Two‐step consecutive transfer of early embryos and blastocysts

Abstract

Aim:  To evaluate and confirm the merit of two consecutive transfer attempts of early embryos and blastocysts.

Methods:  A total of 685 patients underwent routine in vitro fertilization (IVF) cycles. The study population consisted of three groups: (i) Group 1, early stage embryos were transferred (460 patients; 567 cycles); (ii) group 2, blastocysts were transferred (88 patients; 105 cycles); and (iii) group 3, a two‐step (consecutive) transfer was performed on 137 women (141 cycles). Specifically, a standard embryo transfer was performed on day 3, together with a two‐step (consecutive) transfer of blastocysts. After the early embryo transfer, an extended culture of supernumerary embryos was conducted, followed by a second transfer of blastocyst(s).

Results:  No significant differences were found in the three groups with regards to either pregnancy or implantation rates for groups 1, 2, or 3; pregnancy: 34.6, 29.9, and 33.6%, respectively; implantation: 18.6, 15.9, and 15.1%, respectively. The miscarriage rate for each group was also not significant; 20.4, 30.8 and 28.6% for groups 1, 2 and 3, respectively. The multiple pregnancy rate of the three groups was 30.4, 30.8 and 35.7%, respectively, and these were not found to be significant results.

Conclusion:  No significant difference was found between the three groups with regards to the pregnancy, implantation, multiple pregnancy and miscarriage rates. The multiple pregnancy rate was highest in the two‐step (consecutive) transfer group. This difference was not regarded to be highly significant. (Reprod Med Biol 2003; 2: 133–137)

INTRODUCTION

Assisted reproduction technology (ART) has proven to be beneficial for infertile couples. Despite ART advances in recent years, the success rate remains disappointingly low, and one of the major remaining challenges is the attainment of a high implantation rate.

In in vitro fertilization (IVF), intrauterine transfer of embryos is routinely conducted on day 2 (D2) or day 3 (D3), at the four‐ to eight‐cell cleavage stage. Recently, blastocyst transfer has increased in popularity as a clinical application. In general, the blastulation rate with two pronuclei (2 PN)‐stage embryos ranges from 30 to 50%. ¹ , ² Furthermore, most facilities experience at least occasional unsatisfactory results. Recently, sequential culture media were produced specifically to meet the varying requirements of the embryo as it develops from the zygote to the blastocyst stage. Some investigators have reported a high survival rate when only good‐quality embryos are used. The new culture media promotes a high rate of blastocyst development, and more importantly, the blastocysts that develop lead to high implantation and pregnancy rates following transfer. ² , ³ , ⁴ , ⁵ However, leaving all embryos in extended culture until they develop to the blastocyst stage might result in a cancellation of the embryo transfer procedure if none of the embryos reach that stage. If both early stage embryos and blastocysts are transferred using the two‐step (consecutive) procedure, the implantation and pregnancy rate might be increased. Goto et al. ⁶ reported improved results with the two‐step procedure (ASRM/CFAS, abstract, 2001); ⁶ however, the studies of Ashkenazi et al. found no significant benefit in using this method. ⁷ By reason of the efficacy of the two‐step procedure being controversial, the aim of this study was to evaluate the results of the two‐step (consecutive) method, which was modified for the consecutive transfer of early embryos and blastocysts.

MATERIALS AND METHODS

A total of 685 patients who were treated at our ART clinic between January 2001 and December 2002 were subsequently treated with ART including conventional IVF (c‐IVF) and intracytoplasmic sperm injection (ICSI). The patients were divided into three groups: group 1 (control, 460 patients; 567 cycles), an early stage embryo transfer was performed on the women treated during the study period who had had their supernumerary embryos cryopreserved at the 2 PN stage; group 2 (88 patients; 105 cycles), patients had experienced several ART failures and their embryos were suitable for continued culture to the blastocyst stage with subsequent blastocyst transfer on D5; group 3 (137 patients; 141 cycles), a two‐step (consecutive) transfer was performed on women who had experienced several ART failures. Early embryos were transferred, with a subsequent transfer of one or two blastocysts in the same cycle. They had more than five embryos and were offered the option of a two‐step continuous transfer. The present study was a retrospective one as the patient's first choice of treatment was the one undertaken.

The ovarian stimulation protocol, ultrasound examinations, hormonal surveillance methods, timing of human chorionic gonadotropin (hCG) administration, oocyte retrieval, and sperm processing techniques have been described elsewhere. ⁸ , ⁹ The oocytes were fertilized either conventionally or by using intracytoplasmic sperm injection (ICSI).

The sequential media approach was used for the extended culture of embryos to the blastocyst stage. The embryos were initially cultured in 20 µL Cook‐Sydney IVF Culture Medium (K‐SICM medium; Cook, QLD, Australia) until D3. On D3, embryos were transferred for further development into 30 µL drop blastocyst K‐SIBM medium (Cook). On the night prior to transfer, the dishes were prepared and pre‐incubated at 37°C in a 6% CO₂, 5% O₂, 89% N₂ humidified environment.

