A simplified technique for visualizing decondensed sperm heads in the Hamster Egg Penetration Test (HEPT)

P-372 Influence of different gradient separation protocols on percent motile spermatozoa recovery. J. B. Paul, E. L. Seidemann, B. W. Webster, R. A. Cochran. Center for Fertility and Advanced Reproductive Medicine, Woman’s Hospital, Baton Rouge, LA. OBJECTIVE: In an effort to compare products and reduce costs, two sperm gradient brands were evaluated with two protocols for efficacy of recovery. DESIGN: Normal (WHO, 1999) semen specimens presented for intrauterine insemination preparation were randomly assigned to one of four treatments: Treatment 1 (n⫽78), Sage Pureception® bilayer gradient consisting of 2 ml 40% gradient layered over 2 ml 80% gradient, Treatment 2 (n⫽78), Sage Pureception® 80% single layer gradient (2 ml), Treatment 3 (n⫽78), Cook Sydney® bilayer gradient consisting of 2 ml 40% gradient layered over 2 ml 80% gradient, Treatment 4 (n⫽79), Cook Sydney® 80% single layer gradient (2 ml). MATERIALS AND METHODS: No more than 2 ml of liquefied semen was placed on each gradient and centrifuged at 300 ⫻ g for 18 minutes and the supernatant removed. The pellet was washed once for 8 minutes and once for 7 minutes in HEPES-buffered medium containing 5 mg/ml HSA at 300 ⫻ g. Prepared spermatozoa were counted and percent motile sperm recovered (PCTR) was calculated. The statistical model included PCTR as the independent variable with treatment, initial active concentration (ACON), initial active total (ATOT), initial grade (IGRD) and final grade (FGRD) as main effects. RESULTS: Mean (⫾ SE) PCTR for Treatments 1– 4 was 29.8 ⫾ 1.6, 28.1 ⫾ 1.6, 32.7 ⫾ 1.5, and 32.3 ⫾ 1.5, respectively. There were no significant differences in PCTR among treatments and the overall mean PCTR was 30.7 ⫾ 1.9. As expected, ATOT contributed significantly (P⬍0.0001) to the variance, while IGRD also significantly (P⫽0.047) influenced PCTR. CONCLUSION: Data in this trial indicate that sperm separation gradient from the two manufacturers can be utilized interchangeably with similar resulting recoveries, thus allowing product selection to be based on cost/ convenience. Additionally, costs can be greatly reduced by employing a single layer protocol with no detriment to efficiency of recovery.

P-373 A simplified technique for visualizing decondensed sperm heads in the hamster egg penetration test (HEPT). A. L. Wilcox, V. W. Aoki, D. T. Carrell. University of Utah, Salt Lake City, UT. OBJECTIVE: The Hamster Egg Penetration Test (HEPT) is a valuable tool in determining the ability of a patient’s sperm to penetrate an oocyte. One of the challenges of the test is the difficulty in mounting the oocytes to read the test. Oocytes must be flattened to the proper depth to visualize decondensed sperm heads in order to obtain an accurate result without causing the oocytes to lyse. The objective of this study was to evaluate the use of the Makler® chamber and compare it to other methods currently in use for reading the HEPT. DESIGN: A randomized study of the Makler® chamber, CYTOTRACK® 5␮ slide and the standard plain glass slide using paraffin wax technique for visualizing decondensed sperm heads for interpretation of the HEPT. MATERIALS AND METHODS: Hamster oocytes were treated with trypsin to remove the zona pellucida and incubated for 3 hours in a concentration of 10 million progressively motile sperm per ml. The oocytes were then washed in HTF supplemented with 10% human serum before being transferred to one of the slides or chambers being tested. The oocytes were transferred to the slides or chamber in a volume of 2–5␮l of media and the cover slips were then gently placed using standard techniques. The oocytes were observed under phase contrast on an Olympus BH-2 microscope. RESULTS: A total of 1,635 oocytes were observed. Compared to the other two techniques the Makler® chamber had the lowest percentage of lysed cells, ⬍1%, and consistently flattened the oocytes to an appropriate depth for visualizing the decondensed sperm heads. The CYTO-TRACK® and paraffin wax techniques facilitated greater flattening, although all oocytes flattened by the Makler® chamber were readable. CONCLUSION: The Makler® chamber provides an easy and consistent

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method for evaluating the HEPT. The other methods were more difficult to become proficient with and required the use of a greater number of oocytes to compensate for lysed oocytes during the mounting process. Supported by: None.

