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Cytogenetic investigations on human oocytes and early human embryonic stages
FERTILITY AND STERILITY Copyright © 1985 The American Fertility Society
Vol. 43, No.2, February 1985 Printed in U.8A.
Cytogenetic investigations on human oocytes and early human embryonic stages
Hans W. Miehelmann, Dr. se. agr. Liselotte Mettler, Prof. Dr. med. * Department of Obstetrics and Gynecology, University of Kiel, Kiel, Federal Republic of Germany
In the past, the investigation of chromosomes from human oocytes or early embryos was seldom possible. 1 ,2 However, with the advent of in vitro fertilization with embryo culture and embryo replacement, human oocytes and embryos are now available for study. Nevertheless, these oocytes and early embryos can only be used under certain circumstances. Chromosome analysis can only be performed if fertilization has not occurred or a normal embryo is not developing. MATERIALS AND METHODS
The fertilization of a human oocyte occurs during meiosis II. Fertilized and unfertilized eggs will either remain at metaphase II or will pass the pronucleus stage and will enter the first mitosis. If no further cell division takes place, the chromosomes will arrest at metaphase shortly before syngamy. A chromosome investigation according to the air-drying method of Tarkowski3 provides information about the success of fertilization and confirms whether or not a polyspermic reaction has taken place. In addition, the analysis of the chromosomes demonstrates structural and numeric aberrations. If oocytes have more than two pronuclei 16 to 20 hours after insemination, they are considered polyploid. The polyploidy, however, does not prevent the oocyte from under-
Received April 20, 1984; revised and accepted October 19, 1984. *Reprint requests: Professor Dr. med. L. Mettler, Department of Obstetrics and Gynecology, University of Kiel, D-2300 Kiel, Federal Republic of Germany. 320
Michelmann and Mettler Communications-in-brief
going normal cell cleavage and embryonic development. From September 1983 until April 1984, 88 pelviscopic examinations were performed at the Department of Obstetrics and Gynecology, University of Kiel, in patients between 22 and 40 years of age (mean, 33.5 years). The number of oocytes obtained was 285. Fifty-three embryo replacements with an average number of 2.2 embryos resulted: 6 are ongoing pregnancies, 1 pregnancy led to a miscarriage, and 6 replacements resulted in a so-called biochemical pregnancy. RESULTS
Of 285 oocytes, 161 oocytes from 37 patients (mean age, 30.9 years) remained uncleaved. A chromosome analysis was done on 66 undivided human oocytes and 8 polyploid embryos. In 59 cases this analysis was successful (Table 1). Fifteen metaphases could not be analyzed because of inferior quality, e.g., overlapping chromosomes, lack of chromosomes, or chromosome clumping. Thirty-three oocytes were unfertilized. They showed a haploid chromosome set. Reasons for the lack of fertilization cannot be correlated with andrologic parameters or the maturity of the 00cytes. In 15 eggs the diploid chromosome set could be analyzed. The reasons for the discontinuance of the embryonic development after fertilization remain unknown. Eleven of these oocytes showed the separate haploid metaphases shortly before chromosome syngamy. One trisomy was analyzed in the maternal chromosome set of a fertilized oocyte (Fig. 1). Because of the condensation stage of the chromosomes, the trisomic chromosome Fertility and Sterility
Table 1. Chromosome Analysis in Uncleaved Human Oocytes and Polyploid Embryos Haploid
n
=
23,X
n = 25,X,?+b
Oocyte no.
Diploid
32
2n
=
46,?,?a
1
n n
= =
23,? 23,X
n n
= =
23,? 24,X,?+
Oocyte no.
Polyploid
4 10
Oocyte no.
3n
=
69,X,?,?
2
5n
=
115
1
Polyploid
3n
=
69,X,?,?
Embryo no.
8
1
a?, Unidentified chromosome. b?+, Additional unidentified chromosome.
could not be analyzed by banding procedures. Eleven eggs with more than two pronuclei 10 to 20 hours after fertilization had multiple chromosome sets and were classified as polyploid. In spite of the fact that these oocytes had abnormal chromosome numbers, eight of them showed a normal cleavage rate and normal morphologic features.
