Laboratoire d'Eylau, 55 Rue Saint-Didier, 75116 Paris, France
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: implantation rate/multiple pregnancy rate/pregnancy rate/pronuclear morphology/single-embryo transfer
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
If a subpopulation of embryos with a high implantation potential can be recognized by a combined evaluation of pronuclear and cleavage-stage morphology, the application of these criteria in the decision about the number and grade of embryos to be transferred will not only improve pregnancy rates but will also reduce the risk of multiple pregnancies by avoiding the transfer of several high-grade embryos at the same time.
To address these issues, we compare here the pregnancy and implantation rates in fresh embryo transfer cycles in which embryos that show good morphology at the cleavage stages but that had developed from zygotes with different patterns of pronuclear morphology were used. We also show how the relationship between the number of embryos transferred and the incidence of multiple pregnancies is dependent on the pronuclear morphology of the zygotes from which these embryos developed.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Means and percentages were compared by using Student's t-test and 2 test (StatView®; Abacus Concepts, Berkeley, CA, USA) respectively.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Though prospective in terms of the definitive characterization of the study design and the pronuclear morphology evaluation criteria before its beginning, this study is based on a retrospective cohort analysis, since it did not apply these criteria for embryo selection for transfer. Instead, embryos were selected according to the conventional criteria reflecting cleavage speed, blastomere regularity and the volume occupied by anuclear cell fragments. According to these conventional criteria, the morphological grade of the embryos selected for transfer did not differ between the group of embryos that developed from pattern 0 zygotes and those developed from non-pattern 0 zygotes, and the pronuclear grading thus represented an additional, possibly independent evaluation criterion. The inclusion of this additional criterion made it possible to recognize a subpopulation of good morphology embryos that had a very high probability of implanting successfully after transfer. In fact, the implantation rate of the embryos developing from pattern 0 zygotes was almost three-fold higher than that of the embryos developing from non-pattern 0 zygotes although the cleavage speed and morphology did not differ between these two groups of embryos.
In other terms, the developmental inferiority reflected by an abnormal pattern of pronuclear development does not always lead to a visible impairment of cleavage speed or excessive embryo fragmentation and may thus go unnoticed during the early cleavage stages. Because important developmental processes in human pronuclei, including the first zygotic round of DNA replication and an early wave of RNA synthesis, are known to be associated with the development of pronuclear structure and ultrastructure (Tesarik and Kopecny, 1989, 1990
), it may be speculated that asynchrony or alteration of these processes can be at the origin of molecular abnormalities whose morphological consequences appear later in development, perhaps after the major activation of the embryonic genome between the 4-cell and the 8-cell stage (Tesarik, 1987
). It has to be noted, however, that the absence of a difference in the morphological appearance and cleavage speed between these two groups of embryos is due to the fact that both groups consisted of the best embryos available that had been selected for transfer in each treatment cycle. If compared in the whole, unselected group of embryos, those embryos developing from pattern 0 zygotes show better morphology and are less likely to arrest cleavage as compared to embryos developing from non-pattern 0 zygotes (Tesarik and Greco, 1999
). This suggests that the adverse consequences of the pronuclear abnormalities may become patent during prolonged embryo culture to the blastocyst stage. This hypothesis, however, remains to be tested. A prospective study, in which embryos are chosen for transfer exclusively on the basis of pronuclear morphology, is needed in order to decide about the prognostic value of pronuclear morphology by itself as compared to the recently described strict embryo criteria, applied to cleavage-stage embryos, with which high implantation rates can also be achieved (Gerris et al., 1999
; Van Royen et al., 1999
).
It is important to note that the incidence of multiple pregnancies was very high when more than one pattern 0 embryo was replaced in a single transfer cycle, irrespective of the number of non-pattern 0 embryos that accompanied them in mixed transfer cycles. In fact, the incidence of twin pregnancies was almost twice that of singleton pregnancies when two pattern 0 embryos were transferred, and half of the clinical pregnancies developing after the transfer of three pattern 0 embryos were triplet. On the other hand, the overall pregnancy rate did not differ significantly between the transfer cycles in which one, two or three pattern 0 embryos were replaced. These observations have two practical implications. Firstly, it appears sufficient to transfer a single `top quality' embryo (a good morphology cleaving embryo having developed from a pattern 0 zygote) to achieve the optimal chance of pregnancy. In this respect, the value of the pronuclear criteria, used alone or in combination with the previously described criteria for single embryo transfer (Gerris et al., 1999; Van Royen et al., 1999
; Vilska et al., 1999
), remains to be determined by a prospective study. Secondly, the transfer of two or more such embryos entails a great risk of multiple pregnancy. Hence, if more than one embryo is to be transferred, it appears reasonable to avoid a simultaneous transfer of more than one embryo fulfilling, at the same time, the criteria of good morphology at the pronuclear and at the cleavage stage.
This transfer policy would lead to more high quality embryos being cryopreserved, preferentially accompanied by lower grade embryos in individual straws for later common transfer. Because the evaluation criteria concerning pronuclear morphology can also be used alone to select appropriate embryo groups for early cryopreservation at the pronuclear stage (Tesarik and Greco, 1999), and because pronuclear zygotes appear to survive the freezing and thawing procedures better as compared with cleavage-stage embryos (Veeck et al., 1993
; Al-Hassani et al., 1996; Hoover et al., 1997
), this policy may further increase the chance of pregnancy while reducing the risk of multiple implantations.
In conclusion, cleaving embryos developing from zygotes with normal morphology at the pronuclear stage have a better implantation potential than embryos developing from abnormal morphology zygotes, even if the cleavage speed and morphology of the embryos selected for transfer is the same. Even though this study only dealt with ICSI-derived zygotes, the pronuclear criteria used here had been originally defined by analysing a mixed group of embryos of which some originated from ICSI and others from conventional in-vitro fertilization (Tesarik and Greco, 1999). It is thus conceivable that the same selection criteria can be applied in both situations. High implantation rates (>30%) can be achieved by elective transfer of embryos developing from normal morphology zygotes, which may make it possible to transfer only one embryo at a time.
