ICSI population

MATERIALS AND METHODS: Sperm DNA from each individual was isolated and bisulfite converted. The bisulfite converted DNA PCR amplified at 15 regions of interest (identified by previous work from our lab). Amplicons were multiplex sequenced by MiSeq. Each region was analyzed for average fraction methylation and a t test compared between young and aged samples. Additionally, we compared the fraction methylation from each individual read (representing a single sperm in the population) to determine if the entire sperm population is mildly affected by age or if sub-populations of sperm are dramatically altered with age (manifesting as a relatively modest change in total fraction methylation). RESULTS: In general, there are no major outlying populations of sperm that drive the average age-associated methylation alterations. Instead, it appears that most cells in a sperm population have a subtle, but uniform, regional age associated methylation perturbations. While these methylation alterations are subtle they tend to be more significant than those that have been identified in somatic cells based on previous work from Day et al. who showed only a single CpG in somatic tissue that had a yearly change of >0.5%. Our data revealed 13 genomic windows with an average fraction methylation change per year of >0.5%. In regions that undergo hypermethylation with age, the average fraction methylation change was 0.30% per year and in regions undergoing hypomethylation the average fraction methylation change was 0.28% per year. CONCLUSION: Our data support the idea that all cells in a population of sperm (and not just small subpopulations) undergo similar DNA methylation alterations with age, and thus may represent a similar risk to the offspring. These results confirm data previously reported in longitudinally collected donor samples in our laboratory.

CRYOPRESERVATION AND FROZEN EMBRYO TRANSFER I O-296 Wednesday, October 22, 2014 11:15 AM NEONATAL OUTCOMES OF TRANSFER OF BLASTOCYSTS VITRIFIED AND WARMED IN DEFINED SOLUTIONS CONTAINING RECOMBINANT HUMAN ALBUMIN: 354 BABIES BORN FOLLOWING 851 EMBRYO TRANSFERS. M. Murakami, A. Egashira, K. Tanaka, C. Mine, H. Otsubo, T. Kuramoto. Kuramoto Women’s Clinic, Fukuoka, Japan. OBJECTIVE: Our previous pilot study using bovine models and subsequent clinical trials revealed that addition of lower concentrations of recombinant human albumin (recHA), rather than blood-derived human serum albumin (HSA), to our standard vitrification/warming solutions (VS/WS) yielded high embryo viability. Consequently, we have implemented the chemically defined VS/WS with recHA for blastocyst vitrification at our center. Here, we present these clinical data on the novel defined embryo vitrification procedure. DESIGN: Retrospective study. MATERIALS AND METHODS: The base solution used for VS/WS was Dulbecco’s phosphate-buffered saline containing 50 mg/mL HSA (group A) or 2.5 mg/mL recHA (group B). Surplus blastocysts and blastocysts from patients undergoing elective cryopreservation of all embryos at our center were vitrified/warmed using cryotop carriers in our homemade solutions containing either HSA or recHA. Data was analyzed for all warmed cycles performed between March 2006 and June 2013 in both groups. RESULTS: Groups A and B had a total of 1162 and 851 warmed cycles, respectively. Patient age (35.4
3.7 vs. 35.5
3.9 years), embryo survival rates (1258/1275 [98.7%] vs. 901/912 [98.8%]), and the number of embryos transferred (1.08
0.27 vs. 1.06
0.24) were similar for groups A and B, respectively. Embryo transfer (ET) was performed in 1158/1162 (99.7%) and 850/851 (99.9%) of the warmed cycles in groups A and B, respectively. Implantation rates and clinical pregnancy rates/ET were higher (P < 0.05) in group B (493/901 [54.7%] and 475/850 [55.9%], respectively) than in group A (617/1250 [49.4%] and 595/1158 [51.4%], respectively). Groups A and B had similar live delivery rates/pregnancy (428/595 [71.9%] vs. 344/475 [72.4%], respectively) and perinatal outcomes, including birth weight (2987
582 vs. 3043
503 g, respectively). Birth defects occurred in 4/ 444 (0.9%) and 6/354 (1.7%) neonates in groups A and B, respectively. CONCLUSION: Our data demonstrated the efficacy of using recHA as a substitute for HSA in human embryo vitrification procedures and yielded high rates of pregnancy and normal live births after vitrified/warmed ET. Although long-term follow-up studies are required to assure the safety of the procedure, this new approach will help develop defined systems for assisted reproductive technology. Finally, the use of recHA will eliminate the inherent variation and potential risks of using biological products.

