Vitrified-warmed Blastocyst Research Articles

Single vitrified blastocyst transfer maximizes liveborn children per embryo while minimizing preterm birth

To compare live-birth rates, blastocyst to live-birth efficiency, gestational age, and birth weights in a large cohort of patients undergoing single versus double thawed blastocyst transfer. Retrospective cohort study. Assisted reproduction technology (ART) practice. All autologous frozen blastocyst transfers (FBT) of one or two vitrified-warmed blastocysts from January 2009 through April 2012. Single or double FBT. Live birth, blastocyst to live-birth efficiency, preterm birth, low birth weight. Only supernumerary blastocysts with good morphology (grade BB or better) were vitrified, and 1,696 FBTs were analyzed. No differences were observed in patient age, rate of embryo progression, or postthaw blastomere survival. Double FBT yielded a higher live birth per transfer, but 33% of births from double FBT were twins versus only 0.6% of single FBT. Double FBT was associated with statistically significant increases in preterm birth and low birth weight, the latter of which was statistically significant even when the analysis was limited to singletons. Of the blastocysts transferred via single FBT, 38% resulted in a liveborn child versus only 34% with double FBT. This suggests that two single FBTs would result in more liveborn children with significantly fewer preterm births when compared with double FBT. Single FBT greatly decreased multiple and preterm birth risk while providing excellent live-birth rates. Patients should be counseled that a greater overall number of live born children per couple can be expected when thawed blastocysts are transferred one at a time.

Neonatal outcomes after the implantation of human embryos vitrified using a closed-system device.

Closed vitrification poses a risk of adversely affecting embryo development, while it may minimize the risk of contamination. We assessed the effects of closed-system human embryo vitrification on fetal development after implantation, neonatal outcome, and clinical safety. This was a retrospective cohort study conducted at a private fertility clinic. A total of 875 vitrified-warmed blastocysts that were single-transferred under hormone-replacement cycles between November 2011 and December 2013 were randomly divided into two groups (closed vitrification, n 313; open vitrification, n 562) after receiving the patients' consent forms. Developmental competence after implantation, including gestational age, birth weight, sex, Apgar score, and anomalies of newborns, after the transfer of blastocysts vitrified by closing vitrification was compared with that obtained in the case of open vitrification. There were no significant differences between the use of closed and open vitrification systems in embryo development after implantation, gestational age, birth weight, sex ratio, Apgar score, and congenital anomalies of newborns. Human embryos can be vitrified using a closed vitrification system without impairment of neonatal development.

Successful implantation and live birth of a healthy boy after triple biopsy and double vitrification of oocyte-embryo-blastocyst

IntroductionPreimplantation genetic diagnosis and/or screening (PGD/PGS) allow the assessment of the genetic health of an embryo before transferring it into the uterus. These techniques require the removal of cellular material (polar bodies, blastomere(s) or trophectoderm cells) in order to perform the proper genetic analysis. We report the implantation and live birth outcome of a vitrified-warmed blastocyst developed after triple biopsy and double vitrification procedures at oocyte, cleavage embryo and blastocyst stage.Case descriptionAn infertile couple, with family history of β-thalassemia, searched for IVF procedure and PGD. First polar bodies biopsy with subsequent vitrification was uninformative due to meiotic crossing-over, so oocytes were inseminated after warming. Two embryos were obtained and blastomere biopsy was performed on day 3 with inconclusive results on their genetic status. Their culture resulted in one expanded blastocyst stage on day 7 that underwent trophectoderm biopsy and vitrification. This embryo showed to be normal. It was then warmed and transferred in an artificial cycle.Discussion and EvaluationPreconception genetic analysis by removal and analysis of the first polar body is technically possible, but the genetic information that we can obtain at this stage may be limited and the oocytes to be inseminated is not predictable. Compared to blastomere biopsy, trophectoderm biopsy has more diagnostic efficiency with respect to both chromosomal mosaicism and PCR accuracy, reducing the problems of amplification failure and allele drop out. Moreover, embryos biopsied at the cleavage stage seem to have lower implantation rate than biopsied blastocyst.ConclusionsThis is the first case report of a live birth obtained from a three step biopsy and double vitrification procedures of a blastocyst. This case report seems also to suggest the harmlessness of all these procedures if carefully performed by a skilled biologist in an IVF lab with quality management system. Finally, our study highlight that blastocyst cryopreserved on day 7 have clinically important potential and embryos that not reach blastocyst stage on day 6 should not to be discharged because they may result in an ongoing pregnancy.

