Post-thaw Survival Rates Research Articles

When Should We Recommend Egg Freezing and When Is It Too Late?

Social oocyte freezing (SOF), a fast-growing field in ART is the elective cryopreservation of one’s own oocytes at a younger age when oocytes are of higher quality with the goal of prolonging a woman’s childbearing opportunity without any urgent medical reasons. The optimum age for SOF is said to be below the age of 35 years ( ASRM , 2013 ; ESHRE , 2012 ). SOF in women above 35 years of age is associated with lower post-thaw survival rates, a lower live birth rate per thawed oocyte, and an increased overall cost per livebirth as more cycles of oocyte freezing and thawing may be required. Beyond 42 years of age, SOF is unlikely to result in a livebirth due to suboptimal response to stimulation and a higher cycle cancellation rate ( ASRM , 2013 ; Doyle, et al. , 2016 ; Wennberg, et al. , 2019 ). The number of oocytes needed to freeze varies widely between studies but should be individualized according to the woman’s ovarian reserve as well as the IVF center’s own success rate data ( Teo, et al. , 2021 ). The utilization rate of frozen oocyte is low, ranging from 6 to 15%. The main reasons cited are a) women not wanting to start a family without a partner; b) preference for natural conception and, c) refusal to use a sperm donor ( Hammarberg, et al. , 2017 ). With the use of relatively safe IVF techniques and success rates similar to that of fresh oocytes, SOF has become an acceptable reproductive option for women today to extend their reproductive ability to achieve parenthood using their own eggs. It is, however, expensive and more than one cycle of oocyte freezing may be required to improve its efficacy. Thorough counselling must be done to advise women regarding the lack of robust evidence on its efficacy, long-term risks, and costs.

P-138 Protein concentration of 5 mg/mL in recovery medium for thawed blastocysts is equivalent to 10 mg/mL in post-thaw survival and pregnancy rates

Abstract Study question Are comparable survival and pregnancy rates obtained when recovery media are supplemented with 5mg/mL protein versus customary 10mg/mL protein in Frozen Embryo Transfer cycles? Summary answer Recovery medium containing 5mg/mL protein compared to 10mg/mL yielded similar outcomes in blastocyst survival and pregnancy rates. What is known already Accepted practice has been to culture thawed blastocysts in medium with the same protein concentration as the vitrification media (20% protein) to minimize osmotic stress and optimize survival and implantation rates. The potential to acquire similarly positive outcomes with supplementation of only 5 mg protein/ml has been explored and demonstrated, challenging the requirement for high protein levels in recovery media. Study design, size, duration From July through August 2022, a prospective study was conducted in a university laboratory to compare the survival and implantation rates in Frozen Embryo Transfer cycles using vitrified blastocysts recovered in Continuous Single Culture–NX Complete (CSCM-NXC) (5mg/mL Human Serum Albumin (HSA)) or global HP[LW1] (8.8mg/mL HSA and 1.2mg/mL α-and ß-globulins). 50 blastocysts were warmed per condition. Comparative analyses included survival and implantation rates, beta HCG levels, and clinical pregnancy rates by fetal cardiac activity. Participants/materials, setting, methods To evaluate the effect of protein level in recovery medium for vitrified blastocysts, CSCM-NXC and global HP, with 5mg/mL and 10mg/mL total protein, respectively, were compared. Blastocysts were vitrified in Irvine Scientific Vit Kit – Freeze and were warmed in Irvine Scientific Vit Kit – Warm. Minimum recovery time was the same for all blastocysts (30 minutes), and is defined as the time from completion of the warming procedure to the time of embryo transfer. Main results and the role of chance In both study arms, one blastocyst was removed from the data analysis due to embryo transfer cancellation that was not related to embryo survival or suitability for transfer, resulting in N = 49 for each group. Chi-square analysis was performed to assess statistical significance of all compared parameters, and a p value < 0.05 was considered significant. Average patient age (years) for CSCM-NXC was 36.1 and for global HP, 35.0. There was no difference between blastocysts warmed in CSCM-NXC and global HP in average recovery time, i.e., 3.8 and 3.9 hours, respectively, or survival rates, 98.4% and 98.1% respectively. Comparison of patients with blastocysts warmed in CSCM-NXC reflected a trend towards higher outcomes compared to those in global HP with initial positive beta HCG rates of 64.6% vs 56.3% and ongoing clinical pregnancy rates of 52.1% vs 45.8%, respectively. Implantation rates were 56.1% and 57.7% in CSCM-NXC and global HP, respectively. Incidence of monozygotic twinning of 0% in CSM-NXC and 3.3% in Global HP was observed. Limitations, reasons for caution As this is a challenge to accepted formulation of recovery media for vitrified blastocysts, specifically with respect to protein supplementation - a strategy for minimizing osmotic shock and potential damage to warming embryos - more expansive and robust studies must be undertaken to increase the statistical power of this study. Wider implications of the findings As frozen embryo transfers are utilized more routinely in assisted reproductive treatment, the clinical finding that a commercial recovery medium containing of 5mg/mL protein, the standard for routine embryo culture, can support the recovery of vitrified embryos and lead to successful pregnancy could increase the efficiency in the laboratory. Trial registration number na

