Blastocoel Fluid Research Articles

42 DIFFUSION OF THE CRYOPROTECTANTS ETHYLENE GLYCOL AND GLYCEROL INTO DAY 9 TO 11 EQUINE EMBRYOS: PRELIMINARY RESULTS

The impermeability of the capsule to cryoprotectants (CPA) has been blamed for poor cryosurvival of equine embryos. We hypothesised that ethylene glycol, with its lower molecular weight and higher permeability, would be preferred over glycerol for equine embryos. The purpose of this study was to determine concentrations of glycerol and ethylene glycol within equine blastocoele fluid of Day 9 to 11 equine embryos following 30 min of exposure to a 1.5 M solution. Fourteen grade 1 embryos were recovered from May through September, using standard transcervical lavage techniques. Day of ovulation (Day 0) was determined as first appearance of the corpus luteum by daily transrectal ultrasonography, following insemination and administration of an ovulatory induction agent (Chorulon, 2500 IU; Intervet, Canada). Embryos were graded, measured, and washed in holding media (emP3TM Partnar Animal Health, Ontario, Canada), blocked by age, and randomly assigned to either 1.5 M ethylene glycol in holding solution (EG; n = 7) or 1.5 M glycerol in holding solution (Gly; n = 7) each containing 1% dimethyl sulfone as the internal standard, for 30 min at room temperature. Following CPA exposure, embryos were moved to a sterile, dry dish, all fluid was removed with a micropipette, and the dish dried with fine tissue. The embryonic capsule was punctured, and aspirated blastocoele fluid was analysed for CPA concentration by gas chromatography using a CP-3800 gas chromatograph (Varian Canada Inc., Mississauga, Ontario, Canada). The CPA concentration was determined by comparing the internal standard count to the accumulated CPA in millimoles per liter by gas chromatograph. A two-sample Kolmogorov-Smirnov test for equality of distribution was used because data were not normally distributed. Post-hoc 2-way t-test with unequal variances was used to compare mean effective CPA concentration across treatments, with significance set at P = 0.05. Collected embryos ranged from 1470 μm to 9240 μm in diameter, but there was no difference in embryo size between groups (P = 0.59). Mean concentrations of EG from blastocoele fluid were significantly higher than Gly (71.58 ± 18.9 mmol L–1 v. 50.71 ± 11.2 mmol L–1, respectively; P = 0.016). This represents 4.8 and 3.4% of available CPA, respectively. Mean EG concentrations by embryo age were not different (Day 9: 68.3 ± 23.1 mmol L–1; Day 10: 83.0 ± 15.4 mmol L–1; Day 11: 71.0 ± 21.9 mmol L–1; P = 0.75). Mean Gly concentrations by embryo age were not different (Day 9: 47.0 ± 12.7 mmol L–1; Day 10: 46.0 ± 7.1 mmol L–1; Day 11: 56.3 ± 13.6 mmol L–1; P = 0.35). The low concentrations of EG and Gly found in this preliminary study are in agreement with indirect evidence from other studies. Ethylene glycol reached higher concentrations inside the embryo than glycerol because of lower molecular weight and higher permeability. However, permeation appeared to be minimal for both agents despite exposure to solutions of high CPA molarity.

Preimplantation genetic testing: polar bodies, blastomeres, trophectoderm cells, or blastocoelic fluid?

To investigate the blastocoelic fluid (BF) for the presence of DNA that could be amplified and analyzed; the extent to which its chromosomal status corresponds to that found in trophectoderm (TE) cells, polar bodies (PBs), or blastomeres; and the identification of segmental abnormalities. Longitudinal cohort study. In vitro fertilization unit. Fifty-one couples undergoing preimplantation genetic screening or preimplantation genetic diagnosis for translocations by array-comparative genomic hybridization on PBs (n = 21) or blastomeres (n = 30). BFs and TE cells were retrieved from 116 blastocysts, whose chromosome status had already been established by PB or blastomere assessment. Separate chromosome analysis was performed in 70 BFs. Presence of DNA in BFs, evaluation of the chromosome condition, and comparison with the diagnosis made in TE cells and at earlier stage biopsies. DNA detection was 82%, with a net improvement after refinement of the procedure. In 97.1% of BFs, the ploidy condition corresponded to that found in TE cells, with one false positive and one false negative. The rate of concordance per single chromosome was 98.4%. Ploidy and chromosome concordance with PBs were 94% and 97.9%, respectively; with blastomeres, the concordances were 95% and 97.7%, respectively. Segmental abnormalities, which were detected in PBs or blastomeres of 16 blastocysts, were also identified in the corresponding BFs. BF represents to a good extent the blastocyst ploidy condition and chromosome status when compared with TE cells. If the proportion of clinically useful BFs is improved, blastocentesis could become the preferred source of DNA for chromosomal testing.

