- Email: [email protected]
Mechanical biomarkers of oocyte maturation
Clinical Pregnancy Rates
Day 3 ET-Elevated P P-Level Number of Patients Clinical Pregnancy Rate
P-hGCR2 n¼14 7%
P-AUCR4.5 n¼17 0%
objective was to compare the effect of P area under the curve (P–AUC) over multiple days to P–hCG. DESIGN: Retrospective cohort study. MATERIALS AND METHODS: All fresh autologous ART cycles from Aug 2013–Jan 2014 were included. Primary analyses performed with generalized estimating equations (GEE) with nesting for patients with multiple cycles. Univariate GEE assessed variables associated with P–levels and clinical pregnancy with a multivariate GEE model accounting for all significant confounders. The primary outcome was clinical pregnancy rate. RESULTS: 1448 consecutive patients undergoing 1620 fresh autologous ART cycles met inclusion criteria (84 cycles were excluded because of an elevated P–hCG with no embryo transfer). Serum P–hCG, P–levels for each of the 3 days prior to hCG trigger and P-AUC for 1, 2, and 3 days were all negatively associated with pregnancy. The 3 day P-AUC had the strongest negative correlation and persisted in multivariate analysis. In multivariate GEE controlling for age, embryo quality, and day of embryo transfer , 3 day P-AUC remained significantly associated with pregnancy [p ¼ 0.0003, OR 0.83 (0.75 – 0.92)]. Threshold analysis suggested cutoffs of R 2 ng/mL for P-hCG and R 4.5 ng/mL for 3 day P–AUC to predict clinical pregnancy failure. The PPV for pregnancy failure was 69% for elevated P–hCG versus 76% for 3 day P–AUC. Additional analyses demonstrated that 3 day P–AUC was negatively associated with clinical pregnancy rate regardless of day of transfer or embryo grade. CONCLUSION: 3 day P–AUC had a stronger negative association with clinical pregnancy than P–levels on day of hCG, suggesting that chronic elevations in progesterone may be a more useful clinical marker to predict pregnancy failure. Clinicians should consider measuring P when lead follicles are R14 mm in order to identify cycles at risk of pregnancy failure. Supported by: This work was supported, in part, by the Program in Reproductive and Adult Endocrinology (PRAE), Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), Bethesda, MD. O-249 Wednesday, October 22, 2014 11:45 AM OXIDATIVE STRESS BIOMARKERS IN FOLLICULAR FLUID OF WOMEN WITH PCOS AND TUBAL FACTOR INFERTILITY-IS THERE A CORREALTION WITH IN-VITRO-FERTILIZATION OUTCOME? N. Malhotra,a K. Gongadashetti,a R. Dada,b N. Singh.a a ART Center, Department of Obstetrics and Gynecology, All India Institute of Medical Sciences, New Delhi, Delhi, India; bLaboratory for Molecular and Genetics, Department of Anatomy, All India Institute of Medical Sciences, New Delhi, Delhi, India. OBJECTIVE: To compare oxidative stress (OS) biomarkers in follicular fluid of women with PCOS and tubal factor infertility undergoing in-vitrofertilization (IVF) and correlate them with assisted reproductive technique (ART) outcome including oocyte fertilization, embryo cleavage, and clinical pregnancy and miscarriage rate. DESIGN: Cross sectional study. MATERIALS AND METHODS: Follicular fluid collected during oocyte retrieval from 43 women with PCOS (group I) and 57 women with tubal factor infertility (group II) were assessed for OS biomarkers including Reactive oxygen species (ROS), Total anti-oxidant capacity (TAC), and 8-Isoprostane (8-IP) levels. ROS levels were detected by chemiluminesence, TAC and 8-IP by enzyme immunoassay methods. OS biomarkers were compared between both groups using Wilcoxon ranksum test and correlation with ART outcome with Spearman’s rank correlation coefficient. A p value of <0.05 was considered significant. RESULTS: Women in both groups were comparable in age, BMI and duration of infertility. There were no significant difference in the median levels of ROS [(71.7 cpm vs 54.8 cpm) (p¼0.570)] and TAC [(4.4 Mm/ul vs 4.07 Mm/ ul of trolox equivalent) (p¼0.12)] in the follicular fluid from women in group I when compared to group II. Levels of 8-IP were significantly higher in follicular fluid from women in group I as compared to group II [(57.1 pg/ul vs 39.2 pg/ ul) (p¼0.04)]. The levels of TAC were significantly higher in those women who
