- Email: [email protected]
Undisturbed embryo culture in a time-lapse monitoring system improve embryo quality: a prospective cohort study
MATERIALS AND METHODS: Murine zygotes (n¼40) were cultured in commercially available single or sequential media for 5 days. Images (200x, 400x) made using inverted phase contrast microscopes with differential contrast on developmental day 3 (d3) to facilitate quantification of the number of embryos developing to the 8-cell stage or greater in the two media. On d5, images were made and embryos fixed. Comparing and contrasting embryo images from the two types of media facilitated identification of an inner cell mass (ICM), nuclear quality assessment, and quantification of the total number of cells per embryo, ICM and trophoblast. The least square means (LSM) of total nuclei number, ICM nuclear number, and presumptive TE nuclear number between the blastocysts were analyzed using a general linear mixed model. An approximate t-test was used to compare differences in nuclei number (SAS 9.22 PROC MIXED, SAS Institute Inc, Cary, NC). Logistic regression analysis (SAS PROC LOGISTIC) was used to compare the total embryo numbers per stage per day on d3 and d5. Hatching/hatched to unhatched blastocysts, TE: ICM and abnormal nuclei:normal nuclei were compared through this same analytical approach. A P < 0.05 was considered significant. Deubiquitinating enzymes localizing in the ICM served as a point of reference for morphometric analysis. RESULTS: Equivalent numbers of embryos reached the 8C stage on d3 in both single and sequential embryo culture media. More d5 embryos reached the blastocyst stage, had greater numbers of normal nuclei, hatched and had significantly more trophoblast cells, but not ICM cells, when cultured in the single media compared to the sequential media. CONCLUSION: Single medium yields higher quality blastocyst embryos on d5 than sequential media. This supports the idea that single stage medium is more beneficial in human assisted reproduction procedures when compared to sequential media.
P-236 Tuesday, October 21, 2014 FIRST MORPHOKINETIC ANALYSIS OF BLASTOCYST EXPANSION IN HUMAN EMBRYOS OF KNOWN POSITIVE IMPLANTATION USING AN EMBRYOSCOPE. T. T. F. Huang. Pacific IVF Institute, Honolulu, HI. OBJECTIVE: Using an Embryoscope, the objective was to morphokinetically describe features of the expanding human blastocyst cavity in embryos yielding sustained ongoing pregnancies. DESIGN: Retrospective descriptive study in a private practice setting. MATERIALS AND METHODS: Data were obtained from 27 sequential egg donation cycles using blastocysts selected for transfer using an Embryoscope. Day 5 embryos were ranked and selected for transfer by the greatest degree of blastocyst cavity expansion (BE) with otherwise normal cleavage, ICM and TE characteristics. BE was determined from sequential hourly 2D cross sectional area (CSA) measurements starting from blastocyst formation up until ET. Sustained pregnancy was defined as clinical detection of a beating heart. RESULTS: Of 5 single blastocyst transfers (SBT), 4 (80%) resulted in a sustained pregnancy (IR¼80%). Of 21 double blastocyst transfers (DBT), 21 (100%) resulted in a sustained pregnancy with an IR of 80.4%. (One patient had a triple BT but did not become pregnant). Of the DBT’s, 12 (57.1%) were twins (100% IR) and 9 (42.9%) were singletons (50% IR). The study confirmed a correlation between the time of the start of blastocyst cavitation (Tsb) and the time of blastocyst formation (Tb) for both the 100% (r2 ¼ 0.830) and 50% (r2 ¼ 0.893) IR groups. In the 100% IR group, the average hourly rate of BE increased over consecutive 3 hour intervals (557.4 u2 /hr; 916.2 u2 /hr; 1276.3 u2/hr), then tapered to 870.4 u2/ hr during the fourth interval (hours 10-12). This was similar in the 50% IR group. Two morphokinetically distinguishable events characterized BE. The first was a novel pulsatile, oscillatory pattern of accelerations and decelerations with a periodicity of 2-3 hours resulting in continuous BE. The second was an occasional, acute contraction of the cavity, ranging from 5-50% in CSA, often occurring several times in blastocysts monitored for up to 22 hours. Remarkably, contractions recovered completely within 1-3 hours. Interestingly, the rates of expansion and times to reach given CSA milestones at 1, 5, and 10 hours from Tb did not correlate with the initial time of blastocyst formation in the cohort of 100% IR blastocysts. CONCLUSION: This reports the first quantitative description of BE in human embryos yielding sustained pregnancies. Expansion typically accelerates in an oscillatory manner, with occasional but reversible collapses in
FERTILITY & STERILITYÒ
the cavity. Results supports the hypothesis that time-lapse technology identifies novel biological information useful in embryo selection.