Cryopreservation of Day 8 equine embryos after blastocyst micromanipulation and vitrification

Pregnancy rates after cryopreservation of large equine blastocyst stage embryos have remained lower than other domesticated livestock species. It is generally accepted that the embryonic capsule is the primary barrier to cryoprotectant entry into the embryo proper and techniques need to be developed to circumvent this obstacle. Therefore, the objective of this study was to develop an efficient Day 8 equine embryo cryopreservation protocol through blastocyst micromanipulation and vitrification. Grade 1 and 2 embryos recovered from mares (n = 15) 8 days after ovulation were used in these experiments. In experiment 1, the effect of either one- or two-puncture treatments before aspiration of blastocoel fluid and exposure to vitrification solutions was evaluated. No difference was detected in mean embryo volume across treatment groups after exposure to vitrification solutions or after 1, 24, 48, and 72 hours of culture. Percent of embryos re-expanding at 24 hours and percent of embryos showing diameter increase at 48 and 72 hours during in vitro culture were 100%, 83%, and 75% compared with 93%, 67%, and 50% for one- and two-puncture treatment groups, respectively. Capsule loss was 25% for one-puncture and 50% for two-puncture treatment groups. In experiment 2, no difference was detected in mean embryo volume for indirect introduction (aspiration of blastocoel fluid + equilibration) and direct introduction (injection of cryoprotectant into blastocoel cavity) treatment groups, after exposure to dilution solution or to culture medium. There was no difference in mean embryo volume for the indirect and direct introduction treatment groups after 1, 24, 48, and 72 hours of culture. Percent of embryos re-expanding at 24 hours and percent of embryos showing diameter increases at 48 and 72 hours during in vitro culture were 100%, 76.9%, and 69.2%, respectively, for both treatment groups. Those embryos subjected to the direct introduction treatment had a higher (P = 0.05) percent capsule loss (70%) compared with the indirect introduction treatment group (31%). The pregnancy rate after transfer of vitrified expanded Grade 1 blastocysts using the indirect introduction method was 83% (5/6). Three pregnancies were allowed to continue to term and resulted in the birth of three healthy foals. The vitrification protocol used in this study has the potential to become a key tool for the successful cryopreservation of equine expanded blastocysts.

Molecular karyotyping of cell-free DNA from blastocoele fluid as a basis for noninvasive preimplantation genetic screening of aneuploidy

The discovery of DNA fragments in the blastocoele fluid is promising for the development of new noninvasive methods for the preimplantation genetic diagnosis of chromosomal diseases. However, to date there are no data confirming the concordance between the molecular karyotype of cell-free DNA from blastocoele fluid and the blastocyst cells per se. This paper reports on this concordance according to the results of molecular-cytogenetic analysis of the chromosomal set with the use of comparative genomic hybridization.

Extracellular embryo genomic DNA and its potential for genotyping applications.

Background:Preimplantation genetic diagnosis (PGD) currently relies on biopsy of one or few embryo cells. Our aim was to evaluate the embryo extracellular matrices (spent medium and blastocoele fluid) as source of DNA for embryo genotyping.Results/methodology:We first evaluated the amplifiability and the amount of genomic DNA in spent embryo culture media from day 3 (n = 32) and day 5/6 (n = 54). Secondly, we evaluated the possibility to genotype the MTHFR polymorphism C677T from media at day 5/6 (n = 8) and blastocoele fluids (n = 9) by direct sequencing. The C677T polymorphism detection rate was 62.5 and 44.4% in medium and fluid, respectively.Conclusion:A noninvasive approach for embryo genotyping was possible, but still with limitations due to low detection rate and possible allele dropout.

