- Email: [email protected]
Blastocoel proteomic profile and its association with embryo chromosomal status
BFRs for fresh Aut (959/1539; 59%) & OD (2578/3658; 71%) cycles were significantly higher than in age-matched frozen Aut & OD cycles (p< .01). CONCLUSIONS: BFRs in Aut vs. OD OC cycles were not different, although both were lower than in Aut & OD fresh cycles. Of the OC oocytes not reaching BL, most arrested at cleavage while 8% failed to divide at all. >28% peaked at morula or Stage-1 BL, both considered early ‘‘usable’’ embryos resulting in reasonable, albeit lower EI & pregnancy rates. When comparing BL maturity in Aut vs. OC cycles, OD oocytes achieved >BL expansion with higher EI & pregnancy rates, most likely reflecting younger oocyte age.
Embryo development & pregnancy outcomes of OC thaws using Autol vs. OD oocytes.
Autol OC (n¼1098) No. 2PN-Zygotes with No Division (1-cell) No. 2PN-Zygotes with Cleavage Arrest No. Morula (highest stage achieved) No. BL - Stage 1 No. BL - Stage 2 No. BL- Stage 3 No. BL- Stage 4+ ET of Morulas &/or Stage 1 BL: EI ET of Morulas &/or Stage 1 BL: Pregnancy ET of Stage 2 - 6 BL: EI ET of Stage 2 - 6 BL: Pregnancy
P-588 Wednesday, October 21, 2015 WITHDRAWN
OD OC (n¼411)
P-589 Wednesday, October 21, 2015 EXOSOME BOUND MICRORNAS TRANSCRIPTIONALLY REGULATE EMBRYO-ENDOMETRIAL DIALOGUE IMPACTING IMPLANTATION POTENTIAL FOR AMA PATIENTS. A. L. Patton,a B. McCallie,a J. C. Parks,a W. B. Schoolcraft,b M. Katz-Jaffe.b aNational Foundation for Fertility Research, Lone Tree, CO; bColorado Center for Reproductive Medicine, Lone Tree, CO.
P
81 (7%)
39 (9%)
0.2
433 (39%)
139 (34%)
0.04
156 (14%)
60 (15%)
0.9
75 (18%) 181 (42%) 84 (20%) 88 (21%) 7/36 (19%)
22 (13%) 33 (19%) 49 (28%) 68 (39%) 5/17 (29%)
0.2 <0.01 0.02 <0.01 0.5
6/20 (30%)
4/9 (44%)
0.7
32/68 (47%) 23/38 (61%) 0.2 24/49 (49%) 15/26 (58%) 0.6
OBJECTIVE: Advanced maternal age (AMA) is the most significant risk factor associated with implantation failure. Women in their forties exhibit significantly lower implantation rates, less than 10% with their own oocytes, compared to >60% with fertile donor oocytes. The aim of this study was to investigate the impact of maternal age on embryo-endometrial dialogue at the time of implantation. DESIGN: Research study. MATERIALS AND METHODS: Surplus, cryopreserved, good quality blastocysts were donated with IRB consent. Individual blastocysts were warmed for co-culture on a monolayer of fertile endometrial epithelial cells to simulate an in vivo like environment: blastocysts from fertile oocyte donor cycles with no male factor (n¼15) and blastocysts from infertile AMA patients with no other infertility diagnosis (R39 years; n¼14). Exosomes were isolated from co-culture supernatant for microRNA expression using the TaqmanÒ Human MicroRNA Array (Thermo Fisher). Blastocyst and endometrial cell gene transcription was assessed using qRT-PCR. Statistical analysis was performed with RESTÓ statistical software (Qiagen). RESULTS: Isolated exosomes from supernatant collected after co-culture of fertile endometrial epithelial cells with blastocysts from infertile AMA patients revealed an altered miRNAome. A total of 8 miRNAs showed increased expression and 15 displayed reduced expression in association with the maternal age of the blastocyst (P<0.05). Target gene analysis of all 23 altered miRNAs revealed 146 validated target genes (miRTarBase), including VEGFA. VEGFA is a signal protein capable of stimulating angiogenesis as well as acting as a growth factor, secreted by the receiving endometrium as well as the implanting embryo. VEGFA expression was observed to be downregulated in both the corresponding endometrial epithelial cells and blastocysts from infertile AMA patients following co-culture (50% fold change reduction compared to fertile controls; P<0.05). CONCLUSIONS: A viable competent blastocyst and receptive endometrial epithelium are essential for the facilitation of successful implantation. Even though infertile AMA patients can produce morphologically good quality blastocysts, any compromise in the delicate embryo-endometrium dialogue, either autocrine or paracrine, may impact transcription levels of key signaling molecules like VEGFA, resulting in significantly lower implantation potential. In conclusion, exosome bound secreted miRNAs are key transcriptional regulators and potential biomarkers of implantation success.
