The human embryo following biopsy on day 5 versus day 3: viability, ultrastructure and spindle/chromosome configurations.

RESEARCH QUESTION: Are there any differences in viability, spindle abnormalities and mitochondrial and other organelle structures amongst embryos biopsied on day 3 versus day 5 before and after vitrification? DESIGN: A total of 240 day 3 biopsied embryos that developed to blastocysts but were rejected for transfer following preimplantation genetic testing for monogenic/single gene defects (PGT-M) (n = 115) or for aneuploidies (PGT-A) (n = 125) were divided into two groups: (i) 120 blastocysts treated for viability, spindle/chromosome configuration (SCC) analysis and transmission electron microscopy (TEM) analysis (fresh n = 20, n = 20, n = 20 and following vitrification/warming n = 20, n = 20, n = 20); (ii) 120 embryos were re-biopsied at the blastocyst stage and treated for viability, SCC and TEM analysis (fresh n = 20, n = 20, n = 20 and following vitrification/warming n = 20, n = 20, n = 20). Also, 60 vitrified blastocysts biopsied only on day 5 that were rejected for transfer following PGT-M (n = 6) or PGT-A (n = 54) were treated following warming for viability (n = 20), SCC (n = 20) and TEM analysis (n = 20). RESULTS: No differences were observed in SCC and ultrastructure between embryos biopsied on day 5 and day 3 but following vitrification higher numbers of abnormal spindles, distension of mitochondria, multivesicular bodies, lipofuscin droplets, altered cell junctions and occasionally excessive accumulation of glycogen granules were evident. The fresh day 3 biopsied group also had a lower incidence of damaged (propidium iodide-stained) cells compared with the fresh day 3+5 (P = 0.02) and the vitrified day 5 (P = 0.001) biopsied groups. CONCLUSIONS: Biopsies on day 5 and day 3 do not adversely affect embryo viability, SCC or ultrastructure, although following vitrification minimal embryo quality-dependent increases in spindle abnormalities and cell damage are observed.

Delayed development influences the outcome of different grades of D5 and D6 blastocysts during freeze-thaw cycle.

To analyze the effects of blastocysts on the 5th day (D5) and 6th day (D6) of frozen-thawed blastocyst transplantation on pregnancy outcome and provide evidence for further improvement of the strategy. This study included transfers from the Reproductive Medicine Center of the Second Affiliated Hospital of Wenzhou Medical University during freeze-thaw cycles from January 2016 to December 2017. They were divided into D5 group (1616 cases) and D6 group (619 cases) according to blastocyst formation and development. Each group was further divided into 5 groups according to the quality of the blastocyst and the number of transplants, making a total of 10 groups. Following the frozen transplantation cycle, the transplanting rate was significantly higher for D5 (41.73%) than for D6 (23.98%) (P < 0.05); the ongoing pregnancy rate (47,40%) was also significantly higher than that of D6 (28.43%) (P < 0.05).In the frozen-thawed blastocyst resuscitation transplantation, compared to D6 blastocysts, D5 blastocysts were more conducive to blastocyst implantation and could be used to achieve better clinical pregnancy outcome. In blastocyst selection, a single D5 excellent blastocyst transplant is preferred. Only at the 6th day of non-excellent D6, 2 blastocysts are recommended for transplantation.

Increased pregnancy outcome after day 5 versus day 6 transfers of human vitrified-warmed blastocysts.

Vitrification is a highly efficient technique for the cryopreservation of the human embryo. The effect of delayed blastulation may be responsible for implantation failures and negatively affects in vitro fertilization (IVF) outcomes. The current literature displays discordant results; some studies have announced higher pregnancy rates after day 5 (D5) transfer compared with day 6 (D6) transfer, while others have shown equivalent outcomes. In the present study an investigation into the clinical implications of delayed blastulation (D5 versus D6) was carried out. We performed a retrospective study comparing clinical pregnancies and implantation rates following warmed single blastocyst transfer (WSBT). All patients coming for a programmed warmed transfer at Edinburgh Assisted Conception Programme, EFREC, Royal Infirmary of Edinburgh, were included in this study and divided in two groups according to the day of blastocyst vitrification: D5 (n = 1563) and D6 (n = 517). The overall survival rate was 95.0% (1976/2080) with no significant difference between the D5 and D6 groups: 95.3% (1489/1563) and 94.2% (487/517) respectively. WSBT of D6 blastocysts resulted in a lower implantation and clinical pregnancy compared with D5 embryos. The implantation rate (IPR) and clinical pregnancy rate (CPR) were respectively 49.4% and 42.6% for the D5 and 37.4% and 32.2% for the D6 embryos, which was statistically significant. The multiple pregnancy rate was 1.32% (1.14% for D5 vs 1.84% for D6). Although the transfer of D6 vitrified-warmed blastocyst remains a reasonable option, priority to a D5 embryo would reduce the time to successful pregnancy.

Artificial blastocoel collapse of human blastocysts before vitrification and its effect on re-expansion after warming - a prospective observational study using time-lapse microscopy.

