The cytogenetic constitution of embryos derived from immature (metaphase I) oocytes obtained after ovarian hyperstimulation

The cytogenetic constitution of embryos derived from immature (metaphase I) oocytes obtained after ovarian hyperstimulation Deborah Strassburger, Ph.D.,a Alexandra Goldstein, M.Sc.,a,b Shevach Friedler, M.D.,a Aryeh Raziel, M.D.,a Esti Kasterstein, M.Sc.,a Maya Mashevich, Ph.D.,b Mory Schachter, M.D.,a Raphael Ron-El, M.D.,a and Orit Reish, M.D.b a Infertility and IVF Unit; and b Genetics Institute, Assaf Harofeh Medical Center, Zerifin, Israel, affiliated with the Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel

Objective: To examine the chromosomal content of embryos resulting from metaphase I (MI) oocytes obtained after ovarian hyperstimulation for IVF. Design: Prospective cohort study. Setting: University-based IVF Center, Assaf Harofeh Medical Center, Tel Aviv University, Israel. Patient(s): One hundred fifty women undergoing assisted reproduction technique (ART). Intervention(s): Intracytoplasmic sperm injection (ICSI) was performed in MI oocytes that were retrieved after ovarian stimulation. A portion of these oocytes extruded their polar body (rescued in vitro-matured metaphase II [IVM-MII]) after different incubation periods and the remainder did not (arrested MI oocytes). Fluorescence in situ hybridization was performed using probes for chromosomes X, Y, 18. Main Outcome Measure(s): Fertilization rate, number of blastomeres, and embryo euploidy. Result(s): Embryos from rescued IVM-MII oocytes showed significantly higher fertilization rates and more blastomeres per embryo compared with those from arrested MI oocytes (58.5% vs. 43.9% and 5.7 vs. 5.0, respectively). The chromosomal analysis of these embryos revealed a high rate of aberrations (80.6%), mainly complex mosaics. This rate was elevated in embryos from arrested IVM oocytes (97.2%), and after longer incubation periods. No chromosomally normal embryos were found after 24 hours of incubation of their corresponding oocytes. Conclusion(s): Embryos originating from MI oocytes have a high rate of chromosomal aneuploidy, and their replacement should be reconsidered. (Fertil Steril 2010;94:971–8. 2010 by American Society for Reproductive Medicine.) Key Words: In vitro maturation, chromosomal aneuploidy, abnormal embryos, ICSI

A certain percentage of oocytes aspirated from women after stimulation of the ovaries for IVF-embryo transfer fail to resume meiosis in vivo and are thus retrieved in immature stages like metaphase I (MI) or germinal vesicle (GV) stages (1–4). Only mature oocytes are injected by intracytoplasmic sperm injection (ICSI) on a regular basis, whereas the immature oocytes are usually discarded (5). The use of immature oocytes could increase the number of embryos obtained to enhance the chance of pregnancy (6). The MI oocyte performance after ICSI, even after in vitro maturation (IVM), demonstrated impaired results (7), higher degeneration and lower fertilization rates, multipronucleated fertilization, and frequent cleavage blockage or delay (8–10). In addition, reports of normal pregnancies and live births resulting from IVM oocytes from hyperstimulated cycles are mostly limited to Received January 19, 2009; revised March 24, 2009; accepted April 15, 2009; published online June 7, 2009. D.S. has nothing to disclose. A.G. has nothing to disclose. S.F. has nothing to disclose. A.R. has nothing to disclose. E.K. has nothing to disclose. M.M. has nothing to disclose. M.S. has nothing to disclose. R.R.-E. has nothing to disclose. O.R. has nothing to disclose. Reprint requests: Deborah Strassburger, Ph.D., Infertility and IVF Unit, Assaf Harofeh Medical Center, Zerifin 703000, Israel (FAX: 972-8-9779003; E-mail: [email protected]).

