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Day 5 blastocyst morphology correlates with comprehensive chromosome constitution and outcome
Score’’. In this study, we used Receiver Operator Curve (ROC) analysis to compare (i) morphologic grading (ii) the Viability Score and (iii) a combination of morphology and the Viability Score as predictors of implantation potential of Day 5 embryos. DESIGN: Retrospective study. MATERIALS AND METHODS: Embryo culture media samples (n¼198) were collected in 4 IVF centers following Day 5 single embryo transfer (SET), performed using morphologic criteria. Samples were analyzed in two separate sets, using NIR spectroscopy-based metabolomic profiling (Molecular Biometrics, Inc.). First, the samples in the ‘‘Training Set’’ (n¼137) were used to develop and validate the algorithm that generates a Viability Score for each sample. Next, this algorithm was used to analyze the samples in the ‘‘Test Set’’ (n¼ 61) by observers blinded to embryo morphology and IVF outcome. Pregnancy was defined as fetal cardiac activity (FCA) at 12 weeks of gestation. ROC curves were generated and areas under the ROC curve (AUC) were estimated to assess the ability of the Viability Score, morphology, and Composite Score (obtained using a model combining Viability Score with morphologic grading) to predict pregnancy outcome. RESULTS: The AUC was greater for the metabolomic Viability Score compared to morphology [Training set: 0.75 versus 0.55, p ¼ 0.0011; Validation set: 0.68 versus 0.50, P ¼ 0.021], and for the Composite Score compared to morphology alone [0.74 versus 0.50, p ¼ 0.004]. CONCLUSION: Our findings suggest that Viability Score alone or in combination with morphologic grading has the potential to be a better classifier for pregnancy outcome than morphology alone in women undergoing SET on day 5. Supported by: Molecular Biometrics, Inc., Norwood, MA.
O-89 Monday, October 25, 2010 05:30 PM SPERM MOTILITY IS NOT COMPROMISED IF COLLECTED IN MEDIA AND DELIVERED TO THE LABORATORY WITHIN 6 HOURS. R. Pyrzak, R. P. Dickey. The Fertility Institute of New Orleans, Mandeville, LA; Obstetrics and Gynecology, Louisiana State University, New Orleans, LA. OBJECTIVE: To evaluate if diluting semen specimens with media (DM) can extend sperm longevity. DESIGN: Prospective study. MATERIALS AND METHODS: 18 semen samples were evaluated. After collection, each specimen was divided into 3 groups: group 1 (G1), undiluted fresh semen; group 2 (G2), diluted 1:1 and group 3 (G3), diluted 1:20 with DM. In each group, specimens were incubated for 48 hours in the dark at room temperature. An aliquot was taken for evaluation of sperm motility (SM) and progression grade (PG) (range 1-4) after 2, 4, 6, 24 and 48 hours. SM was observed under a microscope slide. Initial SM was used as a base line to calculate the relative change (RSM) expressed as % using correlated ANOVA (VassarStats). RESULTS: Initial sperm count was 101+67x106mL (range 15-269x106), initial SM 62+15 (range 29-81) and PG 3.1+0.4 (range 2-4). There was no significant change in PG among the groups, except in G1 after 24 and 48 hours of incubation. During incubation, the greatest drop in RSM was in G1 and the least in G3. RSM was decreased significantly in G1 and G2 after 6 hours. In G3, there was no significant drop in RSM in the first 6 hours. RSM was decreased significantly in G3 after 24 hours. The decrease in RSM in G3 between 24 and 48 hours was not significant. RSM in G3 at 48 hours was comparable to RSM in G1 after 6 hours. TABLE 1. Change in RSM after using DM and incubation during 48 hr
Incubation (hr) 2 4 6 24 48 a,b,c,d
Group 1
Group 2
Group 3
94 14a 88 18a,b 70 21b 38 26c 16 19d
98 6a 93 11a,b 85 17b 61 24c 42 22d
99 6a 96 6a 92 10a 79 21b 68 21b
superscript indicate significant difference (p<0.01) within column.
CONCLUSION: Diluted semen specimens can be diluted and delivered to the laboratory within six hours after semen collection without compromising sperm motility.