Blastocoel fluid from differentiated blastocysts harbors embryonic genomic material capable of a whole-genome deoxyribonucleic acid amplification and comprehensive chromosome microarray analysis

To obtain embryonic molecular karyotypes from genomic DNA (deoxyribonucleic acid) isolated from blastocoel fluid (BF) and to compare these karyotypes with the karyotypes from the remaining inner cell mass (ICM) and trophectoderm (TE) of the blastocyst. Prospective cohort study. Academic center and preimplantation genetics laboratory. Ninety-six donated cryopreserved embryos. Embryo biopsy, BF aspiration, DNA analysis using a comparative genomic hybridization microarray (aCGH). The aCGH of a single blastomere, BF-DNA, and ICM-TE. The BF-DNA samples resulted in a successful aCGH in 63% of cases. Discordance in karyotypes was found between the BF-DNA and the ICM-TE in 52% of cases. A total of 70% of aneusomic (mosaicism), cleavage-stage embryos differentiated into euploid blastocysts. Probabilities for diagnostic accuracy were calculated and demonstrated the following: sensitivity of 0.88 (95% confidence interval [CI]: 0.62-0.98); specificity of 0.55 (95% CI: 0.39-0.70); positive predictive value of 0.41 (95% CI: 0.25-0.60); negative predictive value of 0.92 (95% CI: 0.75-0.99). Genomic DNA from the BF can be amplified and characterized by comprehensive chromosome microarrays. The results demonstrated that aneusomic cleavage-stage embryos differentiated into euploid blastocysts, possibly using a mechanism that marginalizes aneuploid nuclei into the blastocoel cavity. In addition, owing to the high discordance between the karyotypes obtained from the BF-DNA and the ICM-TE, using BF-DNA for preimplantation genetic testing is not yet advised.

317 SEX DETERMINATION OF EQUINE EMBRYOS BY PCR USING BLASTOCOELE FLUID

Pre-implantation genetic diagnosis (PGD) is used in different species to determine specific genetic traits of early stage embryos. The first part of this technique involves obtaining one or more cells from the embryo. Then, the DNA in the sampled cells is amplified by PCR using specific primers. Recently Palini et al. (2013) performed PGD of human blastocysts using the DNA in blastocoele fluid. The aim of our work was to study if the sex of equine in vivo produced blastocysts could be determined by PGD using blastocoele fluid as a sample for PCR. Thirteen equine blastocysts produced by artificial insemination and uterine flush were used for this study. Once obtained each embryo was placed on a 50-µL droplet of D-PBS without calcium and magnesium supplemented with 10% FBS and 50 µg mL–1 of gentamicin (working medium) under mineral oil, on an inverted microscope equipped with a micromanipulation system. The blastocoele fluid was aspirated using a beveled micropipette (9 µm ID) and then discharged on a 1-µL microdroplet of working medium. The microdroplet containing the blastocoele fluid was transferred to a 0.2-mL DNAse-free tube containing 4 µL of DNAse-free water. A duplex PCR was performed to amplify the Y-encoded testis-specific protein (TSPY) and amelogenin (AMEL) genes. The primer sequences used for this study were: AMEL-F 5′-CCAACCCAACACCACCAGCCAAACCTCCCT-3′, AMEL-R 5′-AGCATAGGGGGCAAGGGCTGCAAGGGGAAT-3′, TSPY-F 5′-GAAGTCAGGCACACCAGTGA-3′ and TSPY-R 5′-TAAGGCTGCAGTTGTCATGC-3′. The PCR was performed twice, using the same primers and cycling protocol. Embryonic cells from embryos which have been previously diagnosed as male and female were used as positive controls for PCR. As a negative control, one tube was included containing all the components for PCR, except the DNA sample was replaced with the same volume of DNAse-free water. The amplification products were electrophoresed on a 2% agarose gel stained with ethidium bromide and visualised under UV light. Embryo viability post-blastocoele fluid aspiration was studied by individually incubating seven aspirated embryos in 50-mL microdroplets of SOFm with 19 mM D-glucose with 10% FBS under mineral oil at 38°C, in 7% O2 and 5% CO2 and observed at 24, 48, and 72 h. The diameter of embryos was registered immediately before blastocoele fluid aspiration and every 24 h during in vitro culture. The sex of 11 out of 13 embryos (84.6%) was determined and they were diagnosed as 8 males and 3 females. Six of the 7 embryos cultured in vitro expanded at 24 h and increased their diameter at 48 and 72 h. Our results demonstrate that PGD of equine embryos can be performed using blastocoele fluid only, avoiding the need of extracting cells from the embryo. The in vitro embryo survival after blastocoele fluid aspiration was high, showing that this technique does not impair the embryo's viability. Further studies are being undertaken using a larger number of embryos to demonstrate if this approach can be used with high efficiency.