Impact of Post-Thaw Incubation Time of Frozen Embryos on Clinical Pregnancy Rate.

Vitrification of embryos following a single-controlled ovarian stimulation has been the strategy practised now in many in vitro fertilisation clinics to minimise the risk of early ovarian hyper stimulation syndrome, to reduce multiple pregnancy rates and to improve cumulative pregnancy rates. In recent years, advances in vitrification techniques and improved culture conditions have led to good post-thaw embryo survival rates, thereby increasing pregnancy rates of frozen embryo transfer (FET) cycles. The aim of this study was to analyse the effect of post-thaw incubation time of frozen embryos on the clinical pregnancy rates (CPRs) of frozen embryo transfer (FET) cycles. This was a retrospective, comparative study done at a teaching hospital in assisted reproductive treatment. Three hundred and ten FET cycles were analysed, of which 125 had day 2 freezing and 185 had day 3 freezing. Depending upon the day of thawing and day of transfer, FET cycles were divided into six groups: Group 1 (day 2 thawing and day 3 transfer), Group 2 (day 2 thawing and day 4 transfer), Group 3 (day 2 thawing and day 5 transfer), Group 4 (day 3 thawing and day 3 transfer), Group 5 (day 3 thawing and day 4 transfer) and Group 6 (day 3 thawing and day 5 transfer). Statistical analysis was performed using version 14 R software version 4.0.1 (2020-06-06) (R foundation for Statistical Computing, Vienna, Austria). A P < 0.05 is taken as significant. The CPR of Group 4 was 42.4% which was more than that of the other groups but it did not reach statistical significance. Short incubation time of 2-4 h is as effective as an extended incubation time in terms of CPRs of FET cycles.

Field performance of cryopreserved seed-derived tomato plants and post-thaw survival of viral-infected meristems

The effectiveness of different cryopreservation techniques of tomato meristems isolated from viral-infected plants ‘Irishka’ cultivar was determined. The pieces of stem were protected with dimethyl sulfoxide and propylene glycol and cooled in vapour phase of liquid nitrogen (–170 °C). For the vitrification and droplet-vitrification protocols, the meristems were treated with loading solution and dehydrated with different plant vitrification solutions (PVS1 modified, PVS2, 88 % PVS3, PVSN). The samples were placed to sterilized aluminum foil pieces, in 1.2 ml cryovials or in 50 µl aluminum pans for differential scanning calorimetry and were directly immersed into liquid nitrogen. Acсording to the dehydration technique, the meristems were dehydrated with sterile airflow for 120 min. The post-thaw survival rates of meristems (from 34.2 to 78.5 %) were observerved only for 50 µl aluminum pans and airflow dehydration. We determined the productivity of plants, obtaned from cryopreserved seeds (‘Seven’, ‘Potiron Ecarlate’ and ‘Druzhba’ cultivars). We observed increasing in total and marketable yields for the plants grown from the cryopreserved seeds for all the cultivars. Total number of diseased plants decreased by 33 % for ‘Seven’, for ‘Potiron Ecarlate’ it did by 6.7 %, for that of ‘Druzhba’ the total percentage of sick and healthy plants did not differ after seeds cryopreservation.

Long-term sperm cryopreservation does not affect post-thaw survival rates.