P-590 Wednesday, October 21, 2015 BLASTOCOEL PROTEOMIC PROFILE AND ITS ASSOCIATION WITH EMBRYO CHROMOSOMAL STATUS. M. Poli,a,b,c A. Ori,d T. Child,a,b S. Jaroudi,c K. Spath,a,c M. Beck,d D. Wells.a,c aNuffield Department of Obstetrics and Gynaecology, University of Oxford, Oxford, United Kingdom; bOxford Fertility Unit, Oxford, United Kingdom; cReprogenetics UK, Oxford, United Kingdom; dEuropean Molecular Biology Laboratory, Heidelberg, Germany. OBJECTIVE: In this study we investigate the relationship between the proteomic profile of single blastocoel fluid samples and the chromosomal status of the corresponding embryo. DESIGN: Retrospective cohort study. MATERIALS AND METHODS: Combining blastocentesis procedure for the collection of blastocoel samples and tandem mass spectrometry analysis, we initially compiled a catalogue of almost 300 proteins represented in the human blastocoel proteome. Based on this information, we created Single Reaction Monitoring (SRM) assays for the identification and measurement of previously identified proteins of interest using targeted mass spectrometry.
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ASRM Abstracts
Vol. 104, No. 3, Supplement, September 2015
Using these SRM assays and aCGH, we performed blastocoel protein profiling and cytogenetic assessment on 14 embryos donated to research. Subsequently, we conducted statistical analysis using logistic regression to reveal any correlations between blastocoel protein profile, embryo morphology, ploidy status, gender, patient’s age. RESULTS: Using levels of a metabolic protein and the detection of a nuclear protein as predictors and embryo’s chromosomal status as class label, the statistical model was able to predict whether an embryo was euploid or chromosomally abnormal. This was achieved with 100% accuracy within the small cohort of embryos investigated. Statistical analysis did not show any association between blastocoel protein profile and any of the other parameters considered. CONCLUSIONS: The application of logistic regression analysis to the data obtained suggests that it may be possible to distinguish chromosomally normal embryos from those affected by aneuploidy using a targeted proteomics approach applied to blastocoel fluid. However, due to the small size of the sample population investigated and the retrospective nature of the analysis, further work will be essential to determine sensitivity and specificity of this promising method of preimplantation aneuploidy detection. If confirmed in larger studies, this minimally invasive proteomic approach could offer a novel methodology for the prediction of embryonic viability and developmental competence. Supported by: Institutional funding. P-591 Wednesday, October 21, 2015 SYMMETRY AT THE 4-CELL STAGE USING TIME-LAPSE IMAGING IS CORRELATED WITH EMBRYO ANEUPLOIDY. C. C. Shenoy, Z. Khan, C. Coddington, J. Jensen, G. S. Daftary, E. A. Stewart, D. Morbeck. Mayo Clinic, Rochester, MN. OBJECTIVE: Embryo selection is important for optimal ART outcomes. Embryo selection has improved over time and the advent of time-lapse (TL) monitoring offers increased information to aid in embryo selection. Nonetheless, TL timing parameters alone do not provide high sensitivity for determining embryo viability or ploidy. We sought to compare euploid prediction using TL timings with traditional morphometric parameters that are easily measured using a TL system. DESIGN: Embryos undergoing preimplantation genetic screening (PGS) that were cultured using TL monitoring were examined in this retrospective study. MATERIALS AND METHODS: All embryos from 2012-2015 undergoing PGS with trophectoderm biopsy were included. The distance between the second and first polar body was determined (PBD). Zona pellucida thickness (ZPT) was measured at the pronuclear stage and at the 2-cell stage in four locations and averaged. Blastomere area was assessed at the 2- and 4cell stages. Symmetry at the 2-cell stage was determined by percent difference between blastomeres (2cSY). Symmetry at the 4-cell stage was the percent difference between the smallest and largest blastomeres (4cSY). Ttest was used to compare group means. RESULTS: Embryos (n¼182) from 21 patients were analyzed. Fouty five percent were euploid. Patient age ranged from 22-43 (avg¼34). The only variable that differed significantly between euploid and aneuploid embryos was symmetry at the 4-cell stage. Aneuploidy rates were 45.5% in the lowest symmetry quartile and 70.5% in the quartile with the most asymmetry at the 4-cell stage. CONCLUSIONS: Poor symmetry at the 4-cell embryo stage is more predictive of aneuploidy than TL parameters, suggesting that embryo selection models using time-lapse parameters should incorporate cleavage-stage morphology. Morphologic and TL Differences Between Euploid and Aneuploid Embryos.