0015-0282/$36.00 doi:10.1016/j.fertnstert.2009.04.035

case reports (6, 8, 11–16). It seems that culture systems adequately support nuclear maturation in immature oocytes, but fail to produce oocytes with cytoplasmic competency. This can impede the formation of bipolar spindles that will restrict subsequent events such as fertilization, cleavage, and probably the creation of embryos with normal chromosomal ploidy (17). Thus, the chromosomal constitution of these oocytes and embryos seems uncertain. The aim of the present study was to examine the chromosomal content of blastomeres of embryos achieved from immature oocytes aspirated after controlled ovarian hyperstimulation (COH).

MATERIALS AND METHODS Source of Oocytes and Embryos The study was approved by our Institutional Review Board (IRB). The study group comprised 150 consenting women from our IVF center undergoing 226 ICSI cycles, between September 2004 and December 2006, after COH. The only inclusion criterion was patients having at least seven mature MII oocytes and in addition, at least one MI oocyte aspirated. The average age of the patients was 30.0  2.48 years.

Fertility and Sterility Vol. 94, No. 3, August 2010 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.

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COH Protocol A long protocol was used: 3.75 mg of triptorelin (Decapeptyl; Ferring, Malmo, Sweden) was used 2 weeks before individualized administration of menotropins. The women had normal gonadotropin levels with a normal response to COH. Human chorionic gonadotropin was administered when the leading follicle was at least 20 mm in diameter. The mean E2 level on the day of hCG administration was 2,414.2  1,318.7 pg/mL, and the mean P level was 1.6  1.9 ng/mL. ICSI Criteria Male factor infertility, when at least two of three semen parameters were pathological, namely sperm concentration %5  106 cells/mL, sperm motility %10%, and %5% sperm with normal morphology (Kruger criteria); in addition, patients with an absent or very low fertilization rate (<20%) in a previous IVF cycle. Oocyte Preparation Modified human tubal fluid (HTF) was used for ovum pick-up (Irvine Scientific, Santa Ana, CA). The cumulus–corona cells were removed after exposure to hyaluronidase (type IV-S; Sigma, Rehovot, Israel) 20 IU/mL in HTF medium (Irvine Scientific) for no more than 1 minute. Oocyte evaluation was performed using an inverted microscope (Diaophot 300; Nikon, Ohi, Japan) with an enlargement of 3,400. The GV oocytes were discarded. The MI state of the oocytes was defined by the absence of a polar body and no discernible GV nucleus. They were separated from the MII oocytes. Two hundred sixty-seven MI oocytes were incubated in 25-mL droplets of medium. They were divided at random into three groups of incubation periods (up to 2, 4–8, and 24 hours) for IVM before their injection. An interim analysis was carried out after obtaining 50 oocytes in each group. The subsequent oocytes were randomly allocated into two incubation time groups: up to 2 and 4–8 hours. The culture medium used was our routine culture medium (G1.2; Vitrolife, Molndalsven, Goeteborg, Sweden), with supplementation of human serum albumin (HSA; Vitrolife). No hormones were added. The droplets were covered under light-weight paraffin oil (Oil for Embryo Culture; Irvine Scientific). ICSI Procedure and Embryo Classification The ICSI was performed, as described previously (7). The central droplet contained polyvinylpyrrolidone (Irvine Scientific) to maintain the sperm movement. The MI oocytes that extruded their polar body, named hereinafter as rescued in-vitro matured MII oocytes, and MI oocytes that did not extrude their polar body were injected. The sperm used for ICSI were from frozen-thawed donor sperm. All sperm donors were karyotyped before their donation. After ICSI, all of the injected oocytes were replaced in the same culture media for 72 hours. Each oocyte was examined 972

