Post-warming Survival Research Articles

Survival rate after vitrification of various stages of cat embryos and blastocyst with and without artificially collapsed blastocoel cavity.

Embryo vitrification is a modern technique for cryopreservation in assisted reproductive programs. From all the embryos, blastocysts are the most challenging during cryostorage due to their size, multicellular structure and the presence of blastocoelic fluid. The aim of this study was to evaluate the suitability for vitrification of various developmental stages of feline embryos and the influence of the artificial shrinkage (AS) of expanded blastocyst on post-vitrification survival rates. The AS procedure is the manual puncture of the trophectoderm allowing for the reduction of blastocoelic fluid prior to vitrification and thus preventing the ice crystal formation. The vitrified embryos were divided into groups of 2-cell, 4- to 8-cell, >8-cell, morulae, compacted and expanded blastocyst, based on morphological assessment and vitrified in groups of 1-3 embryos per Cryotop. The post-warming survival was similar regardless the embryo developmental stage prior vitrification; however, development to blastocysts was only noted in 4- to 8-cell and >8-cell vitrified embryos (13% and 27%, respectively). Following warming, the significantly more viable blastocysts were noted in vitrified compacted versus expanded blastocyst and in expanded blastocyst subjected to AS procedure versus expanded blastocyst without AS (total survival: 58.3% vs. 33.3% and 64.3% vs. 38.5%, respectively; re-expansion rate within 2hr post-warming: 41.7 vs. 6.7% and 50% vs. 7.7%, respectively). One-fifth of vitrified expanded blastocyst showed morphological damage immediately after warming procedure, whereas no visible damage was noted in compacted blastocyst and artificially collapsed expanded ones. The obtained results suggest that the most suitable for vitrification are feline embryos containing four to 16 blastomeres and compacted blastocyst. In addition, the reduction of blastocoel cavity in expanding blastocyst by artificial collapse improved the blastocyst vitrification outcome.

Higher pregnancy rates with zona-free euploid blastocysts

A small percentage of embryos undergoing assisted hatching on Day 3 for Day 5/6 biopsy will hatch completely prior to vitrification, thus resulting in zona pellucida (ZP)-free embryos. Prior studies comparing ZP free to ZP intact blastocyst transfers have found either no difference or only moderate improvement in pregnancy rates with the transfer of ZP-free embryos. No studies have explored the relationship between the loss of the ZP as a result of embryo biopsy and subsequent pregnancy outcomes. The objective of this study was to compare embryo survival, implantation rates and pregnancy rates of thawed, euploid-PGS embryos frozen with and without a zona. A retrospective cohort study Patients who underwent a single embryo transfer (SET) of a euploid embryo previously biopsied and vitrified with or without a ZP between Jan 2013 through Feb 2016 were included in the study. Embryos were laser hatched on day 3, biopsied and vitrified on day 5 or 6. Group 1 consisted of 11 patients (mean age=37, range 31-41) whose embryos were fully hatched with no ZP. Group 2 consisted of 193 patients (mean age=37, range 25-45) with an intact ZP. Post-warming survival, implantation and clinical pregnancy (heartbeat) rates were calculated. Two-sided Fisher Exact test was used. Statistical significance was defined at p<.05. Post warming survival rate was 100% for both groups. Implantation and pregnancy rates (IR and PR) for Group 1 were 81.8% (9/11). All pregnancies were singleton. IR and PR for Group 2 were 49.2% (95/193) and 47.7% (92/193) respectively. Group 2 had three sets of monozygotic twins (MZT). While both IR and PR were better in the ZP-free group, only PR reached statistical difference.Tabled 1Implantation Rate and Pregnancy Rate in Zona Free Group vs. Zona Intact GroupZona Free-Group 1 (n=11)Zona-Intact Group 2 (n=193)p-valueImplantation Rate81.8% (9/11)49.2% (95/193) 3xMZT.06Pregnancy Rate81.8% (9/11)47.7% (92/193).03 Open table in a new tab 1. ZP-free euploid blastocysts resulted in a significantly higher pregnancy rate compared to ZP-intact blastocysts.

Vitrification of in vitro matured oocytes collected from surplus ovarian medulla tissue resulting from fertility preservation of ovarian cortex tissue.

