Method For Cryopreservation Of Embryos Research Articles

P-129 Transferring blastocysts derived from frozen-thawed cleavage embryos versus frozen-thawed blastocysts: A comparative analysis of pregnancy outcomes

Abstract Study question Is there an enhancement in pregnancy outcomes when cleavage-stage embryos are thawed, cultured for transfer as blastocysts, as opposed to transferring thawed blastocysts? Summary answer The strategy of culturing frozen-thawed cleavage embryos for 2 days and transferring them as blastocysts is not inferior to the transfer of frozen-thawed blastocysts. What is known already Embryo cryopreservation in IVF has many advantages, allowing couples to postpone pregnancy for personal reasons and reducing the risk of ovarian hyperstimulation syndrome. In the past, the common practice involved freezing and transferring cleavage embryos. However, a current trend favors single-embryo transfers at the blastocyst stage, promoting potential self-selection during extended culture. Several studies have raised concerns about potential damage during blastocyst vitrification, prompting investigation into whether cryopreserving cleavage-stage embryos for subsequent culturing to blastocysts yields superior outcomes compared to direct transfer of thawed blastocysts. Study design, size, duration This observational study was conducted at Hung Vuong Hospital from January 2022 to December 2023. We included 509 patients who underwent IVF followed by FET cycles. The D3-5 group (n = 167) comprised embryos that were frozen and thawed at the cleavage stage, subsequently extended two more days of culturing to the blastocyst stage for transfer. The D5 group (n = 342) involved frozen and thawed blastocysts. We excluded donor cycles, surrogacy, and cycles with preimplantation genetics testing. Participants/materials, setting, methods We used vitrification method for embryo cryopreservation. The freezing and thawing process were performed using either Kitazato or Cryotec medium kit, following manufacture’s instruction. Regarding the D3-5 group, thawed cleavage embryos were cultured for two more days until blastulation and transfer. Regarding the D5 group, blastocysts transfer was performed at least 2 hours after warming. The embryo transfer procedure was performed under abdominal ultrasonographical guidance. Pregnancy results were recorded and compared between two groups. Main results and the role of chance Regarding the D3-5 group, a noteworthy proportion of 65.3% cycles were unfortunately cancelled. The primary reason for cycle cancellations was the absence of developed blastocysts for transfer (85.3%). The remaining 14.7% of cancellations were attributed to other reasons. Fertilization rates were comparable between the two groups, with 80.8% in the D3-5 group and 81.5% in the D5 group (p = 0.464). However, the blastulation rate in the D3-5 group (40.1%) was lower than in the D5 group (46.4%), (p = 0.016). Patients in the D5 group demonstrated slightly better pregnancy outcomes compared to the D3-5 group: implantation rate (59.9% versus 51.7%, p = 0.302), clinical pregnancy rate (52.3% versus 44.8%, p = 0.36), ongoing pregnancy rate (37.7% versus 34.5%, p = 0746), live birth rate (33.9% versus 31%, p = 0.78), and miscarriage rate (26.3% versus 20.7%, p = 0.46). However, these differences were not statistical significance. Limitations, reasons for caution This is an observational study, we observed that thawing the cleavage-stage embryos then subsequently culture to the blastocyst stage may lead to the lower blastulation rate or even absence of developed blastocysts for transfer. This approach also increases the workload for embryologists. Wider implications of the findings Culturing frozen-thawed cleavage embryos for 2 days and transferring them as blastocysts increases workload for embryologists and poses a risk of cycle cancellation. We propose that the use of frozen-thawed blastocysts may be a more efficient and patient-friendly option. Trial registration number not applicable

Update on the Epigenomic Implication of Embryo Cryopreservation Methods Applied in Assisted Reproductive Technologies With Potential Long-Term Health Effects.

Cryopreservation of embryos has become an efficient method in Assisted Reproductive Technologies (ART) and these methods are currently performed at nearly all fertility centers around the globe. Cryopreservation of supernumerary embryos has contributed to an increase in cumulative pregnancy rates and as a consequence, an increasing number of children are being born through these techniques worldwide. However, long-term follow-up studies of children born through ART are scarce, and concerns about the long-term health effects on individuals conceived through ART have been raised. The relevant genomic transformations that occur at the time cryopreservation is usually applied to embryos may have potential epigenetic risks. With advances in multi-omic single cell technologies, new ways to assess the (epi)genomic status during early embryo development have now become feasible. These novel strategies could provide a revolutionary opportunity to understand the actual impact of ART, but also may help future developments aiming at increase both their efficiency and safety. Here we outline insights in current knowledge and research on the impact of cryopreservation on embryos, the possible consequences at epigenetic level and how emerging new high-throughput technologies can be used for their assessment.

