Trophoblast Research 10:13-18, 1997

CYTOGENETICS OF GAMETES, ZYGOTES A N D EARLY P R E G N A N C Y

Joep P.M. Geraedts

Department of Molecular Cell Biology and Genetics University of Limburg

P.O. Box 616 6200 MD Maastricht, The Netherlands

INTRODUCTION

Of all human conceptions only 25%-30% progress successfully to delivery (Leridon, 1977). Early loss of pregnancy may occur between fertilization and implantation, during the time of implantation, or after implantat ion at various stages of pregnancy. Chromosome abnormalities constitute the major cause of embryonic loss. In chromosome studies of spontaneous abortions abnormalities are encountered in .more than 50% (Bou6 et al., 1975; Eiben et al., 1990). Both abortion rates and chromosome abnormalities are maternal age dependent as a direct result of chromosomal nondisjunction during meiosis (Hook, 1981). Until 1978 the events between ovulation and pregnancy were h idden in a 'black box'. The development of in vitro fertilization (IVF) provided the opportuni ty to determine the frequency of chromosome abnormalities in human gametes and in early stages of embryo development. Because of ethical and legal restrictions in most countries human embryos will only originate in the framework of infertility treatment and therefore will hardly ever become available for investigation. In most studies, including ours, only zygotes and embryos are used which are not suitable for transfer to the uterus. The results of these studies will be presented. Recently it has also become possible to address some questions in the framework of pre- implantat ion diagnosis.

MATERIALS A N D METHODS

Cytogenetic studies were carried out on metaphase chromosomes as well as interphase cells. In the first case classical karyotypes were made following mitotic arrest, while non-dividing cells were studied by in situ hybridization.

For chromosome studies of oocytes and zygotes, Tarkowsky (1966) introduced a method which is used by many authors. For the fixation of oocytes to the microscope slide excellent results were obtained using the technique of Mikamo and Kamigushi (1983).

Protocols used for in situ hybridization, i.e. target cell preparat ion and fixation, probe labeling, hybridization, washing procedures and signal visualization are very diverse.

For fluorescent in situ hybridizat ion (FISH) the DNA from chromosome-specific probes was chemically modif ied by nick-translation (e.g., thymine was replaced by

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deoxyuridine tr iphosphate labeled with biotin or digoxigenin) or by a transamhaation reaction resulting in a sulfone group on the cytosine moieties. Although some probes were directly conjugated with fluorescent molecules, the most widespread approach was the labeling of probes with reporter molecules that, after hybridization, bind fluorescent affinity reagents. In situ hybridizat ion was achieved by the simultaneous denaturation of both the DNA of the target cell and the labeled probe. Incubation of the probe and target DNA together at a temperature below the melting point al lowed the chemically modified probe to anneal to complementary sequences in the target. Following their hybridizat ion with target DNA sequences, these probes were visualized immunocytochemically using fluorochromes such as fluorescein isothiocyanate (yellow-green fluorescence) or tetramethyl rhodamine isothiocyanate (red fluorescence) as reporter molecules. Counterstaining of cells was performed with DNA-specific dyes such as 4'-6-diamidino- 2-phenylindole (DAPI) or propid ium iodide. Binding of several was detected simultaneously by properly employing different probe labels (e.g., biotin and digoxygenin).

The use of FISH on sperm cells is more complex due to the inaccessibility of sperm DNA and the interpretation of the results. Our group has introduced a new method, consisting of a standard, routinely applicable protocol for sperm decondensation, restoring morphology. The detection method is based on brightfield microscopy, allowing counterstaining after ISH. This allows simultaneous nondisjunction and morphology studies (Martini et al., 1995).

RESULTS

Spermatozoa

Aneuplo idy rates have been shown to vary from 0.03% for the X-chromosome to about 1% for chromosome No. 1 (Pieters and Geraedts, 1994). A wide range was observed in the percentages of disomy for the different chromosomes. The median value for all chromosomes reported is in the range of about 0.3%.

In a recent s tudy it was shown by Pang et al. (1995) that the percentage of numerical abnormalities was more than ten-fold increased in a group of patients showing oligoasthenoteratozoospermia (OAT).

