- Email: [email protected]
A new ultra-rapid warming device for vitrified oocytes and embryos
Droplet Size 50ml 200ml P-value
Cases
# Thawed
% Recovered
% Initial Survival
% 3hr Survival
% 2PN
% Cleaved
% Usable Blastocysts
13 14
162 175
98.2 97.7 >0.05
74.2 91.8 <0.0001
71.7 87.7 <0.001
63.2 72 >0.05
80.7 74.7 >0.05
38 32.1 >0.05
Center, Beverly Hills, CA; bSouthern California Reproductive Center, Beverly Hills, CA. OBJECTIVE: Currently, there are a multitude of oocyte warming protocols ranging from large volumes to micro-droplets being used in different laboratories. There are also different procedural steps ranging from use of 3 to 5 droplets with variable timing. The objective of this study was to assess whether laboratory outcomes are affected based on volume of warming media at the time of oocyte thaw. We directly compared the use of 50ml droplets to use of 200ml droplets during our standard 5 step dilution oocyte warming protocol. DESIGN: Retrospective study comparing laboratory outcomes using two oocyte warming micro-droplet volumes. MATERIALS AND METHODS: Oocyte warming consists of a five step protocol using Irvine Scientific Vitrification Thaw media (90137, CA). We compared the laboratory outcomes of 13 cases with use of 50ml droplets to 14 cases with use of 200ml droplets. For both study arms, all oocytes were originally frozen using vitrification. The initial thawing solution used was a 2ml volume droplet at 37 C and the timing for warming was exactly the same in both groups. Micro-drop warming was performed through 5 room-temperature micro-drop dilutions of TS 1min; TS:DS 2min; DS 2min; DS:WS 2min and WS for 5min. Oocyte recovery, initial survival, 3hr survival, 2PN fertilization, cleavage and usable blastocyst rates were determined. Analysis was performed by Chi-square. RESULTS: CONCLUSIONS: In the recent years there has been nearly a 5 fold increase in rate of elective fertility preservation for women. Success rates both in blastocyst formation in the laboratories as well as pregnancy outcomes has improved with the use of vitrification techniques for oocyte cryopreservation and warming. In this study we assessed whether the oocyte warming thaw protocol could affect laboratory outcomes. We demonstrated that initial survival (<0.0001) and survival after 3 hours (<0.001) were significantly improved with the 200ml droplet size. However, the rate of embryo development of the surviving oocytes in either study arm were not different for day 3 cleavage or overall usable blastocysts. These findings suggest that if an oocyte survives the warming and initial 3hr culture, they have the ability to develop equally. Size does matter, oocytes favor a larger droplet during the 5 step warming process. The 200ml study group provided the oocytes an optimal environment to recover and develop into normal embryos. P-181 Tuesday, October 31, 2017 COMPARABLE REPRODUCTIVE OUTCOMES IN OPEN VERSUS CLOSED OOCYTE VITRIFICATION SYSTEMS: A PROSPECTIVE, PAIRED STUDY ON THE SAME GENETIC BACKGROUND AND STIMULATION PROTOCOL. A. Pujol,a M. Zamora,b A. Obradors,c D. Garcıa,b A. Rodrıguez,b R. Vassena.b aCentro de Infertilidad y Reproduccion Humana (CIRH), Barcelona, Spain; bClınica EUGIN, Barcelona, Spain; cFIV Obradors, Girona, Spain. OBJECTIVE: To compare in the same genetic background and stimulation regimen the laboratory and reproductive outcomes of the oocyte vitrification/ warming open system CryotopÒ (Kitazato) to the closed system Rapid-iÒ (Vitrolife). DESIGN: Prospective paired study carried out between July 2014 and November 2016. The study was approved by the local IRB and included a cohort of 83 oocyte donors providing a minimum of 12 mature oocytes (MII) at retrieval. In each case, 6 MII were vitrified using CryotopÒ and assigned to one recipient and other 6 MII were vitrified using Rapid-iÒ and assigned to different recipient. MATERIALS AND METHODS: ICSI was performed in all the cases, and embryos were transferred at D+2 or D+3. Oocyte survival rates, fertilization rate and embryo morphology, as well as reproductive outcomes (biochemical, clinical, ongoing pregnancy and live birth rates) between the two systems were analyzed by t-tests, Mann-Whitney test, and Chi2 tests as appropriate.