FERTILITY & STERILITYÒ

O-297 Wednesday, October 22, 2014 11:30 AM RISK OF PREGNANCY-INDUCED HYPERTENSIVE COMPLICATIONS FOLLOWING ASSISTED REPRODUCTIVE TECHNOLOGY (ART) – A COHORT STUDY FROM THE COMMITTEE ON NORDIC ART AND SAFETY. S. Opdahl,a A. Pinborg,b P. Romundstad,a U.-B. Wennerholm,c M. Gissler,d L. B. Romundstad.a aDepartment of Public Health, Norwegian University of Science and Technology (NTNU), Trondheim, Norway; bDepartment of Obstetrics and Gynecology, Hvidovre Hospital, University of Copenhagen, Hvidovre, Denmark; cDepartment of Obstetrics and Gynecology, University of Gothenburg, Sahlgrenska University Hospital/East, Gothenburg, Sweden; dNational Institute for Health and Welfare, Helsinki, Finland. OBJECTIVE: Women who conceive through ART are at increased risk of pregnancy-induced hypertensive disorders. In this large population based study, we studied whether in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) as well as fresh or frozen cycles differed with respect to risk of hypertensive complications in pregnancy. DESIGN: A population based cohort study including all ART pregnancies in Denmark, Finland, Norway and Sweden throughout 2007. Information on pregnancy complications, type of ART procedure, and potential confounders was obtained from national registries. For singletons, each ART pregnancy was matched with four spontaneously conceived (SC) pregnancies by year and parity. For twins, all pregnancies in the study period were included. MATERIALS AND METHODS: In total, 62 987 ART pregnancies with a gestational age > 22+0 weeks were included. We compared the risk of pregnancy-induced hypertension and preeclampsia (combined) in 51 348 ART singletons and 289 800 SC singletons. 11 639 ART twin pregnancies were compared to 49 618 SC twin pregnancies. We used binomial regression to estimate risk ratios (RR) with 95% confidence intervals (CI) adjusted for parity, year of birth, maternal age, offspring sex and country. RESULTS: For all ART procedures combined, the risk of hypertensive complications was higher in singleton, but not in twin pregnancies (RR 1.15, 95% CI 1.10-1.20 and RR 0.96, 95% CI 0.91-1.02, respectively). For singletons, the risk after frozen cycles was higher (RR 1.36, 95% CI 1.191.55) than in fresh cycles (RR 1.10, 95% CI 1.05-1.16). For twins, a higher risk was observed after frozen cycles (RR 1.39, 95% CI 1.22-1.57), but the risk after fresh cycles was lower than in SC twin pregnancies (RR 0.89, 95% CI 0.84-0.95). Results were similar for IVF and ICSI and consistent across the Nordic countries, time period, offspring sex and parity. CONCLUSION: The higher risk of hypertensive complications in ART pregnancies was observed mainly after frozen cycles. Supported by: The European Society for Human Reproduction and Embryology, the University of Copenhagen, the Danish Agency for Science, Technology and Innovation, the Nordic Society of Obstetrics and Gynecology and the Liaison Committee between the Central Norway Regional Health Authority and NTNU. O-298 Wednesday, October 22, 2014 11:45 AM CRYOPRESERVED EMBRYO TRANSFER (CET) IS AN INDEPENDENT RISK FACTOR FOR PLACENTA ACCRETA IN THE IVF/ ICSI POPULATION. D. J. Kaser, A. Melamed, C. Racowsky, C. L. Bormann, B. W. Walsh, D. A. Carusi. Department of Obstetrics, Gynecology and Reproductive Biology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA. OBJECTIVE: To determine the incidence of placenta accreta in our IVF/ ICSI population and to test the hypothesis that CET is an independent risk factor for placenta accreta. DESIGN: Case-control study. MATERIALS AND METHODS: Among consecutive patients delivering at least one viable infant R24 weeks’ gestational age at our hospital from 2005 to 2011 (n¼54,947), we identified all patients who underwent a day 3 or day 5 transfer at our IVF center (n¼1,569). From this cohort, we confirmed 52 cases of placenta accreta (defined by histology or the clinical finding of an adherent placenta with or without morbid complications such as postpartum hemorrhage, hysterectomy or surgery to remove the placenta). Cases were matched 1:3 by maternal age and history of prior cesarean to IVF/ ICSI patients without accreta. Adjusted odds ratios (aOR) with 95% confidence intervals (CI) were calculated by multivariate analysis, controlling a priori for age (to account for any residual confounding), placenta previa, prior abdominal or laparoscopic myomectomy and uterine factor infertility. RESULTS: The overall incidence of accreta in our IVF/ICSI cohort was 3.31% (52/1,569); among CET patients, the incidence was 7.24%