Colabamento manual da blastocele não foi efetivo para aumentar a viabilidade de embriões equinos vitrificados

Embryo cryopreservation methods have been used for commercialization and formation of genetic banks. Cryopreservation of equine embryos <300 μm in diameter, collected at days 6-6.5 after ovulation, allows satisfactory pregnancy rates. However, higher embryo collection rates in mares are obtained when uterine flush is performed between days 7 and 8 after ovulation when embryos are >300 μm in diameter, needing blastocoel collapse for satisfactory resistance to cryopreservation by vitrification. To evaluate the viability of simplified blastocoel collapse by embryo puncture with low technology and low-cost equipment, 22 embryos, collected at day 8 post-ovulation (D8), were allocated to the following groups: (1) micropuncture with a 30 G needle, assisted by a mechanical micromanipulator, before vitrification (n=4); (2) manual blade microsection before vitrification (n=6); (3) no manipulation prior to vitrification (n=8); and (4) freshly inovulated embryos (n=4). Despite the high re-expansion rates observed after vitrification, embryos manipulated prior to vitrification (groups MP and MS) did not result in pregnancy 25 days after transfer. On the other hand, embryos from groups NM (non-micromanipulated) and FR (freshly inovulated) resulted in pregnancies at 25 days. Under the conditions of the present study, manual blastocoel collapse was not efficient in increasing cryotolerance to vitrification among large embryos, requiring improvements to obtain pregnancies.

P–765 Embryo donation pregnancies are at high risk even in young recipients

Abstract Study question Are pregnancies after embryo donation (ED) at higher risk of complications than those issued from autologous frozen-thawed embryo transfer (FET)? Summary answer Even in young women, the risk of pregnancy induced hypertension (PIH) is four time higher in pregnancies after ED versus controls. What is known already After oocyte donation, a higher risk of PIH is well described. It is more controversial after sperm donation. The risk origin remains uncertain, even though an immunological explanation seems most likely. In ED, the fetus being fully allogeneic to his parents may be less well-tolerated. Very few data are reported about pregnancies after ED. The same allogenic model exists in surrogacies, but pregnancy complications are not well described in the literature. Study design, size, duration This anonymous, multicenter, comparative observational retrospective cohort study included all singleton ED pregnancies diagnosed at 7–8 weeks, from January 2003 to December 2018, in six French ART centers. For each, two controls were matched among autologous FET pregnancies. 209 pregnancies were included: 73 ED and 136 controls. Multiple pregnancies were excluded because of their increased associated obstetrical risks. Participants/materials, setting, methods Controls were matched according to pregnancy date, parity and women’s age. The first two singleton pregnancies after each index case meeting the selection criteria were retained. Each center coordinator collected information on infertility, pregnancy pathologies, outcomes and newborns. Statistical methods included univariate and multivariate analyses. According to French practice, all women were under 44 y/o. The main outcome was the percentage of PIH for ED versus controls. Main results and the role of chance ED was indicated for genetic disease in 17 cases (23.3%), double total infertility in 28 cases (38.3%), and double partial/total infertility in 35 cases (47.9%). Groups were comparable in age (mean age: 34.5 ± 8.6 versus 34.5 ± 4.5; p = 0.68), BMI, except for parity (more nulliparity in ED group: 90.4% vs 79.4%; p = 0.04). Pregnancy outcomes were similar for ED and control groups, the percentages of deliveries being 80.8% and 83.8%, respectively (p = 0.58). PIH occurred significantly more frequently among ED than control pregnancies (24.6% versus 11.9%; P = 0.04), with the difference mainly observed for severe forms: preeclampsia and HELLP (17.5% vs 4.6%; p = 0.01). No eclampsia was reported. In contrast, isolated hypertension frequency was comparable (7.0% vs. 7.3%, p = 0.94). Regarding labor and delivery mode, in ED group C-section was more frequent (47.3% vs 29.2%; p = 0.03). In neonatal data, no difference was found between ED and control group for prematurity, weight and height at birth, Apgar score, Small for gestational age, Large for gestational age and sex ratio. Seven neonatal malformations were recorded in ED group and 3 in the control group (NS). Limitations, reasons for caution Retrospective study in a relatively long period when different endometrial preparation for frozen-thawed embryo transfer and embryo cryopreservation method were used. Relatively limited number of ED because of low practice in France. No analysis of embryo stage at transfer (cleaved embryo or blastocyst). Wider implications of the findings: The PIH risk must be acknowledged to inform couples and provide careful pregnancy monitoring. A special care for gestational carrier should also be done since the allogenic situation is the same than in ED recipients. Trial registration number Not applicable

