First polar body morphology and blastocyst formation rate in ICSI patients

T. Ebner1, M. Moser, M. Sommergruber, C. Yaman, U. Pfleger and G. Tews

Women’s General Hospital, IVF-Unit, Lederergasse 47, A-4010 Linz, Upper Austria, Austria


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: It may be beneficial to identify, at a very early stage of development, concepti that will result in viable blastocysts by using a non-invasive technique. METHODS: Homogeneous groups in terms of first polar body (PB) morphology were analysed with regard to fertilization, embryo quality and blastocyst formation. The strategy was to transfer a maximum of two blastocysts with an adequate inner cell mass deriving from oocytes with identical first PBs in order to obtain information about the actual implantation potential. RESULTS: A significant relationship between first PB morphology and embryo quality was found. Fragmentation after 2 days was increased in embryos derived from oocytes with fragmented first PBs (P < 0.05) in comparison with those derived from oocytes with intact PBs. No similar correlation could be demonstrated for fertilization rate. Embryos in the intact first PB group showed an increased rate of blastocyst formation as compared with the fragmented first PB group (P < 0.05). In addition, a significant difference in implantation rate (48.6 versus 22.0%; P < 0.025) and ongoing pregnancy rate (68.4 versus 34.8%; P < 0.05) was observed for the intact versus fragmented groups respectively. CONCLUSION: In conclusion, the current study provides further evidence that preselection at a very early stage may be helpful in identifying a subgroup of preimplantation embryos with a good prognosis to form blastocysts and, consequently, to implant.

Key words: blastocyst formation/first polar body/ICSI/oocyte quality/non-invasive selection


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Currently, elective embryo transfer at the blastocyst stage (day 5) is the most promising method available for attaining consistently high implantation and pregnancy rates without the risk of higher order multiple pregnancies (Gardner et al., 1998Go). This has been facilitated by the development of a new generation of sequential embryo culture media in assisted reproductive technologies (Gardner and Lane, 1998Go; Ménézo et al., 1998Go).

Nevertheless, many of the blastocysts transferred will not implant. This divergence may at least in part be attributable to culture conditions, which though significantly improved, have not yet been optimized (Gardner and Schoolcraft, 1998Go; Jones and Trounson, 1999Go). It has been suggested that prolonged incubation time may affect the quality of the preimplantation embryo, including alterations to the zona pellucida (De Vos and Van Steirteghem, 1999Go) and to the subsequent hatching process (Fong et al., 2001Go). However, data from Schiewe et al. do not support the concept that additional, abnormal hardening of the zona occurs during extended culturing (Schiewe et al., 1995Go).

It may be of great benefit to the patient if viable blastocyst candidates can be identified earlier in development in order to reduce the time in culture to a minimum. Studies (Scott et al., 2000Go; Balaban et al., 2001Go) have shown that adequate information about viability may be gained without extended incubation by scoring pronuclei at the zygote stage. Consequently, it is likely that the selection of embryos with a good prognosis in terms of implantation may be performed on the day of oocyte collection (Edwards and Beard, 1997Go). This hypothesis has also been supported by a suggested relationship between some morphological features of the oocyte and ICSI outcome (Xia, 1997Go; Ebner et al., 1999Go; Kahraman et al., 2000Go).

Based on our previous results (Ebner et al., 1999Go, 2000Go), the present prospective study was designed to investigate the actual blastocyst formation rate of oocytes showing fragmented or unfragmented first polar bodies (PBs). Homogeneous blastocyst transfers in terms of first PB morphology and subsequent rates of implantation and pregnancy will provide further insight into the prognostic value of the present mode of non-invasive selection.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
During a 3 month period (July to September 2001) a total of 60 consecutive ICSI patients (32.1 ± 3.8 years) considered for blastocyst transfer on day 5 were included in this study. This was approximately one-third (35.4%) of all patients treated during this period. They were admitted to our clinic because of male subfertility. Fifty-eight patients met our inclusion criteria for blastocyst culture and transfer (at least six oocytes collected and three good quality embryos on day 2); two women with one and four oocytes respectively wanted to participate in the study, and accepted the risk of not having transfer at the blastocyst stage.