The cryoaugmentation effect from 1017 autologous fresh elective single blastocyst transfers and 320 vitrified/warmed blastocyst transfers

Throughout the three-decade history of in-vitro-fertilization (IVF) treatment, elective single embryo transfer (eSET) has been the exception, rather than norm. To compensate the low rate of implantation for individual embryos and to achieve pregnancy rates higher than 50%, the general practice is to systematically transfer multiple embryos in the vast majority of patients. Because eSET is the only truly effective means by which to avoid multiple pregnancy in IVF cycles, we looked at our eSET program implemented in 2007 and the cumulative pregnancy rate after transfer of fresh and vitrified-warmed blastocysts (VET) in patients treated with eSET, to evaluate the total potential of a stimulated cycle.

Embryo vitrification using a novel semi-automated closed system yields in vitro outcomes equivalent to the manual Cryotop method.

Can the equilibration steps prior to embryo vitrification be automated? We have developed the 'Gavi' system which automatically performs equilibration steps before closed system vitrification on up to four embryos at a time and gives in vitro outcomes equivalent to the manual Cryotop method. Embryo cryopreservation is an essential component of a successful assisted reproduction clinic, with vitrification providing excellent embryo survival and pregnancy outcomes. However, vitrification is a manual, labour-intensive and highly skilled procedure, and results can vary between embryologists and clinics. A closed system whereby the embryo does not come in direct contact with liquid nitrogen is preferred by many clinics and is a regulatory requirement in some countries. The Gavi system, an automation instrument with a novel closed system device, was used to equilibrate embryos prior to vitrification. Outcomes for embryos automatically processed with the Gavi system were compared with those processed with the manual Cryotop method and with fresh (non-vitrified) controls. The efficacy of the Gavi system (Alpha model) was assessed for mouse (Quackenbush Swiss and F1 C57BL/6J x CBA) zygotes, cleavage stage embryos and blastocysts, and for donated human vitrified-warmed blastocysts. The main outcomes assessed included recovery, survival and in vitro embryo development after vitrification-warming. Cooling and warming rates were measured using a thermocouple probe. Mouse embryos vitrified after processing with the automated Gavi system achieved equivalent in vitro outcomes to that of Cryotop controls. For example, for mouse blastocysts both the Gavi system (n = 176) and manual Cryotop method (n = 172) gave a 99% recovery rate, of which 54 and 50%, respectively, progressed to fully hatched blastocysts 48 h after warming. The outcomes for human blastocysts processed with the Gavi system (n = 23) were also equivalent to Cryotop controls (n = 13) including 100% recovery for both groups, of which 17 and 15%, respectively, progressed to fully hatched blastocysts 48 h after warming. The cooling and warming rates achieved with the Gavi system were 14 136°C/min and 11 239°C/min, respectively. Testing of the Gavi system described here was limited to in vitro development of embryos from two mouse strains and a limited number of human embryos. Validation of Gavi system advanced production models is now required to confirm the success of semi-automated vitrification, including clinical evaluation of pregnancy outcomes from the transfer of Gavi vitrified-warmed human embryos. The Gavi system has the potential to revolutionize and standardize vitrification of embryos and oocytes. The success of the Gavi system shows that it is possible to semi-automate complicated labour-intensive ART methods and processes, and opens up the possibility for further improvements in clinical outcomes and efficiencies in the ART clinic. This study was funded by Genea Ltd. S.B., N.M.T., T.T.P., S.J.M., M.C.B. and T.S. are shareholders of Genea Ltd. E.V., C.H., C.L., S.R.L. and S.M.D. are shareholders of Planet Innovation Pty Ltd. The remaining authors are employees of either Genea Ltd. or Planet Innovation Pty Ltd.