The accuracy of blastocoel fluid comprehensive chromosomal screening (CCS) is dependent on amplification yield and sequencing depth when using nextgen sequencing

The presence of DNA in the blastocoel provides an attractive target for less invasive aneuploidy screening. However, blastocoel fluid provides many challenges including DNA quantity and integrity. To date, a functional screening algorithm that overcomes these challenges and accurately identifies the karyotype of the blastocyst has not been validated. This study seeks to determine consistency of diagnosis between DNA isolated from trophectoderm and blastocoel fluid using NextGen based CCS.

Blastocoel fluid (BF) harbors embryonic DNA that may result from the marginalization of aneuploid cells during embryogenesis

OBJECTIVE: The endoplasmic reticulum (ER) stress is activated in response to an accumulation of unfolded protein in the lumen of the ER. ER stress is critical for cell survival, but chronic or excess ER stress can lead to apoptosis in various cell types. ER stress is known to cause various aging related diseases such as diabetics and neurodegenerative diseases. In this study, the participation of the ER stress in the postovulatory aging of oocyte was examined. DESIGN: Animal model study. MATERIALS AND METHODS: In this study, mouse oocytes released from the oviduct at 12.5 hour and 18.5 hour post-hCG were designated as ‘‘fresh’’ and ‘‘aged’’ oocytes, respectively. Experiments were done as follows: (1) Analysis of expression of BiP (chaperone induced at ER stress) level by western blotting with embryo development in the fresh oocytes, (2) Comparison of expression of BiP at protein level in the fresh and the aged oocytes, (3) Analysis of the effects by ER stress inducer, thapsigargin (Tg: calcium pump inhibitor of ER) on BiP expression and on fertilization rate and embryo development after in vitro fertilization. Animals were treated in accordance with the NIH Guide for the Care and Use of Laboratory Animals, as approved by the Animal Care and Use Committee of Yamagata University. RESULTS: (1) Expression of BiP was the highest in the MII oocytes, and decreased with embryo development after in vitro fertilization. (2) Expression of BiP in the aged oocytes was increased compared with that in the fresh oocytes. (3) Tg treated oocytes showed increase in expression of BiP, low fertilization rate and deteriorated embryo development. Furthermore, Tg treatment in the fresh oocytes deteriorated embryo quality. CONCLUSION: Expression of BiP in mouse oocyte/embryo decreased with the embryo development. The result that excessive expression of BiP in the aged oocyte was observed in association with the poor embryo development suggested that aged oocytes were exposed to excess ER stress. Because excess ER stress induced by Tg leaded low fertilization rate and poor embryo development, these data suggested that ER stress might participate in postovulatory aging process of the mouse oocyte. Supported by: This study was Supported by JSPS KAKEN research grants 20591905 to H.I.

The potential use of blastocoel fluid (BF) from expanded blastocysts as a less invasive form of embryo biopsy for preimplantation genetic testing

To obtain embryonic molecular karyotypes from nuclear DNA obtained from the BF of expanded blastocysts and compare the karyotype with the remaining inner cell mass and trophectoderm (ICM/TE) cells of a differentiated blastocyst.

Proteomic analysis of the early bovine yolk sac fluid and cells from the day 13 ovoid and elongated preimplantation embryos

The bovine blastocyst hatches 8 to 9 days after fertilization, and this is followed by several days of preimplantation development during which the embryo transforms from a spherical over an ovoid to an elongated shape. As the spherical embryo enlarges, the cells of the inner cell mass differentiate into the hypoblast and epiblast, which remain surrounded by the trophectoderm. The formation of the hypoblast epithelium is also accompanied by a change in the fluid within the embryo, i.e., the blastocoel fluid gradually alters to become the primitive yolk sac (YS) fluid. Our previous research describes the protein composition of human and bovine blastocoel fluid, which is surrounded by the trophectoderm and undifferentiated cells of the inner cell mass. In this study, we further examine the changes in the protein composition in both the primitive YS fluid and the embryonic cells during early and slightly later stage cell differentiation in the developing bovine embryo. In vitro–produced Day 6 embryos were transferred into a recipient heifer and after 7 days of further in vivo culture, ovoid and elongated Day 13 embryos were recovered by flushing both uterine horns after slaughter. The primitive YS fluid and cellular components were isolated from 12 ovoid and three elongated embryos and using nano-high-performance liquid chromatography, tandem mass spectrometry, and isobaric tag for relative and absolute quantitation proteomic analysis, a total of 9652 unique proteins were identified. We performed GO term and keyword analyses of differentially expressed proteins in the fluid and the cells of the two embryonic stages, along with a discussion of the biological perspectives of our data with relation to morphogenesis and embryo-maternal communication. Our study thereby provides a considerable contribution to the current knowledge of bovine preimplantation development.

