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A gradual fixation method for chromosomal preparations of human oocytes*
Vol. 41, No.5, May 1984 Printed in U.SA.
FERTILITY AND STERILITY Copyright C> 1984 The American Fertility Society
A gradual fixation method for chromosomal preparations of human oocytes*
H8.kan Wramsby, M.D.t Percy Liedholm, M.D. Department of Obstetrics and Gynecology, General Hospital, University of Lund, Malmo, Sweden
Eleven oocytes were recovered after follicle aspiration carried out in nine women during laparoscopy or laparotomy. All oocytes were classified as preovulatory,judged by the fully dissociated cumulus. After elimination of the cumulus, ten of the oocytes exhibited the first polar body, and one was degenerated. A gradual fixation method was used for the chromosomal preparation of the oocytes. The quality of the preparations was improved, compared with the previously used air-drying technique. Nine of ten preparations from oocytes with first polar body displayed chromosomes. These sets consisted of well-spread meiotic chromosomes, and in six of them the chromosome complements of the first polar body could be seen, although quite distinct from the oocyte complement. Of eight preparations where the chromosomes could be counted, six showed 23 chromosomes, and two were abnormal, showing 21 and 16 chromosomes. Fertil Steril41:736, 1984
A high incidence of chromosomal abnormalities in human spontaneous abortions 1 is considered to be one significant reason for the low estimated human fecundity (- 25%).2 Most of these aberrations originate after abnormal division during oogenesis. 3 These reports and the increasing use of in vitro fertilization in human infertility cases emphasizes the interest of karyotyping human oocytes. However, no reliable method for setting up chromosome preparations of human oocytes has yet been described. The air-drying technique originally developed for mouse eggs was initially suggested to be applicable to eggs of all mam-
Received September 28, 1983; revised and accepted January 18,1984. *Supported by a grant from the Medical Faculty, University of Lund. tReprint requests: HBkan Wramsby, M.D~, Department of Obstetrics and Gynecology, General Hospital, S-214 01 Malmo, Sweden. 736
mals, up to the morula stage. 4 The success rates achieved with such preparations have been generally low. 5 -s Here we report our first results from human oocytes when using a modification of the gradual fixation method described for the Chinese hamster and mouse9 and for the squirrel monkey. 10 MATERIALS AND METHODS
Preovulatory follicles were aspirated at laparoscopy or laparotomy performed because of infertility in nine normally menstruating women in whom ovulation had been stimulated with clomiphene (Clomivid, Draco, Lund, Sweden), 150 mg/ day, on days 3 to 9, and given 4000 IU human chorionic gonadotropin (Gonadex, Leo, Helsingborg, Sweden) 35 to 36 hours before recovery. A Teflon-coated aspiration needle (R6.865, Rocket, Watford Herts, England) was used. The cumulus of each oocyte was assessed in a dissection micro-
Wramsby and Liedholm Chromosomal preparations of human oocytes
Fertility and Sterility
scope, and oocytes with a fully dissociated cumulus were then incubated in 1 ml Earle's medium l l with 10% heat-inactivated serum (EI0 medium) in an incubator gassed with 5% O2 , 5% CO 2 , and 90% N 2 , pH 7.4. In our in vitro fertilization program oocytes are inseminated after 5 hours of preincubation, consistent with the findings of Trounson et al. 12 Thus, in this study the clinical situation was imitated and, after 5 hours, oocytes were transferred to 0.7 ml EI0 medium with hyaluronidase (300 IV/mD and further incubated for 20 minutes to remove cumulus cells. The oocytes were freed from the last cumulus cells mechanically using a micropipette. The presence of the first polar body was checked, whereafter the 00cytes were transferred to 1 ml of 1% sodium citrate for 10 minutes of hypotonic treatment. During this time, fresh fixatives were mixed, fixative A consisting of distilled water, acetic acid, and ethanol (5:1:4) and fixative B consisting of ethanol and acetic acid (3:1). The use of trypsin or other enzymatic treatment of the zona pellucida is not necessary. Each oocyte