PBD (mm) ZPT at PN stage (mm) ZPT at 4-cell stage (mm) 2cSY (% difference) 4cSY (% difference) t2 (h) t5 (h) tsb (h) tb (h) cc2 (h) s2 (h)
Euploid
Aneuploid
43.2 24.8 18.0 2.7 17.9 2.6 11.3 7.5 26.6 11.4 26.4 3.7 51.2 8.3 101.8 9.4 108.9 9.7 11.1 3.1 2.2 4.2
43.6 26.8 18.3 2.4 17.9 2.4 10.0 8.2 31.3 13.4 26.8 2.8 52.2 6.9 103.7 7.9 110.7 8.9 11.6 2.9 1.1 2.4
FERTILITY & STERILITYÒ
p value 0.91 0.45 0.81 0.28 0.01* 0.41 0.39 0.15 0.27 0.20 0.06
P-592 Wednesday, October 21, 2015 FUNCTIONAL CHARACTERIZATION OF CULTURED HUMAN GRANULOSA CELLS IN SERUM FREE CULTURE SYSTEM. Y. Wu,a D. F. Albertini,b Q. Wang,a D. H. Barad,c d a c a V. A. Kushnir, E. Lazzaroni-Tealdi, N. Gleicher. Center for Human Reproduction, New York, NY; bCenter for Human Reproduction & University of Kansas Medical Center, New York, NY; cCenter for Human Reproduction & Foundation for Reproductive Medicine, New York, NY; dCenter for Human Reproduction & Wake Forest University, New York, NY. OBJECTIVE: In vitro culture of human granulosa cells (GCs) is an important research technique for reproductive cell biology and endocrinology studies. However, traditional culture protocols, involving supplementation of serum to culture medium, result in luteinization of GCs, and changes their cell functions during culture. Establishment of a GC culture system that does not cause luteinization would, therefore, be important. DESIGN: Prospective laboratory study. MATERIALS AND METHODS: GCs were collected from follicular fluid after oocytes retrievals. After washed twice by DPBS to remove blood contamination,GCs were seeded into 12-well plates at density of 10x105/ml in DMEM/F12 containing 10% FBS, 2mg/ml of HSA, 2 mM glutamine and 1x Insulin-Transferin-Selenium X and cultured for eight hours at 37 C with 5% CO2 to allow cell attachment. Then the culture medium was replaced by serum free medium (DMEM/F12 with same supplementation but without FBS). After overnight culture, medium was replaced once more. GCs were then cultured for another 96 hours with or without 50ng/ml FSH supplementation. To evaluate GC functions after culture, we examined aromatase and FSH receptor (FSHR) mRNA expression by real-time PCR, cell proliferation by MTT assay, apoptosis by DAPI staining and estradiol (E2) production by analyzing medium hormone concentration after adding testosterone (1mM) to the medium. RESULTS: After 96 hours culture, FSH treatment significantly induced aromatase and FSHR expression in GCs (P<0.05). It also increased E2 production 6 hours post testosterone supplementation (31.71.5ng/ ml vs. 15.51.1ng/ml, P<0.005). The presence of FSH in medium also stimulated GCs proliferation (P<0.01) and decreased GC apoptosis (P<0.01). CONCLUSIONS: We here report a serum free in vitro culture protocol that maintains normal function of GCs by eliminating from serum its luteinizing effect. Our