Zygote transfer on day 1 versus cleavage stage embryo transfer on day 3: a prospective randomized trial

Kamal Jaroudi1, Saad Al-Hassan1, Ulla Sieck1, Hamad Al-Sufyan1, Maya Al-Kabra2 and Serdar Coskun2,3

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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: It has been reported that pronuclear morphology is related to embryo quality and viability, and that zygote stage embryos might establish pregnancies after being transferred to the uterus. The objective of this study was to investigate whether transferring zygotes on day 1 would result in similar pregnancy rates compared to transferring cleavage stage embryos on day 3 in a prospective randomized trial. METHODS: Patients undergoing IVF/ICSI treatments were randomized to either day 1 or day 3 transfers by envelope withdrawal technique. Zygotes were classified as ‘pattern 0’ and ‘non-pattern 0’ according to the size and alignment of pronuclei, the number and distribution of nucleoli. The two best zygotes or embryos were transferred on day 1 or day 3 respectively. The primary outcome measure was pregnancy rate. RESULTS: Pregnancy rates were higher in day 3 group (55/131, 42%) when compared to day 1 (34/123, 28%, P = 0.024). Similarly, implantation rates were higher in day 3 group (P = 0.03). There were more cycles with cryopreservation in the day 1 group (P < 0.001). Embryo quality on day 3 was similar between pattern 0 and non-pattern 0 zygotes. CONCLUSIONS: Day 3 embryo transfers result in better pregnancy and implantation rates compared to day 1 zygote transfers. The present pronuclei scoring cannot reliably select zygotes for transfer on day 1.

Key words: embryo transfer/pronuclear scoring/zygote


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The in vitro growth of human embryos might be supported up to blastocyst stage prior to transfer into its natural environment, the uterine cavity. During this period three distinct phases are noted, the pronuclear stage on day 1, the early cleavage stage on days 2–3 and the blastocyst stage on days 5–6 after oocyte retrieval. During these complex developments, embryologists have only microscopic observations to assess the quality of embryos.

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 2–3, the concept of transferring embryos at blastocyst stage has gained popularity with the emergence of advanced sequential media (Gardner et al., 1998Go). This strategy has been challenged by several prospective randomized trials (Coskun et al., 2000Go; Rienzi et al., 2002Go), and the top quality embryos can be selected with better implantation potential on day 3 (Gerris et al., 1999Go; Van Royen et al., 1999Go).

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, 1998Go). It was demonstrated that embryos transferred at the zygote stage have the ability to implant and end in viable pregnancies (Ahuja et al., 1985Go; Quinn et al., 1990Go). In a recent prospective randomized multicentre study (Dale et al., 2002Go), 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., 2000Go).

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, 1999Go; Wittemer et al., 2000Go; Scott et al., 2000Go; Tesarik et al., 2000Go; Balaban et al., 2001Go; Montag et al., 2001Go; Zollner et al., 2002Go). The nuclear events are dynamic and happen during a certain time-course (Tesarik and Kopecny, 1989Go). Although Scott and Smith (1998Go) performed pronuclear scoring at three different times (16–17, 22 and 22–26 h post insemination), Tesarik and Greco (1999Go) have suggested a successful use of a single static observation (12–20 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study is a prospective randomized trial of day 1 versus day 3 embryo transfers. The day of oocyte retrieval was considered as day 0. The study has been approved by the Clinical Research and Research Ethics Committees of King Faisal Specialist Hospital and Research Center. All couples undergoing oocyte retrieval between December 2001 and October 2002 were informed about the study and asked to sign consent if they wish to participate in the study on the first day of gonadotrophin injections. Patients who consented were randomized to either ‘day 1’ or ‘day 3’ transfer by envelope withdrawal technique by the laboratory personnel 1 day before the oocyte retrieval. To guarantee uniform distribution over the time-period, a block of 14 envelopes (seven for each group) was placed in a box for withdrawal. Patients with no embryo transfer were excluded later from the study.

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., 1998Go, 2000; Jaroudi et al., 1999Go). Culture conditions were the same for both groups. Cumulus–oocyte 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 10–20% fragmentation and poor embryos were heavily fragmented (≥30%).

