immersed in liquid nitrogen. For single sperm cryopreservation, sperm werefirst isolated microscopically and pooled into a microdrop containing cryo-protectant. Cryoloops were placed in a specially designed holder. Individualsperm were selected with a standard ICSI micropipette and depositeddirectly into the cryoprotectant film covering the cryoloop. Cryoloops wereloaded with 5- 10 sperm. Cryoloops with sperm were subsequently thawedby direct immersion into a culture media droplet. Viability testing wasperformed by eosin-nigrosin staining. Sperm motiltiy and recovery wereassessed. Data was compared to control samples frozen in conventionalcryovials. Sperm function testing was also performed. The ability of spermcryopreserved on cryoloops to fertilize oocytes was first tested in the bovinemodel. Subsequently, human sperm frozen on cryoloops were tested fortheir ability to induce pronuclear formation. Intracytoplasmic sperm injec-tion was performed on discarded unfertilized oocytes from the in vitrofertilization laboratory.

Results: Sperm recovery, motility and viability are shown in the table.Sperm function testing demonstrated that both bovine and human spermcryopreserved on loops were capable of fertilizing oocytes. In the bovinemodel, 48 of 100 oocytes injected exhibited two polar bodies and 88% ofthese zygotes cleaved. Thawed human sperm cryopreserved on loops werealso able to induce pronuclear formation.

Conclusion: Individual spermatozoa can be easily cryopreserved andrecovered from cryoloops. The thawed sperm appear to be viable andfunctional. Cryoloops may be an excellent alternative to hamster zonae forcryopreserving small numbers of human sperm. Clinical trials are needed toexplore this possibility.

Tuesday, October 14, 20033:00 P.M.

O-148

Pregnancy derived from human nuclear transfer. John Zhang, Guan-glun Zhuang, Yong Zeng, Carlo Acosta, Yimin Shu, Jamie Grifo. NewYork Univ Sch of Medicine, New York, NY; Sun Yat-Sen Univ of MedicalScience, Guangzhou, China.

Objective: The behavior of an oocyte’s nucleus is closely regulated by itscytoplasmic environment. Nuclear transfer, i.e., the removal and transfer ofthe nucleus (karyoplast) of an oocyte into the cytoplasm (cytoplast) ofanother enucleated oocyte, has shown that, in several mammalian species,embryogenesis and implantation is influenced by cytoplasmic factors inmature oocytes or the zygotes they produce.

Design: Case report of a triplet pregnancy after transfer of pronuclei froma patient’s zygotes into zygotic cytoplasts donated by a fertile female. Workwas approved by Sun Yat-Sen University Hospital IRB.

Materials/Methods: A 30-year-old nulligravida female had two failed IVFcycles characterized by embryo arrest at the 2-cell stage. Controlled ovarianstimulation was synchronized for this patient and her egg donor so that theirMII stage oocytes were retrieved within a 2 h period. Following ICSI, 8 of12 patient oocytes and 12 of 15 donor oocytes were fertilized. All pronuclei(PNs) were then removed from the donor zygotes and discarded. Male andfemale PNs were removed from each patient zygote and transferred sub-zonally into a donor cytoplast (enucleated zygote). Electrofusion of thepatient karyoplast with the donor cytoplast resulted in 7 “reconstructed”zygotes. The 5 “reconstructed” zygotes that cleaved to the 4 cell stage at48 h (after retrieval) were transferred to the patient’s uterus. Nuclear andcytoplasmic DNA profiles were analyzed in bloods from the patient, oocytedonor and fetuses. Nuclear DNA fingerprinting was performed at 5 micro-satellite loci with subtraction of the husband genotypes. Cytoplasmic mito-chondrial (Mt) DNA was analyzed by amplification and sequencing of a524-bp segment in the D-loop region (16024-577).

