Localised assisted hatching effect in cryopreserved blastocyst transfers

acids level by liquid chromatography. The genotypes distribution in case and control groups was tested for deviation from the Hardy-Weinberg Equilibrium (HWE). RESULTS: Only the heterozygous Leiden mutation was detected in 4(4.8%) women of the lot I and non in lot II; no FII mutation were detected in both. The genotype distributions of the polymorphisms and their OR (95% CI) showed that MTRR A66G (GG and AG genotypes) and MTR A2756G (GG genotype) were associated with increased risk of RPL and SA after IVF. The determination of HWE showed that the genotypic distributions were not in equilibrium in lot I for MTRR A66G (X2:10.623, P¼0.001) and MTR A2756G (X2:9.925, P¼0.001). Only 13 from 38 investigated for amino acids women showed a high level(R13mmol\l) of Homocystheine(Hcy) in blood, being associated with the presence of mutations in heterozygous state in following genes MTHFR677–69.2%, MTHFR1298-30.7%, MTR-23.1% and MTRR-76.9% and in homozygous state in MTHFR1298–15.3%. CONCLUSION: Some of the investigated thrombophilic and folate metabolizing genes are associated with RPL and SA after IVF in Moldavian women population; the high Hcy level is associated with heterozygous state in MTHFR677 and MTRR genes. Supported by: institutional scientific project nr. 11.817.09.59A. P-476 Wednesday, October 24, 2012 GENETIC PREDICTION OF ENDOMETRIOSIS IN WOMEN SEEKING ASSISTED REPRODUCTIVE TECHNOLOGIES. K. Ward, R. Chettier, P. Farrington, H. Albertsen. Juneau Biosciences, LLC, Salt Lake City, UT. OBJECTIVE: Endometriosis is a common cause of or co-factor related to infertility. Assisted reproductive technologies (ART) may have more complications and be less successful when endometriosis is present. Since definitive diagnosis requires surgical biopsy, diagnosis is usually delayed or skipped over. We have developed a predictive algorithm for detecting which ART patients have endometriosis. The purpose of this study was to test the performance of this DNA marker panel. DESIGN: Blinded retrospective analysis of DNA samples from infertile women to test performance of a newly-developed panel of predictive markers. MATERIALS AND METHODS: DNA samples form 150 Caucasian infertile women with confirmed endometriosis and 150 with no evidence of endometriosis were genotyped in a blinded fashion using Taqman for 73 single nucleotide polymorphisms associated with endometriosis. Genotype results were weighted using a logistic regression algorithm applied to a large training data set. Prior probability of having endometriosis was calculated based on presence or absence of: early menarche, affected 1st degree relative, primary dysmenorrhea, and parity. Posterior risks were calculated using Bayesean analyses. RESULTS: Using the current model, over 50% of affected women could be correctly classified as having a high (greater than 95%) risk of having endometriosis and the majority of unaffected women were correctly classified as having a low (less than 5%) risk of having endometriosis. The area under the receiver operator characteristic curve was 0.80. CONCLUSION: The panel of DNA markers provides actionable predictions. Additional improvements in the test algorithm are likely as new markers are added and as interaction terms or biologic pathway data are considered. Prospective clinical trials are planned in several bust ART practices. Supported by: Juneau Biosciences. P-477 Wednesday, October 24, 2012 EFFECT OF SEASONAL VARIATIONS IN THE ASSISTED REPRODUCTION (IVF±ICSI): A META-ANALYSIS. S. Vitthala,a P. Makwana,a M. Nissar,a S. V. Iliani,b K. P. Vasundhara.a aFertility Centre, Kamineni Fertility Centre, Hyderabad, AP, India; bFertility Centre, Surya Fertility Centre, Hyderabad, AP, India. OBJECTIVE: Several studies across the globe have reported the seasonal variations can affect the IVFICSI outcomes. However, results are contradictory with respect to clinical pregnancies, number of oocytes retrieved and their fertilisation (outcomes). This meta-analysis aims to evaluate the effect of different seasons on the IVFICSI outcomes. DESIGN: Systematic review and meta-analysis. MATERIALS AND METHODS: We searched MEDLINE, EMBASE, GOOGLE SCHOLOR and SCISEARCH for the studies that reported the effects of seasons on the IVFICSI outcomes.A meta-analysis of proportion was performed using both fixed and random methods on the studies that reported on outcomes in different seasons (summer, spring, winter and autumn) of the year.

