King Faisal Specialist Hospital and Research Center, Departments of 1 Obstetrics and Gynecology and 2 Pathology and Laboratory Medicine, P.O.Box 3354, Riyadh, 11211, Saudi Arabia
3 To whom correspondence should be addressed. e-mail: serdar{at}kfshrc.edu.sa
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Abstract |
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Key words: embryo transfer/pronuclear scoring/zygote
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Introduction |
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After more than two decades practice of assisted reproduction, the best day to transfer embryos to the uterus is still subject to debate. Although the general practice is to transfer at the cleavage stage on day 23, the concept of transferring embryos at blastocyst stage has gained popularity with the emergence of advanced sequential media (Gardner et al., 1998). This strategy has been challenged by several prospective randomized trials (Coskun et al., 2000
; Rienzi et al., 2002
), and the top quality embryos can be selected with better implantation potential on day 3 (Gerris et al., 1999
; Van Royen et al., 1999
).
It seems that the challenge lies in the selection of viable embryos regardless of the day of the transfer. The transfer at pronuclear stage could be justified since the basis for selection according to distinct morphological features of pronuclei has been proposed (Scott and Smith, 1998). It was demonstrated that embryos transferred at the zygote stage have the ability to implant and end in viable pregnancies (Ahuja et al., 1985
; Quinn et al., 1990
). In a recent prospective randomized multicentre study (Dale et al., 2002
), zygotes that were selected according to pronuclear and nucleolar morphology and transferred on day 1 resulted in similar pregnancy and implantation rates compared to embryos transferred on day 2 or 3. Pronuclear stage transfer is appealing because of simplifed laboratory procedures and reduced cost. Moreover, excess zygotes can be cryopreserved regardless of their quality which might result in more cryopreservation and additional pregnancies (Senn et al., 2000
).
It has also been shown that the morphology of pronuclear embryos is correlated to day 3 or 5 embryo morphology and/or pregnancy/implantation (Tesarik and Greco, 1999; Wittemer et al., 2000
; Scott et al., 2000
; Tesarik et al., 2000
; Balaban et al., 2001
; Montag et al., 2001
; Zollner et al., 2002
). The nuclear events are dynamic and happen during a certain time-course (Tesarik and Kopecny, 1989
). Although Scott and Smith (1998
) performed pronuclear scoring at three different times (1617, 22 and 2226 h post insemination), Tesarik and Greco (1999
) have suggested a successful use of a single static observation (1220 h post insemination or ICSI) of pronuclear morphology as a predictor of possible abnormal preimplantation development.
The objective of the present study was to investigate whether the transfer of zygotes selected according to a single observation of pronuclear morphology would result in similar pregnancy and implantation rates compared to those obtained by transferring embryos on day 3.
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Materials and methods |
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IVF/ICSI
Controlled ovarian hyperstimulation, oocyte retrieval, sperm preparation for IVF/ICSI and embryo culture and evaluation were performed as described previously (Coskun et al., 1998, 2000; Jaroudi et al., 1999
). Culture conditions were the same for both groups. Cumulusoocyte complexes after retrieval from the follicular fluid were transferred into 100 µl IVF medium (Medi-Cult, Denmark) under mineral oil (Sigma, USA). They were incubated in 5% CO2 in air with saturated humidity. All the fertilized oocytes were cultured individually in 20 µl Medi-Cult IVF medium under mineral oil until the time of the transfer.
Embryos were graded as good, fair and poor. Good embryos were defined as embryos with <10% fragmentation and 4 blastomeres. Fair embryos had 1020% fragmentation and poor embryos were heavily fragmented (
30%).
Pronuclear scoring
Normal fertilization was confirmed by the presence of two pronuclei and two polar bodies 1518 h following insemination or ICSI. Zygotes were evaluated according to criteria of Tesarik and Greco (1999). Each zygote was checked for the presence of a cytoplasmic halo, alignment and size of pronuclei, nucleolar precursor body (NPB) number and distribution pattern under an inverted microscope with a Hoffman modulation contrast system. All the information was recorded on a pre-designed data sheet and then registered into a database (Microsoft Access). Zygotes were classified according to pattern 0 or non-pattern 0 (Tesarik et al., 2000
). Briefly, pattern 0 zygotes had the following features: (i) the number of NPB between both pronuclei did not differ >3; (ii) NPB in both pronuclei were either polarized or scattered; (iii) in the polarized pronuclei, the number of NPB was
7; (iv) in the scattered pronuclei, the number of NPB was >7; (v) both pronuclei were similar size and aligned.
Embryo transfer
In our unit, a maximum of two embryos is routinely transferred. For day 1 transfers, two pattern 0 zygotes, when available, were chosen to transfer on the same day. If there is only one or no pattern 0 zygote available, non-pattern 0 zygotes were used. When there were at least four extra zygotes, they were cryopreserved. For day 3 transfers, embryos were cultured until day 3 on which the best two embryos were chosen for transfer according to day 3 embryo quality (Coskun et al., 1998). Cryopreservation was performed only when a patient had at least four good quality embryos with <10% fragmentation without any developmental block from day 2 to 3.
