The aim of the present study was to analyze the high-quality blastocyst (HB) rate in all embryo frozen cycles and investigate the pregnancy outcomes for day 5/day 6 (D5/D6) blastocysts with respect to the blastocyst quality in programmed single vitrified-warmed blastocyst transfer (SVBT). We performed a retrospective study comparing D5/D6 HBs in
Extended culture leading to embryo transfer at the blastocyst stage is considered a major advance in
The comparison of pregnancy outcomes between D5 and D6 blastocysts remains controversial. Some reports have concluded that the blastocyst development time is crucial and suggest that D5 is more superior than D6 in terms of implantation rate and live birth rate (
Previous relevant studies only performed simple statistical analyses of the clinical outcomes of vitrified-warmed cycles (
This was a retrospective study carried out at The Center for Reproductive Medicine and Infertility, The Fourth Hospital of Shijiazhuang, China from March 2017 to May 2020. In total, 1,560 (IVF, 1,100 and ICSI 460) all blastocyst frozen cycles were analyzed and 1,161 SVBT cycles (D5 975 and D6 186) were included in the study. The criteria for allocating patients to IVF and ICSI were male semen factors. The women were given IVF protocol in the first assisted reproductive technology (ART) treatment cycle unless accompanied by severe male-factor infertility. Otherwise ICSI treatment was performed. Patients included in the analysis were <35 years at the oocyte collection in their first fresh cycle without fresh embryo transfer, and were undergoing their first autologous FET cycle. The Fourth Hospital of Shijiazhuang Ethics Committee approved (approval no. 20170063; approval date, January 5, 2017) this study.
Cumulus-oocyte-complexes (COC) were isolated from follicular fluid, rinsed in G-IVF™ medium (VitroLife, Sweden) transferred to 0.5 ml G-IVF™ medium and cultured in an incubator under 5% O2, 6% CO2, and 89% N2. Sperm was used for either routine IVF insemination or ICSI procedure using a standard method as described by Jiang
The procedure was always performed using one blastocyst for each straw. An artificial shrinkage (AS), using a laser pulse was performed before vitrification. The blastocyst was then moved at room temperature (22-25˚C) to Kitazato (Japan) equilibration solution (ES). After 6-8 min, the blastocyst was quickly washed in vitrification solution (VS) for 45-60 sec and transferred onto the straw (Kitazato Japan) using a micropipette and immersed vertically into liquid nitrogen.
A Kitazato Thaw Kit Kitazato) was used for warming. The carrier containing the embryo was removed from the straw and placed quickly into the dish containing the thawing medium (thawing solution) preheated at 37˚C. The blastocysts immediately fell from the device and could be easily identified in the medium. After 1 min, the blastocysts were transferred to the DS medium (dilution solution) for 3 min at room temperature (22-25˚C). In the last two step, the blastocysts were placed for 5 min, in the WS1 medium and WS2 (washing solution). The embryo was then returned to G-2 medium for culture until transfer. At this stage, an assessment was performed on an inverted microscope to establish if the embryo survived based on morphological integrity of the ICM and trophectoderm. After 1 or 2 h of culture, the embryo was reassessed again and often the re-expansion of the blastocoel was reported; this indicated that the embryo physiologically survived the warming procedure. Embryo transfer was normally performed within 2 or 3 h. All programmed warmed cycles, both at D5 and D6, were transferred in D5 endometrium.
If the patient had both D5 and D6 blastocysts, the best quality embryo was warmed first. If blastocyst quality was the same, the D5 blastocyst was given priority to transfer. If the embryo did not survive, another embryo was warmed if the patient had any in storage, otherwise the transfer was canceled. Some patient blastocysts were not thawed, did not survived, or two blastocysts transferred were not included in this study.
