Chromosome studies in human unfertilized oocytes and uncleaved zygotes after treatment with gonadotropin-releasing hormone analogs

FERTILITY AND STERILITY

Vol. 56, No. 5, November 1991

Copyright " 1991 The American Fertility Society

Printed on acid-free paper in U.S.A.

Chromosome studies in human unfertilized oocytes and uncleaved zygotes after treatment with gonadotropin-releasing hormone analogs

M. Isabel Tejada, Ph.D.* Rosario Mendoza, B.S. Beatriz Corcostegui, B.S. Jose Andres Benito, M.D. Human Reproduction Unit, Department of Obstetrics and Gynecology, Hospital de Cruces, Baracaldo, Vizcaya, Spain

Objective: To determine the frequency of the anomalies from the cytogenetic point of view in the oocytes remaining from our in vitro fertilization (IVF) program. Two gonadotropin-releasing hormone analogs (GnRH-a) were used (buserelin acetate and leuprolide acetate) in the superovulation treatment. Design: A prospective study was planned in January 1989. Deadline for data and quantitative analysis was to be July 1990. Setting: Hospital de Cruces, a public and tertiary institute. Patients: One hundred thirty-nine IVF patients, yielding 433 oocytes. Selected on the basis of availability of oocytes and staff. Results: Two hundred thirty-eight oocytes (71.25%) exhibited the normal number of metaphase II chromosomes; 64 (19.16%) exhibited aneuploidy; 13 (3.89%) were diploid, hyperdiploid, or hypodiploid; and 19 (5.68%) showed parthenogenetic activation. Of the 99 zygotes, 17 were polyploid and 48 showed prematurely condensed chromosomes, whereas in 31 cases the male and female pronuclei remained separate. Conclusions: It would not appear that the rate of chromosomal anomalies is affected after pituitary suppression with GnRH-a. Fertil Steril56:874, 1991

Cytogenetic studies of human oocytes became routine in the early 1980s1- 3 with the development of in vitro fertilization (IVF) techniques. Several groups4- 17 have reported their results. The aim of these studies has been to assess the contribution of chromosomal aberrations to the preimplantation loss of embryos and to the failure of fertilization produced in IVF programs. Chromosomal anomalies are known to constitute an important factor in human zygotic, embryonic, and fetal losses. 18 It is believed that these abnormalities are more frequent during the meiotic pro-

Received January 23, 1991; revised and accepted June 27, 1991.

* Reprint requests and present address: M. Isabel Tejada, Ph.D., Unidad de Genetica Humana, Santo Hospital Civil de Bilbao, Avda. Montevideo 18, 48013 Bilbao, Spain.

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Chromosomes in oocytes after analogs

cess, especially in females. 19 For unfertilized oocytes, aneuploidy rates ranged from 10% to 50%, 16 depending on technical characteristics and interpretation. Recently, some reports 14•20•21 have shown a higher incidence of chromosomal diploidy and increased detection of prematurely condensed chromosome (PCC) of sperm chromosomes, 22 even in apparently unfertilized oocytes. In the majority of surveys, patients were treated with clomiphene citrate (CC), human menopausal gonadotropin (hMG), and on many occasions both. More recently, Delhanty and Penketh17 have reported on oocytes retrieved after treatment with the gonadotropin-releasing hormone analog (GnRH-a) buserelin acetate. The results reported herein were obtained with 433 oocytes retrieved after treatment with two GnRH -a, buserelin acetate and leuprolide acetate Fertility and Sterility

(LA). The frequency of chromosome anomalies was similar to that reported by investigators working without analogs. 7 •9 •10 There was in addition a high level of postfertilization development failure. MATERIALS AND METHODS