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(16/221). Median age in years (interquartile range) was 39.3 (33.9-41.7) for cases and 39.1 (34.3-41.3) for controls. Because the effect size for CET in the base model did not change by >10% with addition of any potential confounder, none were retained in the final model. Covariates not retained included gravidity, parity, race, body mass index, use of donor oocytes, history of curettage or operative hysteroscopy, intrauterine synechiae, oral contraceptive lead-in, pre-treatment with leuprolide acetate, endometrial thickness %7mm, micromanipulation (ICSI, assisted hatching, embryo biopsy), type of luteal phase support, selective reduction and multifetal delivery. The only significant predictors of accreta in multivariate analysis were CET, prior myomectomy, and placenta previa (Table 1). Restricting analysis to only morbid cases of accreta strengthened the effect of CET on accreta to 2.78 (95% CI 1.11-6.99; P¼.03). TABLE 1. Independent predictors of placenta accreta in IVF/ICSI patients

Covariate

aOR

95% CI

P-value

CET Prior myomectomy Placenta previa

2.33 5.89 11.36

1.04-5.23 1.84-18.81 3.42-37.7

.04 < .01 < .0001

CONCLUSION: CET was a strong independent risk factor for placenta accreta among IVF/ICSI patients, even after controlling for known accreta risk factors. O-299 Wednesday, October 22, 2014 12:00 PM VITRIFICATION OF CLEAVAGE STAGE DAY 3 EMBRYOS RESULTS IN SIGNIFICANTLY HIGHER LIFE BIRTH RATES THAN SLOW FREEZING: A RANDOMIZED CONTROLLED TRIAL. S. Debrock, E. Fernandez Gallardo, K. Peeraer, D. De Neubourg, T. D’Hooghe, C. Spiessens. Department of Development and Regeneration, Leuven University Fertility Center, Leuven, Belgium. OBJECTIVE: To compare vitrification and slow freezing for cryopreservation of day 3 embryos. DESIGN: Randomized controlled trial in an academic tertiary setting. MATERIALS AND METHODS: Patients younger than 40 years using own oocytes in the 1st ART cycle, with embryo transfer (ET) and with available supernumerary embryos on day 3, were randomized at the time of cryopreservation after informed consent. Day 3 embryos with R 6 cells, <25% fragmentation and even to slightly uneven blastomeres were cryopreserved by slow freezing (EmbryoStore, GynemedÒ) or by closed vitrification (Vitr Kit Freeze, Irvine ScientificÒ). Survival was defined as present if R 50% of cells were intact after thawing. Thawed embryos were further cultured overnight. To test the hypothesis that the life birth rate (LBR) per embryo thawed after vitrification was significantly higher (16%) than after slow freezing (6%), power calculation revealed that 184 thawed embryos were needed in each group (b¼0.8, a<0.05). Secondary outcome variables were implantation rate (IR) (presence of fetal sac (intra- or extrauterine) on ultrasound at 6-8 weeks pregnancy) per embryo transferred and survival rate after thawing. Data were analyzed by Mann-Whitney test and Chi square analysis and considered significant if p<0.05. RESULTS: Between September 2011 and March 2013, 307 patients were randomized to slow freezing (155 patients, 480 embryos) or vitrification (152 patients, 495 embryos). In March 2013, 200 embryos were thawed after slow freezing in 95 cycles for 79 patients and 217 embryos were warmed after vitrification in 121 cycles in 90 patients. The LBR per embryo thawed was significantly higher after vitrification (17.1% (37/217) than after slow freezing (5.5% (11/200); p<0.0002)). Similarly, the IR per embryo transferred was higher after vitrification (27.1% (48/177) than after slow freezing (16% (16/100); p¼0.0382). The embryo survival rate was significantly higher after vitrification (84.3% (183/217) than after slow freezing (52.5% (105/200); p<0.0001). Significantly more embryos were fully intact after vitrification (75.4 % (138/183) than after slow freezing (28.6 % (30/105); p<0.0001). The number of ETs was significantly higher after vitrification (90.1% (109/121)) than after slow freezing (73.7% (70/95); p¼0.0019). CONCLUSION: The hypothesis was confirmed that the LBR per embryo thawed is higher after vitrification than after slow freezing on day 3, based on better embryo survival, quality and availability in the vitrification group.