Patients in both groups were supplemented with progesterone (100 mg/daily i.m.; Steris Laboratories Inc., USA) starting from day 3 following the oocyte retrieval. Pregnancies were diagnosed with Tandom Icon urine hCG test (Hybritech, USA), and upon positive urine tests, serum -hCG levels were quantified. The status of pregnancy was checked by ultrasound examinations 5 weeks after embryo transfer.
Statistical analysis
A total of 302 patients was recruited into the study. This number was calculated on the assumption that a 15% lower pregnancy rate compared with the established rate (35%) would be considered as failure for the zygote transfer group. The sample size for the 0.15 difference in proportion with a power of 80% and of 0.05 requires
151 patients in each group. Comparisons between groups were made by using
2-analysis. P < 0.05 was considered statistically significant.
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Results |
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Discussion |
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Earlier studies (Ahuja et al., 1985; Quinn et al., 1990
) did not mention any selection criteria for zygotes to be transferred while Hurst et al. (1998
) gave a brief description of zygote selection. Day 1 transfers resulted in the lowest pregnancy rates when compared to day 23 or 45 transfers in a prospective randomized multicentre study (Margreiter et al., 2003
). However, no detail was given as to how zygotes were selected for transfer. Scott and Smith (1998
) described detailed criteria for zygote scoring in which the early cleavage was also assessed. Tesarik and Greco (1999
) later described a single static observation of pronuclear stage morphology of implanted embryos, which was later used to select zygotes to transfer by Dale et al. (2002
) and also by us in the current study. The main difference between these two studies was the average number of zygotes and embryos that were transferred; 1.85 versus 3.2 on day 1 and 1.91 versus 3.6 on day 3 in our study and that by Dale et al. (2002
) respectively. By transferring more zygotes, Dale et al. (2002
) might have obtained similar pregnancy rates for day 1 and day 3 transfers. Although they have transferred more embryos, pregnancy rates were similar between the two studies for embryo transfer. It has been earlier reported that decreasing the number of embryos did not change the pregnancy rate on day 3 (Templeton and Morris, 1998
).
This might indicate that zygote selection alone might not be sufficient to choose the most viable embryos. In our study, zygotes were selected for transfer according to the pattern 0 (Tesarik and Greco, 1999). Results were also separately analysed according to revised Z-scoring of Scott et al. (2000, data not shown). None of the scoring systems showed any correlation between the use of higher quality zygotes in transfers and pregnancy rates. Although there are several reports relating pregnancies to pronuclear scoring (Montag et al., 2001
; Wittemer et al., 2000
; Tesarik et al., 2000
), Salumets et al. (2001
) also did not find any difference in pregnancy and implantation rates between the pattern 0 and non-pattern 0 groups in single embryo transfer patients.
We analysed the relationship between embryo quality on day 3 and pronuclear morphology of those embryos, and did not find any significant correlation. Similar to our data, Salumets et al. (2001) did not find any significant difference of embryo morphology and zygote scoring. This is in contradiction to others who have reported a positive correlation with zygote morphology and cleavage stage embryo quality (Tesarik and Greco, 1999
; Ludwig et al., 2000
; Balaban et al., 2001
). The literature still remains inconclusive on this issue.
The observation time might have played an important role on the grading of zygotes. The coalescence and polarization of NPB are dynamic events (Tesarik and Kopecny, 1989; Payne et al., 1997
). Timing of pronuclear formation is markedly varied between oocytes after ICSI (Payne et al., 1997
). This individual difference might be further substantiated in IVF since penetration of sperm through cumulus cells, zona pellucida and fusion might require at least an extra 4 h following insemination (Gianaroli et al., 1996
). It has been reported that four stages have been distinguished during pronuclear development and some of the zygotes never reach the final stage (Tesarik and Kopecny, 1989
). A single observation, as has been common practice, including this study, might not reveal the true status of zygote morphology because it cannot determine whether the nuclear development is in progress or arrested at any one of the four stages.
There were significantly more cycles with cryopreservation in the day 1 group. Our criterion is to freeze when there are at least four zygotes or embryos available. Embryos were only frozen if they were classified as good quality while zygotes were frozen regardless of their pronuclear morphology which resulted in more cryopreservation in the day 1 group.
In conclusion, day 3 embryo transfers result in better pregnancy and implantation rates compared to day 1 zygote transfers. Pattern 0 zygotes had similar day 3 embryo quality and pregnancy outcome as compared to zygotes with non-pattern 0 morphology. The present criteria used for zygote scoring alone are not reliable to predict the viability of the embryo. Further studies are required to determine whether a progressive rather than single-point observation could improve the selection criteria for day 1 transfers.
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References |
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Submitted on August 20, 2003; resubmitted on October 6, 2003; accepted on November 4, 2003.
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