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Hypoxia promotes blastocyst hatching and implantation by regulation of e-cadherin and lifr expression via hif-2α in mouse blastocyst cultured in vitro
blastocyst and HQB prediction rate compared to traditional D3M was seen when CES was performed on images combining D1+D3 embryos with an average overall blastocyst prediction rate of 98% vs 86% (p¼0.02) and HQB prediction rate of 86% vs 66% (p¼0.01), respectively. When TLM was compared to CES, the above findings persisted, albeit statistical differences were attenuated. CONCLUSIONS: Simultaneous visual assessment of a patient’s entire embryo cohort on D1+D3 significantly improves selection of embryos destined to become HQB compared to traditional methods which rely on the individual annotation of embryos and subsequent hierarchical ranking of these scores. This novel method is being proposed as a simple and reproducible way of predicting blastocyst formation with a higher blastocyst prediction rate compared with standard D3-morphology and time-lapse imaging. In combination with modern technology, these findings may be used in the development of cost-effective methods for enhanced embryo selection.
tension (20%) on embryo development have been reported in animal models. However, the effect of lower or slightly higher oxygen tension on in vitro embryo development is still under investigation. Our objective is to compare the effect of 4 different oxygen tensions (3%, 5%, 8% and 20 %) on mouse embryo developmental parameters. DESIGN: Experimental animal study. MATERIALS AND METHODS: A total of 407 two-cell stage fresh mouse embryos were harvested from (C57BL/6J) female mice after superovulation and timed natural mating. Embryos were randomly cultured in four different oxygen concentrations; 3%, 5%, 8% ( in triple gas incubator with 6% CO2 and 91%, 89%, 86% N2 respectively) and 20% (in conventional incubator with 6% CO2) at 37 C. Embryos were cultured in groups of 5-10 in a 50 ul droplet of culture media under an oil overlay. Embryo morphology and development were monitored daily until day 4. Chi-square test was used for comparison between the 4 groups. P< 0.05 was considered significant. RESULTS: TABLE 1. Embryos development on day 4
P-146 Tuesday, October 31, 2017 HYPOXIA PROMOTES BLASTOCYST HATCHING AND IMPLANTATION BY REGULATION OF E-CADHERIN AND LIFR EXPRESSION VIA HIF-2a IN MOUSE BLASTOCYST CULTURED IN VITRO. Y. Ma,a C. Chen,b C. Tzeng.b aCenter for Reproductive Medicine & Sciences, Taipei, Taiwan; bDepartment of Obstetrics & Gynecology, Taipei Medical University Hospital, Taipei, Taiwan. OBJECTIVE: In human IVF, many studies have shown that embryos cultured in lower oxygen tension (5% O2) have higher implantation rate, when compared with normaxic condition (20% O2). However the beneficial effect of low oxygen tension in implantation remains unclear. This study aimed to investigate the expression of oxygen-dependent gene and implantation related protein in mouse embryo cultured under hypoxic and normaxic conditions. DESIGN: The 2-cell ICR mouse embryos were cultured to blastocyst stage under 3% O2 tension (N¼186) and 20% O2 tension (N¼189), respectively from four replicate experiments. MATERIALS AND METHODS: The blastocysts were collected from each experiment. The expressions of oxygen-related genes (HIF-1a and HIF-2a) were analyzed by real-time RT PCR. The protein levels of HIF2a, COX-2, E-cadherin and LIFR were validated by Immunofluorescence analysis. Student‘s t test or one-way ANOVA test was used to evaluate statistical significance. RESULTS: The blastocyst formation and hatching rate (Mean SE) was significantly higher in 3% O2 group when compared with 20% O2 group (90.53.3% vs. 77.82.4% and 82.96.9% vs. 70.72.3%, respectively, P<0.5). The transcription levels of HIF-1a and HIF-2a were similar in two groups. Immunofluorescence staining showed the intensity of COX-2 and LIFR was higher in 3% O2 than 20% O2 group, respectively. Protein levels of HIF-2a was detected higher in the nucleus of 3% O2 group. E-cadherin was significantly lower in 3% O2, compared with 20% O2 group. CONCLUSIONS: This study has proposed that lower O2 tension promotes blastocyst hatching and implantation