P-237 Tuesday, October 21, 2014 MICRO-VIBRATION CULTURE OF HUMAN EMBRYOS IMPROVES PREGNANCY AND IMPLANTATION RATES. I. El-Danasouri, N. L. Sandi-Monroy, T. Winkle, K. Ott, C. Krebs, D. H. A. Maas, F. Gagsteiger. Kinderwunsch-Zentrum Ulm/ Stuttgart, Ulm, BW, Germany. OBJECTIVE: Embryos in vivo are not present in a static condition, since in the fallopian tube they are exposed to continuous movement, compression caused by the cilia and peristaltic movements, and shear stress from tubal fluid flow. In this study, we compared the outcome of human oocytes and embryos cultured in a micro-vibration culture system for In Vitro Fertilization (IVF) patients in comparison to the traditional static culture environment. DESIGN: Retrospective analysis. MATERIALS AND METHODS: Oocytes and embryos from 1943 patients enrolled in IVF treatment from January 2010 to February 2013 were cultured in a traditional static culture, while 497 patients’ embryos from March 2013 to March 2014 were cultured in a micro-vibration culture. In the micro-vibration group, culture dishes were placed on the top of a platform producing a three-dimensional vibration of 56 Hz for 5 seconds every 60 minutes. Patients in both groups were compared according to their age (< 30; 30-35; 36-38 and >39 years old). The outcomes of microvibration culturing were compared for fertilization, blastulation, pregnancy and implantation rates. Variables were analyzed by chi-square test and Fisher’s exact test. RESULTS: Pregnancy rates significantly increased in the micro-vibration culture in younger patient groups compared to the static culture groups (< 30: 59.4% vs. 38.4%, P¼0.0006; 30-35: 50% vs. 36.3%, P<¼0.0005; 36-38: 42.7% vs. 31.2%, P¼0.03) as did the implantation rate (<30: 54.2% vs. 25.5%, P<0.0001; 30-35: 48.1% vs. 30.1%, P<0.0001; 36-38: 40.3% vs. 26.5%, P<0.0001). The number of transferred embryos and day of transfer did not differ between these groups. For patients >39-years-old, micro-vibration culture tended to increase both pregnancy and implantation rates over the static culture but did not reach statistical significance (20.6% vs. 17.2%, P¼0.41 and 21.1% vs. 15.3%, P¼0.08, respectively). Micro-vibration culturing produced no effect on the fertilization rate. In addition, the overall blastulation rate was significantly higher in the micro-vibration culture compared to the static culture (40.5% vs. 31.2%, P<0.0001). CONCLUSION: These results demonstrate clearly that the three-dimensional micro-vibration culture of oocytes and embryos significantly increases pregnancy and implantation rates. Mechanical vibration of the embryo may mimic the embryo’s in vivo environment and may cause movement of media around the embryo aiding in refreshing the media surrounding the embryos and diffusion of waste material.