e86
ASRM Abstracts
Day 5 ET-Elevated P P-hGCR2 n¼37 37 %
P-AUCR4.5 n¼61 29 %
Day 5 ET-Normal P P-hGC<2 n¼1176 52 %
P-AUC<4.5 n¼1152 59 %
were pregnant compared to those without pregnancy in both groups. OS biomarkers did not correlate with ART outcome including fertilization, cleavage or pregnancy rate in both PCOS and tubal factor infertility. Ongoing clinical pregnancy were no different in both groups (25.4% vs 29.1%, p¼0.34). Follicular fluid Levels of 8-IP in group I were significantly higher in women who had miscarriage when compared to those with ongoing pregnancy (p¼0.04). There was a positive correlation (0.16, p¼0.01) of 8-IP with miscarriage rate. CONCLUSION: 8- IP an OS biomarker is elevated in follicular fluid from women with PCOS indicating the role of oxidative stress in PCOS. 8-IP levels correlate with miscarriage rate in women with PCOS undergoing IVF. O-250 Wednesday, October 22, 2014 12:00 PM MECHANICAL BIOMARKERS OF OOCYTE MATURATION. L. Zarnescu,a J. Han,b B. Behr,b R. Reijo Pera,c a a D. Camarillo. Department of Bioengineering, Stanford University, Stanford, CA; bDepartment of Obstetrics and Gynecology, Stanford University, Stanford, CA; cDepartment of Chemistry and Biochemistry, Montana State University, Bozeman, MT. OBJECTIVE: In the in vitro fertilization (IVF) clinic, several oocytes are collected from a patient and assessed for maturation. Oocytes which have visibly completed nuclear maturation (MII stage) and extruded a polar body are deemed mature and ready for fertilization. Those which lack a polar body (GV or MI stage) then undergo in vitro maturation (IVM) until they reach the MII stage and are ready for fertilization. Although nuclear maturation is easy to assess by simple observation, oocytes must also undergo cytoplasmic maturation before acquiring optimal developmental competence. Cytoplasmic maturation is still poorly understood and difficult to detect noninvasively. We have developed an approach to measure the mechanical parameters of oocytes, and our objective is to use these parameters to potentially predict cytoplasmic maturation, fertilizability, and viability. DESIGN: We measured the mechanical parameters of mouse oocytes at the GV, MI, and MII stages in order to characterize how these parameters change over the course of nuclear maturation and whether mechanical properties could provide more information about fertilization and viability than visual observation alone. MATERIALS AND METHODS: The mechanical parameters describing the viscous and elastic properties of oocytes were measured by observing their response to micropipette aspiration, and fitting the aspiration depth of each oocyte into the pipette to a 4-parameter bulk mechanical model. RESULTS: Oocytes exhibited large changes in mechanical properties between the GV, MI, and MII stages. We also found many oocytes which did not appear to have reached the MII stage but had mechanical properties similar to MII oocytes, and were capable of fertilization and blastocyst formation. This leads us to believe that mechanical properties of oocytes could provide more information about developmental competence when compared to visual assessment alone, and may be related to cytoplasmic maturation. CONCLUSION: We present a novel technique for demonstrating that the mechanical parameters of oocytes could be used to noninvasively assess both nuclear and cytoplasmic maturation. Supported by: Bio-X seed grant program at Stanford University. O-251 Wednesday, October 22, 2014 12:15 PM A PILOT STUDY EVALUATING PLOIDY PREDICTIVE MODELS VIA TIME LAPSE MICROSCOPY (TLM) MORPHOKINETIC PARAMETERS; EXPOSING UNLIKELY UNIVERSAL PREDICTIVE METHODS. Y. G. Kramer, J. D. Kofinas, K. Melzer, N. Noyes, C. McCaffrey, D. H. McCulloh, J. A. Grifo. NYU Fertility Center, NYU Langone School of Medicine, New York, NY. OBJECTIVE: To determine if Aneuploidy Risk Classification Model presented in the literature is predictive of embryo ploidy at the NYU Fertility Center (NYUFC). DESIGN: Retrospective Cohort Analysis.
