Blastocyst Medium Research Articles

Lipid-rich bovine serum albumin improves the viability and hatching ability of porcine blastocysts produced in vitro.

The effects of lipid-rich bovine serum albumin (LR-BSA) on the development of porcine blastocysts produced in vitro were examined. Addition of 0.5 to 5 mg/ml LR-BSA to porcine blastocyst medium (PBM) from Day 5 (Day 0 = in vitro fertilization) significantly increased the hatching rates of blastocysts on Day 7 and the total cell numbers in Day-7 blastocysts. When Day-5 blastocysts were cultured with PBM alone, PBM containing LR-BSA, recombinant human serum albumin or fatty acid-free BSA, addition of LR-BSA significantly enhanced hatching rates and the cell number in blastocysts that survived compared with other treatments. The diameter, ATP content and numbers of both inner cell mass and total cells in Day-6 and Day-7 blastocysts cultured with PBM containing LR-BSA were significantly higher than in blastocysts cultured with PBM alone, whereas LR-BSA had no effect on mitochondrial membrane potential. The mRNA levels of enzymes involved in fatty acid metabolism and β-oxidation (ACSL1, ACSL3, CPT1, CPT2 and KAT) in Day-7 blastocysts were significantly upregulated by the addition of LR-BSA. The results indicated that LR-BSA enhanced hatching ability and quality of porcine blastocysts produced in vitro, as determined by ATP content, blastocyst diameter and expression levels of the specific genes, suggesting that the stimulatory effects of LR-BSA arise from lipids bound to albumin.

MicroRNAs in spent blastocyst culture medium are derived from trophectoderm cells and can be explored for human embryo reproductive competence assessment

To assess whether extracellular microRNAs (miRNAs) can be accurately profiled from spent blastocyst culture media (SBM) and used as embryonic biomarkers. Prospective cohort study. Private and academic in vitro fertilization centers. Inner cell mass-free trophectoderm (TE) samples and their relative SBM from five good-quality human blastocysts. Protocol for miRNA purification and analysis based on quantitative polymerase chain reaction set and validated on human embryonic stem cells (hESCs) and on SBM with and without biological variability. Analysis of miRNAs in culture media in relation with TE cells and comparison of miRNA profiles between implanted and unimplanted euploid blastocysts. Culture media from embryos in the cleavage, morula, and blastocyst stages were collected to investigate the presence of miRNAs. The SBM were prospectively collected from euploid implanted (n = 25) and unimplanted blastocysts (n = 28) for comparison. We hypothesized that human embryos secrete miRNAs in culture media that can be used as biomarkers. The comparative analysis of TE and SBM samples revealed that 96.6% (57 of 59; 95 CI, 88.3-99.6) of the miRNAs detected in the SBM were expressed from TE cells, suggesting a TE origin. The culture media collected from cleavage and morula stage embryos showed a pattern similar to blanks, suggesting that miRNAs profiling from spent culture media applies only for blastocysts. MicroRNAs analysis of SBM from euploid implanted and unimplanted blastocysts highlighted two miRNAs (miR-20a, miR-30c) that showed increased concentrations in the former and were predicted in silico to be involved in 23 implantation-related pathways. MicroRNAs secreted from human blastocysts in culture media can be profiled with high reproducibility, and this approach can be further explored for noninvasive embryo selection.

Transportation of Preimplantation Porcine Embryos without Cryopreservation Using a Novel Embryo Carrier

Abstract: The objective of this study was to develop a transportation system for fresh porcine embryos in a chemically defined medium without reducing their viability using a novel embryo carrier. When embryos were introduced in 0.25-ml straws with porcine blastocyst medium (PBM) and given a vibration load on a shaker for 20 h to mimic transportation conditions at 38 °C, there was no significant difference in survival rates between the vibration and no-vibration groups. Next, an embryo carrier was developed that can maintain interior conditions of 5% CO2 and 38 °C for the transportation of embryos. The embryos on Day 5 after insemination were divided into three groups. In the first group, the embryos were introduced to straws with PBM and the straws were sealed (sealed group). In the second group, the embryos were transported in a tube with a CO2 pouch (CO2 gas group). They were transported by a door-to-door delivery service. In the last group, the embryos were cultured in an incubator (not-transported gr...