P-238 Tuesday, October 21, 2014 UNDISTURBED EMBRYO CULTURE IN A TIME-LAPSE MONITORING SYSTEM IMPROVE EMBRYO QUALITY: A PROSPECTIVE COHORT STUDY. W. Han, H. L. Wu, H. Ye, N. G. Huang. Chongqing Maternity and Children Health Care Hospi, Chongqing, China. OBJECTIVE: To quantify the benefit of culturing embryos in a time-lapse monitoring system. DESIGN: Prospective cohort study. MATERIALS AND METHODS: One hundred and sixty patients (age % 35 yrs; BMI: 18-25; 5-15 oocytes; pure tubal factor infertility) were included in this prospective cohort study, and were randomly assigned to the control group (80 patients) or the time-lapse(TL) group (80patients) from April 22 to December 25, 2013. Fertilized oocytes in the TL group were cultured in microwell culture dishes and monitored on compact digital inverted microscopes (Primo Vision, Vitrolife). Images were captured every 5 minutes .In the control group, fertilized oocytes were cultured in conventional dishes (one embryo in each microdroplet). In both groups, embryo were selected for transfer on day 3, by traditional morphology assessment. The clinical pregnancy confirmed by the presence of
e217
gestational sacs with fetal heart beat by transvaginal ultrasound examination in week 7. RESULTS: The number of good quality embryo was significantly higher in the TL group compared to control group (4.632.22 VS 3.651.84, p¼0.0014); pregnancy rate and implantation rate showed a tendency to be higher in the TL group compared to control group. TABLE 1. Comparison of culture outcomes between TL group and Control group Time-lapse group(n¼80)
Control group(n¼80)
30.313.53
30.553.06
Oocytes retrieved(n) 2PN(n) good quality embryos(n) Embryos transferred(n) Implantation rate(%) Clinical pregnancy rate(%)
11.353.0 8.212.30 4.632.22 2.020.22 48.77% ( 79/162) 70.00% (56/80)
10.812.78 7.712.33 3.651.84 2.010.19 43.48% (70/161) 61.25% (49/80)
0.12 0.09 0.0014 0.38 0.34 0.24
On-going pregnancy rate(%)
63.75% ( 51/80)
56.25% (45/80)
0.33
Age(yrs)
P value 0.32
P< 0.05 were regarded as significant difference.
CONCLUSION: This prospective cohort study result indicated that culturing embryos by TM significantly improved the embryo quality.The elevated clinical outcome was attributed to a combination of stable culture conditions and the group culture in microwells.
P-239 Tuesday, October 21, 2014 LOW OXYGEN TENSION INCREASE MITOCHONDRIAL MEMBRANE POTENTIAL AND IMPLANTATION ABILITY BY ENHANCED EXPRESSION OF ANTIOXIDANT GENES IN MOUSE BLASTOCYST CULTURED IN VITRO. Y.-I. Ma,a C.-H. Chen,b J.-W. Wang,b C.-R. Tzeng.a aCenter for Reproductive Medicine & SciencesTaipei Medical University, 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 pregnancy rate, when compared with normaxic condition (20% O2). However the beneficial effect of low oxygen tension in embryogenesis remains unclear. This study aimed to investigate the expression of oxygen and antioxidant genes in mouse embryo cultured under hypoxic and normaxic conditions. DESIGN: A control study of animal experiment in a university hospital. MATERIALS AND METHODS: The 2-cell ICR mouse embryos were cultured to blastocyst stage under 3% O2 tension (N¼186) and 20% O2 tension (N¼189), respectively. The blastocysts were collected from each experiment. The expression of oxygen-related genes (HIF-1a and HIF-2a) and antioxidant genes (MnSOD and PRDX5) were analyzed by real-time RT PCR. Protein levels were validated by Immunofluorescence analysis. The apoptosis and mitochondrial membrane potential (mtMP) were assessed by TUNEL and JC-1 stain, respectively. Student‘s t test or one-way ANOVA test was used to evaluate statistical significance. RESULTS: The blastocyst formation and hatching rate (Mean SE) increased significantly in 3% O2 group when compared to 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 MnSOD and PRDX5 were also significantly increased seven to eight fold in 3% O2 group, compared to 20% O2 group (P<0.05). Immunofluorescence staining showed the intensity of MnSOD 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. Although HIF-1a and HIF-2a mRNA level was similar in two groups, apoptotic index was significantly higher in 20% O2, compared with 3% O2 group (P<0.05). The 3% O2 blastocyst also showed a significantly higher mtMP compared with 20% O2 group.
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ASRM Abstracts
CONCLUSION: This study has proposed that lower O2 tension may improve embryo development and viability by increased antioxidant enzymes, it provides more conducive environment by up-regulation of LIFR to increase implantation ability and by increased mtMP to stimulate mitochondrial activity against apoptosis during implantation period. All these effects are initiated and regulated by HIF-2a, which acts as key mediator in hypoxic environment. Supported by: NSC 99-2314-B-038003-MY3.
