Introduction
In a typical ART treatment cycle, fresh cleavage-stage embryos are routinely transferred [
1]. In recent years, with rapid progress in vitrification technology and blastocyst culture protocols, transfer of fresh and vitrified-warmed blastocysts, together with cryopreserved cleavage-stage embryos are now becoming more routine and commonplace in ART clinical practice [
2,
3]. Nevertheless, the optimal timing for embryo transfer has remained controversial to date.
A number of studies, as summarized in a Cochrane review [
4], demonstrated the main advantages of blastocyst transfer, including better correlation between morphology and euploidy status and improved implantation potential due to better synchronization with the endometrium not adversely affected by controlled ovarian stimulation. These in turn translate to higher pregnancy and live birth rates after blastocyst transfer, as compared to cleavage-stage embryo transfer. Nevertheless, conflicting results were reported by a recent Cochrane meta-analysis, which found no evidence of any difference in pregnancy outcomes between Days 2-3 and 5-6 transfers of embryos [
5]. Moreover, the same study [
5] also found that blastocyst transfer was associated with an increase in failure to transfer any embryo in a cycle, as well as a decrease in embryo freezing rates. Interestingly, in a recent study by Langen et al. [
6], it was reported that Asian ART patients (majority of Chinese ethnicity) had significantly poorer clinical outcome compared to Caucasians, even though there was no difference in blastocyst quality between the two populations. Hence, it is plausible to hypothesize that endometrial receptivity is more adversely affected by controlled ovarian stimulation in ethnic Chinese patients, as compared to Caucasian patients.
Embryo cryopreservation following ART cycles provided further possibilities of success, in addition to that achieved with fresh embryo transfer [
7]. Therefore cumulative pregnancy rates after completion of both fresh and additional vitrified embryo transfers per oocyte retrieval cycle should be considered a more accurate measure of clinical outcome, rather than just pregnancy rate per embryo transfer cycle.
Our study retrospectively analyzed the clinical pregnancy, implantation and multiple pregnancy rates after transfer of fresh and vitrified-warmed blastocysts and cleavage-stage embryos in ethnic Chinese women undergoing ART treatment. The aim is to establish an optimized embryo transfer protocol in clinical ART practice, at least for ethnic Chinese patients.
Results
A total of 478 couples under 35 years of age were assigned to either cleavage-stage embryo transfer (n =310 patients) or blastocyst transfer (n =168 patients). There were no significant differences between the two groups with respect to patient clinical characteristics except for age (Table
1). At the same time, a few patients more than 34 years of age were also assigned to undergo embryo transfer of either cleavage-stage embryos (n=52 patients) or blastocysts (n=21 patients). Again, patient clinical characteristics were not statistically different between the two groups for patients above 34 years of age (Table
2).
Table 1
Patient characteristics for blastocyst and cleavage-stage transfer: Jan 2009 ~ Jul 2010 (age < 35 years)
Number of patients | 310 | 168 |
Female patient age (y, mean± SD) | 29.61±3.03 | 28.87±2.70a
|
Duration of infertility (y, mean± SD) | 3.90±1.88 | 3.92±1.90 |
Body mass index (w, mean± SD) | 21.88±2.54 | 21.84±2.24 |
ICSI cycle, no. of patients (%) | 90( 29.0) | 45(26.8) |
Tubal factor, no. of patients (%) | 151( 48.7) | 83(49.4) |
Male factor, no. of patients (%) | 43(13.9 ) | 23(13.7) |
Tubal and male factor, no. of patients (%) | 89( 28.7) | 52(31.0) |
Multiple factors, no. of patients (%) | 27( 8.7) | 10(6.0) |
Table 2
Patient characteristics for blastocyst and cleavage-stage transfer: Jan 2009 ~ Jul 2010 (age between 35 to 39 years)
Number of patients | 52 | 21 |
Female patient age (y, mean± SD) | 36.69±1.45 | 37.05±1.32 |
Duration of infertility (y, mean± SD) | 5.85±3.31 | 6.45±3.20 |
Body mass index (w, mean± SD) | 22.14±2.07 | 22.84±2.47 |
ICSI cycles, no. of patients (%) | 12(23.1) | 5(23.8) |
Tubal factor, no. of patients (%) | 33(63.5) | 10(47.6) |
Male factor, no. of patients (%) | 4(7.7) | 4(19.0) |
Tubal and male factor, no of patients (%) | 13(25) | 5(23.8) |
Multiple factors, no. of patients (%) | 2(3.8) | 2(9.5) |
In fresh embryo transfer cycles, the number of retrieved oocytes in the blastocyst transfer group was significantly higher than the cleavage-stage embryo transfer group (Table
3, younger than 35 years: 11.43±4.01 versus 7.33±3.57, P<0.05; older than 34 years: 11.24±4.55 versus 6.23±3.68, P<0.05). Additionally, the number of embryos transferred was significantly different between the two groups (Table
3) among patients less than 35 years old (1.75±0.34 versus 2.15±0.55, P<0.05), as well as among patients aged older than 34 years old (1.76±0.83 versus 2.67±0.76, P<0.05).