Human Blastocyst Secreted microRNA Regulate Endometrial Epithelial Cell Adhesion

Successful embryo implantation requires synchronous development and communication between the blastocyst and the endometrium, however the mechanisms of communication in humans are virtually unknown. Recent studies have revealed that microRNAs (miRs) are present in bodily fluids and secreted by cells in culture. We have identified that human blastocysts differentially secrete miRs in a pattern associated with their implantation outcome. miR-661 was the most highly expressed miR in blastocyst culture media (BCM) from blastocysts that failed to implant (non-implanted) compared to blastocysts that implanted (implanted). Our results indicate a possible role for Argonaute 1 in the transport of miR-661 in non-implanted BCM and taken up by primary human endometrial epithelial cells (HEECs). miR-661 uptake by HEEC reduced trophoblast cell line spheroid attachment to HEEC via PVRL1. Our results suggest that human blastocysts alter the endometrial epithelial adhesion, the initiating event of implantation, via the secretion of miR, abnormalities in which result in implantation failure.

GlutaMAX prolongs the shelf life of the culture medium for porcine parthenotes

In vitro porcine embryo production systems have been established and well characterized. However, the efficiency of embryo development during IVC is still very low. In the present study, we have investigated the development of parthenogenetic porcine embryos in the well-known PZM-5 medium for porcine embryos, which was modified by replacing glutamine with the GlutaMAX supplement. We revealed that blastocyst apoptosis was significantly lower in the presence of GlutaMAX, which reduced the release of mitochondrial cytochrome c. Furthermore, the expression of apoptosis genes was significantly lower during GlutaMAX treatment (P < 0.05). The modified medium was also examined for the eventual loss of its efficacy in the presence of GlutaMAX. Three, 6, and 12 months after medium preparation, blastocyst formation in the GlutaMAX-supplemented medium was significantly higher than the number of blastocysts in the medium containing glutamine. After a long period of storage, ammonia concentration was significantly increased in the glutamine medium, whereas it was not statistically different in the GlutaMAX medium. Elevated ammonia concentrations reduced the mitochondrial membrane potential and ATP content of blastocysts in the glutamine medium. These results demonstrate that GlutaMAX can reduce blastocyst apoptosis via inhibition of the cytochrome c pathway and significantly extend the shelf life of the culture medium to at least 1 year.

Reduced blastocyst formation in reduced culture volume.

The aim of this prospective sibling oocyte study was to evaluate whether reduced culture volume improves blastocyst formation. Twenty-three patients with extended embryo culture until day 5 were selected for the study. After injection, 345 sibling oocytes were individually cultured in either 25 or 7μl droplets of Origio cleavage medium under oil. On day 3 of development, embryos were transferred to droplets with the corresponding volume of Origio blastocyst culture medium. Fertilization and embryo quality on day 3 and day 5/6 were evaluated. No statistically significant difference (p = 0.326) in fertilization rate was observed (81.3 versus 83.0%). There was no significant difference in terms of the number of excellent and good-quality embryos obtained on day 3 between both groups (p = 0.655). Embryo culture in 25μl droplets led to more embryos with a higher cell number when compared to 7μl culture (p = 0.024). On day 3, 132 and 131 embryos were considered for further culture until day 5/6. Blastulation rates were significantly higher in the 25μl group (75.0 versus 61.6%; p = 0.017) and significantly more day 5 embryos with excellent and good quality were found in this group (54.5 versus 40.5%; p = 0.026). Finally, the utilization rates expressed per mature oocyte (41.4 versus 29.8%; p = 0.043), per fertilized oocyte (50.7 versus 36.6%; p = 0.023), and per day 3 embryo undergoing extended culture to day 5/6 (54.5 versus 39.7%; p = 0.019) were all significantly higher in the 25μl group. Reduced culture volume (7μl) negatively impacts early development by reducing the cell number on day 3 and both blastocyst formation and quality.

