P-403

Carriers of SMN mutation might be prevalent among oligospermicpatients. Rui C. Serafim, Soraya Abdelmassih, Paulo T. Salgueiro, RogerAbdelmassih, Dimitri Dozortsev. Clin e Ctr de Pesquisa em ReproducaoHumana Roger Abdelmassih, Sao Paulo -SP, Brazil.

Objective: Some recessive mutations have clinical manifestations in aheterozygote state. For instance CAVD has been linked to carriers of CisticFibrosis. At the same time, heterozygote manifestations of the second mostfrequent recessive mutation-SMN, to the best of our knowledge, have notbeen described. There are evidences pointing to SMN role in a small nuclearribonucleoprotein (snRNP) assembling and biogenesis. One isoform ofsnRNP (hnRNP A2/B1) is expressed during spermatogenesis, from thespermatogonia to round spermatid stage. Therefore, we decided to evaluatethe prevalence of the most frequent mutations in SMN gene in oligospermicpatients.

Design: Prospective evaluation of patients with oligospermia for SMNmutation.

Materials and Methods: DNA samples were obtained from the blood of 9oligospermic patients (less than 5 millions sperm cells, motility A and B �50%) using GFX Blood DNA. Following DNA purification we performedPCR reaction for SMN exon gene 7 and 8. To separate and distinguishSMN1 from SMN2, enzymatic reactions were used followed by gel elec-trophoresis and bands intensity quantification.

Results: SMN1 (exon 8) heterozygote and SMN2 homozygote (exon 7and 8) mutations were found in 2 and 2 males respectively. (Table 1)

Even though the patients sample is small, the prevalence of SMN1 foundin our study (2/9) is significantly higher than that of a general population1/60 (p�0.05). The prevalence of homozygote SMN2 mutation in thestudied popultaion was not significantly higher (2/9) than that of generalpopulation (1/10). If confirmed on a larger patient’s sample, oligospermiamay become an indication for both, SMN screening and preimplantationgenetics diagnosis.

Table 1. Bands intensity values

Conclusions: Carriers of SMN mutation might be prevalent among oli-gospermic patients.

P-404

Transfering embryos to blastocyst medium 36 hrs post ICSI in prepa-ration for day 3 embryo transfer, improves implantation, and preg-nancy potential. Levent Keskintepe, Jeffery D. Fisch, Mark Adamowicz,Raifa Zody, Geoffery Sher. Sher Institute for Reproductive Medicine, LasVegas, NV.

Objective: To evaluate the effect of transferring day 2 embryos toBlastocyst Medium in preparation for day 3 -ET.

Design: Prospective Randomized study.Setting: Multi-site private practice.Patients: Infertile women �40 years (n�78) undergoing IVF.Interventions: Seventy eight (78) women who underwent ART and had

their oocytes inseminated by Intracytoplasmic Sperm Injection (ICSI), be-tween January, 1st and December 31st, 2002 were studied. Group 1, (n�39)had 119 embryos which were cultured in P1 medium (Irvine Scientific) today 3. Group 2, (n� 39) had 114 embryos which were initially cultured inP1 medium for 48hrs whereupon they were transferred to Blastocyst me-dium (Irvine Scientific) for an additional 24hrs Embryo transfer was per-formed 72 hrs � 6hrs post ICSI.

Main Outcome Measures: Clinical pregnancy and implantation rate (thenumber of ultrasound confirmed gestational sacs) per transferred embryo.

Results: The overall clinical pregnancy and implantation rate for Groups1 & 2 combined was 47% (37/78) and 28% (65 sacs/233 embryos), respec-tively. The clinical pregnancy, and implantation rates in Group 1 were(36%, 19%) respectively, as compared to (59%, 39%) in Group 2.

Conclusions: Standard protocols for culturing embryos to blastocysts,involve initially culturing the embryos in a relatively low glucose environ-ment, whereupon the glucose concentration is significantly increased. In the

process, poor quality embryos are culled out and those that survive toblastocyst seem to have a significantly improved implantation potential. Thefact that we obtained “improved embryo quality ”by culturing embryos in a“higher glucose environment” from day 2 to day 3 suggests that this subtleadjustment in the milieu externa could represent an important protocoladjustment in culturing embryos to day 3 since in doing so we were able todemonstrate a significant improvement in implantation and clinical preg-nancy rate (p�0.05). A larger, randomized study is needed to confirm thesefindings.