The aim of the study was to investigate the maturation rate of immature oocytes collected from ovarian medulla tissue normally discarded during preparation of ovarian cortical tissue for fertility preservation. Further we evaluated survival of derived MII oocytes following vitrification and warming. 36 patients aged from 8 to 41years who had one ovary excised for fertility preservation were included. Oocytes were collected from the medulla tissue and matured in vitro 44-48h followed by vitrification. Number of oocytes collected, the rates of maturation and post-warming survival were assessed. On average, 11 immature oocytes were collected per patient. The overall maturation rate was 29% irrespective of whether the ovary was transported 4-5h on ice or obtained immediately after oophorectomy. The maturation rate in patients below 20years of age (55%) was significantly higher than that of patients aged 20-30 years (29%) and above 30years (26%). The post-warming survival rate was 64%. No significant relationship was observed between the number of collected oocytes and the age of patients. Approximately three MII oocytes were obtained per patient following in vitro maturation (IVM) of immature oocytes collected from medulla tissue, of which two survived vitrification and warming. This approach represents an add-on method to potentially augment the fertility opportunity for cancer patients, especially in young women with cancer where transplantation of cortical tissue may pose a risk of relapse, but the IVM approach is currently too inefficient to be the only method used for fertility preservation.

Effect of cryopreservation technique and season on the survival of in vitro produced cattle embryos

Embryo cryopreservation is a major tool for conservation and propagation of genetically superior animals. However, it adversely affects the survival of embryos. The objective of this study was to determine the effects of cryopreservation technique (vitrification compared with slow freezing) and different seasons in which oocytes were obtained on the post-warming survival of in vitro produced (IVP) cattle morulae. In experiment 1, morulae (Day 6 post-IVF), obtained from abattoir-sourced oocytes during spring, summer, fall and winter over a period of 3.5 years, were subjected to either vitrification (n=271 morulae), slow freezing (n=281 morulae) or no freezing (control; n=249 morulae). After warming, the morulae were cultured to the expanded blastocyst stage (Day 8 post-IVF). Data were compared using Glimmix procedure in SAS®. Blastocyst rate differed (P<0.05) among the treatments: unfrozen control (78±3.6%), vitrification (52±4.6%) and slow freezing (35±4.2%). The re-expansion of vitrified morulae upon warming was not correlated with subsequent blastocyst rate (r=−0.048; P>0.05). The morulae produced during fall season had lesser (P<0.05) cleavage and morula rates (67±1.6%; Day 2 post-IVF and 22±1.4%; Day 6 post-IVF, respectively) than all other seasons (74±1.1 and 30±1.2%, respectively). Blastocyst rate was the least (P<0.05) when oocytes were collected during the summer season in both control and slowly frozen groups. Blastocyst development rate did not change due to season in vitrification group (P>0.05). In conclusion, vitrification is a more desirable technique than slow freezing for cryopreservation of IVP cattle morulae. If the slow freezing method is employed, greater success can be achieved using oocytes collected in the winter and spring with a primary contributing factor being lesser morulae development if oocytes are collected in the fall and also the lesser blastocyst formation of cryopreserved morulae when oocytes are collected in the summer.

Successful elective and medically indicated oocyte vitrification and warming for autologous in vitro fertilization, with predicted birth probabilities for fertility preservation according to number of cryopreserved oocytes and age at retrieval

To evaluate a single treatment center's experience with autologous IVF using vitrified and warmed oocytes, including fertilization, embryonic development, pregnancy, and birth outcomes, and to estimate the likelihood of live birth of at least one, two, or three children according to the number of mature oocytes cryopreserved by elective fertility preservation patients. Retrospective cohort study. Private practice clinic. Women undergoing autologous IVF treatment using vitrified and warmed oocytes. Indications for oocyte vitrification included elective fertility preservation, desire to limit the number of oocytes inseminated and embryos created, and lack of available sperm on the day of oocyte retrieval. Oocyte vitrification, warming, and subsequent IVF treatment. Post-warming survival, fertilization, implantation, clinical pregnancy, and live birth rates. A total of 1,283 vitrified oocytes were warmed for 128 autologous IVF treatment cycles. Postthaw survival, fertilization, implantation, and birth rates were all comparable for the different oocyte cryopreservation indications; fertilization rates were also comparable to fresh autologous intracytoplasmic sperm injection cycles (70% vs. 72%). Implantation rates per embryo transferred (43% vs. 35%) and clinical pregnancy rates per transfer (57% vs. 44%) were significantly higher with vitrified-warmed compared with fresh oocytes. However, there was no statistically significant difference in live birth/ongoing pregnancy (39% vs. 35%). The overall vitrified-warmed oocyte to live born child efficiency was 6.4%. Treatment outcomes using autologous oocyte vitrification and warming are as good as cycles using fresh oocytes. These results are especially reassuring for infertile patients who must cryopreserve oocytes owing to unavailability of sperm or who wish to limit the number of oocytes inseminated. Age-associated estimates of oocyte to live-born child efficiencies are particularly useful in providing more explicit expectations regarding potential births for elective oocyte cryopreservation.

