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An improved fixation technique for fluorescence in situ hybridization for preimplantation genetic diagnosis
FERTILITY AND STERILITY威 VOL. 76, NO. 1, JULY 2001 Copyright ©2001 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
An improved fixation technique for fluorescence in situ hybridization for preimplantation genetic diagnosis Dmitri I. Dozortsev, M.D., Ph.D., H.C.L.D., and Kevin T. McGinnis, M.D. Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Wayne State University, Detroit, Michigan
Objective: To improve existing preimplantation genetic diagnosis fixation techniques. Design: Prospective randomized in vitro study. Setting: Academic medical center. Patient(s): None. Intervention(s): None. Main Outcome Measure(s): The intensity and clarity of fluorescence in situ hybridization (FISH) signals and the percentage of successfully fixed blastomeres. Result(s): The described fixation technique resulted in 100% fixation and 100% adequate FISH signals. Two conventional techniques resulted in 94% and 87% fixation, and in adequate FISH signals in 81% and 87%, respectively. Conclusion(s): This newly developed fixation technique simplifies the process of fixation of blastomeres for preimplantation diagnosis while essentially eliminating the possibility of losing a cell during fixation. It will hopefully allow more IVF programs to offer their patients preimplantation genetic diagnosis using the FISH technique. (Fertil Steril威 2001;76:186 – 8. ©2001 by American Society for Reproductive Medicine.) Key Words: Fixation, FISH, preimplantation genetics, nucleus, blastomere
Received October 20, 2000; revised and accepted January 23, 2001. Presented at the Annual Meeting of the American Society for Reproductive Medicine, San Diego, California, October 21–26, 2000. Reprint requests: Dmitri I. Dozortsev, M.D., Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Hutzel Hospital, Wayne State University, 4707 St. Antoine Boulevard, Detroit, MI 48201 (FAX: 313-7457037; E-mail: [email protected]). 0015-0282/01/$20.00 PII S0015-0282(00)01836-2
186
Fluorescence in situ hybridization (FISH) is a widely accepted approach to the analysis of the chromosomal complement in cells. At present, it is routinely used in preimplantation genetic diagnosis (PGD). Fixation of the cells of interest before FISH is a critical step in the analysis process. Fixation removes cytoplasmic material and makes the cell’s nuclear DNA accessible to the hybridization probes. An ideal fixation technique should completely remove the cytoplasmic proteins, leaving the chromosomal DNA and proteins intact. Traditional cytogenetic staining techniques typically deal with hundreds or even thousands of cells on a single slide. Although they are treated simultaneously, because of minute variations in fixative concentration, temperature, amount of the cytoplasm on the slide, and operator skill, as well as other factors, some of which are not well understood, most cells are usually not fixed appropriately. This is not critical for traditional cytogenetic analysis, as
there are always few perfectly fixed cells that provide sufficient information for diagnosis. At present, PGD borrows fixation techniques from conventional cytogenetics. However the variations and imperfections that are unimportant when numerous cells are present become a serious problem when only a single cell is available for diagnosis. PGD requires careful transfer of a blastomere to a glass slide. The transfer and subsequent treatment is highly sensitive to numerous factors, including operator skill. Transfer and fixation of the blastomere is a critical step because only one or two blastomeres are analyzed and only a small number of embryos, all highly valued by the patient, are available for biopsy. Blastomere fixation for PGD was initially performed using various modifications of a technique by Tarkowski that employed acetic acid/methanol as a fixative agent (1). However, after the publication of Coonen (2), most centers began utilizing Tween 20.
FIGURE 1 FISH results on the blastomeres fixed using different techniques: (A), New technique with probes for chromosomes X, Y, 13, 18, and 21; (B), New technique with probes for chromosomes X and Y; (C), Acetic Acid/Methano with probes for chromosome X and Yl; (D), Tween 20 with probes for chromosome X, Y, 13, 18, and 21. (Magnification, 600⫻.)
Dozortsev. New fixation technique for PGD. Fertil Steril 2001.
