Vitrified-warmed Blastocyst Research Articles

Pregnancy potential and perinatal outcomes of warmed blastocysts: the influence of delayed blastulation prior to vitrification

This study investigated whether embryos that reach blastocysts and cryopreserved at day 5 were more likely to result in a successful pregnancy than where blastulation is delayed until day 6. A retrospective clinical study examined the pregnancy and perinatal outcomes of vitrified blastocysts formed by day 5 compared to day 6. 309 cycles of vitrified-warmed blastocysts transferred in 2009-10 were investigated. 211 cycles had day 5 blastocysts transferred and 98 cycles had day 6 blastocysts. Blastocysts were vitrified and then later warmed prior to transfer. Re-expanded blastocysts were considered to be viable for transfer. Only cycles in which all transferred embryos were vitrified on the same day of development were included. For statistical analysis Fisher's exact test and t test were used. The mean age of the women was not significantly different for day 5 and day 6 groups (34.5 and 34.2, respectively) and the mean number of blastocysts transferred was 1.1 for both groups. There was no significant difference in cryosurvival rate between day 5 and day 6 blastocysts (84.5% and 90.1%), clinical pregnancy rates (31.0% and 26.1%), implantation rates (30.3% and 26.8%), and mean birth weight (3347±642 and 3350±473). 69 babies from vitrified day 5 group were delivered with no reported abnormalities. In day 6 group 24 babies were delivered with 2 reported abnormalities but this was not significantly different to day 5 group (P>0.05). Day 6 group had a significantly higher percentage of female babies compared to day 5 group (75.0% and 43.5%, P<0.05). No significant difference between the day 5 and day 6 vitrified blastocyst transfer groups with respect to cryosurvival rates, pregnancy rates and birth weight was observed. Thus delayed blastulation does not negatively affect pregnancy potential and consequently can contribute to increasing cumulative pregnancy rates in ART. Further monitoring of the differing sex ratio is required to obtain confident results.

A successful birth of healthy monozygotic dichorionic diamniotic (DD) twins of the same gender following a single vitrified-warmed blastocyst transfer

To report the world's first case of live monozygotic dichorionic diamniotic (DD) twins after a single vitrified-warmed blastocyst transfer in a cycle during which hormone supplements were given. The patient was a 39-year-old woman with tubal factor infertility. Six oocytes were retrieved and three blastocysts were vitrified after insemination with her husband's sperm. Two months later, a single warmed blastocyst was transferred on the fifth day after progesterone supplementation during the luteal phase. Two gestational sacs and fetuses with heartbeats showing lambda sign under ultrasonography were recognized at 7 weeks of gestation and the patient gave birth to two female infants at gestational 33 weeks. The pathological examination demonstrated that the DD twin placentas fused in the third trimester. This report suggests that the splitting of the transferred embryo took place after the blastocyst stage and the popular credo that the timing of embryo division governs the placental configuration of monozygotic twins (MZT) must be re-examined as to its veracity.

Clinical outcome of fresh and vitrified-warmed blastocyst and cleavage-stage embryo transfers in ethnic Chinese ART patients

