Human Reproduction, Vol. 16, No. 4, 730-736,
April 2001
© 2001 European Society of Human Reproduction and Embryology
Preliminary findings in germinal vesicle transplantation of immature human oocytes
The Center for Reproductive Medicine and Infertility, Weill Medical College of Cornell University, New York, USA
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Abstract |
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Transplanting a germinal vesicle (GV) from an aged woman's oocyte into a younger ooplasm has been proposed as a possible way to reduce the incidence of oocyte aneuploidy which is considered to be responsible for age-related infertility. In this study, we have assessed the efficiency of each step involved in nuclear transplantation—specifically cell survival, nuclear-cytoplasmic reconstitution, and the capacity of the reconstituted oocytes for in-vitro maturation. In addition, we have evaluated the fertilizability and karyotypic status of the manipulated oocytes by intracytoplasmic sperm injection (ICSI) and fluorescent in-situ hybridization technique respectively. Nuclear transplantation was accomplished with an overall efficiency of 73%. Due to the limited availability of materials, most nuclear transplantation procedures were performed between sibling oocytes. The maturation rate of 62% following reconstitution was comparable with that of control oocytes, as was the incidence of aneuploidy among the reconstituted oocytes. The ICSI results of the reconstituted oocytes yielded a survival rate of 77%, a fertilization rate of 52%, and a satisfactory early embryonic cleavage. Furthermore, in a limited number of observations where the nucleus of an aged oocyte was transferred into a younger ooplasm, there was an appropriate chromosomal segregation. These findings demonstrate that human oocytes reconstituted with GV nuclei are able to undergo maturation, fertilization, and early embryo cleavage, and maintain a normal ploidy. Although in-vitro maturation seems to be a limiting step, this technique would allow us to investigate further the nuclear-ooplasmic relationship during meiotic maturation.
Key words: aneuploidy/human oocytes/ICSI/in-vitro maturation/nuclear transplantation
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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During the last 20 years, assisted reproductive techniques have been evolving to the point that it is now possible to obtain an ~50% pregnancy rate for younger couples regardless of the aetiology of the infertility. Yet, in spite of this high success rate and the innovative techniques being utilized, maternal age remains a limiting factor in achieving a pregnancy for older couples. The effect of oocyte ageing on implantation is considered to be related to an increased incidence of aneuploidy (Munné et al., 1995a
, 1999; Dailey et al., 1996; Gianaroli et al., 1997, 1999), and in turn to an abnormal state of the meiotic spindle (Battaglia et al., 1996; Volarcik et al., 1998). These spindle abnormalities seem to arise primarily during meiosis I (Hassold and Chiu, 1985), but it is not totally clear that abnormal distribution of the chromosomes between the oocyte and the polar body (PB) always results from an error in spindle behaviour per se. Segregation of chromosomes appears to be controlled by the meiotic spindle, but its components are largely supplemented by the ooplasm. Therefore, it has been suggested that dysfunctional cytoplasmic factor(s) are responsible for structural abnormalities of meiotic spindles, which lead to eventual chromosomal malsegregation (Gaulden, 1992; Van Blerkom, 1994; Battaglia et al., 1996).
Attempts to improve ongoing pregnancy/delivery rate in women with age-related infertility, who apparently are at increased risk for oocyte aneuploidy, have been made by selecting oocytes and embryos through preimplantation genetic diagnosis (PGD) (Gianaroli et al., 1997, 1999; Munné et al., 1999; Verlinski et al., 1999). The positive results of those studies with increased implantation and reduced miscarriage rates following PGD prove that oocyte/embryo aneuploidy is responsible for the impaired implantation rate in aged women. However, although the selection approach might enhance the implantation rate by identifying oocytes/embryos with a normal chromosomal content, the drawback is the limited number of embryos that can be transferred.
In view of the key role played by cytoplasmic factors on embryonic development (Muggleton-Harris et al., 1982; Pratt and Muggleton-Harris, 1988; Van Blerkom et al., 1995; Liu et al., 1997), a different approach has been proposed, namely the transfer of ooplasm isolated from oocytes of known developmental capacity into a metaphase II (MII) oocyte of a woman with poor embryo cleavage. This approach has been successful in achieving a pregnancy (Cohen et al., 1998). The injection of `fresh' ooplasm in the above-mentioned case appeared to restore cytoplasmic deficiencies. It has been suggested that healthy ooplasm may restore normal development in `metabolically' compromised embryos, and now accounts for a few offspring (Cohen et al., 1998). This technique, however, does not correct for the occurrence of chromosomal abnormalities due to non-disjunction at meiosis I.