Institutional Review Board approval was not sought since blastocyst culture and subsequent transfer have been introduced as a routine method in our laboratory.

All patients were stimulated according to a long protocol using a combination of a GnRH agonist (Suprecur; Hoechst, Frankfurt, Germany) and an individually adjusted dose of hMG (Menogon; Ferring, Kiel, Germany). At 36 h prior to ultrasound-guided oocyte retrieval, 5000–10 000 IU of hCG (Pregnyl; Organon, Oss, The Netherlands) was administered to induce ovulation.

All oocytes were incubated in BM1 medium (NMS Bio-Medical, Praroman, Switzerland) prior to insemination (3 h, 37°C and 6% CO2). Combining an enzymatic (80 mIU/ml hyaluronidase; MediCult, Copenhagen, Denmark) and a mechanical method, cumulus cells were removed in order to facilitate both evaluation of nuclear maturity and accurate assessment of first PB morphology.

After centrifugation of the ejaculate, a swim-up technique was used to separate progressively motile sperm with optimal morphology from the pellet. These were used for ICSI, which was performed as previously described (Ebner et al., 2001Go). Immediately after sperm injection, oocytes were cultured in groups according to the morphology of the first PB. The previously published grading system in terms of first PB appearance (Ebner et al., 1999Go) was adapted slightly. In detail, only two groups were investigated in the present study, those with intact first PBs (former grades 1 and 2) and those with fragmented first PBs (former grade 3). Based on our previously published data (Ebner et al., 2000Go) we decided not to apply ICSI to oocytes exhibiting large first PBs (former grade 4).

At 16–20 h after injection, fertilization and survival were assessed. The presence of two pronuclei as well as two PBs characterized normal fertilization (2PN). At the zygote stage, the medium was changed to Blastassist System Medium 1 (MediCult). According to their first PB, two groups of gametes were cultured in 75 µl droplets under sterile filtered paraffin (MediCult).

Approximately 42 h post-injection, embryo morphology criteria, i.e. number of blastomeres and degree of fragmentation, were recorded. From day 2 onward, the first sequential medium was replaced by Blastassist System Medium 2 (MediCult) which was changed daily until the day of transfer.

Blastocyst quality, as assessed by the size of the inner cell mass and the degree of expansion (Gardner et al., 2000Go), was recorded prior to transfer which was exclusively performed on day 5.

Our strategy was to retransfer a maximum of two blastocysts with an adequate inner cell mass deriving from oocytes with intact first PBs (n = 19). However, 18 patients had ‘mixed’ blastocyst transfers in terms of first PB morphology (one first PB intact and one fragmented). The remainder did not have any blastocysts which stemmed from oocytes with intact first PB for transfer (n = 23).

All data of this prospective self-controlled investigation were compared using {chi}2-test, Mann–Whitney U-test, and t-test. P < 0.05 was considered as statistically significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A total of 644 MII oocytes could be aspirated from 60 women (mean ± SD of 10.9 ± 3.9 mature oocytes per patient). More than 75% of injected oocytes (492/644) showed regular fertilization on day 1. Out of this cohort, 235 zygotes developed to the blastocyst stage (47.8%).

Table IGo illustrates a significant relationship between first PB morphology and embryo quality on day 2. A higher percentage of fragmentation was seen after 2 days in the group derived from oocytes with a fragmented first PB than in those with an intact PB (P < 0.05). Blastomere number and fertilization rate were similar in both groups. Embryos derived from oocytes with an intact first PB showed an increased rate of blastocyst formation as compared with the fragmented first PB group (P < 0.05).