was transferred in a minimal amount of hypotonic fluid to fixative A and was observed during fixation under a dissection microscope. Immediately the oocyte took on a milky appearance but in 5 to 10 seconds turned more transparent. It was left in fixative A for 30 to 40 seconds and in order to prevent it from sticking to the bottom of the dish, we gently pipetted it up and down with a mouth-controlled micropipette. The oocyte was then moved to a grease-free glass slide. Three drops of fixative B were expelled onto the oocyte, and the drying of this fixative was hastened by placing the slide on a warming plate at 37 0 C. The quality of the preparation was checked immediately in a phase-contrast microscope. Quinacrine mustard staining was performed not earlier than 14 days after fixation. RESULTS
From nine women, 11 oocytes were recovered. The mean amount of follicular fluid was 7.9 ml (range, 1 to 23 mI). All oocytes were judged to be preovulatory from the appearance of their cumulus. After hyaluronidase treatment, all but one oocyte displayed the first polar body. This one oocyte appeared fragmented and was considered degenerated. After chromosomal preparation of the ten oocytes where the presence of meiotic chromosomes could be expected-from the presVol. 41, No.5, May 1984
Figure 1 Chromosomes of a mature human oocyte. Some of the polar body chromosomes are seen at the lower right and are quite distinct from the 23 chromosomes of the oocyte.
ence of the first polar body-nine showed chromosomes, and in these preparations well-spread complements were found. In six preparations, chromosome complements of the first polar body were seen, although in each of these cases quite distinct from the oocyte complement (Fig. 1). In one preparation, the complement of the first polar body interfered with that of the oocyte and made it impossible to determine the number of oocyte chromosomes. Of the eight preparations where chromosomes could be counted, six showed 23 chromosomes, and two were abnormal and showed 21 and 16 chromosomes. DISCUSSION
Although all oocytes included in this study were considered preovulatory from the appearance of the cumulus cell masses, one oocyte was degenerated, and the remaining ten appeared normal after removal of the cumulus cells; i.e., there was a normal ooplasm and one polar body. This finding is in agreement with the observation of Testart et al. 13 and may represent one of the reasons why the rate of fertilization in vitro is never 100%, since the appearance of the cumulus cells surrounding the oocyte is an indirect sign of its condition. The gradual fixation technique used in this study yielded a high rate of well-spread chromosome plates from nine often oocytes where chromosomes could be expected. Of the nine 00cytes with meiotic chromosome complements, six were observed to have an additional metaphase plate belonging to the first polar body. These
Wramsby and Liedholm Chromosomal preparations of human oocytes
737
chromosome plates were easy to discern on the basis of their location, their fuzzy and indistinct appearance, and the extent to which they were spread. When we applied the air-drying technique described by Tarkowsky4 on human oocytes matured in vitro, 5 of 11 preparations could be analyzed. 8 The gradual fixation technique has been reported to allow chromosome preparations of animal oocytes without disruption of the cytoplasmic membranes. This also seems to be true when the method now has been modified for human oocytes and might explain its high reliability. The proportion of normal/abnormal chromosome numbers in this small series does not allow extensive conclusions. The numeric abnormalities found do not correspond to the abnormal karyotypes of spontaneous abortuses where only monosomy, trisomy, or polyploidy has been found.! Thus, oocytes carrying such serious numeric abnormalities will probably not, if fertilized, develop to recognizable pregnancies. The two chromosomally abnormal oocytes were recovered from the same woman, who also yielded one oocyte with 23 chromosomes. This might indicate that ovulation induction may increase the risk of ovulation of abnormal oocytes. However, more extensive studies are needed to find the incidence of chromosomally abnormal human oocytes.