A recent, more aggressive, approach is represented by the replacement of the entire cytoplasm. To be successful in preventing the occurrence of aneuploidy, this technique needs to be performed at an earlier maturational stage. Nuclear transplantation entails the transfer of an isolated germinal vesicle (GV) into a younger enucleated oocyte. Provision of a replacement younger cytoplasm has been suggested as a way of supporting a more normal meiotic spindle formation (Zhang et al., 1999). In the mouse, it has been demonstrated that nuclear transplantation can be accomplished efficiently, and this technique appears not to impair subsequent oocyte maturation or increase the incidence of chromosomal abnormalities (Takeuchi et al., 1999).
In this study, we have transplanted nuclei isolated from GV stage human oocytes, and have assessed the efficiency of this in terms of oocyte survival, nuclear-cytoplasmic reconstitution, subsequent nuclear maturation, fertilizability, and early embryonic development. In addition, some reconstituted oocytes were fixed and processed for cytogenetic analysis.
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Materials and methods |
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Oocyte collection
Immature GV oocytes were obtained from consenting patients undergoing intracytoplasmic sperm injection (ICSI). The research procedures as well as the handling of patient material were performed in accordance with a research protocol approved by the Committee of Human Rights of New York Presbyterian Hospital-Weill Medical College of Cornell University (IRB Number 0198-082). Oocyte retrieval was performed after pituitary desensitization with GnRH agonist and ovulation induction with gonadotrophins as described previously (Palermo et al., 1995
, 1996a,Palermo et al., b).
Immediately prior to ICSI insemination, cumulus-corona cells were removed by enzymatic and mechanical treatment (Palermo et al., 1995, 1996a,Palermo et al., b). The denuded oocytes were then examined under an inverted microscope to assess their integrity and stage of nuclear maturation. In this study, only excess GV stage oocytes of patients undergoing ICSI procedure for male factor infertility were used.
Enucleation procedure
Micromanipulation was performed in a shallow plastic Petri dish (Model: 1006; Falcon Beckton Dickinson Labware, Franklin Lakes, NJ, USA) in 5 µl droplets of HEPES-buffered human tubal fluid (HTF; Irvine Scientific, Santa Ana, CA, USA) medium supplemented with 0.4% of human serum albumin (HSA; Irvine Scientific) (HEPES-HTF-HSA). All microtools were made from borosilicate glass (Drummond Scientific, Broomall, PA, USA) drawn on a horizontal micropuller (Model: 753; Campden Instruments Ltd, Loughborough, UK) and calibrated, cut, and fire-polished on a microforge (Model: MF-9/900; Narishige, New York/New Jersey Scientific Inc., Middlebush, NJ, USA). Tools were controlled by two microinjectors (Model: IM-6; Narishige, New York/New Jersey Scientific Inc.). Micromanipulation procedures were carried out on a heated stage (Eastech Laboratory, Centereach, NY, USA) placed on an inverted microscope (Olympus IX-70; New York/New Jersey Scientific Inc.) which was equipped with two electrical/hydraulic micromanipulators (Model: MM-188 and MO-109, Narishige, New York/New Jersey Scientific Inc.).
The zona pellucida was breached by a glass microneedle tangentially inserted into the perivitelline space, against the holding pipette. Then, the GV surrounded by a small amount of cytoplasm (GV karyoplast) was directly removed by a cylindrical micropipette with a 30 µm inner diameter (Figure 1). Oocytes were exposed to HEPES-HTF-HSA containing 5 µg/ml of cytochalasin B (CCB; Sigma Chemical, St Louis, MO, USA) for at least 5 min prior to and throughout the procedure. The isolated GV karyoplast was immediately inserted with the same tool into the perivitelline space of a previously enucleated oocyte at the same maturational stage (GV cytoplast), so forming a `grafted oocyte' (Figure 2).