Objective:To compare cryosurvival rates of human spermatozoa in a prolonged period of cryopreservation.Methods:This retrospective study involved 33 cryopreserved semen samples from patients with cancer, between 2002 and 2011. The semen sample was obtained by masturbation and initial semen analysis was performed. The cryoprotectant solution was added and samples were frozen in liquid nitrogen in a slow step-wise process. For thawing, the samples were incubated at 25.0ºC for 15 min, followed by incubation at 36.7ºC for 15 min. The cryosurvival rate (CS) was calculate by CS= [(% total motile sperm post-thaw) x100/(% total motile sperm/tube)]. Each study sample was divided into three aliquots (Study Group; n=23): (I) official patient sample, which was kept cryopreserved for subsequent Assisted Reproduction procedure, cryopreserved between 2002 and 2011; (II) sample destined to post-thaw tests, performed after the sample had been kept cryopreserved for 24 hours; and (III) study sample. Only in 2014, after 3-12 years of cryopreservation, the study samples were thawed and evaluated. To validate the study design, a Validation Group was created including 10 samples obtained between 2014 and 2016, using the same methodology in the study samples. The data was analyzed using the T-test, with a significant p-value of 5%.Results:The mean age was 29.93±9.57 years in the Study Group and 21.80±6.49 years in the Validation Group. No significant difference between the Validation and Study Groups was found in the initial semen analysis (p>0.05). After 24 hours of cryopreservation, the cryosurvival rate was 26.11±46.36% in the Study Group and 23.71±57.06% in the Validation Group. Aliquots of the same sample preserved from 3-12 years demonstrated 23.71±57.06% of cryosurvival rate. Thus, no significant difference was found vis-à-vis the cryosurvival rates (p=0.56).Conclusion:We concluded that the method introduced in the late 1990s, which enables the removal of debris, potentially toxic elements and generators of reactive oxygen species from the seminal sample before cryopreservation, exhibited efficiency in maintaining the same cryosurvival rate after an extended period.

Strategies to improve the fertility of frozen-thawed boar semen for artificial insemination

Although cryopreservation of boar semen for artificial insemination (Al) was developed 35 years ago, cryopreservation conditions and Al strategies are still considered sub-optimal. Al with excessive numbers of frozenthawed sperm (5-6 x 109 cells), still does not achieve fertility levels similar to Al using liquid semen because of reduced sperm survival. Frozenthawed (FT) spermatozoa have therefore not been the preferred option for commercial breeding programmes. However, substantial progress has been made regarding boar sperm cryopreservation. Adjustment of cooling and re-warming rates to biophysical properties of boar spermatozoa, new sperm package systems and the achievement of accurately consistent freezing of large numbers of samples using programmable freezers have contributed to post-thaw survival rates above 50%, a threshold similar to that used for bull AI-semen. Moreover, these post-thaw sperm survival rates are consistent within a large population of boars selected for sperm freezability potential, as occurs with Al-bull sires. When such post-thaw boar semen is deposited intra-utero, acceptable fertility (in terms of farrowing rates and litter size) is obtained. Currently, the most effective application of FT-semen for Al is achieved using deep uterine-Al (DUI) which allows placement of a minimal semen dose (in volume 0.5 to 10 mL and sperm number 0.5 to 1 x109 total spermatozoa) into the anterior 1/3 of one uterine horn, with levels of fertility close to Al with liquid semen. However, owing to their shorter life span, FT-boar spermatozoa require an AI-to-ovulation interval not longer than 4-6 h, making peri-ovulatory Al a pre-requisite to obtain the highest possible fertility. Spontaneous ovulation most often occurs when two-thirds of oestrus has passed. Estimation of the duration of oestrus, taking into account the weaning-to-oestrus interval, is helpful when establishing appropriate AI-schedules. However, as the length of oestrus varies within and between farms, different Al strategies should be established a priori. The development of bio-sensors for spontaneous ovulation will widen the use of Al with frozen-thawed frozen semen.

211 EFFECT OF AMNIOTIC FLUID DERIVED STEM CELL-CONDITIONED MEDIUM ON DEVELOPMENT AND POST-THAW SURVIVAL OF IN VITRO-PRODUCED HOLSTEIN EMBRYOS

In vitro-produced (IVP) embryos have altered metabolism with lower blastocyst rates and higher lipid accumulation compared with IVP embryos. Culturing embryos with amniotic fluid derived stem cell-conditioned media (AFS) may help mimic in vivo conditions and improve embryo development. We hypothesised that culturing embryos with AFS will improve in vitro culture conditions and developmental competence. The IVP embryos were produced in 5 replicates by aspirating oocytes (n = 1469) of 2 to 8 mm from abattoir ovaries. Oocytes were matured for 23 h, fertilized with semen from 1 of 2 bulls, and cultured in a novel SOF medium (SOF for conventional freezing media) in 38.5°C in 5% O2, 5% CO2, and 90% N2. Media was supplemented with either 0 (control), 5, or 10% AFS. Stage 7 blastocysts were stained with 1 µg mL−1 of Nile Red and 300 nM Mitotracker Red CMX-Rosamine (Molecular Probes Inc., Eugene, OR, USA) to measure lipid content and mitochondrial polarity, respectively. Ten images per stained embryo were acquired by confocal microscopy using a 5 µM step size at 40× magnification. Remaining blastocysts were slow frozen using 2 min of dehydration in 0.6 M sucrose before equilibration for 10 min in a conventional freezing medium (1.5 M ethylene glycol and 0.5 M sucrose). Embryos were thawed and re-expansion was assessed at 24 and 48 h. Data were analysed by general linear model using a one-way ANOVA with means separated by Tukey’s HSD. Results (Table 1) indicated no difference in development, lipid content, mitochondrial polarity, or post-thaw survival (P &gt; 0.01), suggesting that growth factors present in AFS did not improve culture conditions. Table 1. Main effect of AFS on embryo developmental competence and post-thaw survival rates