Vitrification yields higher cryo-survival rate than slow freezing in biopsied bovine in vitro produced blastocysts

Vitrification and slow freezing are the two commonly used embryo cryopreservation methods. In most studies, vitrification of intact embryos has proven superior in several respects, including cell and embryo survival and pregnancy rate. However, there is a lack of data for comparing these two methods in in vitro produced (IVP) bovine blastocysts, which have been subjected to the retrieval of trophectoderm (TE) biopsy. Day 7 IVP blastocysts were pooled and randomized into four groups: 1) non-biopsy (NB), 2) biopsy (B), 3) biopsy-vitrification (BV), 4) biopsy-slow freeze (BSF). The blastocysts in the B, BV, and BSF groups were subjected to TE biopsy. For the B group, this was followed by 5 hours (h) incubation and subsequent scoring of the biopsy-survival (re-expansion) rate before processing for further analyses. For the BV and BSF groups, the biopsy procedure was followed by 2 h incubation, allowing for a quick re-expansion, after which the blastocysts were subjected to vitrification and slow freezing, respectively. After warming and thawing, respectively, they were then incubated for 5 h followed by scoring the cryo-survival (re-expansion) rates before processing for further analyses. These included quantification of ICM and TE cells, cleaved caspase-3- and TUNEL-positive cells, quantitative PCR on cellular stress markers (SOD1 and PRDX1), and ultrastructural analysis.The biopsy-survival rate in the B group was 94% (307/326). The cryo-survival rate in BV (86%, 138/161) was higher than that in BSF (57%, 81/142; P < 0.001). No differences were noted between the average ICM, TE, and total cell numbers of the groups. The percentages of cleaved caspase-3-positive cells were higher in BV vs. NB (P < 0.05), in BSF vs. NB (P < 0.001), and in BSF vs. B (P < 0.001). The percentages of TUNEL-positive cells were higher in BV vs. NB (P < 0.05) and in BSF vs. NB (P < 0.001). The levels of mRNA abundance for SOD1 and PRDX1 in B, BV, and BSF were not different from that in NB. The ultrastructural analysis of blastocysts in the BV and BSF groups showed distension of extracellular spaces and appearance of intracellular vacuoles in the ICM, distension of mitochondria, and disorganization of mitochondrial cristae in both ICM and TE, and weakened tight junctions between adjacent TE cells. In summary, our findings demonstrate that vitrification yields a higher cryo-survival rate than slow freezing in biopsied bovine IVP blastocysts. However, biopsy-vitrification and biopsy-slow-freeze values are comparable in terms of ICM, TE, and total blastocyst cell numbers, as well as cleaved caspase-3- and TUNEL-positive cell rates. Moreover, biopsy and cryopreservation performed alone had no effect on ICM, TE, total blastocyst cell numbers, or TUNEL-positive cell rates. Biopsy and vitrification performed alone had no effect on the cleaved caspase-3 positive cell rates, whereas slow freezing resulted in an increased rate. Furthermore, double traumatization with a combination of biopsy and cryopreservation, either vitrification or slow freezing, resulted in increased rates of cleaved caspase-3- and TUNEL-positive cells.

Leptin improves the in vitro development of preimplantation rabbit embryos under oxidative stress of cryopreservation.

Vitrification is an economically effective method for embryo cryopreservation in human and livestock animals; however, it carries the risk of damage by the exposure to severe oxidative stress. The present study was conducted to evaluate the effect of leptin at different levels on the in vitro development of fresh and vitrified preimplantation embryos in a rabbit model. Normal embryos at morulae stage were randomly cultured for 2 h with 0, 10, 20 or 100 ng/mL of leptin, then were cultured for further 48 h as freshly or after vitrification. Thereafter, developed blastocysts form the best leptin level in fresh and vitrified embryos along with their controls were allocated to analyze the pro-oxidant (malondialdehyde, MDA; nitric oxide, NO), antioxidant (total antioxidant capacity, TAC; superoxide dismutase, SOD; glutathione peroxidase, GPx), apoptotic (Bcl-2 associated X protein, BAX; heat shock 60kD protein member 1, HSP60; tumor necrosis factor alpha, TNFα) and developmental (sex determining region Y box protein 2, SOX2; Nanog homeobox protein, NANOG; Octamer-binding protein 4, OCT4) biomarkers. Results indicate that expanding and hatching rates of embryos were significantly higher at 20 ng/mL leptin than the other levels, while vitrification had an independent suppression effect on the in vitro development rates. The MDA and NO were significantly higher, while TAC, SOD and GPx were significantly lower in the vitrified than fresh embryos. In contrast, leptin treatment significantly decreased the pro-oxidant biomarkers and increased the antioxidant biomarkers in both fresh and vitrified embryos. Vitrification significantly increased the antiapoptotic biomarkers, and decreased the developmental biomarkers in embryos. In contrast, leptin decreased the BAX and TNFα, increased the HSP60, and moreover, ameliorated the reduction of developmental biomarkers in the vitrified embryos. These results conclude that leptin could be used as antiapoptotic and antioxidant promotor to support the in vitro embryonic development, particularly under oxidative stress emerged from cryopreservation programs.