The effects of season, medium storage at 4°C and sample storage in liquid nitrogen on post-warming survival of vitrified porcine oocytes

The effects of season, medium storage at 4°C and sample storage in liquid nitrogen on post-warming survival of vitrified porcine oocytes

A randomized controlled trial comparing two vitrification methods versus slow-freezing for cryopreservation of human cleavage stage embryos

PurposeTo compare two different vitrification methods to slow freezing method for cryopreservation of human cleavage stage embryos. Design: Prospective randomised trial. Setting: University assisted reproduction centre. Patient(s): 568 patients (mean age 33.4 ± 5.2) from April 2009 to April 2011.Methods1798 supernumerary good-quality cleavage stage embryos in 645 IVF cycles intended to be cryopreserved were randomly allocated to three groups: slow freezing, vitrification with the Irvine® method, vitrification with the Vitrolife® method. Main Outcome Measure(s): Embryo survival and cleavage rates, implantation rate.ResultsA total of 1055 embryos were warmed, 836 (79.2 %) survived and 676 were finally transferred (64.1 %). Post-warming embryos survival rate was significantly higher after vitrification (Irvine: 89.4 %; Vitrolife: 87.6 %) than after slow freezing (63.8 %) (p < 0.001). No differences in survival rates were observed between the two vitrification methods, but a significant higher cleavage rate was observed using Irvine compared to Vitrolife method (p < 0.05). Implantation rate (IR) per embryo replaced and per embryo warmed were respectively 15.8 % (41/259) and 12.4 % (41/330) for Irvine, 17.0 % (40/235) and 12.1 % (40/330) for Vitrolife, 21.4 % (39/182) and 9.9 % (39/395) for slow-freezing (NS).ConclusionsBoth vitrification methods (Irvine and Vitrolife) are more efficient than slow freezing for cryopreservation of human cleavage stage embryos in terms of post-warming survival rate. No significant difference in the implantation rate was observed between the three cryopreservation methods.

Identification of bovine embryos cultured in groups by attachment of barcodes to the zona pellucida.

The low number of oocytes collected from unstimulated donors by ovum pick-up means that embryos produced from each individual female have to be cultured individually or in very small groups. However, it has been demonstrated that single-embryo culture is less efficient than embryo culture in groups. To overcome this limitation, we developed a direct embryo-tagging system, which allows the collective culture of embryos from different origins whilst preserving their pedigree. Presumptive bovine zygotes were tagged with eight wheat-germ agglutinin biofunctionalised polysilicon barcodes attached to the outer surface of the zona pellucida (ZP). Four different barcodes were used to encode groups of 20-25 embryos, which were then cultured in the same drop. Cleavage, Day-7 and Day-8 blastocysts and barcode retention rates were assessed. In addition, Day-7 blastocysts were vitrified and warmed. Barcode attachment to the ZP of bovine embryos affected neither in vitro embryo development nor post-warming survival of the tagged embryos. All the embryos maintained barcodes attached until Day 8 of culture (3.63±0.37 barcodes per embryo) and could be identified. In conclusion, identification of embryos by barcodes attached to the ZP is feasible and will allow the culture of embryos from different donors in the same drop.