Birth of piglets from in vitro–produced porcine blastocysts vitrified and warmed in a chemically defined medium

We examined the effect of different embryo developmental stages, culture periods, and media on the cryotolerance of in vitro–produced porcine blastocysts. All media used for in vitro production, vitrification, and warming were chemically defined. When in vitro–produced embryos at the blastocyst and expanded blastocyst stages were vitrified using the Cryotop method on Day 5 or 6 (Day 0 = the day of IVF), the survival rate and hatching rate of expanded blastocysts after warming were higher than those of blastocysts. The viability after vitrification and warming of Day-6 embryos cultured in porcine blastocyst medium from Day 5 were higher than that of embryos cultured in porcine zygote medium 5. On the other hand, there were no significant differences on the cryotolerance between Day-5 embryos cultured in porcine zygote medium 5 and those replaced with porcine blastocyst medium on Day 4. There were no significant differences in viability between the embryonic ages of 5 and 6 days after vitrification and warming. When expanded blastocysts vitrified on Day 5 or 6 were surgically transferred to recipient gilts, all three recipients became pregnant in the Day-5 group, whereas only one out of three recipients became pregnant in the Day-6 group. These results indicate that the cryotolerance of porcine in vitro–produced blastocysts after vitrification appears to depend on the embryonic stage, culture period, and medium.

Comparison of blastocyst and Sage media for in vitro maturation of human immature oocytes.

In vitro maturation (IVM) of human oocytes is an attractive alternative to conventional assisted reproductive technology (ART) treatment, as it involves no or minimal ovarian stimulation. Currently, commercialized media specifically designed for IVM are often used. These media are expensive, have limited shelf life, and must be ordered in advance. If standard culture media can be used in place of the specialized IVM media, it would simplify management and make IVM more feasible and more widely employed in ART centers around the world, especially in developing countries where resources are scarce. This study was, therefore, conducted to test the hypothesis that blastocyst medium was as good as commercial IVM medium to support maturation and developmental competence of human immature oocytes as previously shown in the mouse system. Immature oocytes were obtained by needle aspiration from 89 pregnant women during cesarean deliveries between April 2012 and February 2013. Sibling oocytes were allocated to Sage IVM media (512 oocytes) or blastocyst medium (520 oocytes) and assessed for maturation 36 hours later. Mature oocytes were inseminated by intracytoplasmic sperm injection and cultured up to 144 hours. There was no difference in maturation rate (65.0% vs 68.7%; P = .218) or fertilization rate (66.9% vs 66.4%; P = .872) of oocytes matured in vitro in both media. There was also no difference in the formation of good-quality blastocysts (46.6% vs 45.9%; P = .889) in the 2 groups. Further study should be done to ascertain implantation and pregnancy potential of these embryos.

Role of Embryo Glue as a transfer medium in the outcome of fresh non-donor in-vitro fertilization cycles.

BACKGROUND:EmbryoGlue is a hyaluronan-enriched embryo transfer (ET) medium which aids in implantation of embryos, hence, improves pregnancy rates in in-vitro fertilization-ET cycles (IVF-ET).AIM:To evaluate the role of EmbryoGlue in improving implantation and pregnancy rates.DESIGN AND SETTING:A prospective case–control study conducted at assisted reproductive center of a tertiary care hospital.METHOD:In 42 women undergoing IVF, embryos were transferred into 50 μL of EmbryoGlue for 10 min prior to transfer inside uterine cavity. In the control group (n = 42), embryos were transferred to conventional blastocyst culture medium. Statistical analysis was performed using SPSS IBM version 19.0.RESULTS:Clinical pregnancy rate in the study group was 7% higher than the control group. The difference, however, was not statistically significant. In addition, no improvement in implantation rates was observed in the study group. However, significant difference (P = 0.04) in clinical pregnancy rate was observed with the EmbryoGlue in patients with previous IVF failure. In the study group, 50% patients (6/12) with previous IVF failure had successful implantation, but in the control group none of the patients (0/11) with previous implantation failure could achieve pregnancy.CONCLUSION:It is difficult to conclude a favourable role of EmbryoGlue in IVF-ET cycles with a good prognosis. However, in patients with recurrent implantation failure, it may be considered as a useful transfer medium.