data indicate that GCs, cultured serum-free, have normal ability to respond to FSH treatment, as indicated by normal gene expression, steroidogenesis and proliferation. This serum-free culture system, therefore, is clearly superior to currently in use GC culture systems. Supported by: Intramural funds from The Center for Human Reproduction and grants from The Foundation for Reproductive Medicine. P-593 Wednesday, October 21, 2015 PRONUCLEAR STAGE ASYNCHRONY FADING INCIDENCE AND RELATIONSHIP WITH REPRODUCTIVE OUTCOME. J. A. Aguilar,a E. Taboas,b M. Ojeda,b M. Perez,b E. Munoz,b M. Meseguer.c aIVF Laboratory, IVI Vigo, Vigo, Spain; bIVI Vigo, Vigo, Spain; cClinical Embryology, Valencia, Spain. OBJECTIVE: Pronuclear fading is an early event of embryo development often synchronically observed (both the male and female pronucleai disappear at the same time) according to the current time lapse data (provided by picture frequencies (every 5 minutes or more). Occasionally this phenomenon is not observed as synchronic. The aim of this research was to evaluate the incidence of asynchronical pronuclear fading in embryos from ICSI cycles using egg donation, and to relate them with morphokinetics parameters. DESIGN: Observational Retrospective study conducted in IVI Vigo. 262 embryos, cultured in an Embryoscope incubator in a 37oC, 6% CO2 and 20% de O2 atmosphere from 28 oocyte donation cycles were analyzed. Pictures of embryos were obtained every 10 minutes in seven different focal planes. The exact timing of the events cited below was evaluated in hours post insemination by ICSI. MATERIALS AND METHODS: The asynchronical fading of pronuclei (PNf asyn) was evaluated. Other morphokinetic events such as time at which both pronuclei disappear (tPNf), and the time for the embryo to reach 2 cells stage (t2), 3cells (t3), 4cells (t4), 5cells (t5), cc2¼t3-t2 and s2¼t4-t3; All these morphokinetic parameters were related with single point morphological evaluation by ASEBIR embryo morphology qualification. T-Student
e309
Embryo development & pregnancy outcomes of OC thaws using Autol vs. OD oocytes.
Autol OC (n¼1098) No. 2PN-Zygotes with No Division (1-cell) No. 2PN-Zygotes with Cleavage Arrest No. Morula (highest stage achieved) No. BL - Stage 1 No. BL - Stage 2 No. BL- Stage 3 No. BL- Stage 4+ ET of Morulas &/or Stage 1 BL: EI ET of Morulas &/or Stage 1 BL: Pregnancy ET of Stage 2 - 6 BL: EI ET of Stage 2 - 6 BL: Pregnancy
P-588 Wednesday, October 21, 2015 WITHDRAWN
OD OC (n¼411)
P-589 Wednesday, October 21, 2015 EXOSOME BOUND MICRORNAS TRANSCRIPTIONALLY REGULATE EMBRYO-ENDOMETRIAL DIALOGUE IMPACTING IMPLANTATION POTENTIAL FOR AMA PATIENTS. A. L. Patton,a B. McCallie,a J. C. Parks,a W. B. Schoolcraft,b M. Katz-Jaffe.b aNational Foundation for Fertility Research, Lone Tree, CO; bColorado Center for Reproductive Medicine, Lone Tree, CO.