Both techniques (as well as their modifications) present an embryologist with problems of premature drying and incomplete removal of cytoplasm, both of which can interfere with hybridization. In addition, both techniques are prone to the problem of scattering of a blastomere’s nuclear material on the slide, which may lead to loss of the specimen. A recent comparison of two techniques for blastomere fixation showed a fixation rate between 78.5% and 93.9% (3). To remove excess cytoplasmic proteins, pepsin is often used. Because amount of remaining cytoplasm varies from slide to slide, and since pepsin activity varies from batch to batch, determination of the duration of pepsin treatment is problematic. If the slide is overexposed to pepsin, DNA degradation may occur rendering the specimen unsuitable for FISH. As a result, blastomere and/or polar body fixation is a cumbersome and stressful process that requires extensive training. The development of a simpler and more reliable fixation technique remains an important goal, and one that we attempt to address in the present study.
MATERIALS AND METHODS Institutional review board approval was obtained to use abnormally fertilized human embryos. Blastomeres removed from eight-cell-stage human embryos (originating from 1PN and 3PN zygotes) were randomly assigned to fixation using three different techniques. After separating the blastomeres, they were individually examined. Blastomeres in which the nucleus was not clearly visible were excluded from further study. The remaining blastomeres were fixed using either one of the current techniques or our new technique. A total of 50 blastomeres from 10 embryos were used in the study.
Technique 1 (Conventional, Tween 20/HCl) Blastomeres were exposed to hypotonic solution (1% sodium citrate in 0.2 mg/mL bovine serum albumin [BSA]) for 2 minutes. After this, blastomeres were transferred onto the microscope slide in a 1–2-L drop of hypotonic solution. FERTILITY & STERILITY威
Just before complete drying, Tween 20 (1% in 0.01 N HCl) was continuously added to the drop until the cell membrane was ruptured and the nucleus separated from the bulk of the cytoplasm. The slide was then placed into a Coplin dish containing a solution of 1% pepsin. The slide was exposed to the pepsin solution for between 5 and 30 seconds, depending on the amount of remaining cytoplasm.
Technique 2 (Conventional, Acetic Acid/Methanol) Blastomeres were exposed to hypotonic solution (1% sodium citrate in 0.2 mg/mL BSA) for 2 minutes. After this, blastomeres were transferred onto the microscope slide in 1–2 L of hypotonic solution. Just before a complete drying, several drops of a 1:3 mixture of acetic acid/methanol were added, until the cell nucleus was freed from the bulk of cytoplasm. The slide was then placed into a Coplin dish containing a solution of 1% pepsin. The slide was exposed to the pepsin solution for between 5 and 30 seconds, depending on the amount of remaining cytoplasm.
Technique 3 (New, Tween 20/HCl ⴙ Acetic Acid/Methanol) Blastomeres were rinsed (5–10 s) in a hypotonic solution (1% sodium citrate in 0.2 mg/mL BSA) and transferred into a Petri dish containing Tween 20 (1% in 0.01 N HCl). Forty seconds later, they were transferred onto a microscope slide in about 3 L of the same solution, which was then allowed to evaporate completely. Thereafter, several drops of a 1:3 mixture of acetic acid/methanol were placed on the slide over the specimen. Slides were processed for hybridization without any additional treatment. Hybridization after either fixation technique was accomplished using a standard Vysis (Downers Grove, IL) protocol with probes for chromosomes X and Y, or X, Y, 13, 18 and 21. Fluorescent signals were analyzed using Vysis imageprocessing software. 187
RESULTS Figure 1 shows representative images from each of the techniques. One of 16 (6%), 2 of 16 (13%) and 0 of 18 blastomeres were lost during fixation using techniques 1, 2, and 3 respectively. Interpretable hybridization results were obtained in 14 of 16 (87%), 13 of 16 (81%) and 18 of 18 (100%) following fixation using techniques 1, 2, and 3 respectively.