View this table:
[in this window]
[in a new window]
 
Table I. Comparison between oocytes with different first polar body (PB) morphology in terms of fertilization, embryo quality and blastocyst formation rate. Values are mean ± SD
 
However, the size of the inner cell mass and developmental stage of the blastocysts were not associated with the morphology of the first PB. In detail, the classification of day 5 blastocysts (Gardner et al., 2000Go) showed that both groups had comparable percentages of early (22.9 versus 27.4%), full (62.7 versus 58.1%), expanded (6.8 versus 8.5%) and hatching blastocysts (7.6 versus 6.0%). No blastocysts had completely escaped from the zona. In addition, development of the inner cell mass and trophectoderm was similar in both patient cohorts.

Most of the women had two blastocysts transferred (n = 54). However, six patients underwent transfer of only one blastocyst because either no more blastocysts were available or there was a decision to limit the number of blastocysts for transfer to just one. This led to a mean number of 1.9 (± 0.3) blastocysts being replaced per patient. The quality of transferred blastocysts did not differ between the two groups.

A total of 114 blastocysts were replaced, out of which 40 implanted, giving an implantation rate of 35.1%. Thirty-four clinical pregnancies were achieved (56.7%). Since five missed abortions occurred (14.7%), the ongoing clinical pregnancy rate was 48.3% (29/60). Six twin pregnancies were detected, giving a multiple pregnancy rate of 17.6% (6/34).

The treatment outcome for both groups is summarized in Table IIGo. There was a significant difference in implantation (P < 0.025) and pregnancy rates (P < 0.05) between the intact and fragmented first PB groups.


View this table:
[in this window]
[in a new window]
 
Table II. Outcome of homogeneous and mixed blastocyst transfers on day 5
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Since there is some evidence that embryo selection on day 2 or 3 based on morphological criteria may be imprecise and result in the transfer of abnormal embryos or those that arrest at later developmental stages (Rijnders and Jansen, 1998Go), culturing embryos past embryonic genome activation and up to the blastocyst stage will allow the identification of those embryos which have no developmental block due to genetic or paternal reasons.

Nevertheless, approximately half of the blastocysts transferred will not proceed to subsequent implantation. One possible cause for this divergence may be aneuploidy (Munné and Cohen, 1998Go), but at least some of the non-viable blastocysts may fail to result in a pregnancy due to suboptimal culture conditions (Gardner and Schoolcraft, 1998Go; Jones and Trounson, 1999Go).

Therefore, it can only be for the benefit of patients if concepti with a good prognosis for implantation can be identified as early as possible. Indeed, there is some evidence that morphology of zygotes on day 1 (Scott et al., 2000Go; Balaban et al., 2001Go) will increase our understanding of subsequent blastulation and implantation.

Consequently, an alternative ‘back to the roots’ step would be to use polarity (Edwards and Beard, 1997Go) or morphological aspects of the oocyte prior to ICSI to obtain information about further development. It is important to mention that not all studies could demonstrate a significant relationship between oocyte morphology and implantation rate (De Sutter et al., 1996Go; Balaban et al., 1998Go) which indicates that, to a certain degree, oocyte dysmorphism is a normal feature. However some authors have reached opposite conclusions, finding that oocyte morphology (vacuoles, granularity of cytoplasm, inclusions) is negatively correlated to outcome (Serhal et al., 1997Go; Loutradis et al., 1999Go; Kahraman et al., 2000Go).

It is well accepted that, due to controlled ovarian stimulation (COS), not all oocytes recruited from the follicles are of the same quality. One possible reason for such a divergence may be a reduced blood supply to the follicles during COS which causes oxygen deficiency. Oocytes growing in such an environment were found to have an affected intracytoplasmic ATP content and show cytoplasmic disorganization (Van Blerkom et al., 1995Go). In addition, hypoxia can result in chromosomal and spindle defects in humans (Van Blerkom et al., 1997Go) and other mammals (Haidri et al., 1971Go).