FERTILITY & STERILITYÒ
RESULTS: Recipient characteristics were similar in both groups with regard to age (41.24.7) and BMI (23.84.0). Oocytes vitrified with Rapid-i had higher survival rates (93.7% vs 86.2 %, p<0.001). The fertilization rate was lower using Rapid-i (58.5% vs 69.2% p<0.001). The number of ICSI with total fertilization failure was 9 (7.2%) for Rapid-i and none for Cryotop (p¼0.035). The percentage of cycles with frozen embryos was higher in Rapid-i (50.60% vs 45.26%, p<0.001). On average, the number of embryos transferred in the two groups was 1.80.4. Biochemical (50.7% vs 41.4%), clinical (45.1% vs 39.1%) and ongoing (45.7% vs 33.3%) pregnancy rates were not different (p>0.05) and were live birth rates (38.2% vs 32.3%, p>0.05). CONCLUSIONS: The participants of this prospective study are oocyte donors, young women in good reproductive health; care should be exerted in translating our results to other populations. Nevertheless, our results suggest that both closed and open oocyte vitrification systems offer comparable laboratory and reproductive outcomes up to live birth. P-182 Tuesday, October 31, 2017 A NEW ULTRA-RAPID WARMING DEVICE FOR VITRIFIED OOCYTES AND EMBRYOS. P. Patrizio,a Y. Natan,b P. E. Levi-Setti,c M. Leong,d A. Arav.e aObstetrics, Gynecology & Reproductive Sciences, Yale Fertility Center & Fertility Preservation, New Haven, CT; bFertilSafe Ltd., Ness Ziona, Israel; c Dept.Gynecology, Division of Gynecology and Reproductive Medicine, Humanitas Fertility Center, Humanitas Research Hospital, Rozzano (Milan), Italy; dThe Womens Clinic, Hong Kong, Hong Kong; eCEO Fertilesafe, Ness Ziona, Israel. OBJECTIVE: Variability in warming rates impacts on the success of vitrification procedures. To assure consistency in warming rates and high reproducibility of results, we report a new device (Helia, FertileSafe, Israel) capable to provide ultra-rapid warming of oocytes and embryos previously vitrified on two available commercial cryocarriers. DESIGN: Basic research experiments evaluating warming rates, survival, hatching rates. MATERIALS AND METHODS: Mice oocytes (n¼96) and blastocysts (n¼40) were vitrified using either an open (Cryotop, Kitazato, Japan) or a closed carrier (HSV,CBS- IMV France). Equilibration was done gradually according to established protocols with up to 5 oocytes or embryos per batch (20 total cryocarriers used for oocytes and 8 for embryos), transferred into vitrification solution (Origio, Denmark) for one minute before placing them on Cryotop or HSV and plunged in LN. Warming was done using the Helia device consisting of a chamber with LN to hold the cryocarriers next to the 5 ml warming solution (Origio) at 37 C. Once removed from the LN the cryocarriers were placed into 0.3ml high security modified sperm straws (CBS, France), kept in the warmed solution, containing a mesh at its distal end. The 0.3ml CBSstraws containing the cryocarriers were then immediately transferred into a second warming solution at RT for 2.5min, followed by transfer into the third holding medium (Origio). At the end of these passages, the distal mesh was cut-removed and oocytes and embryos were transferred into a culture dish for evaluation and culture. Warming rates of each cryocarrier were measured with a thermocouple of 50mm connected to the Cryotop or HSV and to a data logger (PICO, UK). To reproduce routine manual warming procedures two likely distances between LN and the 37 C warming solution (250mm and 500mm) were chosen. These warming rates were compared to those measured in Helia, where the distance between LN and the 37 C warming solution is 10 mm. RESULTS: Warming rates between -196 C and 37 C at a distance of 250 mm were above 31,000C /min. while at 500 mm dropped to 17,000 C /min. In the Helia device, the close proximity of LN to the 37 C solution resulted in warming rates of more than 300,000 C/min. for each experiment (n¼28). The survival of vitrified mice oocytes (Cryotop or HSV) and blastocysts (Cryotop) rewarmed in Helia were 66/68 (97%), 27/28 (96%) and 40/40 (100%) respectively, with 100% hatching.
