Medroxyprogesterone acetate-induced effects on mouse pregnancy

CONCLUSIONS: These data suggest that a subpopulation of CDX2 is phosphorylated and localized in the nucleus in pre-compacted embryos, but that the entire population of CDX2 protein localizes in the nucleus in blastocysts and TS cells. Taken together, the data suggest regulation of nuclear localization of phosphorylated CDX2 at the 2-cell stage and a function unrelated to placental determination. The data also suggest that there are at least two subpopulations of CDX2. One subpopulation is phosphorylated at Serine 60 and does not bind OCT4 and the other is not phosphorylated at Serine 60 and binds OCT4. The presence of subpopulations of CDX2, that bind and that do not bind OCT4, in the 2-cell stage embryo also suggests a CDX2 function unrelated to placental determination. Supported by: This research was supported by grants to DAR from National Institute of Child Health and Human Development, NIH, (R01 HD40972A). P-618 MEDROXYPROGESTERONE ACETATE-INDUCED EFFECTS ON MOUSE PREGNANCY. S. A. Bakry, M. O. Ali, U. A. Sharaf El Dean, Z. O. Merhi, A. A. Fadiel, F. Naftolin. OB/GYN, School of Medicine, NYU, NYC, NY; Center for Genetic Engineering, AlAzhar University, Cairo, Egypt; Ob/Gyn, Maimonides Medical Center, NYC, NY. OBJECTIVE: Little is known about medroxyprogesterone acetate in oil (Depo-Proveraª; DMPA) effects in pregnancy, however, occasionally women are exposed while pregnant. We evaluated DMPA pre- and postimplantation effects on mice embryo-fetuses. DESIGN: Two IRB-approved experiments were performed. I. Sixty mice were divided into three groups, (n ¼ 20/group) on E1 (embryonic day 1). Two groups received 150 mg or 300 mg DMPA (human equivalent by weight per mouse based on doses used for injectable contraception). II. Six groups (n ¼ 10/group) received a single dose of 150 mg- or 300 mg-equivalent DMPA on E6 (post-implantation) and studied on E15 or E19. Two groups were uninjected controls. MATERIALS AND METHODS: On E15 or E19 fetuses and placentas counted, fetuses assessed as dead, alive or resorbed, weighed, crown-rump length measured and fixed for morphology or calcium assessment. Statistical analysis between groups was by A Student’s ‘‘t’’-test (SPSS 10 for Windows). RESULTS: I. There were no implantations in either DMPA-treated group. II. Uteri from DMPA-treated mice had 50% of the control’s numbers of implantation sites with early (0.2 cm) and late (0.5 cm) resorbed embryos present. Surviving embryos’ the crown-rump length and body weight were low compared to controls (all P>0.01). There was decreased ossification of the skull, ribs, sterna brae, vertebral centrae, fore- and hind limbs as well as occurrence of scoliosis of the vertebral column. CONCLUSIONS: In these preliminary studies, DMPA in amounts determined as weight-for-weight equivalent with human contraceptive doses causes blockade of implantation (Experiment 1) and severe impairment of implanted embryo-fetuses (Experiment 2). These findings support the dichotomy of effects of DMPA; while being a progestational agent, DMPA may also be embryo-toxic and teratogenic. It is possible that reduced doses will furnish a model for studies of problems that originate with faulty implantation, such as fetal growth restriction (FGR). However, the disparities between mice and humans in these areas must be reckoned with before wider conclusions can be reached. Supported by: NIH HD 47003 to FN. P-619 THE NOVEL SERINE PROTEASES, PRTN3 AND PRSS23, IN MURINE BLASTOCYST DEVELOPMENT AND HATCHING. H.-W. Chen, C.-R. Tzeng. Dept. Pharmacology, National Yang-Ming University, Taipei, Taiwan; Dept. Obs/Gyn, Taipei Medical University, Taipei, Taiwan. OBJECTIVE: Hatching is a critical process for implantation during early embryo development. Several gene expressions are highly regulated, crosstalked between embryo and maternal endometrium in embryo hatching and implantation process, especially the important role of proteases. In our previous study, we have identified the murine blastocyst hatching-related gens via cDNA microarray technique and T7-based RNA amplification. Among these identical candidate genes, two novel serine proteases, including proteinase 3 (prtn3) and protease, serine, 23 (prss23) have been identified and examined their functions in early embryo development. DESIGN: Prospective basic research study. MATERIALS AND METHODS: The ICR mice embryos were collected and cultured in human tubal fluid (HTF medium; Santa Ana, CA, USA) con-

