Birth of a healthy baby following vitrification of human blastocysts

OBJECTIVE: To assess vitrification of human blastocysts. DESIGN: Retrospective study of blastocyst vitrification. SETTING: A private clinic. PATIENT(S): Twenty couples with different types of infertility. INTERVENTION(S): Blastocysts were frozen with rapid vitrification and then transferred after thawing. We vitrified blastocysts using a modification of Ishimori's vitrification solution of ethylene glycol and dimethyl sulfoxide (VSED). MAIN OUTCOME MEASURE(S): After thawing, survival was defined by the embryo's development morphology after 6 hours or overnight culture. RESULT(S): Eighteen of 20 patients underwent treatment. Of 45 vitrified blastocysts, 36 survived, for a survival rate of 80% (36 of 45). The implantation rate was 21.9% (7 of 32), and the pregnancy rate (per embryo transfer cycle) was 33.3% (6 of 18). One of the pregnancies resulted in the delivery of a healthy baby. CONCLUSION(S): Supernumerary embryos were grown in culture to blastocysts, and the survival rate of vitrified-thawed blastocysts was the same as that for slow freezing of early stage embryos. Blastocyst vitrification should prove effective for clinical treatment. The present results strongly suggest that this rapid and successful vitrification procedure will replace conventional cryopreservation in the future.     

Large-volume vitrification of human biopsied and non-biopsied blastocysts: a simple,robust technique for cryopreservation

Purpose

To evaluate the transition from a proven slow-cooling cryopreservation method to a commercial large-volume vitrification system for human blastocysts.

Methods

Retrospective analysis of de-identified laboratory and clinical data from January 2012 to present date for all frozen embryo replacement (FET) cycles was undertaken. Cryopreservation of trophectoderm-biopsied or non-biopsied blastocysts utilized during this time period was logged as either slow-cooling, small-volume vitrification, or large-volume vitrification. Blastocyst survival post-warm or post-thaw, clinical pregnancy following FET, and implantation rates were identified for each respective cryopreservation method.

Results

Embryo survival was highest for large-volume vitrification compared to micro-volume vitrification and slow-cooling; 187/193 (96.9 %), 27/32 (84.4 %), and 244/272 (89.7 %), respectively. Survival of biopsied and non-biopsied blastocysts vitrified using the large-volume system was 105/109 (96.3 %) and 82/84 (97.6 %), respectively. Survival for micro-volume biopsied and non-biopsied blastocysts was 16/30 (83.3 %) and 2/2 (100.0 %) respectively. Slow-cooling post-thaw embryo survival was 272/244 (89.7 %). Clinical pregnancy and implantation rates outcomes for non-biopsied embryos were similar between large-volume and slow-cooling cryopreservation methods, 18/39 (46.2 %) clinical pregnancy and 24/82 (29.3 %) implantation/embryo, and 52/116 (44.8 %) clinical pregnancy and 67/244 (27.5 %) implantation/embryo, respectively. Comparing outcomes for biopsied embryos, clinical pregnancy and implantation rates were 39/67 (58.2 %) clinical pregnancy and 50/105 (47.6 %) implantation/embryo and 4/16 (25 %) clinical pregnancy and 6/25 (24.0 %) implantation/embryo, respectively.

Conclusions

The LifeGlobal large-volume vitrification system proved to be very reliable, simple to learn and implement in the laboratory. Clinically large-volume vitrification was as, or more effective compared to slow-cooling cryopreservation in terms of recovery of viable embryos in this laboratory.     

Survival,re-expansion and cell survival of human blastocysts following vitrification and warming using two vitrification systems

Purpose

This study evaluated and compared survival, re-expansion, and percentage of live cells of individual Days 5 and 6 human blastocysts that were vitrified and warmed with the Vit Kit Freeze/Thaw (Irvine Scientific, CA), or with two protocols using the Global Fast Freeze/Thaw Kits (LifeGlobal, Canada).

Methods

Frozen/thawed Day 2–3 or discarded embryos were cultured to blastocyst (culture day 5–6). Group 1 blastocysts were vitrified with the Vit Kit (n?=?29) and High Security Vitrification (HSV) devices. Group 2 (n?=?47) and Group 3 (n?=?48) blastocysts were cryopreserved with the Global Fast Freeze Kit and 0.25 ml straws, using a direct plunge or a ?100 °C holding step, respectively. Group 4 (Controls, n?=?30) were not vitrified. Blastocysts were subsequently cultured for 24 h, assessed for survival and expansion, and then stained individually with propidium iodide and Hoechst. Live and total cell number was assessed with ImageJ (NIH), and the percentage of live cells calculated for each blastocyst.