ObjectivesThis study sought to evaluate the outcome of fresh and vitrified-warmed cleavage-stage and blastocyst-stage embryo transfers in patients undergoing ART treatment within an ethnic Chinese population.Study designWe compared the clinical results of embryo transfer on the 3rd (cleavage stage) or 5th (blastocyst stage) day after oocyte retrieval, including clinical pregnancy rates, implantation rates and multiple pregnancy rates.ResultsOur data showed that blastocyst transfer on day 5 did not significantly increase clinical pregnancy rate (41.07% vs 47.08%, p>0.05) and implantation rate (31.8% vs 31.2%, p>0.05) in patients under 35 years of age, in comparison with day 3 cleavage stage embryo transfer. In patients older than 35 years of age, the clinical pregnancy rate after blastocyst transfer was slightly decreased compared with cleavage stage embryo transfer (33.33% vs 42.31%, p>0.05). Unexpectedly, It was found that vitrified-warmed blastocyst transfer resulted in significantly higher clinical pregnancy rate (56.8%) and implantation rate (47%) compared with fresh blastocyst transfer in controlled stimulation cycles (41.07% and 31.8%, respectively). For patients under 35 years of age, the cumulative clinical pregnancy rate combining fresh and vitrified-warmed blastocyst transfer cycles were significantly higher compared to just cleavage-stage embryo transfer (70.1% versus 51.8%, p<0.05). However, the cumulative multiple pregnancy rates showed no significant difference between the two groups.ConclusionsIn an ethnic Chinese patient population, fresh blastocyst transfer does not significantly increase clinical pregnancy rate. However, subsequent vitrified-warmed blastocyst transfer in a non-controlled ovarian hyperstimulation cycle dramatically improves clinical outcomes. Therefore, blastocyst culture in tandem with vitrified-warmed blastocyst transfer is recommended as a favourable and promising protocol in human ART treatment, particularly for ethnic Chinese patients.

Developmental assessment of human vitrified-warmed blastocysts based on oxygen consumption

In this study, we aimed to develop a model for embryo selection based on oxygen consumption following cryopreservation, the relationship between the developmental competence of blastocysts and their oxygen consumption was assessed. Oxygen consumption of vitrified-warmed human blastocysts was measured at 0, 1.5, 3, 4.5, 6, 7.5, 9 and 24 h after warming using scanning electrochemical microscopy. On the basis of their developmental stage at 24 h, blastocysts were classified into four groups (hatched, hatching, arrested and degenerated). Moreover, cytochrome c oxidase (CCO) activity in vitrified-warmed blastocysts was assessed at 0 and 24 h. The oxygen consumption rate of blastocysts just after warming was significantly lower than that of non-vitrified blastocysts (P< 0.05). The oxygen consumption rate of blastocysts was significantly higher in the hatched group than in the arrested and the degenerated groups after 1.5 h (P< 0.05) and than in the hatching group (P< 0.05) at 7.5 and 9 h. Moreover, CCO activity was absent in vitrified-warmed blastocysts at 0 h, but was detected at 24 h. The respiratory rate of vitrified blastocysts after warming was significantly lower than non-cryopreserved blastocysts. Oxygen consumption of blastocysts with high developmental potential was restored earlier than that of blastocysts with low developmental potential. The results of this study suggest that it is possible to select vitrified-warmed blastocysts with high developmental potential based on their respiratory activity.

Effect of energy source during culture on in vitro embryo development, resistance to cryopreservation and sex ratio

The aim of this work was to evaluate whether minimizing the glucose concentration during culture or replacing the hexose with other energy substrates and/or embryotrophic compounds would affect the in vitro development, the resistance to cryopreservation and the sex ratio of bovine embryos. In vitro matured and fertilized oocytes were randomly assigned to 4 groups for in vitro culture, that differed in the energy substrates included: group A) 1.5 mM glucose, as in standard SOF; group B) 0.15 mM glucose; group C) 0.125 mM G3P, in the presence of 0.15 mM glucose and group D) 0.34 mM citrate, in combination with 2.77 mM myo-inositol. Blastocysts were evaluated on day 7, then vitrified by cryotop in 16.5% DMSO, 16.5% EG and 0.5 M sucrose and warmed in decreasing concentration of sucrose (0.25 to 0.15 M sucrose). The survival rates were assessed after 24 h in vitro culture. Finally, the blastocysts produced were sexed by PCR. An increased blastocyst rate was recorded in groups B, C and D, i.e., when glucose concentration was reduced, compared to group A (28.2, 41.0, 35.7 and 35.8, respectively in groups A, B, C and D; P < 0.01). However, the embryos cultured in group D showed the slowest developmental speed, indicated by the lowest percentage of advanced stage-embryos (expanded and hatched blastocysts) out of the total blastocysts (56.1, 45.8, 56.9 and 31.8 %, respectively in groups A, B, C and D; P < 0.01). Furthermore, survival rates after 24 h culture of vitrified-warmed blastocysts also decreased in group D (73.3, 73.1, 71.4 and 58.4%, respectively in groups A, B, C and D; P < 0.01). Interestingly, in group D a higher percentage of female embryos was obtained compared to group A, with intermediate values in groups B and C (45.6, 53.4, 50.0 and 61.5%, respectively in groups A, B, C and D; P < 0.05). In conclusion, it was demonstrated that the energy substrate during in vitro culture affects both the production and the viability of blastocysts. Furthermore, manipulating the metabolic profile of embryos during in vitro culture may have an impact on sex ratio.

