GAMETE AND EMBRYO QUALITY
Role in fertilization failures and reproductive pathology: the contribution of Fluorescent In Situ Hybridization (FISH)
M. Benkhalifa and Y. Menezo
Centre de Biologie médicale - Fondation Marcel Merieux
94 rue Chevreul - B.P. 7322 - 69357 Lyon Cedex, France
One of the main problems in assisted reproductive technology is the quality of gametes and embryos. The possibility that embryos may be maintained and cultivated in vitro is of vital importance, both in clinical medicine and animal husbandry. The concept of embryo quality is difficult to define but we cannot assert that embryo quality is optimal unless two criteria are met: (1) a chronologically and morphologically regular cleavage occurs which is necessary, but not sufficient, and (2) viable offspring develop after embryo transfer.
The future of the embryo is difficult to evaluate on the basis of gamete quality; in some cases, even if fertilization has occurred, no further development may occur. Developmental arrests can be observed at different embryonic stages. It is now clear that most developmental problems are related to chromosome aberrations.
Current research in cytogenetics and molecular biology has demonstrated the role of chromosome aberration in reproductive failure. Abnormalities occurring during gametogenesis, and the first stages of development play a significant role in infertility, in vitro fertilization failure and fetal loss. Consequently, the cytogenetic study of human germ cells and embryos is relevant to the field of human reproduction, as it contributes to elucidate the mechanism of chromosome aberrations. Recently, fluorescent in situ hybridization (FISH) after labelling by non radioactive probes has been introduced in clinical cytogenetic and molecular biology (2,4,5,10,12,16).
The advantages of FISH are short delays and accuracy for chromosomal or partial chromosomal identification. The present study by classical cytogenetic and FISH techniques was designed to estimate chromosome abnormalities in human gametes in order to assess the contribution of oocytes and spermatozoa, the aberration ratio at the zygotic stage, and during the first steps of development following coculture.
Insufficient maturation of spermatozoa may result in defective embryonic development (8). This is very probably true in man for in vitro fertilization (IVF).
Sperm handling and storage techniques may also cause developmental anomalies after fertilization by the artificial ageing of the male gamete (13,15).
Anti-sperm antibodies as well as poor quality sperm may interfere severely with the pregnancy outcome (6).
Biotin labelled probes give a significant chromosome hybridization signal in sperm nuclei. With FISH, we estimate the aneuploidy rate of chromosome 1 at 0.7%. The average proportion of X and Y-bearing sperm nuclei are 49.3% and 50.7% respectively.
The study of aneuploidies in human spermatozoa has been performed by using differential staining methods followed by fertilization of zona-free hamster eggs. On the other hand in situ hybridization has been applied in the same field (3).
FISH appears to be a satisfactory additional tool for the study of aneuploidies and the sex ratio in human sperm. This method is especially suited for the diagnosis of numerical chromosome aberrations. Two hundred and forty human sperm metaphases have been studied by the hamster technique. Results showed that 13.3% karyotypes had chromosomal abnormalities, and 2.6% structural aberrations. The gonosome ratio among spermatozoa was 51.7% X and 48.3% Y. The rate of abnormalities in sperm cells corresponds to data currently available. We observed a discordance between the incidence of structural and numerical abnormalities in human spontaneous abortions and human sperm analyzed by in vitro hamster-egg fertilization.
It has been shown over the last few years that maturation in the follicle may be independent of ovulation. The final stages of maturation of the cytoplasm, membrane and nucleus are sometimes crucial for fertilization, but are always crucial for the subsequent development of the fertilized ovum (19). This is especially the case in gonadotropic stimulation (7,9,11).
Oocyte ageing before fertilization is the main factor responsible for chromosomal anomalies. It generally arises in zootechnical procedures by delaying insemination in relation to ovulation (11). In IVF practice, hazardous manipulation may lead to such anomalies. It is very difficult to estimate the quality of oocytes at the time of ovum retrieval as there is no correlation between cumulus maturation and true oocyte quality.
Fifteen metaphase-II oocytes were studied by FISH with X, 18 and 21 probes: 3 oocytes were aneuploid: 24X, +X; 22X, -18: 24X, +21. Using classical techniques, an interpretable karyotype was obtained from 341 (73.3%) of 465 oocytes. These oocytes were classified according to a cytological criterion of maturity (presence or absence of the first polar body); 39 oocytes displaying no polar body were judged immature. Chromosome analysis showed 8 oocytes in diakinesis, and 31 metaphase-II diploid oocytes, including 10 presenting prematurely condensed paternal chromosomes into single chromatids (PCC).
