Day 5 versus day 3 embryo transfer: a controlled randomized trial

Serdar Coskun1, Johannes Hollanders2, Saad Al-Hassan2, Hamad Al-Sufyan2, Hend Al-Mayman1 and Kamal Jaroudi2,3

1 Departments of Pathology and Laboratory Medicine and 2 Obstetrics and Gynecology, King Faisal Specialist Hospital and Research Center, P.O.Box 3354, Riyadh, Saudi Arabia


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Blastocyst transfer has been suggested to improve implantation rate without affecting pregnancy rate. The aim of this study was to compare the pregnancy and implantation rates of day 3 and 5 transfers in a prospective randomized manner. Patients with four or more zygotes were randomly allocated on day 1 to either day 3 or 5 transfers. Fertilization was achieved through regular IVF or intracytoplasmic sperm injection. Zygotes were kept in Medicult IVF medium for day 3 transfers and transferred into G1.2 and G2.2 on day 1 and 3 respectively for day 5 transfers. The morphologically best two or three embryos or blastocysts were chosen for transfer in both groups. Overall pregnancy rates per embryo transfer were the same (39%) in day 3 and 5 transfers. Implantation rates were 21 and 24% for day 3 and 5 transfers respectively. The pregnancy and implantation rates for day 5 transfers were significantly affected by the availability of at least one blastocyst to transfer and the number of zygotes. The number of good quality embryos on day 3 also significantly affected pregnancy and implantation rates on day 5 transfers. Multiple gestation rate, number of abortions and ongoing pregnancies were similar in both groups. In conclusion, day 3 and 5 transfer had similar pregnancy, implantation and twinning rates. Currently, day 5 transfers have no advantages over day 3 transfers.

Key words: blastocyst/embryo transfer/sequential media


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Human embryos obtained through in-vitro techniques are routinely transferred to the uterus on day 2 or 3 when they are at the 4–8-cell stage. The implantation rates of these embryos are disappointingly low and range between 10 and 20% (Edwards and Brody, 1995Go). Therefore, the standard treatment is to transfer more than one embryo into the uterus to obtain a reasonable pregnancy rate. This results in a high order of multiple pregnancies and twins with increased pregnancy complications such as abortion and premature deliveries (Bergh et al., 1999Go).

It has been suggested that transferring embryos at the blastocyst stage instead of selection at an earlier stage without knowing their developmental capability might enhance the implantation rate by a better embryo selection, thereby reducing the need to transfer more embryos (Gardner et al., 1998aGo). The most competent embryos reaching the blastocyst stage are selected for transfer and the embryos that have arrested their development are identified and hence not transferred. However, under current culture conditions, embryo developmental arrest occurs around the 8-cell stage and formation of the blastocyst is uncommon. To overcome this difficulty, co-culture of embryos with different cell types has been used over the years (Ménézo et al., 1992Go; Van Blerkom, 1993Go; Bongso et al., 1994Go). These cells range from autologous cells, animal cells to cell lines. Although safety was a concern when using these cells, efficiency was not improved over the routine protocols.

It is believed that gene expression of human embryos is switched on around the 8-cell stage immediately before compaction (Braude et al., 1988Go). Therefore, nutrient requirements of embryos are different after this stage. Early embryos can grow in a simple salt solution, whereas they require more complex media after they reach the 8-cell stage. These changes also correspond to environmental changes in vivo since the embryo reaches the uterus from the Fallopian tube at the stage when compaction begins. During recent years, there have been extensive studies on mammalian embryology resulting in the development of two sequential media for blastocyst culture (Bertheussen et al., 1997Go; Jones et al., 1998aGo). Gardner et al. (1998a) reported in a comparative study of embryo culture media that the blastocyst formation rate was significantly enhanced (66%) with the use of new G1 and G2 media in sequence as compared to Ham's F-10 followed by G2 media (38%). In a prospective randomized study, the use of G1 and G2 media with blastocyst transfer resulted in significantly higher implantation and similar pregnancy rates as compared to day 3 transfer in moderate to high responders (Gardner et al., 1998bGo). The availability of these highly specialized media prompted us to conduct the present study.

The objectives of this study were to determine whether transferring blastocysts on day 5 could result in better pregnancy and implantation rates than transferring early embryos on day 3 in a wide patient population selected according to the number of zygotes. The number of embryos to be transferred was kept similar in both groups.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study was a prospective randomized trial of day 3 versus day 5 embryo transfers and was approved by the Clinical Research and Research Ethics Committees of King Faisal Specialist Hospital and Research Center. All the couples undergoing oocyte retrieval were informed about the study and asked to sign an informed consent if they wished to participate in the study.