Vitrified-warmed blastocyst transfer cycles yield higher pregnancy and implantation rates compared with fresh blastocyst transfer cycles—time for a new embryo transfer strategy?

To compare the clinical outcome of fresh versus vitrified-warmed blastocyst transfer (BT) cycles. Retrospective study. Medical university affiliated hospital. Women aged less than 40 years undergoing BT cycles. Vitrification and warming of blastocyst with the Cryotop system. Clinical pregnancy rate (CPR), implantation rate (IR), and multiple pregnancy rate (MPR). In 110 fresh BT cycles versus 136 vitrified-warmed BT cycles performed from January 2007 to March 2010, the IR and CPR of vitrified-warmed BT cycles were 37.0% and 55.1%, respectively, which were statistically significantly higher than the corresponding values of 25.2% and 36.4% obtained for fresh BT cycles. Additionally, the MPR was not statistically significantly different between vitrified-warmed and fresh BT cycles when a similar number of blastocysts was transferred to patients. Vitrified-warmed BT cycles resulted in statistically significantly higher CPR and IR compared with fresh BT cycles. A new embryo transfer strategy is therefore proposed whereby fresh BT would be avoided in the initial ovarian stimulation cycle. Instead, all the patient's available blastocysts would be vitrified-warmed and transferred in subsequent cycles.

Healthy live birth from vitrified blastocysts produced from natural cycle IVF/IVM

Natural-cycle IVF combined with in-vitro maturation (natural-cycle IVF/IVM) was used as a treatment for a 27-year-old woman. She was administered 10,000 IU of human chorionic gonadotrophin intramuscularly about 36 h prior to oocyte collection and oocyte collection was performed on day 11 of her menstrual cycle. One mature oocyte was retrieved from the leading follicle and another five mature oocytes and six immature oocytes were retrieved from the rest of the follicles. Out of 10 fertilized zygotes, eight of them cleaved. Three day-3 embryos derived from in-vivo matured oocytes (one was from the leading follicle) were transferred but failed to conceive. The remaining five embryos were continuously cultured until day 6 and four of them developed to the expanded blastocyst stage and vitrified for the storage. Six months later, two vitrified-warmed blastocysts derived from the immature oocytes were transferred and resulted in the full-term delivery of a healthy female infant. This case report for the first time indicates that blastocysts produced from the immature oocytes retrieved from the small follicles, when a leading follicle exists in the ovaries, can be vitrified to produce a healthy live birth, suggesting that natural-cycle IVF/IVM is an efficient infertility treatment.

