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Abstract |
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Key words: ICSI/implantation/IVF/maternal age/pronuclear morphology
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Introduction |
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The approach to identify human embryos with the highest implantation potential at the pronuclear stage is extremely interesting for countries with a strict embryo protection law, such as Germany and Switzerland. In these countries, the selection of cleavage stages is prohibited by law, and therefore no other embryos are available for later transfer than those selected at the pronuclear stage. At the pronuclear stage it has to be decided which cells are cultured and used for embryo transfer, and which are cryopreserved or discarded. A preliminary study on the use of a pronuclear score under these conditions has been published (Ludwig et al., 2000). These authors assessed several morphological criteria in 74 non-selected ICSI cycles and found a higher pregnancy rate above a certain pronuclear score threshold. In accordance with German embryo protection law, the aim of the present study was to evaluate the potential use of pronuclear selection in IVF and ICSI cycles based on a single morphological observation in a prospective, non-randomized multicentre study.
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Materials and methods |
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Assessment of pronuclear and embryo morphology
Pronuclear morphology was classified 1620 h after IVF or ICSI according to patterns previously described (05; Tesarik and Greco, 1999) with the following exception. In the original description, pattern 0 comprized two sub-patterns, one with >7 NPB showing an equal/symmetric distribution in each pronucleus and another with <7 NPB with polarization in each pronucleus at the area of contact. In this study, these two patterns were specified as pattern 0A (>7 NPB, equally distributed) and 0B (
7 NPB, polarized). Each centre selected preferentially pattern 0A and/or 0B for further culture and transfer. When sufficient pattern 0 pronuclear stage oocytes were not available, other patterns were selected. Each pronuclear-stage oocyte was cultured individually. Embryo morphology was assessed on day 2, and each embryo was given an embryo score according to a published system (Steer et al., 1992
). Embryo transfers were performed on days 25 after follicular aspiration, depending on the transfer policy of each centre. According to the German embryo protection law, no embryos other than those derived from the pronuclear-stage oocytes selected for further culture and transfer were available.
Based on previously published data on the assessment of pronuclear morphology (Scott and Smith, 1998; Tesarik and Greco, 1999
), it was considered unethical to perform a randomization with one group of patients receiving embryos from good prognosis pronuclear-stage oocytes and another group of patients receiving embryos from pronuclear-stage oocytes with a poor prognosis (Ludwig et al., 2000
).
Evaluations
For the calculation of the pregnancy rate only cycles with proven implantation, documented by ultrasound, were considered. The implantation rate was calculated from the number of gestational sacs divided by the total number of embryos transferred. For each pronuclear morphology pattern the mean embryo score was calculated based on all embryos derived from that specific pattern. Clinical pregnancy and implantation rates were calculated for all transfer cycles with at least one embryo derived from pattern 0B versus all remaining transfer cycles without embryos derived from pattern 0B. For all other patterns, pregnancy and implantation rates were calculated for all transfer cycles with at least one embryo derived from that specific pattern X (X = 0A, 1, 2, 3, 4, 5) versus all remaining transfer cycles without embryos derived from pattern X ([0A+1+2+3+4+5] X). For both of these calculations, all transfer cycles with at least one embryo derived from pattern 0B were excluded.
An evaluation of the data was performed for the number of presumable good pronuclear morphology pattern 0A/0B in relation to maternal age (35 years/>35 years) and in relation to assisted reproduction technique (IVF/ICSI).
Statistical analysis
Statistical evaluation was performed with ANOVA and a 2 test where appropriate; a P-value < 0.05 was considered significant.
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Results |
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Embryo development and implantation potential
Out of 1114 embryos which were transferred, 489 were derived from a pronuclear morphology pattern with a presumable good prognosis for implantation (patterns 0A/0B), and 625 from a pronuclear morphology pattern with a presumable poor prognosis for implantation (patterns 15; Table I). Embryos derived from a pronuclear morphology pattern with a presumably good prognosis showed a significant higher embryonic score (12.7 ± 5.0) compared with embryos derived from poor prognosis patterns (11.7 ± 5.3; P < 0.001).