P
81 (7%)
39 (9%)
0.2
433 (39%)
139 (34%)
0.04
156 (14%)
60 (15%)
0.9
75 (18%) 181 (42%) 84 (20%) 88 (21%) 7/36 (19%)
22 (13%) 33 (19%) 49 (28%) 68 (39%) 5/17 (29%)
0.2 <0.01 0.02 <0.01 0.5
6/20 (30%)
4/9 (44%)
0.7
32/68 (47%) 23/38 (61%) 0.2 24/49 (49%) 15/26 (58%) 0.6
OBJECTIVE: Advanced maternal age (AMA) is the most significant risk factor associated with implantation failure. Women in their forties exhibit significantly lower implantation rates, less than 10% with their own oocytes, compared to >60% with fertile donor oocytes. The aim of this study was to investigate the impact of maternal age on embryo-endometrial dialogue at the time of implantation. DESIGN: Research study. MATERIALS AND METHODS: Surplus, cryopreserved, good quality blastocysts were donated with IRB consent. Individual blastocysts were warmed for co-culture on a monolayer of fertile endometrial epithelial cells to simulate an in vivo like environment: blastocysts from fertile oocyte donor cycles with no male factor (n¼15) and blastocysts from infertile AMA patients with no other infertility diagnosis (R39 years; n¼14). Exosomes were isolated from co-culture supernatant for microRNA expression using the TaqmanÒ Human MicroRNA Array (Thermo Fisher). Blastocyst and endometrial cell gene transcription was assessed using qRT-PCR. Statistical analysis was performed with RESTÓ statistical software (Qiagen). RESULTS: Isolated exosomes from supernatant collected after co-culture of fertile endometrial epithelial cells with blastocysts from infertile AMA patients revealed an altered miRNAome. A total of 8 miRNAs showed increased expression and 15 displayed reduced expression in association with the maternal age of the blastocyst (P<0.05). Target gene analysis of all 23 altered miRNAs revealed 146 validated target genes (miRTarBase), including VEGFA. VEGFA is a signal protein capable of stimulating angiogenesis as well as acting as a growth factor, secreted by the receiving endometrium as well as the implanting embryo. VEGFA expression was observed to be downregulated in both the corresponding endometrial epithelial cells and blastocysts from infertile AMA patients following co-culture (50% fold change reduction compared to fertile controls; P<0.05). CONCLUSIONS: A viable competent blastocyst and receptive endometrial epithelium are essential for the facilitation of successful implantation. Even though infertile AMA patients can produce morphologically good quality blastocysts, any compromise in the delicate embryo-endometrium dialogue, either autocrine or paracrine, may impact transcription levels of key signaling molecules like VEGFA, resulting in significantly lower implantation potential. In conclusion, exosome bound secreted miRNAs are key transcriptional regulators and potential biomarkers of implantation success.
P-590 Wednesday, October 21, 2015 BLASTOCOEL PROTEOMIC PROFILE AND ITS ASSOCIATION WITH EMBRYO CHROMOSOMAL STATUS. M. Poli,a,b,c A. Ori,d T. Child,a,b S. Jaroudi,c K. Spath,a,c M. Beck,d D. Wells.a,c aNuffield Department of Obstetrics and Gynaecology, University of Oxford, Oxford, United Kingdom; bOxford Fertility Unit, Oxford, United Kingdom; cReprogenetics UK, Oxford, United Kingdom; dEuropean Molecular Biology Laboratory, Heidelberg, Germany. OBJECTIVE: In this study we investigate the relationship between the proteomic profile of single blastocoel fluid samples and the chromosomal status of the corresponding embryo. DESIGN: Retrospective cohort study. MATERIALS AND METHODS: Combining blastocentesis procedure for the collection of blastocoel samples and tandem mass spectrometry analysis, we initially compiled a catalogue of almost 300 proteins represented in the human blastocoel proteome. Based on this information, we created Single Reaction Monitoring (SRM) assays for the identification and measurement of previously identified proteins of interest using targeted mass spectrometry.