Inclusion criteria
All IVF or intracytoplasmic sperm injection (ICSI) cycles from consenting patients with four or more fertilized oocytes on the day of fertilization check (day 1) were included.

Randomization
Patients were randomized to receive embryo transfer either at day 3 or day 5 (Figure 1Go). An equal number of `day 3' or `day 5' labels placed in sealed envelopes in a block of 14 was kept in a box and an envelope was drawn by the embryologist as soon as a patient qualified for the study. The label was attached with adhesive to the patients' data collection sheet.



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Figure 1. A total of 201 IVF or intracytoplasmic sperm injection patients with four or more fertilized oocytes on the day of fertilization were randomized to receive embryo transfers on day 3 or day 5. All the patients completed the trial; there were no withdrawals.

 
Embryo transfer
The two best quality embryos from both groups were transferred into the uterus on day 3 or 5. When no blastocyst was available on day 5, the two most advanced embryos were transferred into the uterus on day 5 or the embryos were cultured one more day according to the embryologist's judgement based on the availability of the morula. Couples with six or more unsuccessful previous cycles, or a woman aged 36 years or older had three embryos transferred. Blastocyst cryopreservation was not performed since the procedure was not established in our unit.

Patient stimulation and oocyte retrieval
Ovarian suppression was performed by gonadotrophin-releasing hormone agonist long protocol. Patients were given 3.75 mg Lupron Depot (Abbott Laboratories, Chicago, IL, USA) on day 3 of the cycle. Twenty-six days after Lupron injection, ovarian stimulation with human menopausal gonadotrophin (Pergonal; Serono Laboratories, Rome, Italy) started along with Suprefact nasal spray (400 µg; Hoechst UK Ltd, Hounslow, Middlesex, UK) until the day of human chorionic gonadotrophin (HCG). Ten thousand IU of HCG (Pregnyl, Organon, Oss, The Netherlands) were given i.m. when the leading follicle reached 18 mm in diameter and oestradiol concentrations were appropriate. Oocyte retrieval was performed with transvaginal ultrasound guidance using an aspiration needle (Swemed Lab, Billdal, Sweden) under i.v. sedation 36 h after HCG injection. The follicular aspirate was poured into 60 mm Falcon dishes (Beckton Dickinson Labware, Franklin Lakes, NJ, USA) and cumulus–oocyte complexes were transferred into another dish with Medi-Cult flush medium (Medi-Cult, Jillinge, Denmark). Each complex was evaluated for maturity based on cumulus–corona cell morphology. Cumulus–oocyte complexes were transferred into IVF medium (Medi-Cult). They were incubated in 5% CO2 in air with saturated humidity until the time of insemination for IVF or removal of surrounding cumulus oophorus and corona radiata cells for ICSI.

Semen preparation
All the semen samples were allowed to liquefy for 15–30 min. A sperm count and motility analysis were performed, and, depending on the result, the motile sperm fraction was enriched by using discontinuous Percoll (95 and 47.5%; Sigma, St Louis, MO, USA). One-half to 2 ml of raw semen was layered over the Percoll and preparations were centrifuged at 300 g for 20 min. At the end of centrifugation, the pellets from each tube were collected into 5 ml culture medium and centrifuged another 10 min at 300 g for IVF and 1800 g for ICSI. Each drop containing three or four oocytes was inseminated with ~1x106 motile spermatozoa/ml as a final concentration in the drop. Surgical sperm retrieval and preparation and ICSI were performed as described previously (Jaroudi et al., 1999Go). Fertilization was confirmed by checking for the presence of two pronuclei and two polar bodies on day 1. All other types of outcome (i.e. no fertilization, one pronucleus, polyspermia or degeneration) were also recorded.

Embryo culture and development
Zygotes for day 3 transfer were cultured in IVF medium (Medi-Cult) until transfer. Zygotes for day 5 transfer were transferred to G1.2 and G2.2 media (Scandinavian IVF Sciences, Gothenburg, Scandinavia) on day 1 and day 3 respectively. Medi-Cult IVF medium is our standard medium for our daily practice. G1.2 and G2.2 are suggested to be used in a sequential manner for blastocyst culture by the manufacturer. The use of G1.2 for day 3 transfer is not suggested. Therefore Medi-Cult IVF medium was compared with sequential media, just as Ham's F10 for day 3 transfers had previously been compared with sequential media (Gardner et al., 1998bGo).