FERTILITY & STERILITYÒ
O-90 Monday, October 25, 2010 05:45 PM UNCOMPLICATED SINGLETON PREGNANCIES CONCEIVED SPONTANEOUSLY AND THROUGH ASSISTED REPRODUCTION TECHNOLOGIES (ART) HAVE COMPARABLE FIRST TRIMESTER SCREENING PROFILES FOR DOWN SYNDROME. O. Oktem, T. Barut, E. Palaoglu, B. Urman. Women’s Health Center Assisted Reproduction Unit, American Hospital, Istanbul, Turkey; Clinical Laboratory, American Hospital, Istanbul, Turkey. OBJECTIVE: First trimester screening for Down syndrome combines measurement of nuchal translucency (NT), free b-hCG and PAPP-A. We aimed to compare these parameters in the first trimester screening of uncomplicated singleton spontaneous and ART pregnancies. DESIGN: Retrospective case series. MATERIALS AND METHODS: Uncomplicated 972 spontaneous and 71 ART pregnancies conceived during the same period were included in the study. Gestational age was determined using the crown-rump length (CRL). Multiple of Median (MOM) values of NT, free b-hCG and PAPP-A measurements were compared between the groups. All ART pregnancies occurred after intracytoplasmic sperm injection (ICSI) and fresh embryo transfers. All pregnancies followed an uneventful course. RESULTS: The mean age and gestational week were comparable between ART and non-ART pregnancies (33.3 3 vs. 31.5 3 p>0.05, respectively and 12.4 0.6 vs. 12.2 0.7 p>0.05, respectively). Free b-hCG, PAPP-A and NT of ART pregnancies did not differ significantly from non-ART pregnancies (1.3 0.8 vs. 1.2 0.8 p>0.05, respectively; 1.3 0.9 vs. 1.3 0.8 p>0.05, respectively; 1.07 0.2 vs. 1.06 0.4 p>0.05, respectively). Free b-hCG was positively correlated with PAPP-A (r¼0.17 p<0.01), and gestational age was positively correlated with free b-hCG (r¼0.09 p<0.01) and negatively correlated with PAPP-A (r¼-0.14 p<0.01) in non-ART pregnancies. However, such correlations were not found in the ART group. CONCLUSION: These results has shown that singleton uncomplicated ART pregnancies have similar first trimester screening profiles to non-ART pregnancies when age and gestational weeks are comparable. Lack of correlations among gestational age, free b-hCG and PAPP-A levels in ART pregnancies in contrast to spontaneous ones may suggest the effect of the drugs used in ART on these markers. O-91 Monday, October 25, 2010 06:00 PM DAY 5 BLASTOCYST MORPHOLOGY CORRELATES WITH COMPREHENSIVE CHROMOSOME CONSTITUTION AND OUTCOME. S. McCormick, J. Stevens, M. Rawlins, R. Smith, W. B. Schoolcraft, M. G. Katz-Jaffe. Colorado Center for Reproductive Medicine, Lone Tree, CO; Fertility Laboratories of Colorado, Lone Tree, CO. OBJECTIVE: Currently, there is no reliable morphological marker for chromosome aneuploidies during preimplantation embryo development that could be used as selection criteria for transfer. The aim of this study was to investigate D5 blastocyst morphology in association with comprehensive chromosome constitution of all 23 pairs of chromosomes. DESIGN: Retrospective study. MATERIALS AND METHODS: A total of 1,492 blastocysts (RGrade 3BB) were biopsied and vitrified on either D5 or D6 of embryo development. D6 biopsied blastocysts were considered morphologically slow in development on D5 (morula, Grade 1 or Grade 2). Biopsied TE cells were analyzed for comprehensive chromosome screening (CCS) by mCGH (Reprogenetics) or SNP microarray (RMA-NJ). Analysis was performed based on D5 morphology regardless of the day of biopsy. RESULTS: In relation to D5 morphology, similarities in aneuploidy were observed for morula (55%), Grade 1 (56%), Grade 2 (59%) and Grade 3 (57%) blastocysts. In contrast, a significant decrease in aneuploidy was observed for Grade 4 (48%) (P<0.05) and Grade 5 (33%) (P<0.01) D5 blastocysts relative to earlier stages. A significant decrease in aneuploidy was also observed when more TE cells formed a cohesive epithelium compared to fewer TE cells (P<0.05). No significant differences were observed with inner cell mass grading. Ongoing clinical pregnancy rate following a frozen blastocyst transfer with euploid D5 biopsied blastocysts (63/79¼79.8%) was significantly higher than with euploid D6 biopsied blastocysts (35/62¼56.5%) (P<0.05). The CCS technique employed had no bearing on results when analyzed separately. CONCLUSION: Evaluation of D5 blastocyst morphology in relation to comprehensive chromosome constitution has revealed expanded and hatching D5 blastocysts to more likely be euploid. Nevertheless, this morphology indicator was not absolute. In addition, euploid D6 biopsied blastocysts exhibited poorer outcome following transfer, indicating multiple variables influence embryo viability and implantation.