92 DOES BLASTOCENTESIS AFFECT CRYOPRESERVATION SURVIVAL OF IN VITRO-PRODUCED BOVINE EMBRYOS?

The cattle industry primarily employs embryo bisection in order to obtain genetic samples for pre-implantation screening and selection of embryos. Although practical and rapid, bisection is invasive and adversely affects embryo viability and cryopreservation. An alternative biopsy approach is to aspirate the blastocoele fluid (referred to as blastocentesis), which not only provides a genetic sample, but also has the potential to improve cryopreservation (Palini et al. 2013 Repro. Biomed. 26, 603–610). This study investigates blastocentesis as a low impact biopsy procedure to rapidly sample bovine blastocysts with limited effect on embryo cryopreservation survival. In vitro-produced embryos were selected at expanded blastocyst stage and placed in a 50-μL drop of holding media on an inverted microscope. The embryo was held using a glass holding pipette attached to a micromanipulator, oriented so that the inner cell mass was toward the bottom of the view. A 7-μm spiked intracytoplasmic sperm injection pipette attached to the other micro-manipulator was used to pierce the blastocoele cavity and aspirate the blastocoele fluid. Once removed, the aspirate was transferred into 4-μL TE buffer for later genetic analysis. Collapsed blastocysts were then vitrified in ~7 μL 16.5% ethylene glycol, 16.5% dimethyl sulfoxide in TCM-199 (Hanks salts) with 20% FCS and 0.5 M sucrose. Embryos were held for a minimum of 1 week and then thawed and assessed for survival. Post-cryopreservation embryo survival was measured as the proportion of embryos that re-expanded after 48 h in culture. One-way ANOVA was used for statistical testing. A total of 181 control (intact) and 182 blastocentesis embryos were vitrified over 6 replicates. In all but one replicate, non-biopsied control embryos had higher re-expansion rates. Overall, the re-expansion rate was significantly (P = 0.05) higher for control embryos (73.5%) than blastocentesis embryos (61.5%) (Table 1). Initial experiments would suggest embryo survival is affected by the biopsy procedure; however, because this was not the case with every replicate, this may be batch or technician/human error dependent. Further study is required to assess full effect of blastocoele fluid aspiration on embryo cryopreservation, particularly investigating effectiveness for in vivo-produced embryos and subsequent effect on pregnancy rates. Likewise, further investigation is required to assess whether the sample collected is sufficient to allow accuracy over a variety of genetic tests. More than 20 embryos can easily be sampled in an hour using this technique, making it a rapid and efficient process. Given the speed and compatibility with cryopreservation, this sampling procedure may offer an alternative to current techniques used for cattle embryo genetic assessment. Table 1. Post-thaw survival rates of in vitro-produced embryos vitrified after blastocentesis1

Expression of the T85A mutant of zebrafish aquaporin 3b improves post-thaw survival of cryopreserved early mammalian embryos.

While vitrification has become the method of choice for preservation of human oocytes and embryos, cryopreservation of complex tissues and of large yolk-containing cells, remains largely unsuccessful. One critical step in such instances is appropriate permeation while avoiding potentially toxic concentrations of cryoprotectants. Permeation of water and small non-charged solutes, such as those used as cryoprotectants, occurs largely through membrane channel proteins termed aquaporins (AQPs). Substitution of a Thr by an Ala residue in the pore-forming motif of the zebrafish (Dario rerio) Aqp3b paralog resulted in a mutant (DrAqp3b-T85A) that when expressed in Xenopus or porcine oocytes increased their permeability to ethylene glycol at pH 7.5 and 8.5. The main objective of this study was to test whether ectopic expression of DrAqp3b-T85A also conferred higher resistance to cryoinjury. For this, DrAqp3b-T85A + eGFP (reporter) cRNA, or eGFP cRNA alone, was microinjected into in vivo fertilized 1-cell mouse zygotes. Following culture to the 2-cell stage, appropriate membrane expression of DrAqp3b-T85A was confirmed by immunofluorescence microscopy using a primary specific antibody directed against the C-terminus of DrAqp3b. Microinjected 2-cell embryos were then cryopreserved using a fast-freezing rate and low concentration (1.5 M) of ethylene glycol in order to highlight any benefits from DrAqp3b-T85A expression. Notably, post-thaw survival rates were higher (P<0.05) for T85A-eGFP-injected than for -uninjected or eGFP-injected embryos (73±7.3 vs. 28±7.3 or 14±6.7, respectively). We propose that ectopic expression of mutant AQPs may provide an avenue to improve cryopreservation results of large cells and tissues in which current vitrification protocols yield low survival.

