following culture of oocytes immediately post-ICSI (day 0) through ET inG1 versus HS.

Materials and Methods: Oocyte cohorts from 94 retrievals were random-ized to G1 or HS. Oocytes were cultured in HS from retrieval until ICSI(2–4 hrs). Following ICSI oocytes were cultured in G1 or HS until thefertilization check (16–18 hrs post-ICSI). Normally fertilized zygotes weretransferred to fresh drops of the designated media and cultured until ET, themorning of day 2.

Results: Study outcomes are shown below (N-retrievals; average # eggs,# inseminated (ins.) & # 2PN/retrieval; Gr 0-best to 3-poor).

Media Age N#

Eggs#

Ins.#

2PN%

2PN%

1PN%

$3PNPNGr

CellsET

GrET

#Trs.

%IR

%PR

G1 ,35 33 12.2 9.5 7.7 80.3 3.2 2.5 0.70 4.17 0.62 2.62 38.2 58.6

HS ,35 33 16.4 13.1 10.9 83.3 2.5 1.6 0.66 3.75 0.82 2.76 31.9 60.0

G1 35–38 15 10.9 8.5 6.7 78.1 5.5 3.1 0.77 3.58 0.73 3.23 26.2 69.2

HS 35–38 13 13.6 11.1 9.23 67.8 4.5 2.3 0.73 3.73 0.76 3.38 20.5 46.2

Oocytes cultured immediately post-ICSI in G1 and HS had similar rates offertilization, parthenogenetic activation and polyploidy. No differenceswere noted in embryo quality at the PN or cleavage stage or number of cellsper embryo at ET. Patients randomized to HS had significantly moreoocytes retrieved and inseminated (p,0.003) and consequently had agreater number of 2PN embryos (p,0.001) than patients randomized to G1.In spite of the greater number of embryos transferred in the HS groups, IRtended to be higher for G1 than HS, although not significantly different,likely due to the small study size. The pregnancy rate also tended to behigher for G1 than HS in patients 35–38 (p50.23).

Conclusion: These results show that oocytes may be placed into G1immediately following ICSI without decreasing fertilization rates. G1 maybe beneficial for embryo culture even when embryos are to be transferred asearly as the morning of day 2.

P-026

Improved Embryo Quality and Rates of Cryopreservation With Mod-ifications in an In Vitro Fertilization (IVF) Culture System. A. Khabani,S. Shen, V. Y. Fujimoto, B. S. Houmard, C. Rainer, D. E. Battaglia.Department of Obstetrics and Gynecology, University of WashingtonSchool of Medicine, Seattle, WA.

Objectives: To compare embryo morphology and clinical outcome in anIVF laboratory before and after significant modifications were made in theembryo culturing technique.

Design: Implantation rates per transfer, rates of cryopreservation, andembryo morphology were retrospectively analyzed in patients undergoingIVF before and after modifications were made in the embryo culturing system.

Materials and Methods: A total of 174 IVF cycles with d3 embryotransfer were studied from 1/99 to 12/99. A total of 1168 embryos wereincluded for analyzes. From 1/99 to 9/99, our embryo culturing protocol(protocol A) included the following: (1) each embryo was cultured individ-ually in a 50mL drop of culture media and (2) embryo morphology wasassessed on d2 and d3 of culture. In 10/99, modifications were made to ourculturing protocol (protocol B) as follows: (1) elimination of d2 embryomorphology assessment to reduce embryo stress; (2) grouping of embryosduring culture (4–8 embryos in 50mL of culture media per culture dish);and (3) the addition of separate working incubators to reduce incubator dooropenings in the culture incubators. Embryo quality was based on morpho-logical “grade” relating to fragmentation and relative blastomere size, andcleavage rates (embryo quality5 cleavage rate3 grade). Embryo selectioncriteria for cryopreservation was not changed. Implantation rates, cryopre-served embryo number and rates, and embryo morphology were comparedbetween the two protocols using Chi Square analyzes.

Results: Lead (best) embryo quality was significantly improved withprotocol B compared to protocol A (mean score of 11.9 vs. 9.9, P50.016).There was also a significant improvement in both the number of embryoscryopreserved (33 vs. 19%, P,0.0001) and cryopreservation rates pertransfer (47.1 vs. 28.3%, P,0.05) with protocol B compared to protocol A.A trend toward improved implantation rates per transfer with protocol Bwas seen but this did not reach statistical significance (25% for protocolB vs. 20% for protocol A, P50.2). Mean patient age was similar in groupsB and A (37.8 vs. 36.2 years).