by down-regulation of E-cadherin expression via HIF-2a/COX-2 pathway, it also provides more conducive environment by up-regulation of LIFR to facilitate implantation after blastocyst hatching. All these effects may be initiated and regulated by HIF-2a, which acts as key mediator in hypoxic environment. Supported by: Study supported by MOST 104-2314-B-038 -063 -MY2. P-147 Tuesday, October 31, 2017 OXYGEN TENSION INFLUENCES MOUSE EMBRYO DEVELOPMENT WHEN VARIES IN A. Hashem,a CONCENTRATION. M. S. Iews,a,b c d F. AbdelHafez, A. O. Abdelkareem, B. Peng,e M. A. Bedaiwy.b aObstetrics and Gynecology, South Valley University, Qena, Egypt; bDepartment of Obstetrics and Gynecology, BC Women’s Hospital, Vancouver, BC, Canada; cAssiut University, Assiut, Egypt; dObstetrics and Gynecology, Faculty of Medicine, Sohag University, Sohag, Egypt; eDepartment of Obstetrics & Gynaecology, University of British Columbia, CFRI, Vancouver, BC, Canada. OBJECTIVE: Oxygen inside the female genital tract varies from 2-8% which is the optimum oxygen tension needed for embryo development. The beneficial effects of 5% oxygen tension compared to the atmospheric
FERTILITY & STERILITYÒ
Day 4
3%(n¼95) 5%(n¼95) 8%(n¼107) 20%(n¼110)
Total blastocysts 54(57%) n(%) Hatching blastocyst 20% Morula 8 <8 cells 9 Degenerated 24 embryos
72(76%) 81% 3 8 12
89(83%)
67(61%)
19% 3 10 5
30% 11 2 30
On day 2, embryo fragmentation was significantly higher at 3 and 20% oxygen tension compared to that at 5 and 8% tension (P<0.05). On day 4, the total number of blastocysts at 8% oxygen tension was significantly higher than the 3 and 20% groups (P <0.001), but similar to that in the 5% group. However, the number of hatching embryos using 5% oxygen tension was significantly higher compared to all other groups (3%,8%, and 20 %) (P <0.001). CONCLUSIONS: Our preliminary results showed that mouse embryos cultured under 5% oxygen tension have the best development and are more likely to blastulate, hatch and have better morphology compared to both lower and higher oxygen tensions. Further research is recommended with a larger sample size to confirm these findings. P-148 Tuesday, October 31, 2017 THE LIPID PROFILE OF MURINE BLASTOCYST CELLS ORIGINATED FROM IN VITRO FERTILIZATION AND NATURAL FERTILIZATION AS A QUALITY CONTROL TOOL FOR EMBRYO CULTURE. D. F. Moriyama,a D. A. Montani,a A. Rodrigues-Oliveira,b D. Oliveira-Silva,b R. Fraietta,a E. G. Lo Turco.a aDepartment of Surgery, Division of Urology, Human Reproduction Section, Sao Paulo Federal University, Sao Paulo, Brazil; bInstitute of Environmental, Chemical and Pharmaceutical Sciences, Sao Paulo Federal University, Diadema, Brazil. OBJECTIVE: This study compared the lipid composition of murine blastocyst cells, originated from natural fertilization and in vitro fertilization to estimate the biochemical alterations induced by in vitro culture. DESIGN: Prospective study. MATERIALS AND METHODS: Female mouse C57BL/6J (4 weeks) were superovulated with PMSG and hCG. Males (n¼12) and females (n¼24) were placed to mate for natural fertilization (NF group). In the NF group, 128 presumptive blastocysts were recovered. For in vitro fertilization group (IVF group), the oocytes were collected from the tubes, the mature oocytes were inseminated with 1x105 Sptz/ml and cultivated (20 embryos/droplet) for 96 hours in continuous culture media incubated at 37oC, 90% humidity and 5% CO2 (n¼225). Embryos were lysed by sonication, the lipids were extracted using Bligh and Dyer protocol and individually analyzed by electrospray ionization mass spectrometry (ESI MS). Principal Component (PCA) and Partial Least Square Discriminant (PLS-Da) analyses were used to perceive general clustering and to confirm the discriminatory lipids, respectively. The possible lipid biomarkers were estimated using Variable Importance to Projection (VIP) hyper-represented in each group. Moreover, the in silico pathways and metabolic analysis were performed through enrichment analysis using the CytoScape 3.4.0. RESULTS: Fatty acids (m/z313.2677), diacylglycerol (m/z825.838 and m/z791.8453), lysophosphoserine (m/z592.2265) triacylglycerol (m/z893.8229) and phosphatidic acid (m/z927.81835) were hyper-represented in the IVF group.