The freezing method of cleavage stage embryos has no impact on the weight of the newborns.

The aim of this study was to study the effect of the embryo freezing method on the birth weight of newborns from frozen embryo transfer (FET) cycles, and the pregnancy results of cleavage stage embryos cryopreserved by slow freezing or vitrification. This is a retrospective cohort study undertaken in a University Hospital IVF unit using concurrently both the slow-freezing and the vitrification techniques. All frozen-thawed and vitrified-warmed day 2 and day 3 embryo transfers during the time period from 1 April 2009 to 31 November 2013 were included in the study. There was no statistically significant weight difference between newborns from vitrified or slow-frozen embryos (3588 vs 3670 g). A higher post-thaw viability rate was achieved after cryopreservation by the vitrification technique compared to the slow-freezing protocol (83.4 vs 61.4%). The miscarriage rate was lower in the vitrification group (15.7 vs 29.0%). The live birth rates were similar (19.5 vs 19.1%) in the slow-freezing and vitrification groups, respectively. Among vitrified embryos, 7.4 embryos needed to be thawed to produce one delivery; in the slow-freezing group, that number was 11.9. The freezing method has no impact on the weight of the newborn. With lower post-thaw survival rates and higher miscarriage rates, the slow-freezing cryopreservation protocol is inferior to the vitrification technique.

In vitro culture of in vivo Saanen goat embryos by vitrification

The purpose of this study was to investigate the survival rate of high-quality embryos (n: 101) derived in vivo from Saanen goats (n: 15) in culture following their vitrified freezing and thawing during the breeding season. The in vitro postthaw survival rates of 101 vitrified embryos at 24, 48, and 72 h were 59.4%, 33.6%, and 25.7%, respectively. According to the developmental stage of the embryos, the survival rates were 51.3%, 20.5%, and 15.4% in the morula, and 64.5%, 41.9%, and 32.3% in the blastocyst, respectively. The survival rates of the blastocysts and morulae at 48 and 72 h were significantly different (P < 0.05). While the survival rates of the embryos of Grade 1 quality at 24, 48, and 72 h were 78.6%, 46.4%, and 32.1%, Grade 2 quality survival rates were 35.5%, 17.8%, and 17.8%, respectively. The survival rates of Grades 1 and 2 embryos at 24 h (P < 0.001) and 48 h (P < 0.05) were significantly different. As a result, it was concluded that the embryos to be frozen should be selected according to their development stage and quality, and preferably should be of Grade 1 quality in the blastocyst stage for the cryopreservation of in vivo-derived goat embryos by vitrification.

193 IMPROVED POST-THAW SURVIVAL OF BOVINE EMBRYOS PRODUCED IN SERUM-FREE IN VITRO PRODUCTION SYSTEM

Over a few decades the bovine in vitro embryo production (IVP) systems have been improving rapidly. Still, the goal to produce the same quality embryos in vitro as in vivo has not yet been reached. The FCS is usually added to media during IVP to provide growth factors and energy sources. Currently, serum-free culture systems are often preferred due to the lower risk of contamination and prevention of the development of large offspring syndrome. The aim of this study was to establish whether complete elimination of FCS from the bovine IVP system has an effect on blastocyst rates, embryo quality, and embryo survival rates after slow freezing. We replaced our conventional in vitro maturation (IVM) medium [tissue culture medium-199, 10% (v/v) FCS, 10 µg mL–1 epidermal growth factor (EGF), 1500 U mL–1 serum gonadotropin and chorionic gonadotropin (PG600), Na-pyruvate 0.5 mM, gentamycin sulfate 50 µg mL–1 and l-glutamine 1 mM] with SOF (SOFaaci) supplemented with 0.4% fatty acid-free BSA fraction V, 10 µg mL–1 EGF, and 1500 U mL–1 PG600. Matured cumulus-oocyte complexes (COC) from both experimental groups (total of 1145 from serum-free IVP and 687 from our conventional IVP system) were used for in vitro fertilisation and culture. Blastocyst rates were similar in the serum-free and our usual IVP protocol, 18 and 22%, respectively. Seventy-seven Grade 1 (according to IETS) Day 7 blastocysts from the serum-free IVP system and 80 Grade 1 Day 7 blastocysts from our conventional IVP system were frozen in 1.5 M ethylene glycol and 0.1 M sucrose containing cryopreservation medium. The post-thaw survival rates after 24 h of culture and evaluated as percentages of re-expanded embryos were 63.6% for the serum-free IVP and 46.3% for the conventional IVP system (P &lt; 0.05, Z Test for 2 population proportions). These results indicate that it is possible to have a completely serum-free bovine IVP system and based on the slow freezing and thawing results the quality of serum-free IVP embryos might be better than of the embryos matured in our conventional maturation media. However, more experiments and increased sample sizes are needed to confirm the results. This study was supported by Project 3.2.0701.12–0036 of Archimedes Foundation, AP 2.4 of CCRMB, and institutional research funding (IUT 08–01) of the Estonian Ministry of Education and Research.