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Maternal age and pronuclear morphology
The relationship between maternal age and pronuclear morphology was analysed (Table III). Significantly more pronuclear-stage oocytes of pattern 0B and 3 were available in younger patients (aged
35 years; 34.2 and 9.2%) than in older patients (aged >35 years; 25.8 and 5.4%; P < 0.005 and P < 0.05 respectively). For pattern 0A, 4 and 5 no significant differences were found, whereas significantly more pattern 1 and 2 oocytes were observed for transfer in older patients.
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Pronuclear morphology and assisted reproductive technique
The distribution of pronuclear morphology patterns selected for transfer in IVF and ICSI treatment cycles is shown in Table IV. Significantly more 0B pattern were available in ICSI (36.3%) compared with IVF (27.9%; P < 0.025). For pattern 0A and pattern 1, significantly more oocytes were available in IVF, whereas for patterns 25 no significant differences were found. In ICSI, significantly more transfer cycles were noted involving pattern 0B (144/256) compared with IVF (109/239; P < 0.025); however, despite this trend the corresponding pregnancy (35.5 versus 28.9%) and implantation rates (19.3 versus 17.1%) were not significantly different. There was also no significant difference in maternal age (33.0 ± 4.3 versus 33.5 ± 4.1 years).
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Discussion |
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In the current study, transfer cycles involving at least one embryo with pattern 3 versus transfer cycles without pattern 3 gave significantly higher pregnancy and implantation rates. As the primary aim of the study was the selection of pronuclear morphology pattern 0, the number of cycles involving pattern 3 are rather small and cannot be compared with transfer cycles with pattern 0B. It cannot be excluded that pattern 3 pronuclear-stage patterns can develop into pattern 0B with time however, and this point deserves further attention.
Another interesting point with regard to the subclassification of pattern 0 into 0A and 0B is embryo quality. Pattern 0A-derived embryos exhibited on day 2 an embryonic score comparable with that of pattern 0B, and significantly different from patterns 15. However, despite the higher embryonic score, transfers involving pattern 0A gave no higher implantation or pregnancy rates compared with transfers without pattern 0A. This suggests that a possible selection based on embryo quality on day 2 would not necessarily guarantee selection of embryos with the best implantation potential. In other words, at this stage of embryo development there are good quality embryos (derived from pattern 0B) with a high implantation potential, and also good quality embryos with a lower implantation potential (derived from pattern 0A). Consequently, embryo selection should not rely only on embryonic morphology, and other evaluation criteria such as pronuclear morphology or the developmental progression of the embryos must also be considered (Huisman et al., 2000; Racowsky et al., 2000
; Shapiro et al., 2000
).
During the course of this study, another scoring system was published (Scott et al., 2000) which is in fact a revision of the original zygote scoring system established previously by the same group (Scott and Smith, 1998
). The revised system is mainly defined by alignment, number and distribution of NPB. The different zygote morphology patterns were classified as Z1, Z2, Z3 and Z4. Scott and co-workers showed that Z1-scored zygotes possess a high potential to develop to blastocysts on days 56 and that these blastocysts exhibited a high implantation potential. Patterns different from Z1 showed significantly lower blastocyst formation rates and implantation rates. Interestingly, pattern 0B corresponds to zygote morphology Z1, patterns 0A and 1 correspond to Z2, and Z3Z4 (Scott et al., 2000
) may be compared with patterns 25 classified by others (Tesarik and Greco, 1999
) and by the current study. The results from the current prospective multicentre study correlate well with the data presented elsewhere (Scott et al., 2000
) in that preselection by a zygote score can give high implantation rates, although in the current study no double selection was possible by day 3 morphology. However, in contrast to results of the previously cited study (Scott et al., 2000
), a difference was found in the pattern distribution with regard to age and assisted reproduction technique. Therefore, the approach of pronuclear selection is a good option for countries with legal restriction of embryo selection at the blastocyst stage. However, from the patient's viewpoint, a better strategy would be pre-selection of pronuclear morphology patterns 0B/Z1, followed by selection of the best embryos derived from these pronuclear stages on the day of transfer, as proposed by others (Scott et al., 2000
). This treatment option offers undoubtedly the best basis towards single embryo transfer without compromising pregnancy rates.