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ASRM Abstracts

O-300 Wednesday, October 22, 2014 12:15 PM PERINATAL OUTCOME OF CHILDREN BORN AFTER VITRIFICATION OF BLASTOCYSTS (8440 CYCLES WITH 2757 BABIES IN 14 YEARS EXPERIENCES). T. Mukaida,a K. Tanaka,a T. Goto,b C. Oka,b K. Takahashi.a aHiroshima HART Clinic, Hiroshima, Japan; bTokyo HART Clinic, Tokyo, Japan. OBJECTIVE: Vitrification has been already recognized as a useful method for cryopreservation of human blastocysts (BLs). However, slow cooling approach still has been used in many ART facilities, due to not enough perinatal information of vitrified BLs program. Therefore, we analyzed perinatal outcomes after 13 weeks of gestation including the rate of monozygotic twinning(MZT) in infants born after vitrified BL. DESIGN: Retrospective analysis. MATERIALS AND METHODS: Perinatal outcome were summarized for all patients who had vitrified BL transfer cycles between 2000 and 2013. Vitrification involved the exposure DMSO and EG during the cooling step, and 0.25/0.5 M sucrose solution for warming. The blastocoelic cavity was collapsed with laser pulse, prior to vitrification. After warming, zona hatching by laser pulse was performed. MZT was defined that the number of FHB was more than that of embryos transferred. RESULTS: A total of 12941 vitrified BLs in 8440 cycles were warmed and 12339 survived (95.3%). In 8347 transfers, 3948 resulted in pregnancy (47.3%). A total of 11295 vitrified BLs were transferred and 4362 were implanted (38.6%). A total of 2757 babies(1413 boys & 1344 girls) were born in 2483 births. Forty seven clinical pregnancies were confirmed as MZT(1.9%). Thirty three cases had either congenital birth defects or perinatal complications (1.6%), including eight chromosomal abnormalities (two 18, six 21 trisomy), ten multiple anomalies, one stillbirth due to hydropus, six stillbirths of unknown causes(25, 29, 30, 32, 37, 39 weeks of gestations), two anencephaly, one spina bifida, eleven congenital heart or major vessel malformations, one esophageal and one biliary duct obstruction, one Cornelia De Lange, and one Treacher Collins syndrome. Nine hundred and sixty seven pregnant cycles ended in miscarriage(24.5%). CONCLUSION: The perinatal outcomes of 2483 births with 2757 babies, the incidence rates of MZT (1.9%) and rate of congenital defect and perinatal complication (1.6%) that were both similar to fresh BL transfer as we reported previously (2.3% & 2.0%), confirm the safety of vitrified BL program. Also, the high implantation rate encourages us to establish single BL transfer. Further investigation of developmental competence of childrens born after vitrified BL will be necessary. O-301 Wednesday, October 22, 2014 12:30 PM SEMICLOSED VITRIFICATION SYSTEM: UNALTERED CLINICAL OUTCOME AFTER HERMETICAL SEALING FOR STORAGE. A. Cobo, B. Vallejo, P. Campos, E. Sanchez, A. Coello, J. Remohı. IVI Valencia Spain, Valencia, Spain. OBJECTIVE: The value of open devices for oocyte vitrification has been confirmed recently. However the likelihood of cross contamination due to direct contact with liquid nitrogen during storage has been a great drawback related to these devices. This study is aimed to evaluate survival and clinical outcome using a semi-closed (SC) device which allows open vitrification but provides hermetical sealing for storage. DESIGN: Randomized clinical trial. (ClinicalTrials.gov NCT01745536, July 12th 2012). MATERIALS AND METHODS: 370 donors from our ovum donation program (alpha 0.05 and power of 80% for sample size calculation). Randomization was performed 1:1 based on a computer randomization list in two groups: vitrification using the SC and open devices (185 per arm). So far we have been able to perform 159 and 146 donation cycles in SC and open groups respectively. Donor stimulation and endometrial preparation for recipients were performed as described else were. The regular open Cryotop and the SC devices were employed for vitrification. Equilibration, vitrification and loading procedures were performed for both systems as described else were, except that with the SC device, the specially designed cover straw was hermetically sealed for storage. For sealing, the SC Cryotop was introduced into the cover straw after open vitrification by exposing the device to nitrogen vapors. The primary end point was the survival rate. Secondary end points were clinical pregnancy, implantation, fertilization rates (CPR, IR, FR) and embryo quality. c2, student’s ttest and fisher’s exact test were employed. RESULTS: No statistical differences were observed between donors and recipient’s baseline characteristics. Lower survival rate was achieved using the SC system (87.3 vs. 91.7; p<0.05). CPR/cycle (56.0% vs. 46.6%), IR (40.8% vs. 39.3%) and FR (74.6% vs. 74.4%) were comparable between

Vol. 102, No. 3, Supplement, September 2014