47 EFFECT OF L-CARNITINE TREATMENT DURING OOCYTE MATURATION ON THE POST-THAW DEVELOPMENT OF PORCINE EMBRYOS VITRIFIED AT THE PRONUCLEAR STAGE

The extreme cryo-sensitivity of porcine oocytes and embryos is attributed to their endemically high content of cytoplasmic lipid droplets. In attempts to improve the cryo-tolerance of porcine embryos, various strategies have been used to reduce the amount of lipid droplets present in the cytoplasm before vitrification. Recently, the cryo-tolerance of bovine oocytes vitrified at the metaphase II stage was improved by supplementing in vitro maturation (IVM) medium with l-carnitine (LC), a stimulator of lipid metabolism (Chakitisakul et al. 2013 Theriogenology 79, 590–598). The objective of this study was to determine the effect of supplementing IVM medium with LC on the post-thaw development of porcine embryos vitrified at the pronuclear stage. Oocytes recovered from the ovaries of prepubertal gilts were matured in modified porcine oocyte medium supplemented with 0 (control) or 12 mM LC during the final 22 h of IVM. Following IVF, presumptive zygotes were cultured in porcine zygote medium-3. At the pronuclear stage, cohorts of embryos from each group were either vitrified using a solid surface vitrification procedure (Somfai et al. 2009 Biol. Reprod. 80, 42–49) or cultured for 7 d without being vitrified. Vitrified zygotes were subsequently warmed and cultured for 7 d. The rates of cleavage, blastocyst formation, and hatching were recorded, and all blastocysts were stained to determine the total cell numbers. Three replicates were performed. Proportional data were arcsine transformed and subjected to ANOVA, and cell number data were analysed by t-test. The post-thaw survival rates of the embryos that were vitrified did not differ between the groups (control: 95.7%; LC: 97.1%; P &gt; 0.05). There were no significant effects of LC treatment or vitrification on the rates of cleavage, blastocyst formation, and hatching (Table 1). Vitrified embryos derived from LC-treated oocytes produced blastocysts with fewer cells than vitrified embryos derived from untreated oocytes (Table 1; P &lt; 0.05). In contrast to previous findings in other species, the results indicate that supplementing IVM medium with LC did not enhance the post-thaw development of porcine embryos vitrified at the pronuclear stage. Table 1.Effect of l-carnitine (LC) treatment and vitrification on porcine embryo development C. Grupen was supported by an OECD Fellowship.

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.

Freezing of Fresh Wharton's Jelly From Human Umbilical Cords Yields High Post-Thaw Mesenchymal Stem Cell Numbers for Cell-Based Therapies.

Some cord blood banks freeze entire pieces of UC (mixed cord, MC) which after post-thaw yields mixed heterogeneous populations of mesenchymal stem cells (MSCs) from all its microanatomical compartments. Freezing of such entire tissues results in sub-optimal post-thaw cell recovery because of poor cryoprotectant diffusion and intracellular ice-formation, heat and water transport issues, and damage to intercellular junctions. To develop a simple method of harvesting pure homogeneous MSCs for cord blood banks, we compared the post-thaw behavior of three groups of frozen UC tissues: (i) freshly harvested WJ without cell separation; (ii) MSCs isolated from WJ (WJSC); and (iii) MC, WJ, and WJSC produced high post-thaw cell survival rates (93.52 ± 6.12% to 90.83 ± 4.51%) and epithelioid monolayers within 24 h in primary culture whereas post-thaw MC explants showed slow growth with mixed epithelioid and fibroblastic cell outgrowths after several days. Viability and proliferation rates of post-thawed WJ and hWJSC were significantly greater than MC. Post-thaw WJ and WJSC produced significantly greater CD24(+) and CD108(+) fluorescence intensities and significantly lower CD40(+) contaminants. Post-thaw WJ and WJSC produced significantly lesser annexin-V-positive and sub-G1 cells and greater degrees of osteogenic and chondrogenic differentiation compared to MC. qRT-PCR analysis of post-thaw MC showed significant decreases in anti-apoptotic gene expression (SURVIVIN, BCL2) and increases in pro-apoptotic (BAX) and cell cycle regulator genes (P53, P21, ROCK 1) compared to WJ and WJSC. We conclude that freezing of fresh WJ is a simple and reliable method of generating large numbers of clinically utilizable MSCs for cell-based therapies.