Trophectoderm morphology predicts outcomes of pregnancy in vitrified-warmed single-blastocyst transfer cycle in a Chinese population.

In this study, we estimated the effect of blastocoele expansion, ICM and TE quality after warming and culture on the rates of clinical pregnancy, live birth and miscarriage in vitrified-warmed single-blastocyst transfer cycle in a Chinese population. A retrospective analysis of 263 cycles of vitrified-warmed single-blastocyst transfers was performed. The blastocysts with higher TE grade significantly increased the rates of clinical pregnancy (OR = 0.59, 95 % CI, 0.35-0.99, P = 0.045, grade (A + B) vs grade C) and live birth (OR = 0.55, 95 % CI, 0.32-0.94, P = 0.029, grade (A + B) vs grade C). And the association between TE grade and the rate of live birth didn't change after the number of repeated cycles was adjusted (OR = 0.55, 95 % CI, 0.32-0.95, P = 0.033, grade (A + B) vs grade C). The number of repeated cycles was a confounding factor significantly different between the live birth and no live birth groups. By contrast, neither blastocoele expansion nor inner cell mass was statistically related to the rates of clinical pregnancy, live birth and miscarriage. Our data firstly provided the evidence that TE grading, but not ICM grading, was significantly associated with the clinical pregnancy rate and live birth rate in vitrified-warmed blastocyst transfer cycles in a Chinese population. TE morphology may help predict outcomes of pregnancy in single-blastocyst transfer.

Women's age and embryo developmental speed accurately predict clinical pregnancy after single vitrified-warmed blastocyst transfer

The aim of this study was to establish a simple, objective blastocyst grading system using women's age and embryo developmental speed to predict clinical pregnancy after single vitrified-warmed blastocyst transfer. A 6-year retrospective cohort study was conducted in a private infertility centre. A total of 7341 single vitrified-armed blastocyst transfer cycles were included, divided into those carried out between 2006 and 2011 (6046 cycles) and 2012 (1295 cycles). Clinical pregnancy rate, ongoing pregnancy rate and delivery rates were stratified by women's age (<35, 35–37, 38–39, 40–41, 42–45 years) and time to blastocyst expansion (<120, 120–129, 130–139, 140–149, >149 h) as embryo developmental speed. In all the age groups, clinical pregnancy rate, ongoing pregnancy rate and delivery rates decreased as the embryo developmental speed decreased (P < 0.0001). A simple five-grade score based on women's age and embryo developmental speed was determined by actual clinical pregnancy rates observed in the 2006–2011 cohort. Subsequently, the novel grading score was validated in the 2012 cohort (1295 cycles), finding an excellent association. In conclusion, we established a novel blastocyst grading system using women's age and embryo developmental speed as objective parameters.

Maternal age and initial β-hCG levels predict pregnancy outcome after single vitrified-warmed blastocyst transfer.

We retrospectively examined a large cohort of females who underwent single blastocyst transfer to determine if initial β-human chorionic gonadotrophin (β-hCG) levels on day 7 after single vitrified-warmed blastocyst transfer (SVBT) could be used to predict pregnancy outcome. The treatment cycles that gave rise to the early pregnancies included in this study were performed from 2004 to 2011 in a private infertility center. In SVBT cycles, embryos were transferred during a natural cycle or after endometrial preparation with exogenous estrogen and progesterone. A total of 11,458 cycles with β-hCG levels ≥1.0 UI/ml on day 7 after SVBT were evaluated. The proportion of live births per positive β-hCG cycle was established for 10 β-hCG ranges in 3 different age groups (Group A: 21-34 years old; Group B: 35-39 years old; Group C: 40-44 years old). The proportion of live births gradually increased from 1.5 to 93.7%, 0.8 to 87.9%, and 0.6 to 76.2% in Groups A, B, and C, respectively. For each range of β-hCG levels, the proportion of live births was higher for the younger age group, which reflected the increased risk of early pregnancy loss with advancing female age. β-hCG levels on day 7 after SVBT, in conjunction with maternal age, may be used to predict pregnancy outcomes.

