Blastomere transplantation in human embryos may be a treatment for single gene diseases

Size: px

Start display at page:

Download "Blastomere transplantation in human embryos may be a treatment for single gene diseases"

Jesse Justin Lang
5 years ago
Views:

1 FERTILITY AND STERILITY VOL. 81, NO. 4, APRIL 2004 Copyright 2004 American Society for Reproductive Medicine Published by Elsevier Inc. Printed on acid-free paper in U.S.A. Blastomere transplantation in human embryos may be a treatment for single gene diseases Norbert Gleicher, M.D., and Ya Xu Tang, M.D. Centers for Human Reproduction, New York, New York and Chicago, Illinois; and the Foundation for Reproductive Medicine, Chicago, Illinois Received April 17, 2003; revised and accepted August 15, This study was presented in part at the Annual Meeting of ESHRE, Madrid, Spain, July 1, 2003, and was a finalist for the Senior Investigator Award. This study was also presented in part at the Annual Meeting of the ASRM, San Antonio, Texas, October 15, 2003, and was a finalist for the American Society for Reproductive Medicine s General Program Prize Paper Award. Disclosure and Conflict Statement: A patent application for the concept of blastomere transplantation has been submitted by Dr. Gleicher. The publication of this study does not involve any conflicts. Reprint requests: Norbert Gleicher, M.D., Center for Human Reproduction (CHR), 60 East Delaware Place, Suite 1400, Chicago, Illinois (FAX: ; E- mail: chrjournal@aol.com) /04/$30.00 doi: /j.fertnstert Objective: To determine whether human embryos accept blastomere transplants and integrate them normally into the architecture of the developing embryo. Design: A human blastomere transplantation model, involving 44 cryopreserved embryos that were specifically donated to research. Setting: Academically affiliated private infertility center. Patient(s): Forty-four human embryos. Intervention(s): In 21 experiments, one, two, or three blastomeres were transplanted, using standard microsurgical techniques that are widely used in preimplantation genetic diagnosis (PGD). Embryos were thawed and gender was determined, using established PGD techniques. Male (xy) blastomeres were then transplanted into female (xx) day 3 embryos, and the xy cells were tracked through blastocyst stage (days 5 6) and into the hatching period (day 6), using fluorescent in situ hybridization (FISH). Main Outcome Measure(s): Degree and location of xy cell integration into xx embryos. Result(s): High-quality recipient embryos (with 4 to 10 cells) developed uniformly into normal blastocyst stage embryos in 12 of 12 experiments (100%) and integrated donor blastomeres into their architecture, with apparently even distribution of daughter cells; this integration was documented in inner cell mass as well as in trophoectoderm. The intensity of this distribution appeared to correlate with the number of blastomeres transferred. Among nine abnormally developing embryos, only three (33%) demonstrated a normal distribution of offspring donor cells. Conclusion(s): High-quality embryos appear to have the ability to integrate donor blastomeres. Because the treatment of single gene diseases does not require successful treatment of all cells, blastomere transplantation could be explored as a treatment option, which also would greatly enhance efficiency and utilization of preimplantation genetic diagnosis. (Fertil Steril 2004;81: by American Society for Reproductive Medicine.) Key Words: Blastomere transplantation, preimplantation genetic diagnosis, preimplantation genetic treatment, in vitro fertilization, micromanipulation Approximately 3,000 human genetic diseases are believed to be the consequence of single gene defects (1). To cure some, various methods have been used in attempts to introduce missing genes into affected cells (2). Such experimental treatments have suggested that to cure disease, the repair of a genetic defect does not have to be complete (3). This observation led us to speculate that human embryos, which through the use of preimplantation genetic diagnosis (PGD) have been diagnosed with a single gene defect, might be treatable through the transplantation of normal blastomeres, which contain the missing genes. If such transplanted blastomeres were to be integrated normally into the developing recipient embryo, a potential treatment protocol could be devised under which normal embryos, either from a cohort of gender-matched sibling embryos or donor embryos, could serve as blastomere donors to genetically abnormal embryos (Fig. 1). Day 3 (after fertilization) embryos are known to survive well, after removal of blastomeres (4, 5). Whether such transplanted blastomeres are, indeed, integrated into a developing embryo was the subject of this investigation. 977