Effect of N-acetyl cysteine on the quality of blastocyst formation rate using cultured vitrified murine embryos

Vitrification is the best method for embryo cryopreservation although it increases endogenous reactive oxygen species (ROS) production. N-acetylcysteine (NAC) a free radical scavenger may be used for reducing ROS toxic effects. The aim of the present study is to investigate potential beneficial effects of NAC on the developmental embryo competence applying different culture conditions in vitrified-warmed 2-cell embryos derived in vivo or in vitro. Thus, 2-cell embryos were vitrified or cultured fresh in presence or absence of 1 mM of NAC during: a) the entire embryo culture, b) for 24 hours with NAC at days 1.5 (G1) or 2.5 (G2) and returned to basal embryo culture (KSOM) or c) cultured in the presence of NAC for 12 hours at day 3.5 (G3). Despite NAC addition to fresh or vitrified embryos produced in vivo or by IVF, blastocyst rates remained unchanged. In vitrified-warmed IU or IVF-derived embryos, total cell number varied when NAC was added at day 1.5 although differences were not significant (60.1 ± 1.9 vs. 59.4 ± 1.3 for IU G1 and control respectively; and 59.3 ± 1.6 and 52.6 ± 3.0 IVF G1 and control respectively; mean cell number ± SEM, p &gt; 0.05). It seems that the embryo culture medium supplementation with 1 mM of NAC in the first day after vitrification of development improves blastocyst quality of murine embryos and does not exert any beneficial effect at oyher culture points.

The number of oocytes associated with maximum cumulative live birth rates per aspiration depends on female age: a population study of 221 221 treatment cycles.

The number of oocytes associated with maximum cumulative live birth rates per aspiration depends on female age: a population study of 221 221 treatment cycles.

47 A New Device and Method for Successful Vitrification of In Vitro-Produced Ovine Embryos

To advance the use of embryo vitrification technology in veterinary practice, we developed a system in which embryo vitrification, warming, and dilution can be performed within a straw. An in-straw embryo cryopreservation method reduces the need for equipment and technical skills and can facilitate direct embryo transfer to the uterus. This study proposes the use of a new device named “Sarah” that is designed to permit all in-straw embryo cryopreservation procedures. Ovine in vitro-produced (IVP) embryos were vitrified at either early blastocyst stage (EB, n = 65, 6 days post-IVF) or fully expanded blastocyst stage (FB, n = 168, 7 days post-IVF). The vitrification procedure using Sarah constituted a 0.25-mL straw with a capsule having 50-µm pores inserted at one end. Embryos at each stage (EB and FB) were divided into 2 subgroups and vitrified by 1 of 2 methods: (1) multi-step (MS) group-a straw containing 2 embryos was sequentially loaded vertically into 1.5-mL tubes containing 6 different vitrification solutions: 10, 20, 40, 60, 80, or 100% ES (with 100% ES being 7.5% DMSO +7.5% EG + 20% FCS in TCM-199; 90 s each step) followed by 30 s each in 75 and 100% VS (100% VS being 18% DMSO +18% EG + 0.5 M trehalose + BSA in TCM-199); and (2) two-step (TS) group-the straw (2 embryos/straw) was loaded with 100% of ES (5 min), followed by 100% VS solution for 30 s. For both methods, at the end of the preparation steps, the straws were plunged directly into liquid N2. Non-vitrified embryos were maintained in in vitro culture as a control group (n = 102). The warming procedure consisted of placing the straws directly into 5-mL tubes containing 100, 50, 25% WS (WS = 1 M sucrose in TCM-199+ 20% FCS) at 38.6°C (for first solution) and at room temperature for all the rest (5 min each), before being placed into the holding medium. Embryos were recovered from the straws, incubated at 38.6 C in 5% CO2 in air in TCM 199 + 5% FCS, and evaluated for blastocoel re-expansion, embryo survival, and hatching rate at 2, 14, 48 h post-warming. Blastocyst re-expansion (2 h) after warming increased as the developmental stage progressed and was not affected by the vitrification method. In fact, it was significantly (P &lt; 0.05) higher for FB vitrified in the MS and TS methods (77.90% and 71.25%, respectively) compared with the EB method (62.5% and 48.50%, respectively). At 24 h, survival rate of vitrified FB was significantly higher (P &lt; 0.05) in the MS system (95.35%) compared with those in TS (86.25%). Survival rates of FB embryos for both methods (MS and TS) were significantly higher (P &lt; 0.001) than EB embryos vitrified in MS (56.25%) and TS (56.55) methods. After 48 h of culture, the hatching rate for FB vitrified in the MS system (87.21%) was comparable with TS (77.5%) and control (85.3%) groups but significantly higher (P &lt; 0.001) than vitrified EB in MS (43.75%) and TS (36.36%). In conclusion, we showed that a high survival rate of IVP embryos can be achieved by this new in-straw vitrification and warming device (“Sarah”), with hatching rates in vitro comparable with that of control fresh embryos. This method has the potential for use in direct embryo transfer in field conditions.