Pronuclear morphology is a valuable additional criterion for elective embryo transfer. It can be used to make the decision of which embryos will be transferred in the fresh state and which will be frozen as early as the pronuclear stage. As an additional criterion to the criteria reflecting cleavage speed and blastomere morphology, the evaluation of pronuclear morphology is useful to determine the most suitable number of embryos for transfer so as to achieve the optimal chance of conception while reducing the risk of multiple pregnancies.
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Balakier, H., MacLusky, N.J. and Casper, R.F. (1993) Characterization of the first cell cycle in human zygotes: implications for cryopreservation. Fertil. Steril., 59, 359365.[ISI][Medline]
Ebner, T., Moser, M., Yaman, C. et al. (1999) Elective transfer of embryos selected on the basis of first polar body morphology is associated with increased rates of implantation and pregnancy. Fertil. Steril., 72, 599603.[ISI][Medline]
Edwards, R.G. and Beard, H.K. (1997) UK law dictated the destruction of 3000 cryopreserved human embryos. Hum. Reprod., 12, 311.
Evans, D. and Evans, M. (1996) Fertility, infertility and the human embryo: ethics, law and practice of human artificial procreation. Hum. Reprod. Update, 2, 208224.
Garello, C., Baker, H., Rai, J. et al. (1999) Pronuclear orientation, polar body placement, and embryo quality after intracytoplasmic sperm injection and in-vitro fertilization: further evidence for polarity in human oocytes? Hum. Reprod., 14, 25882595.
Gerris, J., De Neubourg, D., Mangelschots, K. et al. (1999) Prevention of twin pregnancy after in-vitro fertilization or intracytoplasmic sperm injection based on strict embryo criteria: a prospective randomized clinical trial. Hum. Reprod., 14, 25812587.
Goud, P., Goud, A., Van Oostveldt, P. et al. (1999) Fertilization abnormalities and pronucleus size asynchrony after intracytoplasmic sperm injection are related to oocyte postmaturity. Fertil. Steril., 72, 245252.[ISI][Medline]
Hoover, L., Baker, A., Check, J.H. et al. (1997) Clinical outcome of cryopreserved human pronuclear stage embryos resulting from intracytoplasmic sperm injection. Fertil. Steril., 67, 621624.[ISI][Medline]
Payne, D., Flaherty, S.P., Barry, M.F. and Matthews, C.D. (1997) Preliminary observations on polar body extrusion and pronuclear formation in human oocytes using time-lapse video cinematography. Hum. Reprod., 12, 532541.[ISI][Medline]
Schäfer, D., Baumann, R. and Kettner, M. (1996) Ethics and reproductive medicine. Hum. Reprod. Update, 2, 447456.
Scott, L.A. and Smith, S. (1998) The successful use of pronuclear embryo transfers the day following oocyte retrieval. Hum. Reprod., 13, 10031013.[Abstract]
Serhal, R.F., Ranieri, D.M., Kinis, A. et al. (1997) Oocyte morphology predicts outcome of intracytoplasmic sperm injection. Hum. Reprod., 12, 12671270.[ISI][Medline]
Staessen, C., Janssenswillen, C., van den Abbeel, E. et al. (1993) Avoidance of triplet pregnancies by elective transfer of two good quality embryos. Hum. Reprod., 8, 16501653.[Abstract]
Tesarik, J. (1987) Gene activation in the human embryo developing in vitro. In Feichtinger, W. and Kemeter, P. (eds), Future Aspects in Human In Vitro Fertilization. Springer Verlag, Berlin, pp. 251261.
Tesarik, J. and Kopecny, V. (1989) Nucleic acid synthesis and development of human male pronucleus. J. Reprod. Fertil., 86, 549558.[Abstract]
Tesarik, J. and Kopecny, V. (1990) Assembly of the nucleolar precursor bodies in human male pronuclei is correlated with an early RNA synthetic activity. Exp. Cell Res., 191, 153156.[ISI][Medline]
Tesarik, J. and Greco, E. (1999) The probability of abnormal preimplantation development can be predicted by a single static observation on pronuclear stage morphology. Hum. Reprod., 14, 13181323.
Van Blerkom, J., Davis, P., Merriam, J. and Sinclair, J. (1995) Nuclear and 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 Royen, E., Mangelschots, K., De Neubourg, D. et al. (1999) Characterization of a top quality embryo, a step towards single-embryo transfer. Hum. Reprod., 14, 23452349.
Veeck, L.L., Amundson, C.H., Brothman, L.J. et al. (1993) Significantly enhanced pregnancy rates per cycle through cryopreservation and thaw of pronuclear stage oocytes. Fertil. Steril., 59, 12021207.[ISI][Medline]
Vilska, S., Tiitinen, A., Hyden-Granskog, C. and Hovatta, O. (1999) Elective transfer of one embryo results in an acceptable pregnancy rate and eliminates the risk of multiple birth. Hum. Reprod., 14, 23922395.
Wright, G., Wiker, S., Elsner, C. et al. (1990) Observations on the morphology of pronuclei and nucleoli in human zygotes and implications for cryopreservation. Hum. Reprod., 5, 109115.[Abstract]
Xia, P. (1997) Intracytoplasmic sperm injection: correlation of oocyte grade based on polar body, perivitelline space and cytoplasmic inclusions with fertilization rate and embryo quality. Hum. Reprod., 12, 17501755.[Abstract]
Submitted on November 29, 1999; accepted on February 24, 2000.