Conclusions: The results of this preliminary study strongly suggest thatan improvement in IVF outcome with respect to embryo quality, cryopreser-vation rate, and possibly implantation rate may result from grouped embryoculture. We cannot exclude the possible roles of reduced embryo observa-tion and increased stabilization of the culture environment in the improvedembryo quality seen in this study. These observations warrant further in-vestigation into the possible role of grouping embryos in culture during IVF.

P-027

Human Sperm and Oocytes Do Not Need Glucose for Normal Fertili-zation and Embryo Development: A Preliminary Report. B. Behr, D.Dasig, A. A. Milki. Department GYN/OB, Stanford University MedicalCenter, Stanford, CA.

Objectives: Much debate still exists regarding the requirement for glucoseduring standard IVF insemination and embryo culture to day 3. It has beenproposed in both the human and mouse models that fertilization in vitro isimpaired by the absence of glucose. The objective of this study was todetermine if the absence of glucose in sperm washing and embryo culturemedia affects fertilization and cleavage rates in patients undergoing stan-dard in-vitro fertilization.

Design: Analysis of 181 oocytes in 12 patients with at least 8 oocyteswere randomly allocated into one of 2 groups: Group A & Group B prior toinsemination. There was no glucose in the culture medium. Patients acted astheir own controls. Outcome measures were 2 PN fertilization rates andsubsequent embryo development.

Materials and Methods: Sperm from 12 consecutive normozospermicpatients were prepared as follows for standard in-vitro fertilization. After aminimum of 2hr incubation at RT in 1:1 mixture of TYB, density gradientseparation of the samples was performed before being divided into 2 groupsfor washing and capacitation: Group A: no glucose exposure (P1 wash);Group B 6 mM glucose exposure (Blastocyst Medium (BM)). Both mediawere supplemented with 5% HSA. Following retrieval, oocytes were ran-domly allocated into droplets of our standard culture medium P1 undermineral oil. Half of the oocytes were inseminated using partner spermwashed and incubated in P11 5% HSA (Group A) and the other half usingpartner sperm washed and incubated in BM1 5% HSA (Group B). Oocyteswere incubated at 37°C in 5% CO2/5% O2/90% N2 gas phase for 18–20 hrspost insemination before fertilization check and then returned to P1 untilday 3 under the same conditions. All media and HSA were obtained fromIrvine Scientific.

Results: Student’s paired t test confirmed no significant difference infertilization or embryo development rates (p.0.1). Sixty five out of 91(71%) oocytes inseminated in Group A fertilized normally displaying 2 PN.Sixty eight of 90 (76%) oocytes inseminated in Group B fertilized normallydisplaying 2 PN. Additionally there was no difference in embryo develop-ment from 2 PN. Sixty three of 65 (97%) zygotes in Group A cleavednormally and 68 of 68 (100%) zygotes in Group B cleaved normally.

Conclusions: In this preliminary report, it appears that the omission ofglucose had no deleterious effects on fertilization and cleavage. We cannotrule out any beneficial effect of the TYB pre-incubation of the semen thatmay mask the requirement of glucose. However, it is possible that theproposed negative effects of the absence of glucose reported by others maybe due to the presence of other culture medium constituents, like EDTA,and not the lack of glucose itself. No data on pregnancy outcome wascollected with respect to the sperm preparation because in most casesa mixed group of embryos were transferred. Data on a larger series willbe presented.

P-028

Effect of Communal In Vitro Culture on Human Embryo Development.1H. I. Higdon III, 1W. R. Boone, 2W. C. Bridges, Jr.,2J. R. Rieck.1Department of Obstetrics and Gynecology, Greenville Hospital System,Greenville, South Carolina, and2Department of Experimental Statistics,Clemson University, Clemson, SC.

Objective: To evaluate the effect of communal in vitro culture on humanembryo development.