738
REFERENCES 1. Boue J, Boue A, Lazar P: Retrospective and prospective epidemiological studies of 1500 karyotyped human abortions. Teratology 12:11, 1975 2. Edmonds DK, Lindsay KS, Miller JF, Williamson E, Wood PJ: Early embryonic mortality in women. Fertil Steril 38:447, 1982 3. Carr DH: Chromosome studies as a cause of spontaneous, abortion. Am J Obstet Gynecol 97:283, 1967 4. Tarkowsky AK: An air-drying method for chromosome preparations from mouse eggs. Cytogenetics 5:394, 1966 5. McGaughey RW, Chang MC: Meiosis of mouse eggs before and after sperm penetration. J Exp Zool 170:397, 1969 6. Donahue RP: Cytogenetic analysis of the first cleavage division in mouse embryos. Proc Natl Acad Sci USA 69:351,,1973 7. Gosden RG: Chromosomal anomalies of preimplantation mouse embryos in relation to maternal age. J Reprod Fertil 35:351, 1973 8. Wramsby H, Hansson A, Liedholm P: Chromosome preparations from in vitro matured human oocytes using a simple air-drying technique. Clin Reprod Fertil 1:323, 1982 9. Kamiguchi Y, Funaki K, Mikamo K: A new technique for chromosome study of murine oocytes. Proc Jpn Acad 52:316, 1976 10. Mizoguchi H, Dukelow WR: Gradual fixation method for chromosomal studies of squirrel monkey oocytes after gonadotropin treatment. J Med Primatol 10:180, 1981 11. Purdy JM: Methods for fertilization and embryo culture in vitro. In Human Conception In Vitro, Edited by RG Edwards, JM Purdy. London, Academic Press, 1982, p 135 12. Trounson AO, Mohr LR, Wood C, Leeton JF: Effect of delayed insemination on in vitro fertilization culture and transfer of human embryos. J Reprod Fertil 64:285, 1982 13. Testart J, Frydman R, DeMouzon J, Lassalle B, Belaisch JC: A study of factors affecting the success of human fertilization in vitro. I. Influence of ovarian stimulation upon the number and condition of oocytes collected. BioI Reprod 28:415, 1983
Wramsby and Liedholm Chromosomal preparations of human oocytes
Fertility and Sterility
FERTILITY AND STERILITY Copyright C> 1984 The American Fertility Society
A gradual fixation method for chromosomal preparations of human oocytes*
H8.kan Wramsby, M.D.t Percy Liedholm, M.D. Department of Obstetrics and Gynecology, General Hospital, University of Lund, Malmo, Sweden
Eleven oocytes were recovered after follicle aspiration carried out in nine women during laparoscopy or laparotomy. All oocytes were classified as preovulatory,judged by the fully dissociated cumulus. After elimination of the cumulus, ten of the oocytes exhibited the first polar body, and one was degenerated. A gradual fixation method was used for the chromosomal preparation of the oocytes. The quality of the preparations was improved, compared with the previously used air-drying technique. Nine of ten preparations from oocytes with first polar body displayed chromosomes. These sets consisted of well-spread meiotic chromosomes, and in six of them the chromosome complements of the first polar body could be seen, although quite distinct from the oocyte complement. Of eight preparations where the chromosomes could be counted, six showed 23 chromosomes, and two were abnormal, showing 21 and 16 chromosomes. Fertil Steril41:736, 1984
A high incidence of chromosomal abnormalities in human spontaneous abortions 1 is considered to be one significant reason for the low estimated human fecundity (- 25%).2 Most of these aberrations originate after abnormal division during oogenesis. 3 These reports and the increasing use of in vitro fertilization in human infertility cases emphasizes the interest of karyotyping human oocytes. However, no reliable method for setting up chromosome preparations of human oocytes has yet been described. The air-drying technique originally developed for mouse eggs was initially suggested to be applicable to eggs of all mam-