Pronuclear scoring
Normal fertilization was confirmed by the presence of two pronuclei and two polar bodies 15–18 h following insemination or ICSI. Zygotes were evaluated according to criteria of Tesarik and Greco (1999Go). 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., 2000Go). 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., 1998Go). 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 {beta}-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 {alpha} of 0.05 requires ~151 patients in each group. Comparisons between groups were made by using {chi}2-analysis. P < 0.05 was considered statistically significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A total of 302 patients was included in the study. Seven patients, all in the day 1 group, withdrew from the study. A total of 41 was excluded from the two groups due to suboptimal stimulation, hyperstimulation, no oocytes or sperm available, no fertilization of the oocytes or no division of the embryo. There were 123 and 131 embryo transfers in day 1 and 3 groups respectively (Figure 1). Patients’ and cycle characteristics were similar between the two groups (Table I).



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Figure 1. Flow chart for randomized study. *Due to cycle cancellation, poor or hyperstimulation, no oocytes or sperm, no fertilization, no embryo division.

 

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Table I. Patients’ characteristics are shown for each group
 
Pregnancy and implantation rates were higher in the day 3 group (55/131, 42%; 64/251, 25%; P < 0.05; Table II) when compared to day 1 (34/123, 28%; 39/227, 17%; P < 0.05) respectively. The abortion and twinning rates were similar between both groups. There were more cycles with cryopreservation in the day 1 group (P < 0.001).


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Table II. Cycle outcome from day 1 and 3 transfers
 
There were 172/593 and 182/700 pattern 0 zygotes in the day 1 and 3 groups respectively. A total of 119 pattern 0 and 105 non-pattern 0 zygotes on day 1 were transferred. Among those, 41 (34.5%) pattern 0 and 25 (23.8) non-pattern 0 zygotes were in pregnant cycles (P = 0.11). On day 3, 77 and 169 embryos generated from pattern 0 and non-pattern 0 zygotes were transferred respectively. There were 39 (50.7%) pattern 0 and 79 (46.8%) non-pattern 0 zygotes in pregnant cycles (P = 0.66, Table III).


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Table III. Pattern 0 distribution of zygotes on day 1 and 3 transfers and their use in cycles that resulted in pregnancies
 
Embryo quality on day 3 was compared to pronuclear morphology on day 1. There were 89 (48.9%) and 212 (40.9%) good quality embryos generated from pattern 0 and non-pattern 0 zygotes (P = 0.075, Table IV).


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Table IV. Distribution of embryo quality on day 3 according to the zygote classification.
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
There is no consensus as to what constitutes the optimal stage for embryos to be transferred to the uterus. Zygote transfers were performed in the early days of assisted reproduction (Ahuja et al., 1985Go). Quinn et al. (1990Go) reported that pregnancy rate after zygote transfers was similar to day 2 embryo transfer during optimal laboratory conditions but superior under suboptimal conditions. The pronuclear embryo transfer resulted in ~40% pregnancy rate after selecting zygotes according to pronuclear morphology (Scott and Smith, 1998Go). However, Hurst et al. (1998Go) reported disappointing results for zygote transfer in a prospective randomized study, which was halted at an interim analysis. Similarly, Margreiter et al. (2003Go) showed that the lowest pregnancy rates were obtained with day 1 transfers. A recent prospective randomized study demonstrated the feasibility of zygote transfers in an unselected population undergoing assisted reproduction (Dale et al., 2002Go). In the present study, zygote transfers resulted in significantly lower pregnancy rates compared to day 3 cleavage stage embryo transfers.

Earlier studies (Ahuja et al., 1985Go; Quinn et al., 1990Go) did not mention any selection criteria for zygotes to be transferred while Hurst et al. (1998Go) gave a brief description of zygote selection. Day 1 transfers resulted in the lowest pregnancy rates when compared to day 2–3 or 4–5 transfers in a prospective randomized multicentre study (Margreiter et al., 2003Go). However, no detail was given as to how zygotes were selected for transfer. Scott and Smith (1998Go) described detailed criteria for zygote scoring in which the early cleavage was also assessed. Tesarik and Greco (1999Go) 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. (2002Go) 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. (2002Go) respectively. By transferring more zygotes, Dale et al. (2002Go) 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, 1998Go).

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, 1999Go). 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., 2001Go; Wittemer et al., 2000Go; Tesarik et al., 2000Go), Salumets et al. (2001Go) 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. (2001Go) 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, 1999Go; Ludwig et al., 2000Go; Balaban et al., 2001Go). 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, 1989Go; Payne et al., 1997Go). Timing of pronuclear formation is markedly varied between oocytes after ICSI (Payne et al., 1997Go). 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., 1996Go). 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, 1989Go). 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.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on August 20, 2003; resubmitted on October 6, 2003; accepted on November 4, 2003.





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