Preimplantation Diagnosis of Common Aneuploidies by the First- and Second-Polar Body FISH Analysis

Purpose: A low pregnancy rate in in vitro fertilization (IVF) patients of advanced maternal age may be caused by aneuploidies originating from non disjunction in the first or second meiotic divisions. We introduced genetic testing of oocytes by sampling and fluorescent in situ hybridization (FISH) analysis of the first and second polar bodies, to avoid fertilization and transfer of aneuploid oocytes in IVF patients of advanced maternal age. Methods: Three hundred and sixty-three IVF patients 34 years and older participated in the study. Using micromanipulation procedures, the first and second polar bodies were removed following their extrusion from the oocytes and studied by FISH, using probes specific for chromosomes 13, 18, and 21 to detect oocytes with common aneuploidies. Results: Of a total of 538 IVF cycles, 3250 oocytes were available for FISH analysis, with conclusive FISH results in 2742 oocytes (84.3%). As many as 1102 (40%) of oocytes were predicted to be aneuploid and not transferred. Of 1640 embryos predicted to be normal, 1145 were transferred in 467 treatment cycles, resulting in 107 pregnancies (23%), from which 67 healthy children have been born, 32 pregnancies spontaneously aborted, and 15pregnancies are ongoing after being confirmed normal by prenatal diagnosis. Conclusions: Preimplantation diagnosis by first- and second-polar body FISH analysis allows us to avoid the age-related risk of common aneuploidies in IVF patients of advanced maternal age.     

Polar body diagnosis of common aneuploidies by FISH

Purpose: The purpose of this work was to investigate the reliability and accuracy of polar body analysis for preimplantation diagnosis of common aneuploidies in IVF patients of advanced maternal age. Design: We have previously introduced polar body analysis as an approach for nondestractive evaluation of the genotype of human oocytes. The method has recently been applied in a clinical trial involving 45 infertile patients, demonstrating the feasibility of preconception diagnosis of common aneuploidies by fluorescent in situ hybridization (FISH). The present paper describes the experience of polar body diagnosis in 135 IVF patients (161 cycles) of advanced maternal age. Results: FISH results of the first and/or second polar bodies were available in 648 (72.4%) of 895 biopsied oocytes subjected to FISH analysis. Of 648 oocytes with FISH results, 208 demonstrated chromosomal abnormalities. Of 440 oocytes predicted to be free from monosomy or trisomy of chromosomes X, 18, and/or 13/21, 314 were normally fertilized, cleaved, and transferred in 122 treatment cycles, resulting in 6 healthy deliveries and 12 ongoing pregnancies following confirmation of the polar body diagnosis by CVS or amniocentesis. Conclusion: The method may be useful for detection of oocytes with common chromosomal trisomies in IVF patients of advanced maternal age.Presented at the 5th Annual Meeting of the International Working Group on Preimplantation Genetics, Hamburg, Germany, June 28, 1995.     

Screening oocytes and preimplantation embryos for aneuploidy.

Pregnancy and live birth rates following in-vitro fertilization decline rapidly with advancing maternal age partly because of an increase in age-related aneuploidies occurring in female meiosis. Screening oocytes or preimplantation embryos for common aneuploidies is now possible by polar body or cleavage stage biopsy and multicolour fluorescence in-situ hybridization.     

Estimation of Second Polar Body Retention Rate After Conventional Insemination and Intracytoplasmic Sperm Injection: In Vitro Observations from More Than 5000 Human Oocytes

Purpose : Tripronucleate (3pn) development after conventional insemination (CONV) or ICSI was analyzed to estimate the rate of second polar body retention giving rise to 3pn formation. Methods : Data from 453 consecutive IVF cycles were reviewed during a 6-month period. Mature oocytes were monitored in ICSI (n = 3195) and CONV (n = 2274) groups by fertilization assessment 16–18 h post-insemination. Ovulation induction protocols and in vitro culture conditions remained constant during the study interval. Results : Normal (2pn) fertilization occurred in 74.2% and 70.5% for CONV and ICSI groups, respectively (p < 0.003). 1pn formation was observed in 4.5% of CONV oocytes, and 2.5% of ICSI oocytes (p < 0.001); 3pn formation was 8.1% in the CONV group, and 2.5% in the ICSI group (p < 0.0001). We observed 4pn formation in 0.4% of oocytes in the CONV group, but in only 0.04% of oocytes fertilized with ICSI (p < 0.007). Cellular degeneration occurred in 2.4% of oocytes inseminated conventionally, and in 3.5% of oocytes fertilized by ICSI (p = 0.02). Maternal age did not impact pronuclear status. Conclusions : We found the 3pn formation rate after ICSI to be approximately one-third that observed in the CONV group. Extrapolating the ICSI data to the CONV data, it may be inferred that 2.5% of 3pn development after CONV was due to second polar body retention. This suggests that 5.6% of CONV oocytes showed dispermic fertilization. Decreasing oocyte quality with increasing maternal age had no apparent influence on any of the fertilization outcomes.     