Table 3
Clinical outcome after fresh blastocyst and cleavage-stage transfer
No. of patients | 274 | 168 | 52 | 21 |
Retrieved oocytes (mean± SD) | 7.33±3.57 | 11.43±4.01a
| 6.23±3.68 | 11.24±4.55a
|
No. of embryos transferred (mean± SD) | 2.15±0.55 | 1.75±0.34a
| 2.67±0.76 | 1.76±0.83a
|
Clinical pregnancy rate, no. (%) | 129/274(47.1) | 69/168(41.1) | 22/52(42.3) | 7/21(33.3) |
Implantation rate, no. (%) | 184/589(31.2) | 97/305(31.8) | 30/139(21.6) | 8/37(21.6) |
Multiple pregnancy rate, no. (%) | 53/129(41.1) | 28/69(40.6) | 7/22(31.8) | 1/7(14.3) |
Singleton pregnancy rate, no. | 76 | 41 | 15 | 6 |
Twin pregnancy rate, no. | 51 | 27 | 6 | 1 |
Triplets pregnancy rate, no. | 2 | 1 | 1 | 0 |
For patients under 35 years of age, the clinical pregnancy rates (Table
3) in the cleavage-stage embryo transfer group versus blastocyst transfer group were 47.1% versus 41.7% ( P>0.05) respectively; while the corresponding implantation rates (Table
3) were 31.2% versus 31.8% ( P>0.05) respectively. For patients more than 34 years of age, the clinical pregnancy and implantation rates after cleavage-stage embryo transfer and blastocyst transfer (Table
3) also exhibited no significant differences (42.3% versus 32.3%, P>0.05; 21.6% versus 21.6%, P>0.05, respectively). The rate of multiple births after cleavage-stage embryo transfer and blastocyst transfer were 41.1% versus 40.6% (< 35 years old), and 31.8% versus 14.3% (> 34 years) respectively, and these were not significantly different for both groups of patients (Table
3).
The clinical outcomes of the blastocyst and cleavage-stage embryo transfer groups (under 35 years of age) with respect to fresh and vitrified-warmed transfer cycles are shown in Table
4. This data set includes 36 of 310 patients who did not undergo fresh cleavage-stage embryo transfer due to poor endometrial receptivity on Day3 (and consequently had all embryos vitrified at the cleavage stage). Out of 168 patients undergoing fresh blastocyst transfer in Table
4, 69 patients became pregnant (41.7%), leaving behind 99 unsuccessful patients. Among these 99 patients, 7 patients did not possess any supernumerary blastocyst for vitrification. Therefore, only 92 patients had vitrified-warmed blastocyst transfer. At the same time, out of a total of 310 patients who had fresh and vitrified-warmed cleavage-stage embryo transfer in Table
4, there were 26 patients who after having failed to get pregnant upon day 3 transfer, still possessed supernumerary embryos that formed blastocysts on day 5. These were subsequently vitrified and transferred in another cycle. Hence, there was a total number of 92 plus 26 = 118 patients having vitrified-warmed blastocyst transfer in Table
4.
