Efficacy of calcium ionophore A23187 oocyte activation for generating parthenotes for human embryo research.

PURPOSE: Our purpose was to examine the efficacy of Ca-A23187 to activate human oocytes and produce parthenotes for research purposes. We examined the feasibility of using fluorescence in situ hybridization (FISH) to study the sex chromosome constitution of activated oocytes. METHODS: One hundred eight nonfertilized oocytes from our IVF program were exposed to Ca-A23187. Oocyte activation was determined by the presence of pronuclear (PN) development. FISH was done on chromosome preparations using X and Y dual-colored probes. Polyploidic and parthenogenetically activated oocytes from our IVF program served as controls. RESULTS: Of the 108 oocytes, 59 (55%) had no PN, 38 (35%) one PN, 10 (9%) two PN, and 1 (0.9%) three PN. Fiftyseven oocytes (53%) were not recovered following spreading and no chromatin was observed on 14 slides (13%) after FISH. This contrasted with 50 of 227 (22%) and 3 of 227 (1.7%) loss rates, respectively, for controls (P < 0.0001). Eight of 49 activated oocytes underwent cleavage. FISH was performed on 37 oocytes. Of 21 zero-PN oocytes, I had no FISH signals, 15 had a single X, 4 had two X's, and I had four X's. For one-PN oocytes, two had no FISH signals, seven had one X, and three had two X's. For two-PN oocytes, two had no FISH signals and two had two X's. FISH results were consistent with a maternal origin of genetic material. CONCLUSIONS: Ca-A23187 resulted in a 45% activation rate, with 16% of oocytes progressing to cleavage before degeneration. Oocyte activation with Ca-A23187 allowed the generation of parthenotes for human embryo research. FISH was useful for evaluation of oocytes and parthenotes.

Maturational asynchrony between oocyte cumulus-coronal morphology and nuclear maturity in gonadotropin-releasing hormone agonist stimulations.

OBJECTIVE: To determine oocyte meiotic maturity and asynchrony between cumulus-coronal morphology and nuclear maturity after gonadotropin-releasing hormone agonist (GnRH-a) and norethindrone-programmed stimulations. DESIGN: Oocyte meiotic maturity was evaluated at follicular aspiration in 4,961 oocytes after GnRH-a/follicle-stimulating hormone (FSH)/human menopausal gonadotropin stimulations (hMG) for in vitro fertilization patients and 299 oocytes after norethindrone-programmed clomiphene citrate (CC)/hMG in oocyte donors. Maturational asynchrony between the oocyte's cumulus-coronal morphology and nuclear maturity was evaluated in 2,336 oocytes. SETTING: In vitro fertilization program at the University of Iowa Hospitals and Clinics; academic tertiary care center. INTERVENTIONS: After evaluating oocyte cumulus-coronal maturity, cumulus masses were spread to determine oocyte nuclear maturity. RESULTS: Fourteen percent, 17%, 50%, 17%, and 2% of oocytes were prophase I, metaphase I, metaphase II, postmature metaphase II, and atretic, respectively. Asynchrony was noted in 28% of prophase I, 71% of metaphase I, 11% of metaphase II, 45% of postmature metaphase II, 32% of atretic, and 28% of all oocytes. Significant differences were not found between GnRH-a and norethindrone-programmed stimulations in asynchrony between cumulus-coronal morphology and nuclear maturity or percentage of prophase I, metaphase I, metaphase II, postmature metaphase II, or atretic oocytes. Sixty-seven percent of oocytes possessed a polar body at retrieval. The rate of fertilization was significantly higher for metaphase II oocytes than postmature metaphase II and metaphase I oocytes > prophase I oocytes. Parthenogenetic activation tended to be highest for postmature metaphase II oocytes. Embryo cleavage was significantly higher for postmature metaphase II, metaphase II, and metaphase I oocytes than for prophase I oocytes. CONCLUSIONS: This is the first report of asynchrony between cumulus-coronal morphology and nuclear maturity at follicular aspiration in GnRH-a and norethindrone-programmed stimulations. Asynchrony was observed in 28% of oocytes. A higher percentage of oocytes possessed a polar body at egg retrieval with these stimulation regimens compared with rates reported previously for FSH, FSH/hMG, and CC/hMG stimulations.

Prediction of nuclear maturity from cumulus-coronal morphology: influence of embryologist experience.

PURPOSE: A majority of in vitro fertilization (IVF) programs continues to evaluate oocyte maturity on the basis of cumulus-coronal morphology (CCM) even though marked asynchrony has been reported between CCM and nuclear maturity. This study was designed to examine changes in embryologists' ability to correctly predict nuclear maturity from CCM as a function of increasing experience. Nuclear maturity was assessed by inverted microscopy with a modified spreading technique at follicular aspiration. A second objective was to determine the percentage of oocytes which displayed asynchrony between CCM and nuclear maturity as assessed by embryologists with extensive experience in oocyte maturity evaluation. RESULTS: The three participating embryologists had directly evaluated 1304, 75, and 0 oocytes for nuclear maturity and CCM at study initiation and correctly predicted nuclear maturity from CCM in 74, 64, and 47% of oocytes, respectively. Embryologist 1 did not significantly change in predictive ability during the 17-month study period. Embryologist 2 significantly improved in predictive ability during the first 9 months of the study (841 oocytes evaluated) and plateaued thereafter, at a similar percentage of correct predictions as embryologist 1. Embryologist 3 continued to improve in predictive ability throughout the study period, reaching 61% correct predictions at the close of the study after evaluating 223 oocytes. Once embryologists had plateaued in their predictive ability, 72% of oocytes evaluated received the correct nuclear maturity classification based on CCM. Significantly fewer oocytes (54%; 375/690) evaluated by embryologists who had not plateaued in their predictive ability received the correct nuclear maturity classification based on CCM. CONCLUSIONS: These results indicate that embryologists' ability to predict oocyte nuclear maturity correctly from CCM continues to change over several months even when pretraining video recordings are used before beginning direct evaluations. After embryologists plateaued in their predictive ability, nuclear maturity still could not be correctly predicted from CCM in 28% of oocytes due to asynchrony between nuclear and CCM maturity. Based upon this, circumstances in which the spreading technique should be used for direct assessment of nuclear maturity as opposed to assessment of CCM only are discussed.