S104 Abstracts Vol. 74, No. 3, Suppl. 1, September 2000

Design: A prospective randomized design. Included in this study were 61patients who (1) were,40 years of age, (2) were undergoing fresh, non-donor oocyte transfers, and (3) had, three IVF-ET cycles.

Materials and Methods: Zygotes were placed into culture media at one ofthe following volumes: Group 1—1 in 10mL; Group 2—2 in 10mL; Group3—3 in 15mL; Group 4—4 in 20mL; Group 5—5 in 25mL. After eitherthree (cultured in Human Tubal Fluid [HTF; Irvine Scientific, Santa Ana,CA]) or six days (cultured three days in HTF and moved into G2.2(Scandinavian IVF Science AB, Gothenburg, Sweden]) of in vitro incu-bation, embryos were evaluated for stage and grade prior to embryotransfer. All calculations for the effect of cohort size and to estimate thecohort size means for stage and grade were performed using the MIXEDprocedure of SAS.

Results:

Effect of grouped culture on human embryo development

Group 1 Group 2 Group 3 Group 4 Group 5 P value

Embryonic stage

Day 3 embryosa 6.266 0.30 6.426 0.25 6.746 0.26 6.516 0.28 6.736 0.33 0.82

N groups/N zygotes 24/24 34/68 31/93 31/124 25/125

Day 6 embryosb 2.686 0.43 2.936 0.68 3.116 0.42 3.026 0.37 3.046 0.45 0.99

N groups/N zygotes 16/16 6/12 17/51 25/100 19/95

Embryonic gradec

Day 3 embryos 1.976 0.14 2.106 0.11 2.006 0.12 2.106 0.13 1.846 0.16 0.50

N groups/N zygotes 23/23 34/68 30/90 29/116 22/110

Day 6 embryos 3.236 0.28 2.986 0.43 2.946 0.27 3.206 0.23 3.396 0.29 0.83

N groups/N zygotes 16/16 6/12 17/51 25/100 19/95

a Day 3 Stage5 number of blastomeres in an embryo.b Day 6 Stage5 0–dead; 1–no change from Day 3 status; 2–morula; 3–earlyblastocyst; 4–blastocyst; 5–expanded blastocyst; 6–hatching blastocyst; 7–hatchedblastocyst.c Embryonic Grade5 1–excellent; 2–good; 3–fair; 4–poor.

Conclusion: These data suggest that cohorting embryos in the zygote:volume ratios investigated in this study had no effect on embryo devel-opment.

P-029

Effects of Operating Room (OR) Disinfection with Quaternary Ammo-nium Salts on Human Embryo Quality. N. Prados, M. J. De los Santos,C. Albert, J. L. Romero, J. Remobi, A. Pellicer. Instituto Valencianode Infertilidad and Dept Ob/Gyn., School of Medicine, Valencia, Spain.

Objective: Embryo culture laboratories are usually set up near OR, whereoocyte retrieval and embryo transfer are performed. These OR are routinelydisinfected, which could be harmful to the nearby cultured embryos. Wehave analyzed the effect on embryo quality and pregnancy rate of disin-fecting with a quarternary ammonium salt aerosol.

Design: Retrospective analysis of cycles of conventional in vitro fertili-zation (IVF) or intracytoplasmic sperm injection (ICSI) performed duringthe OR disinfection in the last six months of 1999. The rate of fertilization,embryo quality and pregnancy was compared with cycles that did notcoincide with the disinfection.

Materials and Methods: The disinfection consisted of a spray of a quar-ternary ammonium salt aerosol (Dynamin-A, KillGerm) during 2 hours inthe late evening of every second Friday. The door connecting the culturelaboratory with the OR and the air conditioning system were sealed withplastic tape and no work with embryos was carried out, including anyopening of incubators, till next morning when work was resumed. The studygroups consisted of 12 cycles of oocyte retrieval the morning before disin-fection (G1), 23 cycles of oocyte retrieval the morning after disinfection(G2) and 20 cycles in which embryos were transferred the day af-ter disinfection (G3). The results were compared with a group of 44cycles (G0)which were performed when no disinfection was done. Fertiliza-tion rate, number of blastomeres, fragmentation score and pregnancy outcomewere compared within the groups. Fisher’s test and t tests were performed asnecessary.

Results: The following table summarizes the main results (means andstandard deviations). No significant differences were found.