Possible selection of viable human blastocysts after vitrification by monitoring morphological changes.

Morphological assessment of human blastocysts has been effective for selecting embryos with high potential. However, they often show repeated shrinkage and expansion toward their hatching. Here we assessed whether capturing morphological changes over time of vitrified-warmed blastocysts could lead to a better selection of viable embryos from shrunken blastocysts. The implantation rates of vitrified-warmed blastocysts that were shrunken or expanded (developing) at the time of loading for transfer were compared among 2,729cycles that were subjected to single blastocyst transfer. Vitrified (107) and fresh blastocysts (17) were donated for the experimental study. To assess the relationship between morphology (expanded vs. shrunken) and the mitochondrial respiration of blastocysts, the oxygen consumption rate (OCR) was analyzed for 55 specimens using an uncoupler of oxidative phosphorylation. The remaining 69 blastocysts were used for recording morphological changes every 15min for 48h after warming. Because there were no surplus embryos, 7% of the vitrified-warmed blastocysts were shrunken and transferred. The shrunken embryos had sufficient implantation ability (40%). The OCR of the shrunken embryos was significantly lower than that of their expanded counterparts. Upon exposure to the uncoupler, the OCR of some shrunken embryos increased to levels similar to the expanded specimens. Time-lapse images revealed some shrunken embryos which formed blastocoel by 5h following warming exhibited developmental competence to the hatched stage. Data of the present study suggest a group of shrunken blastocysts contains many viable and clinically available embryos and time-lapse observation of vitrified-warmed blastocysts is a potential method to distinguish viable embryos from shrunken blastocysts.

Single-embryo transfer of vitrified-warmed blastocysts yields equivalent live-birth rates and improved neonatal outcomes compared with fresh transfers

To compare pregnancy and neonatal outcomes after fresh and vitrified-warmed single-blastocyst transfers. Retrospective study. Private in vitro fertilization (IVF) clinic. 1,209 infertile patients who underwent a total of 1,157 fresh and 645 vitrified-warmed embryo transfers. Day-5 single-blastocyst transfers using fresh or vitrified-warmed (Cryotop method) grade I and grade II embryos. Fetal heart pregnancy rate, live-birth rate, gestational age, and live-birth weight. The overall blastocyst thaw survival rate was 94.4% and was not significantly different between blastocyst grades or developmental stages. Similar clinical outcomes were achieved for fresh and vitrified-warmed blastocyst transfers; for example, grade I blastocysts had a live-birth rate of 52.8% versus 55.3%, respectively, and grade II blastocysts had a rate of 34.9% versus 30.4%, respectively. Significantly improved neonatal outcomes were evident for vitrified-warmed blastocyst transfers for gestational age, being on average 0.3 weeks longer, and for live-birth weight with babies born on average 145 g heavier (3,296 g versus 3,441 g for fresh and vitrified-warmed groups, respectively), as compared with fresh transfers. Embryo transfer of vitrified-warmed blastocysts yields equivalent live-birth rates and improved neonatal outcomes compared with fresh transfers.

Neonatal outcomes after the transfer of vitrified blastocysts: closed versus open vitrification system