Vol. 102, No. 3, Supplement, September 2014
Day 3 ET-Elevated P P-Level Number of Patients Clinical Pregnancy Rate
P-hGCR2 n¼14 7%
P-AUCR4.5 n¼17 0%
objective was to compare the effect of P area under the curve (P–AUC) over multiple days to P–hCG. DESIGN: Retrospective cohort study. MATERIALS AND METHODS: All fresh autologous ART cycles from Aug 2013–Jan 2014 were included. Primary analyses performed with generalized estimating equations (GEE) with nesting for patients with multiple cycles. Univariate GEE assessed variables associated with P–levels and clinical pregnancy with a multivariate GEE model accounting for all significant confounders. The primary outcome was clinical pregnancy rate. RESULTS: 1448 consecutive patients undergoing 1620 fresh autologous ART cycles met inclusion criteria (84 cycles were excluded because of an elevated P–hCG with no embryo transfer). Serum P–hCG, P–levels for each of the 3 days prior to hCG trigger and P-AUC for 1, 2, and 3 days were all negatively associated with pregnancy. The 3 day P-AUC had the strongest negative correlation and persisted in multivariate analysis. In multivariate GEE controlling for age, embryo quality, and day of embryo transfer , 3 day P-AUC remained significantly associated with pregnancy [p ¼ 0.0003, OR 0.83 (0.75 – 0.92)]. Threshold analysis suggested cutoffs of R 2 ng/mL for P-hCG and R 4.5 ng/mL for 3 day P–AUC to predict clinical pregnancy failure. The PPV for pregnancy failure was 69% for elevated P–hCG versus 76% for 3 day P–AUC. Additional analyses demonstrated that 3 day P–AUC was negatively associated with clinical pregnancy rate regardless of day of transfer or embryo grade. CONCLUSION: 3 day P–AUC had a stronger negative association with clinical pregnancy than P–levels on day of hCG, suggesting that chronic elevations in progesterone may be a more useful clinical marker to predict pregnancy failure. Clinicians should consider measuring P when lead follicles are R14 mm in order to identify cycles at risk of pregnancy failure. Supported by: This work was supported, in part, by the Program in Reproductive and Adult Endocrinology (PRAE), Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), Bethesda, MD. O-249 Wednesday, October 22, 2014 11:45 AM OXIDATIVE STRESS BIOMARKERS IN FOLLICULAR FLUID OF WOMEN WITH PCOS AND TUBAL FACTOR INFERTILITY-IS THERE A CORREALTION WITH IN-VITRO-FERTILIZATION OUTCOME? N. Malhotra,a K. Gongadashetti,a R. Dada,b N. Singh.a a ART Center, Department of Obstetrics and Gynecology, All India Institute of Medical Sciences, New Delhi, Delhi, India; bLaboratory for Molecular and Genetics, Department of Anatomy, All India Institute of Medical Sciences, New Delhi, Delhi, India. OBJECTIVE: To compare oxidative stress (OS) biomarkers in follicular fluid of women with PCOS and tubal factor infertility undergoing in-vitrofertilization (IVF) and correlate them with assisted reproductive technique (ART) outcome including oocyte fertilization, embryo cleavage, and clinical pregnancy and miscarriage rate. DESIGN: Cross sectional study. MATERIALS AND METHODS: Follicular fluid collected during oocyte retrieval from 43 women with PCOS (group I) and 57 women with tubal factor infertility (group II) were assessed for OS biomarkers including Reactive oxygen species (ROS), Total anti-oxidant capacity (TAC), and 8-Isoprostane (8-IP) levels. ROS levels were detected by chemiluminesence, TAC and 8-IP by enzyme immunoassay methods. OS biomarkers were compared between both groups using Wilcoxon ranksum test and correlation with ART outcome with Spearman’s rank correlation coefficient. A p value of <0.05 was considered significant. RESULTS: Women in both groups were comparable in age, BMI and duration of infertility. There were no significant difference in the median levels of ROS [(71.7 cpm vs 54.8 cpm) (p¼0.570)] and TAC [(4.4 Mm/ul vs 4.07 Mm/ ul of trolox equivalent) (p¼0.12)] in the follicular fluid from women in group I when compared to group II. Levels of 8-IP were significantly higher in follicular fluid from women in group I as compared to group II [(57.1 pg/ul vs 39.2 pg/ ul) (p¼0.04)]. The levels of TAC were significantly higher in those women who