As yet, it has not been clarified if some of these events may cause fragmentation of the first PB itself or if this scenario may interfere with nuclear and cytoplasmic maturation, which both have to be completed in a co-ordinated manner at the time of collection so as to ensure optimal conditions for subsequent fertilization.

Disturbances or asynchrony in these maturation processes may result in different morphological abnormalities depending on whether cytoplasmic (Loutradis et al., 1999Go; Kahraman et al., 2000Go) or nuclear maturation (Eichenlaub-Ritter et al., 1995Go; Ebner et al., 2000Go) has been affected.

It has been speculated that optimal nuclear maturation may be evidenced by an intact first PB (Xia, 1997Go; Ebner et al., 2000Go; Mikkelsen and Lindenberg, 2001Go). On the other hand, unusual asynchrony in follicular, cytoplasmic and nuclear kinetics may lead to oocytes showing fragmented first PBs. Since it is known that oocytes collected after COS may show an affected cytoplasmic organization and metaphase spindle (Van Blerkom et al., 1995Go, 1997Go), this may have influenced extrusion of the first PB. As a consequence, chromosomal status and/or oocyte polarity would be impaired irreversibly, possibly manifested in decreased fertilization or reduced embryo quality.

In fact, first PB morphology, either in combination with perivitelline space appearance and cytoplasmic inclusions (Xia, 1997Go) or analysed independently (Ebner et al., 1999Go), was found to be correlated with fertilization rate and embryo quality (Ebner et al., 2000Go) as well as with the rates of implantation and pregnancy (Ebner et al., 1999Go).

However, a suspected influence on fertilization rate (Ebner et al., 2000Go) could not be supported in the present prospective study. This discrepancy may be due to the fact that in the present study a simplified grading system has been applied to the oocytes in order to increase the sample number in both study groups. In particular, all ovoid or round first PBs were pooled together irrespective of the appearance of their surface (smooth or rough). The present results are in line with a previous study on in-vitro matured oocytes (Mikkelsen and Lindenberg, 2001Go) in which no relationship between extracytoplasmic abnormalities and fertilization rate could be found.

In terms of embryo quality, our previous results (Ebner et al., 2000Go) were confirmed. Once fertilized, oocytes with intact first PBs led to embryos of superior quality compared with the control group (P < 0.05). In the present study it was possible to show that it is not the number of blastomeres on day 2 which is influenced by the investigated oocyte criterion, but rather the degree of fragmentation (Table IGo). Although this 2.3% difference in fragmentation may be of questionable clinical relevance, it may reflect a possible defect in chromosomal complement of such oocytes and/or embryos, since percentage fragmentation has been associated with chromosomal abnormalities (Plachot et al., 1987Go).

It may be speculated that chromosomal aberrations may also be one of the reasons for a significantly diminished percentage of blastocysts found in the fragmented PB group (P < 0.025) since a strong selection against some clinically relevant chromosomal abnormalities has been reported (Magli et al., 2000Go; Sandalinas et al., 2001Go). However, it has to be stated that these authors provide no data on first PB morphology.

Despite the different percentage of blastocysts, both the homogeneous and the mixed group had the same proportion of high quality blastocysts developed in vitro. Therefore, it was possible to transfer blastocysts of comparable quality in all groups. The differences in the rates of implantation (P < 0.025) and ongoing pregnancy (P < 0.05) may indicate that the viability of blastocysts which stem from oocytes with fragmented PBs may be severely impaired.

Previously, elective transfer of day 2 embryos selected on the basis of first PB morphology was found to be associated with increased pregnancy rates (Ebner et al., 1999Go). As compared with these previous results, the present study appeared to show a trend for higher pregnancy rates in both homogeneous groups (P = 0.09). Although this difference was without statistical significance, it serves to emphasise the importance of a dual selection approach, e.g. preselection at the oocyte stage and a final selection at the blastocyst stage.