Fertilization and pregnancy using metaphase I oocytes in an intracytoplasmic sperm injection program

Purpose: In this study we investigated whether metaphase I oocytes collected in an intracytoplasmic sperm injection program could successfully be matured and fertilized by injecting aged (>20-hr) spermatozoa. Materials and Methods: Metaphase I oocytes aspirated were preincubated for 20 hr to allow the oocytes to reach meiotic maturity. Only metaphase II oocytes were injected. The original sperm sample processed on the day of aspiration was used in the microinjection process. Results: One hundred eighty-three oocytes were collected, of which 42 (23%) were metaphase I oocytes. These were incubated for 20 hr and microinjected with the original sperm sample. Thirty-one (74%) of the metaphase I oocytes reached meiotic maturity (extruded polar body); 67.7% showed two pronuclei 18 hr after injection and 61.3% embryo development 40 hr postinjection. No difference in fertilization and embryo development rate was found in metaphase II oocytes injected 6 hr postaspiration versus 20 hr postaspiration. An ongoing pregnancy was also achieved using only embryos obtained from matured metaphase I oocytes. Conclusions: Metaphase I oocytes can be successfully maturedin vitro and injected using aged (>20-hr) sperm samples. Matured metaphase I oocytes, if successfully injected, produce embryos able to induce pregnancy.     

Accuracy of Preimplantation Diagnosis of Single-Gene Disorders by Polar Body Analysis of Oocytes

Purpose: A number of pitfalls in single-cell DNA analysis, including undetected DNA contamination, undetected allele drop out, and preferential amplification, may lead to misdiagnosis in preimplantation genetic diagnosis of single-gene disorders. Methods: Preimplantation genetic diagnosis was performed by sequential first and second polar body analysis of oocytes in 26 couples at risk for having children with various single-gene disorders. Mutant genes were amplified simultaneously with linked polymorphic markers, and only embryos resulting from the mutation-free oocytes predicted by polar body analysis with confirmation by polymorphic marker testing were transferred back to patients. Results: Overall 529 oocytes from 48 clinical cycles (26 patients) were tested, resulting in the transfer of 106 embryos in 44 clinical cycles. As many as 46 (9.6%) instances of allele dropout were observed, the majority (96%) of which were detected. Seventeen unaffected pregnancies were established, of which nine resulted in the birth of an unaffected child, and the rest are ongoing. Conclusions: A high accuracy of preimplantation genetic diagnosis of single-gene disorders is achieved by application of sequential analysis of the first and second polar body and multiplex polymerase chain reaction.     

Use of PRINS for preconception screening of polar bodies for common aneuploidies

It has been shown that preconceptional screening for oocyte aneuploidies could help increase the pregnancy rate after in vitro fertilization (IVF), particularly in cases of advanced maternal age. The FISH (fluorescent in situ hybridization) technique is usually used to examine the first polar body (I-PB) for oocyte screening and so avoid fertilizing and transferring embryos from aneuploid oocytes. We have tested the feasibility of using another technique, the primed in situ (PRINS) reaction for this purpose. PRINS is a rapid, inexpensive method of labelling chromosomes. Chromosomes were labelled by in situ annealing with chromosome-specific oligonucleotide primers, followed by primer extension with labelled nucleotides using Taq DNA polymerase. A total of 183 PRINS reactions were performed with primers for chromosomes 13, 16, 18, 21 or X on 63 I-PBs removed from oocytes that failed to become fertilized during IVF. Each I-PB underwent three successive double-labelling reactions and intense signals were obtained in less than 40 min. Our data suggest that PRINS may be a useful alternative or a complement to FISH for detecting the main aneuploidies in all oocytes obtained after follicular puncture.     