The effect of single embryo transfer in a human vitrified-warmed program

OBJECTIVE: A multiple embryo transfer increases the chances of conception from vitrified-warmed blastocysts, but it might result in multiple pregnancies. Accordingly, a single embryo transfer is suggested to reduce multiple pregnancies. This study was performed to investigate how a single embryo transfer has an influence on the implantation and pregnancy rates in a vitrified-warmed program. DESIGN: Retrospective study. MATERIALS AND METHODS: The study included 2,126 cycles from November 2002 to June 2007. The embryos developed to the blastocyst stage were vitrified using EM-grid following artificial shrinkage. The solution for vitrification was prepared according to a previously described method (Lee et al., 2007). Embryo warming was conducted by two steps on day 3 after ovulation. The survived blastocysts were transferred into uterus. The clinical pregnancy and implantation rates per ET were examined according to the number of transferred embryos: consisted of the patients in which a single blastocyst was transferred (group1), two blastocysts were transferred (group2) and three blastocysts were transferred (group3). RESULTS: The implantation rate in groups 1, 2 and 3 was 24.2% (58/240), 23.5% (339/1,440), and 23.2% (821/3,536) and did not differ significantly. The corresponding clinical pregnancy rate in groups 1, 2 and 3 was 22.9% (55/240), 38.5% (277/720), and 50.1% (559/1,116), which was statistically significant (p<0.05). The percentage of twin and multiple pregnancies were 22.7% (63/276) and 0.0% (0/276) in group 2 and 24.0% (133/555) and 11.7% (65/555) in group 3. In this study, embryo transfer was not conducted for fifty cycles with no blastocysts survived. CONCLUSIONS: These results suggest that the transfer of two or more vitrified-warmed embryos increase the conception rate, but has no beneficial effect on the implantation rate compared to a single embryo transfer. Furthermore, single embryo transfer minimizes the multiple pregnancy risk due to several chances of a single embryo transfer per ovum pick-up.

Birth of a healthy male infant after transfer of vitrified-warmed blastocysts derived from intracytoplasmic sperm injection with vitrified-warmed oocytes and frozen-thawed spermatozoa

To report a successful delivery of a healthy baby after transfer of vitrified-warmed blastocysts derived from introcytoplasmic sperm injection (ICSI) with vitrified-warmed oocytes and frozen-thawed sperm. A female patient and her husband with non-obstructive azoospermia received a transfer of vitrified-warmed blastocysts from vitrified-warmed oocytes and frozen-thawed sperm. The main outcome measures were fertilization, pregnancy and birth. Nine oocytes were matured and vitrified. When the vitrified oocytes were warmed, six survived with good quality morphology. Using ICSI, frozen-thawed sperm was injected into the six warmed oocytes that survived, and the fertilization rate was 100%. The zygotes were cultured, and five of six early embryos became blastocysts. One of them was transferred, but pregnancy was not achieved. The second time around, two vitrified-warmed blastocysts were transferred resulting in pregnancy, and a healthy boy was delivered. This is a rare case of a successful birth using a vitrified-warmed blastocyst grown after ICSI with a vitrified-warmed oocyte and frozen-thawed sperm.

A birth from the transfer of a single vitrified-warmed blastocyst using intracytoplasmic sperm injection with calcium ionophore oocyte activation in a globozoospermic patient

To present the effectiveness of diagnostic heterologous intracytoplasmic sperm injection (ICSI), mouse oocyte activation test (MOAT), and ICSI combined with assisted oocyte activation (AOA) in a globozoospermic patient. A case report. A private IVF center, Japan. A patient with globozoospermia. MOAT in a mouse and ICSI combined with AOA in a human. Ultrastructure, MOAT, fertilization, and pregnancy. The transmission electron micrographs showed 100% round-headed spermatozoa lacking an acrosome. MOAT showed that the fertilization rate was 68.4% (13/19) when AOA was used but 0% (0/19) when AOA was not used. After the diagnosis of globozoospermia and sperm-related activation deficiency, 17 human mature oocytes were activated with calcium ionophore after ICSI was performed. The fertilization rate was 88.2% (15/17), and 11 blastocysts were cryopreserved using the vitrification method to prevent severe ovarian hyperstimulation syndrome. A single vitrified-warmed blastocyst was transferred. A gestational sac with fetal heart movements was recognized, and a healthy boy weighing 3180 g was born at 40 weeks of gestation by cesarean section without any congenital abnormality. MOAT allows discrimination between sperm- and oocyte-related fertilization failures and shows the effectiveness of AOA.