e86
ASRM Abstracts
Day 5 ET-Elevated P P-hGCR2 n¼37 37 %
P-AUCR4.5 n¼61 29 %
Day 5 ET-Normal P P-hGC<2 n¼1176 52 %
P-AUC<4.5 n¼1152 59 %
were pregnant compared to those without pregnancy in both groups. OS biomarkers did not correlate with ART outcome including fertilization, cleavage or pregnancy rate in both PCOS and tubal factor infertility. Ongoing clinical pregnancy were no different in both groups (25.4% vs 29.1%, p¼0.34). Follicular fluid Levels of 8-IP in group I were significantly higher in women who had miscarriage when compared to those with ongoing pregnancy (p¼0.04). There was a positive correlation (0.16, p¼0.01) of 8-IP with miscarriage rate. CONCLUSION: 8- IP an OS biomarker is elevated in follicular fluid from women with PCOS indicating the role of oxidative stress in PCOS. 8-IP levels correlate with miscarriage rate in women with PCOS undergoing IVF. O-250 Wednesday, October 22, 2014 12:00 PM MECHANICAL BIOMARKERS OF OOCYTE MATURATION. L. Zarnescu,a J. Han,b B. Behr,b R. Reijo Pera,c a a D. Camarillo. Department of Bioengineering, Stanford University, Stanford, CA; bDepartment of Obstetrics and Gynecology, Stanford University, Stanford, CA; cDepartment of Chemistry and Biochemistry, Montana State University, Bozeman, MT. OBJECTIVE: In the in vitro fertilization (IVF) clinic, several oocytes are collected from a patient and assessed for maturation. Oocytes which have visibly completed nuclear maturation (MII stage) and extruded a polar body are deemed mature and ready for fertilization. Those which lack a polar body (GV or MI stage) then undergo in vitro maturation (IVM) until they reach the MII stage and are ready for fertilization. Although nuclear maturation is easy to assess by simple observation, oocytes must also undergo cytoplasmic maturation before acquiring optimal developmental competence. Cytoplasmic maturation is still poorly understood and difficult to detect noninvasively. We have developed an approach to measure the mechanical parameters of oocytes, and our objective is to use these parameters to potentially predict cytoplasmic maturation, fertilizability, and viability. DESIGN: We measured the mechanical parameters of mouse oocytes at the GV, MI, and MII stages in order to characterize how these parameters change over the course of nuclear maturation and whether mechanical properties could provide more information about fertilization and viability than visual observation alone. MATERIALS AND METHODS: The mechanical parameters describing the viscous and elastic properties of oocytes were measured by observing their response to micropipette aspiration, and fitting the aspiration depth of each oocyte into the pipette to a 4-parameter bulk mechanical model. RESULTS: Oocytes exhibited large changes in mechanical properties between the GV, MI, and MII stages. We also found many oocytes which did not appear to have reached the MII stage but had mechanical properties similar to MII oocytes, and were capable of fertilization and blastocyst formation. This leads us to believe that mechanical properties of oocytes could provide more information about developmental competence when compared to visual assessment alone, and may be related to cytoplasmic maturation. CONCLUSION: We present a novel technique for demonstrating that the mechanical parameters of oocytes could be used to noninvasively assess both nuclear and cytoplasmic maturation. Supported by: Bio-X seed grant program at Stanford University. O-251 Wednesday, October 22, 2014 12:15 PM A PILOT STUDY EVALUATING PLOIDY PREDICTIVE MODELS VIA TIME LAPSE MICROSCOPY (TLM) MORPHOKINETIC PARAMETERS; EXPOSING UNLIKELY UNIVERSAL PREDICTIVE METHODS. Y. G. Kramer, J. D. Kofinas, K. Melzer, N. Noyes, C. McCaffrey, D. H. McCulloh, J. A. Grifo. NYU Fertility Center, NYU Langone School of Medicine, New York, NY. OBJECTIVE: To determine if Aneuploidy Risk Classification Model presented in the literature is predictive of embryo ploidy at the NYU Fertility Center (NYUFC). DESIGN: Retrospective Cohort Analysis.
Vol. 102, No. 3, Supplement, September 2014