Clinical evaluation of a new optimized sperm wash medium (Promotor™) on post-wash motion parameters

bias against fresh cycles because fresh failures were apt to return for thaw in a timely fashion); and, Group C) all cycles within Group A with clinical pregnancy in both fresh and thawed attempts (analysis of implantation rates). Chi-square was used for statistical analysis.

bryo score greater than -1 SD of the mean was significantly higher than in patients with a score less than -1 SD of the mean when 2, 3, or 4 preembryos were transferred. This trend was also observed in single preembryo transfers, although differences were not significant because of low numbers. Less than ⫺1 SD

Group A

No. Tr

Fresh Clin Preg/Tr (%)

Fresh Sacs/ Preembryo (%)

Thawed Clin Preg/Tr (%)

Thawed Sacs/ Preembryo (%)

1 5/14 (35.7) 5/14 (35.7) 22/54 (40.7) 22/54 (40.7) 2 188/264 (71.2) 285/528 (54.0) 43/139 (31.0) 56/278 (20.1) 3 555/764 (72.6) 1022/2292 (44.6) 75/183 (41.0) 114/549 (20.8) 4 383/580 (66.0) 756/2320 (32.6) 95/222 (42.8) 150/888 (16.90) 4 53/93 (57.0) 94/489 (19.2) 92/197 (46.7) 169/1259 (13.4) Total 1184/1715 (69.0)* 2162/5643 (38.3)** 326/795 (41.0)* 511/3028 (16.9)** * and ** ⫽ p ⬍ 0.0001

Results: Group A is shown in the table. Significant differences were found for both clinical pregnancy (69.0% vs 41.0%) and implantation (38.3% vs 16.9%) in the group as a unit, and in most subgroups categorized by number of preembryos replaced. Similarly, Group B (711 cycles) demonstrated significantly lower thaw pregnancy and implantation rates (336/ 711 vs 294/711; 47.3% vs 41.4%; p ⬍0.05 and 507/2408 vs 448/2613; 21.1% vs 17.1%; p ⬍0.0005) in matched fresh versus thaw transfers. Group C (147 cycles with clinical pregnancy in each attempt) revealed slightly lower implantation in thawed cycles, but this difference did not reach significance (223/495 vs 233/555; 45.1% vs 42.0%). Conclusions: Cryopreserved conceptuses do not appear to possess the same potentials as their non-cryopreserved siblings. This might be because the best preembryos are selectively chosen for fresh transfer or the result of subtle damage incurred during the freezing/thawing process. Women with cryopreserved preembryos who fail to achieve pregnancy after fresh transfer are at particular risk for low implantation after thaw. Under these circumstances of failure in a fresh attempt, consideration might be given to replacing one additional conceptus after thaw. Supported by: The Center for Reproductive Medicine and Infertility.

P-341 Use of a simple preembryo scoring system for predicting clinical pregnancy and implantation. Rosemary Berrios, Jason Park, Robert N. Clarke, Nikica Zaninovic, Lucinda L. Veeck. Weill Medical Coll of Cornell Univ, New York, NY. Objective: The objective of the study was to evaluate a simple preembryo scoring system for predicting a patient’s chances for becoming pregnant. Design: A retrospective analysis of day 3 preembryo transfers collected over a one-year period in a University, hospital-based system was performed. Donor oocyte recipients and patients over 40 years of age were excluded from the data set. Materials/Methods: Before transfer, preembryos were assigned a score based on blastomere number and morphology. For each preembryo transferred, the number of blastomeres was multiplied by a fragmentation score (1 ⫽ ⬎20% fragmentation; 2 ⫽ 11–20%; 3 ⫽ ⱕ10%). A cumulative preembryo transfer score was calculated by adding the individual score(s) of each preembryo transferred. For data analysis, the mean score of each transfer group (1 transferred, 2 transferred, 3 transferred, or 4 transferred) was calculated. A breakpoint of -1 standard deviation (SD) was found to provide the greatest differences in subsequent pregnancy and implantation results. Interestingly, scores above the mean or greater than ⫹1 SD of the mean were not associated with increasingly higher pregnancy or implantation rates. It was determined that the optimal breakpoint values (mean -1 SD) for cumulative preembryo scores when 1, 2, 3, or 4 preembryos were transferred were 16, 34, 54, and 73, respectively. Data were analyzed using the Fisher’s exact test with significance at p ⬍0.05. Results: The number of patients, patient age, clinical pregnancy rate (CP), and implantation rate (IR) for preembryo scores below and above the breakpoint of -1 SD of the mean is detailed in the table below. These data are categorized based by the number of preembryos transferred. The clinical pregnancy and implantation rates of patients having a cumulative preem-