BackgroundIncreasing evidence indicates that closed vitrification has been successfully used in the cryopreservation of human oocytes and embryos. Little information is available regarding the neonatal outcome of closed blastocysts vitrification. The aim of this study was to evaluate the effectiveness and safety of blastocyst vitrification using a high-security closed vitrification system compared with an open vitrification system.MethodsA total of 332 vitrified-warmed blastocyst transfer cycles between April 2010 and May 2012 were analyzed retrospectively. The post-thaw survival rate, implantation rate, clinical pregnancy rate, live birth rate, and neonatal outcome were recorded.ResultsThere were no significant differences between the open vitrification group and the close vitrification group regarding the post-thaw survival rate (98% versus 95.8%), clinical pregnancy rate (47.6% versus 42.2%), implantation rate (42.9% versus 35.6%), and live birth rate (39.8% versus 32.1%). In total, 332 warming cycles produced 131 healthy babies. There were no significant differences in the mean gestational age, the birth weight, and the birth length between the two groups. No adverse neonatal outcomes were observed in the children born after the transfer of closed vitrified blastocysts compared with the transfer of open vitrified blastocysts.ConclusionsThese data suggest that blastocyst vitrification using a closed vitrification device seems safe and effective with results comparable to those obtained through open vitrification.

Human chorionic gonadotropin levels are equally predictive for pregnancy outcome after fresh and vitrified-warmed blastocyst transfer

The aim of the present study was to evaluate if the live birth predictive values of β-hCG levels differ in fresh and vitrified-warmed blastocyst transfer cycles. In the retrospectively designed study, 775 cycles with positive β-hCG values 13 days after fresh blastocyst transfer (fresh ET; n = 568) or vitrified-warmed blastocyst transfer (FET; n = 207) were selected for analysis. Average β-hCG levels stratified according to pregnancy outcome (biochemical pregnancy, spontaneous abortion, ectopic pregnancy, and singleton or twin birth) were compared between fresh ET and FET cycles. To determine the optimal sensitivity and specificity of β-hCG levels for live birth prediction, a ROC curve was constructed. Fisher's exact test was used to compare the positive predictive values (PPV). Average β-hCG levels stratified according to pregnancy outcome were not statistically different between fresh ET and FET cycles. In fresh ET and in FET group, the β-hCG levels were significantly higher in pregnancies resulting in live birth compared to non-viable pregnancies (1,035 vs. 462 IU/L, p < 0.001 and 968 vs. 411 IU/L, p < 0.001). Optimal cut-off level for live birth prediction was 495 IU/L (sensitivity 83.0 %, specificity 71.8 %) after ET and 527 IU/L (sensitivity 80.0 % and specificity 76.6 %) after FET. The PPV for live birth rate in the groups after ET and FET were 90.6 % and 84.9 % respectively, without statistically significant difference (p > 0.05). Beta-hCG levels after fresh and vitrified-warmed blastocyst transfer are equally predictive for pregnancy outcome. Clinicians can be encouraged to interpret β-hCG results in the same manner.

Elective single cryopreserved embryo transfer of blastocysts cultured in defined media with recombinant human albumin: a randomized study

Human serum albumin (HSA), a commonly used protein source for ART, may cause biological variation and disease transmission. Recombinant human albumin (recHA) could reduce these risks; however, existing literature on the use of recHA is limited. Here, we evaluated the efficacy of chemically defined media containing recHA for blastocyst culture of human embryos. Prospective randomized study. This study included patients from our center undergoing elective cryopreservation of all embryos. Ninety-five patients received IVF/ICSI treatment between July 2012 and October 2012, which resulted in ≥6 2PN embryos 18 h after insemination. They were randomized using sealed envelopes into 2 groups for embryo culture in G1/G2 media (Vitrolife) containing either 5 mg/mL HSA (standard commercial media, group A) or 0.5 mg/mL recHA (group B). We used the same recHA concentration used in our previous study with bovine embryos (Murakami et al., 2012). After culture, only good-quality embryos were vitrified by day 6. We evaluated the clinical data for patients with vitrified blastocysts in groups A (n = 35) and B (n = 33). Patient age (years) was similar for groups A and B (35.5 [3.3] vs. 35.7 [3.7], P = 0.857). A total of 120 and 70 blastocysts were vitrified in groups A and B, respectively. By the end of March 2013, groups A and B underwent a total of 51 and 36 cycles of single ET with the vitrified-warmed blastocysts, respectively. All vitrified blastocysts survived after warming. Clinical pregnancy rate/ET was 24/51 (47.1%) for group A and 25/36 (69.4%) for group B (P = 0.049). Ongoing pregnancy rate/ET was 22/51 (43.1%) for group A and 19/36 (52.8%) for group B (P = 0.632). Culture of human embryos in the defined media with recHA can yield good-quality blastocysts, resulting in a high pregnancy rate after single cryopreserved ET. These results could help develop serum-free defined ART systems. We are currently evaluating the perinatal outcomes after ET with more participants.