Received September 28, 1983; revised and accepted January 18,1984. *Supported by a grant from the Medical Faculty, University of Lund. tReprint requests: HBkan Wramsby, M.D~, Department of Obstetrics and Gynecology, General Hospital, S-214 01 Malmo, Sweden. 736
mals, up to the morula stage. 4 The success rates achieved with such preparations have been generally low. 5 -s Here we report our first results from human oocytes when using a modification of the gradual fixation method described for the Chinese hamster and mouse9 and for the squirrel monkey. 10 MATERIALS AND METHODS
Preovulatory follicles were aspirated at laparoscopy or laparotomy performed because of infertility in nine normally menstruating women in whom ovulation had been stimulated with clomiphene (Clomivid, Draco, Lund, Sweden), 150 mg/ day, on days 3 to 9, and given 4000 IU human chorionic gonadotropin (Gonadex, Leo, Helsingborg, Sweden) 35 to 36 hours before recovery. A Teflon-coated aspiration needle (R6.865, Rocket, Watford Herts, England) was used. The cumulus of each oocyte was assessed in a dissection micro-
Wramsby and Liedholm Chromosomal preparations of human oocytes
Fertility and Sterility
scope, and oocytes with a fully dissociated cumulus were then incubated in 1 ml Earle's medium l l with 10% heat-inactivated serum (EI0 medium) in an incubator gassed with 5% O2 , 5% CO 2 , and 90% N 2 , pH 7.4. In our in vitro fertilization program oocytes are inseminated after 5 hours of preincubation, consistent with the findings of Trounson et al. 12 Thus, in this study the clinical situation was imitated and, after 5 hours, oocytes were transferred to 0.7 ml EI0 medium with hyaluronidase (300 IV/mD and further incubated for 20 minutes to remove cumulus cells. The oocytes were freed from the last cumulus cells mechanically using a micropipette. The presence of the first polar body was checked, whereafter the 00cytes were transferred to 1 ml of 1% sodium citrate for 10 minutes of hypotonic treatment. During this time, fresh fixatives were mixed, fixative A consisting of distilled water, acetic acid, and ethanol (5:1:4) and fixative B consisting of ethanol and acetic acid (3:1). The use of trypsin or other enzymatic treatment of the zona pellucida is not necessary. Each oocyte was transferred in a minimal amount of hypotonic fluid to fixative A and was observed during fixation under a dissection microscope. Immediately the oocyte took on a milky appearance but in 5 to 10 seconds turned more transparent. It was left in fixative A for 30 to 40 seconds and in order to prevent it from sticking to the bottom of the dish, we gently pipetted it up and down with a mouth-controlled micropipette. The oocyte was then moved to a grease-free glass slide. Three drops of fixative B were expelled onto the oocyte, and the drying of this fixative was hastened by placing the slide on a warming plate at 37 0 C. The quality of the preparation was checked immediately in a phase-contrast microscope. Quinacrine mustard staining was performed not earlier than 14 days after fixation. RESULTS
From nine women, 11 oocytes were recovered. The mean amount of follicular fluid was 7.9 ml (range, 1 to 23 mI). All oocytes were judged to be preovulatory from the appearance of their cumulus. After hyaluronidase treatment, all but one oocyte displayed the first polar body. This one oocyte appeared fragmented and was considered degenerated. After chromosomal preparation of the ten oocytes where the presence of meiotic chromosomes could be expected-from the presVol. 41, No.5, May 1984
Figure 1 Chromosomes of a mature human oocyte. Some of the polar body chromosomes are seen at the lower right and are quite distinct from the 23 chromosomes of the oocyte.