Results: A triplet pregnancy with fetal heartbeats was achieved. Fetal

reduction to a twin pregnancy was performed transvaginally at 33 dayspost-transfer. At 24 weeks Fetus B delivered due to premature rupture ofmembranes and died of respiratory distress. At 29 weeks Fetus C deliveredafter intrauterine fetal demise due to cord prolapse. Normal karyotypes werefound in the embryonic tissue (46, XY) and the 24 week (46, XX) and 29week (46, XY) fetuses. Nuclear genetic fingerprinting confirmed that thenuclear DNA from 24 and 29 wk fetuses matched that of the patient’s. MtDNA profiles in fetal red blood cells were similar to those from cytoplastdonor with no detection of patient (karyoplast donor) Mt DNA.

Conclusion: Viable human pregnancies with normal karyotype can beachieved through nuclear transfer. Reconstruction of zygotes by nucleartransfer allows mitochondrial DNA, but not nuclear DNA, to come from thedonor oocyte with no mitochondrial DNA polymorphism. This findingsuggests a unique approach to correct mitochondrial genetic disorders ofmaternal inheritance. Ongoing work to establish the efficacy and safety ofnuclear transfer will result in its use as an aid for human reproduction.

Tuesday, October 14, 20034:00 P.M.

O-149

Junctional zone contractions during zygote intrafallopian transfer.David Levran, Nariman Zahalka, Gustavo Malinger, Jacob Farhi, MarekGlezerman, Ariel Weissman. Wolfson Medical Ctr, Holon, Israel.

Objective: Several studies have focused on junctional zone contractions(JZC) frequency and the outcome of IVF-ET. Fanchin et al. (1988) havenoticed a stepwise decrease in pregnancy and implantation rates withincreasing JZC frequency before ET. One possible mechanism for repeatedimplantation failure (RIF) in IVF-ET could be an abnormal pattern of JZCduring the periovulatory period and at the time of ET, causing the expulsionof embryos from the uterine cavity. ZIFT has been suggested as a powerfulclinical tool for the treatment of patients with RIF (Levran et al., 1998). Ourobjective was to evaluate and characterize the pattern of JZC in patientswith RIF undergoing ZIFT.

Design: Prospective observational study.Materials and Methods: Twenty-three women with a history of � 3 failed

IVF cycles and presence of a patent tube underwent ZIFT. Transvaginalultrasound was performed for 5 minutes immediately before and right aftergeneral anesthesia and during the actual tubal transfer procedure and fiveminutes later. The scan was centered on a sagittal plane of the uterus. Thesignals were recorded and analyzed during fast-forward replay. The fre-quency and direction of contractions were evaluated for each examinationand compared.

Results: The mean age of the patients was 34�5.1 years, they had7.3�3.3 previous IVF cycles and 5.05�0.85 zygotes were transferred. Six(26.1 %) of the patients conceived. JZC were observed in all cases. A highJZC frequency was observed just before anesthesia (4.5�2.4 /min), whichdecreased significantly after anesthesia (2.1�1.6; p � 0.001). Contractionsfrequency was significantly increased again during ZIFT (5.5�2.6 /min) ascompared to the frequency after anesthesia (p � 0.001), and decreased againafter completing the procedure (4.0�1.4; p � 0.002). Interestingly, JZCfrequency before anesthesia was significantly lower in patients who con-ceived as compared to those who did not (2.04�0.6 vs.5.4�226; p �0.004). No correlation was found between the direction of JZC and con-ception.

Conclusion: This is the first report on JZC during ZIFT. Our study clearlydemonstrates that general anesthesia reduces JZC frequency and tubalmanipulation induces JZC. As with conventional ET, a correlation betweenincreased JZC frequency before ZIFT and cycle failure has been established.Measurement of JZC frequency may aid in better selection of patients whomay benefit from ZIFT. The quest for the mechanism responsible for thehigh efficiency of ZIFT in patients with RIF remains ongoing.

Tuesday, October 14, 20034:15 P.M.

O-150

Blastocyst formation after intracytoplasmic sperm injection (ICSI) andculture in two sequential culture media preparations differing withrespect to the method of protein supplementation. Tayyab Rahil, Steven

S56 Abstracts Vol. 80, Suppl. 3, September 2003