e177
CONCLUSIONS: We developed a simple, successful, and highly reproducible method for rapid warming of oocytes and embryos vitrified with various cryocarriers. In addition, warming with Helia eliminates the need of pipetting samples between various solutions. Supported by: FertileSafe Ltd. P-183 Tuesday, October 31, 2017 HUMAN SEMEN PARAMETERS THAT PREDICT SPERM DNA INTEGRITY AFTER CRYOPRESERVATION. G. D. Smith,a W. R. Parker,b a c a da L. Keller, Y. Li, A. Brady, S. Leibo. Ob/Gyn, University of Michigan, Ann Arbor, MI; bUrology, Poughkeepsie, NY; cBiostatistics, University of Michigan, Ann Arbor, MI; dUniverisity of New Orleans, New Orleans, LA. OBJECTIVE: Human semen cryopreservation is important for: i) therapeutic donor insemination, ii) some cases of IVF, and iii) fertility preservation for those at risk of losing fertility. Not only is sperm cryo-survival important, but DNA integrity in motile cryopreserved sperm is essential for optimal outcomes. Objectives of this study were to prospectively investigate pre-cryopreservation semen analysis parameters in relation to post-cryopreservation motile sperm DNA integrity. DESIGN: Prospective clinical laboratory study. MATERIALS AND METHODS: De-identified semen samples (n¼49) underwent semen analysis, motile sperm DNA integrity assessment pre-cryopreservation, cryopreservation, thawing, post-thaw assessment for cryo-survival, and post-thaw evaluation of motile sperm DNA integrity in this IRBapproved study. Pre-freezing semen analysis values assessed by WHO 4th edition were: i) semen volume, ii) semen pH, iii) semen viscosity, iv) sperm total motility (%), v) sperm progressive forward motility, vi) sperm concentration by hemocytometer, and vii) total motile sperm per ejaculate (TMS; concentration x volume x total motility). DNA integrity was performed on motile sperm with the Halo assay. Semen samples were cryopreserved in TEST-yolk buffer with Glycerol and Gentamicin (Irvine Scientific; Irvine, CA) per manufactures protocol. The dependent outcome variable was postthaw motile sperm percent recovery of intact DNA (PTMS-intact DNA; post-thaw percent motile sperm with intact DNA / pre-freezing percent motile sperm with intact DNA x 100). Analysis was performed with SPSS statistics software and a univariate linear regression model. RESULTS: Semen pH (7.5 0.03), semen viscosity (1.2 0.1; arbitrary units), sperm total motility (53 0.7 %), and sperm progressive motility (77 0.4 % of total motile) had no significant predictive value for PTMS-intact DNA (89 1.2 %). However, pre-cryopreservation semen volume (3.8 0.2 mls; p<0.01) and TMS (220 20 x 106; p<0.02) had significant predictive values for PTMS-intact DNA. Pre-cryopreservation semen volume and TMS had an inverse relation to PTMS-intact DNA, whereby per unit increase in volume (0.1ml) or TMS (100 x 106) each independently yielded a reduction of 2% in PTMS-intact DNA. Of note, post-thaw cryo-survival had no predictive value in relation to PTMS-intact DNA. CONCLUSIONS: Semen volume and TMS were prospective PTMS-intact DNA predictive values in the pre-cryopreservation semen analysis. Cryo-survival was not predictive of PTMS-intact DNA. Evaluation of these parameters can assist fertility healthcare providers in counseling men and couples in semen cryopreservation and usage of samples post-thaw. P-184 Tuesday, October 31, 2017 THE DURATION OF CRYOSTORAGE OF BIOPSIED EMBRYOS NEITHER IMPACTS IMPLANTATION POTENTIAL NOR SURROGATE MARKERS OF PLACENTATION. L. Sekhon,a J. A. Lee,b M. Duke,b C. Briton-Jones,b E. Flisser,b A. B. Copperman.c aReproductive Medicine Associates New York, New York, NY; bReproductive Medicine Associates of New York, New York, NY; cObstetrics and Gynecology, RMANY-Mount Sinai, New York, NY. OBJECTIVE: Blastocyst vitrification facilitates embryo banking for successive single embryo transfers (SET) and has become an integral part of IVF treatment. Although storage at -196oC is thought to halt metabolism, the molecular structure of vitrified cells might be dynamic and sensitive to storage temperature variations and impacted by long-term cryostorage. Previous studies of the effect of cryostorage duration on clinical outcome focused on slow-frozen zygote or cleavage stage embryos; and no study to date has assessed whether blastocysts which have undergone trophectoderm biopsy are vulnerable to impaired implantation or
e178
ASRM Abstracts