Results

The percentage of live cells was not different between vitrified and control (non-vitrified) blastocysts, thus vitrification did not affect cell survival. Survival (following thawing and after 24 h culture), re-expansion, and percentage of live cells were not different for blastocysts vitrified and warmed between the two vitrification/warming kits, or between the two protocols for the Global Fast Freeze/Thaw Kits.

Conclusions

Blastocyst vitrification can be achieved with equal success using simplified protocols and cheaper and easy to load freezing straws, providing simultaneously increased safety, and efficiency with lower cost, when compared with vitrification using specialized embryo vitrification devices.

     

Shortened equilibration time can compromise clinical outcomes in human embryo vitrification

Vitrification is an important way to cryopreserve human embryos and the recommended time of embryo exposure to the vitrification solution is 1?min. However, practically speaking, the duration of embryos exposure to equilibration solution can vary from 5 to 15?min. The purpose of this study was to investigate the effect of different equilibration times on the outcomes of frozen-thawed embryo transfer cycles. The data were collected from our medical records from January 2012 to June 2013 and a total of 517 cycles were included. These cycles were divided into four groups according to the equilibration time: (i) 5–6?min; (ii) 7–8?min; (iii) 9–10?min and (iv) 11–12?min. The results show that there were no differences in terms of survival rate and fully intact embryo rate among the four groups. However, lower clinical pregnancy, embryo implantation and live birth rates were observed in the 5–6?min exposure group (54.6%, 31.9% and 48.2%, respectively) compared with the three other groups. The corresponding rates in the 9–10?min group (73.5%, 47.6% and 64.7%) were the highest. This study indicated that different equilibration times influenced the clinical outcomes of human embryo vitrification and vitrification with shortened equilibration time compromised the clinical outcomes. Appropriate prolongation of the equilibrium time would probably improve the clinical outcomes.     

Cryopreservation of human oocytes,zygotes, embryos and blastocysts: A comparison study between slow freezing and ultra rapid (vitrification) methods

Preservation of female genetics is currently done primarily by means of oocyte and embryo cryopreservation. The field has seen much progress during its four-decade history, progress driven predominantly by research in humans. It can also be done by preservation of ovarian tissue or entire ovary for transplantation, followed by oocyte harvesting or natural fertilization. Two basic cryopreservation techniques rule the field, slow-rate freezing, the first to be developed and vitrification which in recent years, has gained a foothold. The slow-rate freezing method previously reported had low survival and pregnancy rates, along with the high cost of cryopreservation. Although there are some recent data indicating better survival rates, cryopreservation by the slow freezing method has started to discontinue. Vitrification of human embryos, especially at early stages, became a more popular alternative to the slow rate freezing method due to reported comparable clinical and laboratory outcomes. In addition, vitrification is relatively simple, requires no expensive programmable freezing equipment, and uses a small amount of liquid nitrogen for freezing. Moreover, oocyte cryopreservation using vitrification has been proposed as a solution to maintain women’s fertility by serving and freezing their oocytes at the optimal time. The aim of this research is to compare slow freezing and vitrification in cryopreservation of oocytes, zygotes, embryos and blastocysts during the last twelve years. Therefore, due to a lot of controversies in this regard, we tried to achieve an exact idea about the subject and the best technique used.     

Modified vitrification of human pronuclear oocytes: efficacy and effect on ultrastructure

The efficacy of cryopreservation by direct plunging into liquid nitrogen (vitrification) of human pronuclear oocytes using open pulled straws with a super-finely pulled tip, as well as the ultrastructural changes caused by cooling and vitrification, were evaluated. Clinical and electron microscopic studies of cooled and vitrified oocytes were performed. Oocytes were cooled to 4 degrees C in the presence and absence of cryoprotectants, vitrified, warmed, cultured and transferred. Abnormally fertilized oocytes were examined by electron microscopy. Vitrified and warmed 2-pronuclear oocytes showed 71.1% survival rate and 83.3% developmental rate. One- and 3-pronuclear oocytes, after cooling without cryoprotectants (presumably non-viable), showed progressive swelling of mitochondrial smooth endoplasmic reticulum (SER). After vitrification, oocytes (presumably viable) showed the formation of large SER vesicles associated with mitochondria. The described protocol of vitrification of human pronuclear oocytes was shown to be effective in producing pregnancy. Normal ultrastructure after undergoing the described vitrification protocol was confirmed.     

Maturation outcomes are improved following Cryoleaf vitrification of immature human oocytes when compared to choline-based slow-freezing

Purpose  

The cryopreservation of immature oocytes permits oocyte banking for patients at risk of losing their fertility. However, the optimum protocol for such fertility preservation remains uncertain.     