40 TRICHOSTATIN A TREATMENT EFFECTS ON IN VITRO DEVELOPMENT OF INTERSPECIES NUCLEAR TRANSFER CAT EMBRYOS DEPEND ON RECIPIENT CYTOPLASM SPECIES

The developmental ability of interspecies somatic cell nuclear transfer (iSCNT) embryos decreases as the taxonomic distance between the donor and recipient species increases. Treatment of cat iSCNT embryos using bovine oocytes with 50 nM of trichostatin A (TSA) improves in vitro embryonic development (Wittayarat et al. 2013 Cell. Reprogram. 15, 301–308). This study investigated whether the TSA treatment effects differ between the development of cat iSCNT embryos reconstructed with porcine and bovine oocytes. Porcine and bovine cumulus-oocyte complexes were in vitro matured for 44 h and 24 h, respectively. After cumulus cell removal, enucleation was performed by aspiration of the metaphase II plate and the first polar body using a piezo-driven pipette. A cat fibroblast cell was then injected into cytoplasm of successfully enucleated oocyte. Reconstructed cybrids were electrically activated by a single 1.5 kV cm–1 pulse for 100 µs (pig-cat embryos), or a 2.3 kV cm–1 pulse for 30 µs (cow-cat embryos). Pig-cat and cow-cat embryos were cultured in porcine zygote medium (PZM)-5 and modified synthetic oviducal fluid medium (mSOF), respectively. After electrical activation, pig-cat and cow-cat embryos were cultured in medium supplemented with 5 µg mL–1 cytochalasin B + 50 nM TSA (TSA group) or without TSA (control group), and the cow-cat embryo medium was also supplemented with 10 µg mL–1 cycloheximide. After 2 h, TSA-treated pig-cat and cow-cat embryos were incubated in medium supplemented with TSA for 22 h, followed by 48 h incubation without TSA. Pig-cat and cow-cat control embryos were cultured in medium without TSA for 70 h after activation. Then, all pig-cat and cow-cat embryos were cultured in porcine blastocyst medium (PBM) or mSOF medium supplemented with 5% fetal bovine serum, respectively, for 5 additional days. Four to seven replicates were performed for each experiment. Data were analysed using Student's t-test. For pig-cat embryos, no difference was observed in cleavage rates between both groups, but development to the blastocyst stage was higher in the pig control group (n = 147, 8.0%) than that of pig TSA group (n = 131, 0.7%; P &lt; 0.05). In contrast, development to the blastocyst stage in cow-cat embryos was not observed in the cow control group (n = 125, 0%), but it was observed in cow TSA group (n = 136, 3.7%). These results indicate that TSA treatment effects are species-specific, but those effects remain to be clarified.