142 ZONA PELLUCIDA TAGGING WITH BARCODES ALLOWS THE TRACEABILITY OF BOVINE EMBRYOS CULTURED IN GROUP

The low number of oocytes collected from unstimulated donors by ovum pick-up means that embryos produced from each female have to be cultured in very small groups. Because embryo quality and development rates are reduced in individual and small group culture, several methods to culture embryos individually but sharing the same medium have been designed. However, these systems prevent embryo movements, interfering with paracrine factors transmission and gradient changes. Here, we present an alternative in vitro culture method to allow the co-culture of embryos from different origins, without movement restriction and preserving their pedigree, by labelling the zygotes with polysilicon barcodes attached to the outer surface of the zona pellucida (ZP). Barcodes (10 × 6 × 1 µm) with 8 rectangular bits of binary codification (256 possible combinations), which can be read under a standard inverted microscope, were fabricated using silicon microtechnologies. To provide the barcodes with a ZP-binding capacity, they were biofunctionalized by self-assembled monolayers with the wheat germ agglutinin (WGA) lectin, which recognizes specific saccharides highly abundant in the ZP of most mammalian species. As a proof of concept, the culture method was tested on bovine zygotes produced from slaughterhouse-derived cow oocytes matured and fertilized in vitro. Using a mouth-controlled pipette, presumptive zygotes were individually rolled over WGA-biofunctionalized barcodes (8 barcodes/embryo) previously placed at the bottom of a drop of manipulation media. Four different barcodes, each one with a different codification, were used to encode 25 embryos (6–7 embryos/barcode codification), which were then cultured together in the same drop of medium. Day 7 (D7) and Day 8 (D8) blastocyst, and barcode retention rates were assessed. In addition, D7 expanded blastocysts were vitrified by the cryotop method and post-warming survival was determined as re-expansion rate at 24 h in culture. Finally, the quality of D8 blastocysts was assessed by differential staining and counting of inner cell mass (ICM) and trophectoderm (TE) cells. In all the experiments, a control group without barcodes was cultured and vitrified-warmed. Data were analyzed by chi-square and Mann–Whitney tests. The presence of barcodes attached to the ZP did not affect in vitro embryo development (D8 blastocysts: 29.7% control n = 309, 36.2% encoded n = 315), post-warming survival (86.4% control n = 66, 80.5% encoded n = 82), or blastocyst quality (IMC/TE: 22.1 ± 1.4/64.5 ± 5.7 control n = 18, 22.2 ± 1.7/64.1 ± 6.1 encoded n = 23). The labelling system was effective until D8 of culture, as all the embryos maintained barcodes attached (4 ± 1.8 barcodes/embryo) and could be identified, even after undergoing vitrification and warming. In conclusion, identification of co-cultured embryos by biofunctionalized barcodes attached to the ZP is feasible and will allow to culture embryos from different donors in the same drop, keeping the benefits of collective culture. Support was provided by Spanish MEC (TEC2011-29140-C03; RZ2010-00015-0-00; AGL2010-19069), Generalitat Catalunya (2009 SGR 282 and 621), and PIF-UAB Fellowship.

Oocyte vitrification in a single woman with diminished ovarian reserve resulting in live birth

Fertility preservation is crucial for women experiencing diminished ovarian reserve (DOR) due to disease treatments or aging. From preserving ovarian tissues to oocytes, scientists are trying to find more convenient, cost-effective ways to meet women’s fertility needs. Since the first successful live birth from a human frozen/thawed oocyte in 1986 [1], more researchers have devoted themselves to this field, developing techniques and applications for oocyte cryopreservation. Progress in human oocyte cryopreservation, especially vitrification along with its obstetric outcome, has been one of the major issues in reproductive medicine recently. Chen et al [2] reported a 75% survival rate, 67% fertilization rate, and 33% pregnancy rate following a new oocyte slow freezing/thawing procedure. Vitrification is thought to be a highly efficient technique for embryo and oocyte cryopreservation [3e5]. The vitrification of oocytes allows higher postwarm survival rates than slow freezing. However, significant improvements have been achieved in both cryopreservation methods [2,6]. Several research studies on the outcome of oocyte cryopreservation have been published. Kim et al [7] reported that in fertile women, vitrification of oocytes produces pregnancy and implantation rates comparable to those of frozen embryos. Although the data were limited, a systematic review of cryopreserved oocytes showed no obvious association with adverse pregnancy outcome [8]. A multicenter, prospective clinical trial to evaluate the efficacy, safety, and outcome of human oocyte cryopreservation is currently ongoing [9]. However, there is still a need for more supportive evidence of oocyte vitrification. Studies in this field have mainly focused on young or fertile women needing cryopreservation for medication purposes [7,10e14], but rarely has there been concern about women who are at an advanced maternal age or infertile [15,16]. Because oocyte number and quality begin to decline from age