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After insertion of the karyoplast, grafted oocytes (couplets of GV karyoplast and cytoplast) were washed in CCB-free medium, then incubated in HTF-HSA at 37°C in 5% CO2 in air for ~30 min to recover before further procedures.
Electrofusion
An Electro Cell Manipulator (BTX 200 and 2001; Genetronics, Inc., San Diego, CA, USA) was used to deliver electrical pulses. Each grafted oocyte was placed between two micro-electrodes (ECF-100; Tokyo Rikakikai Co. Ltd, Tokyo, Japan) and manually aligned to place the karyoplast-cytoplast couplet perpendicular to the axis of the electrodes under micromanipulation control (Figure 3). The diameter of the tip of each micro-electrode was ~100 µm. To induce fusion, a single 1.0–1.5 kV/cm direct current fusion pulse in HEPES-HTF-HSA was delivered for 70–100 µs at 37°C. Fused oocytes were then washed and cultured in HTF-HSA, and studied 30 min later to confirm cell survival and signs of fusion (Figure 4). Up to four electrical pulses were applied at 30 min intervals where fusion did not at first occur.
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In-vitro maturation of reconstituted oocytes
Immature oocytes were allowed to mature by culture (HTF-HSA), and were observed at 24 and 48 h after electrofusion to evaluate nuclear maturation, characterized by germinal vesicle breakdown (GVBD) and subsequent extrusion of the first PB. Control GV oocytes that were not manipulated were cultured in the same conditions.
Cytogenetic evaluation of reconstituted in-vitro matured oocytes
Some oocytes that had extruded the first PB were prepared for chromosome analysis by gradual fixation as previously described (Takeuchi et al., 1999). Slides were then assessed immediately under phase contrast microscopy for presence of nuclei, dehydrated by subsequent passage for 2 min each in increasing concentrations of ethanol (70, 85 and 100%) and either analysed immediately or stored for later analysis at –20°C.
To evaluate as many chromosomes as possible, a sequential fluorescent in-situ hybridization (FISH) was performed on the same oocyte with probes for chromosome X, 13, 16, 18 and 21. The first step included direct label fluorescence probes: Vysis CEP X (Spectrum Aqua); CEP 16 (Spectrum Orange); and CEP 18 (Spectrum Green) (Vysis Inc., Downers Grove, IL, USA), for analysis of chromosomes X, 16 and 18 respectively. The hybridization solution was prepared by mixing 5.8 µl Spectrum CEP Hybridization buffer; 0.4 µl of X Spectrum Aqua; 0.4 µl of 16 Spectrum Orange; 0.4 µl of 18 Spectrum Green. The mixture was vortexed thoroughly, centrifuged for 1–3 s and left at room temperature for a short time. A total of 7 µl of the mixed solution was applied onto the specimen target area, and a coverslip was placed on the solution. Rubber cement was then applied to seal the coverslip on the slides. Denaturation was performed at 80°C for 3 min followed by DNA hybridization at 37°C in a moist dark chamber for at least 6 h. After careful removal of the rubber cement, washing was performed by plunging the slides in 0.3% Nonidet-P40 (Sigma Chemical Co.)/0.4xstandard saline citrate (SSC) at 70°C for 3 min followed by a 1 min wash in 2xSSC/0.1% Nonidet-P40 at room temperature. Nuclei were counterstained and maintained with 14 µl of DAPI in antifade solution (0.5 mg/ml, Vysis Inc.), covered with a coverslip, and observed at x600 with an epifluorescence microscope (Olympus BX 50; New York/New Jersey Scientific Inc.).
After assessment of the first set of chromosomes, the specimen was rinsed three times for 2 min in phosphate-buffered detergent (PBD; Oncor, Gaithersburg, MD, USA). Then the slides were sequentially dehydrated in increasing concentrations of ethanol (70, 85 and 100%), rinsed and denatured in 70% formamide/2xSSC at 70°C for 5 min, then dehydrated in sequential strips in increasing concentration of ethanol at 4°C for 1 min in each solution.
Additional probes were applied [Vysis LSI probes 13 (Spectrum Green) and 21 (Spectrum Orange)] to analyse the corresponding chromosomes.