136 INVESTIGATION OF LEPTIN ON DEVELOPMENT OF MOUSE EMBRYOS

Leptin is a hormone-like protein of 167 amino acids. As an adipocyte-related hormone it has an important role in weight regulation and physical fitness but also has effects on reproductive and other physiological mechanisms. The aim of the present study was to investigate the effects of different concentrations of leptin added to the culture media, the quality, in vitro development rate, and in vivo rate of mouse embryos. Superovulated CB6F1 (C57BL/6XBalb/c) hybrid female mice (5–6 weeks of age) were killed ~18 to 20 h after hCG administration. Single-cell embryos were flushed from the oviducts of the dead mice with human tubal fluid medium supplemented with HEPES and 3 mg mL–1 of BSA. They were cultured in Quinn's cleavage medium supplemented with 4 mg mL–1 of BSA in 5% CO2, 37°C until reaching 2-cell stage. The 2-cell embryos were randomly divided into 4 groups and cultured in Quinn's blastocyst medium supplemented with 4 mg mL–1 BSA + 0, 10, 50, and 100 ng mL–1 leptin (L0, L10, L50, and L100) in 5% CO2, 37°C until the blastocyst stage. Some of the developing blastocysts were used for differential staining for the inner cell mass and trophectoderm (TE) cells [Mallol et al. 2013 Syst. Biol. Reprod. Med. 59,117–122]. Some of them were transferred into pseudopregnant females (CD1) on the 2.5 to 3.5th days and kept until the 13.5th day of pregnancy for the in vivo development rate. The results were evaluated using one-way ANOVA with Bonferroni post-hoc test in SPSS 22.0. The P-values &lt;0.05 were considered statistically significant. Each experiment was repeated at least 4 times. The blastocyst development rates of L0, L10, L50, and L100 were 92.57% (162/175), 97.16% (205/211), 97.80% (178/182), and 97.85% (182/186), respectively. The in vitro development rates were significantly higher in the L10, L50, and L100 compared with L0 (P &lt; 0.05). The inner cell mass cells of L0, L10, L50, and L100 were 13.17, 14, 16, and 15.43. There was no significant difference between the groups in terms of inner cell mass cells (P &gt; 0.05). The TE cells of L0, L10, L50, and L100 were 47, 56.4, 53.7, and 58.57, respectively. The TE numbers were significantly increased in the presence of L10 and L100 compared with L0 (P &lt; 0.05). The in vivo development rates of L0, L10, L50, and L100 were 13.51% (5/37), 48.72% (19/39), 15.38% (6/39), and 41.03% (16/39), respectively. The in vivo development rates of L10 and L100 were significantly higher than for L0 and L50 (P &lt; 0.05). The resorption rates of L0, L10, L50, and L100 were 10.8% (4/37), 30.8% (12/39), 12.8% (5/39), and 20.5% (8/39), respectively. There was no significant difference between the groups in terms of the resorption rates (P &gt; 0.05). This study found that L10, L50, and L100 were supporting the development of the embryos in the in vitro culture. The L10, L50, and L100 significantly increased the total cell numbers. The L10 and L100 were particularly effective on the number of the TE cells. In conclusion, 10 and 100 ng mL–1 leptin have a positive effect on the in vitro, quality and in vivo development of the mouse embryo. Therefore, leptin seems to play an important role on the embryo development and in vivo development. Research supported by TUBITAK-113O223.

Effects of reactive oxygen species levels in prepared culture media on embryo development: A comparison of two media

ObjectiveThis study determined the correlation between the levels of reactive oxygen species (ROS) in prepared culture media and the early development of human embryos. Materials and methodsThis was an autocontrolled comparison study. A total of 159 patients undergoing in vitro fertilization/intracytoplasmic sperm injection treatment were recruited in this study. The pH values, osmolarity pressures, and ROS levels of 15 batches of two culture media were measured. Sibling oocytes or embryos from individual patients were randomly assigned to two culture groups with Quinn's Advantage Cleavage and Blastocyst media (QAC/QAB) or GIII series cleavage and blastocyst media (G1.3/G2.3). The difference between the two culture groups was analyzed using one-sample t test. ResultsThe QAC/QAB and G1.3/G2.3 media exhibited similar pH values and osmolarity pressures. However, the prepared QAC/QAB media were characterized to contain lower amounts of ROS than the G1.3/G2.3 media. Furthermore, the blastocysts that developed under the QAC/QAB media were morphologically superior to those that developed under the G1.3/G2.3 media. ConclusionThe elevated ROS levels in culture media were associated with poor development of blastocyst-stage embryos. Measurement of ROS levels may be a valuable process for medium selection or modification.