Developmental Competence and Embryo Quality of Small Oocytes from Pre‐pubertal Goats Cultured in IVM Medium Supplemented with Low Level of Hormones, Insulin–Transferrin–Selenium and Ascorbic Acid

The aim of this study was to test the effect of insulin-transferrin-selenium (ITS) and L-ascorbic acid (AA) supplementation and the hormonal level during in vitro maturation (IVM) of small oocytes from pre-pubertal goat on the blastocyst yield and quality. Concretely, we used four maturation media: conventional IVM medium (CM), growth medium (GM: CM+ITS+AA and low level of hormones), modified CM (mCM: CM with low level of hormones) and modified GM (mGM: CM+ITS+AA and normal level of hormones). Cumulus-oocyte complexes (COCs) were classified into two categories according to oocyte diameter: <125 μm and ≥ 125 μm. Large oocytes were matured 24 h in CM (Treatment A). Small oocytes were matured randomly in six experimental groups: Treatment B: 24 h in CM; Treatment C: 12 h in GM and 12 h in CM; Treatment D: 24 h in mGM; Treatment E: 12 h in mGM and 12 h in CM; Treatment F: 12 h in mCM and 12 h in CM; and Treatment G: 12 h in GM and 12 h in mGM. After IVM, oocytes were fertilized and cultured for 8 days. The blastocyst quality was assessed by the survival following vitrification/warming and the mean cell number. When different maturation media were combined, the blastocyst rate did not improve. The large oocytes produced the highest blastocysts yield. However, the culture of small oocytes in GM (53.3%) enhanced the post-warming survival of blastocysts compared to large oocytes matured in CM (35.7%). In conclusion, IVM of pre-pubertal goat small oocytes in GM would be useful to improve the quality of in vitro-produced blastocysts.

Oocyte morphology does not affect post-warming survival rate in an egg-cryobanking donation program

To evaluate whether oocyte dysmorphisms affect oocyte survival rates in an egg-cryobanking donation program. This study included 54 patients undergoing intracytoplasmic sperm injection. A total of 415 metaphase II oocytes were vitrified using the Cryotop method. Oocyte morphology was assessed immediately prior to oocyte vitrification under 400× magnification. The influence of dysmorphisms on post-thaw survival rates was assessed using regression analysis. Results were considered to be significant at the 5% critical level. Oocyte survival rate was not affected by the presence of the following analysed oocyte abnormalities: increased cytoplasmic granularity, vacuoles in the ooplasm, aggregates of smooth endoplasmic reticulum in the ooplasm, large perivitelline space size, perivitelline space granularity, fragmented first polar body and zona pellucida abnormalities. Oocyte morphology, observed prior to vitrification, does not predict post-warming survival. The non-invasive identification of predictive markers for oocyte survival potential remains a difficult task.

Effect of antifreeze protein supplementation in vitrification medium on mouse oocyte developmental competence

To investigate the effect of antifreeze protein (AFP) supplementation during mouse oocyte vitrification on the survival, fertilization and embryonic development. Animal study. University laboratory. BDF-1 mice. Invivo-matured metaphase II oocytes were vitrified with the use of CryoTop by two-step exposure to equilibrium and vitrification solution supplemented or not with 500 ng/mL AFP III. Postwarming survival, fertilization, embryonic development up to blastocyst invitro, morphology of spindle and chromosome, membrane integrity, adenosine triphosphate (ATP) contents, and several gene expressions. In the AFP-treated group, blastocyst formation rate was significantly higher and blastomere count with positive caspase was significantly lower compared with the nontreated group. Rate of intact spindle/chromosome, stable membrane, and ATP contents were significantly higher in AFP group. AFP group showed higher Mad2 and lower Eg5 gene expression. Both vitrification groups showed increased Hsf1, Zar1, and Zp1/Zp2 expression and decreased Hook1 and Zp3 expression compared with fresh control samples. Supplementation of AFP in vitrification medium has a protective effect on mouse oocytes for chilling injury; it can preserve spindle/membrane integrity and intracellular ATP contents. More stable spindle integrity in the AFP group may be associated with higher Mad2 and lower Eg5 gene expression.