Preovulatory follicular fluids were obtained from 139 women who had undergone IVF because of a variety of infertility problems, including tubal obstruction, endometriosis, ovulatory dysfunction, and male factor. Their ages ranged from 23 to 40 years, with a mean of 32.3. At present in our IVF program, to stimulate follicular growth, 98% of these patients are treated at random with two protocols involving GnRH-a and gonadotropins. One of these is buserelin acetate/hMG/human chorionic gonadotropin (hCG). The other is LA/follicle-stimulating hormone (FSH)/hMG/hCG, of which the doses have been described. 23 The 139 women whose oocytes and embryos we studied belong to this 98%. The remaining 2% of patients receive the conventional superovulation treatment with CC and hMG/hCG. 24 In the latter case, we did not have sufficient oocytes to use this group as a control, for which reason they are not included in the present study. The hCG (5,000 IU) was administered when follicular development was adequate (at least three follicles between 17 rom and 23 rom), in conjunction with a serum estradiol concentration of at least 1,000 pg/mL. Oocytes were retrieved by ultrasound-guided aspiration 32 to 36 hours after administration ofhCG. Oocytes were preincubated in four-well dishes, the first well being used in each case, for 2 to 4 hours. The medium was Inra Menezo B 2 ; (Api System S.A. 38390, Montalieu-Vercieu, France). The dishes were placed in an incubator gassed with C0 2 • The oocytes were then inseminated with spermatozoa obtained beforehand by the swim-up technique. Sixteen to 20 hours after insemination, the oocytes were transferred to the second well for removal of cumulus cells by means of micropipette. Another 24 hours later, the resulting embryos (oocytes fertilized and well cleaved) were replaced or cryopreserved. The remaining oocytes were prepared for cytogenetic analysis. They fell into two types. One of these comprised oocytes that were apparently unfertilized (because they did not have 2 pronuclei). The other comprised zygotes that, although the pronuclei or second polar body had formed, were still uncleaved on the 2nd day. A total of 562 oocytes/ zygotes were processed from January 1989 to July Vol. 56, No.5, November 1991

1990, selection being based only on availability of both oocytes and staff. A reliable technique was developed as follows for the fixation of oocytes by means of our four-well dish method. Oocytes were transferred by Pasteur pipette from the second well to the third, which contained a hypotonic solution of 1% (wt/vol) trisodium citrate previously warmed to 37°C. There they were kept for 10 minutes. During this period, fresh fixative was prepared (3:1 [wt/vol] methanol:acetic acid) and placed in the fourth well. The oocytes were then transferred to this fourth well with the same Pasteur pipette. Air-dried preparations were quickly made by drawing up the oocytes into the pipette and expelling their contents dropwise onto clean dry slides. After the addition of another drop of fresh fixative solution, the oocytes were allowed to air dry. They were then Giemsa-stained for 15 minutes. RESULTS

A total of 562 oocytes were processed. Chromosomes were absent in 30 oocytes, and 26 were degenerate. Chromosome analyses were performed on 433 oocytes. In the remaining 73 cases, analysis was (1) impossible because of poor spreading or excessive scattering or (2) difficult to interpret because of breaks, fragments, or gaps. Table 1 shows the results obtained with the 334 oocytes that were apparently unfertilized (no pronuclei formation). After fixation, it was confirmed that they were indeed unfertilized. Details of the chromosome anomalies are given in Table 2. Two hundred thirty-eight (71.25%) exhibited a normal 23 metaphase II chromosomes. Of these 238, 125 were fully analyzed and shown to have a normal karyotype (Fig. 1A). Thirty-one (9.28%) were hypohaploid (Fig. 1B). (The complements that had <20 chromosomes were excluded because it was possible that they were overspreading). Hyperhaploidy was evident in 33 oocytes (9.88% ). (One of these is shown in Fig. 1C, with two extra chromosomes.) Six were diploid. Seven were hyperdiploid or hypodiploid with metaphase II chromosomes. It was thus indicated that in 3.89% of the cases, oocytes achieved meiosis without any extrusion of the first polar body. Parthenogenetic activation was observed in 19 cases (5.68%). We fixed 17 zygotes with more than two pronuclei. We fixed 38 with two pronuclei and/or second polar body that remained uncleaved 48 hours after insemination. After fixation, we also found that 44 of the oocytes that had seemed to be unfertilized were in Tejada et al.

Chromosomes in oocytes after analogs

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Table 1

Chromosome Anomalies in Human Unfertilized Oocytes

No. of oocytes No. of haploid oocytes No. of aneuploid oocytes No. of diploid, hyperdiploid, or hypodiploid oocytes with metaphase II chromosomes No. of oocytes exhibiting parthenogenesis a

Buserelin acetate/hMG/hCG

LA/FSH/hMG/hCG

Total

156 111 29

178 127 35

334 238 (71.25)a 64 (19.16)

6

7

13 (3.89)

10

9

19 (5.68)

Values in parentheses are percents of total unfertilized oocytes.

fact fertilized. The results obtained with these 99 zygotes are shown in Table 3. Evidence of fertilization was provided by the presence of two separate decondensed nuclei (Fig. 2), probably before syngamy, of diploid mitotic chromosomes or of sperm chromosomes in an arrested stage ofPCC. 22 Overall (not including the polypenetrated eggs), it was evident that 82 of the oocytes had in fact been fertilized and that they had been arrested in development. DISCUSSION