Table 4
Clinical outcome after vitrified blastocyst and cleavage-stage transfer (age <35 years)
No. of patients | 274 | 36 | 168 | 118 |
No. of embryos transferred (mean± SD) | 2.15±0.55 | 2.19±0.40 | 1.82±0.38 | 1.69±0.58a
|
Clinical pregnancy rate, no. (%) | 129/274 (47.8) | 18/36 (50.0) | 69/168 (41.7) | 67/118 (56.8)b
|
Implantation rate, no. (%) | 184/589 (31.2) | 27/79 (34.2) | 97/305 (31.8) | 94/200 (47.0)b
|
Mutiple pregnancy rate, no. (%) | 53/129 (41.1) | 9/18 (50.0) | 28/69 (40.6) | 26/67 (37.3) |
Singleton pregnancy rate, no. | 76 | 9 | 41 | 41 |
Twin pregnancy rate, no. | 51 | 9 | 27 | 25 |
Triplets pregnancy rate, no. | 2 | 0 | 1 | 1 |
Although different criteria were used in allocating patients to the vitrified blastocyst and vitrified cleavage-stage embryo transfer groups, the two data sets in Table
4 are still comparable, because both cases involved non-controlled ovarian hyperstimulation (non-COH) cycles. Additionally, it can be presumed that the 118 patients in the vitrified blastocyst transfer group were previously unsuccessful due to sub-optimal endometrial receptivity in their COH cycle, which would mirror the situation in the vitrified cleavage-stage embryo transfer group.
As seen in Table
4, neither the clinical pregnancy rate nor implantation rate differed significantly for cleavage-stage embryo transfer in the fresh versus vitrified-warmed group (47.8% versus 50.0%; 31.2% versus 34.2% respectively). In contrast, both clinical pregnancy and implantation rates were significantly different between fresh versus vitrified-warmed blastocyst transfer cycles (41.7% versus 56.8% and 31.8% versus 47.0%, respectively, P<0.05). Multiple pregnancy rates were similar in both groups among either cleavage-stage embryo transfer or blastocyst transfer cycles (Table
4).
The cumulative clinical pregnancy rates for fresh and vitrified-warmed embryo transfers are summarized in Table
5. Out of 310 patients who had day 3 cleavage-stage embryo transfer (Table
4), 26 unsuccessful patients had remaining surplus embryos that were cultured up to the blastocyst stage and vitrified, prior to being transferred in a subsequent vitrified-warmed cycle. This would therefore leave behind a total of 310 – 26 = 284 patients in the cleavage-stage embryo transfer group of Table
5. At the same time, 26 patients were added to 168 patients (Table
4) to give a cumulative total of 194 patients in the blastocyst transfer group of Table
5. In the blastocyst transfer group, 67 additional clinical pregnancies were achieved following vitrified embryo transfers, whereas only 18 additional clinical pregnancies were achieved in the cleavage-stage embryo transfer group (Table
5). Hence, the cumulative clinical pregnancy rate was significantly higher in patients aged less than 35 years after blastocyst transfer, as compared to patients with cleavage-stage embryo transfers (70.1% versus 51.8%, p<0.05). There was however no significant difference in the cumulative multiple pregnancy rates of both groups.
Table 5
Cumulative clinical outcome combining fresh and vitrified-warmed cycles in blastocyst and cleavage-stage transfer (age < 35 years)
No. of couples | 284 | 194 |
Overall clinical pregnancies per couple, no. (%) | 147/284(51.8) | 136/194(70.1)a
|
Fresh embryo transfer, no. | 129 | 69 |
Vitrified-warmed embryo transfer, no. | 18 | 67 |
Cumulative multiple pregnancy rate, n (%) | 62/147(42.2) | 54/136(39.7) |
Singleton pregnancy rate, no. | 85 | 82 |
Twin pregnancy rate, no. | 60 | 52 |
Triplets pregnancy rate, no. | 2 | 2 |
Discussion
The ultimate aim of an ART procedure is to achieve one singleton live birth per stimulated cycle. However, it is still unclear which embryo transfer model will achieve optimal results for ART patients. To try to resolve this question, 551 couples were assigned to fresh or vitrified-warmed embryo transfer at either the cleavage stage or blastocyst stage for their first or second ART attempt.
In accordance with local ART regulations, and due to an anticipated enhancement of implantation rate with blastocyst transfer, we limited the number of embryos transferred per cycle to three or less. Therefore, a maximum of three embryos were placed in each cryotop during vitrification. This is reflected in significantly lesser number of embryos being transferred in the blastocyst transfer group compared to the cleavage-stage transfer group. Hence, a trend towards lower multiple pregnancy rates was therefore observed in blastocyst transfer cycles. Nonetheless, there was no significant difference between the two groups with respect to implantation rate. Meanwhile our retrospective meta-analysis showed that the transfer of fresh blastocysts on day5 did not significantly increase clinical pregnancy rate and implantation rate within the general ART patient population compared with day3 cleavage-stage embryo transfer. In fact, the rates for blastocyst transfer were slightly decreased compared with cleavage-stage transfer, particularly in patients aged older than 34 years.
