Pronuclear stage asynchrony fading incidence and relationship with reproductive outcome

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

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test for numerical variables and chi square test for categorical variables were employed as statistical analysis. P-value lower than 0.05 was considered as statistically significant. RESULTS: The incidence of embryos that were generated from fresh donated oocytes 73.28% (192/262) and 26.72% (70/262) from thawed donated oocytes. PNf asyn rate was 14.5% (38/262) being slightly more frequent in thawed oocytes (17.14% vs 13.54%) without any significant difference (p¼0.42). The average duration between the disappearance of the first pronuclei and the second was 0.33h (IC: 0.17-0.34). The top quality embryo rate (A category from ASEBIR) in those embryos showing PNf asyn was 34.21% (13/38), and in synchronical was 48,86%(86/176); p¼0.10. In Embryos with asynchronical fading, tPNf were significantly later than in those with synchronical (24.664.16 vs 26.564.75, p<0.0001), however, there was not any other statistically significant difference in the remaining morphokinetic parameters: cc2 (10.833.96 vs 11.374.75, p¼0.268) , t5 (50.147.88 vs 54.177.42, p¼0.375) and s2 (2,614.2 vs 2.974.6, p¼0.475). CONCLUSIONS: The incidence of asynchronical fading is reduced. It seems to be more frequent in thawed embryos, although without any significant difference. The variation in disappearance between conjunct pronuclei is short and includes a longer PNf, even though there is no incidence in the embryo quality.

P-594 Wednesday, October 21, 2015 A REVISED BINARY SCORING SYSTEM GENERATED BY THE EEVAÔ TEST’S TIME-LAPSE MICROSCOPY (TLM) SOFTWARE MAY ENHANCE SELECTION AT THE CLEAVAGE K. A. Green,b J. M. Franasiak,c STAGE. M. D. Werner,a b d c e a C. R. Juneau, K. H. Hong, K. M. Upham, R. T. Scott. REI, RMA, NJ, NJ; bRMA, NJ, NJ; cRMANJ, NJ, NJ; dRMA, Basking Ridge, NJ; eRMANJ,Rutgers-RWJ, Basking Ridge, NJ. OBJECTIVE: The Eeva Test’s TLM software automatically generates a binary result (high or low) that prognosticates blastulation rates by day 3 of development. This binary predictive model has previously been studied in patients with normal ovarian responsiveness to assess whether it could aid in selection of euploid blastocysts. Unfortunately, nearly half of the patients studied had all ‘low’ ratings precluding any selection advantage. A revised scoring system was introduced in which embryos previously designated as ‘low’ likelihood of blastulation now may meet requirements to be reclassified as ‘medium’. The goal of the present analysis is to evaluate whether this new scoring system could enhance the utility of the test. DESIGN: Prospective, blinded observational. MATERIALS AND METHODS: Patients with normal ovarian reserve undergoing IVF were recruited. TLM utilizing the EevaTM System (Progyny, Menlo Park, CA) was employed through day 5. No embryologist had access to imaging data prior to performing traditional blastocyst morphology assessment. Two outcomes were evaluated: the proportion of patients for which a single cohort of embryos were designated to have a mix of ‘high’, ‘medium’, and ‘low’ potential for blastulation, as well as whether combining this revised scoring system with morphology on day 3 would have changed selection when compared to morphology at the blastocyst stage. RESULTS: 188 patients and 1,948 fertilized zygotes were included. Blastulation rates were superior for those embryos designated as ‘high’ and ‘medium’ as compared to those in the ‘low’ group (75.1%, 71.1% vs. 53.1%, p<0.0001). However, there was no statistical difference in blastulation rates between the ‘high’ and ‘medium’ designations (p¼0.24). Unfortunately without additional power, this three output scoring system functionally resulted in a binary output. The revision of the scoring system did increase the sensitivity of this test from 23.9% to 53.1%. In the per-patient analysis, this model allowed for selection in 85.6% of cases. Of these cases there were 27 instances in which an embryologist would have chosen a ‘low’ embryo over a ‘medium’ embryo at the blastocyst stage. This would amount to an opportunity to change selection at the cleavage stage for 42.0% (79/188) of the population. CONCLUSIONS: The revised binary predictive model generated by the EevaTM Test enhanced selection at the cleavage stage in comparison to the original commercially available designations. Further development of this technology to overcome the limitation imposed by a binary ranking system could be applied to establish a rank-order amongst a cohort of morphologically normal embryos. It also remains to be determined if this alteration in selection would provide a benefit (or even detriment) compared to traditional selection at the blastocyst stage. Supported by: Equipment supplied by Progyny (formerly Auxogyn).