Oocytes

Since the first report on the study of meiosis in human oocytes in 1968, a large number of publications deal with the cytogenetic analysis of inseminated oocytes that failed to fertilize in the framework of an IVF program. Data on 2434 oocytes, reported by 12 groups have shown that the total incidence of chromosome anomalies ranges from 8.1 to 54.2% with an average of 26.5%: 13.3% hypohaploidy, 8.1% hyperhaploidy, 1.6 % structural abnormalit ies and 3.5% diploidy (Plachot, 1995). These results are different from those obtained by Angell (1994). She studied 400 oocytes from 125 women showing no sign of fertilization or cleavage. Of these 124 were unanalyzable and 79 were normal haploid. The remaining were all abnormal. However, not a single case of hyperhaplo idy was noted.

Cytogenetics of Gametes and Embryos 15

Zygotes

About 20 hours after insemination during IVF the zygotes are studied for the presence of pronuclei. The two main categories of abnormalities are parthenogenetic activation (1 pronucleus and tr iploidy (3 pronuclei). Tripronulear zygotes are eliminated from culture and frequently used for studies. It was shown that the vast majority results from dispermy (Plachot et al., 1989).

Preimplantation Embryos

The first cytogenetic studies of early embryos were carried out using classical techniques. To obtain sufficient metaphases cleaving embryos were required. Only a minority of the embryos studied this way could be analyzed. Furthermore, the proport ion of cases in which results were obtained in all cells was even much smaller (Jamieson et al., 1994; Pellistor et al., 1994). The results of these studies are different with respect to the percentages of abnormalities, which were 22.5% and 90%, respectively.

From the study of preimplantat ion embryos arising from tripronuclear zygotes it has become clear that they display a variety of chromosomal abnormalities (Pieters et al., 1992). They include 1) complete triploidy in all cells after regular division, 2) gross abnormalities in all cells due to chaotic chromosome movement after mult ipolar spindle division, 3) cell subpopulat ions with either a haploid or a diploid chromosomal content because of extrusion of a haploid nucleus during the first cleavage division and 4) cell subpopulat ions with a diploid or a triploid chromosomal content as a result of extrusion of a haploid nucleus dur ing the first cleavage division and subsequent incorporation in one of the two nuclei. FISH studies performed on nuclei of embryos resulting from abnormal fertilization revealed mostly mosaic chromosome complements (Coonen et aI., 1994; Harper et al., 1994).

The development of FISH technology resulted in the use of arrested human embryos as study material. Since interphase cells could be s tudied with high efficiency it was possible to draw conclusions about the presence of normal and abnormal chromosome copy numbers. Besides these, in quite a proport ion of embryos it was shown that more than one cell type was present. These so called mosaicisms are generated in almost all cases at the second or later divisions when they are monospermic diploid. However, when they are polyploid or haploid mosaic they usually originate at the first divisions (Munn6 et al., 1994).

DISCUSSION

Cytogenetic analysis of human gametes, zygotes and early embryos is an important tool for the investigation of formation, transmission and etiology of chromosomal investigations. However, for cytogenetic analysis, metaphase-arresteci nuclei are essential. In contrast, when using appropria te DNA probes, in situ hybridizat ion methodologies can rapidly and efficiently be applied for the identification of numerical and even structural abnormalities in interphase nuclei. ISH and FISH play an increasingly important role in a variety of research areas, including cytogenetics, prenatal diagnosis, tumor biology, gene amplification and gene mapping.

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In situ hybridizat ion of sperm nuclei proved to be efficient and promising with respect to the detection of a broad variety of studies, including the effects of environmental, occupational and therapeutic agents.

It can also be applied to study the sex chromosome constitution of spermatozoa, for example to control the results obtained with sperm separation methods.

The frequencies of disomic sperm cells show large variations between studies. This can be attributed to the following technical factors: decondensation and denaturation of the DNA in the specimen, distortion of the morphology after pretreatment leading to difficulties in the interpretat ion of the distinction between disomic and diploid sperm. This problem can be circumvented or solved by the combined use of an ISH method and a morphological staining method or double hybridizat ion (Martini et al., 1995).