Cryopreservation of immature and in-vitro matured human oocytes by vitrification

The objective of this study was to determine the efficacy of vitrification of human oocytes before and after in-vitro maturation (IVM). The immature oocytes recovered (n = 472) were divided into two groups: (i) immature oocytes (n = 219) vitrified at the germinal vesicle (GV) stage; and (ii) immature GV-stage oocytes (n = 253) that were firstly matured in vitro (MII-stage oocytes; n = 178), then vitrified (n = 79). The remaining oocytes (n = 99), which were not vitrified, were processed as controls. After warming, the oocyte survival, maturation and fertilization rates, as well as embryonic development, were compared. The results showed no significant difference between the survival rates of the oocytes vitrified at GV stage and those vitrified at MII stage (85.4% versus 86.1%). However, oocyte maturation rates were significantly reduced (P < 0.05) when oocytes were vitrified at immature GV stage followed by IVM (50.8%) in comparison with the control group (70.4%). Following insemination by intracytoplasmic sperm injection, there was no difference in the fertilization (62.1% versus 58.8%), cleavage (69.5% versus 67.5%) and blastocyst development (0.0% versus 0.0%) rates between these two groups. However, these results were significantly lower (P < 0.05) than those achieved in the control group. This suggests that better results can be achieved by vitrifying mature oocytes rather than immature oocytes.     

Highly efficient vitrification method for cryopreservation of human oocytes

Two experiments were performed to develop a method to cryopreserve MII human oocytes. In the first experiment, three vitrification methods were compared using bovine MII oocytes with regard to their developmental competence after cryopreservation: (i) vitrification within 0.25-ml plastic straws followed by in-straw dilution after warming (ISD method); (ii) vitrification in open-pulled straws (OPS method); and (iii) vitrification in <0.1 microl medium droplet on the surface of a specially constructed fine polypropylene strip attached to a plastic handle (Cryotop method). In the second experiment, the Cryotop method, which had yielded the best results, was used to vitrify human oocytes. Out of 64 vitrified oocytes, 58 (91%) exhibited normal morphology after warming. After intracytoplasmic sperm injection, 52 became fertilized, and 32 (50%) developed to the blastocyst stage in vitro. Analysis by fluorescence in-situ hybridization of five blastocysts showed that all were normal diploid embryos. Twenty-nine embryo transfers with a mean number of 2.2 embryos per transfer on days 2 and 5 resulted in 12 initial pregnancies, seven healthy babies and three ongoing pregnancies. The results suggest that vitrification using the Cryotop is the most efficient method for human oocyte cryopreservation.     

Vitrification of human hatched blastocysts: a report of 4 cases

BACKGROUND: Vitrification is a fairly well-established technique for cryopreservation of human zona-intact blastocysts. However, little is known about the efficacy of the vitrification technique for zona-free hatched blastocysts. CASE: A total of 4 hatched blastocysts from 4 healthy, infertile women undergoing in vitro fertilization were vitrified and warmed. All 4 hatched blastocysts expanded after warming and were transferred to 4 patients. Three of the women became clinically pregnant. Two healthy infants were born in 2 deliveries, and 1 pregnancy had progressed to 35 weeks at this writing. CONCLUSION: Our results suggest that vitrification is a useful cryopreservation technique not only for zona-intact blastocysts but also for zona-free hatched blastocysts.     

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

Purpose

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.

Methods

The implantation rates of vitrified–warmed blastocysts that were shrunken or expanded (developing) at the time of loading for transfer were compared among 2,729 cycles 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 15 min for 48 h after warming.

Results

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 5 h following warming exhibited developmental competence to the hatched stage.

Conclusions

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.     

玻璃化法冻融小鼠桑葚胚期和囊胚期胚胎的效果观察

目的 比较玻璃化法冻融小鼠桑葚胚期、囊胚早期和囊胚期胚胎后,胚胎存活及继续发育的能力。方法应用6 mol/L乙二醇和1 mol/L蔗糖的玻璃化冷冻液,冻融小鼠142个桑葚胚期、135个囊胚早期和148个囊胚期胚胎,观察冻融后胚胎存活及继续发育的情况。结果 小鼠桑葚胚期、囊胚早期和囊胚期胚胎的存活率分别为88.0％、73.3％和60.1％,囊胚孵出率分别为73.9%、61.5％和49.3％。桑葚胚期的胚胎存活率及囊胚孵出率均高于囊胚早期,囊胚早期高于囊胚期,各期胚胎存活率、囊胚孵出率比较,差异均有显著性(P<0.05)。结论 玻璃化法冻融桑葚胚期的效果好于囊胚早期及囊胚期,桑葚胚期是卵裂期后胚胎玻璃化冻融的最佳阶段。