DISCUSSION This new technique has several major advantages over existing methods of fixation. Whether acetic acid/methanol or Tween was used, it was always necessary to keep adding additional solution to prevent drying of the specimen until the cytoplasm was removed. During this process, the dissolving blastomere’s nuclear material is frequently lost as a result of the turbulent flow as the added solution mixes with the solution already on the slide. With the new technique nothing is added to the drop with the blastomere while the solution is allowed to evaporate completely. Therefore, the blastomere can always be found at the same location where it was originally placed. This essentially eliminates the risk of losing a specimen. This technique may also prove to be of value in the fixation of polar bodies, which are particularly prone to being lost because of their small size. Furthermore, with the conventional techniques, a varying amount of cytoplasm often remains on the slide covering the specimen. This cytoplasm is typically removed with pepsin or another peptidase. The removal of this cytoplasm is critical. If treatment with pepsin is not sufficient, the remaining cytoplasmic proteins will prevent or decrease the binding of
188
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the fluorescent probe to the nuclear material and may also reduce the quantum efficiency of the fluorescent probe either through quenching or absorption. On the other hand, overexposure to pepsin, may lead to target DNA degradation. This delicate balance is not easy to achieve due to variability in the amount of cytoplasm and in the activity of the peptidase. Consequently, the intensity of the hybridization signal may vary greatly from slide to slide. Our new protocol doesn’t involve enzymatic treatment and removes the cytoplasmic remnants completely and uniformly. This allows the analyst to obtain very consistent hybridization results. The exact mechanism by which this combination technique works is not completely clear. It appears that the acidified mixture of Tween 20 denatures cytoplasmic proteins without cross-linking them so that they can subsequently be easily solubilized in a mixture of acetic acid and methanol and dispersed across the slide in a uniform manner.
CONCLUSION This new fixation technique removes steps contributing to variability in PGD results and virtually eliminates the chance of losing a specimen. This new technique does not require special training to achieve consistent results. It may even allow development of a completely automated cell fixation system in the future. References 1. Tarkowski AK. An air-drying method for chromosome preparation from mouse eggs. Cytogenetics 1966;5:394 – 400. 2. Coonen E, Dumoulin JC, Ramaekers FC, Hopman AH. Optimal preparation of preimplantation embryo interphase nuclei for analysis by fluorescence in-situ hybridization. Hum Reprod 1994;9:533–7. 3. Xu K, Huang T, Liu T, Shi Z, Rosenwaks Z. Improving the fixation method for preimplantation genetic diagnosis by fluorescent in situ hybridization. J Assist Reprod Genet 1998;15:570 – 4.
New fixation technique for PGD
Vol. 76, No. 1, July 2001
An improved fixation technique for fluorescence in situ hybridization for preimplantation genetic diagnosis Dmitri I. Dozortsev, M.D., Ph.D., H.C.L.D., and Kevin T. McGinnis, M.D. Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Wayne State University, Detroit, Michigan
Objective: To improve existing preimplantation genetic diagnosis fixation techniques. Design: Prospective randomized in vitro study. Setting: Academic medical center. Patient(s): None. Intervention(s): None. Main Outcome Measure(s): The intensity and clarity of fluorescence in situ hybridization (FISH) signals and the percentage of successfully fixed blastomeres. Result(s): The described fixation technique resulted in 100% fixation and 100% adequate FISH signals. Two conventional techniques resulted in 94% and 87% fixation, and in adequate FISH signals in 81% and 87%, respectively. Conclusion(s): This newly developed fixation technique simplifies the process of fixation of blastomeres for preimplantation diagnosis while essentially eliminating the possibility of losing a cell during fixation. It will hopefully allow more IVF programs to offer their patients preimplantation genetic diagnosis using the FISH technique. (Fertil Steril威 2001;76:186 – 8. ©2001 by American Society for Reproductive Medicine.) Key Words: Fixation, FISH, preimplantation genetics, nucleus, blastomere
Received October 20, 2000; revised and accepted January 23, 2001. Presented at the Annual Meeting of the American Society for Reproductive Medicine, San Diego, California, October 21–26, 2000. Reprint requests: Dmitri I. Dozortsev, M.D., Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Hutzel Hospital, Wayne State University, 4707 St. Antoine Boulevard, Detroit, MI 48201 (FAX: 313-7457037; E-mail: [email protected]). 0015-0282/01/$20.00 PII S0015-0282(00)01836-2
186