All vitrified-warmed cycles of endometrium preparation were natural cycle (NC) or artificial cycle [hormone replacement therapy (HRT)] based on the implantation programs. NC was applicable for patients with a regular menstrual cycle. Follicular development was monitored using B ultrasound on days 8-10 of menstruation. The follicular and endometrial development conditions were assessed and combined with the estradiol (E2) and luteinizing hormone (LH) levels to confirm the ovulation time. Embryo transfer was performed on D5 of ovulation. HRT was applicable for patients with an irregular menstrual cycle, ovulation disorder, or poor endometrial and follicular development in NC. Starting from days 2-3 of menstruation, 2-6 mg/day of estradiol valerate (Progynova, Bayer) was administered, and the endometrial thickness and serum E2 levels were monitored using B ultrasound. When the endometrial thickness was at least 8 mm, progesterone 60 mg/day was additionally administered. Embryo transfer was performed on day 6 of progesterone injection. All warmed blastocysts, both vitrified on D5 or D6 were replaced in the D5 endometrium. All embryo transfers were performed using transabdominal ultrasound guidance.
Observation of the gestational sac and fetal heart by B ultrasound at 35 days after implantation was diagnosed as clinical pregnancy. The implantation rate was defined as the ratio between the number of gestational sacs and fetal heart observed under B ultrasound and the number of transferred blastocysts. Implantation rates, pregnancy rates, and twinning of D5/D6 SVBT were analyzed.
Statistical analyses were performed using SPSS 19.0 statistical software (SPSS Inc.). The data are presented as the mean ± standard deviation (SD). The mean values of two groups were compared using the independent samples t-test. Percentages were compared using the χ2 test and P<0.05 was considered statistically significant.
The total HB rate was 50.5% (2,688/5,328) for which IVF was higher than ICSI (52.7% vs. 42.6%; P<0.05). The D5 HB rate was much higher than the D6 HB rate (61.6% vs. 29.4%; P<0.05). There were 22.4% (349/1560) cycles of only cultured D6 blastocysts, in which IVF was lower than ICSI (19.8% vs. 28.5%; P<0.05) (
In total, 1,161 SVBT cycles (D5 975 and D6 186) were analyzed (
Extending embryo culture to the blastocyst stage has become a routine in many
Whether there are differences in the pregnancy outcomes of blastocysts cryopreserved during different developmental stages remains under debate because the results among studies are inconsistent. A meta-analysis of clinical outcomes showed that in day 5 (D5) vs. day 6 (D6) blastocyst transfers, clinical pregnancy rate and live birth rates were significantly higher following D5 compared to D6 blastocyst transfers (
In addition, blastocyst grade plays an important role in pregnancy outcomes. Blastocysts with trophectoderm grades A, B, and C were found to have euploidy rates of 71.43, 60.00 and 19.67%, respectively (P<0.05) (
In previous research, patients who underwent single vitrified-warmed blastocyst transfer (SVBT) cycles were able to obtain optimal pregnancy outcomes, especially in the <35 year age group (
While the previous studies focused on warming embryo of D5/D6 frozen embryo transfer (FET) cycles, the present study was the first to consider the high-quality blastocyst (HB) rate in fresh
From this research, we know that in IVF, the cultured blastocysts and HBs per cycle were more than these parameters in ICSI, and cultured blastocysts in IVF were earlier than ICSI. We concluded that the fertilization method directly influenced HB and blastocyst development rates. Therefore, the IVF/ICSI ratio needs to be considered when analyzing D5/D6 SVBT. In the present SVBT study, D5 (IVF 71.4% and ICSI 28.6%) cycles and D6 (IVF 68.8% and ICSI 31.2%) IVF/ICSI ratios were not significantly difference. Speyer
Most patients prefer to use D5 HB in their first FET cycle, and finally choose D6 blastocysts when none of the thawed D5 blastocysts have resulted in successful pregnancy. Therefore, the inclusion criteria were patients who were in their first fresh cycle without fresh embryo transfer and who were undergoing their first SVBT cycle.
In conclusion, following control of patient age, transfer frequency, and endometrium on day 5, it is not the development stage (D5/D6) but the transfer blastocyst quality that plays an important role in achieving the optimal pregnancy outcomes. The D5 HB rate was found to be 2-times higher than D6, and the IVF HB rate was also higher than ICSI, which may be the reason for the current debate in the literature regarding the pregnancy outcomes of D5/D6 SVBT.
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
YJ conceived the study and wrote the paper. YJ and GS performed the experiments and analyzed the data. YJ and XHZ contributed to design and conception. SBM and XHW contributed to acquisition and interpretation of data. GS, XHZ, SBM and XHW confirmed the authenticity of all of the data. XHW supervised the study. All authors read and approved the final manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved.