There is no longer any doubt regarding the importance of the cytogenetic study of the oocytes and embryos remaining from IVF programs. First, the high percentage of anomalies found (up to 60 in some authors8 •13 ) has served to explain why not all oocytes are fertilized (which fact was at first attributed to imperfections in the laboratory or in its methods). Second, it has been shown that the fixation of oo-

Table 2

a b

Karyotype of Abnormal Unfertilized Human Oocytes

No. of hypohaploid (n = 31)

No. of hyperhaploid (n = 33)

=20 chr (5)a 20,X,-E,-E,-D (1) 20,X,-E,-C,-C(X?) (1) 2l,X,-G,-E (2) 2l,X,-G,-F (1) 21,X,-F,-E (1) 2l,X,-F,-D (2) 22,X,-G (2) 22,X,-F (2) 22,X,-E (3) 22,X,-18 (1) 22,X,-D (5)d

24,X,+l chr (4) 24,X,+frag or mark (4)b 24,X,+G (6) 24,X,+16 (1) 24,X,+D (1) 24,X, +C(X?) (2) 24,X,+A (1) 25,X,+2 mark (2)• 25,X, +2 chr (2) 26,X,+3 chr (5) 28,X,+5 chr (2) 27 to 30 chr (3)

chr, unidentified chromosome; no. of oocytes in parentheses. frag or mark, chromosome smaller than group G chromosomes.

876

cytes serves to reveal not only aneuploidies but also failure of the oocytes to mature, 5•10 anomalies in their physiology/3 and flaws in the fertilization that from the clinical point of view could be of prognostic importance for the patients in succeeding cycles. 20 The more and more frequent use of GnRH -a in IVF programs is because of not only the greater number of retrieved oocytes and of pregnancies25 but also because of the fact that it is more practical for the programming of the cycles. 23 These were the reasons that led our team to use GnRH-a on 98% of the patients. The aim of our study was to determine if, in the unfertilized oocytes and in the zygotes that were arrested in their development after these stimulation protocols, there are more or fewer anomalies than those described to date with conventional protocols. Unfortunately, we were not able to use the women treated with CC-hMG as a control group because they constituted only 2% of our patients. We did

Tejada et al.

Chromosomes in oocytes after analogs

No. of diploid, hyperdiploid, or hypodiploid metaphase II chromosomes (n = 13) 46,XX (6) 34,XX?,-12 chr (1) 35,XX?,-11 chr (1) 43,XX,-G,-C,-C(X?) (1) 42,XX,-4 chr (1) 48,XX,+2 chr (1) 53,XX,+3 chr,+4 mark (1) 66,XXX? (1)

• See Figure lC. d See Figure lB.

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Figure 1 Karyotypes prepared from three unfertilized oocytes. The chromosomes have been arranged in the conventional groups A through G. Panel A: A normal human oocyte (n = 23). Panel B: A hypohaploid complement (n = 22) with one chromosome missing in group D. Panel C: A hyperhaploid complement (n = 25) with two extra chromosomes smaller than those of group G. Vol. 56, No.5, November 1991

not have a sufficient number of many fixed oocytes, and even if we had, the statistical comparison with 98% would be meaningless. Accordingly, we decided to compare our data with that in the literature. We were aware of the limitations involved, i.e., the variation in protocol regimens and the details of methodology in the handling of oocytes before chromosome analyses in the various centers. As to the technical problems, it is difficult (1) to obtain adequately dispersed chromosomes and to avoid random loss and (2) to interpret certain structural anomalies (gaps, breaks, fragments) that are frequent. 11 •13 •15 •16 It is for these reasons that in 73 cases we were unable to analyze the oocytes. Our experience leads us to believe that the culture conditions and the handling of the oocytes are fundamental for the preservation of chromosomes and for chromatid integrity. In ideal circumstances, a control group of spontaneously ovulated oocytes, selected for fertilization failure and analyzed by the same methods as those used in the case of the study group, might resolve some of these dilemmas. In the absence of such circumstances, however, we concentrated on describing the chromosomal analyses of the 433 oocytes or uncleaved embryos that did not involve problems of interpretation from numerical analysis or from questioning in each case whether fertilization had occurred. Of the 334 unfertilized oocytes analyzed, 96 (28.74%) (Table 1) exhibited chromosomal anomalies. This seems to agree with the majority of studies carried out on oocytes without analogs. 7•9 •10 In addition, the European multicenter study published in 198811 reported a very similar frequency (26%), and it is noteworthy that the authors did indeed work with oocytes obtained by means of stimulation after analogs. Of the four types of stimulation used in the various centers that collaborated, significant differences were not found. Of these 96 anomalies, 64 (19.16%) were aneuploidies. Such findings are consistent with the values published to date. 7 •9 - 11 •13 We calculated this frequency strictly while taking into account the hypohaploid oocytes. We counted only those oocytes in which chromosomes were together, the chromosomes were within a perfectly visible cytoplasm, and there were at least 20 in each oocyte. Delhanty and Penketh, 17 working with oocytes obtained after buserelin acetate treatment, found 10% aneuploidy. This frequency is a little lower than the frequencies published but not significantly. The authors comment that the difference might be because of the fact that their method of calculating frequency was that of doubling the number of hyperhaploids, which Tejada et al.