2 FIGURE 1 Theoretical model of blastomere transplantation to treat single gene defects in affected embryos. *In potential clinical application, in contrast to the here-reported study, embryos would be gender-matched to prevent male female microchimerism. Indeed, if sibling embryos were used, parentage of donor and recipient embryos would be the same. Normal and abnormal are boldface for graphic reasons. MATERIALS AND METHODS This study was initiated, after approval of the study protocol by an institutional review board, after legal review by an outside law firm. The study only involved human embryos that, by written consent, were expressly donated to research. This represents one of three possible options for couples who no longer wish to maintain their cryopreserved embryos and follows ethics guidelines published by the American Society for Reproductive Medicine (6). To investigate the acceptance of transplanted blastomeres into recipient embryos, a chimera model was used: male embryos, with an xy-chromosomal complement, served as blastomere donors, whereas female embryos, with an xxchromosomal complement, served as recipients. Integration of xy-donor blastomeres into the recipient xx-embryo was then traced by staining the day 5 or 6 blastocyst stage embryo with a y-chromosome marker, as is routinely done in PGD studies for the determination of embryonic gender [see below in description of fluorescent in situ hybridization (FISH)]. 978 Gleicher and Tang Blastomere transplantation Vol. 81, No. 4, April 2004

3 Forty-four embryos that were previously cryopreserved on day 3 (4- to 10-cell stage) were thawed in routine fashion. Thirty-seven (84%) survived the thaw and underwent embryo biopsy for PGD, as previously described, to determine the gender of each embryo. Specifically, the biopsy was performed on a Nikon Diaphot (Nikon, Inc., Melville, NY) inverted microscope, using Narishige manipulators (Narishige International USA, Inc., East Meadow, NY), with microtools from Humagen (Humagen Fertility Diagnostics, Inc., Charlottesville, VA). Day 3 embryos at the 4- to 10-cell stage were placed in a drop of biopsy medium under mineral oil by holding them with a holding pipette. The zona pellucida was locally digested, by releasing acidified Tyrode s solution (Sigma, St. Louis, MO) through the assistant hatching pipette. Through a small opening created, one blastomere was aspirated and removed from the embryo by micromanipulation, using a biopsy pipette. The embryo was then backed to culture, and the blastomere s gender was determined by fluorescent in situ hybridization (FISH) (7). To perform FISH, the blastomere was placed in a drop of hypotonic solution for 5 minutes and transferred to a glass slide. Carnoy s fixative solution (Sigma; 3:1 methanol and acetic acid) was then dropped onto the blastomere cell under an inverted microscope. All slides were dehydrated through an ethanol series (70%, 85%, and 100% ethanol) and then air dried. Probes CEP Y (DYZI, satellite III spectrumgreen) and CEP X (DXZI, alpha satellite spectrum orange) were used to identify chromosome y and x, respectively (Vysis, Inc., Downers Grove, IL). The two-color hybridization solutions were applied to the target area, and a coverslip was placed. Embryonic DNA was denatured with the probe mixture at 78 C for 10 minutes, followed by hybridization at 37 C for 2 hours in a HYBrite unit (Vysis). Slides were washed with 0.4% saline sodium citrate (SSC)/ 0.3% Nonidet P-40 (NP40) at 73 C for 5 minutes in a water bath and 2 SSC/0.1% NP40 (2 SSC solution containing 0.1% NP-40) for 1 minute at room temperature and air dried; a counter stain (DAPI II; Vysis, Inc.) was applied. Specific