Oocytes with rio further development will arrest in most cases during the metaphase of the first mitotic division. Therefore, during this phase of embryonic development a unique opportunity to check fertilization and to trace genome mutations back to their origins is created. The results of the . chromosome analysis of 51 uncleaved human 00cytes prove that there is no correlation between the fertilization progress, sperm parameters, oocyte maturity, and maternal age. Even the combination of mature eggs from young patients with sperm from normal donors does not necessarily lead to fertilization. Because of different condensation stages, maternal and paternal chromosomes differ in size and shape if they are investigated before syngamy. It is only during this phase that chromosomes of mature sperm can be analyzed. The presence of more than two pronuclei in connection with normal cell division has been ob-
DISCUSSION
Up-to-date chromosome analysis in human 00cytes or preimplantation embryos could be performed in some rare cases only. Angell et al.,! as well as Edwards, 2 examined only a few oocytes cytogenetically. So, for the first time, the present cytogenetic investigation deals with a high number of human eggs and preimplantation embryos. The technique used is established, easy to handle, and can be performed even by untrained personnel. The application of Colcemid is not necessary.
7r>" 8r> "" . -:. . . 11
2r> . •L'
1
31>. 4f>.
I.
91>. 14r>
-r.;;. 12 A
15
13
A
6
10f>'
16t>·. 17t>
18 r>'"
22t>I
•
20f>-
23t>
21 t> 24
Figure 1 Maternal metaphase with a chromosome set of 24 in a fer· tilized but uncleaved human oocyte.
Vol. 43, No.2, February 1985
Michelmann and Mettler Communications-in-brief
321
served in eight oocytes. A chromosome analysis could be performed and confirmed polyploidy. Normal-looking but polyploid 4- or 8-cell embryos therefore should not be replaced. SUMMARY
REFERENCES 1. Angell RO, Aitken RJ, van Look PFA, Lumsden MA, Templeton AA: Chromosome abnormalities in human embryos after in vitro fertilization. Nature 303:336,1983 2. Edwards RG: Chromosomal abnormalities in human embryos. Nature 303:283, 1983 3. Tarkowski AK: An air-drying method for chromosome preparations from mouse eggs. Cytogenetics 5:394, 1966
The chromosome analysis of undivided oocytes and polyploid embryos within a human in vitro fertilization and embryo replacement program offers a unique opportunity to develop correlations between a successful or unsuccessful fertilization and the maturity of the oocytes or the quality of the sperm. Furthermore, it provides information concerning the origin of chromosomal defects.
322
Michelmann and Mettler Communications-in-brief
Fertility and Sterility
Vol. 43, No.2, February 1985 Printed in U.8A.
Cytogenetic investigations on human oocytes and early human embryonic stages
Hans W. Miehelmann, Dr. se. agr. Liselotte Mettler, Prof. Dr. med. * Department of Obstetrics and Gynecology, University of Kiel, Kiel, Federal Republic of Germany
In the past, the investigation of chromosomes from human oocytes or early embryos was seldom possible. 1 ,2 However, with the advent of in vitro fertilization with embryo culture and embryo replacement, human oocytes and embryos are now available for study. Nevertheless, these oocytes and early embryos can only be used under certain circumstances. Chromosome analysis can only be performed if fertilization has not occurred or a normal embryo is not developing. MATERIALS AND METHODS
The fertilization of a human oocyte occurs during meiosis II. Fertilized and unfertilized eggs will either remain at metaphase II or will pass the pronucleus stage and will enter the first mitosis. If no further cell division takes place, the chromosomes will arrest at metaphase shortly before syngamy. A chromosome investigation according to the air-drying method of Tarkowski3 provides information about the success of fertilization and confirms whether or not a polyspermic reaction has taken place. In addition, the analysis of the chromosomes demonstrates structural and numeric aberrations. If oocytes have more than two pronuclei 16 to 20 hours after insemination, they are considered polyploid. The polyploidy, however, does not prevent the oocyte from under-
Received April 20, 1984; revised and accepted October 19, 1984. *Reprint requests: Professor Dr. med. L. Mettler, Department of Obstetrics and Gynecology, University of Kiel, D-2300 Kiel, Federal Republic of Germany. 320
Michelmann and Mettler Communications-in-brief
going normal cell cleavage and embryonic development. From September 1983 until April 1984, 88 pelviscopic examinations were performed at the Department of Obstetrics and Gynecology, University of Kiel, in patients between 22 and 40 years of age (mean, 33.5 years). The number of oocytes obtained was 285. Fifty-three embryo replacements with an average number of 2.2 embryos resulted: 6 are ongoing pregnancies, 1 pregnancy led to a miscarriage, and 6 replacements resulted in a so-called biochemical pregnancy. RESULTS
Of 285 oocytes, 161 oocytes from 37 patients (mean age, 30.9 years) remained uncleaved. A chromosome analysis was done on 66 undivided human oocytes and 8 polyploid embryos. In 59 cases this analysis was successful (Table 1). Fifteen metaphases could not be analyzed because of inferior quality, e.g., overlapping chromosomes, lack of chromosomes, or chromosome clumping. Thirty-three oocytes were unfertilized. They showed a haploid chromosome set. Reasons for the lack of fertilization cannot be correlated with andrologic parameters or the maturity of the 00cytes. In 15 eggs the diploid chromosome set could be analyzed. The reasons for the discontinuance of the embryonic development after fertilization remain unknown. Eleven of these oocytes showed the separate haploid metaphases shortly before chromosome syngamy. One trisomy was analyzed in the maternal chromosome set of a fertilized oocyte (Fig. 1). Because of the condensation stage of the chromosomes, the trisomic chromosome Fertility and Sterility
Table 1. Chromosome Analysis in Uncleaved Human Oocytes and Polyploid Embryos Haploid
n
=
23,X
n = 25,X,?+b
Oocyte no.