Label-free characterization of vitrification-induced morphology changes in single-cell embryos with full-field optical coherence tomography.

Vitrification is an increasingly popular method of embryo cryopreservation that is used in assisted reproductive technology. Although vitrification has high post-thaw survival rates compared to other freezing techniques, its long-term effects on embryo development are still poorly understood. We demonstrate an application of full-field optical coherence tomography (FF-OCT) to visualize the effects of vitrification on live single-cell (2 pronuclear) mouse embryos without harmful labels. Using FF-OCT, we observed that vitrification causes a significant increase in the aggregation of structures within the embryo cytoplasm, consistent with reports in literature based on fluorescence techniques. We quantify the degree of aggregation with an objective metric, the cytoplasmic aggregation (CA) score, and observe a high degree of correlation between the CA scores of FF-OCT images of embryos and of fluorescence images of their mitochondria. Our results indicate that FF-OCT shows promise as a label-free assessment of the effects of vitrification on embryo mitochondria distribution. The CA score provides a quantitative metric to describe the degree to which embryos have been affected by vitrification and could aid clinicians in selecting embryos for transfer.

Human embryo cryopreservation-methods, timing, and other considerations for optimizing an embryo cryopreservation program.

The contribution of embryo cryopreservation to the birth rate per in vitro fertilization cycle has escalated from a rare subsidy to a vital tool that is called upon to augment the cycle outcome. Embryology laboratories must identify the embryo stage, quality criteria and methodology that will optimize their ability to preserve each embryo's reproductive potential. This chapter reviews the principles of cryopreservation, outcomes based on embryo stage and cryopreservation method and benchmarks that may be employed by the laboratory to measure the performance of their embryo cryopreservation program.

Pregnancies after Transfer of Vitrified Biopsied Blastocysts

Objective To show two cases describing the successful pregnancies after transplantation of vitrified biopsied embryos after preimplantation genetic diagnosis (PGD). Methods PGD by day 3 embryo biopsy was performed. Excess embryos were frozen using vitrification method. Four months later, the patient elected to undergo a frozen-thawed embryo transfer (FET) cycle. Results These two patients became pregnant and both of them delivered healthy PGD baby respectively. Conclusion This report shows that vitrification is an efficient and practical method for embryo cryopreservation during PGD. Frozen-thawed blastocysts that do survive obtained from vitrified D3 biopsied embryos are able to implant.

