. Genet. 15, 253 – 257. Verlinsky, Y., Kuliev, A. (Eds.), 1993. Preimplantation Diagnosis of Genetic Disorders: a New Technique for Assisted Reproduction. Wiley, New York. Verlinsky, Y., Milayeva, S., Evsikov, S., et al., 1992. Preconception and preimplantation diagnosis for cystic fibrosis. Prenat. Diagn. 12, 103 – 110. Verlinsky, Y., Rechitsky, S., Cieslak, J., et al., 1997. Preimplantation diagnosis of single gene disorders by two-step oocyte analysis using first and second polar body. Biochem. Mol. Med. 62, 182 – 187. Verlinsky, Y., Rechitsky, S., Verlinsky, O., et al., 1999. Prepregnancy testing for single gene disorders by polar body analysis. Genet. Testing 3, 185 – 190.
Preembryonic diagnosis for sickle cell disease Anver Kuliev *, Svetlana Rechitsky, Oleg Verlinsky, Charles Strom, Yury Verlinsky Reproducti6e Genetics Institute, Chicago, IL 60657, USA
Abstract Embryos found to be abnormal during preimplantation genetic diagnosis are discarded or analyzed to confirm the diagnosis. The destruction of affected embryos is ethically unacceptable to some couples. We developed a preembryonic genetic diagnosis, that uses sequential first and second polar body removal, followed by oocyte freezing at the pronuclear stage. This was applied in a patient at risk of having a child with sickle cell disease, who suffered hyper-stimulation syndrome. Fourteen oocytes were obtained and tested for the maternal sickle cell allele by PCR analysis of the first and second polar body. Immediately after procedure of polar body removal, the pronuclear-stage oocytes were frozen. Six mutation-free oocytes detected by polar body analysis were then thawed, allowed to cleave, and transferred in the two consecutive clinical cycles, both resulting in clinical pregnancies, one of which resulted in birth of a healthy child. The oocytes predicted to contain abnormal b-globin gene were not further cultured, to avoid formation and discard of the affected embryos. The results demonstrate feasibility of preembryonic diagnosis for single gene disorders, avoiding the establishment and destruction of mutant embryos. © 2001 Published by Elsevier Science Ireland Ltd. Keywords: First and second polar body; Multiplex nested PCR; Preembryonic diagnosis; Preimplantation genetic diagnosis; Sickle cell disease
1. Introduction Preimplantation genetic diagnosis (PGD) has been used for several hundred patients at risk of having children with genetic and chromosomal disorders. Two different approaches have been used; including cleavage-stage embryo biopsy and polar body (PB) sampling, both of which have resulted in the births of unaffected children (IWGPG, 1998). In cleavage-stage embryo biopsy, unaffected embryos are selected for transfer or frozen for subsequent transfer. Affected embryos may be discarded, frozen indefinitely, or destroyed during diagnostic confirmation. In the standard sequential PB approach, the first PB (PB1) is removed prior to fertilization, the second PB (PB2) is removed after fertilization and all fertilized embryos are cultured pending the availability of the genotype results. Therefore, both affected and unaffected oocytes are fertilized and developed to a cleavage-stage, so the same situation occurs as in blastomere biopsy, where embryos have been established that are later discovered to be affected. * Corresponding author.
The destruction or indefinite frozen storage of affected embryos is ethically unacceptable to some groups and couples on ethical and/or religious grounds. For these couples, there is a need for an ethically more palatable technique. We developed a method to complete the PB1 and PB2 removal prior to the fusion of the male and female pronucleus, followed by freezing all oocytes in the pronuclear phase. After analysis, in a subsequent menstrual cycle, only the oocytes predicted to have inherited the normal maternal allele are thawed and cultured to allow fusion of the pronuclei, embryo development, and transfer. Since zygotes prior to pronuclear fusion are not yet considered embryos and no abnormal oocytes are thawed and cultured, no affected embryos are established, causing this technique to be ethically more acceptable to many couples. Therefore, this technique creates a new class of genetic diagnosis, preembryonic genetic diagnosis, which pushes the frontier of genotyping to an even earlier stage. The present paper describes the case of preembryonic diagnosis performed for sickle cell disease, resulting in two unaffected singleton pregnancies and the birth of a healthy child.