In conclusion, the current study provides further evidence that preselection at very early stages may be helpful to identify a special subgroup of preimplantation embryos with good prognosis to form blastocysts and, consequently, to implant.


    Notes
 
1 To whom correspondence should be addressed. E-mail: thomas.ebner{at}gespag.at Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Balaban, B., Urman, B., Sertac, A., Alatas, C., Aksoy, S. and Mercan, R. (1998) Oocyte morphology does not affect fertilization rate, embryo quality and implantation rate after intracytoplasmic sperm injection. Hum. Reprod., 13, 3431–3433.[Abstract]

Balaban, B., Urman, B., Isiklar, A., Alatas, C., Aksoy, S., Mercan, R., Mumcu, A. and Nuhoglu, A. (2001) The effect of pronuclear morphology on embryo quality parameters and blastocyst transfer outcome. Hum. Reprod., 16, 2357–2361.[Abstract/Free Full Text]

De Sutter, P., Dozortsev, D., Qian, C. and Dhont, M. (1996) Oocyte morphology does not correlate with fertilization rate and embryo quality after intracytoplasmic sperm injection. Hum. Reprod., 11, 595–597.[Abstract]

De Vos, A. and Van Steirteghem, A. (1999) Zona hardening, zona drilling and assisted hatching: new achievements in assisted reproduction. Cells Tissues Organs, 166, 220–227.[ISI]

Ebner, T., Moser, M., Yaman, C., Feichtinger, O., Hartl, J. and Tews, G. (1999) Elective embryo transfer selected on the basis of first polar body morphology is associated with increased rates of implantation and pregnancy. Fertil. Steril., 72, 599–603.[ISI][Medline]

Ebner, T., Yaman, C., Moser, M., Sommergruber, M., Feichtinger, O. and Tews, G. (2000) Prognostic value of first polar body morphology on fertilization rate and embryo quality in intracytoplasmic sperm injection. Hum. Reprod., 15, 427–430.[Abstract/Free Full Text]

Ebner, T., Yaman, C., Moser, M., Sommergruber, M., Jesacher, K. and Tews G. (2001) A prospective study on oocyte survival rate after ICSI: influence of morphological features and injection technique. J. Assist. Reprod. Genet., 18, 601–606.

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

Eichenlaub-Ritter, U., Schmiady, H., Kentenich, H. and Soewarto, D. (1995) Recurrent failure in polar body formation and premature chromosome condensation in oocytes from a human patient: indicators of asynchrony in nuclear and cytoplasmic maturation. Hum. Reprod., 10, 2343–2349.[Abstract]

Fong, C.Y., Bongso, A., Sathananthan, H., Ho, J. and Ng, S.C. (2001) Ultrastructural observations of enzymatically treated human blastocysts: zona-free blastocyst transfer and rescue of blastocysts with hatching difficulties. Hum. Reprod., 16, 540–546.[Abstract/Free Full Text]

Gardner, D.K. and Lane, M. (1998) Culture of viable human blastocysts in defined sequential serum-free media. Hum. Reprod., 13 (Suppl. 3), 148–159.[Medline]

Gardner, D.K. and Schoolcraft, W.B. (1998) No longer neglected: the human blastocyst. Hum. Reprod., 13, 3289–3292.[ISI][Medline]

Gardner, D.K., Schoolcraft, W.B., Wagley, L., Schlenker, T., Stevens, J. and Hesla, J. (1998) A prospective randomized trial of blastocyst culture and transfer in in-vitro fertilization. Hum. Reprod., 13, 3434–3440.[Abstract]

Gardner, D.K., Lane, M., Stevens, J., Schlenker, T. and Schoolcraft, W.B. (2000) Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer. Fertil. Steril., 73, 1155–1158.[ISI][Medline]

Haidri, A.A., Miller, I.M. and Gwatkin, R.B. (1971) Culture of mouse oocytes in vitro, using a system without oil or protein. J. Reprod. Fertil., 26, 409–411.[Medline]