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for structural integrity and fertilization 16–18 hours after injection. Fertilization was confirmed if two distinct pronuclei and two polar bodies were observed under the inverted microscope. Cleavage was evaluated 40–44 hours after oocyte injection followed by a second evaluation 68–72 hours after injection. The number of blastomeres and the extent of fragmentation were recorded. Embryos were classified according to their morphological appearance: grade I, symmetrical blastomeres with no enucleated fragments; grade II, asymmetrical blastomeres with or without <20% of the volume filled with enucleated fragments; grade III, with 20%–50% of the volume covered with fragments; and grade IV, extremely asymmetrical blastomeres and >50% of the volume filled with enucleated fragments. All the embryos included in the study had normal fertilization (two pronuclear) and additional cleavage. Multinucleated embryos were excluded from the study. Fixation and Fluorescence In Situ Hybridization The day 3 embryos were placed in acid tyrode solution (Irvine Scientific) for 2 minutes for the removal of the zona pellucida (ZP). They were then transferred into a 2-mL droplet of spreading solution (0.01 N HCl/0.1% and 5 mL of Tween 20) placed on a slide, and spread during constant observation under an inverted-phase contrast microscope (Olympus, London, United Kingdom; CK2 with 5, 10, and 20 magnification objectives). The number of fixed nuclei for each blastomere was recorded .The slides were air-dried, washed in phosphate-buffered saline (PBS) for 5 minutes, and dehydrated with an ethanol series. Triple-target fluorescence in situ hybridization (FISH) was performed with the use of directly labeled DNA probes for chromosomes X (Vysis, Downers Grove, IL; alpha-satellite DNA probe, spectrum green), Y (Vysis; satellite III DNA probe, spectrum aqua), and 18 (Vysis;, satellite II/III DNA probe, spectrum orange). The nuclei were counterstained with 4, 6 diamidino-2-phenylindole (1.25 ng/mL), and then analyzed under a Zeiss Axioskop fluorescence microscope (Zeiss, Jena, Germany) with the relevant filter sets (18). Statistical Analysis To analyze differences in distribution between the two groups, c2 tests were performed. A value P<.05 was considered significant. RESULTS Of the 3,030 oocytes retrieved from the 150 women participating in this study, 88 (2.9%) degenerated either before or due to denudation. Two thousand five hundred oocytes (82.5%) were mature (at the MII stage), 130 (4.3%) at the GV stage, and 312 (10.3%) were at the MI stage. Of the 267 MI oocytes donated to our study, 111 (41.5%) were injected within 2 hours, 102 (38.3%) after 4–8 hours, and 54 (20.2%) after 24 hours. The number of oocytes that matured in vitro and extruded their polar body (rescued IVM-MII)

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TABLE 1 Intracytoplasmic sperm injection results in arrested metaphase I and rescued in vitro matured metaphase II oocytes. Incubation time All incubation periods Arrested MI Rescued IVM-MII 2h Arrested MI Rescued IVM-MII 4 h–8 h Arrested MI Rescued IVM-MII 24 h Arrested MI Rescued IVM-MII

Injected oocytes

2PN fertility rate (%)

Cleavage rate (%)

No. of day 3 cells

Morphology day 3

267 132 135 111 78 33 102 39 63 54 15 39

137 (51.3) 58 (43.9)a 79 (58.5)a 56 (50.4) 36 (46.1) 20 (60.6) 54 (52.9) 15 (38.4)b 39 (61.9)b 27 (50.0) 7 (46.7) 20 (51.2)

128 (93.4) 53 (91.3) 75 (94.9) 52 (92.8) 33 (91.7) 19 (95.0) 51 (91.4) 13 (86.7) 38 (95.0) 25 (92.5) 7 (100.0) 18 (90.0)

5.5  2.05 5.0  1.79c 5.7  1.81c 5.5  2.00e 4.9  1.86d 6.7  1.90d 5.4  1.37f 5.8  1.36 5.5  1.36 4.5  184e,f 4.0  1.73 4.9  1.84

2.5  0.60 2.5  0.58 2.4  0.65 2.5  0.59 2.6  0.57 2.4  0.64 2.7  0.52 2.5  0.51 2.6  1.48 2.2  0.21 2.0  0.57 2.4  0.66

Note: MI ¼ metaphase I; IVM ¼ in vitro matured; MII ¼ metaphase II. All other cross-comparisons within and between subgroups were not statistically significant. a,c P¼ .01. b P¼ .03. d P¼ .002. e,f P< .05. Strassburger. Euploidy in embryos from MI and oocytes. Fertil Steril 2010.