Slides were viewed under a fluorescent microscope using single-bandpass filter sets including aqua (Vysis Inc.), rhodamine, fluorescein isothiocyanate (FITC) and DAPI (Olympus New York/New Jersey Scientific Inc.), and a triple bandpass filter set, DAPI/FITC/rhodamine (Vysis Inc.). After direct analysis, FISH images were captured and analysed with an imaging software (Quips; Vysis Inc.) (Figure 5). Scoring criteria were as previously described (Munné et al., 1995b; Dailey et al., 1996; Palermo et al., 1997).
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Fertilization assay
Some of the reconstituted in-vitro matured oocytes were injected with a single spermatozoon (ICSI) in a standard fashion (Palermo et al., 1995
, 1996b). In most of these, ICSI was performed 2–3 h after observation of the first PB extrusion. Approximately 16–20 h after the injection, the oocytes were observed for the presence of two distinct pronuclei and two clear PB, criteria of normal fertilization. Fertilized oocytes were then incubated further in HTF-HSA medium, for up to 72 h to evaluate their developmental capacity. The day 3 observation period was chosen according to our routine ICSI-embryo transfer protocol.
Data analysis
The 
2-test was utilized to identify differences in maturation rate observed between oocytes manipulated and non-manipulated control. Significance levels were at P = 0.05. All statistical computations were conducted using the Statistical Analysis System (SAS Institute, Cary, North Carolina, USA).
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Results |
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Reconstitution of oocytes after GV transplantation
A total of 197 GV human oocytes was obtained from 81 patients having a mean age of 34.3 ± 4 years (range, 25–43). Due to the limited availability of this human material, in most cases enucleated GVs were exchanged between sibling oocytes. In a preliminary experiment, nine GV oocytes which had arrested for at least 24 h were subjected to nuclear transplantation. Seven of these nine were successfully enucleated (77.8%) and then their GVs grafted in five oocytes (71.4%). Although subsequent electrofusion generated five reconstituted oocytes (100%), as expected none extruded the first PB. This preliminary experiment led us to improve the enucleation technique by increasing the inner diameter of enucleation pipettes, from 20 to 30 µm, and by adopting a gentler aspiration to avoid the rupture of the nuclear membrane. While we were aware of the importance of a control group, the majority of the available GV stage oocytes were subjected to nuclear transplantation, in order to maximize the information that can be obtained from this aggressive technique. The results of nuclear transplantation in 158 freshly retrieved oocytes are shown in Table I
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The overall incidence of reconstitution was 72.8% (115/158), and among 115 reconstituted oocytes, 71 matured spontaneously and extruded the first PB (61.7%) within 48 h in HTF-HSA. The remaining 44 oocytes remained at the GV (13/115, 11.3%) or metaphase I (31/115, 27.0%) stage. Among the GV stage control oocytes that were simply cultured in vitro, and not subject to either micromanipulation or electrostimulation, 19 of 30 (63.3%) matured to MII stage. Thus, the maturation rate of the reconstituted oocytes was not significantly different from the controls. After electrofusion, 26 out of 141 grafted oocytes failed to reconstitute. From these 26 oocytes, six remained intact and none underwent GVBD following 24 h culture.
Cytogenetic analysis of reconstituted oocytes
The average maternal age for the oocytes analysed cytogenetically was 32.4 ± 3 years (range, 28–42). A total of 21 reconstituted oocytes and 17 control oocytes were processed for FISH. Among 38 oocytes each fixed on an individual glass slide for FISH analysis, one slide (2.6%) was not analysable because of poor chromosomal spread. FISH errors were evident on three slides (7.9%), where there were discrepancies in FISH signals between the oocyte and the corresponding PB. Therefore, the overall efficiency of this FISH method was 89.5%. The karyotypes of the reconstituted oocytes generated by exchanging GV between sibling oocytes are shown in Table II. Normal complements of X, 13, 16, 18 and 21 were observed in 11 out of 14 oocytes (the average maternal age was 31.6 ± 3 years, range 29–41), with three having abnormalities (21.4%). Non-disjunction of a bivalent chromosome 13 was observed in one oocyte obtained from a 31 year old woman. Another (34 years old) showed unbalanced predivision of chromatid 18 and balanced predivision of chromatid X, while a third (29 years old) was diagnosed as unbalanced predivision of chromatid 16 and X, and balanced predivision of chromatid 21. Since only 11 out of 15 (73.3%) oocytes showed normal karyotypes in the control group (the average maternal age 33.7 ± 4, range 28–42 years), there was no significant difference in the incidence of chromosomal abnormality between the two groups.