The embryo quality was evaluated prior to the early transfer. Embryos with equal‐sized blastomeres, an ideal cleavage rate (eight cells on D3), and <15% fragmentation were defined as grade A. ¹⁰ , ¹¹ Cleaving embryos were selected for early transfer based on their morphologic score. At the early trasfer stage, the bext embryo along with others were transferred. The supernumerary embryos from the study group were reserved for extended culture. Blastocysts, which developed from a second grade score at the early stage embryo, were transferred on D5, and after transfer, supernumerary blastocysts were frozen by vitrification. ¹² The quality of blastocyst morphology was evaluated by the criteria proposed by Gardner and Lane. ¹³

The total number of embryos transferred was consistent, and based on the individual woman's age and number of previously failed IVF attempts. However, less than three embryos were transferred. Transfers were performed in our laboratory atraumatically with the use of soft‐pass catheters; Wallace (Simcare Ltd, Lancing, West Susser, England, UK) catheters were used, but this depended on their availability.

Clinical pregnancy was defined as the visualization of a gestational sac with the use of transvaginal ultrasound, in association with rising β‐hCG levels.

Informed consent for the procedures was obtained from all couples.

For statistical analysis, the Chi‐squared test, and Fisher's exact test were used; P < 0.05 was considered to be significant.

RESULTS

A total of 685 patients who underwent 813 treatment cycles were included in the present study; early embryo transfer was performed on 460 patients (567 cycles), blastocyst transfer was performed on 88 patients (105 cycles), and a two‐step consecutive transfer was conducted on 137 patients (141 cycles).

Seventy‐three treatment cycles were performed with the use of conventional IVF (c‐IVF), and 602 cycles were conducted with the use of ICSI. The remaining 138 cycles were performed by using both ICSI and c‐IVF on sibling oocytes; in this group, both types of embryos were transferred. The blastulation rate for group 2 was 56.6% (300 of 530). For group 3, the blastulation rate from supernumerary embryos, which were left in culture after the early transfer, was 47.7% (296 of 621).

The findings for the three groups are presented in Table 1. The mean age was similar in all three groups: group 1, 35.5 ± 4.8; group 2, 35.5 ± 4.5; and group 3, 35.4 ± 4.5 years old, respectively. A significant difference in the number of previously treated IVF cycles was found; group 1 versus group 2 (P < 0.05); group 1 versus group 3 (P < 0.05) and group 2 versus group 3 (P < 0.05).

Table 1.

Findings for the three study groups

	Group 1 (n = 460)	Group 2 (n = 88)	Group 3 (n = 137)
No. cycles	567	105	141
Age (years)	35.5 ± 4.8	35.5 ± 4.5	35.4 ± 4.5
No. previous IVF cycles	2.6 ± 2.5	4.0 ± 2.7**	2.0 ± 2.2*
No. oocytes retrieved	8.2 ± 5.8	11.2 ± 7.2	10.9 ± 5.9
No. matured oocytes	6.5 ± 4.9	8.9 ± 5.5*	8.7 ± 4.4*
No. fertilized oocytes	5.0 ± 3.9	7.0 ± 4.0	6.9 ± 3.4
No. embryo transferred on D3	2.5 ± 1.0	–	1.3 ± 0.6
No. embryo transferred on D5	–	2.5 ± 0.9	2.0 ± 0.8
Blastocyst development (%)	–	56.6 (300/530)	47.7 (296/621)
Cancellation rate (%)	8.4 (44/567)	17.1 (18/105)	11.3 (16/141)
Pregnancy rate (%)	34.6 (181/523)	29.9 (26/87)	33.6 (42/125)
Multiple gestation rate (%)	30.4 (55/181)	30.8 (8/26)	35.7 (15/42)
Miscarriage rate (%)	20.4 (37/181)	34.6 (9/26)	28.6 (12/42)
Implantation rate (%)	18.6 (239/1284)	15.9 (35/220)	15.1 (56/371)

D, day; IVF, in vitro fertilization. A significant difference in the number of previously treated IVF cycles was found: group 1 versus group 2 (P < 0.01); group 1 versus group 3 (P < 0.05). The average number of retrieved mature oocytes from groups 1, 2, and 3 was 6.5, 8.9, and 8.7, respectively. A significant difference was found with both group 1 versus group 2 (P < 0.05), and group 1 versus group 3 (P < 0.05). * P < 0.05, **P < 0.01.