The number of blastomeres, the degree of fragmentation and evenness of blastomere size for each embryo were recorded on days 2 and 3 for day 3 transfers, and continued to be monitored on days 4 and 5 for day 5 transfers. Embryos were graded as good, fair and poor as described previously (Coskun et al., 1998Go). Good embryos included those with even-sized blastomeres with no obvious fragmentation or even-sized blastomeres with <10% fragmentation or uneven-sized blastomeres with no obvious or <10% fragmentation. Fair embryos had 10–30% fragmentation and poor embryos were heavily fragmented (>30%). Blastocysts were graded according to the size of blastocele cavity as early, full or expanded.

Patients were supplemented with progesterone (100 mg/daily i.m., Steris Laboratories Inc., Phoenix, AZ, USA). Pregnancy was confirmed first with Tandom Icon urine HCG test (Hybritech, San Diego, CA, USA) and serum ßHCG concentrations at 13 days, and with ultrasound 5 weeks after embryo transfer respectively.

Statistical analysis
Comparisons between groups were made by using Fisher's exact test or {chi}2 analysis. P <= 0.05 was considered as significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The distribution of patients with day 3 and day 5 transfers was similar between the groups (n = 101 and 100 respectively) and there were no significant differences in terms of age, infertility diagnosis, fertilization rate, number of ICSI cases or number of embryos transferred (Table IGo).


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Table 1. Distribution between patients with day 3 versus 5 transfers with respect to age, diagnosis, fertilization rate, intracytoplasmic sperm injection (ICSI) and number of embryos transferred
 
Overall pregnancy rates were the same (39%) in day 3 and day 5 transfers. Implantation rates were 21 and 24% for day 3 and day 5 transfers respectively (Table IIGo). The difference was not statistically significant.


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Table II. Pregnancy and implantation rates for day 3 and 5 transfers
 
Data were analysed according to the age, infertility diagnosis, the number of zygotes on day 1, and the number of 8-cell and good quality embryos on day 3. There was no statistical difference between day 3 and day 5 transfers in any of the categories analysed. Implantation rates were significantly lower with increasing age in both day 3 and day 5 transfers (P = 0.025, Table IIGo). Blastocyst formation was similar among different age groups (Table IIIGo). Patients who were eligible for transfer of two embryos had 41 and 44% pregnancy and 26 and 29% implantation rates for day 3 and day 5 transfers respectively. The differences were not statistically significant.


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Table III. Blastocyst formation and patients with at least one blastocyst to transfer for day 5 transfer group
 
Effects of number of zygotes and day 3 embryo quality on blastocyst formation, pregnancy and implantation rates
There were significant differences in some subcategories in day 5 transfers whereas there were no such significant differences in the corresponding subcategories in day 3 transfers. Pregnancy and implantation rates were significantly lower when there were four or five zygotes compared to six or more zygotes to culture in sequential media for day 5 transfers (P = 0.006 and 0.000 respectively, Table IIGo). The number of zygotes did not have any effect on blastocyst formation or patients with blastocyst transfer (Table IIIGo).

The availability of 8-cell embryos on day 3 had a significant effect on blastocyst formation (P < 0.0001 for >=2 versus 0 on day 3, Table IIIGo) without affecting pregnancy and implantation (Table IIGo). Nonetheless, the number of good quality embryos on day 3 not only significantly affected the blastocyst formation (P < 0.0001 for >=5 versus 0 good quality embryos on day 3, Table IIIGo) and the number of patients having blastocyst transfer (Table IIIGo) but also pregnancy and implantation rates on day 5 (P = 0.011 and 0.015 respectively, Table IIGo). When no good quality embryos were available on day 3, no pregnancy was achieved in day 5 transfers but 33% achieved pregnancy in day 3 transfers (Table IIGo).

Blastocyst development and its effect on pregnancy
The blastocyst formation rate was 28% (197/703) and 77% of the patients had at least one blastocyst to transfer on day 5 (Table IIIGo). The pregnancy and implantation rates were significantly lower in patients with no blastocyst (9 and 5% respectively) compared to those with at least one blastocyst to transfer (48 and 30%, P = 0.001 and P < 0.0001 respectively, Table IIIGo). Blastocyst formation was significantly higher in tubal infertility patients (41%) compared to male infertility patients (26%, P < 0.0001, Table IIIGo). However, both groups had similar pregnancy and implantation rates (Table IIGo).