Chromosomes in oocytes after analogs

877

Table 3

Cytogenetic Results in Human Uncleaved Zygotes Buserelin acetate/hMG/hCG

LA/FSH/hMG/hCG

Total

38

61

99

16

15 1 37 8 0

31 3 48 14 3

No. of arrested zygotes No. of eggs showing male and female pronuclei separated No. of eggs showing arrested division• No. of PCC-G 1 No. of triploid No. of polyploid (>3 pronuclei)

2 11

6 3

• Arrested division was evidenced by presence of diploid mitotic chromosomes.

they did because the true hypohaploids would be confused with others deriving from the methodology. Because the likelihood of such confusion is very difficult to assess, we believe that perhaps it is immaterial which method is used. In our case, the two values would be similar because we found almost the same number of hypo haploid oocytes (31) ashyperhaploid ones (33) (Table 2). If we had calculated the number of aneuploids in the same way as the other authors, we would have obtained 62 (31 X 2), i.e., 18.56% of the sample. An incidence of 3.89% of unreduced oocytes in metaphase II (these oocytes being because of failure of the first meiotic division) is not significantly different from the figure of 6.8% obtained by other authors.7 In the above-m(:1ntioned work of Delhanty and Penketh/ 7 the percentage obtained is 8, although there are authors who report up to 21.5% 15 of unreduced oocytes.

With regard to parthenogenesis, there seems in the European cooperative study to be a slight correlation between (1) the increase in this phenomenon and (2) idiopathic sterility (4.2% in sterility of unknown origin versus 1.2% in tubal factor sterility). Having found a somewhat higher frequency (5.68% ), we did not corroborate these results: 16 of our 19 oocytes with parthenogenesis came from patients with bilateral tubal factor sterility. In regard to clinical parameters, no correlations were found between the type of infertility, the stimulation treatment, the type of GnRH-a (Table 1), the dose of gonadotropins administered, and the frequency or type of chromosome anomaly in unfertilized oocytes. No maternal effect was found. (Mean age of patients with anomalies was 32.7 years.) Some authors have suggested that there could be decreased maternal selection against affected conceptus when the mother is of advanced age. It

Figure 2 Panel A: Oocyte fertilized normally 19 hours after insemination. Panel B: On the following day, the same oocyte, uncleaved, was arrested in its development. The figure shows the two pronuclei (PN) decondensed and the two polar body (PB) after fixation. 878

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may also be that our 96 oocytes with anomalies were insufficient for the drawing of sound correlations. Moreover, this mean age, above that considered ideal for reproduction, can result in an overall increase in the percentage of chromosomal anomalies. The problem is common because the patients who participate in IVF programs have mainly been unsterile for years. Accordingly, the average age in the literature is high. Failure of postfertilization development was observed in 99 of the 433 cases studied (22.8%) (Table 3). Fertilization in oocytes thought to be unfertilized has also been found by other investigators, and their rates are consistent with our figure of 22.8%: 30.7% 14; 36% 4; 21.7%. 21 In 31 cases, there were two nuclei observed, one male and one female, before syngamy. Some of these exhibited different condensations, as in former studies 4·16; in some cases, the condensations were equal (Fig. 2). In these latter cases, we believe that the oocytes had been normally fertilized and then arrested (for unknown reasons) in their development. As for those with unequal condensations, cessation of development was perhaps because of failure of the male and female nuclei to synchronize properly, and perhaps this failure was in turn because of the cytoplasmatic immaturity of the oocyte. 22 In three zygotes, there were mitotic chromosomes found, two in metaphase and one in telophase, all of them from the first mitosis, probably before cell division. These three oocytes exhibited no visible pronuclei. Similar cases have been reported.13·16 Possibly, they resulted from a retarded oocyte fertilization that showed the two pronuclei at night or at other moments outside the laboratory schedule and failed to divide within 48 hours. This possibility leads us to suspect again that the oocytes were not sufficiently mature, even though appearances were morphologically otherwise at the moment of insemination. We are thus reminded once again that there is no direct relation between morphological appearance and fertilization, 5•12 and also that it is necessary to analyze patients' oocytes for prognostic purposes. In our series, the frequency of polypenetrated oocytes was 3.9% (17 /433), which is close to others so far reported (4.3%, 3 5% 8 ). Plachot et al.U report a somewhat higher three pronuclei frequency in oocytes retrieved after analogs than in the case without analogs (7% versus 2.4%), our frequency being intermediate. Further work will be necessary to determine if these differences owe more to the smallness of the samples than to reality. Vol. 56, No.5, November 1991