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tension (20%) on embryo development have been reported in animal models. However, the effect of lower or slightly higher oxygen tension on in vitro embryo development is still under investigation. Our objective is to compare the effect of 4 different oxygen tensions (3%, 5%, 8% and 20 %) on mouse embryo developmental parameters. DESIGN: Experimental animal study. MATERIALS AND METHODS: A total of 407 two-cell stage fresh mouse embryos were harvested from (C57BL/6J) female mice after superovulation and timed natural mating. Embryos were randomly cultured in four different oxygen concentrations; 3%, 5%, 8% ( in triple gas incubator with 6% CO2 and 91%, 89%, 86% N2 respectively) and 20% (in conventional incubator with 6% CO2) at 37 C. Embryos were cultured in groups of 5-10 in a 50 ul droplet of culture media under an oil overlay. Embryo morphology and development were monitored daily until day 4. Chi-square test was used for comparison between the 4 groups. P< 0.05 was considered significant. RESULTS: TABLE 1. Embryos development on day 4
P-146 Tuesday, October 31, 2017 HYPOXIA PROMOTES BLASTOCYST HATCHING AND IMPLANTATION BY REGULATION OF E-CADHERIN AND LIFR EXPRESSION VIA HIF-2a IN MOUSE BLASTOCYST CULTURED IN VITRO. Y. Ma,a C. Chen,b C. Tzeng.b aCenter for Reproductive Medicine & Sciences, Taipei, Taiwan; bDepartment of Obstetrics & Gynecology, Taipei Medical University Hospital, Taipei, Taiwan. OBJECTIVE: In human IVF, many studies have shown that embryos cultured in lower oxygen tension (5% O2) have higher implantation rate, when compared with normaxic condition (20% O2). However the beneficial effect of low oxygen tension in implantation remains unclear. This study aimed to investigate the expression of oxygen-dependent gene and implantation related protein in mouse embryo cultured under hypoxic and normaxic conditions. DESIGN: The 2-cell ICR mouse embryos were cultured to blastocyst stage under 3% O2 tension (N¼186) and 20% O2 tension (N¼189), respectively from four replicate experiments. MATERIALS AND METHODS: The blastocysts were collected from each experiment. The expressions of oxygen-related genes (HIF-1a and HIF-2a) were analyzed by real-time RT PCR. The protein levels of HIF2a, COX-2, E-cadherin and LIFR were validated by Immunofluorescence analysis. Student‘s t test or one-way ANOVA test was used to evaluate statistical significance. RESULTS: The blastocyst formation and hatching rate (Mean SE) was significantly higher in 3% O2 group when compared with 20% O2 group (90.53.3% vs. 77.82.4% and 82.96.9% vs. 70.72.3%, respectively, P<0.5). The transcription levels of HIF-1a and HIF-2a were similar in two groups. Immunofluorescence staining showed the intensity of COX-2 and LIFR was higher in 3% O2 than 20% O2 group, respectively. Protein levels of HIF-2a was detected higher in the nucleus of 3% O2 group. E-cadherin was significantly lower in 3% O2, compared with 20% O2 group. CONCLUSIONS: This study has proposed that lower O2 tension promotes blastocyst hatching and implantation by down-regulation of E-cadherin expression via HIF-2a/COX-2 pathway, it also provides more conducive environment by up-regulation of LIFR to facilitate implantation after blastocyst hatching. All these effects may be initiated and regulated by HIF-2a, which acts as key mediator in hypoxic environment. Supported by: Study supported by MOST 104-2314-B-038 -063 -MY2. P-147 Tuesday, October 31, 2017 OXYGEN TENSION INFLUENCES MOUSE EMBRYO DEVELOPMENT WHEN VARIES IN A. Hashem,a CONCENTRATION. M. S. Iews,a,b c d F. AbdelHafez, A. O. Abdelkareem, B. Peng,e M. A. Bedaiwy.b aObstetrics and Gynecology, South Valley University, Qena, Egypt; bDepartment of Obstetrics and Gynecology, BC Women’s Hospital, Vancouver, BC, Canada; cAssiut University, Assiut, Egypt; dObstetrics and Gynecology, Faculty of Medicine, Sohag University, Sohag, Egypt; eDepartment of Obstetrics & Gynaecology, University of British Columbia, CFRI, Vancouver, BC, Canada. OBJECTIVE: Oxygen inside the female genital tract varies from 2-8% which is the optimum oxygen tension needed for embryo development. The beneficial effects of 5% oxygen tension compared to the atmospheric