The average number of retrieved mature oocytes from groups 1, 2, and 3 were 6.5, 8.9, and 8.7, respectively. Significant differences were found with both group 1 versus group 2 (P < 0.05), and group 1 versus group 3 (P < 0.05). No significant difference in the implantation rate was observed, with percentages varying only slightly; 18.6% (239 of 1284), 15.9% (35 of 220) and 15.1% (56 of 371) in groups 1, 2 and 3, respectively. The pregnancy rate also varied slightly between the three groups: 34.6% (181 of 523), 29.9% (26 of 87) and 33.6% (42 of 125) for group 1, 2 and 3, respectively. Furthermore, there were no statistical differences between the groups with regards to the incidence of multiple gestation: group 1, 30.4% (55 of 181); group 2, 30.8% (eight of 26); and group 3, 35.7% (15 of 42), respectively.

There was significant difference in the blastulation rate for the supernumerary embryos, which were reserved for extended culture after early transfer on D3 and D5 (group 3, 47.7% (296 of 621); group 2, 56.6% (300 of 530); P < 0.05).

The incidence of multiple pregnancies in group 3 was higher than that in groups 1 and 2. The miscarriage rate of groups 2 and 3 were comparative higher than that of group 1; however, these differences are not significant.

DISCUSSION

In our continuing efforts to achieve successful pregnancies for patients who have experienced IVF failures, it is prudent to explore new technologies and media. The introduction of new culture media formulations, which facilitate the growth of blastocysts with high implantation potential without the use of coculture, has regenerated an interest in transferring embryos at the blastocyst stage. The major advantage of the blastocyst transfer approach is the presumed high probability of implantation. If a two‐step consecutive embryo transfer can be conducted during the same treatment cycle, a higher pregnancy rate than c‐IVF embryo transfer might be achieved. The early stage embryo insertion into the uterus might stimulate an unknown adhesion factor, which can promote implantation. Therefore, the two‐step (consecutive) transfer of embryos at the early stage and at the blastocyst stage can be especially beneficial in specific clinical situations with patients who have experienced repeated implantation failure. ¹⁴ , ¹⁵ Good results have been reported using this procedure (ASRM/CFAS, abstract, 2001); ⁶ however, Ashkenazi et al. reported that they found no significant advantage in using the two‐step (consecutive) transfer of early embryos and blastocysts when compared with the traditional early transfer. ⁷

We found that supernumerary embryos matured to the blastocyst stage in 40–50% of embryos; however, it should be remembered that not all patients will be able to undergo embryo transfer if it is deferred to the blastocyst stage. Obviously, a two‐step (consecutive) transfer did not have a blastocyst available for the second transfer.

Our two‐step (consecutive) transfer approach was used to evaluate the possible advantages of blastocyst transfer in patients who were offered this procedure. The results show that the two‐step (consecutive) transfer does not improve either pregnancy or implantation rates compared with the traditional early stage embryo (D3) transfer. The pregnancy rate of group 1 patients was higher than that of groups 2 and 3 patients. However, group 1 and 3 patients had a lower number of treatments compared with patients in group 2 who had had four lots of treatment. Group 2 had many patients who had had several previous IVF cycle treatments, and who wanted to have blastocyst embryos transferred, therefore, the number of treatments was higher compared with the other two groups.

A good reason for using the two‐step consecutive transfer might be because of the enhanced immune response triggered by the previously transferred embryo(s); ¹⁴ , ¹⁵ however, our results indicate that the first embryo(s) transferred did not improve the implantation environment for the embryos transferred at the second stage. We did not observe any benefits using the two‐step transfer method even though the second transfer process might select the best embryos; those embryos left in culture must further develop to the blastocyst stage for transfer on D5. However, our results are from a retrospective study, and therefore, it is necessary to evaluate the two‐step method by using a random‐sampling prospective study. Rijuders and Jansen have shown that the predictive value of embryo morphology for subsequent blastocyst formation is limited because of the full activation of the embryonic genome. ¹⁰ In their study, 49% of the embryos preselected on D3 were not transferred on D5, partly because there were better embryos present on D5 that had not been preselected, and primarily because despite their good quality on D3, these embryos underwent a developmental arrest or degradation.

Another possible explanation is an adverse effect of the second transfer on the implantation process. A second insertion of a catheter through the cervix might traumatize the endometrium or stimulate secretion of prostaglandins; thus inducing uterine contractions. ¹⁶ In the present study, it was observed that the patient's condition after one and two treatments was the same, and thus it can be concluded that the twice‐transfer method did not mechanically damage the uterus.

One positive aspect about the secondary transfer is that it is performed more smoothly because of the fact that the angle of uterine cervix‐body, the length of the uterine cavity, and the best embryo transfer catheter during the first transfer has already been confirmed.

In conclusion, we found that the two‐step (consecutive) transfer of early embryos and blastocyst(s) does not improve pregnancy or implantation rates compared with early‐stage embryo transfer. However, our data is limited because it is a retrospective study, and, therefore, the possible benefits of the two‐step procedure are open to further discussion and research.