FERTILITY & STERILITYÒ

RESULTS: The search revealed 37 publications reporting on the IVFICSI outcomes in different seasons.10 studies met the inclusion criteria and were included in the meta-analysis.The proportions of oocytes retrieved in different seasons are autumn:0. 29; spring:0. 27; summer:0. 23; winter:0. 22. Similarly, the proportions of fertilization rate in different seasons are autumn:0. 46; spring:0. 45; summer:0. 44; winter:0. 43.The highest oocyte retrieval and fertilisation rates were noted in autumn and the lowest in the winter.The proportions of clinical pregnancy rates in different seasons are spring: 0.27; autumn: 0.24; winter:0.21; summer:0.18.The highest pregnancies noted in the spring and the lowest in summer. Seasonal Variations in IVFICSI

Outcomes No. of oocytes retrieved Fertilisation rate Clinical Pregnancy Rate

Autumn

Spring

Summer

Winter

0.29 0.46 0.24

0.27 0.45 0.27

0.23 0.44 0.18

0.22 0.43 0.21

Proportion Meta-Analysis. CONCLUSION: The seasonal variations do influence the IVFICSI outcomes. Outcomes are better in autumn/spring in comparison to winter/summer. The extremes of weather, less sun light in the winters and the hot and humid conditions in the summer adversely affect the outcomes. Large multi-centered studies in different continents are needed to confirm our findings. P-478 Wednesday, October 24, 2012 PREVENTING OOCYTE AGING BY ASEPTIC VITRIFICATION FOR PATIENTS WITH UNEXPECTED DELAY OF SPERM PRODUCTION. B. Wirleitner,a P. Vanderzwalmen,b B. Lejeune,b N. H. Zech.a aIVF Centers Prof Zech, Bregenz GmbH, Bregenz, Austria; b CHIREC, Braine-l’Alleud, Belgium. OBJECTIVE: One of the critical points after oocytes pick-up (OPU) is the aging of the oocytes due to inability to obtain sperm at the right time (<3 hrs post OPU) in patients with erectile dysfunctions or in cases of azoospermia where testicular biopsy has to be applied. Oocytes age when intracytoplasmic sperm injection (ICSI) or intracytoplasmic morphologically selected sperm injection (IMSI) has to be postponed, impacting on their capacity to fertilize and impairing embryo development. One strategy to avoid oocyte aging is to vitrify and thaw them in a cryo-cycle when sperm collection is possible. DESIGN: In this study we present clinical data from oocyte vitrification due to absence of sperm at the day of pick-up in 56 IVF-patients. MATERIALS AND METHODS: In all patients no sperm could be obtained until 3 hrs after the OPU. Therefore, oocytes were denuded and incubated in 5 steps in non-vitrifying and vitrifying solutions and vitrified in an aseptic vitrification device (VitriSafe) ensuring aseptic conditions during vitrification and storage. For warming a 4-step protocol in sucrose solutions was applied. As main outcome live-birth rate was evaluated. RESULTS: A total of 455 oocytes were vitrified from which 416 were viable and fertilized after warming. A fertilization rate of 71.4% and a cleavage rate of 95.3% on d3 were observed. After transfer of blastocysts an ongoing pregnancy rate of 43.8% and a birth rate of 37.5% were reported, 28 babies were born. CONCLUSION: High birth rates can be obtained after aseptic vitrification of oocytes. Our results suggest that in all centres with a good, standardized protocol, oocyte cryopreservation using vitrification in hermetically closed devices should be applied in cases of unexpended failure of sperm production. These encouraging data suggest applying this strategy not only in cases of no sperm production but also when the sperm of IMSI candidate patients show 100% of sperm with large nuclear defects.

