Women's General Hospital, IVF-Unit, Linz, Austria
Email: Thomas.ebner{at}gespag.at
Sir,
We read the manuscript dealing with the developmental potential of zygotes of different pronuclear orientation (Kattera and Chen, 2004) with great interest. To our amazement, one of the pioneering papers on this topic (Garello et al., 1999
) was not included in the list of references.
This shortcoming is all the more regrettable as Garello et al. (1999) support one important hypothesis of Kattera and Chen. Specifically, both concluded that the intracytoplasmic placement of a spermatozoon in a fixed plane relative to the first polar body does not result in an altered pronuclear orientation compared with the situation in conventional IVF in which sperm entry is unrestricted.
On the other hand, the results and subsequent conclusions differed in one major point, a fact deserving closer critical reflection. Most interestingly, Garello et al. (1999) reported a worsening of embryo quality with an increasing angle between the axis given by the pronuclei and the furthest polar body. According to this, an optimal situation can be assumed if the pronuclear axis and adjacent polar bodies are aligned. In order to restore oocyte polarity, this orientation can be achieved by active clockwise rotation of the ooplasm regulated by the sperm centrosome and aster (Edwards and Beard, 1997
).
However, this polar arrangement in pronuclear zygotes clearly resembles oocyte type PN3 as classified by Kattera and Chen, but, in contrast to previous data (Garello et al., 1999), it was obviously associated with an impaired preimplantation development. On the other hand, a significant increase in early cleavage and grade I embryos was observed in classes PN1 and PN4, indicating that a more parallel orientation of the pronuclei to the polar bodies might be advantageous. The authors' explanantion refers to the spatial requirements of the fertilized oocyte prior to meridional cleavage; however, it has to be taken into consideration that some spindle rotation occurs after pronuclear membrane breakdown (Scott, 2000
) and, consequently, any pronuclear orientation class has the possibility to arrange perpendicularly to the longitudinal axis prior to first cleavage. There is video-based evidence (Edwards and Beard, 1997
; Payne et al., 1997
) that in the vast majority of zygotes, the rotation is not anticlockwise, as assumed by the authors.
Some possible reasons may account for these contradictory findings. One would be inaccuracy in identifying both polar bodies. Though the first polar body in eutherian mammals generally has a shorter life than the second one, many first polar bodies in humans remain intact for >20 h after ovulation (Ortiz et al., 1983). Therefore, intactness and non-fragmentation may not be adequate markers to distinguish the first from the second polar body. The latter is typically nucleated (Gardner, 2002
), a morphological characteristic which may not serve as a clear distinctive feature at the light microscopic level. As mentioned by the authors, granularity of the cytoplasm (Wakayama and Yanagimachi, 1998
), indicating apoptotic processes (Choi et al., 1996
), would be the appropriate morphological feature in order to identify the first polar body in zygotes. However, the actual influence of such an error on further morphology may be considered as minimal since zygotes were excluded from the present analysis if both polar bodies were not in close proximity. Exclusion may have ranged up to one-third of all 2PN zygotes, since data from Garello et al. (1999)
suggest that only 64% of IVF and ICSI zygotes have both their polar bodies within an angle of 30°. In the light of more recent data (Gianaroli et al., 2003
), it may be argued that those zygotes with remote polar bodies represent a cohort characterized by chromosomal aberrations, though embryo morphology was not affected at all (Garello et al., 1999
).
Presuming that Kattera and Chen (2004) correctly orientated most of the zygotes, with the second polar body at the 6 o'clock position, an additional problem arose, which is identification of the female pronucleus in order to classify all zygotes accurately according to their guidelines. Smaller size, closer proximity to the second polar body and a smaller number of nucleoli may contribute to the identification of the female pronucleus, but usually less than half of the oocytes meet all three criteria (Payne et al., 1997
). In the present study, proper identification of the smaller female pronucleus could only be guaranteed in 70% of the fertilized oocytes, which is rather low compared with previous data (Payne et al., 1997
).
In the light of these potential inconsistencies, the predictive value of the study of Kattera and Chen (2004) may be somewhat limited. Considering their data, it is likely that early cleavage as well as implantation behaviour better represent the pronuclear pattern (Tesarik and Greco, 1999
) than pronuclear orientation.
To conclude, we consider this manuscript to be an important contribution to non-invasive selection in IVF laboratories, but in order to develop its entire prognostic potential a more accurate literature search would have been desirable.
Notes
The reply to this letter (deh300) written by Dr Kutluk Oktay was published in Human Reproduction Volume 19, Number 7 July 2004, pages 16811683
References
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Edwards RG and Beard H (1997) Oocyte polarity and cell determination in early mammalian embryos. Mol Hum Reprod 3, 863905.[Abstract]
Gardner RL (2002) Experimental analysis of second cleavage in the mouse. Hum Reprod 17, 31783189.
Garello C, Baker H, Rai J, Montgomery S, Wilson P, Kennedy CR and Hartshorne GM (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.
Gianaroli L, Magli MC, Ferraretti AP, Fortini D and Grieco N (2003) Pronuclear morphology and chromosomal abnormalities as scoring criteria for embryo selection. Fertil Steril 80, 341349.[CrossRef][Medline]
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Ortiz ME, Lucero P and Croxatto HB (1983) Postovulatory aging of human ova: II spontaneous division of the first polar body. Gamete Res 7, 269276.
Payne D, Flaherty SP, Barry MF and Mathews CD (1997) Preliminary observations on polar body extrusion and pronuclear formation in human oocytes using time-lapse video cinematography. Hum Reprod 12, 532541.[CrossRef][Medline]
Scott LA (2000) Oocyte and embryo polarity. Semin Reprod Med 18, 171183.[CrossRef][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.
Wakayama T and Yanagimachi R (1998) The first polar body can be used for the production of normal offsplring in mice. Biol Reprod 59, 100104.
Submitted on April 27, 2004; accepted on April 29, 2004.