1 Infertility Clinic, The Family Federation of Finland, Kalevankatu 16A, FIN-00100 Helsinki, and 2 Department of Obstetrics and Gynaecology, Helsinki University Central Hospital, FIN-00290 Helsinki, Finland
3 To whom correspondence should be addressed. e-mail: andres.salumets{at}vaestoliitto.fi
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Abstract |
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Key words: early cleavage/embryo viability/ICSI/IVF/single embryo transfer
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Introduction |
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Materials and methods |
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Ovarian stimulation regimen, oocyte collection, IVF and ICSI procedures and embryo transfer were similar at both clinics and have been reported elsewhere (Vilska et al., 1999; Tiitinen et al., 2001
). Briefly, the patients underwent pituitary down-regulation with GnRH agonist (Synarela; Syntex Nordica AB, Sweden) commenced in the mid-luteal phase of the previous menstrual cycle. When suppression was achieved, ovarian stimulation was performed using recombinant FSH (Gonal-F; Serono, Switzerland; or Puregon; Organon; The Netherlands). When two or more follicles reached the size of
17 mm in diameter, hCG (Pregnyl; Organon, or Profasi; Serono) was administered. Transvaginal oocyte retrieval was performed after 36 h.
Culture media for oocytes and embryos were either IVF medium (MediCult, Denmark) or Sydney IVF Fertilisation and Cleavage Medium (Cook IVF, Australia). The spermatozoa were prepared using both density gradient centrifugation (PureSperm; NidaCon, Sweden) and swim-up. Insemination or microinjection was carried out 46 h after oocyte retrieval. The oocytes were checked for the presence of pronuclei and polar bodies 1618 h after insemination. The zygotes were examined again 2527 h after insemination or ICSI to see whether they had undergone the first mitotic cleavage. Embryos that possessed two cells at 2527 h after insemination were designated as early cleavage (EC) embryos and those that had not yet cleaved were classified as no early cleavage (NEC) embryos.
The embryo quality was evaluated 4446 h after insemination or ICSI, considering the blastomere number, the degree of fragmentation, the uniformity of blastomeres and the presence of multinucleated blastomeres. The degree of fragmentation was expressed as a percentage of the perivitelline space occupied by anucleate cytoplasmic fragments. Embryos were considered evenly cleaved when the difference in size between blastomeres was 10%. Only embryos with mononucleated blastomeres were accepted for eSET. The embryo selection for transfer on day 2 was not influenced by early cleavage, but instead was based on embryo morphology and growth rate (as measured by blastomere numbers).
After embryo transfer, micronized vaginal progesterone (Lugesteron; Leiras, Finland) was used for luteal support. A positive serum hCG test (>10 mIU/ml) performed 2 weeks after embryo transfer confirmed pregnancy. The clinical pregnancy was documented by the presence of a positive fetal heart activity on transvaginal sonography at the sixth or seventh week of pregnancy. All eSET procedures with transient elevation of hCG level but without recognized clinical pregnancy were classified as preclinical abortions.
Different clinical parameters were compared between EC and NEC embryo transfers. The effects of different factors on the clinical pregnancy rate were also estimated. In both of these calculations, 2-test and independent-samples Students t-test were used. The independence of different factors influencing the clinical pregnancy rate was tested applying generalized linear analysis using SAS system (Release 8.1.) procedure GENMOD, binominal distribution and standard logit link. The significance level was considered at P < 0.05.
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Results |
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Discussion |
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As only good quality embryo transfers were included in the current study, the comparison with previous studies, which also covered the analysis of transfers of poor quality embryos, has its limitations. However, the results of our study correspond with prior reports (Shoukir et al., 1997; Sakkas et al., 1998
; Lundin et al., 2001
) and support the view that EC embryos possess significantly higher developmental competence than NEC embryos. In the study by Shoukir et al. (1997
), the pregnancy rate for EC embryo transfers (33%) was more than double that of NEC embryo transfers (15%) in IVF procedures (Shoukir et al., 1997
). An even bigger difference between EC and NEC embryos was found in ICSI procedures as patients who received EC embryos exhibited almost 4-fold higher clinical pregnancy rate (26%) than those having NEC embryos transferred (6%) (Sakkas et al., 1998
). In most of these earlier studies, however, EC embryos had been preferentially transferred. When not enough EC embryos were available, the embryo transfer group was completed with NEC embryos, making it impossible to ascertain which of the transferred embryos led to the pregnancy. Transferring only a single embryo enabled us to conclusively demonstrate the positive effect of early cleavage on embryo implantation.
