Fundaço Universitária de Endocrinologia e Fertilidade (FUEFE), Rua Alcides Cruz 101, CEP: 90.630160, Porto Alegre, RS, Brazil
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Abstract |
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Key words: assessment times/early cleavage/embryo selection/pregnancy rates
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Introduction |
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More recently, assessment of the time of cleavage to the 2-cell stage has proven to be a reliable parameter for the selection of embryos with the highest capability of implantation and successful pregnancy after transfer (Shoukir et al., 1997; Sakkas et al., 1998
; Jimenez et al., 1999
). These studies used different threshold times to assess early cleavage: 25 (Shoukir et al., 1997
), 26 (Jimenez et al., 1999
) and 27 h (Sakkas et al., 1998
) post-insemination/intracytoplasmic sperm injection (ICSI). All three reports recognized that the evaluation of early-cleaving 2-cell embryos is an effective method of assessment of embryo viability. Furthermore, pregnancy rates were highest when the number of early cleavage embryos transferred was increased from one to three.
We have observed that the frequency of embryos reaching the 2-cell stage at 25 h post-insemination/ICSI is very low and few patients would benefit from such an early assessment and selection prior to transfer. The earliest time the human embryo can reach the 2-cell stage ranges between 2027 h post-insemination (Trounson et al., 1982; Balakier et al., 1993
; Capmany et al., 1996
). It is not known, however, at which time point of completion of the first cell cycle embryo viability starts to drop. In the present study, we have investigated whether a similar positive effect on pregnancy rate is observed when cleavage to the 2-cell stage is also evaluated at 29 h post-insemination/ICSI. The reason for performing this later assessment is to maximize the number of potentially good embryos available to be transferred, and to increase the number of patients receiving selected embryos. We therefore assessed early cleavage at three time points, namely 25, 27 and 29 h post-insemination/ICSI, and called all these embryos `early-cleavage' (EC). Pregnancy rates were compared between the group of patients who received at least one EC embryo and patients who received none.
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Material and methods |
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A standard long protocol of ovarian stimulation was adopted throughout the study. All patients were desensitized with gonadotrophin releasing hormon (GnRH) agonist (Lupron; TAP Pharmaceuticals, Chicago, IL, USA) followed by follicular growth stimulation with recombinant FSH (Gonal-F; Serono, Aubonne, Switzerland). Human chorionic gonadotrophin (HCG; Profasi; Serono) at a 10 000 IU dose was administered i.m., when the diameter of two or more follicles reached 17 mm. Oocyte retrieval was scheduled 36 h after HCG injection. Oocytes were recovered transvaginally with ultrasound guidance. The luteal phase was supported by progesterone (Utrogestan; Laboratories Besins-Iscovesco, Paris, France) administered vaginally at a dose of 400 mg/day from the day of oocyte retrieval.
After retrieval, oocytes were maintained in human tubal fluid (HTF; Irvine Scientific, Santa Ana, CA, USA ) medium plus 10% synthetic serum substitute (SSS; Irvine Scientific) for ~2 h before insemination or ICSI.
For the IVF procedure, oocytes were inseminated with 0.30.5x106/ml motile spermatozoa in HTF medium.
For ICSI, oocytes were first incubated in 80 IU/ml hyaluronidase for <30 s and corona-cumulus cells were stripped off the oocyte with gentle pipetting. The ICSI procedure was performed 2 h later (Palermo et al., 1992). Injected oocytes were rinsed twice and placed for culture in HTF supplemented with 10% SSS.
Approximately 18 h after insemination in conventional IVF or ICSI procedures, fertilization was confirmed by the presence of two pronuclei. Zygotes were placed individually in 20 µl fresh HTF medium under oil for culture. On the same day, zygotes were examined to see whether cleavage to the 2-cell stage had occurred at 25, 27 and 29 h post-insemination/ICSI. Embryos were maintained in separate culture drops until transfer on day 3. Routinely, a maximum of four embryos were transferred to the patient. When there were only one, two or three EC embryos, the embryo transfer group was completed by adding one, two or three morphologically best non-early cleavage (NEC) embryos. In 12 cycles, patients received five or six embryos due to repeated failure in previous cycles, advanced maternal age or poor quality embryos.
Pregnancy was determined by serum HCG measurement on day 1415 after transfer and confirmed by ultrasound at 5 weeks post-oocyte retrieval.
Statistical analysis used Student's t-test and MannWhitney Rank Sum test for comparing means and medians between two groups, and 2 analysis with Yate's continuity correction to compare rates. Statistical significance was set at P < 0.05.
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Results |
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EC embryos presented an implantation rate two times higher than NEC embryos (P = 0.024) (Table I). Pregnancy rate in the EC group was more than double than in the NEC group (P = 0.020) (Table I
).
In 50% of cycles (21/42), EC embryos were detected at 29 h post-insemination/ICSI. Four out of 42 (9.5%) cycles presented EC embryos at 25 or 27 h post-insemination/ICSI. Ten cycles showed early cleavage to the 2-cell stage at both 27 and 29 h (24%), and three cycles presented EC embryos at all three time points (7%).
Table II shows the number of EC embryos transferred per patient and the corresponding pregnancy rates. Interestingly, increasing the number of EC embryos transferred did not correlate with an increase in pregnancy or multiple gestation rates. All pregnancies in the NEC group of patients were singleton pregnancies.