Super Open Pulled Straw Vitrification of Porcine Blastocysts: Effect of Centrifugation and Cytoskeletal Stabilization.

The objective of the present study was to evaluate the effect of centrifugation and cytoskeletal stabilization with cytochalasin-B before vitrification on the survival and cytoskeleton integrity of vitrified porcine blastocysts. Unhatched blastocysts were collected by laparotomy from weaned crossbred sows (n=10) on day 6 of the estrous cycle. A subset of blastocysts (n=22) was not vitrified and cultured to evaluate the in vitro development of fresh embryos. The remaining blastocysts were randomly allocated to different experimental groups: a) blastocysts vitrified without pre-treatments (n=25); b) blastocysts centrifuged at 13.000 x g for 12 min (n=28); c) blastocysts equilibrated in 7.5 µg/ml cytochalasin-B for 20 min (n=30) and d) blastocysts equilibrated in cytochalasin-B for 20 min and then centrifuged at 13.000 × g for 12 min. Pretreated and non-pretreated blastocysts were vitrified (n=28). The basic medium for vitrification and warming (TCM-NBCS) was TCM199 Hepes supplemented with 20% (v/v) of newborn calf serum (NBCS) and 0.5 mM of L-Glutamine. Groups of 4 to 6 embryos were equilibrated in TCM-NBCS with 7.5% (v/v) dimethyl-sulfoxide (DMSO) and 7.5% (v/v) ethylene glycol (EG) for 3 min and in TCM-NBCS with 17% (v/v) DMSO, 17% (v/v) EG and 0.4 M sucrose for 1 min. During the last step, embryos were loaded into the narrow end of a superfine open pulled straw (SOPS) by capillary action and plunged into the liquid nitrogen. Straws were warmed using a one step dilution procedure. The narrow of the straw was immersed vertically in a well containing 800 µL TCM-NBCS with 0.13 M sucrose for 5 min. Fresh and vitrified-warmed blastocysts were cultured for 24 h in TCM199 supplemented with 10% fetal calf serum at 39°C, in 5% CO2 in humidified air. After the culture, embryos were assessed by stereomicroscopy and classified as viable or degenerated. Blastocysts that reformed their blastocoelic cavities after warming, displaying a normal zona pellucida and excellent appearance, were considered viable. The in vitro survival rate was defined as the ratio of viable embryos divided by the total number of embryos cultured. The hatching rate was determined as the ratio of the number of embryos hatched to the total number embryos cultured. Some vitrified and fresh embryos were labeled after culture, with Alexa Fluor 488 phalloidin and wheat germ agglutinin-Alexa Fluor 594 conjugate, to assess cytoskeleton quality. Data were analyzed by Chi-square test and expressed as mean values ± SEM. The embryo survival (81.8 ± 1.2%) and the hatching rate (36.4 ± 8.6) for fresh embryos were not significantly different to those obtained with vitrified embryos (ranging from 75.0±8.3% to 80.0±8.2% and from 20.0 ± 8.2% to 39.3 ± 9.4%, respectively). No differences were found for in vitro suvirval, hatching rates and cytoskeleton quality among vitrification groups Embryos classified as degenerated by stereomicroscopy displayed wide cytoskeletal disruption. Vitrified embryos classified as viable by stereomicroscopy displayed similar cytoskeleton quality than fresh blastocysts, showing only few cells with microfilament disruption. In conclusion, the pretreatment with cytochalasin-B and/or the centrifugation prior vitrification did not improve the blastocysts developmental competence and the cytoskeletal quality after SOPS-vitrification. Supported by CICYT-FEDER (AGL2004-07546), CARM (2I05SU0012), MEC (BES-2005-8894) and Seneca Foundation (04543/GERM/07).

