Department of Gynecology and Obstetrics, Medical University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany
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Abstract |
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Key words: embryo selection/multiple pregnancy rates/pronuclear stage oocytes
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Introduction |
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Several authors have tried to find a reliable scoring system for PN stage oocytes which has the same value for the prediction of implantation and pregnancy rates as have the ones for embryo selection at later cleaving stages. These scores are clearly indicated, as shown in recent articles because there is a definite polarity present, even as early as the PN stage, for different factors, e.g. gene expression (Edwards and Beard, 1997, 1999
). This tendency to polarization is the basis for the alignment of pronuclei as well as nucleoli during the fertilization process.
A maturation process of pronuclei in human oocytes following cryopreservation has been proposed (Wright et al.. 1990). These workers observed an ongoing association of pronuclei and the process of alignment of nucleoli which takes up to 26 h following insemination. The observation of aligned pronuclei was correlated with increased pregnancy rates. Similiar changes have been observed using time-lapse cinematography (Payne et al., 1997
), who also described a defined time-course of events as well as a great variation in the timing of these processes.
Based on these observations, Scott and Smith (1998) recently published a new scoring system for oocytes at the PN stage. This score is based on the fact that a faster developmental process after fertilization demonstrates a better quality of the oocytes and resulting embryos. Their score included not only the morphological appearance of the pronuclei, but also the further development up to the PN membrane breakdown and first cleavage division. This last item (PN membrane breakdown and first cleavage division within 2426 h post oocyte retrieval) constituted 2/5 of the maximum score. In Germany, however, this item cannot be included, since only selection at the PN stage is allowed and supernumerary PN oocytes must be cryopreserved at the PN stage or discarded.
In this study we applied the scoring system on the quality assessment of PN stages, based only on the morphological appearance of the PN oocytes, in order to evaluate its impact on the pregnancy rate under the conditions of the ESchG.
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Materials and methods |
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Scoring was done at 1618 h post ICSI according to (i) the position of the PN, (ii) the alignment of nucleoli at the junction of the two pronuclei, and (iii) the appearance of the cytoplasm, as shown in Table I. Each PN oocyte was evaluated at x400 magnification under an inverted microscope.
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Ovarian stimulation and ICSI
All patients were stimulated according to the long luteal protocol, using a gonadotrophin releasing hormone agonist depot preparation (Decapeptyl Gyn Depot; Ferring Arzneimittel GmbH, Kiel, Germany) and either human menopausal gonadotrophin (HMG, Menogon; Ferring Arzneimittel GmbH) or recombinant FSH (recFSH, Gonal F; Serono GmbH, München, Germany). Ovulation induction was begun when at least three follicles had a diameter of 18 mm, using 10 000 IU human chorionic gonadotrophin (HCG, Choragon; Ferring Arzneimittel GmbH). Transvaginal oocyte retrieval was performed by ultrasound guidance under general anaesthesia or slight sedation. ICSI was performed in each of these cycles as previously described (Al-Hasani et al., 1999
). In 62 cycles the indication for an ICSI procedure was male factor infertility; in 12 cases other indications, including previously failed or low fertilization rate or a thick zona was present.
Luteal phase support was performed using either vaginal progesterone alone (Utrogest; Kade, Berlin, Germany), in combination with a single injection of HCG (Choragon, Ferring Arzneimittel GmbH) on the day of embryo transfer, or several injections of HCG on the day of embryo transfer (5000 IU), and 3 days (5000 IU) and 6 days later (2500 IU).
Only clinical pregnancies with positive fetal heart beats were registered.
Cumulative embryo score
The cumulative embryo score (Steer et al., 1992) was slightly modified, since we did not use four but three different grades of embryo quality. The quality score of a single embryo was calculated as the product of the number of its blastomeres and the degree of its quality grade (1: fair; 2: moderate; 3: ideal). The cumulative embryo score was calculated as the sum of all single embryo scores.
Statistics
All statistics were done using a two-sided heteroscedastic t-test using the Microsoft Office Excel 7.0 software.
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Results |
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The mean and cumulative PN scores for all cycles are shown in Table II. Table III
shows the scores for those cycles in which surplus PN oocytes were available and therefore could be selected for further embryo culture, whilst supernumerary PN oocytes were cryopreserved, as well as for those cycles where a maximum of three 2PN oocytes was present and therefore no selection was possible.
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The mean PN score was not different in those cycles with and without male factor infertility (12.83 ± 1.47, n = 62 and 12.97 ± 1.28, n = 12 respectively). The data arranged according to different female ages are shown in Table IV, and revealed an apparent trend towards lower cumulative PN scores for older women, but this was not significant.