after 2, 4–8, and 24 hours of incubation were, 33/111 (29.7%), 63/102 (61.7%), and 39/54 (72.2%), respectively. The 2 pronuclei (2PN) fertilization rate after ICSI was significantly lower in embryos originated from both arrested MI and rescued IVM-MII oocytes for all incubation periods when compared with that of mature sibling MII oocytes from the same patients (137/267; 51.3% vs. 1,848/2,583; 71.5%; P¼ .001). However, the fertilization rate was higher in all rescued IVM-MII oocytes compared with all arrested MI oocytes (58.5% vs. 43.9%; P¼ .01) (Table 1). This tendency had a statistical difference after 4–8 incubation hours. Embryo cleavage rates were not affected by incubation times or the maturational status of the oocyte and were uniformly high in all groups (86%–100%). The quality of all embryos originating from MI oocytes, in terms of number of cells, but not of morphology, were lower from embryos of the sibling MII oocytes (number of cells 5.5  2.05 vs. 7.1  1.2 cells; P¼ .001) and morphological grade of 2.5  0.60 vs. 2.6  0.52 on day 3). The number of blastomeres in all embryos originating from rescued IVM-MII oocytes was, however, higher compared with the number of cells in embryos from arrested MI oocytes (5.7  1.81 vs. 5.0  1.79; P¼ .01). This was particularly significant in the 2-hour incubation group (P¼ .002). The number of cells in all embryos in the 24-hour incubation group was, however, significantly lower in comparison with the number of cells after 2- and 4 to 8-hour incubation periods (P<.05) (Table 1). Ninety-three embryos originating from MI oocytes were successfully genetically analyzed. Altogether 521 blastoFertility and Sterility

meres were counted before spreading (5.2  1.7 nuclei per embryo). Successful genetic evaluation for three chromosomes was obtained in 303 nuclei, representing 3.2  1.73 nuclei per embryo. The difference between the number of spread blastomeres and the number of analyzed nuclei was partially due to the loss of some nuclei and partially to the presence of enucleated blastomeres. The overall rate of pathological embryos was 80.6 % (Table 2). There were significantly more pathological embryos from arrested MI ova compared with rescued IVM-MII (97.2% vs. 70.1%; P<.01). Percentages of pathological embryos from arrested MI oocytes were similar for all incubation periods. However, the number of abnormal embryos from rescued IVM-MII oocytes increased gradually in correlation with the duration of the incubation periods (40%, 67%, and 100%). In the 24-hour incubation group, all the embryos were abnormal. For the FISH analysis, we grouped the results in Table 3 as follows: [1] uniformly diploid embryos; [2] uniformly haploid embryos, in which all the blastomeres analyzed clearly showed one sex chromosome and one chromosome 18; [3] uniformly triploid embryos, in which all the blastomeres analyzed clearly showed three sex chromosomes and three chromosomes 18; [4] uniformly tetraploid embryos, having four sex chromosomes and four chromosomes 18; [5] mosaicism, which comprised mosaicism of abnormal/normal cells when R50% of the blastomeres were normal, and complex mosaicism when all nuclei had a different chromosome constitution—abnormal/abnormal; [6] nullisomy, when the set of sex chromosomes or the two chromosomes 18 were missing; 973

TABLE 2 Chromosomal status of embryos originated from arrested metaphase I and rescued in vitro-matured metaphase II oocytes after various incubation periods (%). Pathological embryos All embryos Arrested MI Rescued IVM-MII Injection 2 h Arrested MI Rescued IVM-MII Injection 4–8 h Arrested MI Rescued IVM-MII Injection 24 h Arrested MI Rescued IVM-MII

75/93 (80.6) 35/36 (97.2)a 40/57 (70.1)a 25/32 (78.1) 21/22 (95.4)b 4/10 (40.0)b,c 32/43 (74.4)d 10/10 (100.0) 22/33 (66.6)e 18/18 (100.0)d 4/4 (100.0) 14/14 (100)c,e

Note: MI ¼ metaphase I; IVM ¼ in vitro matured; MII ¼ metaphase II. All other cross-comparisons within and between subgroups were not statistically significant. a,b P< .01. c P¼ .003. d,e P< .02. Strassburger. Euploidy in embryos from MI and oocytes. Fertil Steril 2010.