The current study revealed that for patients with advanced maternal age (>35 years) significantly fewer pronuclear morphology pattern 0B were available for further culture and transfer. It should be noted that this analysis did not include all day 1 pronuclear oocytes, but only those selected for further culture and transfer. The reason for choosing the age of 35 years was due to an existing guideline in Germany, according to which only two embryos should be transferred in patients aged 35 years, and three embryos in those aged >35 years. A similar evaluation in choosing a cut-off at 38 years has already been made for 262 transfer cycles (Wittemer et al., 2000
). These authors found no significant difference for pattern 0 (0A + 0B) with regard to maternal age, although a trend was detectable towards reduced numbers of pattern 0 (39.3 versus 52.3%) in older patients (aged >38 years). The higher number of transfer cycles in this study (495 versus 262) may explain why statistical significance was reached. The current data also show that the implantation rate of cycles with pattern 0B is lower for older patients (>35 years; 15.8%) compared with younger patients (
35 years; 22.6%). These differences were not significant, but such a trend shows that in older patients the success of implantation is also influenced by other factors.
The current study reports a significant difference between IVF and ICSI cycles with regard to the number of pattern 0B for further culture and transfer. In ICSI cycles, more pattern 0B were available compared with IVF cycles (36.3 versus 27.9%; P < 0.025), and this phenomenon may be explained by the difference in the course of development which was reported to be accelerated after ICSI (Nagy et al., 1998; Sakkas et al., 1998
). Once the pronuclei are formed, further changes occur in the nucleus which involve polarization of chromatin (Van Blerkom et al., 1995
) and NPB (Tesarik and Kopecny, 1989
; Payne et al., 1997
). NPB are markers for chromosomes hosting rDNA-genes (Goessens, 1984
; Tesarik and Kopecny, 1990
), and consequently pattern 0B/Z1 characterizes the most advanced stage of nuclear polarization, which is also part of embryonic axis formation in the zygote (Edwards and Beard, 1997
). Zygotes reach this stage earlier after ICSI than after IVF. All centres participating in this study usually screened the pronuclear stages at 1620 h after insemination of oocytes, independently of the type of assisted reproductive technique used. It may be worthwhile exploring whether the difference observed would still exist if zygotes derived from IVF were to be screened later (1820 h) than those from ICSI (1618 h).
In conclusion, the current study provides further evidence that pronuclear morphology is an important addition to current assisted reproduction technique practice. In countries with legal restrictions of embryo selection, pronuclear morphology screening appears to be a useful and easy technique. In all other countries, pronuclear morphology offers an additional screening method which, in combination with extended embryo culture and a second selection based on blastocyst evaluation, represents a major step towards single embryo transfer.
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Acknowledgements |
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Notes |
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* Participating centres: V.Blumenauer, J.Schuster, F.A.Hmeidan, Zentrum f. Reproduktionsmedizin, Goldschmidstr. 30, D-04103 Leipzig; C.Balko, R.Sudik, Klinik f. Gynäkologie u. Geburtshilfe, Salvador-Allende-Str. 30, D-17036 Neubrandenburg; E.Schanze, K.Marx, R.J.van Kooij, U.Weidner, Praxis Verhoeven Scholtes Marx Wohlers Schulte, Neusser Str. 111, D-40219 Düsseldorf; J.Hirchenhain, J.S.Krüssel, P.Bielfeld, Universitäts-Frauenklinik, Moorenstr. 5, D-40225 Düsseldorf; B.Herrmann, J.Tigges, Praxis Dres Tigges Doerner Tuchel, Rheydter Str. 143, D-41515 Grevenbroich; P.Gaßner, A.Schepers, H.M.Behre, Institut f. Reproduktionsmedizin, Universität Münster, Domagkstr. 11, D-48129 Münster; M.Montag, M.Kupka, K.van der Ven, H.van der Ven, Abt. Gyn. Endokrinol. u. Reproduktionsmedizin, Universitäts-Frauenklinik, Sigmund-Freud-Str. 25, D-53105 Bonn; M.Greuner, S.Roth, M.Thaele, Praxis Happel Thaele Happel, Kaiserstr. 7, 66111 Saarbrücken; I.Eberhardt, D.Seehaus, T.Strowitzki, Abt. Gyn. Endokrinol. u. Fertilitätsstörungen, Universitäts-Frauenklinik, Voßstr. 9, D-69115 Heidelberg; D.Ecke, C.Keck, Breckwoldt, Universitäts-Frauenklinik, Hugstetter Str. 55, D-79106 Freiburg
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Submitted on May 15, 2001; accepted on August 10, 2001.