signals were viewed and documented under the Olympus Ax70 fluorescence microscope with appropriate filter sets. Images were captured using a DKC-5000 digital camera (Scientific, Inc., Middlebush, NJ). After the gender of an embryo was determined, male embryos, serving as blastomere donors, were placed into a drop of biopsy medium, covered by mineral oil, and held in place with a holding pipette. The embryo was then touched gently with a biopsy pipette to find the earlier established biopsy site. Additional blastomeres were then removed through the original biopsy opening in the zona pellucida, in the same fashion as just described, and the embryo was put back into culture. Female embryos were treated similarly, though served as recipients. They, too, were placed into a drop of biopsy medium and covered by mineral oil; the prior biopsy site was identified. Donor blastomeres were then picked up with the biopsy pipette and gently inserted through the initial biopsy opening in the zona pellucida of the recipient embryo, in a step reversing the prior blastomere removal process. Once the blastomere transplant had taken place, the embryo was returned to routine culture. Recipient embryos were uniformly cultured to day 5 or 6, whereas donor embryos were only selectively cultured to days5or6,toconfirm persistent viability after removal of up to three blastomeres. On day 5, normal embryos reached blastocyst stage. Later on day 5, or early on day 6, normal embryos started hatching. Later on day 6, some embryos were observed until fully hatched. Embryos were then, at all of those blastocyst stages, investigated by FISH, for the presence of x and y chromosomes, using the same techniques as previously described for the gender determination of individual blastomeres. Embryos were considered to develop abnormally if they failed to reach blastocyst stage by day 5 or early day 6. In the chimera model used here, after blastomere transplantation, the appearance of a green marker (y chromosome) against a red background (x chromosome) would thus represent one half of the donor genotype accepted and integrated by the recipient embryo, because the other half (the x chromosome) cannot be differentiated against the recipient embryo s own chromosomal background. Y penetration was classified as negative (no signal), minor (1 15 signals), moderate (16 30 signals), and profound ( 31 signals) for each blastocyst stage embryo. Recipient embryos received one, two, or three blastomeres. Donor embryos were either completely dismantled, with all blastomeres used for transplantation, or had one to three blastomeres removed (beyond the original blastomere removed for gender determination) when they were cultured to blastocyst stage. Sixteen male embryos served as donor embryos, and 21 female embryos received blastomere transplants. Among recipient embryos, four (19%) arrested their development before reaching blastocyst stage. However, only 12 (57%) were considered to have reached normal blastocyst stage. Nine embryos were allowed to hatch either partially or completely, before staining. RESULTS Figure 2 demonstrates a normally developing day 5 blastocyst stage recipient embryo. As can be seen, there is a considerable level of integration of the y chromosome (green) seen against the recipient background, throughout the inner cell mass and the trophectoderm. Figure 3 demonstrates a completely hatched recipient embryo on day 6. Once again, the evidence of a y chromosome can be seen throughout the recipient embryo. FERTILITY & STERILITY 979

The anatomy of this blastocyst stage embryo is recognizable, because of counterstaining of nuclei with DAPI II (blue color).

