A reply to ‘Developmental potential of human pronuclear zygotes in relation to their pronuclear orientation’

Suresh Kattera and Christopher Chen

Centre for Reproductive Medicine and Gleneagles IVF Centre, Gleneagles Hospital, Singapore 258500

Email: suresh{at}cccrm.com

Sir,

We thank Ebner et al. for their interest in our study and the issues that they raised in their letter.

In our study, we encountered a small percentage of oocytes with polar bodies far apart. This could have occurred due to movement of the first polar body during dissection prior to ICSI or during dissection following the fertilization check in IVF. It is also possible that these oocytes represent a cohort with chromosomal aberrations (Gianaroli et al., 2003Go). In our study, we excluded all those zygotes with polar bodies far apart. We classified zygotes into four types PN1, PN2, PN3 and PN4 based on the orientation of the pronucleus in relation to the second polar body where both the polar bodies were close together. On the other hand, Garello et al. (1999)Go measured the angles between the PN axis and the nearest polar body (Þ), between the PN axis and the farthest polar body ({beta}) and between the two polar bodies (Y). Thus the designs of the two studies were different and the conclusions drawn were obviously different. However, we agree that both Garello et al. (1999)Go and ourselves (Kattera and Chen, 2004Go) examined the possibility that the position of sperm entry into the oocytes is responsible for a particular orientation of PN. Both studies concluded that the sperm entry position has no role in the orientation of PN.

Ebner et al. in their letter point out contradictory findings in our work compared with the work of others (Edwards and Beard, 1997Go; Payne et al., 1997Go; Scott, 2000Go). First, we agree that the first polar body undergoes fragmentation and the parameters that are employed to differentiate the first and second polar body may be inadequate. We classified zygotes into four types when they were observed with the second polar body at the 6 o'clock position. However, in order to classify the zygotes, it was not absolutely necessary to identify first and second polar bodies as long as they were close together. This is because the PN orientation of a particular zygote will be similar whether it is measured in relation to the first or second polar body. The classification of the zygotes will remain the same. Thus it is not important to differentiate accurately between the first and the second polar body as long as they are together.

Secondly, Ebner et al. suggest errors in our classification of zygotes for PN orientation due to inaccurate identification of female pronuclei. In our study, classification of PN orientation was not dependent on identification of the female pronucleus. Nevertheless, we recorded the size of PN because criteria to identify the female PN include proximity to the polar bodies and size (Rawlins et al., 1988Go; Palermo et al., 1994Go; Feng and Gordon, 1996Go). The use of nucleoli to identify the female PN may lead to errors in some zygotes because the nucleoli are known to coalesce in some oocytes that develop rapidly and sometimes the number of nucleoli is equal in the two PN (Gossens, 1984Go; Tesarik and Kopecny, 1989Go, 1990Go). We measured the size of PN using a digital camera and image analysis software (Image ProPlus) in 2714 zygotes. We found that in 70% of the oocytes, the PN nearer the second polar body was smaller than the other, in 22% it was slightly larger and in 8% both PN were the same size. It is difficult to compare these data with those of Payne et al. (1997)Go who measured the PN in 38 zygotes using video cinematography. Differences in conclusions on the relative size of the female PN may be attributable to the methods of measurement and/or the vastly different number of observations.

Finally, Ebneret al. argue that there is video-based evidence (Edwards and Beard, 1997Go; Payne et al., 1997Go) that in the vast majority of zygotes, the rotation of the cytoplasm is not anti-clockwise. The video-based evidence is provided by Payne et al. (1997)Go not by Edwards and Beard (1997)Go as assumed by Ebneret al. We do not contest the video findings of Payne et al. (1997)Go. They reported clockwise movement of cytoplasmic waves in 95% of the oocytes that extruded the second polar body. The cytoplasmic waves ceased after extrusion of the second polar body and were not observed during PN formation and development. We did not study cytoplasm movement in oocytes prior to PN formation and development. We specifically looked at the orientation of PN, their possible rotation and their relationship to early cleavage. We did not observe any change in the orientation of PN in any of the four types of zygotes prior to their disappearance and cleavage. In our study, PN4 type zygotes with PN already oriented parallel to the polar bodies had a higher percentage of early cleavage than did PN2 and PN3 type zygotes. Between the disappearance of the PN and the onset of cleavage, the PN and spindle may rotate in preparation for meridional cleavage. This is based on observations (Edwards and Beard, 1997Go) of human pronuclear eggs in vitro, which show that the two PN can rotate to a polarized position. The probability that this occurs in an anticlockwise manner is supported by our observation of a higher percentage of early cleavage observed among PN4 type zygotes. However, further research in this area would prove our presumption.

References

Edwards RG and Beard H (1997) Oocyte polarity and cell determination in early mammalian embryos. Mol Hum Reprod 3, 863–905.[Abstract]

Feng YL and Gordon JW (1996) Birth of normal mice after removal of the supernumerary male pronucleus from polyspermic zygotes. Hum Reprod 11, 341–344.[Abstract]

Garello C, Baker H, Rai J, Montogomery 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, 2588–2595.[Abstract/Free Full Text]

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, 341–349.[CrossRef][Medline]

Gossens G (1984) Nucleolar structure. Int Rev Cytol 87, 107–108.[Medline]

Kattera S and Chen C (2004) Developmental potential of human pronuclear zygotes in relation to their pronuclear orientation. Hum Reprod 19, 294–299.[Abstract/Free Full Text]

Palermo G, Munne S and Cohen J (1994) The human zygote inherits its mitotic potential from the male gamete. Hum Reprod 9, 1220–1225.[Abstract]

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, 532–541.[CrossRef][Medline]

Rawlins GG, Binor Z, Radwanska E and Dmowski WP (1988) Microsurgical enucleation of tripronuclear human zygotes. Fertil Steril 50, 266–272.[Medline]

Scott LA (2000) Oocyte and embryo polarity. Semin Reprod Med 18, 171–183.[CrossRef][Medline]

Tesarik J and Kopecny V (1989) Development of human male pronucleus. Ultrastructure and timing. Gamete Res 24, 135–149.[Medline]

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, 153–156.[Medline]

Submitted on April 27, 2004; accepted on April 29, 2004.





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