Prospective randomized comparison of non-cryopreserved and vitrified sibling oocyte function and resulting embryo development

Vitrification of donor oocytes and establishment of a “Donor Oocyte Bank” has practical, quarantine/disease-testing, and economic advantages. Study aims were to compare oocyte function and embryo development in a pool of sibling oocytes used both as non-cryopreserved oocytes and following vitrification and warming. Prospective randomized clinical study Couples undergoing ICSI elected to participate in this IRB-approved study. Sibling MII oocytes were randomly used in a fresh cycle (NoCryo) or vitrified (Vit). Oocytes were vitrified with dimethyl sulfoxide/ethylene glycol/sucrose with a Cryotip (Irvine Sci). After warming oocytes were observed for survival, incubated for 4h, and inseminated by ICSI. No/normal/abnormal fertilization and oocyte lysis were assessed. Zygotes were placed in G1 media (VitroLife) for growth and assessed for cleavage and embryo development. Statistical analyses were performed by paired Student's t-test. Sixty-nine patients (age: 34 ± 1; ave ± sem) with an average of 18 MII oocytes participated in this study. Post-warming survival was 83 ± 3%. Following ICSI, no fertilization (14 ± 2% vs 11 ± 2%) or abnormal fertilization (1 ± 0.2% vs 2 ± 0.6%) were not significantly different between NoCryo and Vit, respectively. Normal fertilization was significantly higher in the NoCryo group (82 ± 2%) compared to Vit (76 ± 2%; P<0.05), due to increased incidence of post-ICSI oocyte lysis (4 ± 1% vs 9 ± 2%, respectively; P<0.01). Cleavage rate was similar between groups. Day 3 embryos had significantly more cells (6.4 ± 0.1 vs 6.1 ± 0.1; P<0.05) in NoCryo compared to Vit, respectively. Degree of embryo fragmentation was similar between groups. In an infertile population, vitrification of oocytes resulted in acceptable fertilization and embryo development, yet not equivalent to non-cryopreserved oocytes. Knowledge of these oocyte functional and embryo developmental differences will assist in future planning for efficient donor oocyte cryo-banking.

Cryopreservation of In Vitro-Produced Bovine Embryos for Direct Transfer.

Our objective was to identify a method for efficacious cryopreservation of in vitro-produced bovine embryos that would enable direct embryo transfer from 0.25 mL straws used as containers for cryopreservation. Although not a method for direct transfer, cryotops were chosen for controls (CON), as they are becoming the industry standard for vitrification of human embryos. Embryos were cryopreserved by vitrification with either a cryotop (CON; n = 118 ) or using an aluminum block (BLK; n = 128), or by slow freezing (SLF; n = 131). Procedures were done at room temperature, 22° ± 2°C. For vitrification, embryos were exposed to 5 M ethylene glycol in SynGro base medium for 3 min, and then moved to 6.5 M ethylene glycol + 0.5 M galactose + 18% Ficoll in SynGro base medium (V2). CON embryos were placed in < 1 µl V2 onto cryotops, and after 35 s, vitrified by plunging directly into liquid nitrogen. BLK embryos were loaded into 0.25 mL straws, in 20 µl V2, between two columns of 1 M galactose in SynGro. After 35 s in V2, embryos were vitrified by cooling for 2 min via contact of straw walls with columns drilled into an aluminum block immersed in liquid nitrogen, and then directly plunged into liquid nitrogen. Embryos cryopreserved via SLF were exposed to 1.36 M glycerol in modified Dulbecco's PBS + 0.4% BSA (PBS) for 10 min, loaded into 0.25 mL straws, and placed into a freezing machine. Straws were cooled to -6°C at 4°C per min, held at -6°C for 5 min, seeded, held at -6°C for an additional 10 min, and then cooled to -30°C at 0.5°C per min and plunged into liquid nitrogen. After storage for at least 24 h in liquid nitrogen, embryos were warmed/thawed. CON embryos were removed from cryotops by direct placement into a 200 µl drop of 1 M galactose in SynGro for 2 min. BLK/SLF embryos were warmed/thawed by holding straws in air for 8 s and placing them in a water bath at 37°C for 20 s. By flicking straws, BLK embryos were mixed with 1 M galactose in SynGro in the straw and held for 2 min. SLF embryos were expelled from straws, and glycerol was removed in three 6 min steps: 0.8 M glycerol + 0.3 M sucrose; 0.4 M glycerol + 0.3 M sucrose; and 0.3 M sucrose followed by PBS for 2 min. After all embryos were recovered, they were rinsed through holding medium, and cultured in chemically defined medium similar to SOF for 24 h before being evaluated for survival. Post warming survival was greater (P < 0.01) for CON than BLK (83.9%, 67.2%, respectively); BLK was greater (P < 0.01) than SLF (51.9%). Although BLK resulted in lower post-warming survival than CON, it may be an acceptable method for direct transfer, which yielded greater post-warming survival than the current SLF method used for cryopreservation of bovine embryos. (poster)