These findings are consistent with another recent study [
15], which also reported that blastocyst culture and transfer reduced implantation and pregnancy rates in the general ART patient population compared to cleavage-stage embryo transfer. Nevertheless, positive results of blastocyst transfer have been reported by many previous studies, which all revealed higher implantation and pregnancy rates with blastocyst transfer compared to cleavage-stage embryo transfer [
16‐
20]. It is difficult to explain why the results of our study demonstrated negligible advantages of blastocyst transfer, despite comparable baseline clinical characteristics, other than the patient age being significantly younger and the number of oocytes retrieved being significantly higher in the blastocyst transfer group. Nevertheless, while all of the above differences should be considered advantageous, being good prognostic factors for both blastocyst formation and pregnancy [
21], this potential advantage did not translate into higher implantation and pregnancy rates for the blastocyst transfer group in this study. Several studies have underlined the difficulties of correctly selecting the best embryo on Day 2-3 [
22,
23]. The aim of extending embryo culture to Day 5/6 was to select an embryo with increased probability of implantation rather than just to improve embryo quality [
19]. Another recent meta-analysis [
24] suggested that the putative clinical superiority of blastocyst transfer protocols needs to be further verified and that the cumulative clinical pregnancy rate combining both fresh and cryopreserved embryo transfer cycles would be the more reliable measurable outcome for comparison.
In this study, it was surprisingly observed that vitrified-warmed blastocyst transfer for patients aged less than 35 years resulted in significantly higher clinical pregnancy and implantation rates compared with fresh blastocyst transfer. Our results are consistent with a previous study that reported significantly higher ongoing pregnancy, clinical pregnancy, and implantation rates in cryopreserved versus fresh embryo transfer cycles [
25]. Differences in implantation rates between the two groups may reflect different endometrial receptivity and a higher degree of synchronization between endometrial development and the transferred blastocysts in vitrified-warmed cycles [
26]. Our overall results demonstrated that in women undergoing their first or second ART attempt with vitrified-warmed embryo transfers, the cumulative clinical pregnancy rate was significantly increased upon blastocyst transfer, as compared to the transfer of cleavage-stage embryos. It is suggested that if embryo-endometrium synchrony was suboptimal, the embryos should be cryopreserved for a subsequent transfer under more optimal conditions [
27]. Therefore, the concept of cryopreserving all available embryos and transferring them in subsequent non-stimulated cycles may enhance embryo-endometrium synchrony and clinical outcomes of ART cycles in some patients. This in turn may provide a number of clinical benefits, including increasing the cumulative pregnancy rates, reducing the risk of OHSS and decreasing patient discomfort and cost without the need for superovulation. Although a trend towards lower cumulative multiple pregnancy rate was also noticed in blastocyst transfer cycles, this still needs to be further improved and diminished to a minimum by modifying the embryo transfer strategy.
The results of this study therefore demonstrate that blastocyst culture and transfer is not suited for all ART patients. This could be because later embryonic development to the blastocyst stage and beyond is also dependent on maternally transcribed mRNA stored within the oocyte [
28]; and we hypothesize that some patient with poorer quality oocytes (i.e. older women) may have abnormally low levels of such stored mRNA transcripts, leading to arrest of blastocyst formation. Examples of maternally transcribed mRNA that play important roles in blastocyst formation and beyond include CDX2 involved in trophectoderm function and maintenance of the blastocoelic cavity [
29], and STK40 implicated in extra-embryonic endoderm differentiation [
30].
Hence, blastocyst culture and transfer should be offered primarily to younger patients (less than 35 years) with better prognosis, in tandem with blastocyst vitrification. Undoubtedly, blastocyst transfer will still remain a favourable and promising option in ART. In future, the efficacy of embryo transfer at the blastocyst stage could be fully optimized through further refinement and improvement of current blastocyst cryopreservation protocols.
In any case, the ultimate aim of reproductive medicine practitioners would still be the improvement of ART efficacy in terms of take-home-baby rates. However, the optimal outcome in current ART practice is the delivery of singleton infants rather than multiple births. To this end, blastocyst culture in tandem with blastocyst vitrification and single-blastocyst transfer emerges as a highly efficient protocol in human ART treatment.
Competing interest
All co-authors hereby declare that they have no competing interests in the production of this manuscript.
Authors’ contribution
G.Q.T and X.F.L contributed to data collection and analysis, as well as manuscript writing. S.R.C, X.W, J.Q.Z and J.C contributed to data collection and analysis. B.C.H contributed to manuscript writing.