Jones, G.M. and Trounson, A. (1999) Blastocyst stage transfer: pitfalls and benefits. The benefits of extended culture. Hum. Reprod., 14, 1405–1408.[Free Full Text]

Kahraman, S., Yakin, K., Dönmez, E., Samli, H., Bahce, M., Cengiz, C., Sertyel, S., Samli, M. and Imirzalioglu, N. (2000) Relationship between granular cytoplasm of oocytes and pregnancy outcome following intracytoplasmic sperm injection. Hum. Reprod., 15, 2390–2393.[Abstract/Free Full Text]

Loutradis, D., Drakakis, P., Kallianidis, K., Milingos, S., Dendrinos, S. and Michalas, S. (1999) Oocyte morphology correlates with embryo quality and pregnancy rate after intracytoplasmic sperm injection. Fertil. Steril., 72, 240–244.[ISI][Medline]

Magli, M.C., Jones, G.M., Gras, L., Gianaroli, L., Korman, I. and Trounson, A.O. (2000) Chromosome mosaicism in day 3 aneuploid embryos that develop to morphologically normal blastocysts in vitro. Hum. Reprod., 15, 1781–1786.[Abstract/Free Full Text]

Ménézo, Y.J.R., Hamamah, S., Hazout, A. and Dale B. (1998) Time to switch from co-culture to sequential defined media for transfer at the blastocyst stage. Hum. Reprod., 13, 2043–2044.[Free Full Text]

Mikkelsen, A.L. and Lindenberg, S. (2001) Morphology of in-vitro matured oocytes: impact on fertility potential and embryo quality. Hum. Reprod., 16, 1714–1718.[Abstract/Free Full Text]

Munné, S. and Cohen, J. (1998) Chromosome abnormalities in human embryos. Hum. Reprod., Update, 4, 842–855.[Abstract/Free Full Text]

Plachot, M., Junca, A.M., Mandelbaum, J., de Grouchy, J., Salat-Baroux, J. and Cohen, J. (1987) Chromosome investigations in early life. II. Human preimplantation embryos. Hum. Reprod., 1, 29–35.[Abstract]

Rijnders, P.M. and Jansen, C.A.M. (1998) The predictive value of day 3 embryo morphology regarding blastocyst formation, pregnancy and implantation rate after day 5 transfer following in-vitro fertilization or intracytoplasmic sperm injection. Hum. Reprod., 13, 2869–2873.[Abstract/Free Full Text]

Sandalinas, M., Sadowy, S., Alikani, M., Calderon, G., Cohen, J. and Munné, S. (2001) Developmental ability of chromosomal abnormal human embryos to develop to the blastocyst stage. Hum. Reprod., 16, 1954–1958.[Abstract/Free Full Text]

Schiewe M.C., Araujo, E., Asch, R.H. and Balmaceda, J.P. (1995) Enzymatic characterization of zona pellucida hardening in human eggs and embryos. J. Assist. Reprod. Genet., 12, 2–7.[ISI][Medline]

Scott, L.A., Alvero, R., Leondires, M. and Miller, B. (2000) The morphology of human pronuclear embryos is positively related to blastocyst development and implantation. Hum. Reprod., 15, 2394–2403.[Abstract/Free Full Text]

Serhal, P.F., Ranieri, D.M. and Kinis, A. (1997) Oocyte morphology predicts outcome of intracytoplasmic sperm injection. Hum. Reprod., 12, 1267–1270.[ISI][Medline]

Van Blerkom, J., Davis, P.W. and Lee, J. (1995) ATP content of human oocytes and developmental potential and outcome after in-vitro fertilization and embryo transfer. Hum. Reprod., 10, 415–424.[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, 1047–1055.[ISI][Medline]

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, 1750–1755.[Abstract]

Submitted on January 9, 2002; resubmitted on March 15, 2002; accepted on May 21, 2002.