88 IS STEREOMICROSCOPY AN EFFICIENT METHOD FOR EVALUATING THE VITRIFIED PORCINE EMBRYO QUALITY?

The development of the open pulled straw vitrification has provided excellent results of in vitro porcine embryo development. Embryo quality evaluation after vitrification has been traditionally focused on morphological assessment performed by stereomicroscopy. The objective of this experiment was to evaluate the efficiency of the stereomicroscopic evaluation of vitrified-warmed (V) porcine blastocysts. Unhatched blastocysts were obtained after slaughter from Large-White gilts (n = 9). Blastocysts (n = 75) were vitrified and warmed using the protocol described by Cuello et al. (2004 Theriogenology 61, 353-361). After warming, vitrified blastocysts were cultured for 24 h. Then blastocysts were morphologically assessed for their progression and morphology by stereomicroscopy. Blastocysts that reformed their blastocoelic cavities showing an excellent appearance were considered viable. Some of the viable blastocysts kept their zonae pellucidae (V viable expanded blastocysts) and others hatched during the in vitro culture (V viable hatched blastocysts). The remaining blastocysts were classified as degenerated embryos. A group of fresh blastocysts was not vitrified and cultured in vitro for 24 h (control group). All of the control blastocysts were considered viable by stereomicroscopy. Some fresh, V viable expanded, V viable hatched, and V degenerated blastocysts (n = 13, n = 19, n = 9, and n = 9, respectively) were processed for ultrastructural study by light and transmission electron microscopy or stained with Hoechst-33342 and TUNEL for cell death evaluation (n = 16, n = 21, n = 11, and n = 6, respectively). All V hatched blastocysts showed ultrastructure similar to that of control hatched blastocysts. However, 26.3% of the V viable expanded blastocysts revealed important ultrastructural alterations in comparison with control expanded blastocysts. These observations suggest that stereomicroscopic evaluation was not efficient enough for V expanded blastocysts. As expected, degenerated blastocysts showed ultrastructural disintegration and disorganization. Hatched V blastocysts did not differ (P &lt; 0.05) from control hatched blastocysts with regard to the total cell number and ratio of death cells (173 � 4.8 vs. 202.1 � 10.9 and 2.8 � 0.5% vs. 1.9 � 0.3%, respectively). However, V expanded blastocysts a had higher (P &lt; 0.01) cell death level (4.3 � 3.4%) than that observed in the control expanded blastocysts (1.1 � 0.3%). Degenerated embryos showed the lowest (P &lt; 0.01) total cell number (45.7 � 4.0). The 66.7% of the degenerated blastocysts exhibited wide TUNEL-labeled areas, and the remaining 33.3% showed TUNEL label over 19.4 � 6.3% of the cells. In conclusion, the hatching rate assessed by stereomicroscopy is a more efficient parameter than assessing the in vitro viability (ratio of blastocysts that reformed their blastocoelic cavities after warming) for estimating the quality of V blastocysts. This work was supported by CICYT (AGL2004-07546) and S�neca (01287/PD/04).

Pregnancies and improved early embryonic development with bovine oocytes matured in vitro with 9-cis-retinoic acid

Retinoids have an important role in cell growth, morphogenesis and differentiation. In the present study the developmental potential of bovine oocytes was examined after in vitro maturation in the presence of 9-cis-retinoic acid, a vitamin A metabolite, at 5 nmol l(-1) in chemically defined conditions. Experiments studied early in vitro development, blastocyst differential cell counts and the capacity of embryos to establish pregnancy after transfer to recipients. After in vitro fertilization and culture in simple medium, blastocyst development and hatching rates increased in oocytes matured with 9-cis-retinoic acid. Addition of ethanol (used as a solvent for 9-cis-retinoic acid) resulted in higher cell counts and proportions of cells in the inner mass of day 7 blastocysts. Day 8 blastocysts represented most differences observed in the number of cells. In these embryos, ethanol and 9-cis-retinoic acid increased both the number of cells and proportions in the inner mass. However, while ethanol treatment reduced the number of cells in the trophectoderm, 9-cis-retinoic acid did not. The total number of cells was unaffected by treatment within 1 day, although untreated oocytes lead to day 8 blastocysts with reduced total cell counts. Once transferred to recipients, both fresh and vitrified-warmed blastocysts derived from oocytes matured with 9-cis-retinoic acid yielded more pregnancies at day 60. Modifications of retinoid metabolism affect development and trophectoderm differentiation, and in vitro maturation with 9-cis-retinoic acid increased the developmental competence of the oocyte.