placentation as a result of long-term cryostorage. This study examined whether the duration of cryostorage of euploid blastocysts adversely impacts reproductive potential or perinatal outcome. DESIGN: Retrospective, observational study. MATERIALS AND METHODS: The study included patients that underwent autologous IVF and aneuploidy screening of blastocysts by preimplantation genetic testing (PGT), followed by vitrification, warming and SET, from 2011 to 2016. Donor oocyte cycles were excluded. Cryostorage duration was calculated as the interval (days) elapsed from when the blastocyst was vitrified to when it was warmed and transferred. Multivariate logistic and linear regression analyses were performed. Main outcomes included implantation, clinical pregnancy, live birth and early pregnancy loss. Secondary outcomes included infant birthweight and gestational age at delivery. RESULTS: Vitrified-warmed, euploid blastocysts, derived from autologous IVF, were transferred in 2,320 SET cycles. The average cryostorage duration was 4 months, with a maximum duration of up to 4.9 years (mean 126.4 197.3 days; range 21-1794 days). Controlling for patient age at IVF and embryo transfer, BMI, endometrial thickness and embryo biopsy day, the duration of cryostorage did not impact the odds of implantation (OR 1.0 [95% CI 0.99-1.01]), clinical pregnancy (OR 1.0 [95% CI 0.9981.004]), live birth (OR 0.998 [95% CI 0.99-1.003]), biochemical pregnancy loss (OR 0.998 [95% CI 0.99-1.002]), and clinical pregnancy loss (OR 1.01 [95% CI 0.98-1.05]). The duration of cryostorage did not impact birthweight (b¼ -0.01, p¼0.96) or gestational age at delivery (b¼ -0.22, p¼0.51). CONCLUSIONS: Modern advances in cryopreservation and PGT have facilitated the opportunity for patients to bank embryos with confidence. This emerging treatment strategy will result in the increased storage of biopsied embryos for longer durations. This study is the first to demonstrate that long-term cryostorage of vitrified, biopsied blastocysts does not adversely affect reproductive potential, fetal growth or the likelihood of preterm delivery. Based on these findings, patients can be reassured regarding the efficacy, efficiency and safety of embryo banking. FERTILITY PRESERVATION P-185 Tuesday, October 31, 2017 BLASTOCYST COLLAPSE AND DOWNGRADING OF INNER CELL MASS MORPHOLOGY SCORE AFTER VITRIFICATION-WARMING IS PREDICTIVE OF REDUCED IMPLANTATION AND INCREASED EARLY PREGNANCY LOSS. L. Sekhon,a C. Briton-Jones,a J. A. Lee,a R. Slifkin,a M. Duke,a A. B. Copperman,b T. Mukherjee.a aReproductive Medicine Associates of New York, New York, NY; bObstetrics and Gynecology, RMANY-Mount Sinai, New York, NY. OBJECTIVE: Embryo vitrification has increased confidence in embryo banking. Whether the effect of freeze-thaw on euploid blastocyst morphology impacts implantation potential is not well characterized. This study examines whether change in pre- and post-vitrification morphology correlates with clinical outcome, controlling for endometrial environment and embryo ploidy. DESIGN: Retrospective. MATERIALS AND METHODS: The study included patients that underwent single, euploid FET from 2011 to 2017. Blastocyst morphology was graded by a modified Gardner’s scale: 1) prior to vitrification and 2) after warming, 1 to 3 hours prior to FET. Patients were stratified according to whether morphology 1) improved, 2) downgraded, 3) remained the same or 4) inner cell mass (ICM)/trophectoderm (TE) could not be assessed due to blastocyst collapse. Chi-square and multivariate binary logistic regression (controlling for oocyte age, age at transfer, BMI, endometrial thickness and day of biopsy) was performed. RESULTS: A total of 2,026 single, euploid FET cycles were included. Blastocysts with discordant changes in pre- and post-freeze morphology (n¼135) were excluded. Compared with blastocysts with unchanged morphology, those that downgraded and could not be assessed had lower implantation and clinical pregnancy rate. Improved morphology led to comparable outcomes (Table 1). Downgrading and inability to assess ICM grade were associated with reduced implantation (OR 0.4 [95% CI 0.2-0.7]; OR 0.4 [95% CI 0.2-0.7], p<0.01) and clinical pregnancy rate (OR 0.4 [95% CI 0.3-0.8]; OR 0.4 [95% CI 0.2-0.7], p<0.01) and increased EPL (OR 2.3 [95% CI 1.21-5.0]; OR 2.8 [95% CI 1.3-6.2], p<0.05). TE improvement/ downgrade did not impact clinical outcome. Inability to assess TE was associated with reduced implantation (OR 0.4 [95% CI 0.2-0.8], p<0.01), clinical pregnancy (OR 0.4 [95% CI 0.2-0.8], p<0.01) and increased EPL (OR 2.8 [95% CI 1.2-6.1], p<0.05).