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Abstracts

No. No. Avg Transf Cycles Age 1 2 3 4 ⫽ NS; analysis.

a,e

12 29 64 74 b,d,f

35.1 33.5 32.3 35.8

Greater than ⫺1 SD

CP/ Transf (%)

IR/ Transf No. (%) Cycles

Avg Age

CP/ Transf (%)

IR/ Transf (%)

16.7a 34.5b 40.6c 48.7d

16.7e 20.7f 19.8g 17.6h

34.2 33.1 32.1 36.2

30.0a 59.5b 67.3c 62.0d

30.0e 37.6f 37.6g 27.5h

60 153 407 447

⫽ p ⬍ 0.05; h ⫽ p ⬍ 0.0005;

c,g

⫽ p ⬍ 0.0001; chi-square

Conclusions: A simple cumulative preembryo transfer score based on blastomere number and percent fragmentation proves effective in predicting acceptable or reduced chances for clinical pregnancy when -1 SD of the mean is used as a breakpoint value. Pregnancy and implantation were not enhanced with scores above the mean or above ⫹1 SD of the mean, suggesting that day 3 preembryos with good morphology and greater than average blastomere number do not contribute at higher rates to positive outcomes. Supported by: The Center for Reproductive Medicine and Infertility, Weill Medical College of Cornell University.

P-342 Clinical evaluation of a new optimized sperm wash medium (Promotor™) on post-wash motion parameters. Scott M. Slayden, Hilton I. Kort, Joe B. Massey, Carlene W. Elsner, Andrew A. Toledo, William E. Roudebush. Reproductive Biology Assoc, Atlanta, GA. Objective: Sperm prior to any assisted reproductive technique (e.g. intrauterine insemination and in vitro fertilization) must be washed in order to remove the seminal fluid and to insure that the most normal motile sperm are available for conception. Typically, density type washings (e.g. silane coated colloidal silica bead suspensions) are used, followed by washing and resuspension in a modified basic salt solution (sperm wash medium). The study objective was to compare commercially available sperm wash media on motility motion parameters. Design: Prospective randomized comparison of six commercially available sperm wash media on post-wash sperm motility via computer assisted semen analysis (CASA) parameters. Materials/Methods: Semen samples were divided into one of six sperm wash treatment groups: IS-Sperm Wash Medium ($25/100mL; Irvine Scientific, Santa Ana, CA), PureSpermTM ($85/100mL; Nidacon Laboratories AB, Gothenburg, Sweden), Quinn⬘s Sperm WashTM ($27/100mL; SAGE Biopharma, Bedminster, NJ; Enhance-WTM ($33/100mL; Conception Technologies, San Diego, CA) IVC-Sperm Wash MediumTM ($35/100mL; InVitroCare, San Diego, CA), PromotorTM Sperm Rinse ($40/100mL; CERES Fertility, San Diego, CA) prior to isolation (400g, 12 minutes) through a silane-coated silica bead processing method (PromotorTM, CERES Fertility). Sperm were then washed (4mL;300g, 8 minutes) and resuspended (0.5– 0.8mL final volume) in the appropriate medium. CASA (IVOSv10.9i, Hamilton-Thorne Research, Beverly, MA) motion parameters (including percent hyperactivation) were obtained before and after sperm washing (Mortimer ST, J Androl 2000;21:515–24). Data were analyzed by analysis of variance and the Tukey test. Results: A total of 201 semen samples were processed as described. Mean path velocity (VAP), progressive velocity (VSL), track speed (VCL), lateral amplitude (ALH), beat frequency (BCF) and percent hyperactivation (HAV), before and after processing via CASA are presented in Table 1. Whereas all sperm wash media significantly (p ⬍0.05) improved most sperm motion parameters over the initial-raw values, Promotor⬘s improvement was significantly (p ⬍0.01) greater than all other media in all CASA categories. Additionally, the percent of hyperactivated (as defined by the CASA categories VCL, ALH, and linearity) sperm was significantly (p ⬍0.01) greater in Promotor than all other media.