Pregnancies and improved early embryonic development with bovine oocytes matured in vitro with 9-cis-retinoic acid

Retinoids have an important role in cell growth, morphogenesis and differentiation. In the present study the developmental potential of bovine oocytes was examined after in vitro maturation in the presence of 9-cis-retinoic acid, a vitamin A metabolite, at 5 nmol l(-1) in chemically defined conditions. Experiments studied early in vitro development, blastocyst differential cell counts and the capacity of embryos to establish pregnancy after transfer to recipients. After in vitro fertilization and culture in simple medium, blastocyst development and hatching rates increased in oocytes matured with 9-cis-retinoic acid. Addition of ethanol (used as a solvent for 9-cis-retinoic acid) resulted in higher cell counts and proportions of cells in the inner mass of day 7 blastocysts. Day 8 blastocysts represented most differences observed in the number of cells. In these embryos, ethanol and 9-cis-retinoic acid increased both the number of cells and proportions in the inner mass. However, while ethanol treatment reduced the number of cells in the trophectoderm, 9-cis-retinoic acid did not. The total number of cells was unaffected by treatment within 1 day, although untreated oocytes lead to day 8 blastocysts with reduced total cell counts. Once transferred to recipients, both fresh and vitrified-warmed blastocysts derived from oocytes matured with 9-cis-retinoic acid yielded more pregnancies at day 60. Modifications of retinoid metabolism affect development and trophectoderm differentiation, and in vitro maturation with 9-cis-retinoic acid increased the developmental competence of the oocyte.

The effect of media, serum and temperature on in vitro survival of bovine blastocysts after Open Pulled Straw (OPS) vitrification

The recently introduced Open Pulled Straw (OPS) vitrification technique has successfully been used for cryopreserving porcine embryos as well as for bovine embryos and oocytes. The aim of this work is to investigate several factors on the in vitro survival of bovine blastocysts. In 5 experiments, a total of 862 in vitro produced blastocysts and expanded blastocysts was vitrified and wanned using the OPS technology, then cultured in vitro for an additional 3 days. The culture medium in Experiments 1 to 4 was SOFaa with supplements and 5% calf serum (CS). In Experiment 1, the replacement of TCM-199+20% CS with PBS+20% CS in the holding medium during vitrification and warming did not result in significant differences in the re-expansion (92 vs 95%) and hatching rates (79 vs 72%). In Experiment 2, the PBS holding medium was supplemented with either 20% CS, 5 mg/mL bovine serum albumin (BSA) or 3 mg/mL polyvinyl-alcohol (PVA). Although the re-expansion rates did not differ (98, 95 and 93%, respectively), there was a decrease in the hatching rate after vitrification with PVA (77 and 78 vs 51%, respectively). In Experiment 3, the influence of temperature of equilibration media prior to and rehydration media after the vitrification was investigated. When the temperature of these media was adjusted to 20 °C instead of the standard 35 °C, both the re-expansion and the hatching rates decreased markedly. However, increasing the time of equilibration with the diluted cryoprotectant solution at 20 °C eliminated these differences. In Experiment 4, the ethylene-glycol and dimethyl sulfoxide cryoprotectant mixture was replaced with ethylene glycol-ficoll-trehalose solution. No difference in the re-expansion (89 vs 96%, respectively) or hatching rate (79 vs 84%, respectively) was detected. In Experiment 5, the vitrified-warmed blastocysts were cultured in SOFaa medium supplemented with 5% CS or 5 mg/mL BSA. Although the re-expansion rates were identical in the 2 groups (95%), the hatching rates were lower when embryos were cultured in BSA (71 and 47%, respectively). These findings indicated the possible broader application for OPS, as they demonstrated that the physical advantages of rapid cooling and warming may be accompanied by different chemical composition (holding media, cryoprotective additives) according to the requirements of the biological structure. Our study also shows the need for serum supplementation of the medium for hatching to occur after OPS vitrification.