Detection of aneuploidy in human oocytes and corresponding first polar bodies by fluorescent in situ hybridization

Purpose: The purpose of the study was to investigate the reliability of the fluorescent in situ hybridization (FISH) analysis of the first polar body (IPB) for cytogenetic evaluation of human oocytes as a method of choice in preimplantation diagnosis of chromosomal aneuploidies. Design: Human unfertilized oocytes and their extruded IPB were analyzed using the directly labeled fluorescence alpha-satellite DNA probes to chromosomes X and 18. Results: Paired signals for chromosomes X and 18 were observed in the second meiotic prophase (MII) of unfertilized oocytes and their extruded IPB. In the series of 156 unfertilized oocytes in which the number of X chromosome-and chromosome 18-specific signals were analyzed in both MII and IPB, five nondisjunction events have been detected, with corresponding signals in MII and their IPB: missing signals in MII corresponded to extra signals in their IPB and extra signals in MII corresponded to missing signals in IPB. In one oocyte chromosome 18 nondisjunction was detected, with both chromosome 18 signals in MII and no chromosome 18 signal in IPB. In four oocytes chromatid malsegregations for chromosome X or chromosome 18 were detected: in two oocytes, three of four chromosome 18 signals were present in MII, with only one in IPB, and in the other two oocytes, three of four chromosome signals were present in MII, with only one left in IPB. Conclusions: The data suggest the possibility of detecting chromosomal aneuploidy in oocytes through cytogenetic analysis of their corresponding IPB by FISH as a possible approach for preimplantation diagnosis of major chromosomal trisomies.Presented in part at the IXth World Congress on In Vitro Fertilization and Alternate Assisted Reproduction, Vienna, Austria, April 3–7, 1995.     

Chromosomal abnormalities in a series of 6,733 human oocytes in preimplantation diagnosis for age-related aneuploidies

Most chromosomal abnormalities originate from female meiosis and contribute significantly to pregnancy failures, particularly in women of advanced maternal age. A total of 8,382 oocytes were obtained in 1,297 IVF cycles from patients of advanced maternal age (mean 38.5 years). Following a standard IVF protocol, oocytes were tested following removal and fluorescence in-situ hybridization (FISH) analysis of the first (PB1) and second polar bodies (PB2), using probes specific for chromosomes 13, 16, 18, 21 and 22 (Vysis). FISH results were available in 67,33 (80.3%) oocytes tested, 3,509 (52.1%) of which were aneuploid, with the remaining 3,224 (47.9%) normal oocytes available for transfer. In all, 41.7% of oocytes had meiosis I errors, compared to 35.1% with meiosis II errors. Abnormalities in meiosis I were represented by extra chromatids in 15.4%, missing chromatids in 48.1%, missing chromosomes in 5.9%, extra chromosomes in 0.5%, and complex abnormalities in 30.1%. The proportions of abnormal oocytes with missing or extra chromatids in meiosis II were 36.6 and 41.2% respectively, with the remaining oocytes having complex abnormalities, involving missing or extra chromatids of different chromosomes (22.1%) following meiosis II. Overall, 41.8% oocytes had meiosis I, 30.7% meiosis II, and 27.6% both meiotic division errors. A total of 45.1% of the abnormal oocytes had complex errors, involving the same chromosome in both meiotic divisions (21.5%), or different chromosomes (78.5%), of which 74.8% were with abnormalities of two, and 25.2% with abnormalities of three chromosomes studied. Of 3,224 detected aneuploidy-free zygotes, 2,587 were transferred in 1,100 treatment cycles (2.35 embryos per transfer), resulting in 241 (21.9%) clinical pregnancies and 176 healthy children born, suggesting a positive clinical outcome following aneuploidy testing of oocytes in a group of IVF patients of average age 38.5 years.     

The role of preimplantation genetic diagnosis in women of advanced reproductive age

PURPOSE OF REVIEW: More than half of in-vitro fertilization patients are of advanced reproductive age and at risk for producing offspring with age-related aneuploidies, which contribute significantly to spontaneous abortions and implantation failure. RECENT FINDINGS: Fluorescent in-situ hybridization analysis of thousands of oocytes and preimplantation embryos obtained from these patients revealed an aneuploidy rate of over 50%, suggesting practical relevance of preimplantation genetic diagnosis for aneuploidy to women of advanced reproductive age. The overall preimplantation genetic diagnosis experience for age-related aneuploidies comprising more than 3000 clinical cycles indicates the positive impact of preselection and transfer of aneuploidy-free embryos on implantation and pregnancy rates and outcome of pregnancies in women of advanced reproductive age. SUMMARY: These patients will need to be informed about preimplantation genetic diagnosis availability, in order use this option to improve their relatively poor chances of becoming pregnant, especially with the current tendency of limiting the number of transferred embryos to avoid complications due to multiple pregnancies. This may contribute significantly to improving standards of assisted reproduction technology, substituting the current practice of selection of embryos for transfer using morphological parameters with the preselection of aneuploidy-free embryos with a higher potential to result in pregnancy.     