e308
ASRM Abstracts
Vol. 104, No. 3, Supplement, September 2015
Using these SRM assays and aCGH, we performed blastocoel protein profiling and cytogenetic assessment on 14 embryos donated to research. Subsequently, we conducted statistical analysis using logistic regression to reveal any correlations between blastocoel protein profile, embryo morphology, ploidy status, gender, patient’s age. RESULTS: Using levels of a metabolic protein and the detection of a nuclear protein as predictors and embryo’s chromosomal status as class label, the statistical model was able to predict whether an embryo was euploid or chromosomally abnormal. This was achieved with 100% accuracy within the small cohort of embryos investigated. Statistical analysis did not show any association between blastocoel protein profile and any of the other parameters considered. CONCLUSIONS: The application of logistic regression analysis to the data obtained suggests that it may be possible to distinguish chromosomally normal embryos from those affected by aneuploidy using a targeted proteomics approach applied to blastocoel fluid. However, due to the small size of the sample population investigated and the retrospective nature of the analysis, further work will be essential to determine sensitivity and specificity of this promising method of preimplantation aneuploidy detection. If confirmed in larger studies, this minimally invasive proteomic approach could offer a novel methodology for the prediction of embryonic viability and developmental competence. Supported by: Institutional funding. P-591 Wednesday, October 21, 2015 SYMMETRY AT THE 4-CELL STAGE USING TIME-LAPSE IMAGING IS CORRELATED WITH EMBRYO ANEUPLOIDY. C. C. Shenoy, Z. Khan, C. Coddington, J. Jensen, G. S. Daftary, E. A. Stewart, D. Morbeck. Mayo Clinic, Rochester, MN. OBJECTIVE: Embryo selection is important for optimal ART outcomes. Embryo selection has improved over time and the advent of time-lapse (TL) monitoring offers increased information to aid in embryo selection. Nonetheless, TL timing parameters alone do not provide high sensitivity for determining embryo viability or ploidy. We sought to compare euploid prediction using TL timings with traditional morphometric parameters that are easily measured using a TL system. DESIGN: Embryos undergoing preimplantation genetic screening (PGS) that were cultured using TL monitoring were examined in this retrospective study. MATERIALS AND METHODS: All embryos from 2012-2015 undergoing PGS with trophectoderm biopsy were included. The distance between the second and first polar body was determined (PBD). Zona pellucida thickness (ZPT) was measured at the pronuclear stage and at the 2-cell stage in four locations and averaged. Blastomere area was assessed at the 2- and 4cell stages. Symmetry at the 2-cell stage was determined by percent difference between blastomeres (2cSY). Symmetry at the 4-cell stage was the percent difference between the smallest and largest blastomeres (4cSY). Ttest was used to compare group means. RESULTS: Embryos (n¼182) from 21 patients were analyzed. Fouty five percent were euploid. Patient age ranged from 22-43 (avg¼34). The only variable that differed significantly between euploid and aneuploid embryos was symmetry at the 4-cell stage. Aneuploidy rates were 45.5% in the lowest symmetry quartile and 70.5% in the quartile with the most asymmetry at the 4-cell stage. CONCLUSIONS: Poor symmetry at the 4-cell embryo stage is more predictive of aneuploidy than TL parameters, suggesting that embryo selection models using time-lapse parameters should incorporate cleavage-stage morphology. Morphologic and TL Differences Between Euploid and Aneuploid Embryos.
PBD (mm) ZPT at PN stage (mm) ZPT at 4-cell stage (mm) 2cSY (% difference) 4cSY (% difference) t2 (h) t5 (h) tsb (h) tb (h) cc2 (h) s2 (h)
Euploid
Aneuploid
43.2 24.8 18.0 2.7 17.9 2.6 11.3 7.5 26.6 11.4 26.4 3.7 51.2 8.3 101.8 9.4 108.9 9.7 11.1 3.1 2.2 4.2
43.6 26.8 18.3 2.4 17.9 2.4 10.0 8.2 31.3 13.4 26.8 2.8 52.2 6.9 103.7 7.9 110.7 8.9 11.6 2.9 1.1 2.4
FERTILITY & STERILITYÒ
p value 0.91 0.45 0.81 0.28 0.01* 0.41 0.39 0.15 0.27 0.20 0.06