P-240 Tuesday, October 21, 2014 GROWTH FACTORS ASSOCIATED WITH EMBRYO CO-CULTURE AND AUTOLOGOUS GRANULOSA CELL CLUSTERS COMPARED TO REGULAR EMBRYO CULTURE IN IVF. A. Vithoulkas,a M. Levanduski,b V. T. Goudas,a,c K. Illmensee.a aGenesis Fertility Center, Patra, Achaia, Greece; bEmbryoserv Corp, Riverdale, NJ; cAdvanced Fertility Services, New York, NY. OBJECTIVE: To investigate and compare the presence of various growth factors in human embryo culture with and without autologous granulosa cell supplementation. DESIGN: Randomized prospective comparative study. MATERIALS AND METHODS: 17 IVF couples participated in the study. In each case, half of MII-oocytes were randomly assigned to co-culture (group I) and regular culture (group II), treated for ICSI and cultured in microdrops of Irvine Cleavage Medium (15% HSA). On day-3, 50ml of the culture supernatants from group I, II and autologous granulosa clusters cultured alone (control group C), were collected and stored frozen. Supernatants were analysed for 7 growth factors (EGF, FGF-1, FGF-2, VEGF-A, VEGF-C, VEGF-D and Leptin) with the Luminex method using Milliplex panels by Lab Supplies Inc., Athens, Greece. RESULTS: VEGF-A and VEGF-C were found in statistically significant levels in groups I and C (control), whereas not detectable in group II. For VEGF-A groups I and C the p values are < 0.005 and < 0.01respectively. For VEGF-C group I and C the p values are < 0.05 and < 0.01 respectively (two-tailed t-test). FGF-2 concentrations are presented in Table 1 but differences between group I and C are not statistically significant. TABLE 1. Growth Factor Case # Group I (pg/ml) Group II (pg/ml) Group C (pg/ml) Growth Factor
VEGF-A 1 2086.5 7.5 680.5 VEGF-C
Case # Group I (pg/ml) Group II (pg/ml) Group C (pg/ml) Growth Factor Case # Group I (pg/ml) Group II (pg/ml) Group C (pg/ml)
2 4013.5 14.5 2159.5
3 3451.5 5 1262.5
4 1455.5 57.5 901.5
5 2520.5 6.5 912.5
6 3806.5 55.5 1340.5
1 99 0.1 20 FGF-2 1
2 158 0.1 34
3 93 0.1 29
4 6 0.1 6
5 16 0.1 1.5
6 35.5 0.1 1
2
3
4
5
6
114 0.5 455
98.5 0.5 446.5
4.5 0.5 19.5
22.5 0.5 103.5
26.5 0.5 30.5
9.5 0.5 483.5
Concentration (netMFI ¼ pg/ml) of growth factors VEGF-A, VEGF-C and FGF-2 in supernatants of human embryo culture supplemented with (group I) and without (group II) granulosa cell clusters, and in controls (group C). Differences were observed at the levels of all 3 growth factors VEGF-A, VEGF-C and FGF-2 and also between the 17 cases investigated (see Table 1, six cases presented). EGF and FGF-1 were detectable in very low concentrations in group I and C. VEGF-D and Leptin could not be detected. CONCLUSION: Autologous granulosa cell clusters added to oocyte culture contribute measurable levels of growth factors VEGF-A VEGF -C and FGF-2. This may explain the improved embryo development previously reported (Fertil Steril, 96, 3 suppl, S59, 2011). Supported by: Ferring Hellas and Genesis Fertility Center.
Vol. 102, No. 3, Supplement, September 2014
P-236 Tuesday, October 21, 2014 FIRST MORPHOKINETIC ANALYSIS OF BLASTOCYST EXPANSION IN HUMAN EMBRYOS OF KNOWN POSITIVE IMPLANTATION USING AN EMBRYOSCOPE. T. T. F. Huang. Pacific IVF Institute, Honolulu, HI. OBJECTIVE: Using an Embryoscope, the objective was to morphokinetically describe features of the expanding human blastocyst cavity in embryos yielding sustained ongoing pregnancies. DESIGN: Retrospective descriptive study in a private practice setting. MATERIALS AND METHODS: Data were obtained from 27 sequential egg donation cycles using blastocysts selected for transfer using an Embryoscope. Day 5 embryos were ranked and selected for transfer by the greatest degree of blastocyst cavity expansion (BE) with otherwise normal cleavage, ICM and TE characteristics. BE was determined from sequential hourly 2D cross sectional area (CSA) measurements starting from blastocyst formation up until ET. Sustained pregnancy was defined as clinical detection of a beating heart. RESULTS: Of 5 single blastocyst transfers (SBT), 4 (80%) resulted in a sustained pregnancy (IR¼80%). Of 21 double blastocyst transfers (DBT), 21 (100%) resulted in a sustained pregnancy with an IR of 80.4%. (One patient had a triple BT but did not become pregnant). Of the DBT’s, 12 (57.1%) were twins (100% IR) and 9 (42.9%) were singletons (50% IR). The study confirmed a correlation between the time of the start of blastocyst cavitation (Tsb) and the time of blastocyst formation (Tb) for both the 100% (r2 ¼ 0.830) and 50% (r2 ¼ 0.893) IR groups. In the 100% IR group, the average hourly rate of BE increased over consecutive 3 hour intervals (557.4 u2 /hr; 916.2 u2 /hr; 1276.3 u2/hr), then tapered to 870.4 u2/ hr during the fourth interval (hours 10-12). This was similar in the 50% IR group. Two morphokinetically distinguishable events characterized BE. The first was a novel pulsatile, oscillatory pattern of accelerations and decelerations with a periodicity of 2-3 hours resulting in continuous BE. The second was an occasional, acute contraction of the cavity, ranging from 5-50% in CSA, often occurring several times in blastocysts monitored for up to 22 hours. Remarkably, contractions recovered completely within 1-3 hours. Interestingly, the rates of expansion and times to reach given CSA milestones at 1, 5, and 10 hours from Tb did not correlate with the initial time of blastocyst formation in the cohort of 100% IR blastocysts. CONCLUSION: This reports the first quantitative description of BE in human embryos yielding sustained pregnancies. Expansion typically accelerates in an oscillatory manner, with occasional but reversible collapses in
FERTILITY & STERILITYÒ
the cavity. Results supports the hypothesis that time-lapse technology identifies novel biological information useful in embryo selection.
P-237 Tuesday, October 21, 2014 MICRO-VIBRATION CULTURE OF HUMAN EMBRYOS IMPROVES PREGNANCY AND IMPLANTATION RATES. I. El-Danasouri, N. L. Sandi-Monroy, T. Winkle, K. Ott, C. Krebs, D. H. A. Maas, F. Gagsteiger. Kinderwunsch-Zentrum Ulm/ Stuttgart, Ulm, BW, Germany. OBJECTIVE: Embryos in vivo are not present in a static condition, since in the fallopian tube they are exposed to continuous movement, compression caused by the cilia and peristaltic movements, and shear stress from tubal fluid flow. In this study, we compared the outcome of human oocytes and embryos cultured in a micro-vibration culture system for In Vitro Fertilization (IVF) patients in comparison to the traditional static culture environment. DESIGN: Retrospective analysis. MATERIALS AND METHODS: Oocytes and embryos from 1943 patients enrolled in IVF treatment from January 2010 to February 2013 were cultured in a traditional static culture, while 497 patients’ embryos from March 2013 to March 2014 were cultured in a micro-vibration culture. In the micro-vibration group, culture dishes were placed on the top of a platform producing a three-dimensional vibration of 56 Hz for 5 seconds every 60 minutes. Patients in both groups were compared according to their age (< 30; 30-35; 36-38 and >39 years old). The outcomes of microvibration culturing were compared for fertilization, blastulation, pregnancy and implantation rates. Variables were analyzed by chi-square test and Fisher’s exact test. RESULTS: Pregnancy rates significantly increased in the micro-vibration culture in younger patient groups compared to the static culture groups (< 30: 59.4% vs. 38.4%, P¼0.0006; 30-35: 50% vs. 36.3%, P<¼0.0005; 36-38: 42.7% vs. 31.2%, P¼0.03) as did the implantation rate (<30: 54.2% vs. 25.5%, P<0.0001; 30-35: 48.1% vs. 30.1%, P<0.0001; 36-38: 40.3% vs. 26.5%, P<0.0001). The number of transferred embryos and day of transfer did not differ between these groups. For patients >39-years-old, micro-vibration culture tended to increase both pregnancy and implantation rates over the static culture but did not reach statistical significance (20.6% vs. 17.2%, P¼0.41 and 21.1% vs. 15.3%, P¼0.08, respectively). Micro-vibration culturing produced no effect on the fertilization rate. In addition, the overall blastulation rate was significantly higher in the micro-vibration culture compared to the static culture (40.5% vs. 31.2%, P<0.0001). CONCLUSION: These results demonstrate clearly that the three-dimensional micro-vibration culture of oocytes and embryos significantly increases pregnancy and implantation rates. Mechanical vibration of the embryo may mimic the embryo’s in vivo environment and may cause movement of media around the embryo aiding in refreshing the media surrounding the embryos and diffusion of waste material.