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In order to assess the real contribution of the cytoplasmic replacement, in a total of eight oocytes, GV karyoplasts generated from patients of the `aged' and `young' groups were reciprocally cross-transplanted into cytoplasts of the opposite age (Figure 6A and B
). Seven out of eight oocytes (87.5%) were reconstituted by nuclear transplantation, and five eventually extruded a first PB (62.5%). Both oocytes (n = 2) reconstituted by placement of an aged karyoplast (37.5 ± 2 years, mean ± SD) in a young ooplast (30.0 ± 3 years) displayed a normal karyotype, whereas two of three karyotypes of young GV (31.3 ± 3 years) maturing in older ooplasm (36.0 ± 1 years) showed abnormal karyotypes (one oocyte had an unbalanced predivision of chromatids 13, and the other showed unbalanced predivision of chromatids 16 and 18).
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Fertilization and early embryonic development after ICSI
Reconstituted MII oocytes (n = 31) were inseminated by ICSI to assess their capacity to fertilize and develop. Their survival and fertilization characteristics are shown in Table III
. The average maternal age of this experimental group was 33.2 ± 4 years. In an initial study of nine oocytes, the oolemma was pierced by an ICSI needle inserted through the area previously used for nuclear removal and cell fusion; only five oocytes survived (56%) and only one fertilized (11%). In a later series of 22 oocytes, the sperm injection avoided the area where cell fusion had probably occurred, and both survival and fertilization rates were remarkably higher, 86% (19/22) and 68% (15/22), respectively. The fertilized oocytes with 2PN were allowed to cleave up to day 3 of culture, and showed an overall cleavage rate of 93.8% (15/16). At 3 days, the mean blastomere number and the frequency of anucleate fragments was 4.9 ± 2 (mean ± SD) and 25.0 ± 15 (mean % ± SD), respectively. Of two embryos kept further in culture up to day 5, one arrested at 8 cells and the other reached the compacted morula stage.
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Discussion |
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Our results demonstrate that nuclear transplantation of immature human oocytes can be accomplished efficiently, at a rate of >80% for each step. Since the maturation rate of the manipulated oocytes of 62% was similar to that in the non-manipulated control oocytes, it appears that in-vitro maturation (IVM) is the limiting step for a successful nuclear transplantation procedure. In previous investigations of IVM of cumulus-free GV oocytes isolated from stimulated ovaries, up to 38% matured during culture in simple defined media with or without serum (Janssenswillen et al., 1995
; Goud et al., 1998).
No GV karyoplasts which had failed to reconstitute underwent GVBD during 24 h incubation. This is in agreement with previous reports in the mouse that demonstrated an inverse relationship between spontaneous nuclear maturation and cytoplasmic volume (Karnikova et al., 1998; Takeuchi et al., 1999).
The incidence of chromosomal aneuploidy appears to be 25–35% in human oocytes matured in vivo (Plachot et al., 1988; Pellestor, 1991; Van Blerkom, 1994), and recently the incidence of aneuploidy was reported as 25% also among cumulus-enclosed immature oocytes cultured in vitro in a specific medium supplemented with gonadotrophins (Park et al., 1997). Our preliminary cytogenetic evaluation here has indicated that nuclear transplantation itself does not increase the incidence of aneuploidy after IVM compared to that in control oocytes. Although this observation includes only a limited number of cases, it indicates that younger ooplasm can support normal meiotic division of older GV, and, conversely, that older ooplasm tends to induce an abnormal segregation of meiotic chromosomes. This finding agrees with an earlier report in which younger ooplasm supported normal meiotic divisions in 80% (four out of five) of the transplanted older GV nuclei (Zhang et al., 1999).