Effect of knockout serum replacement supplementation to culture medium on porcine blastocyst development and piglet production

We have previously developed chemically defined media suitable for in vitro production (IVP) of porcine embryos and subsequently generated piglets by nonsurgical embryo transfer. In this study, to further improve the culture conditions for IVP of porcine embryos, we evaluated the effect of knockout serum replacement (KSR), a substitute for serum or albumin, on the viability and development of porcine blastocysts. The addition of 5% (v:v) KSR to porcine blastocyst medium (PBM) on Day 5 (Day 0 = IVF) significantly increased the survival and hatching rates of blastocysts and the total cell number of Day-7 blastocysts compared with those in cultures without KSR or addition of 10% fetal bovine serum. Furthermore, the number of cells in the trophectoderm of Day-6 blastocysts and the ATP content of Day-7 blastocysts cultured with 5% KSR were significantly higher than those of blastocysts cultured without KSR. The mRNA expression of a rate-limiting enzyme in β-oxidation, carnitine palmitoyltransferase 1, in Day-6 blastocysts, and a serine proteinase, urokinase-type plasminogen activator, in Day-7 blastocysts cultured in 5% KSR-PBM was significantly higher than that of blastocysts cultured in PBM alone. Four of eight recipients (50%), in which Day-5 blastocysts treated with 5% KSR were transferred nonsurgically, became pregnant. However, the efficiency of piglet production (percentage of piglets born based on the number of embryos transferred) was similar to recipients with transferred blastocysts treated without KSR. The present study demonstrated that the addition of KSR to PBM enhanced the in vitro viability of porcine blastocysts. In addition, our data suggest that KSR improved development to the hatching stage and blastocyst quality by increasing ATP content and hatching-related mRNA expression of blastocysts.

“No Transfer Rates” In Vitrified and Fresh Donor Oocytes Cycles – Implication for Fertility Preservation

“No Transfer Rates” In Vitrified and Fresh Donor Oocytes Cycles – Implication for Fertility Preservation

Developmental competence of human embryos derived from in vitro maturation of immature oocytes retrieved during cesarean section

To investigate the developmental competence of human embryos that originated from in vitro matured oocytes retrieved during cesarean section. Immature oocytes were collected from 95 pregnant women, who underwent cesarean section at Buddhachinaraj Hospital Medical School and consented to participate in the study. Retrieved oocytes were cultured in blastocyst medium supplemented with 75 IU/L of human menopausal gonadotropin. Oocyte maturation was assessed at 30 and 48 h after culture. In vitro matured oocytes were inseminated by intracytoplasmic sperm injection and cultured up to 144 h. The fertilization, cleavage and blastocyst formation rates were observed. Maturation rate of oocytes after 30 h of culture was 67.9% compared with 13.1% at 48 h (P < 0.0001). Insemination of oocytes in both groups resulted in similar fertilization, cleavage and blastocyst formation rates. A large proportion of oocytes retrieved at the time of cesarean section exhibited the capacity to undergo maturation in vitro. They can be fertilized and developed into good-quality blastocyst stage.

Identification of Galectin-9 on exosomes from human blastocyst culture medium

Exosomes are small membrane vesicles secreted by various cells by inward budding of endosomes into multivesicular bodies. They are important for communication between cells, delivery of functional RNA to other cells, cell adhesion and antigen presentation. Exosomes released by the blastocyst may be an important mediator for implantation. Galectin-9, a β-galactoside binding lectin, has been found in human endometrial epithelial cells with significant upregulation during the window of implantation.