Fluorescence in situ hybridization (FISH) is a widely accepted approach to the analysis of the chromosomal complement in cells. At present, it is routinely used in preimplantation genetic diagnosis (PGD). Fixation of the cells of interest before FISH is a critical step in the analysis process. Fixation removes cytoplasmic material and makes the cell’s nuclear DNA accessible to the hybridization probes. An ideal fixation technique should completely remove the cytoplasmic proteins, leaving the chromosomal DNA and proteins intact. Traditional cytogenetic staining techniques typically deal with hundreds or even thousands of cells on a single slide. Although they are treated simultaneously, because of minute variations in fixative concentration, temperature, amount of the cytoplasm on the slide, and operator skill, as well as other factors, some of which are not well understood, most cells are usually not fixed appropriately. This is not critical for traditional cytogenetic analysis, as
there are always few perfectly fixed cells that provide sufficient information for diagnosis. At present, PGD borrows fixation techniques from conventional cytogenetics. However the variations and imperfections that are unimportant when numerous cells are present become a serious problem when only a single cell is available for diagnosis. PGD requires careful transfer of a blastomere to a glass slide. The transfer and subsequent treatment is highly sensitive to numerous factors, including operator skill. Transfer and fixation of the blastomere is a critical step because only one or two blastomeres are analyzed and only a small number of embryos, all highly valued by the patient, are available for biopsy. Blastomere fixation for PGD was initially performed using various modifications of a technique by Tarkowski that employed acetic acid/methanol as a fixative agent (1). However, after the publication of Coonen (2), most centers began utilizing Tween 20.
FIGURE 1 FISH results on the blastomeres fixed using different techniques: (A), New technique with probes for chromosomes X, Y, 13, 18, and 21; (B), New technique with probes for chromosomes X and Y; (C), Acetic Acid/Methano with probes for chromosome X and Yl; (D), Tween 20 with probes for chromosome X, Y, 13, 18, and 21. (Magnification, 600⫻.)
Dozortsev. New fixation technique for PGD. Fertil Steril 2001.
Both techniques (as well as their modifications) present an embryologist with problems of premature drying and incomplete removal of cytoplasm, both of which can interfere with hybridization. In addition, both techniques are prone to the problem of scattering of a blastomere’s nuclear material on the slide, which may lead to loss of the specimen. A recent comparison of two techniques for blastomere fixation showed a fixation rate between 78.5% and 93.9% (3). To remove excess cytoplasmic proteins, pepsin is often used. Because amount of remaining cytoplasm varies from slide to slide, and since pepsin activity varies from batch to batch, determination of the duration of pepsin treatment is problematic. If the slide is overexposed to pepsin, DNA degradation may occur rendering the specimen unsuitable for FISH. As a result, blastomere and/or polar body fixation is a cumbersome and stressful process that requires extensive training. The development of a simpler and more reliable fixation technique remains an important goal, and one that we attempt to address in the present study.
MATERIALS AND METHODS Institutional review board approval was obtained to use abnormally fertilized human embryos. Blastomeres removed from eight-cell-stage human embryos (originating from 1PN and 3PN zygotes) were randomly assigned to fixation using three different techniques. After separating the blastomeres, they were individually examined. Blastomeres in which the nucleus was not clearly visible were excluded from further study. The remaining blastomeres were fixed using either one of the current techniques or our new technique. A total of 50 blastomeres from 10 embryos were used in the study.
Technique 1 (Conventional, Tween 20/HCl) Blastomeres were exposed to hypotonic solution (1% sodium citrate in 0.2 mg/mL bovine serum albumin [BSA]) for 2 minutes. After this, blastomeres were transferred onto the microscope slide in a 1–2-L drop of hypotonic solution. FERTILITY & STERILITY威
Just before complete drying, Tween 20 (1% in 0.01 N HCl) was continuously added to the drop until the cell membrane was ruptured and the nucleus separated from the bulk of the cytoplasm. The slide was then placed into a Coplin dish containing a solution of 1% pepsin. The slide was exposed to the pepsin solution for between 5 and 30 seconds, depending on the amount of remaining cytoplasm.
Technique 2 (Conventional, Acetic Acid/Methanol) Blastomeres were exposed to hypotonic solution (1% sodium citrate in 0.2 mg/mL BSA) for 2 minutes. After this, blastomeres were transferred onto the microscope slide in 1–2 L of hypotonic solution. Just before a complete drying, several drops of a 1:3 mixture of acetic acid/methanol were added, until the cell nucleus was freed from the bulk of cytoplasm. The slide was then placed into a Coplin dish containing a solution of 1% pepsin. The slide was exposed to the pepsin solution for between 5 and 30 seconds, depending on the amount of remaining cytoplasm.