P-238 Tuesday, October 21, 2014 UNDISTURBED EMBRYO CULTURE IN A TIME-LAPSE MONITORING SYSTEM IMPROVE EMBRYO QUALITY: A PROSPECTIVE COHORT STUDY. W. Han, H. L. Wu, H. Ye, N. G. Huang. Chongqing Maternity and Children Health Care Hospi, Chongqing, China. OBJECTIVE: To quantify the benefit of culturing embryos in a time-lapse monitoring system. DESIGN: Prospective cohort study. MATERIALS AND METHODS: One hundred and sixty patients (age % 35 yrs; BMI: 18-25; 5-15 oocytes; pure tubal factor infertility) were included in this prospective cohort study, and were randomly assigned to the control group (80 patients) or the time-lapse(TL) group (80patients) from April 22 to December 25, 2013. Fertilized oocytes in the TL group were cultured in microwell culture dishes and monitored on compact digital inverted microscopes (Primo Vision, Vitrolife). Images were captured every 5 minutes .In the control group, fertilized oocytes were cultured in conventional dishes (one embryo in each microdroplet). In both groups, embryo were selected for transfer on day 3, by traditional morphology assessment. The clinical pregnancy confirmed by the presence of
e217
gestational sacs with fetal heart beat by transvaginal ultrasound examination in week 7. RESULTS: The number of good quality embryo was significantly higher in the TL group compared to control group (4.632.22 VS 3.651.84, p¼0.0014); pregnancy rate and implantation rate showed a tendency to be higher in the TL group compared to control group. TABLE 1. Comparison of culture outcomes between TL group and Control group Time-lapse group(n¼80)
Control group(n¼80)
30.313.53
30.553.06
Oocytes retrieved(n) 2PN(n) good quality embryos(n) Embryos transferred(n) Implantation rate(%) Clinical pregnancy rate(%)
11.353.0 8.212.30 4.632.22 2.020.22 48.77% ( 79/162) 70.00% (56/80)
10.812.78 7.712.33 3.651.84 2.010.19 43.48% (70/161) 61.25% (49/80)
0.12 0.09 0.0014 0.38 0.34 0.24
On-going pregnancy rate(%)
63.75% ( 51/80)
56.25% (45/80)
0.33
Age(yrs)
P value 0.32
P< 0.05 were regarded as significant difference.
CONCLUSION: This prospective cohort study result indicated that culturing embryos by TM significantly improved the embryo quality.The elevated clinical outcome was attributed to a combination of stable culture conditions and the group culture in microwells.
P-239 Tuesday, October 21, 2014 LOW OXYGEN TENSION INCREASE MITOCHONDRIAL MEMBRANE POTENTIAL AND IMPLANTATION ABILITY BY ENHANCED EXPRESSION OF ANTIOXIDANT GENES IN MOUSE BLASTOCYST CULTURED IN VITRO. Y.-I. Ma,a C.-H. Chen,b J.-W. Wang,b C.-R. Tzeng.a aCenter for Reproductive Medicine & SciencesTaipei Medical University, 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 pregnancy rate, when compared with normaxic condition (20% O2). However the beneficial effect of low oxygen tension in embryogenesis remains unclear. This study aimed to investigate the expression of oxygen and antioxidant genes in mouse embryo cultured under hypoxic and normaxic conditions. DESIGN: A control study of animal experiment in a university hospital. MATERIALS AND METHODS: The 2-cell ICR mouse embryos were cultured to blastocyst stage under 3% O2 tension (N¼186) and 20% O2 tension (N¼189), respectively. The blastocysts were collected from each experiment. The expression of oxygen-related genes (HIF-1a and HIF-2a) and antioxidant genes (MnSOD and PRDX5) were analyzed by real-time RT PCR. Protein levels were validated by Immunofluorescence analysis. The apoptosis and mitochondrial membrane potential (mtMP) were assessed by TUNEL and JC-1 stain, respectively. Student‘s t test or one-way ANOVA test was used to evaluate statistical significance. RESULTS: The blastocyst formation and hatching rate (Mean SE) increased significantly in 3% O2 group when compared to 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 MnSOD and PRDX5 were also significantly increased seven to eight fold in 3% O2 group, compared to 20% O2 group (P<0.05). Immunofluorescence staining showed the intensity of MnSOD 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. Although HIF-1a and HIF-2a mRNA level was similar in two groups, apoptotic index was significantly higher in 20% O2, compared with 3% O2 group (P<0.05). The 3% O2 blastocyst also showed a significantly higher mtMP compared with 20% O2 group.