Relationship of the Human Cumulus-Free Oocyte Maturational Profile with In Vitro Outcome Parameters After Intracytoplasmic Sperm Injection

Purpose: We investigated whether the human oocyte maturational profile at the removal of cumulus/corona cells affects the fertilization rate and subsequent embryo quality after intracytoplasmic sperm injection. Methods: A total of 1011 oocytes from 150 cycles was included in this retrospective analysis. Cumulus-free oocytes that were in prophase or metaphase I of meiosis at the removal of cumulus/corona cells were incubated in vitro until they reached metaphase II (in vitro-matured oocytes) and were then immediately injected with a single spermatozoa. Oocytes that were in metaphase II at the removal of cumulus/corona cells (MII oocytes) received sperm injection after 3–4 hr of preinjection incubation. Results: The fertilization rate of the MII oocytes was significantly higher than that of in vitro-matured oocytes (81 vs 62%; P < 0.001). The cleavage rates were similar in the two groups (MII oocytes, 94%; in vitro-matured oocytes, 91%). However, MII oocytes had significantly higher percentages of good-quality embryos (grade 1–3 embryos, 87 vs 58%, P < 0.001) and embryos with high cumulative embryo scores (score 10–32 embryos, 62 vs 33%, P < 0.001). The mean cumulative embryo score of MII oocytes after fertilization was also higher than that of in vitro-matured oocytes (12.1 ± 3.8 vs 8.8 ± 3.4; P = 0.014). Conclusions: MII oocytes that extruded the first polar body at the removal of cumulus/corona cells had better fertilization rates and embryo morphology than in vitro-matured oocytes that extruded the first polar body following the removal of cumulus/corona cells and in vitro culture.     

Resumption of meiosis-I tissue to enucleated preovulatory oocytes: a preliminary report

Purpose : Ovarian tissue banking may be the best strategy to preserve female fertility. But optimal method to obtain viable mature oocytes remains challenging. In order to bypass the long in vitro oocyte growth period, we developed this study to test whether reconstruction of thawed primordial oocytes with enucleated preovulatory germinal vesicle (GV) oocytes could induce dictyate nuclei to undergo chromosomal condensation and meiotic maturation. Methods : Isolated primordial oocytes from thawed mouse ovarian tissue were reconstructed with enucleated GV oocytes. After electrofusion and in vitro maturation, the reconstituted oocytes were assessed for first polar body extrusion, cytoskeleton configuration, and chromosome abnormalities. Results : Primordial oocytes from thawed ovarian tissue showed a high survival rate. Following transfer and electrofusion, they could be fused with enucleated GV oocytes (35.6%, 36/101) and extruded a first polar body (52.8%, 19/36). These mature oocytes showed a normal spindle configuration and chromosome number. Conclusions : We successfully established a mouse cell model to prove that omitting the whole growth and maturation period by transfer of primordial oocytes to developmentally older enucleated oocytes would bypass the long growth period required to the preovulatory stage. Polar body extrusion could also ensue after in vitro growth. This study provided an alternative approach for future investigations in oocyte maturation.     