Clinical outcome of emergency egg vitrification for women when sperm extraction from the testicular tissues of the male partner is not successful

The development of an effective oocyte cryopreservation system will have a significant impact on the clinical practice of reproductive medicine. However, the important option of emergency oocyte cryopreservation has yet to be well documented. In this report, we review the cases of 15 women with male partners who were diagnosed with nonobstructive azoospermia and for whom testicular sperm extraction on the day of oocyte retrieval failed. Emergency oocyte vitrification was performed and after two months, the vitrified oocytes were warmed and the surviving oocytes inseminated with frozen-thawed donor sperm by intracytoplasmic sperm injection (ICSI). A total of 117 mature oocytes from the 15 women were vitrified and warmed. The post-warming survival rate was 84.6% (99/117), and the fertilization rate following ICSI was 83.8% (83/99). We selected 30 embryos for transfer to 15 patients, 8 of whom became pregnant. The clinical pregnancy rate was 53.3% (8/15) and the implantation rate was 30.0% (9/30). Nine healthy live births resulted from 8 pregnancies. These results indicate that emergency oocyte vitrification is an effective rescue technique that can be applied clinically with acceptable pregnancy and live birth rates when testicular sperm extraction from the male partner failed on the day of oocyte retrieval. These results also highlight another important option for oocyte cryopreservation through the use of vitrification technology.

Mouse ovarian follicle cryopreservation using vitrification or slow programmed cooling: Assessment of in vitro development, maturation, ultra-structure and meiotic spindle organization

To compare different outcomes of vitrification and slow freezing of isolated pre-antral follicles and to evaluate different cryo-devices for vitrification of isolated follicles. Pre-antral follicles were isolated from mouse ovaries and cryopreserved using vitrification and slow freezing. A preliminary experiment was carried out to select the optimal cryo-device for vitrification of isolated follicles. A total of 414 follicles were randomly distributed among four groups: control (CT) fresh (n=100), nylon mesh (n=96), electron microscopy grid (n=102), and micro-capillary tips (n=116). Subsequently, a total of 979 follicles were randomly assigned to three different groups: CT fresh (n=256), vitrification (n=399) and slow freezing (n=324). CT and cryopreserved/thawed follicles were cultured in vitro and examined daily for development. Final maturation was triggered with human chorionic gonadotrophin and rates of oocyte maturation were calculated. The ultra-structure of cryopreserved/thawed follicles was studied using electron microscopy. Meiotic spindle presence and organization in mature oocytes were examined using the Oosight imaging system. Micro-capillary tips resulted in poor immediate post-warming survival but no differences were observed in the subsequent in vitro development characteristics between different cryo-devices. Nylon mesh proved to be the easiest carrier, particularly when large numbers of follicles were to be vitrified. Compared to vitrification, slow freezing resulted in a significantly lower number of intact follicles at the end of the culture period (P<0.0001). However all other outcome measures were comparable between both techniques. Isolated follicles were more vulnerable to cryodamage after slow freezing as compared to vitrification.

Stress for stress tolerance: improving cell survival by sublethal stress treatment of eggs before vitrification – pilot study

OBJECTIVE: Sublethal environmental stress induces general adaptation of cells and makes them more resistant during subsequent interventions such as cryopreservation. In the present study sublethal hydrostatic pressure (HP) stress was used to treat mouse and human oocytes before vitrification in order to increase post-warming survival and developmental competence.