Vol. 108, No. 3, Supplement, September 2017
Cases
# Thawed
% Recovered
% Initial Survival
% 3hr Survival
% 2PN
% Cleaved
% Usable Blastocysts
13 14
162 175
98.2 97.7 >0.05
74.2 91.8 <0.0001
71.7 87.7 <0.001
63.2 72 >0.05
80.7 74.7 >0.05
38 32.1 >0.05
Center, Beverly Hills, CA; bSouthern California Reproductive Center, Beverly Hills, CA. OBJECTIVE: Currently, there are a multitude of oocyte warming protocols ranging from large volumes to micro-droplets being used in different laboratories. There are also different procedural steps ranging from use of 3 to 5 droplets with variable timing. The objective of this study was to assess whether laboratory outcomes are affected based on volume of warming media at the time of oocyte thaw. We directly compared the use of 50ml droplets to use of 200ml droplets during our standard 5 step dilution oocyte warming protocol. DESIGN: Retrospective study comparing laboratory outcomes using two oocyte warming micro-droplet volumes. MATERIALS AND METHODS: Oocyte warming consists of a five step protocol using Irvine Scientific Vitrification Thaw media (90137, CA). We compared the laboratory outcomes of 13 cases with use of 50ml droplets to 14 cases with use of 200ml droplets. For both study arms, all oocytes were originally frozen using vitrification. The initial thawing solution used was a 2ml volume droplet at 37 C and the timing for warming was exactly the same in both groups. Micro-drop warming was performed through 5 room-temperature micro-drop dilutions of TS 1min; TS:DS 2min; DS 2min; DS:WS 2min and WS for 5min. Oocyte recovery, initial survival, 3hr survival, 2PN fertilization, cleavage and usable blastocyst rates were determined. Analysis was performed by Chi-square. RESULTS: CONCLUSIONS: In the recent years there has been nearly a 5 fold increase in rate of elective fertility preservation for women. Success rates both in blastocyst formation in the laboratories as well as pregnancy outcomes has improved with the use of vitrification techniques for oocyte cryopreservation and warming. In this study we assessed whether the oocyte warming thaw protocol could affect laboratory outcomes. We demonstrated that initial survival (<0.0001) and survival after 3 hours (<0.001) were significantly improved with the 200ml droplet size. However, the rate of embryo development of the surviving oocytes in either study arm were not different for day 3 cleavage or overall usable blastocysts. These findings suggest that if an oocyte survives the warming and initial 3hr culture, they have the ability to develop equally. Size does matter, oocytes favor a larger droplet during the 5 step warming process. The 200ml study group provided the oocytes an optimal environment to recover and develop into normal embryos. P-181 Tuesday, October 31, 2017 COMPARABLE REPRODUCTIVE OUTCOMES IN OPEN VERSUS CLOSED OOCYTE VITRIFICATION SYSTEMS: A PROSPECTIVE, PAIRED STUDY ON THE SAME GENETIC BACKGROUND AND STIMULATION PROTOCOL. A. Pujol,a M. Zamora,b A. Obradors,c D. Garcıa,b A. Rodrıguez,b R. Vassena.b aCentro de Infertilidad y Reproduccion Humana (CIRH), Barcelona, Spain; bClınica EUGIN, Barcelona, Spain; cFIV Obradors, Girona, Spain. OBJECTIVE: To compare in the same genetic background and stimulation regimen the laboratory and reproductive outcomes of the oocyte vitrification/ warming open system CryotopÒ (Kitazato) to the closed system Rapid-iÒ (Vitrolife). DESIGN: Prospective paired study carried out between July 2014 and November 2016. The study was approved by the local IRB and included a cohort of 83 oocyte donors providing a minimum of 12 mature oocytes (MII) at retrieval. In each case, 6 MII were vitrified using CryotopÒ and assigned to one recipient and other 6 MII were vitrified using Rapid-iÒ and assigned to different recipient. MATERIALS AND METHODS: ICSI was performed in all the cases, and embryos were transferred at D+2 or D+3. Oocyte survival rates, fertilization rate and embryo morphology, as well as reproductive outcomes (biochemical, clinical, ongoing pregnancy and live birth rates) between the two systems were analyzed by t-tests, Mann-Whitney test, and Chi2 tests as appropriate.