Vitrified human blastocysts show varied re-expansion capacity after warming. This prospective observational study compared behaviour of artificially collapsed blastocysts (study group patients, n = 69) to that of blastocysts that were vitrified without artificial collapse (control group patients, n = 72). Warmed blastocysts were monitored by time-lapse microscopy and blastocoel re-expansion speed and growth patterns compared between study and control groups. These parameters were also retrospectively compared between blastocysts that resulted in live birth and those that failed. Artificially collapsed blastocysts re-expanded on average 15.01 µm2/min faster than control blastocysts (P = 0.0013). Warmed blastocysts expressed four different patterns of blastocoel growth. The pattern showing contractions at the end of culture was observed to have a lower prevalence in control blastocysts, which coincided with the lower incidence of hatching in this group. Re-expansion speed and prevalence of growth patterns were comparable between blastocysts that did and did not result in a live birth. This was seen in the study and control groups. Despite faster re-expansion and different growth patterns of artificially collapsed blastocysts, live birth rate did not differ between groups. However, this result should be interpreted with caution due to the small sample size and high risk of bias.

Blastocyst vitrification, cryostorage and warming does not affect live birth rate, infant birth weight or timing of delivery.

RESEARCH QUESTION: Does vitrification and warming affect live birth rate, infant birth weight and timing of delivery? DESIGN: Retrospective, cohort study comparing outcomes of donor oocyte recipient fresh (n = 25) versus vitrified (n = 86) euploid blastocyst transfers; donor oocyte recipient singleton live births from fresh (n = 100) versus vitrified (n = 102) single embryo transfers (SET); and autologous vitrified euploid SET (n = 1760) (cryostored 21-1671 days). RESULTS: Group 1: fresh and vitrified-warmed blastocysts had similar live birth (OR 1.7; 95% CI 0.5 to 5.9), implantation (OR 0.9; 95% CI 0.2 to 3.9), clinical pregnancy (OR 3.4; 95% CI 0.9 to 13.0) and pregnancy loss (OR 1.2; 95% CI 0.98 to 1.4); group 2: low birth weight (OR 0.44; 95% CI 0.1 to 1.6) and preterm delivery (0.99; 95% CI 0.4 to 2.3) rates were similar in fresh and vitrified-warmed blastocyst transfers; group 3: cryostorage duration did not affect live birth (OR 1.0; 95% CI 1.0 to 1.0), implantation (OR 1.0; 95% CI 0.99 to 1.01), clinical pregnancy (OR 1.0; 95% CI 1.0 to 1.0]), pregnancy loss (OR 0.99; 95% CI 1.0 to 1.0), birth weight (ß = -15.7) or gestational age at delivery (ß = -0.996). CONCLUSIONS: Vitrification and cryostorage (up to 4 years) are safe and effective practices that do not significantly affect clinical outcome after embryo transfer.

Single blastocyst transfer (SET) and pregnancy outcome of day 5 and day 6 human blastocysts vitrified using a closed device.

This study investigates the utility of the Rapid-i closed device for vitrification of human blastocysts on day-5 (D5) and day-6 (D6) of development and the implantation and pregnancy rate following single blastocyst transfer (SBT) of warmed D5/D6 blastocysts. This retrospective cohort study was performed at Edinburgh Assisted Conception Programme, EFREC, Royal Infirmary of Edinburgh between January 2013 and January 2017. Good quality blastocysts were vitrified on D5 or D6 using Irvine Vitrification medium (Irvine Scientific-USA) and the Rapid-I closed Vitrification System™ (Vitrolife, Sweden). After warming, blastocysts were cultured in G-TL™ medium (Vitrolife) supplemented with 20% HSA-solution™ (Human Serum Albumin) for 2 h before the transfer. The survival, pregnancy and implantation rates were compared in relation to the day of culture at the time of vitrification (D5/D6) in 1090 cryopreserved cycles. The overall survival rate was 93.4% (1018/1090) with no significant difference between the D5 and D6 groups: 93.9% (712/758) and 92.2% (306/332) respectively. Single embryo transfers of D6 vitrified/warmed blastocysts resulted in a lower implantation and clinical pregnancy rate compared to D5 embryos. The implantation rate (IPR) and clinical pregnancy rate (CPR) were respectively 49.6% and 43.0% for the D5 and 37.0% and 33.0% for the D6 embryos, which was statistically significant. The multiple pregnancy rate was 1.08% (0.98% for D5 vs 1.3% for day 6).

Morphokinetics of vitrified and warmed blastocysts predicts implantation potential.