Comparison of clinical outcomes between single and double vitrified-warmed blastocyst embryo transfer according to the day of vitrification.

To compare the efficacy of single vitrified-warmed blastocyst embryo transfer (SVBT) versus double vitrified-warmed blastocyst embryo transfer (DVBT) according to the day of vitrification. This retrospective study included a total of 1,051 cycles in women less than 37 years of age with their autologous SVBT cryopreserved on day 5 (5d-SVBT, n = 737) or day 6 (6d-SVBT, n = 154) and DVBT on day 5 (5d-DVBT, n = 129) or day 6 (6d-DVBT, n = 31) from January 2009 to December 2011. The clinical pregnancy rate (41.8 % vs. 48.1 %, p = 0.184) and ongoing pregnancy rate (36.6 % vs. 45.0 %, p = 0.072) were not significantly different between the 5d-SVBT group and the 5d-DVBT group. However, the clinical pregnancy (29.9 % vs. 58.1 %, p = 0.003) and ongoing pregnancy rates (23.4 % vs. 51.6 %, p = 0.001) were significantly lower in the 6d-SVBT group compared with those in the 6d-DVBT group. The implantation rate (42.2 % vs. 34.5 %, p = 0.03) of the 5d-SVBT group was significantly higher than that of the 5d-DVBT group, while the implantation rate (29.9 % vs. 37.1 %, p = 0.303) of the 6d-SVBT group was not statistically different compared with that in the 6d-DVBT group. The multiple pregnancy rates (1.0 % in the 5d-SVBT group vs. 38.7 % in the 5d-DVBT group, p < 0.001 and 0 % in the 6d-SVBT group vs. 22.2 % in the 6d-DVBT group, p = 0.001) were statistically significantly lower in the SVBT group compared with those in the DVBT group regardless of the day of vitrification. This study showed that the 5d-SVBT resulted in comparable clinical outcomes compared to the 5d-DVBT while the 6d-SVBT yielded significantly lower clinical outcomes compared to the 6d-DVBT.

Effect of the site of assisted hatching on vitrified-warmed blastocyst transfer cycles: a prospective randomized study

To assess the effect of assisted hatching (AH) site on the clinical outcomes in vitrified-warmed blastocyst transfer cycles. A total of 160 women who underwent vitrified-warmed blastocyst transfer cycles were randomized to either the ICM group (AH performing at the site near the inner cell mess, ICM), or the TE group (AH performing at the site opposite to the ICM). AH with laser zona drilling was performed 20 or 30 min after thawing once the ICM can be detected. Clinical pregnancy rate, implantation rate, live birth rate and the occurrence rate of monozygotic twins (MZT) pregnancy after transfer of these two groups were compared. No significant difference was found in the clinical pregnancy rate (63.8% vs. 67.5%), implantation rate (51.7% vs. 53.6%) and live birth rate (57.5% vs. 62.5%) between the ICM group and the TE group. The occurrence rate of MZT was comparable between the two groups (3.9% vs. 5.6%). The site of assisted hatching has no influence on the implantation, pregnancy and live birth rate in human vitrified-warmed blastocyst transfer cycles.