ence of the first polar body-nine showed chromosomes, and in these preparations well-spread complements were found. In six preparations, chromosome complements of the first polar body were seen, although in each of these cases quite distinct from the oocyte complement (Fig. 1). In one preparation, the complement of the first polar body interfered with that of the oocyte and made it impossible to determine the number of oocyte chromosomes. Of the eight preparations where chromosomes could be counted, six showed 23 chromosomes, and two were abnormal and showed 21 and 16 chromosomes. DISCUSSION
Although all oocytes included in this study were considered preovulatory from the appearance of the cumulus cell masses, one oocyte was degenerated, and the remaining ten appeared normal after removal of the cumulus cells; i.e., there was a normal ooplasm and one polar body. This finding is in agreement with the observation of Testart et al. 13 and may represent one of the reasons why the rate of fertilization in vitro is never 100%, since the appearance of the cumulus cells surrounding the oocyte is an indirect sign of its condition. The gradual fixation technique used in this study yielded a high rate of well-spread chromosome plates from nine often oocytes where chromosomes could be expected. Of the nine 00cytes with meiotic chromosome complements, six were observed to have an additional metaphase plate belonging to the first polar body. These
Wramsby and Liedholm Chromosomal preparations of human oocytes
737
chromosome plates were easy to discern on the basis of their location, their fuzzy and indistinct appearance, and the extent to which they were spread. When we applied the air-drying technique described by Tarkowsky4 on human oocytes matured in vitro, 5 of 11 preparations could be analyzed. 8 The gradual fixation technique has been reported to allow chromosome preparations of animal oocytes without disruption of the cytoplasmic membranes. This also seems to be true when the method now has been modified for human oocytes and might explain its high reliability. The proportion of normal/abnormal chromosome numbers in this small series does not allow extensive conclusions. The numeric abnormalities found do not correspond to the abnormal karyotypes of spontaneous abortuses where only monosomy, trisomy, or polyploidy has been found.! Thus, oocytes carrying such serious numeric abnormalities will probably not, if fertilized, develop to recognizable pregnancies. The two chromosomally abnormal oocytes were recovered from the same woman, who also yielded one oocyte with 23 chromosomes. This might indicate that ovulation induction may increase the risk of ovulation of abnormal oocytes. However, more extensive studies are needed to find the incidence of chromosomally abnormal human oocytes.
738
REFERENCES 1. Boue J, Boue A, Lazar P: Retrospective and prospective epidemiological studies of 1500 karyotyped human abortions. Teratology 12:11, 1975 2. Edmonds DK, Lindsay KS, Miller JF, Williamson E, Wood PJ: Early embryonic mortality in women. Fertil Steril 38:447, 1982 3. Carr DH: Chromosome studies as a cause of spontaneous, abortion. Am J Obstet Gynecol 97:283, 1967 4. Tarkowsky AK: An air-drying method for chromosome preparations from mouse eggs. Cytogenetics 5:394, 1966 5. McGaughey RW, Chang MC: Meiosis of mouse eggs before and after sperm penetration. J Exp Zool 170:397, 1969 6. Donahue RP: Cytogenetic analysis of the first cleavage division in mouse embryos. Proc Natl Acad Sci USA 69:351,,1973 7. Gosden RG: Chromosomal anomalies of preimplantation mouse embryos in relation to maternal age. J Reprod Fertil 35:351, 1973 8. Wramsby H, Hansson A, Liedholm P: Chromosome preparations from in vitro matured human oocytes using a simple air-drying technique. Clin Reprod Fertil 1:323, 1982 9. Kamiguchi Y, Funaki K, Mikamo K: A new technique for chromosome study of murine oocytes. Proc Jpn Acad 52:316, 1976 10. Mizoguchi H, Dukelow WR: Gradual fixation method for chromosomal studies of squirrel monkey oocytes after gonadotropin treatment. J Med Primatol 10:180, 1981 11. Purdy JM: Methods for fertilization and embryo culture in vitro. In Human Conception In Vitro, Edited by RG Edwards, JM Purdy. London, Academic Press, 1982, p 135 12. Trounson AO, Mohr LR, Wood C, Leeton JF: Effect of delayed insemination on in vitro fertilization culture and transfer of human embryos. J Reprod Fertil 64:285, 1982 13. Testart J, Frydman R, DeMouzon J, Lassalle B, Belaisch JC: A study of factors affecting the success of human fertilization in vitro. I. Influence of ovarian stimulation upon the number and condition of oocytes collected. BioI Reprod 28:415, 1983
Wramsby and Liedholm Chromosomal preparations of human oocytes
Fertility and Sterility