0303-7207/01/$ - see front matter © 2001 Published by Elsevier Science Ireland Ltd. PII: S 0 3 0 3 - 7 2 0 7 ( 0 1 ) 0 0 5 6 9 - X
S20
A. Kulie6 et al. / Molecular and Cellular Endocrinology 183 (2001) S19–S22
2. Material and methods A 33-year old women and spouse at risk for the birth of a child with sickle cell disease were referred for PGD to avoid a possible termination of a pregnancy following prenatal diagnosis. A standard IVF protocol was initiated but the patient suffered hyper-stimulation syndrome, which precluded transfer of embryos in the current cycle. Twenty-eight mature oocytes were aspirated and placed in culture medium. Of the 28 aspirated oocytes, 14 extruded PB1 that were removed by micromanipulation as described elsewhere (Verlinsky and Kuliev, 1993). The oocytes were then fertilized by intracytoplasmic sperm injection. As soon as the PB2 was removed, and prior to the fusion of the male and female pronuclei, all embryos were frozen. The PB1 and PB2 were analyzed by multiplex nested PCR as described elsewhere (Rechitsky et al., 1998). Genotyping was performed using multiplex PCR because the most important source of diagnostic error in PGD occurs when only one allele of a heterozygous cell amplifies in a process known as allele-drop out (ADO). This occurs in approximately 5– 10% of PB analyses in the beta globin gene (Rechitsky et al., 1998). We developed a technique to detect ADO to prevent misdiagnosis. This involves nested, multiplex PCR with completely separate primer sets for the sickle cell mutation and two linked short tandem repeats (STR) markers; one located at the 5% end of the b-globin gene (5% STR) and the other in the human thyrosin hydroxylase gene (THO-STR). The mother was heterozygous for both linked markers and the phase of the linkage is known by family analysis using her affected child. To detect potential contamination with extraneous DNA and identify the embryo that implanted and established pregnancy, three additional non-linked STRs were amplified, including STR at the 5% untranslated region of human coagulation factor A subunit gene (HUMF13A01), STR for Von Willibrands Disease (vWF), and an STR for chromosome 21 (D21S11). Primers were synthesized on an Applied Biosystems 381 A Synthesizer. The list of primer sequences, reaction conditions and details of the nested PCR have been described earlier (Rechitsky et al., 1998; Kuliev et al., 1998). In brief, the first round PCR (25 cycles) was performed with a mixture of outside primers for bglobin gene and STRs, and then the resulting PCR products were aliquotted and amplified separately with inside primers for each individual locus in the second PCR round. For sickle cell mutation analysis, the resulting PCR products were restriction digested for 3 h overnight with DdeI (Promega) under the conditions recommended by the manufacturer, followed by polyacrylamide gel electrophoresis (PAGE) as described elsewhere (Rechitsky et al., 1998). For STR fragment
analyses, the PCR products were analyzed by PAGE alone. The pronuclear-stage oocytes predicted to be normal were thawed, cultured to develop into the cleaving embryos of 6–8 cell stage and transferred back to the patient in the two subsequent clinical cycles. The oocytes predicted to contain the mutant maternal gene were not thawed, but analyzed directly at the pronuclear-stage for the confirmation of PB diagnosis.
3. Results and discussion Of 28 aspirated oocytes, 14 extruded their PB1 and were studied for the presence of sickle cell mutation. Following intracytoplasmic sperm injection, PB2s were extruded from 13 of them and studied. Results of both PB1 and PB2 were available in 12 of these 13 oocytes (Fig. 1). Overall, six oocytes were predicted to contain a normal allele (Fig. 1, oocyte number 3, 5, 9, 10, 13 and 14) based on heterozygous status of PB1 and hemizygous mutant status of PB2. As seen from Fig. 1, a probable case of ADO was detected in oocyte number 6. Although the sickle cell analysis of PB1 showed only the normal allele and the 5%-STR showed only six repeats, it was heterozygous for the THO-STR, suggesting that this is a case of ADO. Therefore, without simultaneous amplification of linked STRs, a misdiagnosis of the heterozygous oocyte as homozygous due to ADO would have occurred leading to a misdiagnosis of the maternal contribution to the zygote. In this particular instance, the error would have caused an unaffected zygote to be misdiagnosed as affected, but the reverse could also occur. Subsequently, the patient was prepared for a frozen embryo transfer. In the first frozen cycle, four zygotes determined to have the maternal unaffected allele were thawed and cultured. Three developed into cleaving embryos of acceptable quality and were transferred (Fig. 1, embryo number 3, 10, 14), resulting in a singleton pregnancy, which was spontaneously aborted (Verlinsky et al., 1999). In the second frozen cycle, two unaffected embryos were transferred (Fig. 1, embryo number 5 and 9) and resulted in a singleton pregnancy and birth of an unaffected child, following confirmation of PB diagnosis by chorionic villus sampling (CVS). The results of the application of non-linked markers allowed not only the exclusion of a possible DNA contamination but also the identification of the embryo (number 9) that implanted yielding a clinical pregnancy. All the remaining oocytes predicted to contain an abnormal gene were not further cultured, but exposed directly to PCR analysis at the pronuclear stage for confirmation of diagnosis as frozen samples, and were shown to be abnormal as predicted by PB analysis.