体内成熟与体外成熟的卵母细胞纺锤体位置及其与胚胎发育的关系

目的研究体内成熟与体外成熟卵母细胞的纺锤体位置及其与胚胎发育的关系。方法对134个体内成熟卵母细胞在卵母细胞胞质内单精子注射法（ICSI）操作时用纺锤体实时观察仪进行纺锤体位置的观察,体内成熟卵母细胞来自单纯因男性不育而进行ICSI治疗的患者15例（体内成熟组）。另外对45个体外成熟的卵母细胞观察纺锤体位置,体外成熟卵母细胞来自因多囊卵巢综合征致不孕而进行治疗的患者5例（体外成熟组）。纺锤体的位置按照其与第一极体之间的角度不同分为Ⅰ、Ⅱ、Ⅲ、Ⅳ和Ⅴ级。并观察两组成熟卵母细胞的受精及其胚胎发育情况。结果体内成熟组和体外成熟组患者的卵母细胞中可观察到纺锤体的分别占83．6％（112／134）和82．2％（37／45）。体内成熟组患者卵母细胞纺锤体的位置Ⅰ、Ⅱ、Ⅲ、Ⅳ和Ⅴ级分别为22．4％、55．2％、3．O％、3．O％、16．4％,体外成熟组则分别为17．8％、51．1％、8．9％、4．4％、17．8％,两组各级间分别比较,差异均无统计学意义（P〉0．05）。在体内成熟组卵母细胞中,纺锤体离第一极体较近（Ⅰ级）者受精率较高（93．3％）,显著高于其他各级（分别为73．0％、2／4、1／4、63．6％,P〈0．05）。结论体内成熟与体外成熟卵母细胞间纺锤体位置未见显著差异;纺锤体的位置与卵母细胞受精率有一定相关性。     

Outcome of laser-assisted polar body biopsy and aneuploidy testing

Polar body biopsy and subsequent fluorescence in-situ hybridization (FISH) analysis allows detection of maternally derived chromosomal aneuploidies in human oocytes during IVF treatment. The development of a diode laser technique for the partial opening of the zona pellucida has stimulated the use of this technique to assist polar body biopsy. Laser-assisted polar body biopsy was performed in 140 IVF cycles from patients of advanced maternal age (> or =35 years). A total of 921 oocytes were treated by a laser for partial zona opening and polar body removal. FISH was performed for chromosomes 13, 16, 18, 21 and 22 and results were available for 903 oocytes (98%). In all, 443 oocytes (49.1%) were euploid and of these, 293 were fertilized. A total of 214 embryos were transferred in 120 embryo transfer cycles (1.78 per embryo transfer) resulting in 27 clinical pregnancies (22.5% per embryo transfer) with an implantation rate of 15.4%. Subsequently, five women aborted (18.5%) and 24 healthy children were born from the remaining 22 pregnancies, which gives a take home baby rate of 18.3% per transfer cycle. It is concluded that polar body biopsy using a diode laser system is as efficient as standard polar body biopsy using zona drilling.     

Cryopreserved Immature Mouse Oocytes: A Chromosomal and Spindle Study

Purpose: Cryopreservation of human oocytes might provide an alternative approach to freezing supernumerary embryos obtained during IVF. This process, performed on immature denuded prophase I mouse oocytes, was investigated. Methods: We first investigated the capacity of frozen, immature, murine oocytes to continue in vitro maturation after thawing. We then evaluated the risk to offspring from chromosomal damage by cytogenetical and cytological (spindle) analysis. Finally, we attempted to determine the reasons for and the stage of maturation failure. Results: A total of 700 immature oocytes was frozen, 629 (90%) were recovered intact after thawing, and 53% extruded the first polar body, versus 74% for the control group. Freezing was not accompanied by an increase in aneuploidy in maturing oocytes (18 and 15% for thawed and control oocytes, respectively). Consequently, the first meiotic division occurred normally, without an increase in nondisjunction. Spindle analysis demonstrated only a few abnormalities (15 and 2% for thawed and control oocytes, respectively) incompatible with further development. Oocytes arrested during in vitro maturation were mainly at the metaphase I stage (64 and 76% for thawed and control oocytes, respectively). Whereas 17% of thawed oocytes were blocked before the formation of the first meiotic spindle, this never occurred in the control group. Conclusions: Immature murine oocytes can withstand cryopreservation, which is encouraging for future human application of this technique.     

Intracytoplasmic Sperm Injection and Conventional In Vitro Fertilization Are Complementary Techniques in Management of Unexplained Infertility

Purpose : To evaluate the role of ICSI in unexplained infertility. Methods : In 125 cycles with six or more oocytes retrieved per cycle, sibling oocytes were randomly allocated to IVF or ICSI (group A). In 74 cycles with less than six oocytes retrieved per cycle, cycles were allocated to IVF or ICSI (group B). Results : In group A, ICSI fertilization rate of 61% per allocated oocyte was higher than IVF fertilization rate of 51.6% (P < 0.001). Complete fertilization failure occurred in 19.2 and 0.8% of cycles in IVF and ICSI, respectively (P < 0.001). In group B, fertilization rate in IVF cycles was 53.3% as compared to 60.7% per allocated oocyte in the ICSI cycles (P = 0.29). Complete fertilization failure was higher (P = 0.02) in conventional IVF (34.3%) than ICSI cycles (10.3%). Conclusions : Allocation of sibling oocytes to IVF and ICSI in the first cycle minimizes risk of fertilization failure. For patients with limited number of oocytes, ICSI technique is recommended.     