49 PRACTICAL APPLICATION OF THE HOLLOW FIBER VITRIFICATION METHOD FOR CRYOPRESERVATION OF MAMMALIAN EMBRYOS

We recently developed the hollow fibro vitrification (HFV) method, which is a novel, high-performance embryo cryopreservation method (Matsunari et al., 2012). In this study, we aimed to verify the applicability of the HFV method for cryopreserving various types of embryos; BDF1 mouse embryos at the 2-cell stage, porcine parthenogenetic morulae derived from in vitro-matured oocytes, bovine morulae produced by in vitro maturation/fertilization (LIAJ Animal Biotechnology Center, Tokyo, Japan), and in vivo-derived blastocysts of common marmosets were vitrified, and their survival was assessed by culture or transfer. The embryos were vitrified using 20 mM HEPES-buffered TCM-199 containing 20% calf serum as a base medium. Cellulose acetate hollow fibres (25 mm) containing 1 to 20 embryos were placed in an equilibration solution containing 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for 5 to 7 min, followed by incubation for 1 min in vitrification solution containing 15% EG, 15% DMSO, and 0.5 M sucrose. The embryos were then vitrified by immersion in LN. The embryos were devitrified by immersing the hollow fibre in a 1 M sucrose solution at 38.5°C, which was followed by stepwise dilution of the cryoprotectants and washing. For a subset of the vitrified mouse embryos, rewarming in a non-ultra-rapid manner by melting a hollow fibre in air at room temperature for 5 s was tested. Embryo transfer was performed to assess the viability of the vitrified mouse embryos. For porcine embryos, vitrification in LN vapor (–150°C) was tested. Development of the vitrified mouse embryos to blastocysts was equal to that of the non-vitrified embryos [105/110 (95.5%) v. 109/110 (99.1%)]. Post-transfer development to fetuses was also equal between the vitrified and non-vitrified embryos [pregnancy rates: 4/4 v. 2/2; developmental rates: 55/80 (68.8%) v. 35/40 (87.5%)]. Non-ultra-rapid rewarming did not decrease the survival of the vitrified mouse embryos [blastocysts: 94/100 (94.0%); pregnancy: 4/4; fetuses: 55/80 (68.8%)]. Blastocyst formation was equivalent for vitrification of porcine embryos in LN vapor [27/34 (79.4%)], direct immersion into LN [28/35 (80.0%)], and the non-vitrified control [31/32 (96.9%)]. Vitrification of 191 bovine morulae resulted in 153 (80.1%) blastocysts. In preliminary experiments, survival of marmoset blastocysts was 100% (n = 6). These data demonstrate that the HFV method is (1) effective for embryos of various species and production methods; (2) effective even for porcine in vitro-derived morulae, which are highly cryosensitive; and (3) amenable to modifications such as non-ultra-rapid warming and cooling in LN vapor, increasing the potential applicability of the HFV method. For instance, vitrification in LN vapor may allow embryo cryopreservation with high hygienic standards. This study was supported by JST, ERATO, Nakauchi Stem Cell and Organ Regeneration Project.

Perinatal outcomes after ART with transfer of cryopreserved embryos

The number of children born after ART, which includes cryopreservation methods, is steadily rising worldwide. Data on perinatal outcomes after transfer of cryopreserved embryos is presented in the article. Analysis of the influence of fertilization method — IVF/ICSI, embryo cryopreservation method — slow freezing method and vitrification is composed.

67 A HOLLOW FIBER VITRIFICATION METHOD ENABLES CRYOBANKING OF IN VITRO-MATURATION/IN VITRO-FERTILIZATION-DERIVED TRANSGENIC PIG EMBRYOS

We have recently developed a novel high-performance embryo cryopreservation method: the hollow fiber vitrification (HFV) method (Matsunari et al. 2012 J. Reprod. Dev., in press). In this study, we aimed to demonstrate the utility of the HFV method for the cryopreservation of transgenic pig embryos produced by in vitro oocyte maturation/fertilization (IVM/IVF). In vitro-matured oocytes were inseminated with cryopreserved epididymal sperm (Kikuchi et al. 1998 Theriogenology 50, 615–623) from a transgenic pig carrying the humanized Kusabira-Orange gene (Matsunari et al. 2008 Cloning Stem Cell 10, 313–323) and then cultured for 96 h. Morulae with normal morphology were divided into the vitrification and nonvitrification groups. The vitrification of embryos was performed by the HFV method using 20-mM HEPES-buffered TCM199 containing 20% calf serum as a base medium. Cellulose acetate hollow fibers (25 mm), each containing 10 to 20 embryos, were placed in an equilibration solution containing 7.5% ethylene glycol and 7.5% dimethyl sulfoxide for 5 to 7 min and were then placed for 1 min in the vitrification solution containing 15% ethylene glycol, 15% dimethyl sulfoxide, and 0.5 M sucrose. The embryos were then vitrified by immersion in liquid nitrogen and held there for 5 to 10 min. The embryos were warmed by immersing the hollow fiber in a 1-M sucrose solution at 38.5°C, followed by a stepwise dilution of the cryoprotectants using 0.5-M sucrose solution (3 min) and the base medium (10 min). Vitrified and nonvitrified embryos were cultured for 40 h, and their development into blastocysts was evaluated. The in vitro development of vitrified embryos to the blastocyst stage was compared with that of the nonvitrified controls on Day 6. In the embryo-transfer experiments, blastocysts at either Day 5 or Day 6 from both the vitrification and nonvitrification groups were transferred to 3 recipient gilts per group (25–32 blastocysts/gilt), and their development through farrowing was compared. To test long-term preservation, some of the vitrified morulae were kept in liquid nitrogen for 43 days, and their development to Day 30 fetuses was evaluated after transfer to an additional recipient. The differences in proportional data between the 2 groups were analyzed with the χ2-test. Of the 393 putative zygotes obtained by IVM/IVF, 169 (43.0%) developed into morulae. In vitro development of the vitrified morulae to blastocysts (66/85, 77.6%) was comparable with that of the nonvitrified morulae (67/84, 79.8%, not significant: NS). The embryo-transfer experiments resulted in pregnancy in all 6 of the recipients. The production efficiency of piglets (piglets/embryos transferred) was 17/88 (19.3%) for the vitrification group and 27/88 (27.7%, NS) for the nonvitrification group. Approximately 50% of the offspring in both groups were transgenic. Long-term cryopreservation using the HFV method resulted in similar piglet production efficiency (7 piglets produced out of 32 embryos transferred). This study demonstrated for the first time that the HFV method effectively cryopreserves IVM/IVF-derived transgenic pig embryos. Supported by the JST CREST program.

