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First results of human embryo culture in a novel incubator and integrated cell culture observation device
P-720 FIRST RESULTS OF HUMAN EMBRYO CULTURE IN A NOVEL INCUBATOR AND INTEGRATED CELL CULTURE OBSERVATION DEVICE. R. Janssens, R. Souffreau, H. Van de Velde, A. Verloes, J. Van der Elst. Centre for Reproductive Medicine, UZ Brussel, Brussel, Belgium; Gezondheidszorg Vesalius, Hogeschool Gent, Gent, Belgium; Haematology Laboratory, UZ Brussel, Brussel, Belgium; Centre for Reproductive Medicine, Brussel, Belgium. OBJECTIVE: Since the start of IVF, technology for embryo culture and observations has not changed majorly. Embryos are taken out of their controlled environment for regular inspection. Recent developments in digital equipment and automation has led to a total new incubator with integrated embryo monitoring where culture dishes remain incubated. We report on the first results of human embryo culture in this device. DESIGN: Laboratory technology assessment. MATERIALS AND METHODS: The BioStationCT (Nikon Instruments) is a system for observing and recording cell growth and morphology in culture. Before the system could be used for IVF, a CODA incubator unit filter and cell tracking software were installed. Culture dishes are placed in the incubator through an interlock port. A robotic arm moves each vessel carrier to an integrated microscope. The unit can hold up to 30 individual carriers. A high performance cooled scientific grade CCD camera is incorporated for time-lapse sequences. The optical system delivers high resolution images (magnifications of 2X, 4X, 10X, 20X, and 40X with phase contrast). The software allows to select live observation or saved time-lapsed image sequences even from remote locations. The software is being upgraded to tailor the specific needs for IVF. RESULTS: The unit was validated for temperature, pH, CO2 and O2. All parameters are stable over time and there is limited or no effect of opening the interlock port. 21 donated oocytes from two patients were injected and randomly distributed for culture in a standard incubator (n¼10) or in the Biostation CT (n¼11). Embryos from the first patient cultured in a standard incubator were inspected at the usual timepoints for fertilisation and embryo development on an inverted microscope. The embryos in the Biostation CT were documented by digital images every 20 minutes for 5 days with medium change on day 3. Embryos from the second patient were cultured for 3 days. Embryo and blastocyst development were similar. We obtained 4 blastocysts out of 7 embryos in a standard incubator and 4 out of 8 in the Biostation CT for patient 1. The blastocysts were fixed for immunocytic staining for ICM and TE markers. CONCLUSIONS: The unit opens the possibility to keep track of fertilisation and embryo development in culture. Embryologists can have easy access to continuous images, allowing embryo scoring and selection from remote locations (office, home) with far more information concerning the kinetics of embryo development than now in regular practice where the number of evaluations are limited. Supported by: None.
P-721 REDUCED OXYGEN TENSION HELPS INCREASE THE QUALITY OF BLASTOCYST AVAILABLE ON D5. A. Hoff, A. Khabani, C. Khabani, L. Hickok, L. Marshall. Pacific Northwest Fertility and IVF Specialists, Seattle, WA. OBJECTIVE: It has been suggested that a low oxygen environment that mimics physiological conditions may improve embryo morphology. Since the oxygen tension in the uterus of most mammalian species is around 58%, these conditions may be favorable for embryo development in vitro.The objective of this retrospective analysis was to compare the quality of blastocysts formed from donor oocytes before (20%O2 atmospheric conditions) and after (5% O2 physiological conditions) the implementation of low oxygen culture conditions in our program. DESIGN: A retrospective analysis of patients whose embryos were cultured prior to and after the implementation of reduced oxygen tension invitro. MATERIALS AND METHODS: Donor Recipients undergoing a D5 ET whose embryos were cultured in 5%CO2 and atmospheric O2 were compared with those whose embryos were cultured under physiological conditions (5%CO2, 5%O2, 90%N2) after the implementation of low oxygen culture in our embryology lab. Embryo culture was performed using a sequential microdrop system under oil in Sages Cleavage Media from D1-D3. On D3, embryos were transferred to Invitro Care Blastocyst culture media until D5. This culture system was used throughout the analysis. Day 5 blastocysts were evaluated using the Gardner scoring system and good quality embryos in our program consist of AA or AB graded blastocysts.