If the observed 0.3% is truly representative for the d isomy rate among sperm, an overall percentage of 6.9 would be found using the FISH technique. This figure is in the range of the findings using the zona free hamster technique (Martin et al., 1990; Pellestor, 1991).

Besides technical factors explaining a proport ion of the variation observed, it should not be forgotten that also differences might be observed between patients. The finding of the frequency of abnormalities in the patients showing OAT has to be considered as an important complication for ICSI, which nowadays is the method of choice for the treatment of the infertility problem of these patients.

For ethical reasons only polypronuclear embryos can be studied. As expected the majority of these is chromosomally abnormal. However, about 18% of the cells might be normal (Pellestor, 1995).

With respect to the abnormali ty rate of embryos a discrepancy is clearly present when one compares the results obtained by Jamieson et al. (1994) and Pellestor et al. (1994). This seems to result from one important factor being the morphology. The percentage of abnormalities was more than two times higher in poor quality embryos than in good quality embryos (Pellestor, 1995).

Furthermore it was shown that aneuploidy increases significantly (p<0.001) with maternal age. This corroborates the hypothesis that oocytes of older women are more prone to non-disjunction caused by meiotic errors at the gamete level (Munn6 et al., 1995).

The observation that mosaicism is present in a large proport ion of embryos has consequences for preimplantat ion diagnosis. It is recommended to use two blastomeres if available. In case results from both cells are conflicting, the embryo should not be considered suitable for transfer. However, it should be kept in mind that if no mosaicism has been detected, chromosomal mosaicism in the remaining cells can never be totally excluded. Furthermore, the presence of mosaicism also depends on the developmental capacity.

Cytogenetics of Gametes and Embryos 17

SUMMARY

By the introduction of in vitro fertilization and other artificial reproductive technology methods it has become possible to study, gametes, zygotes and preimplantation embryos. It has been estimated that the contribution of aneuploid oocytes and spermatozoa is about 26% and 7%, respectively. Furthermore, between 5 and 10% of all in vitro fertilizations result in a zygote showing three in stead of two pronuclei after 20 hours. In most cases the origin of triploidy is dispermy. In total about 40% of all zygotes is chromosomally abnormal. Also after the first cleavage division abnormalities might occur. These will result in an early embryo showing mosaicism.

REFERENCES

Angell, R.R. (1994) Cytogenetic analysis of unfertilized human oocytes. In: Gamete and Embryo Quality (ed.) L. Mastroianni Jr., H.J.T. Coding Bermink, S. Suzuki, and H.M. Vemer, London. Parthenon, pp. 47-62.

Bou6, J., Bou6, A., and Lazar, P. (1975) Retrospective and prospective epidemiological studies of 1500 karyotyped abortions. Teratology 12, 11-26.

Coonen, E., Harper, J.C., Delhanty, J.D.A., Ramaekers, F.C.S., Geraedts, J.P.M., Hopman, A.H.N. and Handyside, A.H. (1994) Presence of chromosomal mosaicism in abnormal preimplantation embryos detected by fluorescence in situ hybridization. Hum. Genet. 94, 609-615.

Eiben, B., Bartels, I., Bfihr-Porsch, S., Borgmann, S., Gatz, G., Gellert, G., Goebel, R., Hammans, W., Hentemann, M., Osmers, R., Rauskolb, R., and Hansmann, I. (1990) Cytogenetic analysis of 750 spontaneous abortions with the direct preparation method of chorionic villi and its implications for studying genetic causes of pregnancy wastage. Am. J. Hum. Genet. 47, 656-663.

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Hook, E.B. (1981) Rates of chromosome abnormalities at different maternal ages. Obstet. GynaecoI. 58, 282-285.

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Mikamo, K. and Kamiguchi, Y. (1983) A new assessment system for chromosomal mutagenicity using oocytes and early zygotes of the Chinese hamster. In: Radiation-induced Damage in Man, (eds.) T. Ishihara, T. and M. Sasaki, New York: Alan R. Liss, pp. 411-432.

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