FERTILITY & STERILITYÒ
RESULTS: Recipient characteristics were similar in both groups with regard to age (41.24.7) and BMI (23.84.0). Oocytes vitrified with Rapid-i had higher survival rates (93.7% vs 86.2 %, p<0.001). The fertilization rate was lower using Rapid-i (58.5% vs 69.2% p<0.001). The number of ICSI with total fertilization failure was 9 (7.2%) for Rapid-i and none for Cryotop (p¼0.035). The percentage of cycles with frozen embryos was higher in Rapid-i (50.60% vs 45.26%, p<0.001). On average, the number of embryos transferred in the two groups was 1.80.4. Biochemical (50.7% vs 41.4%), clinical (45.1% vs 39.1%) and ongoing (45.7% vs 33.3%) pregnancy rates were not different (p>0.05) and were live birth rates (38.2% vs 32.3%, p>0.05). CONCLUSIONS: The participants of this prospective study are oocyte donors, young women in good reproductive health; care should be exerted in translating our results to other populations. Nevertheless, our results suggest that both closed and open oocyte vitrification systems offer comparable laboratory and reproductive outcomes up to live birth. P-182 Tuesday, October 31, 2017 A NEW ULTRA-RAPID WARMING DEVICE FOR VITRIFIED OOCYTES AND EMBRYOS. P. Patrizio,a Y. Natan,b P. E. Levi-Setti,c M. Leong,d A. Arav.e aObstetrics, Gynecology & Reproductive Sciences, Yale Fertility Center & Fertility Preservation, New Haven, CT; bFertilSafe Ltd., Ness Ziona, Israel; c Dept.Gynecology, Division of Gynecology and Reproductive Medicine, Humanitas Fertility Center, Humanitas Research Hospital, Rozzano (Milan), Italy; dThe Womens Clinic, Hong Kong, Hong Kong; eCEO Fertilesafe, Ness Ziona, Israel. OBJECTIVE: Variability in warming rates impacts on the success of vitrification procedures. To assure consistency in warming rates and high reproducibility of results, we report a new device (Helia, FertileSafe, Israel) capable to provide ultra-rapid warming of oocytes and embryos previously vitrified on two available commercial cryocarriers. DESIGN: Basic research experiments evaluating warming rates, survival, hatching rates. MATERIALS AND METHODS: Mice oocytes (n¼96) and blastocysts (n¼40) were vitrified using either an open (Cryotop, Kitazato, Japan) or a closed carrier (HSV,CBS- IMV France). Equilibration was done gradually according to established protocols with up to 5 oocytes or embryos per batch (20 total cryocarriers used for oocytes and 8 for embryos), transferred into vitrification solution (Origio, Denmark) for one minute before placing them on Cryotop or HSV and plunged in LN. Warming was done using the Helia device consisting of a chamber with LN to hold the cryocarriers next to the 5 ml warming solution (Origio) at 37 C. Once removed from the LN the cryocarriers were placed into 0.3ml high security modified sperm straws (CBS, France), kept in the warmed solution, containing a mesh at its distal end. The 0.3ml CBSstraws containing the cryocarriers were then immediately transferred into a second warming solution at RT for 2.5min, followed by transfer into the third holding medium (Origio). At the end of these passages, the distal mesh was cut-removed and oocytes and embryos were transferred into a culture dish for evaluation and culture. Warming rates of each cryocarrier were measured with a thermocouple of 50mm connected to the Cryotop or HSV and to a data logger (PICO, UK). To reproduce routine manual warming procedures two likely distances between LN and the 37 C warming solution (250mm and 500mm) were chosen. These warming rates were compared to those measured in Helia, where the distance between LN and the 37 C warming solution is 10 mm. RESULTS: Warming rates between -196 C and 37 C at a distance of 250 mm were above 31,000C /min. while at 500 mm dropped to 17,000 C /min. In the Helia device, the close proximity of LN to the 37 C solution resulted in warming rates of more than 300,000 C/min. for each experiment (n¼28). The survival of vitrified mice oocytes (Cryotop or HSV) and blastocysts (Cryotop) rewarmed in Helia were 66/68 (97%), 27/28 (96%) and 40/40 (100%) respectively, with 100% hatching.