Tail structure is formed when blastocoel roof contacts blastocoel floor in Xenopus laevis

The tail organizer has been assessed by such transplantation methods as the Einsteck procedure. However, we found that simple wounding of blastocoel roof (BCR) made it possible to form secondary tails without any transplantation in Xenopus laevis. We revealed that the ectopic expression of Xbra was blocked by inhibiting the contact between BCR and blastocoel floor (BCF), and wounding per se seemed to be not directly related to the secondary tail formation. Therefore, the secondary tail might be induced by the contact between BCR and BCF due to the leak of blastocoel fluid from the wound. This secondary tail was similar to the original tail in the expression pattern of tail genes, and in the fact that the inhibition of fibroblast growth factor signaling prevented the secondary tail induction. Our results imply that the secondary tail formation reflects the developmental processes of the original tail, indicating that simple wounding of BCR is useful for the analysis of tail formation in normal development.

Proteomic analysis of bovine blastocoel fluid and blastocyst cells

The understanding of the early mammalian development is a prerequisite for the advancement of in vitro fertilization and improvement of derivation and culturing of embryonic stem cells. While, whole genome transcriptomic analysis on bovine blastocysts has identified genes active in early development, little information is available about the protein complement of early embryos. Modern, sensitive proteomic technology (nano HPLC tandem mass spectrometry) allowed us to describe the proteome of the scarce blastocoel fluid and cell material of expanded bovine blastocysts isolated by micromanipulation. From two independent replicates, 23 proteins were identified in the blastocoel fluid while 803 proteins were identified in the remaining cell material. The proteins were grouped into categories according to their gene ontology (GO) terms by which proteins involved in cell differentiation, cell proliferation, development, and reproduction could be derived. Proteins classified in these categories could be candidates for further functional studies to understand pluripotency and early mammalian development.

Comparative Genomic Hybridization Microarray (aCGH) Analysis of DNA Isolated from the Blastocoel Fluid from 26 Blastocysts

Currently the only sources of embryologic DNA used to perform preimplantation genetic testing (PGT) includes a cleavage stage blastomere or trophectoderm (TE) cells from a differentiated blastocyst. The potential use of DNA isolated from blastocoel fluid for PGT has not been fully explored.

Vitrification solution without sucrose for cryopreservation in mouse blastocysts

ObjectiveThis study was designed to investigate the survival rate of vitrified mouse blastocysts depending on the presence or absence of sucrose in vitrification solution.MethodsMouse two-cell embryos were collected and cultured to blastocysts. Two vitrification solutions were prepared. The control solution was composed of 25% glycerol, 25% ethylene glycol, and 0.5 M sucrose (G25E250.5S) containing 2.5 mL glycerol, 2.5 mL ethylene glycol, 2 mL SSS, and 0.855 g sucrose in 5 mL PB1. The experimental solution was composed of 25% glycerol and 25% ethylene glycol (G25E25) and contained 2.5 mL glycerol and 2.5 mL ethylene glycol in 5 mL PB1. Artificial shrinkage was conducted by aspirating the blastocoelic fluid using an ICSI pipette. To examine the effect of sucrose in the vitrification solution on the survival rate of mouse blastocysts, the shrunken-equilibrated blastocysts were rehydrated or vitrified after being exposed to one of the two vitrification solutions. After exposure and the vitrification-thawing process, the re-expansion rate and hatching rate were evaluated after 6 hours of in vitro culture.ResultsThe re-expansion rate of mouse blastocysts exposed to vitrification solution with and without sucrose were not different in the experimental solution (without sucrose) (98%) and the control solution (with sucrose) (92%) (p>0.05). The hatching rate was higher in the experimental solution (95%) than in the control solution (88%), but did not differ across two treatments (p>0.05). The re-expansion rate of mouse blastocysts vitrified in the control solution was 92% and 94%, respectively (p>0.05), and the hatching rate was higher in the experimental solution (90%) than in the control solution (74%) (p<0.05).ConclusionSucrose need not be added in vitrification solution for freezing of artificially shrunken mouse blastocysts.