4 FIGURE 2 Day 5 blastocyst stage embryo. This xx-embryo received on day 3, at seven-cell stage, a one-cell xy-blastomere transplant. It now demonstrates a symmetric dissemination of the y-signal (green) throughout inner cell mass and trophoectoderm of the embryo. The anatomy of this blastocyst stage embryo is recognizable, because of counterstaining of nuclei with DAPI II (blue color). The y-signal in this embryo was classified as minor, because only 14 signals were counted. Twelve normally developing embryos uniformly demonstrated an even and well-distributed staining pattern for the y chromosome, whereas among nine abnormally developing or arrested embryos, most demonstrated an uneven, spotty, and usually diminished staining pattern. Indeed, among such embryos, only three (33%) demonstrated a y distribution pattern that was considered normal. FIGURE 3 Completely hatched day 6 recipient embryo. This embryo received three donor blastomeres as a six-cell embryo on day 3. The penetration by y-marker (green) was classified as profound, because 90 y-signals were counted. TABLE 1 Correlation between y-signal intensity and number of blastomeres transferred. No. of blastomeres transplanted No. of embryos Blastocyst quality No. of y-signals Penetration grading 1 3 Poor 3 Minor Poor 22 Moderate Good 14 Minor 2 16 Excellent 50 Profound Excellent 50 Profound Good 11 Minor Poor 18 Moderate Arrested 12 Minor Excellent 60 Profound Excellent 50 Profound Good 14 Minor Good 12 Minor Good 11 Minor Good 12 Minor Poor 0 Negative Poor 0 Negative Arrested 3 Poor Arrested 9 Poor Arrested 7 Poor 3 2 Excellent 90 Profound Good 38 Profound Table 1 summarizes the intensity of the y signal in transplant recipient embryos. As can be seen, the number of blastomeres transferred generally appears to correlate with the intensity of y penetration into the embryo, though the small number of experiments precludes the establishment of valid statistical correlations. DISCUSSION The here-reported experiments represent only a very preliminary step in the potential utilization of blastomere transplants to treat genetically abnormal embryos. The fact that transplanted blastomeres demonstrate an apparently normal distribution of daughter cells throughout inner cell mass and trophoectoderm suggests that these transplanted blastomeres are, indeed, integrated into the early development of recipient embryos. What remains to be demonstrated is that this integration is normal and would result in the birth of an anatomically, genetically, and functionally normal child. That the amount of y signal apparently increases with an increasing number of blastomeres transferred is encouraging, because this observation suggests that the amount of gene therapy can potentially be titrated. Yet, nothing in these experiments conclusively proves that blastomere transplantation can repair single-gene defects in a clinically relevant way. Indeed, the concept of blastomere transplantation raises considerable potential eth- 980 Gleicher and Tang Blastomere transplantation Vol. 81, No. 4, April 2004