e177
CONCLUSIONS: We developed a simple, successful, and highly reproducible method for rapid warming of oocytes and embryos vitrified with various cryocarriers. In addition, warming with Helia eliminates the need of pipetting samples between various solutions. Supported by: FertileSafe Ltd. P-183 Tuesday, October 31, 2017 HUMAN SEMEN PARAMETERS THAT PREDICT SPERM DNA INTEGRITY AFTER CRYOPRESERVATION. G. D. Smith,a W. R. Parker,b a c a da L. Keller, Y. Li, A. Brady, S. Leibo. Ob/Gyn, University of Michigan, Ann Arbor, MI; bUrology, Poughkeepsie, NY; cBiostatistics, University of Michigan, Ann Arbor, MI; dUniverisity of New Orleans, New Orleans, LA. OBJECTIVE: Human semen cryopreservation is important for: i) therapeutic donor insemination, ii) some cases of IVF, and iii) fertility preservation for those at risk of losing fertility. Not only is sperm cryo-survival important, but DNA integrity in motile cryopreserved sperm is essential for optimal outcomes. Objectives of this study were to prospectively investigate pre-cryopreservation semen analysis parameters in relation to post-cryopreservation motile sperm DNA integrity. DESIGN: Prospective clinical laboratory study. MATERIALS AND METHODS: De-identified semen samples (n¼49) underwent semen analysis, motile sperm DNA integrity assessment pre-cryopreservation, cryopreservation, thawing, post-thaw assessment for cryo-survival, and post-thaw evaluation of motile sperm DNA integrity in this IRBapproved study. Pre-freezing semen analysis values assessed by WHO 4th edition were: i) semen volume, ii) semen pH, iii) semen viscosity, iv) sperm total motility (%), v) sperm progressive forward motility, vi) sperm concentration by hemocytometer, and vii) total motile sperm per ejaculate (TMS; concentration x volume x total motility). DNA integrity was performed on motile sperm with the Halo assay. Semen samples were cryopreserved in TEST-yolk buffer with Glycerol and Gentamicin (Irvine Scientific; Irvine, CA) per manufactures protocol. The dependent outcome variable was postthaw motile sperm percent recovery of intact DNA (PTMS-intact DNA; post-thaw percent motile sperm with intact DNA / pre-freezing percent motile sperm with intact DNA x 100). Analysis was performed with SPSS statistics software and a univariate linear regression model. RESULTS: Semen pH (7.5 0.03), semen viscosity (1.2 0.1; arbitrary units), sperm total motility (53 0.7 %), and sperm progressive motility (77 0.4 % of total motile) had no significant predictive value for PTMS-intact DNA (89 1.2 %). However, pre-cryopreservation semen volume (3.8 0.2 mls; p<0.01) and TMS (220 20 x 106; p<0.02) had significant predictive values for PTMS-intact DNA. Pre-cryopreservation semen volume and TMS had an inverse relation to PTMS-intact DNA, whereby per unit increase in volume (0.1ml) or TMS (100 x 106) each independently yielded a reduction of 2% in PTMS-intact DNA. Of note, post-thaw cryo-survival had no predictive value in relation to PTMS-intact DNA. CONCLUSIONS: Semen volume and TMS were prospective PTMS-intact DNA predictive values in the pre-cryopreservation semen analysis. Cryo-survival was not predictive of PTMS-intact DNA. Evaluation of these parameters can assist fertility healthcare providers in counseling men and couples in semen cryopreservation and usage of samples post-thaw. P-184 Tuesday, October 31, 2017 THE DURATION OF CRYOSTORAGE OF BIOPSIED EMBRYOS NEITHER IMPACTS IMPLANTATION POTENTIAL NOR SURROGATE MARKERS OF PLACENTATION. L. Sekhon,a J. A. Lee,b M. Duke,b C. Briton-Jones,b E. Flisser,b A. B. Copperman.c aReproductive Medicine Associates New York, New York, NY; bReproductive Medicine Associates of New York, New York, NY; cObstetrics and Gynecology, RMANY-Mount Sinai, New York, NY. OBJECTIVE: Blastocyst vitrification facilitates embryo banking for successive single embryo transfers (SET) and has become an integral part of IVF treatment. Although storage at -196oC is thought to halt metabolism, the molecular structure of vitrified cells might be dynamic and sensitive to storage temperature variations and impacted by long-term cryostorage. Previous studies of the effect of cryostorage duration on clinical outcome focused on slow-frozen zygote or cleavage stage embryos; and no study to date has assessed whether blastocysts which have undergone trophectoderm biopsy are vulnerable to impaired implantation or
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ASRM Abstracts