The analysis of possible factors predicting the clinical pregnancy rate after eSET revealed that both early cleavage and regularity of blastomere divisions are important determinants of embryo competence. These findings are in agreement with studies (Giorgetti et al., 1995; Hardarson et al., 2001
) showing the relationship between even cleavage of embryos and better capacity for implantation. However, further analysis of our data indicated that these two factors influence independently the embryo viability. A possible reason why we did not observe the effect of age of patients and routine embryo parameters (the number of blastomeres and the degree of fragmentation) on the success rate of eSET was that single embryo was predominantly transferred to younger patients (85% of all patients were
37 years old) having good quality embryos.
The results of this study have also led us to revise our embryo selection criteria and currently EC embryo is preferentially selected for transfer in eSET procedures when at least two good quality embryos are available. The question whether to transfer one or two embryos is, however, more complex and cannot be solved solely based on embryo quality, albeit two-embryo transfer could be considered more seriously when no good quality EC embryo is available.
It has been demonstrated that EC embryos have better morphology (Lundin et al., 2001) and a higher number of blastomeres (Sakkas et al., 2001
) 2 days after insemination or ICSI. In addition, a better blastocyst formation rate has been demonstrated for EC than NEC embryos (Fenwick et al., 2002
). Our findings confirm and complement the previous studies, as, in addition to better quality on day 2, EC embryos had also lower incidence of MNB when compared with NEC embryos. The reasons for better quality and viability of EC embryos remain largely unknown. The previous evaluation of the timing of the first cleavage may allow an embryologist to discern on the day of embryo transfer whether a 4-cell embryo had just cleaved to the 4-cell stage or had reached the 4-cell stage hours earlier (Sakkas et al., 2001
). In addition, early cleavage of zygotes may depend on the quality of oocytes (Lundin et al., 2001
), but the paternal effect cannot be ruled out (Salumets et al., 2002
). However, the semen characteristics have not been shown to have any effect on early cleavage of either IVF or ICSI zygotes (Shoukir et al., 1997
; Sakkas et al., 1998
). Therefore, more detailed studies are needed to specify these putative biological mechanisms underlying this phenomenon. Future studies should also concentrate on the incidence of chromosomal abnormalities in EC and NEC embryos.
The reduction of multiple pregnancies by using eSET requires critical and careful selection of the embryo for transfer. Traditionally, cleavage stage embryos are selected for transfer 2 or 3 days after insemination considering simultaneously their morphological appearance and growth rate (Puissant et al., 1987; Steer et al., 1992
). According to some authors, the selection of cleavage stage embryos for transfer could be substantially improved by evaluation of pronuclear morphology (Scott and Smith, 1998
; Tesarik and Greco, 1999
). However, in the recent study (Salumets et al., 2001
), we failed to show any beneficial effect of the evaluation of pronuclear morphology on clinical pregnancy outcome following eSET procedures.
Postponing the transfer until embryos have reached the blastocyst stage has also been used as a possible way to select the most viable embryos for transfer. Although high pregnancy (5070%) and implantation (3050%) rates have been reported for blastocysts transfers (Gardner et al., 1998; Marek et al., 1999
; Langley et al., 2001
) the clinical evidence on the preference of blastocyst transfers to cleavage stage embryo transfers is still controversial. Several prospective studies have shown the superiority of blastocyst transfers over day 2/3 embryo transfers (Gardner et al., 1998
; Van Der Auwera et al., 2002
), while others have been unable to confirm these findings (Scholtes and Zeilmaker, 1996
; Coskun et al., 2000
; Huisman et al., 2000
; Lundqvist et al., 2002
). In a recent study (Rienzi et al., 2002
), day 3 embryo transfers with combined evaluation at pronuclear and cleavage stages demonstrated similar results to blastocyst transfers. The clinical pregnancy rate for EC embryos (50%) found in the current study is also comparable to that usually reported for blastocysts. Though the basis of our embryo selection differed from that of Rienzi et al. (2002
) (Rienzi et al., 2002
), both of these studies suggest that embryo selection could be successfully accomplished on days 2/3, making the extended culture and blastocyst transfer redundant.
In conclusion, the current study provides compelling evidence that early cleavage is a strong indicator of embryo competence. However, larger prospective studies are needed to confirm the usefulness of early cleavage in embryo selection for eSET.
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Acknowledgements |
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References |
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Submitted on September 26, 2002; resubmitted on November 27, 2002; accepted on January 8, 2003.