Vol. 78, No. 3, Suppl. 1, September 2002

Table 1. CASA motion parameters, before and after semen processing. Sperm Wash

VAPa

VSLb

VCLc

ALHd

BCFe

HAVf

Raw Conception SAGE Nidacon InVitroCare CERES Irvine

53.5g 59.9h 60.7i 71.4j 61.5k 89.2g-l 63.4l

45.5m 50.5n 49.9o 61.4 53.3 74.1m-o 56.5

73.4p 92.9q 95.0r 112.2s 93.1t 145.9p-u 98.3u

1.7 4.2w 4.4x 4.6y 4.2z 5.6v-A 4.1A

25.8B 24.4C 23.9D 28.3 27.1 31.9B-D 27.5

0.9E 1.2F 1.6G 7.3H 0.4I 19.6E-J 2.3J

Conclusions: 1. Frozen-thawed blastocysts resulting from previously biopsied embryos can remain viable and result in a normal pregnancy. 2. Due to its cumbersome nature, PGD testing must be performed by a highly specialized genetics laboratory, rather than be incorporated into every IVF laboratory’s protocol. In our experience, collaboration between an IVF program and an outside genetics laboratory has proven efficient for PGD case management. Supported by: n/a.

P-344

Due to space limitations, the exhaustive statistical comparisons cannot be presented. Therefore, only the major differences are included (i.e. CERES vs. ‘others’). Similar superscripts are significantly different, ANOVA: a-f , P ⬍ 0.01; Tukey Test: g-z; A-J, P ⬍ 0.01.

Relationship between the survival rate of thawed embryos and clinical outcome in frozen thawed embryo transfer cycles. Ying Zhou, Xuefeng Huang, Junju Lin, Bilu Ye. The First Affiliated Hosp of Wenzhou Medical Coll, Wenzhou, China.

Conclusions: While most sperm washing products significantly improved sperm performance over the raw specimens, PromotorTM consistently had superior end products across every CASA category. Considering the substantial improvement of sperm motion characteristics and cost, PromotorTM is an ideal medium for routine sperm washing for assisted reproduction. Supported by: CERES Fertility, San Diego, CA; IVFonline.com, Guelph, Ontario, Canada; International, Guilford, CT; SAGE Biopharma, Bedminster, NJ; Irvine Scientific, Santa Ana, CA.

Objective: Embryo cryopreservation is a well established technique in most IVF clinics. However, it has been shown that not all human embryos survive after cryopreservation. As a result, it is common practice in our centre for frozen thawed fully intact embryos to be used together with partially damaged ones for subsequent transfer. This study was undertaken to analyze the influence of the survival rate of thawed embryos on clinical outcome in frozen thawed embryo transfer cycles. Design: A retrospective analysis of frozen embryo transfer results from June 2000 and December 2001. Materials/Methods: Embryos were cryopreserved on day 2 or on day 3 with 1,2 propanediol sucrose by slow freezing protocol (Cohen et al.,1988) and were thawed using rapid thawed protocol. Survival was defined as at least 50% of the blastomeres being intact. Frozen thawed embryos were replaced in natural cycles or artificial cycles (hormone replacement treatment HRT). Results: A total of 294 frozen thawed embryos were transferred in 106 transferred procedures, including 138 fully intact embryos (47%) and 156 embryos with damaged blastomeres (53%). Overall, clinical pregnancy rate was 22% (23/106); the embryo implantation rate was 8.8%. (26/294). There were 16 transfer cycles with fully intact embryos (39 embryos) and 20 transfer cycles with partially damaged embryos (45 embryos). Other transfer cycles were mixed transfers of fully intact embryos together with partially damaged ones (70 cycles). The percentage of gestational sacs with fetal heartbeat and pregnancy cycle obtained after only transfer of fully intact embryos was almost 9 times (18% versus 2%) and 6 times (31% versus 5%) higher than those after only transfer of partially damaged embryos (p ⬍0.05) (table I). There was significant difference (p ⬍0.05) of implantation rates (4%; 9%; 12.3%; 28.6% respectively) when 0, 1, 2 and 3 fully intact 4 cells or 8 cells embryos after thawing were transferred.