[7] monosomy, which included all blastomeres that contained one less sex chromosome or one less chromosome 18; [8] triploidy, which defined an extra sex chromosome or chromosome 18 (19). Seventy-two percent (54 of 75) of the total pathological embryos were mosaic, of them 90% (49 of 54) were complex mosaics (chaotic) with aberrations of more than one chromosome (abnormal/abnormal), and five embryos were abnormal/normal with an average of 5.0  1.5 analyzed nuclei. There was no significant difference between the rates of mosaic embryos achieved from oocytes after the various incubation times (Table 3) and those achieved from rescued mature MII oocytes (29/57; 50.8%) or arrested MI oocytes (25/36; 69.4%) (Table 4). A correlation was found between good embryo quality and normal chromosomal status, as all embryos from rescued IVM-MII oocytes or after short incubation periods (2–8 hours) had a significantly higher number of blastomeres per embryo and also having significantly fewer abnormal embryos. DISCUSSION Studying the outcome of IVM of MI oocytes after COH is of great interest to evaluate their clinical potential (5). The major objective of our study was to examine the chromosomal status of such embryos originating from MI oocytes after COH. Approximately 5%–20% of human oocytes collected during IVF procedures are unresponsive to the maturation trigger 974

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in vivo and remain meiotically immature (8, 10, 15, 20, 21). The maturation status of MI oocytes was checked after 2, 4– 8, and 24 hours of incubation in ICSI cycles. These incubation periods seem suitable for enabling proper nuclear and cytoplasm maturation. The maturation rates in our study of 29.7%, 61.7% and 72.2%, after 2, 4–8, and 24 hours of incubation, respectively, are in line with the maturation rates published earlier (24%–67% and 64%–75% after 2–9 and 24–26 hours of incubation (6–8, 15, 21–24). Considering fertilization of IVM oocytes, lower fertilization rates compared with IVM-MII oocytes has been reported (ranges of 40%–62% and 69%–81%, respectively) (7–10, 15, 21). Also, in the current study, we show a lower fertilization rate for rescued IVM-MII oocytes compared with retrieved MII oocytes (58.5% vs. 71.5%) and only a 43.9% fertilization rate in arrested MI oocytes. No significant statistical difference in fertilization rates was found among the different IVM incubation time interval groups in either the rescued IVM-MII or the arrested MI oocytes, in the current data or those of Vanhoutte et al. (6). Embryo quality in this condition is not clear (7, 8, 15). We found a lower mean number of cells in embryos from MI oocytes compared with MII oocytes (5.5 vs. 7.1). After a 24-hour incubation period, the number of cells was 4.5 per embryo, which agreed with the study of Chen et al. (15), who related these low rates to the aging of the oocyte. At present, few studies have checked the degree of aneuploidy in embryos originated from IVM oocytes in hyperstimulated cycles. Nogueira et al. (25) indicated a 78% aneuploidy rate in 14 embryos originating from GV oocytes, some of which were arrested at the two-cell stage. De Scisciolo et al. (26) found only 3% normal embryos from GV oocytes after IVM of 24 hours compared to 23% in mature MII oocytes (MII and MI at aspiration) after COH. Emery et al. (27) found a similar increase in aneuploidy in rescued ICSI oocytes after maturation of 16–24 hours compared with control embryos (79.7% vs. 60.5%), but they did not distinguish between MI and GV oocytes. In the present study, a high rate of 80.6% embryos from MI oocytes was found to be genetically abnormal: 97.2% in the arrested MI ova and 70.2% in the rescued IVM-MII oocytes. This is in comparison with the 40.3% abnormal embryo result in our routine preimplantation genetic diagnosis program. This result of 70% aneuploidy is in line with the 70%–75% genetic abnormality confirmed by FISH studies and comparative genomic hybridization on morphologically normal embryos after IVF (28–33). In our study, probes for only three chromosomes were used due to budget restrain. A higher rate of aneuploidy might have been detected had more analytic probes been used. The frequency of abnormal embryos increased among those originating from IVM-MII oocytes with the prolongation of incubation periods. After 24 hours of incubation no embryo was diploid, probably due to oocyte aging, which causes the vulnerability of its spindle. It has been shown