PURPOSE: It was studied whether morphokinetics of blastocoele re-expansion and hatching in vitrified-warmed blastocysts is predictive of implantation, clinical pregnancy, and live birth. METHODS: In 144 patients aiming for single warmed blastocyst transfer, blastocysts were cultured in a new time-lapse system (Miri® TL) immediately after warming. Video sequences with an image interval of 5 min were annotated and the corresponding morphokinetic variables were correlated with pregnancy outcome. In detail, tRE (start of re-expansion), tCRE (completion of re-expansion), tAH (hatching from the manipulated zona pellucida), and presence of collapses were recorded. RESULTS: In the pregnant group, tRE and tCRE were significantly lower (0.69 ± 0.45 h and 2.16 ± 0.94 h) as compared to the non-pregnant group (1.23 ± 1.08 h and 2.70 ± 1.20 h). Both variables and the duration of re-expansion (tCRE-tRE) allowed for distinction between "non-pregnant," "loss of pregnancy," and live birth/ongoing pregnancy. Presence and number of collapses showed no correlation with outcome. CONCLUSIONS: Time-lapse imaging of vitrified-warmed blastocysts offers additional selection criteria allowing for prediction of implantation potential. As a consequence, cumulative pregnancy rate could be increased and time-to-pregnancy reduced.

Pregnancy and birth outcomes following fresh or vitrified embryo transfer according to blastocyst morphology and expansion stage, and culturing strategy for delayed development.

STUDY QUESTION: How do live birth rates (LBRs), following fresh and vitrified/warmed embryo transfer, compare according to morphological grade, developmental stage and culturing strategy of human blastocysts in vitro? SUMMARY ANSWER: Equivalent LBRs were obtained after fresh embryo transfer and after vitrified/warmed embryo transfer of blastocysts of top or non-top quality, while vitrification after prolonged embryo culture of blastocysts with delayed development had a positive impact on LBR. WHAT IS KNOWN ALREADY: Blastocyst morphology correlates with clinical outcome; however, few data are available on vitrified/warmed embryo transfer using non-top quality blastocysts. The aim of this study was to determine clinical outcomes of non-top quality blastocysts and blastocysts with delayed development that underwent vitrified/warmed embryo transfer. STUDY DESIGN, SIZE, DURATION: This retrospective, single-centre study (conducted January 2009 to June 2013) compared 1010 fresh embryo transfer and 1270 vitrified/warmed embryo transfer of blastocysts originating from the same stimulation cycle. Furthermore, 636 fresh embryo transfers and 304 vitrified/warmed embryo transfer after delayed expansion or blastulation in the same period were also analysed. PARTICIPANTS/MATERIALS, SETTING, METHODS: Clinical outcomes after fresh and vitrified/warmed embryo transfer according to blastocyst morphology were compared in both groups. MAIN RESULTS AND THE ROLE OF CHANCE: Similar LBRs after fresh embryo transfer or after vitrified/warmed embryo transfer of top or non-top quality blastocysts were observed. A statistically significant improvement in clinical outcomes was obtained after vitrified/warmed embryo transfer of Day 5 embryos with delayed expansion or blastulation when applying prolonged culture. Our study suggests that vitrification of non-top quality blastocysts as well as delayed cavitating and blastulating Day 5 embryos should be considered in autologous IVF cycles. LIMITATIONS AND REASONS FOR CAUTION: Given that the present retrospective study used aseptic vitrification of blastocysts, the results, particularly the survival rates, may not be fully applicable to other vitrification protocols. The retrospective nature of the study has to be mentioned. WIDER IMPLICATIONS OF THE FINDINGS: Restriction of vitrification to top quality blastocysts may result in discarding potentially viable embryos. STUDY FUNDING AND COMPETING INTERESTS: This study was not externally funded. There are no conflicts of interest to declare.

D6 high-quality expanded blastocysts and D5 expanded blastocysts have similar pregnancy and perinatal outcomes following single frozen blastocyst transfer.

Objective: We compared the pregnancy and perinatal outcomes between expanded blastocysts vitrified on D5 versus D6 following single frozen blastocyst transfer. Methods: Clinical data on 7,606 cycles of frozen-thawed blastocyst implantations were retrospectively analyzed. Depending on whether blastocysts were vitrified on D5 or D6 and the transferred blastocysts, the blastocysts were divided into 6 groups: HQB-D5, HQB-D6, 4XC-D5, 4XC-D6, 4CX-D5, and 4CX-D6 groups. The differences in clinical pregnancy rate, live birth rate, first trimester abortion rate, preterm birth rate, gestational age, birth weight, and sex ratio at birth among the groups were compared. Results: Our study showed that there was no difference in pregnancy and perinatal outcomes between the delayed formation of D6 high-quality expanded blastocysts and D5 expanded blastocysts, whether they were high-quality blastocysts or not. For low-quality blastocysts, the clinical pregnancy rate of D5 was higher than that of D6, and D5 was also better than D6 in live birth rate for those with inner cell mass rating B or above, while there was no difference between D5 and D6 for those with inner cell mass rating C. Conclusion: Based on our research, we suggest that when we are developing the implantation strategy, we give priority to the selection of high-quality expanded blastocysts, regardless of D5 and D6, whose clinical outcomes are not different. For low-quality blastocysts, D5 expanded blastocysts are preferred for transfer.

Appendix B: Solid Surface Vitrification.

Solid surface vitrification method involves direct contact of carrier loaded with droplet containing gametes or embryos with precooled metal surface. Over the years, following certain modifications, solid surface vitrification has emerged as an efficient method for vitrifying human gametes and embryos. Here, we describe the principle and methodology of solid surface vitrification.