P-374 Average spindle retardance observed using the PolScope predicts cell number in day 3 embryos. J. R. Trimarchi, R. A. Karin, D. L. Keefe. Women & Infants’ Hospital, Providence, RI; Brown University, Providence, RI. OBJECTIVE: The meiosis II (MII) spindle is a barrel-shaped assembly of microtubules that organizes and directs movements of sister chromatids allowing distribution of genetic material into the second polar body and the mature ovum. Uneven chromosome segregation as a result of spindle dysfunction leads to aneuploidy, the most commonly identified chromosome abnormality in clinically recorded pregnancies. We sought to determine whether the PolScope can reliably and accurately quantify the morphometry of the spindle of living human oocytes and determine whether spindle structure predicts subsequent embryo development. DESIGN: The PolScope was employed to examine and quantify the morphometry of the Meiosis II spindle of human eggs prior to ICSI. MATERIALS AND METHODS: 138 eggs from 21 consenting patients who were undergoing ICSI were imaged (218 images) at an equatorial plane using an Axiovert S100TC microscope (0.55 condenser, 40X 0.6 NA objective) (Zeiss) fitted with the PolScope (Cambridge Research & Instrumentation, Inc). Eggs were placed in 5␮l drops of modified HEPESbuffered HTF covered with warm oil in a WilCo glass bottom petri dish (World Precision Instruments) and maintained at 37 ⫾ 0.5°C during examination with a TakaiHit Thermo Plate (Zander Medical Supplies) and objective heater (CRI). Spindle morphometry was measured using Metamorph software (Universal Imaging Corp) and compared using the f-test, ANOVA and logistic regression (Minitab software). RESULTS: Of the 218 images, 125 images (57.3%) from 78 eggs were of sufficient quality for spindle morphometric analysis as defined by PolScope calibration and clear demarcation of spindle boundaries. The repeatability of twenty-four morphometric parameters was calculated using pairs of images from 20 eggs and yielded 8 parameters that represented structural features of the spindle with sufficient repeatability for comparison between eggs. Using these parameters we quantitatively describe the morphometry of MII spindles. Overall, spindle metrics were not significantly predictive of day 3 embryo development, however when only images in which the polar body lay in the same focal plane as the spindle were considered, average spindle retardance was predictive of 24.7% of the variance in day 3 cell number. The degree of embryo fragmentation was not predicted by spindle metrics. CONCLUSION: This is the first study quantitatively describing the morphometry of MII spindles in living human occytes using the PolScope. Of the spindle metrics analyzed, spindle retardance was most predictive of subsequent embryo development suggesting that high microtubule density as seen with the PolScope may be critical for proper spindle function. Supported by: None.

P-375 Assisted hatching of embryos at the cleavage stage (d3) followed by embryo transfer at the blastocyst stage (d5). R. E. Stones, A. Coates, R. K. Matteri, J. S. Hesla. Portland Center for Reproductive Medicine, Portland, OR. OBJECTIVE: There has been some controversy surrounding the value of assisted hatching at the cleavage stage of embryo development. Many clinics hatch embryos on day 2 or 3 of culture and transfer on the same day. Studies have shown there may be an advantage for some patients to extend the culture of these embryos to the blastocyst stage. This study compares the pregnancy and implantation rates of patients who had assisted hatching on day 3 followed by day 5 embryo transfer with a similar group of patients who had blastocyst transfer without assisted hatching. DESIGN: A retrospective study comparing the outcome of blastocyst transfers with or without assisted hatching on day 3. All cycles were carried out at The Portland Center for Reproductive Medicine between 2001–2004.

Vol. 82, Suppl. 2, September 2004