Human preimplantation embryo development in vitro: a morphological assessment of sibling zygotes cultured in a single medium or in sequential media

A comparison was made of the development of human zygotes in either a one-step (Global® medium) or two-step culture system (Quinn's Advantage®). A total of 257 normally fertilized 2PN zygotes from 28 patients were used in the study. The study was broken down into two parts: the first concerned the development of embryos from Days 1 to 3 in Global® medium and Quinn's Advantage® cleavage medium; the second consisted of a comparison of the development of embryos from Day 3 to 5/6 in Global® medium and Quinn's Advantage® blastocyst medium. There were no significant differences between the two culture media with respect to embryo quality throughout the preimplantation phase of human embryo development as determined by the extent and variability of the cell counts, fragmentation, and nucleation. A difference was noted in the blastomere symmetry of Day 2 embryos in the two media, but was no longer apparent on examination of Day 3 embryos. No differences were noted in the rates of blastocyst development, inner cell mass (ICM), and trophectoderm (TE) scores in the two culture media. Finally, no significant differences were noted with either the proportion of blastocysts chosen for transfer or cryopreservation (vitrification). The findings support the view that two-step sequential media protocols are sufficient but not necessary to support the complete in vitro development of human preimplantation embryos.

150 IN VITRO AND IN VIVO DEVELOPMENT OF EMBRYO FOLLOWING BIOPSY WITH DIFFERENT TECHNIQUES IN MOUSE EMBRYOS

In vitro development ratios, quality evaluation, in vivo implantation, and fetal development ratios were investigated following aspiration biopsy in 8-cell mouse embryos and trophectoderm biopsy in blastocyst developed from 8-cell stage embryos in vitro. Superovulated CB6F1 hybrid female mice (5–6 weeks) were sacrificed 68 to 72 hours after hCG administration. Eight-cell embryos were flushed from oviducts of the sacrificed mouse with HTF medium supplemented with HEPES and 3 mg mL–1 BSA. Embryos were randomly divided into two groups. In the first group, embryos at 8-cell stage were used for a single cell blastomer aspiration; in the second group, embryos were cultured in vitro until blastocyst stage. Trophectoderm cells (15% of trophoblastic cells) were biopsied from developing blastocysts. There were also control groups for both groups. Biopsy procedures for both groups were applied in 50 µL drops of Ca2+/Mg2+ free HTF medium containing HEPES+3 mg mL–1 BSA+5 µg mL–1 cytochalasine B. After biopsy, embryos were cultured in Quinn’s blastocyst medium supplemented with 4 mg mL–1 BSA and incubated in 5% CO2 and 5% O2 incubator at 37°C for 48 and 24 hours for blastomer aspiration and trophectoderm biopsy groups, respectively. While some developing blastocysts were used for determining total cell number, some of them were transferred to the recipients. Results were evaluated by independent t-test and ANOVA of SPSS 17.0 statistic program (SPSS Inc., Chicago, IL, USA). In blastomere biopsy and control groups, development rates were determined as 81.02% (121/152) and 96.37% (62/63), while the total cell numbers were determined as 50 and 50, respectively. There was no significant difference between groups in terms of development ratios and total cells. In blastomer biopsy and control groups, the implantation sites and fetal development rates were found as 25% (9/36) and 26% (8/30), and 19.44% (7/36) and 20% (6/30), respectively. No significant difference was observed between groups in terms of implantation sites and fetal development rates. In trophectoderm biopsy and control groups, while the development rates were found as 86.96% (69/79) and 93.33% (23/28), the total cell numbers were 26.66 and 55.33, respectively. Although there was not any significant difference between groups in terms of development rates, there was a significant difference between groups in terms of total cell numbers (P &lt; 0.05). In trophectoderm biopsy and control groups, the implantation sites and fetal development rates were determined as 21.88% (7/32) and 59.09% (13/22), and 0% (0/32) and 18.18% (4/22), respectively. Although there was not any significant difference between groups in terms of implantation sites, there was a significant difference between groups in terms of fetal development rates (P &lt; 0.05). Therefore, it was concluded that biopsy applied at early stage of embryonic development does not affect embryo development negatively and biopsy procedures applied at early developmental stages have more advantages especially in embryos developing faster with low total cell numbers such as mouse species. Supported by TUBITAK KAMAG-107G027).