P-479 Wednesday, October 24, 2012 LOCALISED ASSISTED HATCHING EFFECT IN CRYOPRESERVED BLASTOCYST TRANSFERS. D. Gumbao,a B. Amorocho,a A. Sanchez Leon,a J. Marcos,a M. Molla,a J. Landeras.b aIVF Laboratory, IVI Murcia, Murcia, Spain; bDepartment of Gynecology, IVI Murcia, Murcia, Spain. OBJECTIVE: Evaluate the clinical outcome in oocyte donation cycles of cryopreserved blastocysts which had previously undergone a localised assisted hatching process.

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DESIGN: The study took place at IVI Murcia Clinic between November 2009 and January 2012. To differentiate between the assisted hatching (AH) effect carried out at the level of the inner cell mass (ICM) and that which was carried out at the level of the trophectoderm—opposite the ICM (TRP) — 60 vitrified blastocyst cases included in an oocyte donation program on day five of the embryo development cycle—were randomly selected to undergo the different AH treatments. MATERIALS AND METHODS: Vitrified-thawed blastocysts were randomly subjected to localised assisted hatching on the same day of the transfer using the laser ZP thinning technique and after their re–expansion had taken place. The clinical results were analysed statistically using the Fisher test (P<0.05) in order to observe significant differences between both groups. RESULTS: TABLE 1. Fisher test, P<0.05

n Age Transferred embryo average Pregnancy rate Implantation rate Biochemical miscarriage Clinical miscarriage

AH ICM

AH TRP

P

29 37 1.3 60.7% 52.8% 5.6% 10.7%

31 38 1.5 41.9% 35.6% 23.5% 25.8%

0.75 0.16 0.38 0.24

CONCLUSION: The results appear to point towards a favourable trend in applying assisted hatching techniques at the level of the inner cell mass; this result would agree with the results obtained by other groups which also defend the role played by inner cell mass in the embryo hatching process. Supported by: IVI Murcia Fertility Clinic, Murcia, Spain.

P-480 Wednesday, October 24, 2012 EFFECT OF PIOGLITAZONE ON TUMOR NECROSIS-ALPHA AND INTERLEUKIN-6 IN FOLLICULAR FLUID AND IVF/ICSI OUTCOMES IN INFERTILE PATIENTS WITH ENDOMETRIOSIS. C. H. Kim,a S.-K. Kwon,a J.-W. Ahn,b S.-Y. Choi,a K.-H. Lee,a B.-M. Kang.a aObstetrics and Gynecology, College of Medicine, University of Ulsan, Asan Medical Center, Seoul, Korea; bObstetrics and Gynecology, College of Medicine, University of Ulsan, Ulsan University Hospital, Ulsan, Korea. OBJECTIVE: To investigate the effect of pioglitazone on follicular fluid (FF) tumor necrosis factor-a (TNF-a) and interleukin-6 (IL-6) concentrations and IVF/ICSI outcomes in infertile patients with stage III or IV endometriosis. DESIGN: Prospective, randomized controlled trial. MATERIALS AND METHODS: A total of one hundred infertile patients with stage III or IV endometriosis were randomly assigned either to pioglitazone group (n¼50) or control group (n¼50). The long protocol of GnRH agonist (GnRH-a) was used for controlled ovarian stimulation (COS) in all subjects. Those in pioglitazone group were treated with pioglitazone at a dose of 30mg/day orally from the starting day of GnRH-a administration to the day of human chorionic gonadotropin (hCG) injection. Oocyte retrieval was performed 35-36 h after hCG injection and one to three embryos were transferred into the uterus on the third day after oocyte retrieval. RESULTS: There were no significant differences in patient’s characteristics between pioglitazone and control groups. There were also no differences in total dose and days of rhFSH administered, and numbers of retrieved oocytes, mature oocytes, fertilized oocytes, and embryos transferred between the two groups. However, number of garde 1 or 2 embryos was significantly higher in pioglitazone group of 3.21.9 compared with 2.01.6 ml in control group (P¼0.001). FF TNF-a and IL-6 concentrations at oocyte retrieval were also significantly lower in pioglitazone group (P<0.001, P<0.001). Embryo implantation rate was significantly higher in pioglitazone group of 23.3% compared with 13.7% in control group (P¼0.025). There were no differences in the clinical pregnancy rate, miscarriage rate and multiple pregnancy rate between the two groups. CONCLUSION: Pioglitazone treatment can reduce FF TNF-a and IL-6 levels in COS cycles, and therefore may be beneficial in improving IVF results and embryo implantation rate in patients with stage III or IV endometriosis undergoing IVF/ICSI.

