Equine non-invasive time-lapse imaging and blastocyst development.

In this study we examined the timeline of mitotic events of invitro-produced equine embryos that progressed to blastocyst stage using non-invasive time-lapse microscopy (TLM). Intracytoplasmic sperm injection (ICSI) embryos were cultured using a self-contained imaging incubator system (Miri®TL; Esco Technologies) that captured brightfield images at 5-min intervals that were then generated into video for retrospective analysis. For all embryos that progressed to the blastocyst stage, the initial event of extrusion of acellular debris preceded all first cleavages and occurred at mean (±s.e.m.) time of 20.0±1.1h after ICSI, whereas 19 of 24 embryos that did not reach the blastocyst stage demonstrated debris extrusion that occurred at 23.8±1.1h, on average 4h longer for this initial premitotic event (P<0.05). Embryos that failed to reach the blastocyst stage demonstrated a 4-h delay compared with those that reached the blastocyst stage to reach the 2-cell stage (P<0.05). All embryos that reached the blastocyst stage expressed pulsation of the blastocyst with visible expansion and contraction at approximate 10-min intervals, or five to six times per hour. Using a logit probability method, we determined that 2- and 8-cell stage embryos could reasonably predict which embryos progressed to the blastocyst stage. Together, the results indicate that TLM for equine embryo development is a dynamic tool with promise for predicting successful embryo development.

Paternal effects from methamidophos administration in mice.

In this study, the mouse was used to evaluate paternal germline exposure to the organophosphate methamidophos for its potential to produce adverse effects on spermatozoa and in the offspring. There have been reports that organophosphate exposure can increase abnormal sperm morphology in mice. However, effects transmitted to the offspring following paternal exposure have not been reported previously. The maximum tolerated dose (MTD) was 7.5 mg kg(-1) body weight and this dose resulted in no deaths, although blood plasma cholinesterase activity was still decreased. Males were euthanized 4 weeks after an acute intraperitoneal injection of methamidophos (0.5, 3.75, 5.0, and 7.5 mg kg(-1) body wt) and the number of spermatids per gram testes and sperm morphology were analyzed. In this study, abnormal sperm morphology on a per group basis exhibited a dose-response significantly related to increased methamidophos exposure as indicated by regression analysis and a nested ANOVA (p < 0.0001). Preimplantation embryos that were conceived 6 weeks after paternal methamidophos exposure (5 mg kg(-1) body wt) exhibited a significant increase in cleavage arrest. Fertility of males was also affected as shown by a decrease in the number of two- to four-cell embryos per male (postexposure week 6) and an increase in the number of degenerated embryos (postexposure weeks 4-6). We conclude that methamidophos may have the potential to produce transmissible adverse embryonic effects following an acute paternal germline exposure.

In vitro fertilization rate of mouse oocytes with spermatozoa from the F1 offspring of males irradiated with 1.0 Gy 137Cs gamma-rays.

Previous studies suggest that the spermatozoa from acutely irradiated male mice exhibit a reduced fertilization rate in vitro with the maximum decrease occurring for spermatozoa produced 6 weeks after irradiation (Y. Matsuda et al., Mutation Res. 142 (1985) 59-63). We have found that spermatozoa from unirradiated F1 males conceived 6 weeks after paternal F0 irradiation also exhibit a significantly reduced fertilization rate in vitro. After acute 137Cs gamma-irradiation yielding an absorbed dose of 1.0 Gy, adult CD1 F0 male mice were mated at weekly intervals with unirradiated female CD1 mice. Unirradiated adult males from F1 litters conceived 5 and 6 weeks after paternal F0 irradiation were allowed to mature. Their epididymal spermatozoa were evaluated for in vitro fertilization rates using oocytes from unirradiated 8-12-week-old CD1 females. The mean fertilization rate for spermatozoa from F1 males conceived 5 weeks after paternal F0 irradiation (80.74 +/- 15.74 SD %, n = 5) did not differ significantly from the control fertilization rate (89.40 +/- 10.94 SD %, n = 8). However, the fertilization rate for spermatozoa from F1 males conceived 6 weeks after paternal F0 irradiation (56.14 +/- 21.93 SD %, n = 5) was significantly less than the fertilization rate for control spermatozoa (p < 0.006) or for that of the F1 males conceived 5 weeks after paternal F0 irradiation (p < 0.04). These data suggest that spermatozoa obtained 6 weeks after paternal F0 irradiation can transmit a decrease in fertilization rate to the F1 generation males as well as exhibit decreased fertilization rate themselves when tested directly in vitro.

Normal mice develop from oocytes injected with spermatozoa with grossly misshapen heads.

Because in the mouse some structurally abnormal spermatozoa can penetrate oocytes, we decided to determine whether mouse oocytes fertilized by spermatozoa with grossly misshapen heads are able to develop normally. We injected such spermatozoa from BALB/c mice individually into mature oocytes of hybrid mice B6D2F1. Eighty to ninety percent of the oocytes were activated and developed into blastocysts in vitro and, when transferred to foster mothers, some developed into apparently normal fertile adults. This finding indicates that a proportion of abnormal spermatozoa carry all the genome and organelles necessary for normal embryonic development and growth to fertile maturity.

The effect of pituitary suppression and the women's age on embryo viability and uterine receptivity.

OBJECTIVE: To examine the effect of pituitary suppression and the women's age on embryo viability and uterine receptivity. DESIGN: Retrospective analysis of 394 embryo transfers (ET) after in vitro fertilization (IVF). SETTING: Community hospital IVF program from 1986 to 1990. PATIENTS: Three groups were studied: women less than 40 years with pituitary suppression (group 1) and without pituitary suppression (group 2); women 40 years of age and older with pituitary suppression (group 3). INTERVENTIONS: Pituitary suppression was achieved in groups 1 and 3 with daily leuprolide acetate starting in the luteal phase; human menopausal gonadotropin and progesterone were given intramuscularly. MAIN OUTCOME MEASURES: Ongoing and multiple ongoing pregnancy rates (PRs) were compared in the three groups. A mathematical model of implantation was used to estimate embryo viability and uterine receptivity. RESULTS: Ongoing and multiple ongoing PRs per ET in group 1 (28.6% and 12.3%) were significantly higher than the corresponding rates in group 2 (16.9% and 2.4%) and in group 3 (16.9% and 3.4%). Implantation analysis revealed higher embryo viability without change in uterine receptivity with pituitary suppression (group 1 versus 2). Decrease in both embryo viability and uterine receptivity was noted in women greater than 40 (group 1 versus 3). CONCLUSIONS: (1) Pituitary suppression improved implantation outcome by increasing embryo viability with no change in uterine receptivity and was associated with a high multiple PR in women less than 40; (2) in women greater than 40 both embryo viability and, to a lesser extent, uterine receptivity were decreased; (3) transfer of a larger number of embryos in older patients may improve IVF outcome without excessive risk of multiple pregnancy.