Single-step vs. two-step media: are there any differences in embryonic development?

The two-step protocol is thought to mimic the physiological interaction between specific developmental stages of the embryo within the oviduct and uterus. The opposing system is based on the ‘let the embryo choose’ principal that is analogous to a single step culture medium. By culturing sibling oocytes in both media under the same culture conditions, we compared embryo development until day 6, as well as blastocyst formation and utilization. A retrospective analysis. Group embryo culture was done in 4-well dishes under oil and 5% oxygen. On day-1, following 2pn-check, sibling zygote cohorts from 181 patients (average age of 33.5±4.5) were randomly equally allocated into the SAGE (Trumbull, CT) or GLOBAL (IVFonline, Guilford, CT) culture media. A combined total of 2002 embryos were cultured in groups of up to five in 0.5ml volumes of their assigned medium. Embryos cultured in the SAGE were transferred from cleavage to blastocyst medium early on day-4 of culture. Embryos cultured in GLOBAL were transferred in fresh media on day-4 culture also. Chi square test was used for statistical analysis. Day 3 embryo development (≥6 blastomeres) was not significant between both media. On day 4, a significant higher number of compacting embryos was observed in the GLOBAL (63.5% vs. 51.5%; P<.025). Consequently, a significant higher number of blastocysts (day 5 and day 6 combined) grew in the GLOBAL (73.7% vs. 64.4%; P<.02). Looking at the blastocyst utilization rate (BUR), both media had similar BUR's (38.1% vs. 38.6%; P<.92). However, a significant higher portion of day 5 blastocysts were vitrified from the GLOBAL (69.4% vs. 53.4%; P<.025). This trial demonstrates that a single step culture medium protocol with media replacement on day 4 supports the development of extended preimplantation embryos through day-6 of in-vitro culture. Although the BUR between both media was similar, a higher percentage of good-quality day 5 blastocysts were cryopreserved from the Global.

Trophectoderm biopsy. a simplified mechanical method

Trophectoderm biopsy is an emerging approach in preimplantaion genetic diagnosis. The most recent introduction of laser technology has facilitated this step considerably.Worldwide the majority of the IVF clinics performing trophectoderm biopsy are using the laser for zona drilling. But, the cost of laser is considered high. Blastocyst trophectoderm biopsy using micromanipulation methods was first reported by Dorkas et al. (1990); however, new improvements are still needed. The objective was to evaluate a simplified, inexpensive method to remove trophectoderm cells. Discarded day-6 expanded or hatching blastocysts of more than 140 hours post insemination from IVF/ICSI were used to evaluate a simplified mechanical method for removal of trophectoderm cells. Trophectoderm biopsies were conducted under mineral oil in Falcon 1006 Petri dishes in 10 μl drops of G-MOPS medium without protein (Vitrolife Sweden AB). An Olympus IX70 equipped with integra Ti micromanipulation system and a tool holder with an opthalmic surgery micro blade (Micro Feather Blade # K-722) were used to perform biopsies. Blastocysts were placed individually in G-MOPS medium without holding pipette. The protruding trophectoderm was at 12 o'clock position. Excision of the trophectoderm cells was achieved with the assistance of the micro blade. Ten microliters of blastocyst medium with 20% of protein was added to the biopsy drops. Embryos and cells were rinsed and moved to the culture medium for a short evaluation. A total of 42 hatching blastocysts were biopsed. All the blastocysts survived the biopsy procedure. Trophectoderm cells were removed from the biopsy dish to the rinse dish with no problem. Further development of manipulated embryos or cells were not apparently impaired. All biopsies were performed in less than 90 seconds. These results demonstrate the feasibility of the trophectoderm biopsy using a practical mechanical method.