P-343 Single in vitro fertilization (IVF) cycle with blastomere biopsy for preimplantation genetic diagnosis (PGD) of Huntington’s disease, assisted hatching and cryopreservation results in healthy baby and subsequent ongoing pregnancy. Marina Gvakharia, Julie L. Hubbard, G. David Adamson, Mark Hughes. Fertility Physicians of Northern CA, San Jose, CA; Wayne State Univ, Detroit, MI. Objective: The invasive nature of embryo biopsy causes legitimate concerns about its safety and the viability of embryos undergoing this procedure for preimplantation genetic diagnosis. The objective of this report is to communicate a healthy singleton delivery from a single IVF cycle with fresh embryo transfer followed by a clinical pregnancy from a cryopreserved embryo transfer using embryos that were subjected to assisted hatching, embryo biopsy and culture to the blastocyst stage. Furthermore, a private IVF program’s approach to the management of PGD case will be described. Design: A 28 old woman with family history of Huntington’s disease was enrolled as a participant in an IRB-approved embryo biopsy/PGD study. The couple was fertile and their goal was to obtain unaffected embryos for transfer and possibly cryopreservation. The patient’s own carrier status was unknown to either the couple or the IVF center and the patient explicitly did not want to know her Huntington’s disease status. Materials/Methods: Ovarian hyperstimulation, transvaginal oocyte retrieval, in vitro fertilization, and embryo biopsy were performed at Fertility Physicians of Northern California (Palo Alto and San Jose, CA) (FPNC). Based on the couple’s request of non-disclosure of their results, information pertaining to the performance of the couple’s gametes and embryos (e.g. number of oocytes, fertilization rate, embryo biopsy results, etc) is to be kept confidential and, therefore, is not included in this report. One or two blastomeres were removed from 6 – 8 cell embryos 6 – 69 hours after oocyte retrieval. Obtained blastomeres were then placed in 200 ␮l PCR tubes containing 5 ␮l of lysing buffer and tubes were hand-delivered within 12 hours to HughesLab at Wayne State University (Detroit, MI) for genetic analysis. Results of PCR analysis became available 36 hour later and were sent to FPNC via fax transmission. Results: Four blastocysts became available following extended culture of biopsied embryos. Two blastocysts were transferred to patient’s uterus on day 5 of embryo culture. Two remaining blastocysts were cryopreserved in 9% glycerol solution using slow freezing protocol. The patient became pregnant with a singleton fetus. Amniocentesis and confirmatory prenatal testing were performed at 12 weeks of pregnancy. This pregnancy resulted in a term delivery of a healthy boy. Approximately two years later, the patient returned to FPNC for frozen embryo transfer. Two blastocysts were thawed and transferred in a standard protocol unstimulated cycle. This transfer resulted in a currently ongoing normal singleton pregnancy.

FERTILITY & STERILITY威

Table I Outcome of embryo transfer according to the survival after cryopreservation

Number Number Number Number

of of of of

embryos transfers transferred cycles clinical pregnancies embryos implantation

Intact embryos only

Damaged embryos only

Mixed transfers

39 16 5 (31%)a 7 (18%)b

45 20 1 (5%)a 1 (2%)b

211 70 16 (23%) 18 (9%)

Chi square test a p ⬍ 0.05 b p ⬍ 0.05 Conclusions: Although clinical pregnancy has been obtained after transfer of partially damaged embryos, their implantation rate was lower greatly than that after transfer of partially damaged embryos. The survival rate of thawed embryos and clinical outcome in frozen thawed embryo transfer cycles were showed distinctly correlation. The aim of a cryopreservation program must be to obtain fully intact embryos after thawing. Supported by: Local foundation.

P-345 Comparison of two embryo culture systems: The upgraded B2 INRA Medium威(CCD, France) & the Universal IVF Medium威(Medicult, Denmark). Gautam Allahbadia, Goral Gandhi, Rubina Merchant, Kaushal

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