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.

In vitro bovine embryo production in a synthetic medium: Embryo development, cryosurvival, and establishment of pregnancy

The aim of this study was to develop an in vitro embryo culture medium without either fetal calf serum or BSA, using various growth factors and cytokines (GFs-CYKs; IGF-I, IGF-II, bFGF, LIF, GM-CSF, TGF-β1, and PDGF-BB), and other molecules with surfactant and embryotrophic properties, such as recombinant albumin (RA) and hyaluronan (HA). The first part of the study was dedicated to define the best combination of GFs-CYKs + RA + HA for optimal embryonic development. Next, we compared development rates and embryo quality (inner cell mass [ICM]-to-total cell number [TCN] ratio), and postthaw survival and hatching rates using this synthetic medium (T1) and a control medium: synthetic oviduct fluid + BSA + ITS (insulin, transferrin, and selenium). The blastocyst rates were significantly higher with T1 than those with the control at 7 and 8 days after fertilization. There was no significant difference in TCN or the ICM/TCN ratio between the two treatments. Survival and hatching rates 48 hours after thawing were similar for both treatments. Finally, nine embryo transfers were conducted using fresh and previously frozen Day-7 blastocysts to evaluate the in vivo viability of embryos produced in this synthetic medium; four gestations were obtained from six fresh embryos and one gestation from three frozen embryos. In conclusion, the fetal calf serum and BSA-free medium, supplemented with GFs-CYKs + RA + HA, improved embryo development and gave comparable ICM/TCN ratios and postthaw survival rates to the control with BSA. Fresh and frozen embryos produced in this medium are viable for embryo transfer. This fully synthetic method of embryo culture is a useful means of reducing the risk of disease transmission via embryo transfer.

59 POST-THAW SURVIVAL OF VITRIFIED, IN VITRO-PRODUCED PORCINE EMBRYOS: COMPARISONS OF VITRIFICATION SOLUTIONS, SOLUTION TEMPERATURE, AND BLASTOCOELE COLLAPSE METHODS

Current post-thaw survival rates of vitrified in vitro-produced porcine blastocysts are much lower than those of other domestic species. The main reason for this is the suboptimal development of in vitro-produced porcine embryos, and the endemically high lipid content of porcine oocytes. Currently, there are several vitrification protocols that have been used to successfully vitrify blastocysts from other species, but many of these have not been used to vitrify porcine embryos. Furthermore, the practice of collapsing the blastocoele cavity before vitrification is used routinely in human clinics and in other domestic animals; however, there has been little data published regarding the collapse of porcine blastocysts before vitrification. In this study we compared several different vitrification protocols containing different constituents and altered constituent concentrations. We also compared 2 methods of blastocoele collapse before vitrification. The aim of this study was to determine the optimum conditions for cryopreserving porcine embryos. All experiments were performed on Day 7 in vitro-produced porcine blastocysts. In experiment 1 embryos were vitrified in either a standard solution (17% ethylene glycol + 17% dimethyl sulfoxide + 0.4 M sucrose) or 17% ethylene glycol + 17% propandiol + 0.4 M sucrose. In experiment 2 embryos were vitrified in either the standard solution or 17% ethylene glycol + 17% dimethyl sulfoxide + 0.4 M trehalose. In experiment 3 embryos were vitrified in the standard solution at 38.5°C or at room temperature. In experiment 4 embryos were collapsed before vitrification by micro-pipetting or via 2 sucrose solutions. Survival of embryos was determined by the presence of a blastocoele cavity 24 h after thaw. Data were subjected to ANOVA and an appropriate post-hoc test when differences were found. The post-thaw survival of embryos vitrified in the standard solution (58%) and the propandiol solution (61%) did not differ. Also, the survival rates of blastocysts vitrified using sucrose (72%) and trehalose (72%) were the same. The survival rate of embryos vitrified in warmed media was significantly lower than embryos vitrified in media at room temperature (54 and 71%, respectively; P &lt; 0.05). In experiment 4 the post-thaw survival of noncollapsed and embryos collapsed via micro-pipetting was similar (55 and 44%, respectively); however, survival tended to decrease if embryos were collapsed using sucrose before vitrification (25%; P = 0.093). These findings indicate there was little impact on post-thaw survival rates due to the vitrification solutions used. However, the temperature of the solutions appears to influence post-thaw survival. Contrary to findings in other species, collapsing the blastocoele cavity before vitrification did not improve cryosurvivability; it had no effect or, in the case of sucrose, a negative effect on survival. This may be due to the inherent sensitivity of in vitro-produced porcine embryos to manipulations.