Growth retardation in human blastocysts increases the incidence of abnormal spindles and decreases implantation potential after vitrification

Does the human embryo growth rate affect the outcome of vitrified-warmed blastocyst transfer? Following vitrification, the incidence of abnormal spindle morphology was increased and the implantation competence was decreased in growth-retarded embryos compared with normally developing embryos. Various types of spindle abnormality occur in human cleavage- and blastocyst-stage embryos. However, the incidence of abnormal spindle morphology in growth-retarded blastocysts is not known. Furthermore, there is conflicting data about the implantation potential of such blastocysts. This was a retrospective cohort study including 878 single vitrified-warmed blastocyst transfers between 9 January 2010 and 10 July 2012, and an experimental study using 121 vitrified-warmed blastocysts donated to research. A comparison on the implantation potential and spindle shape of vitrified-warmed blastocysts was made between normally developing and growth-retarded blastocysts. In the clinical study, we compared the implantation rates of vitrified-warmed embryos that developed to the blastocyst stage on Day 5 after insemination (normally developing embryos) with those that required culture to Day 6 (growth-retarded embryo). In the experimental study, donated vitrified-warmed blastocysts were immunostained with an anti-α-tubulin antibody to visualize microtubules, an anti-γ-tubulin antibody to image centrosomes and Hoechst 33342 or 4,6-diamidino-2-phenylindole to visualize DNA. Confocal image analysis captured a z-series stack of 0.5-µm-thick optical sections encompassing the entire blastocyst. Only spindles with fusiform poles and with chromosomes aligned at the equator were classified as normal. The implantation rate of growth-retarded embryos (47%, n = 270) was significantly lower (P < 0.05) than that of normally developing embryos (57%, n = 608). A total of 533 spindles were analyzed in Day 5 and 6 vitrified-warmed blastocysts. The incidence of abnormal spindles in the growth-retarded embryos (47%, n = 274) was significantly higher (P < 0.01) than in the normally developing embryos (30%, n = 259). Further studies are required to clarify the link between an increase in abnormal spindle formation and a decrease in embryonic implantation potential. This study provided new insights into the possible implications of abnormalities in spindle formation in growth-retarded human blastocysts.

A closed system supports the developmental competence of human embryos after vitrification : Closed vitrification of human embryos.

Closed-system vitrification may enable the risk of contamination to be minimised. We performed three studies to compare the developmental competence of human embryos vitrified using either a closed vitrification system (CVS; Rapid-i®) or an open vitrification system (OVS; Cryo-top®). The first study was performed in vitro using 66 zygotes previously vitrified at pronuclear stage. These were warmed and randomised 1:1 to revitrification using either the OVS or the CVS. After re-warming, embryo development and blastocyst cell number were assessed. For the second study, also performed in vitro, 60 vitrified-warmed blastocysts were randomised 1:1:1 into three groups (OVS or CVS revitrification, or no revitrification). The proportion of dead cells was assessed by staining. The third study was performed in vivo, using 263 high-grade blastocysts randomly assigned to vitrification using either the CVS (n = 100) or the OVS (n = 163). After warming, single blastocyst transfer was performed. There were no differences between the CVS and the OVS in survival rate (100% vs. 97%), blastulation rate (96h: 50% vs. 50%; 120h: 68% vs. 56%), proportion of good blastocysts (96h: 32% vs. 22%, 120h: 47% vs. 41%), or mean number of cells (137 vs. 138). The proportion of dead cells in blastocysts re-vitrified by CVS (31%) was similar to that for OVS (38%) and non-revitrification (32%). In vivo, the implantation rate for blastocysts vitrified using the CVS (54%) was similar to that with the OVS (53%). Our studies consistently indicate that human embryos may be vitrified using a CVS without impairment of developmental competence.

Clinical outcomes of vitrified-thawed embryo transfer using a pull and cut straw method

ObjectiveTo compare the clinical outcomes of patients with vitrified-thawed embryos transferred using either the 0.25 mL straw method and the pull and cut straw (PNC) method. To evaluate the clinical outcomes of patients with transferred embryos that underwent assisted hatching at the cleaved embryo (day 3) or the blastocyst (day 5) stage.MethodsThe study population consisted of women who underwent vitrified-warmed embryo transfer between May 2000 and December 2011 and assisted hatching was performed after warming of embryos. Cycles of thawing between assisted hatching treated and non treated groups were compared for survival and pregnancy rates.ResultsThe PNC vitrification method improved survival and pregnancy rates in partial lysed embryos. While assisted hatching did not affect the developmental and clinical pregnancy rates of the vitrified-warmed blastocyst group, it did increase the pregnancy rate of poor quality vitrified-warmed cleaved embryos.ConclusionThese results suggest that PNC may increase the number of clinical pregnancies via the vitrification of both cleaved embryos and blastocysts. In addition, selective assisted hatching treatment of embryos that show a poor prognosis after warming may increase the rate of clinical pregnancy.