P-592 Wednesday, October 21, 2015 FUNCTIONAL CHARACTERIZATION OF CULTURED HUMAN GRANULOSA CELLS IN SERUM FREE CULTURE SYSTEM. Y. Wu,a D. F. Albertini,b Q. Wang,a D. H. Barad,c d a c a V. A. Kushnir, E. Lazzaroni-Tealdi, N. Gleicher. Center for Human Reproduction, New York, NY; bCenter for Human Reproduction & University of Kansas Medical Center, New York, NY; cCenter for Human Reproduction & Foundation for Reproductive Medicine, New York, NY; dCenter for Human Reproduction & Wake Forest University, New York, NY. OBJECTIVE: In vitro culture of human granulosa cells (GCs) is an important research technique for reproductive cell biology and endocrinology studies. However, traditional culture protocols, involving supplementation of serum to culture medium, result in luteinization of GCs, and changes their cell functions during culture. Establishment of a GC culture system that does not cause luteinization would, therefore, be important. DESIGN: Prospective laboratory study. MATERIALS AND METHODS: GCs were collected from follicular fluid after oocytes retrievals. After washed twice by DPBS to remove blood contamination,GCs were seeded into 12-well plates at density of 10x105/ml in DMEM/F12 containing 10% FBS, 2mg/ml of HSA, 2 mM glutamine and 1x Insulin-Transferin-Selenium X and cultured for eight hours at 37 C with 5% CO2 to allow cell attachment. Then the culture medium was replaced by serum free medium (DMEM/F12 with same supplementation but without FBS). After overnight culture, medium was replaced once more. GCs were then cultured for another 96 hours with or without 50ng/ml FSH supplementation. To evaluate GC functions after culture, we examined aromatase and FSH receptor (FSHR) mRNA expression by real-time PCR, cell proliferation by MTT assay, apoptosis by DAPI staining and estradiol (E2) production by analyzing medium hormone concentration after adding testosterone (1mM) to the medium. RESULTS: After 96 hours culture, FSH treatment significantly induced aromatase and FSHR expression in GCs (P<0.05). It also increased E2 production 6 hours post testosterone supplementation (31.71.5ng/ ml vs. 15.51.1ng/ml, P<0.005). The presence of FSH in medium also stimulated GCs proliferation (P<0.01) and decreased GC apoptosis (P<0.01). CONCLUSIONS: We here report a serum free in vitro culture protocol that maintains normal function of GCs by eliminating from serum its luteinizing effect. Our data indicate that GCs, cultured serum-free, have normal ability to respond to FSH treatment, as indicated by normal gene expression, steroidogenesis and proliferation. This serum-free culture system, therefore, is clearly superior to currently in use GC culture systems. Supported by: Intramural funds from The Center for Human Reproduction and grants from The Foundation for Reproductive Medicine. P-593 Wednesday, October 21, 2015 PRONUCLEAR STAGE ASYNCHRONY FADING INCIDENCE AND RELATIONSHIP WITH REPRODUCTIVE OUTCOME. J. A. Aguilar,a E. Taboas,b M. Ojeda,b M. Perez,b E. Munoz,b M. Meseguer.c aIVF Laboratory, IVI Vigo, Vigo, Spain; bIVI Vigo, Vigo, Spain; cClinical Embryology, Valencia, Spain. OBJECTIVE: Pronuclear fading is an early event of embryo development often synchronically observed (both the male and female pronucleai disappear at the same time) according to the current time lapse data (provided by picture frequencies (every 5 minutes or more). Occasionally this phenomenon is not observed as synchronic. The aim of this research was to evaluate the incidence of asynchronical pronuclear fading in embryos from ICSI cycles using egg donation, and to relate them with morphokinetics parameters. DESIGN: Observational Retrospective study conducted in IVI Vigo. 262 embryos, cultured in an Embryoscope incubator in a 37oC, 6% CO2 and 20% de O2 atmosphere from 28 oocyte donation cycles were analyzed. Pictures of embryos were obtained every 10 minutes in seven different focal planes. The exact timing of the events cited below was evaluated in hours post insemination by ICSI. MATERIALS AND METHODS: The asynchronical fading of pronuclei (PNf asyn) was evaluated. Other morphokinetic events such as time at which both pronuclei disappear (tPNf), and the time for the embryo to reach 2 cells stage (t2), 3cells (t3), 4cells (t4), 5cells (t5), cc2¼t3-t2 and s2¼t4-t3; All these morphokinetic parameters were related with single point morphological evaluation by ASEBIR embryo morphology qualification. T-Student
e309