Seminal factors initiate an epithelial cell-mediated inflammatory reaction in the pre-implantation mouse uterus

The aim of this work was to evaluate whether minimizing the glucose concentration during culture or replacing the hexose with other energy substrates and/or embryotrophic compounds would affect the in vitro development, the resistance to cryopreservation and the sex ratio of bovine embryos. In vitro matured and fertilized oocytes were randomly assigned to 4 groups for in vitro culture, that differed in the energy substrates included: group A) 1.5 mM glucose, as in standard SOF; group B) 0.15 mM glucose; group C) 0.125 mM G3P, in the presence of 0.15 mM glucose and group D) 0.34 mM citrate, in combination with 2.77 mM myo-inositol. Blastocysts were evaluated on day 7, then vitrified by cryotop in 16.5% DMSO, 16.5% EG and 0.5 M sucrose and warmed in decreasing concentration of sucrose (0.25 to 0.15 M sucrose). The survival rates were assessed after 24 h in vitro culture. Finally, the blastocysts produced were sexed by PCR. An increased blastocyst rate was recorded in groups B, C and D, i.e., when glucose concentration was reduced, compared to group A (28.2, 41.0, 35.7 and 35.8, respectively in groups A, B, C and D; P < 0.01). However, the embryos cultured in group D showed the slowest developmental speed, indicated by the lowest percentage of advanced stage-embryos (expanded and hatched blastocysts) out of the total blastocysts (56.1, 45.8, 56.9 and 31.8 %, respectively in groups A, B, C and D; P < 0.01). Furthermore, survival rates after 24 h culture of vitrified-warmed blastocysts also decreased in group D (73.3, 73.1, 71.4 and 58.4%, respectively in groups A, B, C and D; P < 0.01). Interestingly, in group D a higher percentage of female embryos was obtained compared to group A, with intermediate values in groups B and C (45.6, 53.4, 50.0 and 61.5%, respectively in groups A, B, C and D; P < 0.05). In conclusion, it was demonstrated that the energy substrate during in vitro culture affects both the production and the viability of blastocysts. Furthermore, manipulating the metabolic profile of embryos during in vitro culture may have an impact on sex ratio.

7 - Non-rhesus (D) Blood Group Isoimmunization in Obstetrics

In this study the clinical and neo-natal outcomes after transfer of blastocysts derived from oocytes containing aggregates of smooth endoplasmic reticulum (SER) were compared between IVF and intracytoplasmic sperm injection (ICSI) cycles. Clinical and neo-natal outcomes of blastocysts in cycles with at least one SER metaphase II oocyte (SER + MII; SER + cycles) did not significantly differ between the two insemination methods. When SER + MII were cultured to day 5/6, fertilization, embryo cleavage and blastocyst rates were not significantly different between IVF and ICSI cycles. In vitrified-warmed blastocyst transfer cycles, the clinical pregnancy rates from SER + MII in IVF and ICSI did not significantly differ. In this study, 52 blastocysts (27 IVF and 25 ICSI) derived from SER + MII were transferred, yielding 15 newborns (5 IVF and 10 ICSI) and no malformations. Moreover, 300 blastocysts (175 IVF and 125 ICSI) derived from SER–MII were transferred, yielding 55 newborns (24 IVF and 31 ICSI cycles). Thus, blastocysts derived from SER + cycles exhibited an acceptable ongoing pregnancy rate after IVF (n = 125) or ICSI (n = 117) cycles. In conclusion, blastocysts from SER + MII in both IVF and ICSI cycles yield adequate ongoing pregnancy rates with neo-natal outcomes that do not differ from SER–MII.