1 Division of Reproductive Medicine and Infertility, Department of Obstetrics and Gynecology, Women & Infants Hospital of Rhode Island, Brown University School of Medicine, Providence, RI, 2 Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, New England Medical Center, Boston, MA and 3 Marine Biological Laboratory, Woods Hole, MA, USA
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: embryo development/human oocytes/Polscope/spindles
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Since most aneuploidies arise in the oocytes, studies of oocyte and particularly spindle structure, might help identify normal from abnormal embryos. Until now, no satisfactory method existed for clinical IVF to distinguish morphologically normal spindles. Conventional methods to image the spindle rely on fixation (Pickering et al., 1988; Pickering et al., 1990
; Almeida and Bolton, 1995
; Battaglia et al., 1996
), so they provide limited value to clinical IVF. Recently, we found that spindles in living human oocytes could be imaged with a new orientation-independent polarized microscope (Polscope) (Wang et al., 2001
). The Polscope employs novel electro-optical hardware and digital processing to enhance the sensitivity of polarized optics to image macromolecular structures within cells based on their birefringence (Oldenburge, 1999
). Since it illuminates specimens with the same intensity of light as differential interference contrast (DIC), and since DIC, which also employs a form of polarized light, has been used safely during IVF for over 20 years, the Polscope should not be detrimental to human oocytes. We have demonstrated no detrimental effects on mouse (Liu et al., 2000
) and human (W.H.Wang et al., unpublished data) oocytes and embryos when they were exposed to the Polscope, so the Polscope has potential for application in human IVF. Our previous studies also indicated that birefringent spindles in living human oocytes predicted higher fertilization rates after ICSI (Wang et al., 2001
) and that human spindles were easily destroyed during in-vitro manipulation (Wang et al., 2000a
) or during oocyte meiotic maturation (Wang et al., 2000b
). Therefore, changes in spindle structure may reflect cytoplasmic dysfunction or other damage to the oocytes. In the present study, we examined our hypothesis that spindle structure may predict the rate of embryo development in vitro.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Spindle examination in oocytes with the Polscope and ICSI
For imaging spindles and performing ICSI, each oocyte was placed in a 5 µl drop of modified HTF covered with warm paraffin oil (Gallard-Schleserger, Coral Place, NY, USA) in a Bioptechs Delta T.C.O. Culture Dish System (Bioptechs Inc., Butler, PA, USA). The system comprises a temperature controller, a stage adapter and the Delta T culture dish that has a specially coated clear glass (0.15 mm thick) bottom. The dishes were maintained at 37 ± 0.5°C during examination and ICSI. Oocytes were examined under a Zeiss Axiovert 100 with a Neofluar x40 strain-free objective with a Bioptechs Objective Heating System (Bioptechs Inc.) and LC Polscope optics and controller (CRI, Cambridge, MA, USA), combined with a computerized image analysis system (MetaMorph Universal Imaging System, West Chester, PA, USA). After imaging, ICSI was performed. Since the first polar body position in many oocytes does not predict accurately the spindle position (Wang et al., 2001), in oocytes with spindles, ICSI was performed after oocytes were rotated to place the spindle at the 6 or 12 o'clock position relative to the injection needle. In oocytes without birefringent spindles, ICSI was performed after the first polar body was placed at 6 or 12 o'clock relative to the injection needle. After ICSI, oocytes with or without spindles were washed and cultured separately in P1 medium (Irvine Scientific) supplemented with 6% SSS for examination of fertilization.
Embryo culture and examination
Sixteen to 18 h after ICSI, fertilization was evaluated and oocytes with 2 pronuclei and a second polar body were considered as normally fertilized. Fertilized oocytes were washed 34 times and cultured in freshly prepared growth medium (P1 + 10% SSS), which had been equilibrated in a CO2 incubator (3% CO2 in air, 37°C) overnight. On day 3 (the day of examination of fertilization was considered to be day 1), embryo cleavage and viability were evaluated according to laboratory protocols. If patients had fewer than seven fertilized oocytes, embryo transfer was conducted on day 3 and the remaining embryos cultured until day 5 or day 6, with excess viable embryos being frozen. If patients had seven or more normal fertilized oocytes, embryos were transferred on day 3 to blastocyst medium (Irvine Scientific) containing 10% SSS until embryo transfer on day 5 or day 6.
Experimental design
Imaging of birefringent spindles with the Polscope, ICSI on day 0 (oocyte retrieval day), fertilization examination on day 1 (1618 h after ICSI) and cleavage evaluation on day 3 were conducted on all oocytes from 136 cycles. As 73 patients received embryo transfer on day 3, no embryo evaluation was available on days 5 and 6 for these cycles. The 63 patients who received embryo transfer on day 5 (22 cycles) and day 6 (41 cycles) were evaluated on days 5 and 6 respectively.
Statistical analysis
Comparisons of data between oocytes with or without spindles were conducted by 2 test. Probability of P < 0.05 was considered to be statistically significant.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Spindle birefringence is an inherent physical property of the microtubule based structure. In contrast to fluorescence microscope imaging of spindles, polarized light imaging does not require invasive preparative techniques, such as fixation or staining. In addition to providing information about the presence or absence of the spindles, the Polscope also gives quantitative information about the morphological structure of the spindles, which was not analysed in this study. Further studies will need to evaluate the value of this quantitative, morphological information on spindle birefringence. Spindle structure may reflect oocyte quality by serving as a marker for reproductive senescence, cytoplasmic maturation and/or of pH and/or temperature stress during handling.
