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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Key words: aneuploidy/human oocytes/ICSI/in-vitro maturation/nuclear transplantation
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Introduction |
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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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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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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 1.01.5 kV/cm direct current fusion pulse in HEPES-HTF-HSA was delivered for 70100 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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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 13 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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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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Cytogenetic analysis of reconstituted oocytes
The average maternal age for the oocytes analysed cytogenetically was 32.4 ± 3 years (range, 2842). 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 2941), 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 2842 years), there was no significant difference in the incidence of chromosomal abnormality between the two groups.
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Discussion |
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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 2535% 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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Notes |
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References |
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Battaglia, D.E., Goodwin, P., Klein, N.A. et al. (1996) Influence of maternal age on meiotic spindle assembly in oocytes from naturally cycling women. Hum. Reprod., 11, 22172222.[Abstract]
Cohen, J., Scott, R., Alikani, M. et al. (1998) Ooplasmic transfer in mature human oocytes. Mol. Hum. Reprod., 4, 269280.[Abstract]
Dailey, T., Dale, B., Cohen, J. et al. (1996) Association between nondisjunction and maternal age in meiosis-II human oocytes. Am. J. Hum. Genet., 59, 176184.[ISI][Medline]
de Loos, F., Kastrop, P., Van Maurile, P. et al. (1991) Heterologous cell contacts and metabolic coupling in bovine cumulus oocyte complexes. Mol. Reprod. Dev., 28, 255259.[ISI][Medline]
Edirisinghe, W.R., Junk, S.M., Matson, P.L. and Yovich, J.L. (1997) Birth from cryopreserved embryos following in-vitro maturation of oocytes and intracytoplasmic sperm injection. Hum. Reprod., 12, 10561058.[ISI][Medline]
Gaulden, M.E. (1992) Maternal age effect: the enigma of Down syndrome and other trisomic conditions. Mutat. Res., 296, 6988.[ISI][Medline]
Gianaroli, L., Magli, M.C., Ferraretti, A.P. et al. (1997) Preimplantation genetic diagnosis increases the implantation rate in human in vitro fertilization by avoiding the transfer of chromosomally abnormal embryos. Fertil. Steril., 68, 11281131.[ISI][Medline]
Gianaroli, L., Magli, M.C., Ferraretti, A.P. et al. (1999) Preimplantation diagnosis for aneuploidies in patients undergoing in vitro fertilization with a poor prognosis: identification of the categories for which it should be proposed. Fertil. Steril., 72, 837844.[ISI][Medline]
Goud, P.T., Goud, A.P., Qian, C. et al. (1998) In-vitro maturation of human germinal vesicle stage oocytes: role of cumulus cells and epidermal growth factor in the culture medium. Hum. Reprod., 13, 16381644.[Abstract]
Hassold, T. and Chiu, D. (1985) Maternal age-specific rates of numerical chromosome abnormalities with special reference to trisomy. Hum. Genet., 70, 1117.[ISI][Medline]
Janssenswillen, C., Nagy, Z.P. and Van Steirteghem, A. (1995) Maturation of human cumulus-free germinal vesicle-stage oocytes to metaphase II by coculture with monolayer Vero cells. Hum. Reprod., 10, 375378.[Abstract]
Karnikova, L., Urban, F., Moor, R. and Fulka, J. Jr (1998) Mouse oocyte maturation: the effect of modified nucleocytoplasmic ratio. Reprod. Nutr. Dev., 38, 665670.[ISI][Medline]
Kono, T., Obata, Y., Yoshimzu, T. et al. (1996) Epigenetic modifications during oocyte growth correlates with extended parthenogenetic development in the mouse. Nat. Genet., 13, 9194.[ISI][Medline]
Liu, L., Day, Y. and Moor, R.M. (1997) Nuclear transfer in sheep embryos: the effect of cell-cycle coordination between nucleus and cytoplasm and the use of in vitro matured oocytes. Mol. Reprod. Dev., 47, 255264.[ISI][Medline]
Muggleton-Harris, A., Whittingham, D.G. and Wilson, L. (1982) Cytoplasmic control of preimplantation development in-vitro in the mouse. Nature, 299, 460461.[ISI][Medline]
Munné, S., Alikani, M., Tomkin, G. et al. (1995a) Embryo morphology, developmental rates, and maternal age are correlated with chromosome abnormalities. Fertil. Steril., 64, 382391.[ISI][Medline]
Munné, S., Dailey, T., Sultan, K.M. et al. (1995b) The use of first polar bodies for preimplantation diagnosis of aneuploidy. Mol. Hum. Reprod., 1, see Hum. Reprod., 10, 10141020.
Munné, S., Magli, C., Cohen, J. et al. (1999) Positive outcome after preimplantation diagnosis of aneuploidy in human embryos. Hum. Reprod., 14, 21912199.