Diploid
32
2n
=
46,?,?a
1
n n
= =
23,? 23,X
n n
= =
23,? 24,X,?+
Oocyte no.
Polyploid
4 10
Oocyte no.
3n
=
69,X,?,?
2
5n
=
115
1
Polyploid
3n
=
69,X,?,?
Embryo no.
8
1
a?, Unidentified chromosome. b?+, Additional unidentified chromosome.
could not be analyzed by banding procedures. Eleven eggs with more than two pronuclei 10 to 20 hours after fertilization had multiple chromosome sets and were classified as polyploid. In spite of the fact that these oocytes had abnormal chromosome numbers, eight of them showed a normal cleavage rate and normal morphologic features.
Oocytes with rio further development will arrest in most cases during the metaphase of the first mitotic division. Therefore, during this phase of embryonic development a unique opportunity to check fertilization and to trace genome mutations back to their origins is created. The results of the . chromosome analysis of 51 uncleaved human 00cytes prove that there is no correlation between the fertilization progress, sperm parameters, oocyte maturity, and maternal age. Even the combination of mature eggs from young patients with sperm from normal donors does not necessarily lead to fertilization. Because of different condensation stages, maternal and paternal chromosomes differ in size and shape if they are investigated before syngamy. It is only during this phase that chromosomes of mature sperm can be analyzed. The presence of more than two pronuclei in connection with normal cell division has been ob-
DISCUSSION
Up-to-date chromosome analysis in human 00cytes or preimplantation embryos could be performed in some rare cases only. Angell et al.,! as well as Edwards, 2 examined only a few oocytes cytogenetically. So, for the first time, the present cytogenetic investigation deals with a high number of human eggs and preimplantation embryos. The technique used is established, easy to handle, and can be performed even by untrained personnel. The application of Colcemid is not necessary.
7r>" 8r> "" . -:. . . 11
2r> . •L'
1
31>. 4f>.
I.
91>. 14r>
-r.;;. 12 A
15
13
A
6
10f>'
16t>·. 17t>
18 r>'"
22t>I
•
20f>-
23t>
21 t> 24
Figure 1 Maternal metaphase with a chromosome set of 24 in a fer· tilized but uncleaved human oocyte.
Vol. 43, No.2, February 1985
Michelmann and Mettler Communications-in-brief
321
served in eight oocytes. A chromosome analysis could be performed and confirmed polyploidy. Normal-looking but polyploid 4- or 8-cell embryos therefore should not be replaced. SUMMARY
REFERENCES 1. Angell RO, Aitken RJ, van Look PFA, Lumsden MA, Templeton AA: Chromosome abnormalities in human embryos after in vitro fertilization. Nature 303:336,1983 2. Edwards RG: Chromosomal abnormalities in human embryos. Nature 303:283, 1983 3. Tarkowski AK: An air-drying method for chromosome preparations from mouse eggs. Cytogenetics 5:394, 1966
The chromosome analysis of undivided oocytes and polyploid embryos within a human in vitro fertilization and embryo replacement program offers a unique opportunity to develop correlations between a successful or unsuccessful fertilization and the maturity of the oocytes or the quality of the sperm. Furthermore, it provides information concerning the origin of chromosomal defects.
322
Michelmann and Mettler Communications-in-brief
Fertility and Sterility