Study group G0 G1 G2 G3

Number of cycles 44 12 23 20Percentage of 2PN

per cycle73.16 27.1 66.26 17.6 69.36 20.7 75.76 23.8

Mean blastomerenumber per cycle

3.56 0.8 3.86 1.1 3.66 0.5 3.56 0.9

Mean fragmentationscore per cycle

1.66 0.4 1.66 0.8 1.66 0.6 1.66 0.4

Pregnancy rate 51% (20/39) 50% (5/10) 52% (12/23) 57% (11/20)

Conclusion: Disinfecting the OR with the described precautions does notseem to affect the fertilization rate, the embryo quality or the pregnancyrate.

P-030

Short Term Sperm-Oocyte Co-Incubation During In Vitro Fertilization(IVF) Does Not Seem to Improve Total Fertilization Rate and EmbryoQuality. S. De Vincentiis, A. G. Martinez, F. Nodar, C. F. Chillik, S.Brugo-Olmedo. Center of Studies in Gynecology and Reproduction -CE-GyR- Buenos Aires, Argentina.

Objective: The purpose of this study was to compare IVF outcome whenshort (1 hour) and standard (16–18 hours) sperm-oocyte co-incubationprotocols were used in our Center.

Design: Prospective, randomized study.Materials and Methods: Forty-two patients (336 4.4 years old), under-

going IVF procedures were included in this study. All males provid-ed normozoospermic samples including normal morphology according toKruger’s Strict Criteria. Unclassified oocytes from each patient (10.026 3.8oocytes per patient) were randomly assigned into two groups. Oocytes wereincubated in HTF supplemented with 15% of SSS (HTF-SSS) for 5–6 hoursuntil insemination was performed. Sperm recovery was carried out usingdiscontinuous Percoll gradients. Group I oocytes (N5 212) were incu-bated with spermatozoa only for one hour (short exposure) and thenrinsed in HTF-SSS and cultured in fresh HTF-SSS medium. Group IIoocytes (N5 209) (standard protocol) were incubated with spermatozoafor 16 –18 hours. Fertilization was checked in both groups 16 –18 hoursafter insemination. Cleavage and embryo quality were evaluated at40 – 42 hours after insemination. The best embryos, from short andstandard incubation, were selected for transfer. Differences in percent-ages of total, normal and abnormal fertilization and embryo cleavagewere compared using Mann-Whitney Test. Embryo quality was com-pared using Fisher’s Exact Test.

Results: Abnormal fertilization differed significantly (Group I: 6.1% vs.Group II: 7.2%, p,0.0001) although normal fertilization remained the same(Group I: 81.6% vs. Group II: 83.7%, not significant). Embryo cleavage(Group I: 3.36 1.5 cells vs. Group II: 3.46 1.9 cells) and embryo quality(Group I: 88.7% vs. Group II: 85.3% of Class III-IV embryos) were notsignificantly different. As we transferred the best embryos from each group,this enabled us to evaluate the effect of shortened exposure on pregnancy,implantation and miscarriage rates. In 16 patients there were embryostransferred corresponding only to one of the groups (short or standard). Theparameters evaluated in these 16 females were not different (Group I: N59, 31.26 3.8 years old, 96 9 oocytes, 2.76 0.7 embryos transferred perpatient, 100% Class III-IV embryos transferred, and Group II: N5 7,326 2.8 years old, 196 12.9 oocytes, 2.96 0.4 embryos transferred perpatient, 95% Class III-IV embryos transferred). Although the numbers areinsufficient to apply a statistical analysis, implantation rate appears to begreater when embryos corresponding only to standard insemination condi-tions were transferred (Group I: 20.8% vs. Group II: 40%) while pregnancyrate was similar (Group I: 44.4% vs. Group II: 42.8%).

Conclusions: Short co-incubation of spermatozoa and oocytes in IVFdoes not improve fertilization rate and embryo quality in our Center.

P-31

Failure of the American Association of Bioanalysts Embryology Cul-ture Proficiency Test to Perform Under Certain Culture Conditions.K. F. Miller, K. L. Fry. Section of Reproductive Endocrinology and Infer-tility, Department of Gynecology and Obstetrics, Cleveland Clinic Founda-tion, Cleveland, OH.

FERTILITY & STERILITY t S105