Human Reproduction, Vol. 14, No. 5, 1403-1404,
May 1999
© 1999 European Society of Human Reproduction and Embryology
Letters to the Editor |
Techniques for sorting X and Y spermatozoa may adversely affect histone-associated regions in human spermatozoa
Embryology Unit, Children's Medical Research Institute, Westmead, New South Wales 2145, Australia
Dear Sir,
The recent paper (Fugger et al., 1998
) which demonstrates that sorting of X and Y spermatozoa can be used to produce apparently healthy babies of a chosen sex is a milestone in assisted reproduction technology in humans. Previously, this technique has been used successfully only for several other animal species, primarily cattle.
Over the past few years, concerns have been raised concerning the safety of a technique which subjects a sperm cell to a potentially mutagenic agent such as Hoechst 33342 dye (bisbenzimide). Fugger's group have addressed these concerns in detail. For instance, they argue that although some somatic cells are sensitive to bisbenzimide (Durand and Olive, 1982; Van Zandt and Fry, 1983) this may not be true for sperm cells whose DNA is compacted and stabilized, compared with somatic cells. This argument may indeed hold for cattle whose somatic cell DNA is relatively loosely packaged with histones in nucleosomes and whose sperm DNA has almost all the histones replaced with protamine molecules (Palmer et al, 1991). However, I consider that the same reasoning does not hold for human spermatozoa, since the displacement of histones by protamines is incomplete in human spermatozoa. In human spermatozoa, about 15% of the DNA remains complexed with histones (Tanphaichitr et al., 1978; Gatewood et al., 1987) and nucleosome-like structures have been observed (Banerjee et al., 1995). This raises the uncomfortable possibility that the bisbenzimide could adversely affect DNA in the histone-associated regions of human spermatozoa in the same way it does in somatic cells.
As discussed by Fugger et al., successful animal trials have been carried out in rabbit, swine, sheep and cattle (Fugger et al., 1998). However, none of these species has been demonstrated to have a human-like form of sperm DNA packaging. Further work needs to be carried out on the sperm chromatin structure in these test species before one can infer from the animal trials that human offspring will never be adversely affected. I consider that it would be unwise to continue with human trials without first finding an animal species with a similar sperm chromatin structure to ourselves and conducting animal trials using this species.
Fugger and coworkers argue that bisbenzimide is safe because it did not cause mutations within the ß-globin gene in human spermatozoa (Watkins et al., 1996). Our recent study shows that the ß-globin gene in spermatozoa is protamine-associated at the sites tested and that it is probably more compact and protected than other genes (Gardiner-Garden et al., 1998). This makes it an unfortunate choice for mutagenicity studies. In our study, other genes such as the 
- and 
-globin genes, which are expressed earlier in human development, were shown to contain histone-associated regions in spermatozoa (Gardiner-Garden et al., 1998). Such histone-associated genes would form better safety controls. Many more studies of mutagenicity on a variety of genes in human spermatozoa are required to prove that the detrimental affects of bisbenzimide observed in somatic cells will not also occur in important genes in sperm cells.
References
Banerjee, S., Smallwood, A. and Hulten, M. (1995) ATP-dependent reorganization of human sperm nuclear chromatin. J. Cell. Sci., 108, 755–765.[Abstract]
Durand, R.E. and Olive, P.L. (1982) Cytotoxicity, mutagenicity and DNA damage by Hoechst 33342. J. Histochem, Cytochem., 30, 111–116.[Abstract]
Fugger, E.F., Black S.H., Keyvanfar, K. and Schulman, J.D. (1998) Births of normal daughters after MicroSort sperm separation and intrauterine insemination, in vitro fertilization, or intracytoplasmic sperm injection. Hum. Reprod., 13, 2367–2370.
Gardiner-Garden, M., Ballesteros, M., Gordon, M. and Tam, P.P.L (1998) Histone- and protamine-DNA association: conservation of different patterns within the ß-globin domain in human sperm. Mol. Cell. Biol., 18, 3350–3356.
Gatewood, J.M., Cook, G.R., Balhorn, R. et al. (1987) Sequence-specific packaging of DNA in human sperm chromatin. Science, 236, 962–964.
Palmer, D.K., O'Day, K., Trong, H.L. et al. (1991) Purification of the centromere-specific protein CENP-A and demonstration that it is a distinctive histone. Proc. Natl. Acad. Sci. USA, 88, 3734–3738.
Tanphaichitr, N., Sobhon, P., Taluppeth, N and Chalermisarachai, P. (1978) Basic nuclear proteins in testicular cells and ejaculated spermatozoa in man. Exp. Cell Res., 117, 347–356.[Web of Science][Medline]
Van Zandt, G. and Fry, C.G. (1983) H33342 staining of mouse bone cells: effects on colony-forming cells. Cytometry, 4, 40–46.[Web of Science][Medline]
Watkins, A.M., Chan, P.J. and Kalugdan, T.H. (1996) Analysis of flow cytometer stain Hoechst 33342 on human spermatozoa. Mol. Hum. Reprod., 2, 709–712.
1 Genetics and IVF Institute, 3020 Javier Road, Fairfax, Virginia 22031, 2 Department of Obstetrics and Gynecology, and 3 Department of Human Genetics, Medical College of Virginia, Richmond, Virginia, USA
Dear Sir,
We appreciate Dr. Gardiner-Garden's interest in our clinical research study for flow cytometric separation of human X- and Y-bearing spermatozoa. It is currently unproven whether histone- versus protamine-associated gene packaging of DNA in spermatozoa is differentially influenced by Hoechst 33342, if at all. Catt et al. (1997) found no difference in the incidence of endogenous DNA nicks in decondensed human sperm DNA exposed to Hoechst 33342 and UV compared with controls.
Dr. Gardiner-Garden makes the point that the - and -globin gene in human spermatozoa contain histone-associated regions and should be tested. We agree that it may be appropriate to test histone as well as other protamine-associated genes in human spermatozoa. We welcome and encourage this and other kinds of investigations that add to the knowledge base on this subject.
Nevertheless, the preponderance of pre-clinical experimental evidence delineated in our paper point to a lack of mutagenicity. There is no evidence of which we are aware indicating Hoechst mutagenicity in spermatozoa and regardless of the offered theory regarding histone binding, there is now extensive evidence from actual births in humans as well as several other mammalian species that numerous offspring are normal. The clinical trial data currently show no difference from the animal data. To date, 32 babies have been born as a result from flow cytometric separation of spermatozoa, all normal and healthy.
Notes
4 To whom correspondence should be addressed 
References
Catt, S.L., Sakkas, D., Bizzaro, et al. (1997) Hoechst staining and exposure to UV laser during flow cytometric sorting does not affect the frequency of detected endogenous DNA nicks in abnormal and normal human spermatozoa. Mol. Hum. Reprod., 3, 821–825.
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