P-405

Oocytes and embryos assessment using GnRH agonists and antagonists.Raffaella Depalo, Giuseppe Loverro, Filomenamila Lorusso, MariateresaCapotorto, Margherita Vacca, Luigi Selvaggi. II Clin Ostetrica e gineco-logica, Bari, Italy; III Clin Ostetrica e ginecologica, Bari, Italy.

Objective: Gonadotropin releasing hormone (GnRH) agonist suppressesthe LH surge and decreases cancellation rate in protocols of controlledovarian stimulation.

Recently the use of GnRh antagonist instead of its agonist has beenclaimed to be easier, safer and more comfortable for patients, but fewinformation are available on the oocytes and embryos quality using thesetwo different protocols.

This study aimed to compare the number and maturity of retrievedoocytes and the quality of clived embryos using GnRH agonist or antago-nist.

Design: Prospective study.Materials and Methods: 68 consecutive patients selected for IVF received

GnRH antagonist (Cetrorelix) (Group A, 31 patients) or GnRH agonist(Triptorelin) (Group B, 37 patients). Group A received Cetrorelix 0.25mg/die starting when the leading follicle was � 14 mm, until the day beforehCG administration. Group B received triptorelin 0.1 mg/die from the 21stof the cycle, and 0.5 mg/die on the second day of the next cycle until the dayof hCG. Ovarian stimulation started on day 3 of the cycle with FSHrecombinant 225 UI. Ovulation was induced by 10.000 UI hCG. Oocytesmaturity and embryos quality were assessed according to L. Veeck.

Results: Mean age was comparable in the two groups. Lower number ofoocytes was retrieved in Group A compared to group B (8.66-63;6.85 vs12.4-63;6.6; p �0.05). The total number of oocytes was inversely correlatedwith mean age in Group A (r2�0.13) and in Group B (r2�0.12). Higherproportion of mature/metaphase II oocytes were observed in Group B(9.53-63;6.9 vs 6.1-63;4.1; p �0.05). The total number of clived embryoswas higher in Group B compared to Group A (7.53-63;5.6 vs 4.35-63;4.7;p �0.05). No statistical differences were observed in good embryo qualityin Group A versus Group B (4.5 vs 5.1). No statistical differences werefound in Pregancy Rate (PR) (28% vs 29%).

Conclusion: GnRH agonists provides an higher overall number of re-trieved oocytes, mature/metaphase II oocytes and clived embryos in aunselected population of women undergoing IVF. No difference was ob-served about embryo quality and PR between both the groups.

P-407

Histological characteristics of the female genital tract after high doseandrogen therapy: An in vivo model. Ami Shah, Robert Weiss. BostonUniv Medical Ctr, Boston, MA.

Objective: Transsexual patients undergo therapy with high dose of an-drogens for the expressed purpose of gender reassignment. These patientsthen undergo oophorectomy with or without hysterectomy for the purposesof their gender reassignment. This treatment provides an in vivo model ofthe effects of a supra-physiological dose of androgens on the normal femalegenital tract-thus, providing the opportunity to further understand the role ofandrogens in modulating hormone receptor expression, changes in cellularproliferation, cell atrophy, ovulation, and the propensity to dysplasticchange.

Design: This study is designed as a retrospective analysis of tissuehistology and pathology over a three year time interval. Included patientswere treated with testosterone 200 mg intra-muscular injections every weekfor at least one year for the purpose of gender reassignment. Patients thenunderwent a hysterectomy with bilateral salpingo-oophorectomy (BSO), ora laparoscopic BSO.

FERTILITY & STERILITY� S255

Materials and Methods: Pathological and histological evaluation of thecervix, endometrium, uterus, and ovaries was reviewed. The data is pre-sented as a case series.