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ASRM Abstracts

P-481 Wednesday, October 24, 2012 THE IMPACT OF HEALTH CARE REFORM ON ACCESS TO ART IN THE U.S.. A. Y. Armstrong, H. Eltoukhi, J. H. Segars. Program in Reproductive and Adult Endocrinology, NICHD/NIH, Bethesda, MD. OBJECTIVE: 1% of couples in the U.S. who experience infertility pursue ART. The average cost of ART varies from $10-15,000 per attempt (2012 USD). Live birth rates average 25% to 30% and multiple cycles are frequently required to achieve a pregnancy. As a result of the substantial cost of ART treatment, many couples may not be able to afford this care. In this investigation we assessed the potential impact of healthcare legislation on access to ART. DESIGN: Retrospective review. MATERIALS AND METHODS: A PubMed search was conducted from 2002-2012 using search terms, ‘‘in vitro fertilization, insurance, & access.’’ The sections of the current healthcare legislation relating to reproductive health were also reviewed. RESULTS: Three types of insurance were identified: 1) universal mandates, insurance covers ART; 2) restricted, some specified types of insurers must cover ART; and 3) other, infertility treatment but not ART is covered. Only 15 States have passed legislation that mandates insurance coverage for infertility treatments. A minimum benefits package has not been established under the Patient Protection and Affordable Care Act (ACA). Universal mandates led to a substantial increase in access to ART services. Most importantly, patients in universal mandate markets had a lower number of embryos transferred (P<0.01) This decrease in embryo number led to a decrease (P<0.01) in high order multiples (HOM). CONCLUSION: The current healthcare policy will increase ART access if ART is included in the minimum benefits package; however, a minimum benefits package has not been established. Currently, the strategies which are likely to have the greatest impact on access to ART services would be to protect existing mandates and support additional mandates. A federal mandate that includes ART would markedly increase ART access and may have the long term effect of decreasing cost due to HOM. Supported by: This research was supported, in part, by the Intramural Program in Reproductive and Adult Endocrinology, NICHD, NIH. P-482 Wednesday, October 24, 2012 ARE THE ANNEXIN V MICROBEADS, A REAL SOLUTION TO SPERM DNA FRAGMENTATION?. A. Domingo, C. Anarte, J. A. Agirregoikoa, I. Calvo, J. L. De Pablo, G. Barrenetxea. Quiron Bilbao Assisted Reproduction Center, Bilbao, Bizkaia, Spain. OBJECTIVE: The aim of our study was to assess the effect of annexin V microbeads on cycles with abnormal sperm DNA fragmentation (>20%), in fertilization, embryo quality and success rate. DESIGN: Analytic Retrospective Observational study. MATERIALS AND METHODS: We analyzed 88 cycles of Assisted Reproduction treatments.All patients had abnormal sperm DNA fragmentation and previously had undergone one or more unsuccessful cycles of IVF/ICSI.The cycles were divided into two groups: Group 1: unsuccessful cycles of IVF/ICSI. Group 2: cycles with sperm sorting with annexin V columns (36 cycles) from these same partners. The sperm DNA fragmentation was assessed by TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling).The embryos were evaluated according to criteria of the Spanish Association for Reproductive Biology (ASEBIR). The transfer was performed on day 3 of embryo development. RESULTS: No significant differences were found between groups or age, nor in the rates of fertilization (P>0.05). However, embryo quality did show statistically significant differences, being higher the number of top quality embryos (A, P¼0.004. A+B, P¼0.02) in group 2. CONCLUSION: The results suggest that the use of annexin V columns improves embryo quality in couples with previous failure in IVF/ICSI. So it could be used, as a useful tool, in patients with previous IVF failures. The fertilization rate shows no significant differences. This may be because sperm with fragmented DNA were capable of fertilizing the egg as effectively as sperm without DNA fragmentation. Supported by: Institutional.

Vol. 98, No. 3, Supplement, September 2012