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ASRM Abstracts
CONCLUSION: This study has proposed that lower O2 tension may improve embryo development and viability by increased antioxidant enzymes, it provides more conducive environment by up-regulation of LIFR to increase implantation ability and by increased mtMP to stimulate mitochondrial activity against apoptosis during implantation period. All these effects are initiated and regulated by HIF-2a, which acts as key mediator in hypoxic environment. Supported by: NSC 99-2314-B-038003-MY3.
P-240 Tuesday, October 21, 2014 GROWTH FACTORS ASSOCIATED WITH EMBRYO CO-CULTURE AND AUTOLOGOUS GRANULOSA CELL CLUSTERS COMPARED TO REGULAR EMBRYO CULTURE IN IVF. A. Vithoulkas,a M. Levanduski,b V. T. Goudas,a,c K. Illmensee.a aGenesis Fertility Center, Patra, Achaia, Greece; bEmbryoserv Corp, Riverdale, NJ; cAdvanced Fertility Services, New York, NY. OBJECTIVE: To investigate and compare the presence of various growth factors in human embryo culture with and without autologous granulosa cell supplementation. DESIGN: Randomized prospective comparative study. MATERIALS AND METHODS: 17 IVF couples participated in the study. In each case, half of MII-oocytes were randomly assigned to co-culture (group I) and regular culture (group II), treated for ICSI and cultured in microdrops of Irvine Cleavage Medium (15% HSA). On day-3, 50ml of the culture supernatants from group I, II and autologous granulosa clusters cultured alone (control group C), were collected and stored frozen. Supernatants were analysed for 7 growth factors (EGF, FGF-1, FGF-2, VEGF-A, VEGF-C, VEGF-D and Leptin) with the Luminex method using Milliplex panels by Lab Supplies Inc., Athens, Greece. RESULTS: VEGF-A and VEGF-C were found in statistically significant levels in groups I and C (control), whereas not detectable in group II. For VEGF-A groups I and C the p values are < 0.005 and < 0.01respectively. For VEGF-C group I and C the p values are < 0.05 and < 0.01 respectively (two-tailed t-test). FGF-2 concentrations are presented in Table 1 but differences between group I and C are not statistically significant. TABLE 1. Growth Factor Case # Group I (pg/ml) Group II (pg/ml) Group C (pg/ml) Growth Factor
VEGF-A 1 2086.5 7.5 680.5 VEGF-C
Case # Group I (pg/ml) Group II (pg/ml) Group C (pg/ml) Growth Factor Case # Group I (pg/ml) Group II (pg/ml) Group C (pg/ml)
2 4013.5 14.5 2159.5
3 3451.5 5 1262.5
4 1455.5 57.5 901.5
5 2520.5 6.5 912.5
6 3806.5 55.5 1340.5
1 99 0.1 20 FGF-2 1
2 158 0.1 34
3 93 0.1 29
4 6 0.1 6
5 16 0.1 1.5
6 35.5 0.1 1
2
3
4
5
6
114 0.5 455
98.5 0.5 446.5
4.5 0.5 19.5
22.5 0.5 103.5
26.5 0.5 30.5
9.5 0.5 483.5
Concentration (netMFI ¼ pg/ml) of growth factors VEGF-A, VEGF-C and FGF-2 in supernatants of human embryo culture supplemented with (group I) and without (group II) granulosa cell clusters, and in controls (group C). Differences were observed at the levels of all 3 growth factors VEGF-A, VEGF-C and FGF-2 and also between the 17 cases investigated (see Table 1, six cases presented). EGF and FGF-1 were detectable in very low concentrations in group I and C. VEGF-D and Leptin could not be detected. CONCLUSION: Autologous granulosa cell clusters added to oocyte culture contribute measurable levels of growth factors VEGF-A VEGF -C and FGF-2. This may explain the improved embryo development previously reported (Fertil Steril, 96, 3 suppl, S59, 2011). Supported by: Ferring Hellas and Genesis Fertility Center.
Vol. 102, No. 3, Supplement, September 2014