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TABLE 3 Results of fluorescent in-situ hybridization analysis for all embryos originated from both arrested metaphase I and rescued in vitro-matured metaphase II oocytes after various incubation periods. Incubation time

2h

4–8 h

24 h

No. of embryos analyzed Uniformly diploid X,X,18,18 X,Y,18,18

32

43

18

6 1 21.8%

9 2 25.5%

0 0 0

1 0 3.1%

0 3 6.9%

0 0 0

1 3.1%

0 0

0 0

2 6.2% 18 56.2%

0 0 24 (3 N/A) 55.8%

0 0 12 (2 N/A) 66.7%

1 1 6.2%

4 0 15.4%

2 1 16.7%

1 3.1% 0 0 0 0

1 2.3% 0 0 0 0

0 0 2 11.1% 1 5.5%

Uniformly haploid Y,18 X,18 Uniformly triploid

Uniformly tetraploid X,X,X,X,18,18,18,18, Mosaic Nullisomy 18 X Trisomy X Monosomy X, nullisomy 18 Monosomy X

Note: All cross-comparisons within and between subgroups were not statistically significant. Strassburger. Euploidy in embryos from MI and oocytes. Fertil Steril 2010.

already that in aged oocytes, the metaphase spindle is not located on the periphery and moves to the center of the oocyte (34). Embryos from arrested MI oocytes showed a high rate of aneuploidy in all incubation groups. It is possible that arrested MI oocytes include a cohort of oocytes that have incorrect spindle formation or function (35, 36), which interferes with their transition from MI to MII (37). It has already been shown that such oocytes can fertilize properly, but develop into aneuploid embryos (38, 39). Different embryonic abnormalities were found in our study. The complex chaotic mosaics with aberrations of more than one chromosome had a dominance of 58%. These results resemble the abnormalities found in the preimplantation genetic diagnosis program of embryos emerging from mature oocytes (40). The cause of abnormal embryos could be oocyte aneuploidy. Scarce information is available concerning aneuploidy in MI oocytes. Golbus (41) reported that rescued IVM mouse oocytes had a higher level of aneuFertility and Sterility

ploidy (possibly resulting from aberrant nuclear maturation). Cekleniak et al. (24) reported 62% and 75% aneuploidy in rescued IVM-MII oocytes after incubation in two different culture media. However, they included GV oocytes. Clyde et al. (42) demonstrated hyperploidy by Multifluor-FISH in GV-stage oocytes (from a woman who underwent ovarian stimulation) matured to MII in vitro. In a previous study, we found 20% (4 of 20) intact MI oocytes with chromosomal aberrations (2 nullisomies, 1 disomy, and 1 trisomy), similar to the rate of aneuploidies (26%) found in unfertilized oocytes after classic IVF programs (43–46). Aged oocytes or sperm abnormal karyotypes were suggested as the cause of aneuploidy. These possibilities are irrelevant in our study (46–50) as the average age of our patients was young and we strictly adhered to using the same donor sperm. Aneuploidy could arise during meiosis or fertilization. In this case, the first cell division may be hit by mitotic events leading to chromosome loss or gain 975

TABLE 4 Results of fluorescent in-situ hybridization analysis for all embryos originated from arrested metaphase I compared to embryos from rescued in vitro-matured metaphase II oocytes.