A. Kulie6 et al. / Molecular and Cellular Endocrinology 183 (2001) S19–S22
S21
Fig. 1. Diagnostic scheme and sequential polar body analysis for sickle cell disease. Upper panel, schematic representation of beta globin locus and linked markers on chromosome 11p15.5 used for this study. STR, short tandem repeat in 5% untranslated region of b-globin gene. SCA-Dde 1, Dde 1 restriction site at the sickle cell mutation (A T missense mutation). THO-STR, short tandem repeat in thyrosin hydroxylase gene on chromosome 11p15.5. i, intron. Arrows represent primer sets for nested PCR. The lower panels represent series which all include the first polar body (PB1) followed by the second polar body (PB2) in the lane to its immediate right for single zygotes. The oocyte number that appears at the bottom of the figure signifies the oocyte number for all analyses shown. The upper panel represents the Dde1 restriction digestion for the sickle cell mutation genotyping. The middle panel represents the 5% STR analysis. The lower panel represents the THO-STR analysis. ET1, embryos that were thawed and cultured for transfer in the first frozen cycle. ET2, embryos that were thawed and cultured for transfer in the second frozen cycle. Chorionic villus sampling (CVS), genotype of embryo resulting in pregnancy, prenatal diagnosis and birth of unaffected child. ADO, notes analysis in which ADO was detected by linked marker analysis.
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The presented data demonstrate the feasibility of performing preembryonic diagnosis for single gene disorders, which has resulted in obtaining two unaffected pregnancies and the birth of a healthy, unaffected child. PGD by PB1 testing has also been described for the maternally derived translocations (Munne et al., 1998). In our earlier work, we have attempted to perform PGD for single gene disorders by the use of PB1 analysis alone (Verlinsky et al., 1992). Although this allowed pre-selection of a few mutation-free oocytes inferred from the homozygous abnormal status of PB1, the majority of oocytes were heterozygous after the first meiotic division, so the genotype of the resulting embryos could not be predicted, thus limiting the number of normal embryos for transfer. To overcome this problem, a two-step oocyte testing process by sequential analysis of both PB1 and PB2 has been introduced, resulting in pregnancies and births of a number of children free of genetic disorders, including the clinical pregnancy in the case of sickle cell disease (Verlinsky et al., 1997). However, in most of these cases, the oocytes tested by PB analysis were kept in culture, so that they developed into a cleavage stage embryo by the time the genotyping results were obtained. To avoid discarding these preimplantation embryos, we have introduced freezing of oocytes immediately following fertilization and extrusion of PB2 and prior to fusion of the male and female pronuclei, which is considered the beginning of the embryonic period (Larsen, 1994). In fact, freezing may be omitted in the near future as recent developments in PCR analysis would allow com-
pleting the genetic diagnosis before pronuclei fusion. This opens a possibility for application of PGD for those couples who cannot accept any intervention or discarding of the human embryos.
References International Working Group on Preimplantation Genetics, 1998. Preimplantation diagnosis: an alternative to prenatal diagnosis of genetic and chromosomal disorders. Report of the Eight Annual Meeting in Association with International Conference on Prenatal Diagnosis and Therapy, Los Angeles, June 1998. J. Assist. Reprod. Genet. 16 161 – 164. Kuliev, A., Rechitsky, S., Verlinsky, O., et al., 1998. Preimplantation diagnosis of thalassemias. J. Assist. Reprod. Genet. 15, 219 – 225. Larsen, W.J., 1994. Human Embryology, Churchill Livingstone. Munne, S., Morrison, L., Fung, J., et al., 1998. Spontaneous abortions are reduced after preconception diagnosis of translocations. Assist. Reprod. Genet. 15, 290 – 296. Rechitsky, S., Strom, C., Verlinsky, O., et al., 1998. Allele drop-out in polar bodies and blastomeres. J. Assist. Reprod. Genet. 15, 253 – 257. Verlinsky, Y., Kuliev, A. (Eds.), 1993. Preimplantation Diagnosis of Genetic Disorders: a New Technique for Assisted Reproduction. Wiley, New York. Verlinsky, Y., Milayeva, S., Evsikov, S., et al., 1992. Preconception and preimplantation diagnosis for cystic fibrosis. Prenat. Diagn. 12, 103 – 110. Verlinsky, Y., Rechitsky, S., Cieslak, J., et al., 1997. Preimplantation diagnosis of single gene disorders by two-step oocyte analysis using first and second polar body. Biochem. Mol. Med. 62, 182 – 187. Verlinsky, Y., Rechitsky, S., Verlinsky, O., et al., 1999. Prepregnancy testing for single gene disorders by polar body analysis. Genet. Testing 3, 185 – 190.