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O-89 Monday, October 25, 2010 05:30 PM SPERM MOTILITY IS NOT COMPROMISED IF COLLECTED IN MEDIA AND DELIVERED TO THE LABORATORY WITHIN 6 HOURS. R. Pyrzak, R. P. Dickey. The Fertility Institute of New Orleans, Mandeville, LA; Obstetrics and Gynecology, Louisiana State University, New Orleans, LA. OBJECTIVE: To evaluate if diluting semen specimens with media (DM) can extend sperm longevity. DESIGN: Prospective study. MATERIALS AND METHODS: 18 semen samples were evaluated. After collection, each specimen was divided into 3 groups: group 1 (G1), undiluted fresh semen; group 2 (G2), diluted 1:1 and group 3 (G3), diluted 1:20 with DM. In each group, specimens were incubated for 48 hours in the dark at room temperature. An aliquot was taken for evaluation of sperm motility (SM) and progression grade (PG) (range 1-4) after 2, 4, 6, 24 and 48 hours. SM was observed under a microscope slide. Initial SM was used as a base line to calculate the relative change (RSM) expressed as % using correlated ANOVA (VassarStats). RESULTS: Initial sperm count was 101+67x106mL (range 15-269x106), initial SM 62+15 (range 29-81) and PG 3.1+0.4 (range 2-4). There was no significant change in PG among the groups, except in G1 after 24 and 48 hours of incubation. During incubation, the greatest drop in RSM was in G1 and the least in G3. RSM was decreased significantly in G1 and G2 after 6 hours. In G3, there was no significant drop in RSM in the first 6 hours. RSM was decreased significantly in G3 after 24 hours. The decrease in RSM in G3 between 24 and 48 hours was not significant. RSM in G3 at 48 hours was comparable to RSM in G1 after 6 hours. TABLE 1. Change in RSM after using DM and incubation during 48 hr
Incubation (hr) 2 4 6 24 48 a,b,c,d
Group 1
Group 2
Group 3
94 14a 88 18a,b 70 21b 38 26c 16 19d
98 6a 93 11a,b 85 17b 61 24c 42 22d
99 6a 96 6a 92 10a 79 21b 68 21b
superscript indicate significant difference (p<0.01) within column.
CONCLUSION: Diluted semen specimens can be diluted and delivered to the laboratory within six hours after semen collection without compromising sperm motility.