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Discussion |
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Identification of the best embryos for transfer has been made using different parameters since the original observation that 4-cell stage embryos after 2-day culture are the optimal cleavage stage to implant (Ziebe et al., 1997). Scott and Smith found that pronuclear embryos can be successfully selected for transfer after an `embryo score' (ES) based on positioning of their pronuclei, the alignment of nucleoli and appearance of the cytoplasm (Scott and Smith, 1998
). When ES was corrected (CS), according to the number of cleaved embryos that were transferred, the authors found a strong correlation between CS and implantation and delivery rates. Subsequent reports confirmed that selection of embryos with highest implantation rates can be made as early as the pronuclear stage (Tesarik and Greco, 1999
; Ludwig et al., 2000
; Tesarik et al., 2000
).
The advantage of such an early evaluation and selection would be to overcome ethical and administrative problems associated with the fate of supernumerary embryos. If selection of embryos to be transferred can be made at the pronuclear stage, the remaining ones can be cryopreserved or discarded at a stage when both genomes are still physically separated and can thus hardly be considered as embryos. However, pronuclear assessment has some drawbacks which makes it less attractive to the embryologist than 2-cell cleavage analysis. Pronuclear morphology is a very dynamic process consisting of assembly, growth and mutual fusion of nucleolar precursor bodies (Tesarik and Kopecny, 1989). An embryo may show a specific nucleolar morphology at a certain time point and a different one at a subsequent evaluation. Furthermore, accurate pronuclear assessment requires considerable manipulation of the zygotes until the two pronuclei are clearly visible, a time consuming process performed outside the incubator. In contrast, 2-cell division takes just a few seconds to evaluate, even in a large number of embryos.
Blastocyst culture has also been successfully used as a means of embryo selection (Gardner et al., 1998). Considering that only 4050% of all zygotes placed in culture will reach the blastocyst stage by day 56 of culture (Schoolcraft et al., 1999
; Scott et al., 2000
), embryo culture selects the most developmentally competent embryos for transfer, and eliminates the ones pre-destined to arrest. However, extended in-vitro embryo culture requires daily medium changes and the use of different sequential media, which makes the whole procedure more difficult. Not forgetting the human factor, one has to weight the psychological impact on those patients who did not get any transferable blastocysts after extended culture. These patients would probably have preferred an earlier transfer, using less selected embryos, despite their low chances of pregnancy.
Also of note is the fact that one twin and one triplet gestation happened in the group of patients who received only one EC embryo, suggesting that in the group of embryos from these patients, other embryos were of as good quality as the selected EC one. This observation emphasizes the idea that embryo quality cannot be based on only one evaluation parameter.
Our observation that significantly more EC embryos occurred in younger women contrasts with previous reports where this difference was not detected (Shoukir et al., 1997; Sakkas et al., 1998
). However, it has been shown that proportionally less zygotes presenting ideal pronuclear morphology occur in women >38 years old (Wittemer et al., 2000
). Older women are less responsive to the hormonal treatments used to induce ovulation and produce less oocytes, and pregnancy rates drop dramatically after the age of 40 years (Van Kooij et al., 1996
; Lass et al., 1998
; Schoolcraft et al., 1999
; Scott et al., 2000
).
There was no difference in timing for 2-cell cleavage between embryos generated by IVF or ICSI. This result agrees with the observation that no difference was detected in the timing of pronuclear formation between IVF and ICSI zygotes (Wittemer et al., 2000). On the other hand, Sakkas and colleagues found a greater proportion of early cleavage embryos after ICSI than when they assessed IVF embryos in a previous study (Shoukir et al., 1997
; Sakkas et al., 1998
). However, the group used two different times to evaluate early cleavage to the 2-cell stage, and it is likely that if the same time point were used in both studies, the proportion of selected embryos would also have been similar between the two procedures.
Our observation that the majority of EC embryos have undergone first cleavage 2729 h post-insemination/ICSI contrasts with previous reports (Sakkas et al., 1998) where early cleavage was detected in 61% of cycles by 27 h post-ICSI. The discrepant results may reflect differences in culture media and stimulation protocols. In their study, Sakkas and colleagues used Whittingham's T6 medium (Sakkas et al., 1998
), whereas we used HTF medium. Their stimulation protocol used HMG (Pergonal) (Sakkas et al., 1994
), which contains LH, whereas we used continuously recombinant FSH, which is completely devoid of LH. The role of LH in ovarian stimulation protocols is a matter of great debate (Levy et al., 2000
). The influence of the presence of LH during ovarian stimulation on embryo quality was demonstrated when blastocysts developed from oocytes that were exposed to FSH and LH had a higher implantation potential than blastocysts that developed from oocytes exposed to pure FSH (Schoolcraft et al., 1999
).
In conclusion, our results are in agreement with earlier reports suggesting that early cleavage embryos should be selected from the embryo pool and preferentially transferred. We recommend that observations and selection can be extended up to 29 h post-insemination/ICSI, to detect EC embryos in a larger group of patients and to increase the number of high quality embryos for transfer, without compromising embryonic potential to implant and to establish a successful pregnancy. Finally, we believe that embryo selection should be based on more than one criteria allowing the fresh transfer of only one embryo of the highest quality, without compromising the chances of pregnancy, and cryopreservation of other good quality embryos in appropriate groups resulting in an overall increase in pregnancy rates.
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Acknowledgements |
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Notes |
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References |
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Submitted on May 29, 2001; accepted on September 6, 2001.