Technique 3 (New, Tween 20/HCl ⴙ Acetic Acid/Methanol) Blastomeres were rinsed (5–10 s) in a hypotonic solution (1% sodium citrate in 0.2 mg/mL BSA) and transferred into a Petri dish containing Tween 20 (1% in 0.01 N HCl). Forty seconds later, they were transferred onto a microscope slide in about 3 L of the same solution, which was then allowed to evaporate completely. Thereafter, several drops of a 1:3 mixture of acetic acid/methanol were placed on the slide over the specimen. Slides were processed for hybridization without any additional treatment. Hybridization after either fixation technique was accomplished using a standard Vysis (Downers Grove, IL) protocol with probes for chromosomes X and Y, or X, Y, 13, 18 and 21. Fluorescent signals were analyzed using Vysis imageprocessing software. 187
RESULTS Figure 1 shows representative images from each of the techniques. One of 16 (6%), 2 of 16 (13%) and 0 of 18 blastomeres were lost during fixation using techniques 1, 2, and 3 respectively. Interpretable hybridization results were obtained in 14 of 16 (87%), 13 of 16 (81%) and 18 of 18 (100%) following fixation using techniques 1, 2, and 3 respectively.
DISCUSSION This new technique has several major advantages over existing methods of fixation. Whether acetic acid/methanol or Tween was used, it was always necessary to keep adding additional solution to prevent drying of the specimen until the cytoplasm was removed. During this process, the dissolving blastomere’s nuclear material is frequently lost as a result of the turbulent flow as the added solution mixes with the solution already on the slide. With the new technique nothing is added to the drop with the blastomere while the solution is allowed to evaporate completely. Therefore, the blastomere can always be found at the same location where it was originally placed. This essentially eliminates the risk of losing a specimen. This technique may also prove to be of value in the fixation of polar bodies, which are particularly prone to being lost because of their small size. Furthermore, with the conventional techniques, a varying amount of cytoplasm often remains on the slide covering the specimen. This cytoplasm is typically removed with pepsin or another peptidase. The removal of this cytoplasm is critical. If treatment with pepsin is not sufficient, the remaining cytoplasmic proteins will prevent or decrease the binding of
188
Dozortsev and McGinnis
the fluorescent probe to the nuclear material and may also reduce the quantum efficiency of the fluorescent probe either through quenching or absorption. On the other hand, overexposure to pepsin, may lead to target DNA degradation. This delicate balance is not easy to achieve due to variability in the amount of cytoplasm and in the activity of the peptidase. Consequently, the intensity of the hybridization signal may vary greatly from slide to slide. Our new protocol doesn’t involve enzymatic treatment and removes the cytoplasmic remnants completely and uniformly. This allows the analyst to obtain very consistent hybridization results. The exact mechanism by which this combination technique works is not completely clear. It appears that the acidified mixture of Tween 20 denatures cytoplasmic proteins without cross-linking them so that they can subsequently be easily solubilized in a mixture of acetic acid and methanol and dispersed across the slide in a uniform manner.
CONCLUSION This new fixation technique removes steps contributing to variability in PGD results and virtually eliminates the chance of losing a specimen. This new technique does not require special training to achieve consistent results. It may even allow development of a completely automated cell fixation system in the future. References 1. Tarkowski AK. An air-drying method for chromosome preparation from mouse eggs. Cytogenetics 1966;5:394 – 400. 2. Coonen E, Dumoulin JC, Ramaekers FC, Hopman AH. Optimal preparation of preimplantation embryo interphase nuclei for analysis by fluorescence in-situ hybridization. Hum Reprod 1994;9:533–7. 3. Xu K, Huang T, Liu T, Shi Z, Rosenwaks Z. Improving the fixation method for preimplantation genetic diagnosis by fluorescent in situ hybridization. J Assist Reprod Genet 1998;15:570 – 4.
New fixation technique for PGD
Vol. 76, No. 1, July 2001