74 MECHANICAL DELIPIDATION IMPROVES CRYOSURVIVAL AND IN VITRO DEVELOPMENT OF VITRIFIED CAT OOCYTES

Although considerable progress has been made in the development of successful methods for cryopreservation of embryos, oocytes are much less cryotolerant. There appears to be an inverse relationship between cryosurvival and intracellular lipid levels. For example, cat oocytes, which appear microscopically as coffee-coloured, nearly opaque spheres due to their high lipid content, are extremely sensitive to cryopreservation. Oocyte delipidation thus represents a potential approach to improving cryosurvival. The objectives of the present study were to examine 1) the effects of calcium (Ca2+, 0 v. 10 nM), FBS (0 v. 10%), and cytochalasin B (CB, 7.5 v. 20.0 μg mL–1) during mechanical delipidation by high-speed centrifugation on in vitro development of IVM cat oocytes, and 2) the influence of centrifugation, degree of lipid polarization (partial v. full), and co-culture with cat fetal fibroblasts (CFF) on in vitro development of vitrified IVM cat oocytes. In Experiment 1, oocytes were randomly allocated to each centrifugation medium and centrifuged at 12 000 × g for 20 min. Oocytes were then fertilized with epididymal sperm (motile sperm mL–1) and cultured until Day 8 (Pope et al. 2006 Theriogenology 66, 59–71). In Experiment 2, oocytes were centrifuged with the optimal centrifugation medium obtained in experiment 1, allocated to each treatment and vitrified in a solution of 15% DMSO, 15% ethylene glycol, and 18% sucrose (2008 Reprod. Fertil. Dev. 20, 188). Liquified oocytes were fertilized and cultured until Day 8. In both experiments, cleavage and degeneration rates were determined on Day 2 and blastocyst development on Day 8. Data were analysed by 2-way ANOVA and chi-square tests. In Experiment 1, of 939 oocytes that were centrifuged and fertilized, 40% of those treated in 0 nM Ca2+ cleaved and 22% developed into blastocysts, v. 33 and 6%, respectively, in 10 nM Ca2+ (P &lt; 0.05). The respective cleavage and degeneration frequencies for oocytes treated in 10% FBS were 43 and 19% v. 19 and 3% in 0% FBS (P &lt; 0.05). Cleavage and blastocyst development after treatment with 7.5 and 20.0 μg mL–1 CB were 36 and 15% v. 42 and 22%, respectively. In Experiment 2, 493 oocytes were vitrified/liquified and fertilized. The degeneration, cleavage, and blastocyst rates of non-centrifuged oocytes were 49, 21, and 0% v. 31 (P &lt; 0.05), 38 (P &lt; 0.05), and 7%, respectively, of centrifuged oocytes. Of centrifuged oocytes with partially extruded lipids, 34% degenerated, 34% cleaved, and 4% developed into blastocysts v. 29, 42, and 10%, respectively, of oocytes with fully extruded lipids. Degeneration, cleavage and blastocyst rates of co-cultured v. control oocytes were 18, 36, and 10%, v. 26 (P &lt; 0.05), 34, and 3%, respectively. In summary, cryotolerance of domestic cat oocytes to vitrification was 1) affected by their lipid content, and 2) improved by mechanical reduction of intracellular lipids. When oocytes were fully delipidated in Ca2+-free medium containing 10% FBS and 20.0 μg mL–1 CB before vitrification and co-cultured after IVF with CFF, blastocyst development was similar to that of control, non-vitrified oocytes.