Distribution of pregnancies
Both day 3 and day 5 transfers had 39 pregnancies and each group had one biochemical pregnancy (Table IVGo). Multiple pregnancy rates were similar (33 and 38%) for day 3 and day 5 transfers respectively. The numbers of abortions and ongoing pregnancies were similar in both groups (Table IVGo).


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Table IV. Distribution of pregnancies
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The selection of an embryo with a high chance of implantation is a difficult task in assisted human reproduction. Therefore, several embryos are transferred to increase the pregnancy rate but this entails an associated risk of multiple gestation. Gardner et al. (1998b) have recently demonstrated an increased implantation rate with blastocyst transfer on day 5 compared to cleavage stage embryo transfer on day 3 in patients who responded well to gonadotrophin stimulation (Gardner et al., 1998bGo). Pregnancy rates were similar in both groups. Scholtes and Zeilmaker (1996) have reported comparable overall results of pregnancy and implantation rates after day 3 and day 5 transfers in their prospective randomized study (Scholtes and Zeilmaker, 1996Go). In that study, a single medium was used for blastocyst culture rather than sequential media. However, on analysis of the subgroup blastocyst formation, the transfer of one or more cavitating embryos produced better pregnancy and implantation rates in day 5 compared to day 3 transfers (Scholtes and Zeilmaker, 1996Go). Our prospective randomized study showed that both pregnancy and implantation rates were similar between day 3 and day 5 transfers.

There are two major differences between our study and that of Gardner et al. (1998b). First, the average number of zygotes for each patient was lower (7.0 versus 11.6) in our study. This is due to differences in inclusion criteria and patient selection. Gardner et al. (1998b) included `good responder' patients with at least 10 follicles >=12 mm in diameter on the day of HCG injection. We tried to keep the inclusion criteria as wide as possible by including all patients with four or more fertilized oocytes. Patients with three or fewer zygotes were excluded since most embryos needed to be transferred in these patients regardless of the developmental stage.

Second, the number of embryos transferred was similar for both groups in our study whereas a significantly higher number of embryos was transferred in the day 3 group in the Gardner et al. (1998b) study. The validity of comparing implantation rates is therefore questionable since the denominator is different between the groups. Moreover, there could have been dilution of the best embryos in the cohort for day 3 transfers in the Gardner et al. (1998b) study since more embryos were transferred. This means that the day 3 group were at a disadvantage as a result of the experimental design and the number of embryos available for cryopreservation was inevitably reduced.

Assessment of embryo quality at different stages could help to determine the embryos with the best potential for implantation. Transfer of high score pronuclear embryos judged by the closeness of pronuclei, alignment of nucleoli and appearance of cytoplasm resulted in 28% implantation rate (Scott and Smith, 1998Go). In another retrospective study (Van Royen et al., 1999Go) it was reported that transfer of two top quality embryos could result in 63% pregnancy, 57% twinning and 49% ongoing implantation rates. A follow-up prospective randomized study between single and double embryo transfers also confirmed that top quality cleaving embryos could have over 40% implantation rate including single embryo transfers (Gerris et al., 1999Go). In fact, it has been proposed to combine pronucleate embryo scoring with blastocyst culture in order to determine which embryos grow into blastocysts in vitro (Edwards and Beard, 1999Go).

Earlier results (Scholtes and Zeilmaker, 1996Go) and ours indicate that day 5 transfer might not benefit all patients. Therefore, the data were analysed according to different subgroups. Age did not have any effect on blastocyst formation. The effect of age on blastocyst transfers in the literature is not conclusive. While Gardner et al. (1998b) did not see any significant relationship between age and blastocyst formation, others reported reduced blastocyst development with increasing age (Janny and Ménézo, 1996Go; Pantos et al., 1999Go). The chance of blastocyst transfer has been reported to depend on the number of oocytes retrieved rather than age (Scholtes and Zeilmaker, 1998Go). Advanced age was reported to be associated with the decline in pregnancy and implantation rates (Pantos et al. 1999Go; Janny and Ménézo, 1996Go). In the current study, significantly lower implantation rates were obtained with patients older than 35 years regardless of the day of transfer. Age above 35 years was also associated with a lower pregnancy rate but this was not significant. We believe that with a higher number of cycles significance could have been reached, as was the case with the implantation rate.