We have demonstrated that the oocyte reconstituted by nuclear transplantation can retain its ability to mature in vitro and to be normally fertilized after ICSI. Although the oocyte survival rate and the embryo quality may appear to be poor, nevertheless these aspects and the patterns of fertilization were not different from those obtained using oocytes maturing in vivo (Spandorfer et al., 1998). The poor embryonic development observed in this study may have been due to suboptimal culture conditions utilized for IVM. It should also be noted that all the oocytes utilized in this study were exposed to HCG administered in vivo before collection, and were denuded of corona cells prior to GVBD; therefore, they were not isolated and cultured in an ideal setting for IVM (Janssenswillen et al., 1995; Goud et al., 1998). Moreover, IVM is still experimental and the object of several investigations; in fact only a few deliveries from such immature oocytes have been reported so far (Nagy et al., 1996; Edirisinghe et al., 1997).
Although the birth of mouse offspring following sequential nuclear transfer in two different maturational stage, ooplasm has been reported (Kono et al., 1996), further studies need to determine whether normal offspring can be obtained by single nuclear transfer of a GV stage oocyte.
Although there is no doubt that IVM of oocytes is a promising technique, its optimization remains a challenge (Trounson et al., 1994, 1998). Furthermore, although cumulus cells have been shown to benefit oocyte maturation and early development, it is necessary to remove them to perform nuclear transplantation. Even co-culture of immature oocytes with a cumulus-corona complex does not overcome the effect of its premature removal (de Loos et al., 1991; Allworth and Albertini, 1993). We need more information on the inter-relationship between the oocytes and somatic cells during the first meiotic division.
The effect of the electrofusion procedure on further development of the reconstituted oocytes must also be evaluated, since it has been suggested that the developmental capacity of the reconstituted oocytes is impaired by electrofusion (Cohen et al., 1998). However, it has been recently reported that electrical stimulation had a beneficial effect in inducing oocyte activation and resulted in successful births in patients whose oocytes failed to fertilize after ICSI (Yanagida et al., 1999). In this study, we used micro-electrodes controlled by a micromanipulator that allowed us to avoid the use of alternating current for cell alignment. Thus by eliminating the application of alternating current, we are able to minimize any unpredictable and undesirable harmful effects during the electrofusion step (Rickords and White, 1992; Takeuchi et al., 1999).
In summary, our findings provide further support to current hypotheses indicating that ooplasmic factors are finally responsible for the equal distribution of bivalent chromosomes between the ooplasm and the first PB during the completion of meiosis I. An explanation for this may lie in reactive oxygen species damage of mitochondrial function with consequent reduction in ATP levels (Tarín, 1995; Tarín et al., 1998). Another possibility may be a compromised perifollicular microcirculation attributed to an age-related follicular hypoxia that would induce a lower intracellular pH (pHi) (Gaulden, 1992; Van Blerkom et al., 1997). Such a decrease in pHi by a mitochondrial impairment would compromise the meiotic spindle of maturing oocytes. Due to paucity of human oocytes where inter-patient nuclear transplantation was performed, it is not possible to draw definite conclusions. We have demonstrated that our nuclear transplantation technique is highly effective in reconstituting hybrid immature human oocytes at a rate of 73%, while subsequent maturation rate is ~62%. Even though the limiting maturational step might be improved by utilizing better culture conditions, the acquisition of a sufficient number of oocytes from an aged woman, i.e. >40 years, to treat by nuclear transplantation remains the limiting factor, as proved by the limited number of oocytes available for cross-exchange in the present study.
However, there are other aspects of nuclear transplantation that also need to be evaluated, such as the role of mitochondrial DNA of donor/recipient oocytes on the development and phenotype of the resulting embryos (Tsai et al., 1999), and also the availability and viability of donor ooplasm.
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Acknowledgements |
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We thank the oocyte donors for their generous donations; the staff of the Center for Reproductive Medicine and Infertility for assisting with this study. We are also grateful for Prof. J.Michael Bedford for his critical review of the manuscript, Ms Queenie Neri for editing the manuscript, and Richard S.LaRocco for producing the illustrations.
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1 To whom correspondence should be addressed at: The Center for Reproductive Medicine and Infertility, Weill Medical College of Cornell University, 505 East 70th Street, HT-336, New York, NY 10021, USA. E-mail: gdpalerm{at}med.cornell.edu
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Submitted on August 17, 2000; accepted on January 4, 2001.
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