Abnormal chromosome behavior in human oocytes which remained unfertilized during human in vitro fertilization

Chromosomal abnormalities and abnormal embryonic development have previously been observed after human in vitro fertilization (IVF). Chromosomal abnormalities may arise not only after fertilization but even earlier during meiotic maturation of human oocytes in culture. Since chromosomal analysis is simple in oocytes during meiotic maturation, the chromosomal status was analyzed in oocytes which remained unfertilized in a human in vitro fertilization program. In 50 fertilization attempts the chromosomes of 62 unfertilized oocytes could be analyzed; 45 of them were in the process of meiotic maturation. In three oocytes two small polar bodies were observed 16–18 hr after insemination in the absence of fertilization. In one oocyte abnormal chromosome behavior was found during the first meiotic division, and in four oocytes during metaphase of the second meiotic division. These data suggest that chromosomal analysis of unfertilized oocytes in human IVF may improve the understanding of human oocyte maturation and fertilization.     

Cytogenetics of uncleaved oocytes and arrested zygotes in IVF programs

Purpose: Cytogenetic studies of arrested oocytes and zygotes were used to understand in vitro fertilization (IVF) failures. Methods: We investigated the cytogenetics (Giemsa banding and FISH) of 710 uncleaved oocytes and 94 arrested zygotes from 208 patients undergoing IVF procedures. Results and Conclusions: Of uncleaved oocytes without a polar body, 39% were judged cytogenetically abnormal (17% unbalanced predivision and 21.5% diploid). Of 575 oocytes with a polar body, 124 (21.5%) showed numerical or structural chromosome aberrations. In arrested zygotes, approximately equal cases were found with separate condensed haploid complements (no syngamy), nuclear asynchrony and pulverized DNA, and apparently cytogenetically normal zygotes arrested at mitosis. These data on chromosome abnormalities were also analyzed with respect to two ovarian stimulation protocols and to maternal age. Both ovarian stimulation protocols showed the same levels of chromosome abnormalities. Overall chromosome abnormalities and premature chromosome condensation were also unchanged with maternal age. These data illustrate the significance of chromosome aberrations in IVF failures.Presented at the 5th Annual Meeting of the International Working Group on Preimplantation Genetics, Hamburg, Germany, June 28, 1995.     

采用第一极体进行植入前诊断的实验研究

目的 :建立采用第一极体植入前染色体非整倍体诊断的方法。方法 :取试管婴儿后的未受精卵细胞 ,激光打孔法行第一极体活检 ,固定后行多色荧光原位杂交 (FISH) ,分析极体中 13,16 ,18,2 1和 2 2号染色体的核型情况。结果 :活检成功率为 94 .7% ,FISH成功率为 86 .7% ,6 1.5 %的极体核型正常 ,38.5 %的极体为非整倍体。结论 :激光打孔结合FISH法是一种快速有效的极体植入前诊断方法     

Cytogenetic analysis of human oocytes parthenogenetically activated by puromycin

Purpose

Treatment of aged human oocytes by puromycin allows a high rate of parthenogenetic activation and development until the first cleavage division. This technique was used for the study of the chromosome complement of oocytes which remained unfertilized after in vitro fertilization. Three hundred four unfertilized oocytes were treated with 10 Μg/ml puromycin for 6–8 hr and further cultured for 12–15 hr.

Results

Activation occurred in 90.5% of the oocytes. Heterozygous diploids with two pronuclei predominated (61%), which is in contrast to the mouse, where the majority of oocytes activated by puromycin are uniform haploids (89%).

Conclusions

Therefore we conclude that puromycin treatment induces retention of the second polar body in human oocytes, unlike in mouse oocytes treated in the same way. Chromosome analysis performed on 182 oocytes suggested a nondisjunction (ND) rate for the second meiotic division of 12.7%. This is a low figure considering the fact that puromycin itself has been reported to induce nondisjunction. For the first meiotic division a ND rate of only 5.6% was found. This rate is lower than the one found in metaphase II arrested oocytes and we believe that this difference is due to the technical differences between the study of meiotic and that of mitotic chromosomes.