FERTILITY & STERILITYÒ
Day 4
3%(n¼95) 5%(n¼95) 8%(n¼107) 20%(n¼110)
Total blastocysts 54(57%) n(%) Hatching blastocyst 20% Morula 8 <8 cells 9 Degenerated 24 embryos
72(76%) 81% 3 8 12
89(83%)
67(61%)
19% 3 10 5
30% 11 2 30
On day 2, embryo fragmentation was significantly higher at 3 and 20% oxygen tension compared to that at 5 and 8% tension (P<0.05). On day 4, the total number of blastocysts at 8% oxygen tension was significantly higher than the 3 and 20% groups (P <0.001), but similar to that in the 5% group. However, the number of hatching embryos using 5% oxygen tension was significantly higher compared to all other groups (3%,8%, and 20 %) (P <0.001). CONCLUSIONS: Our preliminary results showed that mouse embryos cultured under 5% oxygen tension have the best development and are more likely to blastulate, hatch and have better morphology compared to both lower and higher oxygen tensions. Further research is recommended with a larger sample size to confirm these findings. P-148 Tuesday, October 31, 2017 THE LIPID PROFILE OF MURINE BLASTOCYST CELLS ORIGINATED FROM IN VITRO FERTILIZATION AND NATURAL FERTILIZATION AS A QUALITY CONTROL TOOL FOR EMBRYO CULTURE. D. F. Moriyama,a D. A. Montani,a A. Rodrigues-Oliveira,b D. Oliveira-Silva,b R. Fraietta,a E. G. Lo Turco.a aDepartment of Surgery, Division of Urology, Human Reproduction Section, Sao Paulo Federal University, Sao Paulo, Brazil; bInstitute of Environmental, Chemical and Pharmaceutical Sciences, Sao Paulo Federal University, Diadema, Brazil. OBJECTIVE: This study compared the lipid composition of murine blastocyst cells, originated from natural fertilization and in vitro fertilization to estimate the biochemical alterations induced by in vitro culture. DESIGN: Prospective study. MATERIALS AND METHODS: Female mouse C57BL/6J (4 weeks) were superovulated with PMSG and hCG. Males (n¼12) and females (n¼24) were placed to mate for natural fertilization (NF group). In the NF group, 128 presumptive blastocysts were recovered. For in vitro fertilization group (IVF group), the oocytes were collected from the tubes, the mature oocytes were inseminated with 1x105 Sptz/ml and cultivated (20 embryos/droplet) for 96 hours in continuous culture media incubated at 37oC, 90% humidity and 5% CO2 (n¼225). Embryos were lysed by sonication, the lipids were extracted using Bligh and Dyer protocol and individually analyzed by electrospray ionization mass spectrometry (ESI MS). Principal Component (PCA) and Partial Least Square Discriminant (PLS-Da) analyses were used to perceive general clustering and to confirm the discriminatory lipids, respectively. The possible lipid biomarkers were estimated using Variable Importance to Projection (VIP) hyper-represented in each group. Moreover, the in silico pathways and metabolic analysis were performed through enrichment analysis using the CytoScape 3.4.0. RESULTS: Fatty acids (m/z313.2677), diacylglycerol (m/z825.838 and m/z791.8453), lysophosphoserine (m/z592.2265) triacylglycerol (m/z893.8229) and phosphatidic acid (m/z927.81835) were hyper-represented in the IVF group.
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