What induces embryo developmental blocks in oocytes lacking a spindle is unknown. Some explanations for the low developmental ability of oocytes without birefringent spindles can be suggested. First, it has been estimated that around a quarter of human oocytes from stimulated cycles are aneuploid and the frequency of this abnormality increases with age (Janny and Ménézo, 1996). Early studies indicated that genomic activation was necessary for blastocyst formation in the human. Failure of genetic activation may result in arrest of human embryos at the 8-cell to morula stages (Tesarik, 1989
). In this study, we found that oocytes without spindles during imaging released the second polar body if they were fertilized. These results may suggest that oocytes did not show a spindle due to temporary depolymerization of microtubules by some environmental changes, such as temperature (Wang et al., 2000a
). However, these oocytes recovered the spindles after being returned to the incubator. We do not know whether these oocytes will be aneuploid after fertilization. It is possible that oocytes without birefringent spindles may have abundant chromosomal abnormalities, which in turn might induce cell cycle arrest although direct confirmation of the association between aneuploidy and spindle structure is needed.
Secondly, because the first polar body position does not always accurately predict the spindle position (Wang et al., 2001), the damage to the spindles caused by ICSI is more likely to occur in oocytes without visible spindles.
Finally, oocytes without a birefringent spindle may be of poorer quality, thus developing also poorly as compared with those with birefringent spindles.
Battaglia et al. reported that the proportion of oocytes with abnormal spindles was significantly higher in older than in younger women, and abnormal spindles were associated with abnormal chromosome distribution (Battaglia et al., 1996). In the present study, although we found patient-dependent effects on the proportion of oocytes with or without birefringent spindles, we did not find any relationship between birefringent spindles and patient age. The difference between our study and that of Battaglia et al. 1996, in addition to the methods employed to image spindles, is that they obtained oocytes from `normal volunteers' while all our oocytes were retrieved from infertile patients. This is presumably why many young infertile patients may suffer spindle abnormalities. Other factors may also have influenced the presence of spindle birefringence in our study. For example, as the cytoskeleton is quite sensitive to environmental changes (Pickering et al., 1988
; Pickering et al., 1990
; Almeida and Bolton, 1995
; Wang et al., 2000a
), disruption of spindle architecture may have arisen during in-vitro manipulation. Also, the small number of cycles from older patient limits the ability to make conclusions about the effects of maternal age on spindle birefringence.
In the present study, we used objective heater in the x40 objective lens during oocyte examination and ICSI to maintain the medium temperature at 37°C, which we found to improve spindle stability and embryo quality. Despite this, a proportion of oocytes (<20%) still did not show birefringent spindles. As mentioned above, imaging of spindles in living oocytes under the Polscope is based on birefringence, an inherent physical property of the microtubules. Thus, oocytes not exhibiting spindle birefringence probably sustained insult during oocyte development, maturation and/or other in-vitro conditions. Furthermore, oocyte age, maternal age and other patient-dependent factors may disrupt spindles.
In this study, we found that the outcome of oocytes with spindles (from fertilization to blastocyst formation) were superior to those of oocytes without spindles. It is possible that such a tendency continues during subsequent development during implantation and pregnancy, although further studies are needed. It also is not clear at present whether decreased development in oocytes without spindles is related specifically to aneuploidies, so future studies should examine this possibility, since the spindle plays such a crucial role during meiotic maturation and fertilization.
In conclusion, our results indicate that spindle birefringence can predict fertilization and embryo development. Further studies are needed to examine the relationship between spindle birefringence and the formation of aneuploidies and also to examine whether quantitative assessment of spindle structure and/or birefringence can further predict embryo development as well as pregnancies.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Battaglia, D.G., Goodwin, P., Klein, N.A. et al. (1996) Influence of maternal age on meiotic spindle in oocytes from naturally cycling women. Hum. Reprod., 11, 22172222.[Abstract]
Janny, L. and Ménézo, Y.J. (1996) Maternal age effect on early human embryonic development and blastocyst formation. Mol. Reprod. Dev., 45, 31.[ISI][Medline]
Liu, L., Oldenbourg, R., Trimarchi, J.R. and Keefe, D.L. (2000) A reliable, noninvasive technique for spindle imaging and enucleation of mammalian oocytes. Nature Biotech., 18, 223225.[ISI][Medline]
Munné, S., Alikani, M., Tomkin, G. et al. (1995) Embryo morphology, developmental rates and maternal age are correlated with chromosome abnormalities. Fertil. Steril., 64, 382391.[ISI][Medline]
Oldenburge, R. (1999) Polarized light microscopy of spindles. Methods Cell Biol., 61, 175208.[ISI][Medline]
Pickering, S.J., Johnson, M.H. and Braude, P.R. (1988) Cytoskeletal organization in fresh, aged and spontaneously activated human oocytes. Hum. Reprod., 3, 978989.[Abstract]
Pickering, S.J., Braude, P.R., Johnson, M.H. et al. (1990) Transient cooling to room temperature can cause irreversible disruption the meiotic spindle in the human oocytes. Fertil. Steril., 54, 102108.[ISI][Medline]
Tesarik, J. (1989) Influence oocyte coded message in cell differentiation control of human embryos. Development, 105, 119.[Abstract]
Wang, W.H., Meng, L., Hackett, R.J. et al. (2000a) Cooling induced spindle disassembly and the recovery after warming in living human eggs. Biol. Reprod. Suppl., 66, 191.
Wang, W.H., Meng, L., Hackett, R.J. et al. (2000b) Selection of morphologically normal human oocytes by the living spindle Polscope images. Fertil. Steril. (Suppl.), 74, S146.
Wang, W.H., Hackett, R.J., Meng, L. et al. (2001) Spindle observation and its relationship with fertilization after ICSI in living human oocytes. Fertil. Steril., 75, 348353.[ISI][Medline]
Submitted on December 11, 2000; accepted on April 4, 2001.