Outcomes of vitrified–warmed day-4 embryos after day-3 cleavage-stage biopsy

The objective of this study was to compare the post-warming survival rates of biopsied and non-biopsied day-3 embryos vitrified on day 4 and to evaluate the clinical outcomes of following transfers. This study included 18 preimplantation genetic diagnosis (PGD) patients and 18 non-PGD patients treated between January 2005 and January 2009 who had not achieved live births during their fresh embryo-transfer cycles and whose surplus embryos were cryopreserved on day 4. The embryo survival rate after warming in the PGD and non-PGD groups was similar (53/59, 89.8% versus 55/64, 85.9%, respectively; difference of 3.9% 95% CI -7.3 to 13.4). Vitrified embryo-transfer cycles yielded no significant differences between PGD and non-PGD groups in implantation rates (12/46, 26.1% versus 9/47, 19.1%, respectively; difference of 6.9%, 95% CI -9.7 to 22.2), clinical pregnancy rates (11/18, 61.1% versus 9/18, 50%, respectively; difference of 11.1%, 95% CI -20.6 to 40.6) and live birth rates (9/18, 50% versus 6/18, 33.3%, respectively; difference of 16.7%, 95% CI -15.1 to 44.8). These results showed that, in PGD cycles, embryos can be vitrified effectively on day 4 after biopsy on day 3. The objective of this study was to compare the post-warming survival rates of biopsied and non-biopsied day-3 embryos that vitrified on day 4 and to evaluate the clinical outcomes of following transfers. This retrospective study included 18 preimplantation genetic diagnosis (PGD) and 18 non-PGD patients treated between January 2005 and January 2009 who had not achieved live births during their fresh embryo transfer cycles and whose surplus were frozen on day 4. After warming in frozen embryo-transfer cycles, embryo survival with respect to embryo grades, implantation, clinical pregnancy and live birth rates were compared. The embryo survival rate after warming in the PGD group was similar to the survival rate in the non-PGD group (53/59, 89.8% versus 55/64, 85.9%, respectively; difference of 3.9%, 95% CI -7.3 to 13.4, P=0.701). Frozen embryo transfer yielded no significant differences between PGD and non-PGD groups in implantation rates (12/46, 26.1% versus 9/47, 19.1%, respectively; difference of 6.9%, 95% CI -9.7 to 22.2, P=0.581), clinical pregnancy rates (11/18, 61.1% versus 9/18, 50%, respectively; difference of 11.1%, 95% CI -20.6 to 40.6, P=0.737) or live birth rates (9/18, 50% versus 6/18, 33.3%, respectively; difference of 16.7%, 95% CI -15.1 to 44.8, P=0.499). These results showed that, in PGD cycles, embryos can be vitrified effectively on day 4 after biopsy on day 3.

Vitrification of ICSI- and IVF-derived bovine blastocysts by minimum volume cooling procedure: effect of developmental stage and age

The objective was to investigate the effects of developmental stage (fully-expanded or expanding blastocysts) and/or age (harvested on Days 7 or 8) on post-vitrification in vitro survival of bovine blastocysts derived from intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF). Post-warming survival (re-expansion of blastocoele within 24 h) of ICSI-derived fully-expanded blastocysts (80%) was similar to that of their IVF-derived counterparts (88%). However, the ability of ICSI-derived expanding blastocysts to survive vitrification procedures (61%) was lower than that of IVF-derived blastocysts (85%; P < 0.05), although the ICSI- and IVF-derived fresh blastocysts were of similar quality. The age of the blastocysts before vitrification did not affect cryotolerance for either ICSI-derived (73 and 59% for Days 7 and 8 embryos, respectively) or IVF-derived blastocysts (86% for both Days 7 and 8 embryos). At 24 h of post-warming culture, ICSI-derived blastocysts surviving vitrification contained a higher proportion of dead cells than their IVF-derived counterparts (5–13% vs. 2–4%; P < 0.05), but these proportions were not different from those of fresh control embryos. There was an adverse effect of vitrification on the ability of blastocysts to hatch within 72 h of culture only in IVF-derived Day 8 blastocysts (41 and 70% in vitrified and fresh control groups, respectively). In conclusion, the proportion of blastocysts that survived vitrification procedures was similar for ICSI- and IVF-derived bovine blastocysts if the former were cultured to the fully-expanded stage prior to vitrification, with no significant difference between embryos harvested on Day 7 versus Day 8.