Nagy, Z.P., Cecile, J., Liu, J. et al. (1996) Pregnancy and birth after intracytoplasmic sperm injection of in vitro matured germinal-vesicle stage oocytes: case report. Fertil. Steril., 65, 10471050.[ISI][Medline]
Palermo, G.D., Cohen, J., Alikani, M. et al. (1995) Intracytoplasmic sperm injection: a novel treatment for all forms of male factor infertility. Fertil. Steril., 63, 12311240.[ISI][Medline]
Palermo, G.D., Alikani, M., Bertoli, M. et al. (1996a) Oolemma characteristics in relation to survival and fertilization patterns of oocytes treated by intracytoplasmic sperm injection. Hum. Reprod., 11, 172176.[Abstract]
Palermo, G.D., Schlegel, P.N., Colombero, L.T. et al. (1996b) Aggressive sperm immobilization prior to intracytoplasmic sperm injection with immature spermatozoa improves fertilization and pregnancy rates. Hum. Reprod., 11, 10231029.[Abstract]
Palermo, G.D., Avrech, O.M., Colombero, L.T. et al. (1997) Human sperm cytosolic factor triggers Ca2+ oscillations and overcomes activation failure of mammalian oocytes. Mol. Hum. Reprod., 3, 367374.[Abstract]
Park, S.E., Son, W.Y., Lee, S.H. et al. (1997) Chromosome and spindle configurations of human oocytes matured in vitro after cryopreservation at the germinal vesicle stage. Fertil. Steril., 68, 920926.[ISI][Medline]
Pellestor, F. (1991) Frequency and distribution of aneuploidy in human female gametes. Hum. Genet., 86, 283288.[ISI][Medline]
Plachot, M., Veiga, A., Montagut, J. et al. (1988) Are clinical and biological IVF parameters correlated with chromosomal disorders in early life: a multicentric study. Hum Reprod., 3, 627635.[Abstract]
Pratt, H.P. and Muggleton-Harris, A.L. (1988) Cycling cytoplasmic factors that promote mitosis in the cultured 2-cell mouse embryo. Development, 104, 115120.[Abstract]
Rickords, L.F. and White, K.L. (1992) Effect of electrofusion pulse in either electrolyte or non-electrolyte fusion medium on subsequent murine embryonic development. Mol. Reprod. Dev., 32, 259264.[ISI][Medline]
Spandorfer, S.D., Avrech, O.M., Colombero, L.T. et al. (1998) Effect of parental age on fertilization and pregnancy characteristics in couples treated by intracytoplasmic sperm injection. Hum. Reprod., 13, 334338.[Medline]
Takeuchi, T., Ergün, B., Huang, T.H. et al. (1999) A reliable technique of nuclear transplantation for immature mammalian oocytes. Hum. Reprod., 14, 13121317.
Tarín, J.J. (1995) Etiology of age-associated aneuploidy: a mechanism based on the `free radical theory of aging'. Hum. Reprod., 10, 15631565.[Abstract]
Tarín, J.J., Vendrell, F.J., Ten, J. et al. (1998) Antioxidant therapy counteracts the disturbing effects of diamide and maternal ageing on meiotic division and chromosomal segregation in mouse oocytes. Mol. Hum. Reprod., 4, 281288.[Abstract]
Trounson, A., Wood, C. and Kausche, A. (1994) In vitro maturation and the fertilization and developmental competence of oocytes recovered from untreated polycystic ovarian patients. Fertil. Steril., 62, 353362.[ISI][Medline]
Trounson, A., Anderiesz, C., Jones, G.M. et al. (1998) Oocyte maturation. Hum. Reprod., 13 (Suppl. 3), 5262.[Medline]
Tsai, M.C., Takeuchi, T., Rosenwaks, Z. and Palermo, G.D. (1999) The mitochondrial status of karyoplasts used for nuclear transplantation. Hum. Reprod., 14 (abstract book), 92.
Van Blerkom, J. (1994) Developmental failure in human reproduction associated with chromosomal abnormalities and cytoplasmic pathologies in meiotically mature oocytes. In Van Blerkom, J. (ed.), The Biological Basis of Early Human Reproductive Failure. Oxford University Press, New York, p. 283.
Van Blerkom, J., Davis, P., Merriam, J. et al. (1995) Nuclear cytoplasmic dynamics of sperm penetration, pronuclear formation and microtubule organization during fertilization and early preimplantation development in the human. Hum. Reprod. Update, 1, 429461.[Abstract]
Van Blerkom, J., Antczak, M. and Schrader, R. (1997) The developmental potential of the human oocyte is related to the dissolved oxygen content of follicular fluid: association with vascular endothelial growth factor levels and perifollicular blood flow characteristics. Hum. Reprod., 12, 10471055.[ISI][Medline]
Verlinsky, Y., Cieslak, J., Ivakhnenko, V. et al. (1999) Prevention of age-related aneuploidies by polar body testing of oocytes. J. Assist. Reprod. Genet., 16, 165169.[ISI][Medline]
Volarcik, K., Sheean, L., Goldfarb, J. et al. (1998) The meiotic competence of in-vitro matured human oocytes is influenced by donor age: evidence that folliculogenesis is compromised in the reproductively aged ovary. Hum. Reprod., 13, 154160.[Abstract]
Yanagida, K., Katayose, H., Yazawa, H. et al. (1999) Successful fertilization and pregnancy following ICSI and electrical oocyte activation. Hum. Reprod., 14, 13071311.
Zhang, J., Wang, C.W., Krey, L. et al. (1999) In vitro maturation of human preovulatory oocytes reconstructed by germinal vesicle transfer. Fertil. Steril., 71, 726731.[ISI][Medline]
Submitted on August 17, 2000; accepted on January 4, 2001.