Clinical outcomes following cryopreservation of blastocysts by vitrification or slow freezing: a population-based cohort study.

What are the clinical efficacy and perinatal outcomes following transfer of vitrified blastocysts compared with transfer of fresh or of slow frozen blastocysts? Compared with slow frozen blastocysts, vitrified blastocysts resulted in significantly higher clinical pregnancy and live delivery rates with similar perinatal outcomes at population level. Although vitrification has been reported to be associated with significantly increased post-thaw survival rates compared with slow freezing, there has been a lack of general consensus over which method of cryopreservation (vitrification versus slow freezing) is most appropriate for blastocysts. A population-based cohort of autologous fresh and initiated thaw cycles (a cycle where embryos were thawed with intention to transfer) performed between January 2009 and December 2011 in Australia and New Zealand was evaluated retrospectively. A total of 46 890 fresh blastocyst transfer cycles, 12 852 initiated slow frozen blastocyst thaw cycles and 20 887 initiated vitrified blastocyst warming cycles were included in the data analysis. Pairwise comparisons were made between the vitrified blastocyst group and slow frozen or fresh blastocyst group. A Chi-square test was used for categorical variables and t-test was used for continuous variables. Cox regression was used to examine the pregnancy outcomes (clinical pregnancy rate, miscarriage rate and live delivery rate) and perinatal outcomes (preterm delivery, low birthweight births, small for gestational age (SGA) births, large for gestational age (LGA) births and perinatal mortality) following transfer of fresh, slow frozen and vitrified blastocysts. The 46 890 fresh blastocyst transfers, 11 644 slow frozen blastocyst transfers and 19 978 vitrified blastocyst transfers resulted in 16 845, 2766 and 6537 clinical pregnancies, which led to 13 049, 2065 and 4955 live deliveries, respectively. Compared with slow frozen blastocyst transfer cycles, vitrified blastocyst transfer cycles resulted in a significantly higher clinical pregnancy rate (adjusted relative risk (ARR): 1.47, 95% confidence intervals (CI): 1.39-1.55) and live delivery rate (ARR: 1.41, 95% CI: 1.34-1.49). Compared with singletons born after transfer of fresh blastocysts, singletons born after transfer of vitrified blastocysts were at 14% less risk of being born preterm (ARR: 0.86, 95% CI: 0.77-0.96), 33% less risk of being low birthweight (ARR: 0.67, 95% CI: 0.58-0.78) and 40% less risk of being SGA (ARR: 0.60, 95% CI: 0.53-0.68). A limitation of this population-based study is the lack of information available on clinic-specific cryopreservation protocols and processes for slow freezing-thaw and vitrification-warm of blastocysts and the potential impact on outcomes. This study presents population-based evidence on clinical efficacy and perinatal outcomes associated with transfer of fresh, slow frozen and vitrified blastocysts. Vitrified blastocyst transfer resulted in significantly higher clinical pregnancy and live delivery rates with similar perinatal outcomes compared with slow frozen blastocyst transfer. Comparably better perinatal outcomes were reported for singletons born after transfer of vitrified blastocysts than singletons born after transfer of fresh blastocysts. Elective vitrification could be considered as an alternative embryo transfer strategy to achieve better perinatal outcomes following Assisted Reproduction Technology (ART) treatment. No specific funding was obtained. The authors have no conflicts of interest to declare.

Assessing the effects of vitrification on embryo viability using label-free imaging

Embryo vitrification enables patients to undergo multiple cycles of in vitro fertilization (IVF) without repeated ovarian stimulation and oocyte retrieval. Although vitrification has been shown to have fairly high post-thaw embryo survival rates, its impact on embryo viability remains poorly understood, as damage due to vitrification is difficult to detect using routine microscopy techniques. We have constructed a non-invasive, high-resolution, label-free microscope based on optical coherence tomography (OCT) that is able to observe dramatic changes in embryo appearance immediately after vitrification and thawing that are not visible under brightfield microscopy.