FERTILITY & STERILITYÒ
O-90 Monday, October 25, 2010 05:45 PM UNCOMPLICATED SINGLETON PREGNANCIES CONCEIVED SPONTANEOUSLY AND THROUGH ASSISTED REPRODUCTION TECHNOLOGIES (ART) HAVE COMPARABLE FIRST TRIMESTER SCREENING PROFILES FOR DOWN SYNDROME. O. Oktem, T. Barut, E. Palaoglu, B. Urman. Women’s Health Center Assisted Reproduction Unit, American Hospital, Istanbul, Turkey; Clinical Laboratory, American Hospital, Istanbul, Turkey. OBJECTIVE: First trimester screening for Down syndrome combines measurement of nuchal translucency (NT), free b-hCG and PAPP-A. We aimed to compare these parameters in the first trimester screening of uncomplicated singleton spontaneous and ART pregnancies. DESIGN: Retrospective case series. MATERIALS AND METHODS: Uncomplicated 972 spontaneous and 71 ART pregnancies conceived during the same period were included in the study. Gestational age was determined using the crown-rump length (CRL). Multiple of Median (MOM) values of NT, free b-hCG and PAPP-A measurements were compared between the groups. All ART pregnancies occurred after intracytoplasmic sperm injection (ICSI) and fresh embryo transfers. All pregnancies followed an uneventful course. RESULTS: The mean age and gestational week were comparable between ART and non-ART pregnancies (33.3 3 vs. 31.5 3 p>0.05, respectively and 12.4 0.6 vs. 12.2 0.7 p>0.05, respectively). Free b-hCG, PAPP-A and NT of ART pregnancies did not differ significantly from non-ART pregnancies (1.3 0.8 vs. 1.2 0.8 p>0.05, respectively; 1.3 0.9 vs. 1.3 0.8 p>0.05, respectively; 1.07 0.2 vs. 1.06 0.4 p>0.05, respectively). Free b-hCG was positively correlated with PAPP-A (r¼0.17 p<0.01), and gestational age was positively correlated with free b-hCG (r¼0.09 p<0.01) and negatively correlated with PAPP-A (r¼-0.14 p<0.01) in non-ART pregnancies. However, such correlations were not found in the ART group. CONCLUSION: These results has shown that singleton uncomplicated ART pregnancies have similar first trimester screening profiles to non-ART pregnancies when age and gestational weeks are comparable. Lack of correlations among gestational age, free b-hCG and PAPP-A levels in ART pregnancies in contrast to spontaneous ones may suggest the effect of the drugs used in ART on these markers. O-91 Monday, October 25, 2010 06:00 PM DAY 5 BLASTOCYST MORPHOLOGY CORRELATES WITH COMPREHENSIVE CHROMOSOME CONSTITUTION AND OUTCOME. S. McCormick, J. Stevens, M. Rawlins, R. Smith, W. B. Schoolcraft, M. G. Katz-Jaffe. Colorado Center for Reproductive Medicine, Lone Tree, CO; Fertility Laboratories of Colorado, Lone Tree, CO. OBJECTIVE: Currently, there is no reliable morphological marker for chromosome aneuploidies during preimplantation embryo development that could be used as selection criteria for transfer. The aim of this study was to investigate D5 blastocyst morphology in association with comprehensive chromosome constitution of all 23 pairs of chromosomes. DESIGN: Retrospective study. MATERIALS AND METHODS: A total of 1,492 blastocysts (RGrade 3BB) were biopsied and vitrified on either D5 or D6 of embryo development. D6 biopsied blastocysts were considered morphologically slow in development on D5 (morula, Grade 1 or Grade 2). Biopsied TE cells were analyzed for comprehensive chromosome screening (CCS) by mCGH (Reprogenetics) or SNP microarray (RMA-NJ). Analysis was performed based on D5 morphology regardless of the day of biopsy. RESULTS: In relation to D5 morphology, similarities in aneuploidy were observed for morula (55%), Grade 1 (56%), Grade 2 (59%) and Grade 3 (57%) blastocysts. In contrast, a significant decrease in aneuploidy was observed for Grade 4 (48%) (P<0.05) and Grade 5 (33%) (P<0.01) D5 blastocysts relative to earlier stages. A significant decrease in aneuploidy was also observed when more TE cells formed a cohesive epithelium compared to fewer TE cells (P<0.05). No significant differences were observed with inner cell mass grading. Ongoing clinical pregnancy rate following a frozen blastocyst transfer with euploid D5 biopsied blastocysts (63/79¼79.8%) was significantly higher than with euploid D6 biopsied blastocysts (35/62¼56.5%) (P<0.05). The CCS technique employed had no bearing on results when analyzed separately. CONCLUSION: Evaluation of D5 blastocyst morphology in relation to comprehensive chromosome constitution has revealed expanded and hatching D5 blastocysts to more likely be euploid. Nevertheless, this morphology indicator was not absolute. In addition, euploid D6 biopsied blastocysts exhibited poorer outcome following transfer, indicating multiple variables influence embryo viability and implantation.
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