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Discussion |
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This is highly relevant to German IVF programmes, which are governed by the ESchG prohibiting embryo selection: from our results, it should be possible to select the two best PN oocytes and therefore limit the number of embryos for transfer to two, without reduction of pregnancy rates. The mean PN score might be advantageous over the cumulative PN score, since only the former can be directly related to pregnancy rates in cycles in which only two PN oocytes are selected, since the mean PN score is independent from the total number of selected PN oocytes.
A reason for the lower positive preditive value in our series, compared to that of Scott and Smith (1998) may be that we exclusively used ICSI and not conventional IVF. It is known that the fertilization process after ICSI is accelerated compared to conventional IVF (Nagy et al., 1994, 1998
). Therefore it might be necessary to evalute the PN score earlier in ICSI than in conventional IVF.
Furthermore, it has been shown that early cleavage of embryos [the item of the Scott and Smith score (1998) which we by law had to exclude] is one of the most important predictors of embryo quality and pregnancy rates (Edwards et al., 1984; Sakkas et al., 1998
).
Finally, the transfer procedure in the original series (Scott and Smith, 1998) is responsible for the difference in pregnancy rates: three, four, five and six embryos were transferred in patients <30, 3035, 3639 and
40 years of age respectively. In our own series the mean number of embryos was of necessity below three because in Germany only a maximum of three embryos is allowed to be transferred (Ludwig and Diedrich, 1999
).
Scott and Smith (1998) were the first to propose a score for clinical application. Recently, however, Tesarik and Greco (1999) used the assembly, growth and mutual fusion of nucleolar precursor bodies (NPB) as a parameter for PN oocyte quality assessment. Evaluation was done 12 to 20 h post insemination or post ICSI, and six different patterns of PN stage morphology were proposed. A normal pattern was defined as either the alignment of a mean of four NPB at the contact of the two pronuclei, or a random pattern of more than seven NPB in each pronucleus (pattern 0). These zygotes showed the lowest significant rate of cleavage arrest during further development over the next 12 days. Abnormal patterns were defined by a difference >3 between the number of NPB in the two pronuclei (pattern 1), or a reduced number of randomly scattered NPB (<7) (pattern 2), a large number (>7) of polarized NPB at the contact of the two pronuclei (pattern 3), a small number of NPB (<3) in at least one pronucleus (pattern 4), or a polarized distribution of NPB in one pronucleus and a non-polarized distribution in the other (pattern 5). This categorization, however, did not allow a good prediction of embryo morphology. The authors therefore proposed combining several scoring systems, e.g. from Scott and Smith (1998) and their own (Tesarik and Greco, 1999). Since this scoring system did not include the further development of the resulting embryos as an essential element, it might well be helpful for the PN scoring required in Germany. For this, however, another, prospective study is required.
The question remains as to which are the best culture conditions for human embryos. This is especially true since sequential media were proposed (Gardner and Lane 1997), thus reviving the idea of blastocyst transfer in the minds of most embryologists and clinicians in reproductive medicine. This may, however, work only in a setting where further selection of embryos is possible. Therefore, the practice of blastocyst transfer does not seem to be a good approach under the conditions of the German embryo protection law. It is also an interesting observation from our own study that the mean and cumulative PN scores were significantly different between the group of pregnant and non-pregnant patients, but that the cumulative embryo score was not significantly different between these two groups. This might indicate that in fact the further culture of qualitatively excellent PN oocytes leads to a loss in embryo quality. Consequently, in Germany the transfer at the PN stage would be the best way to circumvent these possibly negative effects of in-vitro culture. To our knowledge, only one prospective randomized study has been done which addressed this topic (Prietl et al., 1993
). Either PN stages or embryos were transferred at different time points. There was no significant difference between the pregnancy rates achieved by PN transfer or by embryo transfer on day 2. The fact that embryo quality did not improve during in-vitro culture after pre-selection at the PN stage confirms the results of others, who have demonstrated the highly efficient strategy of self-selection of embryos. By that natural strategy at day 3 or day 5, embryos with a much higher implantation rate can be achieved (Dawson et al., 1995
; Gardner et al., 1998
).
The use of the PN scoring may help to improve the pregnancy rate even under the conditions of the ESchG. To evaluate this possibility further, a prospective study would be necessary with a score-assisted selection of PN oocyte in one group and a random selection of PN oocytes in another. It is questionable if such an approach is ethical. However, even prospective use of the PN oocyte score in Germany combined with retrospective analysis of a matched control group should make it possible to show whether this score improves pregnancy rates in Germany after selection of the two best PN oocytes.
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Notes |
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References |
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Submitted on May 24, 1999; accepted on October 15, 1999.