Cryopreservation of Embryos in Laboratory Species

Cryopreservation of embryos is an important strategy for the conservation of species and valuable strains of laboratory species. Historically, in the mouse, the technical development of cryopreservation started with slow freezing methods in the 1970's, which was then followed by vitrification method developed in the 1980's. Vitrification is advantageous in its quickness and simplicity, because it does not need programmable controlled-rate freezing machines. Furthermore, the survivability of embryos recovered after vitrification is significantly improved by avoiding chilling injury and intra- and extra-cellular ice formation. Recently, a series of new vitrification methods using a minimal volume of cryopreservation agent (CPA) and extremely high osmolality CPAs have been developed. For laboratory rats and mice, there is a long history of embryo cryopreservation and huge numbers of embryos have been kept frozen in cryobanks. In contrast, the techniques have only recently been optimized for other laboratory animals, e.g., rabbits, Syrian hamsters, Mongolian gerbils and mastomys (African rodent). Besides safety in cr yopreservation, simple transportation of vitrified embryos on dry ice has been a challenging issue. By using the extremely high osmolality CPAs, we are now examining the feasibility and effectiveness of a simple method for the transportation of vitrified embryos. Development of new cryopreservation methods for embryos of laboratory species should be an integral part of the technological logistics supporting the development of biomedical

Cryopreservation of Mammalian Oocyte for Conservation of Animal Genetics

The preservation of the female portion of livestock genetics has become an international priority; however, in situ conservation strategies are extremely expensive. Therefore, efforts are increasingly focusing on the development of a reliable cryopreservation method for oocytes, in order to establish ova banks. Slow freezing, a common method for cryopreservation of oocytes, causes osmotic shock (solution effect) and intracellular ice crystallization leading to cell damage. Vitrification is an alternative method for cryopreservation in which cells are exposed to a higher concentration of cryoprotectants and frozen with an ultra rapid freezing velocity, resulting in an ice crystal free, solid glass-like structure. Presently, vitrification is a popular method for cryopreservation of embryos. However, vitrification of oocytes is still challenging due to their complex structure and sensitivity to chilling.

Slow freezing, vitrification and ultra-rapid freezing of human embryos: a systematic review and meta-analysis

Embryo cryopreservation is an important aspect of assisted reproduction. Many methods have been described, but they have been poorly investigated in randomized trials, highlighting the need for a systematic review of the literature. Meticulous electronic/hand searches were performed to locate randomized trials (RCT) comparing embryo cryopreservation methods. Primary outcomes were clinical pregnancy rate (CPR) and incidence of congenital abnormalities. Secondary outcomes included live-birth (LBR), ongoing pregnancy (OPR), implantation (IR), and miscarriage (MR) rates. Data were extracted to allow for an intention-to-treat analysis and analysed using a random-effects model. Literature search revealed 11 RCT, of which five were excluded. The quality of the included studies was variable, but generally poor. There was a significantly higher CPR, OPR and IR with vitrification compared with slow freezing (odds ratio (OR)=1.55, 95% confidence interval (CI)=1.03-2.32, OR=1.82, 95% CI=1.04-3.20 and OR=1.49, 95% CI=1.03-2.15, respectively). In addition, there was a significantly lower CPR and OPR with embryo ultra-rapid freezing compared with slow freezing (OR=0.35, 95% CI=0.16-0.76 and OR=0.37, 95% CI=0.17-0.81, respectively). Vitrification is superior to slow freezing, which in turn is superior to ultra-rapid freezing. However, more well-designed and powered studies are needed to further corroborate these findings.

Vitrification is a highly efficient method to cryopreserve human embryos in in vitro fertilization patients at high risk of developing ovarian hyperstimulation syndrome

This study aimed to evaluate the efficacy of embryo vitrification as an emergency procedure for patients at high risk of developing ovarian hyperstimulation syndrome (OHSS). A total of 69 embryos, derived from 24 patients for whom embryo transfer could not be performed because of the risk of developing OHSS, were vitrified and warmed for deferred embryo transfer. Surviving embryos were transferred, resulting in 10 clinical pregnancies, of which 4 were successfully delivered and the remaining 6 are still ongoing.