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ASRM Abstracts

P-595 Wednesday, October 21, 2015 COMPUTER-AUTOMATED TIME-LAPSE MICROSCOPY (TLM) RESULTS ENHANCE THE OPPORTUNITY TO SELECT EMBRYOS THAT BLASTULATE WITHIN THE WINDOW OF ENDOMETRIAL RECEPTIVITY. M. D. Werner, J. M. Franasiak, C. R. Juneau, K. A. Green, K. H. Hong, K. M. Upham, R. T. Scott. RMA, NJ, NJ. OBJECTIVE: The EevaTM Test, a computer automated test enabled by TLM generates a binary result (high or low) that prognosticates which embryos on day 3 are more likely to reach the blastocyst stage of development. However, some embryos grow more slowly, reaching the blastocyst stage on day 6, rather than day 5. It has been shown that slowly blastulating embryos have better outcomes when cryopreserved and transferred during a cycle with synchronous endometrial development than when transferred in a fresh IVF cycle. This study seeks to characterize if this outcome predictor can refine selection by performing a paired analysis to identify which embryos blastulate on day 5 versus day 6. DESIGN: Prospective, observational. MATERIALS AND METHODS: Patients with normal ovarian reserve undergoing IVF were recruited. TLM utilizing the EevaTM System (Progyny, Menlo Park, CA) was performed through day 5 of development and the automated results were retrieved after cycle completion. Imaging data were not used for any clinical decisions. Standard morphologic grading was performed on day 5 and day 6 of development. The day on which embryos reached the usable blastocyst stage (appropriate for cryopreservation or transfer based on traditional morphologic grading) was recorded. The EevaTM Test result was compared to the day of usable blastocyst development. Additionally, a paired analysis was then performed between sibling zygotes to predict whether this would impact a patient when used clinically. The Wilcoxon rank sum test was used to compare the number of ‘high’ embryos that became usable blasts to the number of ‘low’ embryos becoming a usable blastocyst within a single patient’s cohort. RESULTS: 190 Patients were enrolled and 1932 fertilized zygotes were imaged and analyzed. 790 zygotes arrested in development prior to the blastocyst stage, 739 zygotes reached the blastocyst stage by day 5, and 403 zygotes did not blastulate until day 6. Embryos scored as ‘high’ were more likely to blastulate on day 5 in comparison to day 6 (60.2% vs. 36.2%, p<0.0001). In the per-patient analysis, 122 patients had a mix of embryos that blastulated on day 5 and day 6 and a paired analysis could be performed. Within this cohort, the Eeva Test could have enhanced selection for 61.5% of patients by identifying an embryo that was more likely to blastulate on day 5 of development and was therefore synchronous with endometrial development (p<0.0001). CONCLUSIONS: Automated TLM enhanced the opportunity for cleavage stage selection of an embryo that would become a usable blastocyst on day 5 of development, therefore decreasing the risk for failure due to embryo-endometrial dyssynchrony. It remains to be demonstrated if the accuracy provided by this assessment can equate to extended culture to the blastocyst stage. Supported by: Equipment supplied by Progyny (formerly Auxogyn). P-596 Wednesday, October 21, 2015 NON-INVASIVE PREDICTION OF EMBRYO DEVELOPMENTAL POTENTIAL BY EMBRYO CULTURE MEDIUM QUANTITATIVE SECRETOMIC: A PILOT STUDY. J. Camillo,a A. B. Victorino,a A. A. de Melo,a F. B. Cordeiro,a D. P. Braga,b E. Borges, Jr.,b E. G. Lo Turco.a aDepartment of Surgery, Division of Urology, Human Reproduction Section, Sao Paulo Federal University, Sao Paulo, Brazil; bFertility Medical Group, Sao Paulo, Brazil. OBJECTIVE: To quantify endogenous metabolites in spent embryo culture medium and correlate it with embryo developmental competence. DESIGN: Case-control study. MATERIALS AND METHODS: For this study, embryo culture media samples were harvested on the day three, from patients undergoing assisted reproduction techniques. Samples were split into groups based on the embryo developmental status: (i) poor quality embryos on cleavage stage (GroupPoor Quality, n¼110), (ii) high quality embryos on cleavage stage (GroupHigh Quality, n¼35), (iii) embryos achieving the blastocyst stage (GroupBlast, n¼35), and (iv) embryos that failed to achieve the blastocyst stage (Group-Non-Blast, n¼100). Samples were analysed by triple quadrupole mass spectrometer using the Absolute IDQÒ p180 Kit (BIOCRATES Life Sciences, Innsbruck, Austria). In order to accurate the analysis the groups Poor quality and Non-Blast were pooled together and the groups High Quality and Blast were pooled together. Data were analysed using the principal

Vol. 104, No. 3, Supplement, September 2015