No. of embryos analyzed Uniformly diploid X,X,18,18 X,Y,18,18 Uniformly haploid Y,18 X,18 Uniformly triploid X,X,X,18,18,18 Uniformly tetraploid X,X,X,X,18,18,18,18, Mosaic Nullisomy 18 X Trisomy X Monosomy X, nullisomy 18 Monosomy X

Arrested MI

Rescued IVM-MII oocytes

36

57

1 0 2.8%

14 3 29.8%

0 1 2.8%

1 2 5.3%

1 2.8%

0 0

2 5.6% 25(3 N/A) 69.4%

0 0 29 (2 N/A) 50.8%

4 1 13.9%

3 1 7.1%

1 2.8% 0

1 1.8% 2

0 0 0

3.6% 1 1.8%

Note: All cross-comparisons within and between subgroups were not statistically significant. Strassburger. Euploidy in embryos from MI and oocytes. Fertil Steril 2010.

and both blastomeres will show the same chromosomal abnormality, either as a single aneuploidy or in combination with other abnormalities (10, 51–53). Chaotic cells are probably the result of postzygotic cleavages, involving multiple chromosomes simultaneously (50, 54). At the MII stage, the normal metaphase plate within the spindle is distinct, with the chromosomes in compact alignment within it (55). Aberrant nucleus or cytoplasmic maturation can cause damage to the nucleus DNA rearrangement and disformation of bipolar spindles. Chromosome 976

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alignments in the oocytes are indicators of a predisposition to nondisjunction and aneuploidy (17, 55–58). Various spindle abnormalities including abnormal shape, length, and chromosome loss from the spindle, presumably leading to chromosome malsegregation to the daughter cells was recently observed in a study using confocal laser scanning microscopy in embryos immunolabeled with antibodies against tubulin (54). These abnormal cell divisions can persist as long as the embryonic genome is not fully active and the cell cycle control is absent (59, 60). Low level mosaicism could result in a viable fetus if a core of abnormal cells that form the fetus is overgrown by normal cells (32, 40, 61). In our study, most mosaic embryos showed extensive aneuploidy (only 5 of 54 embryos had some normal cells) and thus, the possibility of the predominance of the normal cells and the correction of the mosaic is unlikely. It should be mentioned that although blastomeres with complex chromosome abnormalities are characteristic of preimplantation embryos (40), the conformation rate in mosaicism is low, as the removal of blastomeres is not random, and the chance of removing the reciprocal daughter cells is 25%. In addition, the poor representations of one-cell or two-cell biopsies for the seven- to six-cell postbiopsy embryo points to a high rate of false-negative results. In our study, however, the possibility of falsenegative results was reduced, as 3.2 blastomeres per embryo were fixed. To conclude, a significant difference between rescued IVM and arrested MI oocytes and their derivatives after ICSI was confirmed in this study. Rescued IVM oocytes showed higher fertilization rates, more blastomeres per embryo, and fewer abnormal embryos compared with embryos from arrested MI oocytes. In addition, rescued IVM-MII oocytes seem to be dependent on incubation periods, and their ICSI results are closer to the ones obtained from MII oocytes. Our study shows a high rate of chromosomal aberrations, mainly complex mosaics in embryos originating from arrested IVM or rescued IVM-MI oocytes that were incubated for longer than 8 hours. Because the general consensus is that mosaic embryos with <50% normal cells would be unlikely to survive beyond the implantation stage, a very limited potential value exists for the use of these embryos in the clinical context. In cases of a low number of MII oocytes, the use of rescued IVM-MI oocytes after a short incubation period could be an alternative. Preimplantation genetic diagnosis can be considered in these cases given our findings of a high rate of aneuploid embryos. However, the actual benefits of such testing have not been studied. REFERENCES 1. Cha KY, Chian RC. Maturation in vitro of immature human oocytes for clinical use. Hum Reprod Update 1998;66:103–20. 2. Kim BK, Lee SC, Kim KJ, Han CH, Kim JH. In vitro maturation, fertilization, and development of human germinal vesicle oocytes collected from stimulated cycles. Fertil Steril 2000;74:1153–8. 3. Smith SD, Mikkelsen A, Lindenberg S. Development of human oocytes matured in vitro for 28 or 36 hours. Fertil Steril 2000;73:541–5.

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Euploidy in embryos from MI and oocytes

Vol. 94, No. 3, August 2010