5 ical as well as medical concerns, because it involves the creation of genetic chimeras, and the potential clinical, as well as genetic, consequences of such chimeras are currently largely unknown. Before blastomere transplantation in humans can, therefore, be further considered, it would appear essential to establish appropriate animal models to investigate potential adverse consequences. Human embryos have demonstrated remarkable resistance to damage and a strong ability for self-repair. For example, although animal experiments had suggested otherwise, intracytoplasmic sperm injection (ICSI) in the human has been remarkably successful (8). Early human embryos are now well recognized to demonstrate a high degree of genetic mosaicism (9), but they often self-repair by the time blastocyst stage is reached (10). Moreover, chimerism occurs in humans naturally and has, indeed, been shown to represent an essential feature of successful organ transplantation (11, 12). The here-reported finding should, therefore, not surprise. Indeed, Alikani and Willadsen (13) recently reported that they were able to assemble embryos in donor zona pellucidas, from single blastomeres from different donor embryos, and observed such embryos to develop to blastocyst stage. Our observation of apparently highly successful grafting of donor blastomeres into developing recipient embryos supports the contention that early embryos are amenable to successful treatment. This observation offers the possibility of blastomere transplantation as a treatment tool. Use of PGD to diagnose embryos affected by single gene diseases is currently increasing. Indeed, more than 30 such diseases were diagnosed in embryos, in Europe alone, during the year The process is, however, technically difficult and inefficient in establishing pregnancies, in that pregnancy rates after PGD have been reported as significantly lower than those after IVF without PGD. This difference is probably at least partially due to the greatly diminished number of embryos that are available for embryo transfer after PGD. In interpreting the here-reported results, one also has to consider that all embryos, used in these experiments, had been cryopreserved. Survival and embryo quality of thawed embryos can, of course, be affected by cryopreservation (14). It is, therefore, possible that identical experiments, performed on freshly produced embryos, may demonstrate even better results. Because of unavoidable technical difficulties with PGD, the potential outcome will always result in embryos considered to be affected, normal, or undetermined (i.e., no clear diagnosis can be reached). Under current practice guidelines, only normal embryos will be transferred into the uterus, with embryos in the other two groups being discarded. Often, this leaves only few (or no) embryos for transfer and thus turns PGD into a rather inefficient and costly procedure. Our potential ability to treat affected embryos could greatly increase the efficiency of this procedure and, consequently, also make it more cost effective. More important, however, the potential ability to treat affected embryos removes the principal argument against the utilization of PGD. Many European countries legally prohibit the utilization of PGD, mostly because of ethical objections toward the discarding of diseased embryos (15). The potential of treating diseased embryos, after their diagnosis by PGD, potentially equates their situation ethically to that of transplants after birth and may, therefore, spawn a reevaluation of current ethical and legal standards in countries that currently prohibit the use of PGD under all circumstances. A wider utilization of PGD to prevent single gene diseases could have a significant public health impact. Finally, it is also conceivable that the technique of blastomere transplantation may benefit current methods of stem cell production from human embryos by expanding their genetic repertoire. In summary, although the ultimate utilities of human blastomere transplantation at the early embryo stage remain to be established, the here-presented study strongly suggests that such transplantations are technically feasible. References 1. Sachs BP, Uorf B. The Human Genome Project: implications for the practicing obstetrician. Obstet Gynecol 1993;81: Stribley JM, Rehman RS, Niv H, Christman GM. Gene therapy and reproductive medicine. Fertil Steril 2002;77: Hacien-Bey-Abina S, Le Deist F, Carlier F, Bouneaud C, Hue C, De Villartay JP, et al. Sustained correction of X-linked severe combined immunodeficiency by ex vivo gene therapy. N Engl J Med 2002;346: American Society of Reproductive Medicine and Society for Assisted Reproductive Technology. A Practice Committee report: preimplantation genetic diagnosis. Birmingham, AL: American Society for Reproductive Medicine, ESHRE PGD Consortium Sharing Committee. ESHRE Preimplantation Genetic Diagnosis Consortium: data collection III. Hum Reprod 2001; 17: The American Society for Reproductive Medicine guidelines for gamete and embryo donation. Guidelines for cryopreserved embryo donation. Fertil Steril 2002;77(Suppl):S Handyside AH, Kontogianni EH, Handy R, Winston RM. Pregnancies from biopsied human preimplantation embryos sexed by Y specific DNA amplification. Nature 1990;344: Palermo G, Joris H, Devroey P, Van Steirteghem AC. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 1992;340: Delhanty JD, Harper JL, Ao A, Handyside AH, Winston RM. Multicolour FISH detects frequent chromosomal mosaicism and chaotic division in normal preimplantation embryos from fertile patients. Hum Genet 1997;99: Bielanska M, Lin Tan S, Ao A. Different probe combinations for assessment of postzygotic chromosomal imbalances in human embryos. J Assist Reprod Genet 2002;19: Fuchimoto Y, Yamada K, Shimizu A, Yasumoto A, Sawada T, Huang CH, et al. Relationship between chimerism and tolerance in a kidney transplantation model. J Immunol 1999;162: Quaini F, Urbanek K, Beltrami AP, Finato N, Beltrami CA, Nadal- Ginard B, et al. Chimerism of the transplanted heart. N Engl J Med 2002;346: Alikani M, Willadsen SM. Human blastocysts from aggregated mononucleated cells of two or more non-viable zygote-derived embryos. Reprod Biomed Online 2002;5: Elias S. Preimplantation genetic diagnosis by comparative genomic hybridization. N Engl J Med 2001;345: Orellana C. German ethics group advises against pre-implantation genetic diagnosis. Lancet 2002;359:1926. FERTILITY & STERILITY 981

Preimplantation genetic diagnosis: polar body and embryo biopsy

Human Reproduction, Vol. 15, (Suppl. 4), pp. 69-75, 2000 Preimplantation genetic diagnosis: polar body and embryo biopsy Luca Gianaroli SISMER, Via Mazzini 12, 40138 Bologna, Italy Scientific Director