GYNOB2 Effect of hydroxyurea on in-vitro embryo development. (Gynecology & Obstetrics).

GYNOB2 EFFECT OF HYDROXYUREA ON IN-VITRO EMBRYO DEVELOPMENT. Anthony E. Archibong, PhD, Brandon Strange, MD, and Edward R. Hills, MD. Department of Obstetrics & Gynecology, Meharry Medical College, Nashville, Tenn.

The presence of high levels of fetal hemoglobin (HbF) in red blood cells decreases the severity of clinical manifestations associated with sickle cell disease. Hydroxyurea (HU), among tested drugs, has been shown to increase levels of HbF and decrease the incidence of vaso-occlusive pain episodes by approximately 50% in adults with sickle cell anemia. However, this compound has been demonstrated to have an inhibitory effect on DNA synthesis, thus provoking our belief that exposure of preimplantation embryos (a stage of active DNA synthesis and cell proliferation) to bioavailable HU can retard and/or inhibit their development. To test this hypothesis, preimplantation mouse embryos were cultured using two different experimental methods: Expt. 1 - continuous exposure and Expt. 2 - intermittent exposure (to mimic normal physiological dynamics), to peak bioavailable HU. Twenty-one-day-old C57BL/6J female mice were injected intraperitoneally (IP) with 2.5 IU of pregnant mare serum gonadotropin followed 48 hours lat er by an IP injection of 2.5 IU of human chorionic gonadotropin (hCG). Immediately following hCG, females were co-caged with adult males of similar strain in 1:1 ratio, to be mated. Males were allowed to remain with females overnight and at approximately 35 hours post hCG, mated females were sacrificed and oviducts excised and minced in Whitten's medium (WM) for the recovery of two-cell embryos. Recovered embryos were washed twice in VIM and only apparently normal 2-cell embryos were selected for in vitro culture. Embryos were considered normal if blastomeres of equal size were present within the boundaries of the vitelline membrane. In Expt. 1, embryos were randomly assigned to be cultured in WM supplemented with CZBt medium containing growth factors and glucose (control medium: CM) or CM + HU (18.3 ug/mL) for 5 days. The level of FLU used for embryo culture is reflective of the bioavailable peak HU in patients on 10 mg HU per day. Experiment 2 was similar to Expt. 1 except that embryor were exposed to bioav ailable peak HU for one hour, after which they were transferred to CM. This process was repeated every day for five days. Data on percentage of stages attained by embryos at the end of culture were compared by chi-square. All embryos, regardless of treatment and experiment, progressed to 4-cell stage of within 36 hours of incubation. By day 5 of culture, 40% of embryos in CM attained blastocyst stage, higher than 5% blastocyst formation for embryos exposed to FLU (P < .05). More morulae were observed (P < .05) among embryos in CM versus those exposed to FLU at the end of in vitro culture (35% vs. 0%). The remaining 25% of embryos in CM were either 4-cell or degenerate, whereas more embryos (P < .01) in the HU group (95%) were degenerate at the end of in vitro culture. In experiment 2, 15% of embryos exposed intermittently to HU attained blastocyst stage and 24% remained at morula stage. The percentage of HU-exposed embryos that attained blastocyst stage in Expt. 2 was higher (P < .05) than that of their count erparts in Expt. 1. Although HU is a beneficial drug for the alleviation of the clinical manifestations of sickle cell disease, it is highly toxic to preimplantation embryos. That some embryos exposed to HU developed to blastocyst stage indicates resilience to xenobiotics by some embryos. However, subtle damage may still occur in the surviving embryos and may be expressed post partum.