The number of zygotes did not significantly influence the pregnancy and implantation rates in the day 3 group. On the other hand, both pregnancy and implantation rates were significantly lower when four or five zygotes were available compared to six or more zygotes in day 5 group. However, blastocyst formation rates were similar regardless of the number of zygotes. Gardner et al. (1998b) reported that even though the percentage blastocyst development was not affected by the number of pronuclear embryos, there was a significant linear relationship between the number of the blastocysts formed and the number of pronuclear embryos. Thus, there will be the opportunity to choose even better blastocysts in patients with a higher number of pronuclear embryos.

Embryo quality on day 3 did not have any influence on pregnancy and implantation rates of day 3 transfers. On the contrary, in day 5 transfers both pregnancy and implantation rates were significantly lower when there was no good embryo on day 3 as compared to when at least one good embryo was available. Lack of good quality embryos on day 3 resulted in no pregnancy for day 5 transfer versus 33% pregnancy for day 3 transfers in our study. The number of 8-cell embryos on day 3 did not have any impact on pregnancy and implantation rates in either group. The predictive value of embryo morphology for blastocyst formation has been limited in one study (Rijnders and Jansen, 1998Go). However, Racowsky et al. (1999) reported that the number of 8-cell embryos could be a determining factor for the optimal day of transfer. Interestingly, lack of 8-cell embryos on day 3 resulted in no pregnancy for day 5 transfer versus 33% pregnancy for day 3 transfers (Racowsky et al., 1999Go). These results in combination with ours strongly suggest that some suboptimal quality embryos may be rescued in the uterine environment and that extended culture might be a cause of arrest for further development of such embryos.

The blastocyst formation rate in this study is relatively low (28%). Our patient population consisted mainly of male factor patients, which might have contributed to the overall low rate of blastocyst formation as compared to earlier published reports (Gardner et al., 1998bGo; Schoolcraft et al., 1999Go) with higher rate of blastocyst formation. Patients with tubal factor infertility had significantly higher blastocyst formation (41%) than male factor patients (26%) though pregnancy rates were similar in the two groups. Since blastocyst formation rates in tubal patients were comparable to earlier studies (Gardner et al., 1998bGo; Schoolcraft et al., 1999Go), the overall low blastocyst formation might again be related to the high number of male factor patients in our population. It has been suggested previously that there could be a paternal effect on human preimplantation embryo development and blastocyst formation (Janny and Ménézo, 1994Go; Jones et al., 1998bGo; Shoukir et al., 1998Go). Patients with normal sperm parameters had higher blastocyst formation than patients with severely impaired sperm quality (Janny and Ménézo, 1994Go). The number of patients in whom at least one blastocyst was available in our study is also relatively low (77%). This could be due to our wide inclusion criteria in contrast to those of Gardner et al. (1998b) resulting in a selected group of moderate to high responders in whom they reported that 91% of patients had blastocyst transfers.

The most important parameter was the availability of a blastocyst on day 5 for transfer. Blastocyst transfer on day 5 resulted in 48% pregnancy rate whereas less advanced embryo transfer on day 5 resulted in 9% pregnancy rate. Similarly, implantation rates were significantly higher in blastocyst transfers (30%) as compared to embryos with less advanced development in the day 5 group (5%). It has also been reported previously that blastocysts have a significantly higher implantation rate than non-cavitating embryos on day 5 (Scholtes and Zeilmaker, 1996Go). It seems fair to say that blastocyst transfers could result in better implantation rates than transfer of randomly selected early embryos if at least one blastocyst is available to transfer on day 5. However, not all the patients will have at least one blastocyst to transfer under current culture conditions. We have to stress that those cleaving embryos that have the potential to reach the blastocyst stage may have a similar implantation rate at day 3 and day 5. Gerris et al. (1999) prospectively identified top quality embryos for single transfer on day 3 resulting in an implantation rate of 42.3%.

Although the most important purpose of blastocyst culture or transferring two embryos on day 3 was to eliminate multiple gestations, a high incidence of twin pregnancy was established during this study. Further efforts should be made towards reducing multiple gestations as far as possible by transferring a single embryo, zygote or blastocyst but without reducing too severely the chance for pregnancy.

Due to this high twinning rate, the final proof of any superiority in selection at any particular stage of embryo development should be in comparing the result of transfer of the best single embryo at any stage in a randomized trial.


    Notes
 
3 To whom correspondence should be addressed at: King Faisal Specialist Hospital and Research Center, P.O.Box 3354 MBC#52, Riyadh, Saudi Arabia. E-mail: jaroudi{at}kfshrc.edu.sa Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on February 28, 2000; accepted on May 25, 2000.