The Fourth Hospital of Shijiazhuang Ethics Committee approved (approval no. 20170063; approval date, January 5, 2017) this study. The procedures used in this study adhered to the tenets of the Declaration of Helsinki. Informed consent was obtained from all individual participants included in the study.
Not applicable.
The authors declare that they have no competing interests.
Comparison of the HB rate in D5 and D6 blastocysts of fresh IVF/ICSI cycles. HB, high-quality blastocyst; IVF,
Clinical pregnancy rate of HB/LB SVBT on D5 and D6 in IVF/ICSI. HB, high-quality blastocyst; LB, low-quality blastocyst; SVBT, single vitrified-warmed blastocyst transfer; D5, day 5; D6, day 6; IVF,
HB/LB pregnancy data between the day 5 (D5) and day 6 (D6) groups.
Variables | Groups | Day 5 | Day 6 | χ2 | P-value |
---|---|---|---|---|---|
No. of patients | 975 | 186 | |||
No. of high-quality blastocyst (HBs) | 705 | 112 | |||
No. of low-quality blastocysts (LBs) | 270 | 74 | |||
Clinical pregnancy rate (%) | Total | 57.4 (560/975) | 46.2 (86/186) |
7.94 | 0.0045 |
HBs | 60 (426/705) | 54.5 (61/112) | 1.43 | 0.2320 | |
LBs | 49.6 (134/270) | 33.8 (25/74) |
5.87 | 0.0150 | |
Implantation rate (%) | Total | 58.9 (574/975) | 47.3 (88/186) |
8.52 | 0.0025 |
HBs | 62.0 (437/705) | 56.3 (63/112) | 1.34 | 0.2470 | |
LBs | 50.7(137/270) | 33.8 (25/74) |
6.70 | 0.0100 | |
Multiple pregnancy rate (%) | Total | 1.44 (14/975) | 1.08 (2/186) | 0.149 | 0.7150 |
HBs | 1.56 (11/705) | 1.78 (2/112) | - | 0.6960 | |
LBs | 1.11 (3/270) | (0/74) | - | - | |
Male rate (%) | Total | 54.0 (233/431) | 48.4 (31/64) | 0.708 | 0.3500 |
HBs | 53.9 (181/336) | 50.0 (24/48) | 0.253 | 0.6150 | |
LBs | 54.7 (52/95) | 43.8 (7/16) | 0.664 | 0.4150 |
aP<0.05, significant difference.
IVF/ICSI pregnancy data between the day 5 (D5) and day 6 (D6) groups of SVBT.
Variables | Groups | IVF | ICSI | χ2 | P-value |
---|---|---|---|---|---|
No. of patients | 824 | 337 | |||
No. of D5 blastocysts | 696 | 279 | |||
No. of D6 blastocysts | 128 | 58 | |||
Clinical pregnancy rate (%) | Total | 56.3 (464/824) | 54.0 (182/337) | 0.515 | 0.470 |
D5 | 58.0 (404/696) | 55.9 (156/279) | 0.370 | 0.543 | |
D6 | 46.8 (60/128) | 44.8 (26/58) | 0.067 | 0.795 | |
Implantation rate (%) | Total | 57.0 (473/824) | 56.1 (189/337) | 0.035 | 0.860 |
D5 | 59.2 (412/696) | 58.1 (162/279) | 0.105 | 0.746 | |
D6 | 47.7 (61/128) | 46.6 (27/58) | 0.020 | 0.889 | |
Multiple pregnancy rate (%) | Total | 1.09 (9/824) | 2.08 (7/337) | 1.71 | 0.175 |
D5 | 1.15 (8/696) | 2.15 (6/279) | 0.792 | 0.374 | |
D6 | 0.78 (1/128) | 1.72 (1/58) | - | 0.528 | |
Male rate (%) | Total | 52.2 (189/362) | 56.4 (75/133) | 0.68 | 0.430 |
D5 | 52.8 (167/316) | 57.4 (66/115) | 0.701 | 0.403 | |
D6 | 47.8 (22/46) | 50.0 (9/18) | 0.024 | 0.876 |
IVF,