136 BLASTOCOELE COLLAPSE IMPROVES POST-THAW SURVIVAL OF SLOW FROZEN AND VITRIFIED IN VITRO-PRODUCED BOVINE EMBRYOS

Slow-freeze cryopreservation of in vitro-produced bovine embryos often results in poor post-thaw embryo viability. Results with vitrification tend to be better but do not always produce consistent results for a variety of reasons. Experiments with human and equine embryos have demonstrated improved post-thaw survival when blastocoele fluid is removed before vitrification. We hypothesised that removing blastocoele fluid before vitrification or slow-freeze cryopreservation would improve post-thaw survival of in vitro-produced bovine embryos. Three replicates of embryos were generated from abattoir-derived ovaries using standard techniques. Seven days post-fertilization, embryos were evaluated for quality, and grade 1 or 2 embryos were allocated to 1 of 4 treatments: slow-freeze control (n = 103); slow-freeze collapsed (n = 106); vitrified control (n = 117); vitrified collapsed (n = 99). Blastocoele fluid was removed by aspiration with a pipette advanced to the centre of the blastocoele while the embryos were stabilised with a holding pipette. After collapse, embryos were immediately slow frozen in commercial medium [1.5 M ethylene glycol (EG) + 0.1 M sucrose, Bioniche] in 0.25-mL straws or vitrified in 2 steps: 3 min in 1.5 M EG, 30 s in 7 M EG + 0.6 M galactose + 18% Ficoll in open-pulled straws. After thawing or warming, embryos were cultured in chemically defined medium-2 + 5% FCS in 5% CO2, 5% O2, 90% N2 at 38.5°C. Embryos were evaluated for survival based on re-expansion of the blastocoele after 24 h in culture. Data were analysed by GLIMMIX (SAS Institute Inc., Cary, NC, USA). There were significant replicate effects but there were no significant differences in survival between embryos that were vitrified or slow frozen. Approximately twice as many collapsed embryos survived compared to intact control embryos regardless of whether the embryos were vitrified or slow frozen (collapsed: 50.5 ± 4.1%, control: 26.3 ± 3.5%; P &lt; 0.001). Advanced blastocysts (fully expanded and hatched blastocysts) survived better than early blastocysts (pre-expansion blastocysts and expanding blastocysts, 45.9% ± 3.9 v. 30.0% ± 4.4, respectively; P &lt; 0.01). The only significant interaction (P &lt; 0.05) was that vitrification was superior to freezing for early blastocysts (39.5 ± 5.9% v. 22.1 ± 5.0% survival) but not advanced blastocysts (44.4 ± 5.3% v. 47.4 ± 5.8% survival). Removal of blastocoele fluid before cryopreservation of in vitro-produced bovine embryos may be a useful technique for improving post-thaw survival regardless of cryopreservation method or blastocyst stage.

52 VITRIFICATION OF DAY-8 EQUINE EXPANDED BLASTOCYST: AN IN VITRO COMPARISON OF DIRECT VERSUS INDIRECT MECHANICAL INTRODUCTION OF CRYOPROTECTANT