Among the 302 oocytes having extruded the first polar body, and presumably in metaphase-II, 24.8% were aneuploid (9.9% hypohaploid, 14.9% hyperhaploid). Mainly small chromosomes were involved. Sperm chromosomes presenting single chromatids were present in 28.1% of the 302 oocytes.
These studies point out a higher rate of hyperhaploidy in the oocytes in metaphase-II. These findings have been confirmed in our own research. Our observations of the first group of 39 oocytes also corroborated these results and demonstrated the possibility of metaphase-II diploidy without extrusion of the first polar body, and the more frequent occurrence of hyperhaploidy for small chromosomes which are more exposed to non-disjunction.
The results of this last study and our data are inconsistent with the correlation reported between maternal age and the rate of abnormalities. Our two techniques of stimulation resulted in a similar rate of aneuploidy, and did not affect the pre-existing chromosome equilibrium of the oocyte.
In IVF protocols, approximately 25% of the collected oocytes after ovarian stimulation fail to cleave 48 hours after insemination. Cytogenetic analysis of these human eggs is a useful tool to assess the rate of aneuploidy at different stages of female meiosis.
The two most useful predictors of viability of preimplantation embryos are (i) normal morphology, and (ii) fast cleavage rate. As there are often faults in assessing these morphological parameters, a good understanding of metabolism becomes an additional prerequisite for any test of embryo quality. Theoretically, any molecule incorporated by the embryo may serve as a basis for a quality test. Because of the size of the embryo, this approach is not that easy. The problem is then to develop a sufficiently sensitive system to quantify the transport of these molecules between the egg and its culture medium (generally by using radioisotopes). For human eggs no tests are presently available, as the use of radioisotopes is not possible. In animals, several experiments have demonstrated that hexose uptake is useful to determine embryo quality in farm animals (17) (Table 1).
Another approach is the study of chemical messages released by the egg (early pregnancy factors [EPF], cytokins and other luteotrophic factors). These specific factors influence ovum transport and ovarian activity in vivo.
To increase embryonic quality in vitro, several coculture systems have recently been designed (14). These systems allow a selection in vitro of the best embryos. It is not easy to determine the future of the embryo in vivo as genital tract secretions may interfere positively, but also negatively with embryo development and implantation.
Early stage embryos.
Non transferable, non freezable embryos with four to eight blastomeres presenting morphological or cytological abnormalities were elected from IVF programs. A culture with colcemid was performed during 7 hours. Chromosome preparations were obtained using a variant of the air drying method of Tarkowsky (18).
Of 78 embryos processed, 47 (60%) were diploid after FISH with chromosome 13 and 21 probes. FISH with X probes displayed 21 male and 26 female embryos. Four embryos (5%) were haploid after FISH with a X probe. Twelve embryos (15%) were aneuploid after GIEMSA staining: FISH with 9 and 18 probes revealed one 9 trisomy, one 18 trisomy and one 9 monosomy. Fifteen embryos (19%) appeared polyploid after FISH with X or Y probes. This study confirmed that the development of zygotes may be interrupted at different stages of the first somatic division. This event may be correlated with cytoplasmic immaturity (1). The presence of PCC in 33% of these zygotes is noteworthy.
The biotin labelled DNA probes give significant chromosome hybridization signals in metaphase and interphase nuclei with all immunological systems used, both with phase contrast and fluorescence microscopy. In early embryos, we observed aneuploidies.
We recently demonstrated (4), that mixoploidy appears to be a normal feature in preimplantation embryos and that it occurs very early in human embryo development (at least at the morula blastocyst transition).
A viable embryo must pass harmoniously through several critical steps: maturation (mainly of the oocyte), fertilization, with anomalies that may only be manifest much later during development, and interactions between the egg and the female genital tract. The oviduct environment may sometimes have to compensate for certain temporary deficiencies of the new genome; the uterine regulations (both positive and negative) allow growth towards implantation.
Biotin labelled DNA probes allow precise identification of condensed metaphase chromosomes of oocytes, and direct genetic study of interphase nuclei of spermatozoa without the need of heterospecific fertilization (hamster test). In future, FISH will be a valuable tool for preimplantation diagnosis of chromosome abnormalities.
Investigation of the molecular mechanism of early embryonic defects, however interesting they may be, probably provide only a partial explanation of pregnancy success rates. Better knowledge of embryo quality is a prerequisite to better understanding of the extraordinary and complex series of interactions (male and female) that permit harmonious embryonic development from fertilization to birth.
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