placentation as a result of long-term cryostorage. This study examined whether the duration of cryostorage of euploid blastocysts adversely impacts reproductive potential or perinatal outcome. DESIGN: Retrospective, observational study. MATERIALS AND METHODS: The study included patients that underwent autologous IVF and aneuploidy screening of blastocysts by preimplantation genetic testing (PGT), followed by vitrification, warming and SET, from 2011 to 2016. Donor oocyte cycles were excluded. Cryostorage duration was calculated as the interval (days) elapsed from when the blastocyst was vitrified to when it was warmed and transferred. Multivariate logistic and linear regression analyses were performed. Main outcomes included implantation, clinical pregnancy, live birth and early pregnancy loss. Secondary outcomes included infant birthweight and gestational age at delivery. RESULTS: Vitrified-warmed, euploid blastocysts, derived from autologous IVF, were transferred in 2,320 SET cycles. The average cryostorage duration was 4 months, with a maximum duration of up to 4.9 years (mean 126.4 197.3 days; range 21-1794 days). Controlling for patient age at IVF and embryo transfer, BMI, endometrial thickness and embryo biopsy day, the duration of cryostorage did not impact the odds of implantation (OR 1.0 [95% CI 0.99-1.01]), clinical pregnancy (OR 1.0 [95% CI 0.9981.004]), live birth (OR 0.998 [95% CI 0.99-1.003]), biochemical pregnancy loss (OR 0.998 [95% CI 0.99-1.002]), and clinical pregnancy loss (OR 1.01 [95% CI 0.98-1.05]). The duration of cryostorage did not impact birthweight (b¼ -0.01, p¼0.96) or gestational age at delivery (b¼ -0.22, p¼0.51). CONCLUSIONS: Modern advances in cryopreservation and PGT have facilitated the opportunity for patients to bank embryos with confidence. This emerging treatment strategy will result in the increased storage of biopsied embryos for longer durations. This study is the first to demonstrate that long-term cryostorage of vitrified, biopsied blastocysts does not adversely affect reproductive potential, fetal growth or the likelihood of preterm delivery. Based on these findings, patients can be reassured regarding the efficacy, efficiency and safety of embryo banking. FERTILITY PRESERVATION P-185 Tuesday, October 31, 2017 BLASTOCYST COLLAPSE AND DOWNGRADING OF INNER CELL MASS MORPHOLOGY SCORE AFTER VITRIFICATION-WARMING IS PREDICTIVE OF REDUCED IMPLANTATION AND INCREASED EARLY PREGNANCY LOSS. L. Sekhon,a C. Briton-Jones,a J. A. Lee,a R. Slifkin,a M. Duke,a A. B. Copperman,b T. Mukherjee.a aReproductive Medicine Associates of New York, New York, NY; bObstetrics and Gynecology, RMANY-Mount Sinai, New York, NY. OBJECTIVE: Embryo vitrification has increased confidence in embryo banking. Whether the effect of freeze-thaw on euploid blastocyst morphology impacts implantation potential is not well characterized. This study examines whether change in pre- and post-vitrification morphology correlates with clinical outcome, controlling for endometrial environment and embryo ploidy. DESIGN: Retrospective. MATERIALS AND METHODS: The study included patients that underwent single, euploid FET from 2011 to 2017. Blastocyst morphology was graded by a modified Gardner’s scale: 1) prior to vitrification and 2) after warming, 1 to 3 hours prior to FET. Patients were stratified according to whether morphology 1) improved, 2) downgraded, 3) remained the same or 4) inner cell mass (ICM)/trophectoderm (TE) could not be assessed due to blastocyst collapse. Chi-square and multivariate binary logistic regression (controlling for oocyte age, age at transfer, BMI, endometrial thickness and day of biopsy) was performed. RESULTS: A total of 2,026 single, euploid FET cycles were included. Blastocysts with discordant changes in pre- and post-freeze morphology (n¼135) were excluded. Compared with blastocysts with unchanged morphology, those that downgraded and could not be assessed had lower implantation and clinical pregnancy rate. Improved morphology led to comparable outcomes (Table 1). Downgrading and inability to assess ICM grade were associated with reduced implantation (OR 0.4 [95% CI 0.2-0.7]; OR 0.4 [95% CI 0.2-0.7], p<0.01) and clinical pregnancy rate (OR 0.4 [95% CI 0.3-0.8]; OR 0.4 [95% CI 0.2-0.7], p<0.01) and increased EPL (OR 2.3 [95% CI 1.21-5.0]; OR 2.8 [95% CI 1.3-6.2], p<0.05). TE improvement/ downgrade did not impact clinical outcome. Inability to assess TE was associated with reduced implantation (OR 0.4 [95% CI 0.2-0.8], p<0.01), clinical pregnancy (OR 0.4 [95% CI 0.2-0.8], p<0.01) and increased EPL (OR 2.8 [95% CI 1.2-6.1], p<0.05).
Vol. 108, No. 3, Supplement, September 2017