Results: Ten patients met the inclusion criteria. Six patients underwenthysterectomies with BSO, and four patients underwent laparoscopic BSO.Seven patients had follicular cysts, the largest being 0.6 cm. Four patientshad pathologic evidence of a fibroid, the largest being 3cm. Two hadcervical squamos metaplasia with extensive atypical atrophic changes. Sixof patients had proliferative endometrial tissue.

Conclusion: High dose testosterone therapy in transsexual patients whomthen undergo a hysterectomy and BSO provides an in vivo model of theeffects of supra-physiologic doses of testosterone on the female genitaltract. There is histological evidence that the ovaries continue to function andovulation may occur. The endometrium typically revealed a proliferativepattern, potentially in response to testosterone’s effect on endometrial cells.

P-408

Sperm motility is related to platelet-activating factor-acetylhydrolasecontent in semen. William E. Roudebush, Jim Zhu, Dorothy Mitchell-Leef, Andrew A. Toledo, Joe B. Massey, Hilton I. Kort. ReproductiveBiology Assoc, Atlanta, GA.

Objective: Platelet-activating factor [1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF] is present in human sperm and its’ content has asignificant and positive relationship with motility. PAF-acetylhydrolase(PAFah), the enzyme that removes the acetyl group (responsible for PAF’sactivity), is present in semen and may serve as a sperm decapacitatingfactor. However, there are no reports on the levels of PAFah in semen andsperm motility. Therefore the study objective was to determine the relation-ship between PAFah content in semen and sperm motility.

Design: PAF-AH levels in semen were measured and correlated withsperm motility.

Materials and Methods: Human semen was obtained from healthy maturemales (n�25) and sperm motility (WHO, 1999) recorded prior to measure-ment of PAFah activity by spectrophotometric analysis (AZWELL Inc,Japan). Data were analyzed by linear regression and Student’s t-test.

Results: Seminal PAFah content ranged from a low of 252 IU/L cells toa high of 1,469 IU/L. The overall mean PAFah content in semen was 807.38IU/L (range 1,217 IU/L). Linear regression analysis revealed a significant(P�0.001) relationship [Motility�56.76�Log(PAFah)] between PAF-AHcontent in semen and sperm motility. Semen specimens presenting with anormal (�50%) percent motility (695.06 IU/L) had a significantly (P�0.01)lower PAF-AH content than specimens with an abnormal (�50%) percentmotility (1097.11 IU/L).

Conclusion: The data confirms the presence of PAFah in human semenand that levels are related to sperm cell motility. Additional studies willelucidate the role of PAFah in semen on sperm motility and the significancePAFah plays in sperm capacitation and human fertility.

P-409

Is serum level of vascular endothelial growth factor 165 related tofibroid volume in patients before and after hysterectomy? Da-ChungChen, Jah-Yao Liu, Gwo-Jang Wu, Chih-Hung Ku, Chi-Huang Chen. Deptof OBS/GYN, Tri-Service Gen Hosp, National Defense Medical Ctr, Taipei,Taiwan Republic of China; Sch of Public Health, National Defense MedicalCtr, Taipei, Taiwan Republic of China.

Objective: Vascular endothelial growth factor (VEGF) has been shown toplay a vital role in preservation and restoration of endothelial integrity.Uterus is reported to be an important source of VEGF. Several immuno-histochemical studies demonstrated increased VEGF expression in fibroids.And reduction of serum VEGF165 was also reported in association withestrogen replacement after hysterectomy. The relatioship of fibroids andserum VEGF165 is still unknown. The aim of this study was to investigatethe alteration of serum VEGF165 before and after hysterectomy.

Design: Prospective, repeated-measurement study.Materials and Methods: Forty-four women with fibroids were indicated

for hysterectomy to be enrolled. Four hours before and forty eight hoursafter vaginal hysterectomy without thermal injuries to ovaries, serum sam-ples were collected, centrifuged and stored at -80°C until assayed. Serum

samples were thawed to room temperature and assayed for VEGF using acommercially available enzyme linked immuno-sorbent assay kit (HumanVEGF Quantikine™ ELISA, R&D system, Catalogue No. DVE00). Theantibody contained in this kit was raised against recombinant VEGF165, thepredominantly secreted isoform. Statistic analysis was performed by usingSAS 8.2 version by multiple logistic regression to evaluate age, body massindex (BMI), parity, serum VEGF165 level and uterine weight; and by pairedstudent-t test to compare serum VEGF165 level before and after operation.