S350
Abstracts
RESULTS: Blastocyst formation, quality of blastocysts and number of blastocyst cryopreserved were compared on D5. No difference was seen in the overall blastocyst formation of either group though there were a slightly higher number of blastocysts formed by D5 in the reduced oxygen group. There was a higher number of good quality blastocyst (AA or AB) available on D5 in the reduced oxygen group as well as a higher number blastocyst available for cryopreservation on D5 in the reduced oxygen group. TABLE 1. Blastocyst Development D5 results
n 20% O2 124 5%O2 75 P value*
total blast Blast formation D5 Blast D5 Blasts formation on D5 quality AA,AB Cryopreserved 55% 58% NS
57% 61% NS
37% 48% .004
21% 36% .001
* chi square; NS not significant CONCLUSIONS: After the implementation of reduced oxygen tension in our culture system, an increased in blastocyst quality has been observed on D5. This has led to a larger cohort of good quality embryos available for blastocys transfer and cryopreservation. Supported by: None.
P-722 TILTING EMBRYO CULTURE SYSTEM IMPROVED MOUSE EMBRYO DEVELOPMENT. Y. Kuroda, K. Mastuura, M. Takenami, H. Funahashi, K. Naruse. Graduate School of Medicine, Okayama University, Okayama, Japan. OBJECTIVE: Mammalian embryos in vivo are exposed to mechanical stimulation from oviduct such as compression and shear stress. Moderate mechanical stimulation would be important for the enhancement of embryonic developments. We have developed tilting embryo culture system (TECS) that can make moderate mechanical stimulation by tilting dishes. The operations of the device are M1: rotating the plate and M2: tilting and holding the plate. M1 and M2 are repeated. We will report the evaluation of development of mouse embryo by TECS and the estimation of shear stress based on the image of tilting dish. DESIGN: Animal-model experiments. MATERIALS AND METHODS: The tilting angle of TECS (STREX Inc. Japan) was 10 degree. The velocity of the tilting and the holding time were 1 degree/second and 60 second, respectively. Frozen 2 cell stage of ICR (Arc Resources Inc. Japan) were melted and cultured in mw medium (Daiya Shiyaku Inc. Japan) for 3 days in a humidified environment of 5% CO2 in air at 37 C. The volume of the medium in a micro-drop covered with mineral-oil was 0.05 ml. To count cells of the blastocyst, the cells were stained with Hochest 33342. Nonparametric and parametric statistics were performed with c-square and Student’s t-test, respectively. RESULTS: Blastocyst developments was significantly improved by TECS (TECS 46% (n¼151); Static Control (CTRL) 59% (n¼145) P<0.05). The blastocysts (n¼34) by TECS had 36111 cells, while those of CTRL (n¼26) had 2994 cells. There was significant difference in the averages of the cell numbers between the two groups (TECS: 77 cells; CTRL: 66 cells P<0.05). These results suggest that number of cells in the blastocyst by TECS is increased, and that TECS could enhance cell division of mouse embryos. We observed blastomere during the tilt and hold by our originally designed microscope. By setting the tilt angle from 0 to 10 deg within 6 sec (corresponding to TECS motion M1), the embryo was rolling along the slope (velocity: 1.5 mm/min.). When the tilt angle was hold to 10 deg (corresponding to M2), the embryo was sliding with velocity of 0.03 mm/min. Our calculation of shear-stress (SS) in M1 and M2 were 7.5x10-3, and 1.5 x10-4 dynes/cm2, respectively. These values were quite lower than the SS that induces lethal effect (1.2 dynes/cm2). CONCLUSIONS: We can suggest that the moderate mechanical stimulation by TECS contribute to the improvement. Supported by: A grant-in-aid for Scientific Research on Priority Areas (no. 17076006 to K.N.) from the Ministry of Education Science Sports and Culture, Japan.
Vol. 90, Suppl 1, September 2008
P-721 REDUCED OXYGEN TENSION HELPS INCREASE THE QUALITY OF BLASTOCYST AVAILABLE ON D5. A. Hoff, A. Khabani, C. Khabani, L. Hickok, L. Marshall. Pacific Northwest Fertility and IVF Specialists, Seattle, WA. OBJECTIVE: It has been suggested that a low oxygen environment that mimics physiological conditions may improve embryo morphology. Since the oxygen tension in the uterus of most mammalian species is around 58%, these conditions may be favorable for embryo development in vitro.The objective of this retrospective analysis was to compare the quality of blastocysts formed from donor oocytes before (20%O2 atmospheric conditions) and after (5% O2 physiological conditions) the implementation of low oxygen culture conditions in our program. DESIGN: A retrospective analysis of patients whose embryos were cultured prior to and after the implementation of reduced oxygen tension invitro. MATERIALS AND METHODS: Donor Recipients undergoing a D5 ET whose embryos were cultured in 5%CO2 and atmospheric O2 were compared with those whose embryos were cultured under physiological conditions (5%CO2, 5%O2, 90%N2) after the implementation of low oxygen culture in our embryology lab. Embryo culture was performed using a sequential microdrop system under oil in Sages Cleavage Media from D1-D3. On D3, embryos were transferred to Invitro Care Blastocyst culture media until D5. This culture system was used throughout the analysis. Day 5 blastocysts were evaluated using the Gardner scoring system and good quality embryos in our program consist of AA or AB graded blastocysts.