Prematurely condensed chromosome has been reported previously in human oocytesY-17·20 It is a phenomenon in which the chromosomes of the oocyte are arrested in metaphase II, whereas those of the spermatozoon undergo a premature condensation. The frequency of this phenomenon is variable (from 3% to 16.9% 16 ), but the manner of calculating the percentages is not always the same. In our investigation, the frequency (i.e., 48 PCC cases of 433 fixed oocytes) was 11.08%, but if we count the 48 PCC cases as fertilized oocytes, the frequency is 58.2% (48 PCC cases of82 fertilized oocytes in which the development was arrested). We thus have an idea of the importance of this phenomenon_as a factor when postfertilization embryonic development is not as it should be. 22 We find that in the LA/ FSH/hMG/hCG treatment the number of zygotes with PCC is greater. This will be the subject of another study. 22 If, in conclusion, we bring together (1) chromosomal anomalies in unfertilized oocytes and (2) failures in embryonic development subsequent to fer· tilization, we find that in 45% of the cases of IVF failure the causes are genetic or physiological and lie with the oocyte. It would not appear that this rate is affected by the new stimulation protocols after pituitary suppression with GnRH -a.

Acknowledgments. We are very grateful to Josep Santalo, Ph.D., of the Department de Biologia cellular de Ia U niversitat Auto noma de Barcelona for his scientific advice and to Mr. Luis Tejada for the excellent figures he produced.

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16. Macas E, Floersheim Y, HotzE, Imthurn B, Keller PJ, Walt H: Abnormal chromosomal arrangements in human oocytes. Hum Reprod 5:703, 1990 17. Delhanty JDA, Penketh RJA: Cytogenetic analysis of unfertilized oocytes retrieved after treatment with the LHRH analogue, buserelin. Hum Reprod 5:699, 1990 18. Bom~ A, Boue J, Gropp A: Cytogenetics of pregnancy wastage. In Advances in Human Genetics, Edited by H Harris, K Hirschhorn. New York, Plenum Press, 1985, p 1 19. Hassold T, Matsuyama A: Origin of trisomies in human spontaneous abortions. Hum Genet 46:285, 1979 20. Schmiady H, Kentenich H: Premature chromosome condensation after in-vitro fertilization. Hum Reprod 4:689, 1989 21. SelvaJ, Martin-PontB, HuguesJN, Rince P, Fillion C, Herve F, Tamboise A, Tanboise E: Cytogenetic analysis ofuncleaved human oocytes after in vitro fertilization (IVF). (Abstr. P95) Presented at the 22nd Annual Symposium of The European Society of Human Genetics, Korru, Greece, May 16 to 19, 1990. Published by The European Society of Human Genetics, in the Program Supplement, 1990 22. Tejada MI, Mendoza MR, Corcostegui B, Benito JA: Factors associated with premature chromosome condensation (PCC) following in vitro fertilization. Work in preparation 23. Benito JA, Carbonero JL, Echanoja6.regui AD, Montoya F, Ramon 0, Aparicio V, Corcostegui B, Mendoza R, Tejada I: i,Es programable el dia de Ia puncion folicular para FIVTE/ GIFT/ZIFT con el uso de anruogos de Ia GnRH?. Prog Obstet Gynecol 33:34, 1990 24. Lopata A, Brown JB, Leeton JF, McTalbot J, Wood C: In vitro fertilization of preovulatory oocytes and embryo transfer in infertile patients treated with clomiphene and human chorionic gonadotropin. Fertil Steril30:27, 1978 25. Chetkowski RJ, Kruse LR, Nass TE: Improved pregnancy outcome with the addition of leuprolide acetate to gonadotropins for in vitro fertilization. Fertil Steril 52:250, 1989

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