Results: The mean age, BMI, uterine weight were 45� 4.6 years old(range 36-56), 24.99� 4.2 kg/m2 and 300.16� 219.95 gm (range 80-1095gm), respectively. The serum VEGF165 level was increased in 14 (31.8%)and decreased in 30 (68.2%) patients postoperatively. The mean serumVEGF165 levels declined from 556.69� 52.3 pg/ml to 447� 49.2 pg/mlafter operation. This decrease was statistically significant (p�0.00035).After controlling for age, BMI, uterine weight, and parity by time, we foundthat nulliparous patients (n�11) have significantly 351.5� 119.7 pg/mlhigher VEGF165 level as compared to the multiparous patients (n�33)(p�0.0033). However, the concentrations of VEGF165 were 109.7 pg/mldecreased after operation in both groups.

Conclusion: Serum VEGF165 demonstrated significantly decline potentialafter hysterectomy, but not positively related to fibroid volume beforeoperation. Nulliparous patients with fibroids have higher serum VEGF165

level than in multiparous patients.

REPRODUCTIVE BIOLOGY: ANIMAL ANDEXPERIMENTAL MODELS

P-410

Sequential media used in human IVF do not affect imprinting of theH19 gene in mouse blastocysts. David K. Gardner, Elizabeth A. Hewitt,Michelle Lane. Colorado Ctr for Reproductive Medicine, Englewood, CO.

Objective: Several imprinted genes have been identified that are ex-pressed from only one parental allele. One of these genes, H19 is expressedonly from the maternal allele. However, it has been reported that culture ofmouse embryos in Whitten’s medium or KSOMAA, two media developedfor mouse embryo culture, result in expression of H19 from both thematernal and paternal allele. Whitten’s medium is a simple medium lackingkey regulators of development such as amino acids, chelators and vitaminsand medium KSOMAA produces high amounts of ammonium when incu-bated at 37°C due to the presence of glutamine. These media are notroutinely used in human IVF. The objective of this study was to examine theeffects of media used in human IVF on the imprinting of H19. Further, theeffect of serum supplementation was determined.

Design: Mouse embryos were cultured in different media and the im-printing of H19 determined in individual blastocysts.

Materials and Methods: Pronucleate embryos derived from CAST fe-males and F1 hybrid (C57Bl/6 x CBA) males were cultured in eitherKSOMAA, Whitten’s medium, sequential media G1/G2 (version III), orG1/G2 supplemented with 20% human serum. In vivo developed blasto-cysts were flushed from the reproductive tract as controls. The reciprocalcross of F1 females and CAST males was also analyzed. Parental specificexpression of H19 was assessed in single blastocysts by RT-PCR followedby sequencing an area of the gene containing 6 polymorphisms.

Results: All in vivo developed blastocysts expressed H19 only from thematernal allele. Similarly, blastocysts cultured in G1/G2 (version III) alldisplayed correct imprinted expression from the maternal allele. However,supplementation of G1/G2 with 20% serum resulted in 29% of blastocystshaving incorrect allelic expression. Culture of embryos in KSOMAA re-sulted in 57% incorrect imprinting, while culture in Whitten’s mediumresulted in 90% incorrect imprinting.

Conclusion: Physiologically-based media developed to support the hu-man embryo in culture do not induce aberrant imprinting of H19 in mouseblastocysts. Supplementation of such media with serum leads to alterationsin imprinting. Similar to previous reports, the imprinting of H19 in mouseblastocysts was affected by culture in either KSOMAA or Whitten’s media.

P-411

Ammonium alters gene expression and imprinting of H19 in culturedmouse blastocysts. David K. Gardner, Elizabeth A. Hewitt, Michelle Lane.

S256 Abstracts Vol. 80, Suppl. 3, September 2003