The cryopreservation of equine expanded blastocysts (&gt;300 μm) has been largely unsuccessful primarily due to the low permeability of the embryo to cryoprotectants. This low permeability has been attributed to the acellular glycoprotein capsule that develops when an embryo approaches approximately 300 μm in diameter. Mechanical alternatives may provide a means to overcome the capsule barrier and the relative large embryo size to successfully cryopreserve equine embryos. The objective of this experiment was to compare re-expansion rates of vitrified equine expanded blastocysts following either direct or indirect mechanical introduction of cryoprotectants using a coaxial microinjection system (Dracula pipette). Twenty-six Day-8 expanded blastocysts were subjected to capsule puncture, cryoprotectant injection, and blastocoele fluid extraction (direct treatment) or capsule puncture and blastocoele fluid extraction (indirect treatment) before cryopreservation. The Dracula pipette incorporates the injection pipette within the holding pipette, facilitating aspiration of blastocoele fluid or injection of cryoprotectant in a single unit. A standard vitrification protocol using a final concentration of 3.4 M glycerol and 4.6 M ethylene glycol at cryopreservation was used. Vitrified embryo re-expansion was assessed following in vitro culture at 24, 48, and 72 h post-warming. Differences across treatments were analysed using the Student's t-test for re-expansion and the chi-squared test of independence for capsule loss. Pre-vitrification embryo mean diameter (mean ± standard error) for direct and indirect treatment groups were not different, 979 ± 85.6 μm and 912 ± 101.4 μm, respectively (P = 0.62). Post-vitrification embryo mean diameters were not different for the direct and indirect treatments (688 ± 63 and 662 ± 75 μm, respectively; P = 0.79). Following 72 h of in vitro culture, there was no difference in mean embryo diameter (1813.16 ± 209 μm v. 1383.88 ± 198 μm; P = 0.21), or re-expansion rates (69 v. 69%) for direct and indirect treatment groups, respectively. However, partial or total capsule loss was 69% (9/13) for direct treatment embryos compared with 30% (4/13) for indirect treatment embryos (P = 0.049). Results from this experiment demonstrate that capsule puncture and blastocoele fluid extraction before vitrification resulted in high re-expansion rates of Day-8 equine expanded blastocysts after warming. More importantly, the relatively large percentage of capsule failure when directly introducing cryoprotectant into the embryo may interfere with maternal recognition of pregnancy following embryo transfer. Nonetheless, based on the embryonic re-expansion rate of vitrified equine embryos following the indirect technique, we anticipate that a relatively high pregnancy rate can be obtained if this technique is used.

Validation of blastocoele fluid aspiration for preimplantation genetic screening using array comparative genomic hybridization (aCGH)

Trophectoderm (TE) biopsy at the blastocyst stage is increasingly used in conjunction with preimplantation genetic screening (PGS). Recently, one study reported evidence of DNA within the blastocoele fluid (Palini et al. 2013), raising the possibility that sampling of genetic material could be simplified. This study tested whether blastocoele fluid aspiration provides an adequate source of DNA for chromosome analysis.

The blastocoel fluid as a source of DNA for preimplantation genetic diagnosis and screening

The blastocoel fluid as a source of DNA for preimplantation genetic diagnosis and screening

Sucrose needs not to be added in vitrification solution for freezing the artificial shrunken mouse blastocysts

Sucrose included in freezing medium causes embryos to shrink by losing intracellular water, so that intracellular ice formation is reduced and embryos can be frozen rapidly. Blastocyst consists of trophoblast and inner cell mass. It may be considered that the artificial shrunken blastocyst is not embryo but tightly packed somatic cells. This study was carried out to examine the necessary of sucrose addition to vitrification solution for vitrifying the artificial shrunken mouse blastocysts. Laboratory study using mice blastocyst. To investigate the toxicity of vitrification solutions and the availability of artificial shrinkage technique with ICSI pipette, blastocoel fluid was aspirated in the expanded mouse blastocysts using ICSI pipette and shrunken blastocysts were equilibrated in EBS1 and EBS2 for 1.5 min at room temperature, reapectively. Then the equilibrated blastocysts were exposed to vitrification solution for maximum of 20 sec. Two vitrification solutions with and without sucrose were prepared. Control and treatment were composed of 25%glycerol + 25% ethyleneglycol and 25%glycerol + 25%ethyleneglycero +0.5 mol/l sucrose, respectively. Assisted hatching was performed by partial zona dissection method using assisted hatching pipette after thawing procedure or rehydration. Rates of re-expanding and hatching were examined after culture for 6h. Re-expanding rate of mouse blastocyst exposed to VS(without and with 0.5 mol sucrose) were not different in G25E25(98%) and G25E25S0.5(92%) (P>0.05) and hatching rate was higher in G25E25(95%) than in G25E25S0.5(88%) but did not differ in two treatments (P>0.05). Re-expanding rate of mouse blastocyst vitrified in G25E25 and G25E25S0.5 was 95% and 90%, respectively (P=0.05), and hatching rate was higher in G25E25(90%) than in G25E25S0.5(76%) (P>0.05). Our results showed that sucrose needs not to be added in vitrification solution for freezing the artificial shrunken mouse blastocysts.