Effect of MEM vitamins and forskolin on embryo development and vitrification tolerance of in vitro-produced pig embryos

The aims of this study were (1) to determine the effect of in vitro maturation (IVM) medium supplementation with MEM vitamins on in vitro embryo development and sensitivity to vitrification of Day 6 blastocysts and (2) to evaluate whether the addition of forskolin to in vitro culture (IVC) medium enhances blastocyst survival following Super Open Pulled Straw (SOPS) vitrification. Cumulus–oocyte complexes (COCs; n=4000) were matured with 0.0% or 0.05% (v/v) MEM vitamins. After 44h of IVM, the oocytes were in vitro fertilized, and presumptive zygotes were cultured. At Day 5 of IVC, embryos from both experimental groups were cultured for 24h with 0 or 10μM forskolin, achieving a 2×2 factorial design. The blastocyst formation rate was assessed on Day 6, and subsets of samples from the four experimental groups were vitrified (n=469) or kept fresh (n=546). Fresh and vitrified-warmed blastocysts were cultured for 24h prior to embryo survival and total blastocyst cell number assessment. The MEM vitamins increased (P<0.001) the blastocyst formation rate at Day 6, but they did not affect embryo survival after vitrification. In contrast, the addition of forskolin to the culture medium enhanced (P<0.05) the blastocyst vitrification tolerance. The total blastocyst cell number was similar among the groups. In conclusion, supplementation with 0.05% MEM vitamins improved the blastocyst formation rate, and the addition of 10μM forskolin to the culture medium increased survival in Day 6 in vitro-produced blastocysts after SOPS vitrification.

A closed vitrification system enables a safe and an aseptic vitrification without impairing the developmental competence of human embryos

To avoid the risk of contamination by bacteria, virus and prions, we compared the developmental competence of human embryos vitrified using a closed vitrification system (CVS) with that using an open vitrification system (OVS). Prospective randomized human study. This study was approved by a local IRB of IVF Namba Clinic. In experiment 1 and 2, vitrified embryos donated from patients who completed fertility treatment and gave informed consent were used. In experiment 1, 66 embryos at pronuclear stage were divided randomly into 2 groups after warming: CVS (Rapid-I, Vitrolife) and OVS (Cryotop, Kitazato medical) and were re-vitrified using CVS or OVS. After warming, embryos development and blastocysts cell number were assessed. In experiment 2, 60 vitrified-warmed blastocysts were divided randomly into 3 groups (CVS, OVS, and non-revitrified) and were assessed the proportion of dead cell by staining with Hoechst and Propidium iodide. In experiment 3, 27 high grade blastocysts were randomly divided into two groups and were vitrified using CVS (14) and OVS (13). After warming, single blastocyst transfer was performed. There were no differences between CVS and OVS in the survival rates (100% vs. 100%, respectively), the blastulation rates (Day 5: 50% vs. 50%; Day 6: 68% vs. 56%, respectively), the rate of good blastocyst (Day 5: 32% vs. 22%, Day 6: 47% vs. 41%, respectively), and the mean cell numbers (137 vs. 138) in experiment 1. In experiment 2, the proportion of dead cells in re-vitrified blastocysts were similar at 31%, 38% and 32% in the groups CVS, OVS and non-revitrified respectively. In experiment 3, the implantation rate of blastocysts vitrified using CVS (73%) was comparable to that for the OVS group (54%). The closed vitrification system overcomes the concerns associated with direct liquid nitrogen contact in the open system without impairing the developmental competence of human embryos.