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taining 0.3% of bovine serum albumin (BSA, Sigma). Specific siRNA (shRNA) for prtn3 and prss23 with microinjection technique was used to knock-down the genes in mice embryo. RESULTS: The cDNA microarray data indicated that prtn3 and prss23 have higher expression levels in hatched blastocyst (3.32- and 4.52-folds, comparing with pre-hatching blastocyst). By confirming with real time quantitative RT-PCR, the gene expressions of prtn3 and prss23 showed very different expression dynamic changes during early embryo development from 2-cells to hatched blastocyst. The mRNA level of prtn3 was increased after 2-cells stage and highly expressed in both pre-hatching and hatched blastocyst. However, prss23 only expressed in hatched blastocyst, whereas, almost undetectable from 2-cells to pre-hatching blastocyst. At least three shRNA constructs for prtn3 or prss23 were microinjected into zygotes and then the embryo development were monitored. The results showed that the development of embryos to blastocyst was significantly reduced in prtn3 shRNA injected group as comparing with mock construct control group. The hatching rates were both decreased in prtn3 (34.1% reduced) and prss23 (57.4% reduced) shRNA injected groups. CONCLUSIONS: This study indicated that the novel serine proteases, prtn3 and prss23 might play important roles in pre-implantation embryo development and blastocyst hatching. These findings might be very helpful not only in improving our understanding how these serine proteinases works during embryo hatching and implantation process, but also proving the candidate targets for us to control fertility. Supported by: This study was supported by the grants from the National Science Council (NSC95–2314-B-038–048-), Taiwan. P-620 PREGNANCYAND DELIVERYAFTER FREEZING AND THAWING OF BLASTOCYSTS DERIVED FROM BIOPSIED EMBRYOS. J. Hernandez, V. Sanabria, E. Chinea, V. Pe~na, M. Sandalinas, A. Palumbo. Centro de Asistencia a la Reproduccio´n Humana de Canarias, La Laguna, Tenerife, Spain; Reprogenetics Spain, Barcelona, Spain. OBJECTIVE: Preimplantation genetic diagnosis (PGD) is a labour intensive process which places a burden, both psychologically and economically on the couple undergoing treatment. When supernumerary healthy embryos are obtained they can be frozen, thus allowing one more chance for pregnancy with much less effort. However, disappointing results have been reported with the freezing of biopsied embryos. DESIGN: This is a case report of 2 pregnancies obtained after transferring frozen-thawed blastocysts that had been previously biopsied on day 3. MATERIALS AND METHODS: Between January 2004 and January 2007 we have performed 19 PGD cycles in 12 patents; in 3 cycles we were able to freeze supernumerary blastocysts. Embryo biopsy was performed on day 3 and one cell was analyzed in embryos with six or more cells. All embyo transfers were performed on day 4 with ultrasound guidance. One to three embryos were transferred. Supernumerary normal embryos were grown to the blastocyst stage (day 5) and then were frozen. The cryopreservation solution was Freeze-kit blast (Vitrolife, Kungsbacka, Sweden) and slow freezing was used for all blastocysts. For thawing we used the Thawkit blast (Vitrolife, Kungsbacka, Sweden). Patients received either oral estradiol valerate or transdermal estradiol until they had an endometrial thickness R8 mm and an estradiol value between 150–300 pg/ml. Vaginal progesterone suppositories were started 6 days before the thaw. All transfers were performed 4 hours after the thaw. RESULTS: A total of 6 blastocysts were frozen and 5 of them survived the thawing process. One of the patients had one embryo transferred and the other two had 2 embryos transferred. Pregnancy was achieved in the latter two patients. The first patient was a sex selection case for hemophilia, which was delivered in January 2005. The second patient was a recurrent aborter who underwent aneuploidy screening and this pregnancy is currently ongoing. Overall, in this small series 7 out of 12 (58.3%) patients obtained a pregnancy, and two of these came from cryotransfers. CONCLUSIONS: These data indicate that it is always worth freezing supernumerary embryos after performing PGD, since this adds a substantial chance of pregnancy. Supported by: None. P-621 THE USE OF sHLA-G (SOLUBLE HUMAN LEUKOCYTE ANTIGEN CLASS G) AS A TOOL FOR EMBRYO SELECTION. C. C. Rocha, L. L. Salgueiro, C. Carizza, V. Abdelmassih, S. Abdelmassih, R. Abdelmassih. ART Lab, Clinic and Research Center for Human

Vol. 88, Suppl 1, September 2007