S350
Abstracts
RESULTS: Blastocyst formation, quality of blastocysts and number of blastocyst cryopreserved were compared on D5. No difference was seen in the overall blastocyst formation of either group though there were a slightly higher number of blastocysts formed by D5 in the reduced oxygen group. There was a higher number of good quality blastocyst (AA or AB) available on D5 in the reduced oxygen group as well as a higher number blastocyst available for cryopreservation on D5 in the reduced oxygen group. TABLE 1. Blastocyst Development D5 results
n 20% O2 124 5%O2 75 P value*
total blast Blast formation D5 Blast D5 Blasts formation on D5 quality AA,AB Cryopreserved 55% 58% NS
57% 61% NS
37% 48% .004
21% 36% .001
* chi square; NS not significant CONCLUSIONS: After the implementation of reduced oxygen tension in our culture system, an increased in blastocyst quality has been observed on D5. This has led to a larger cohort of good quality embryos available for blastocys transfer and cryopreservation. Supported by: None.
P-722 TILTING EMBRYO CULTURE SYSTEM IMPROVED MOUSE EMBRYO DEVELOPMENT. Y. Kuroda, K. Mastuura, M. Takenami, H. Funahashi, K. Naruse. Graduate School of Medicine, Okayama University, Okayama, Japan. OBJECTIVE: Mammalian embryos in vivo are exposed to mechanical stimulation from oviduct such as compression and shear stress. Moderate mechanical stimulation would be important for the enhancement of embryonic developments. We have developed tilting embryo culture system (TECS) that can make moderate mechanical stimulation by tilting dishes. The operations of the device are M1: rotating the plate and M2: tilting and holding the plate. M1 and M2 are repeated. We will report the evaluation of development of mouse embryo by TECS and the estimation of shear stress based on the image of tilting dish. DESIGN: Animal-model experiments. MATERIALS AND METHODS: The tilting angle of TECS (STREX Inc. Japan) was 10 degree. The velocity of the tilting and the holding time were 1 degree/second and 60 second, respectively. Frozen 2 cell stage of ICR (Arc Resources Inc. Japan) were melted and cultured in mw medium (Daiya Shiyaku Inc. Japan) for 3 days in a humidified environment of 5% CO2 in air at 37 C. The volume of the medium in a micro-drop covered with mineral-oil was 0.05 ml. To count cells of the blastocyst, the cells were stained with Hochest 33342. Nonparametric and parametric statistics were performed with c-square and Student’s t-test, respectively. RESULTS: Blastocyst developments was significantly improved by TECS (TECS 46% (n¼151); Static Control (CTRL) 59% (n¼145) P<0.05). The blastocysts (n¼34) by TECS had 36111 cells, while those of CTRL (n¼26) had 2994 cells. There was significant difference in the averages of the cell numbers between the two groups (TECS: 77 cells; CTRL: 66 cells P<0.05). These results suggest that number of cells in the blastocyst by TECS is increased, and that TECS could enhance cell division of mouse embryos. We observed blastomere during the tilt and hold by our originally designed microscope. By setting the tilt angle from 0 to 10 deg within 6 sec (corresponding to TECS motion M1), the embryo was rolling along the slope (velocity: 1.5 mm/min.). When the tilt angle was hold to 10 deg (corresponding to M2), the embryo was sliding with velocity of 0.03 mm/min. Our calculation of shear-stress (SS) in M1 and M2 were 7.5x10-3, and 1.5 x10-4 dynes/cm2, respectively. These values were quite lower than the SS that induces lethal effect (1.2 dynes/cm2). CONCLUSIONS: We can suggest that the moderate mechanical stimulation by TECS contribute to the improvement. Supported by: A grant-in-aid for Scientific Research on Priority Areas (no. 17076006 to K.N.) from the Ministry of Education Science Sports and Culture, Japan.
Vol. 90, Suppl 1, September 2008