The potentiality of two-dimensional preantral follicle culture as an in vitro model in predicting premature ovarian failure.

The purpose of this study is to identify the potential of a two-dimensional preantral follicle culture as an in vitro model of predicting premature ovarian failure. The two-dimensional preantral follicles culture method was established by cultivating preantral follicles collected from ICR F1 hybrids (aged 12-14days) for 12days. The preantral follicles were incubated with 0.54mg/ml cyclophosphamide, 0.5mg/ml busulfan, 0.12mg/ml cisplatin, 3.12mg/ml 4-vinylcyclohexene diepoxide, 5mg/ml D (+) galactose, and 0.5mg/ml hydrocortisone for 24h at culture days 2, 6 and 11. The diameter of follicles, the cumulus-oocyte complex number and the maturity of oocytes were recorded as the parameters to detect follicular maturation induced by the culture agents. The results indicated that, except for busulfan, D (+) galactose, and hydrocortisone, such test articles could significantly decrease follicular growth (p<0.05 or p<0.01), and induce oocyte degeneration and follicle atresia when the follicles were treated at day 2. With the exception of hydrocortisone, such agents also gradually decreased follicular development (p<0.05 or p<0.01) when the follicles were treated at day 6. All of the test articles but hydrocortisone can interfere with the ovulation, the cumulus-oocyte complex discharge and oocyte maturation of follicles when treated at days 2, 6 and 11. It is suggested that two-dimensional preantral follicle culture could be utilized as a potential in vitro system to mimic the POF model. It may also be employed in screening potential ovarian toxic agents, reducing laboratory animal use and promoting animal welfare.

Development and application of an analytical method for curdione quantification in pregnant Sprague-Dawley rats by LC-MS/MS.

The vaginal administration route suffers from relatively low absorption efficiency, which may hinder the identification of the toxicokinetics of curdione in pregnant women. A sensitive analytical method for determining the plasma concentration of curdione was developed and applied in the determination of curdione in pregnant Sprague-Dawley rats as a simulated model. Glimepiride was used as an internal standard and chromatographic separation was achieved on a Capcell Pak C18 MGIII column. A gradient elution profile with 0.5% formic acid (A)-0.5% formic acid-acetonitrile (B) was selected as mobile phase. The selected reaction monitoring mode was used for quantification based on the target fragment ions m/z 237.2 to m/z 135.1 for curdione and m/z 491.3 to m/z 352.1 for the glimepiride. The standard curve was linear over the range of 0.5-500 ng/mL for curdione in rat plasma and yielded a consistent peak pattern, even at the lower limit of quantitation of 0.5 ng/mL. The retention times of curdione and IS were 6.55 and 6.59 min, respectively. The mean recovery of curdione in rat plasma was 95.5-101.1%. The intra-day and inter-day precisions were between 2.35 and 9.08%. This LC-MS/MS method provides a simple and sensitive means for determining the plasma concentration.

The toxicokinetic profile of curdione in pregnant SD rats and its transference in a placental barrier system detected by LC-MS/MS.

The objective of this study was to determine the toxicokinetic profile of curdione in pregnant SD rats as well as the transference of curdione into the fetus through the placental barrier system using LC-MS/MS. Thirteen pregnant SD rats were treated with 7, 21 and 63 mg/kg curdione once daily from gestational day 6 (GD6) to GD15. Blood samples were collected at different time points on GD6 and GD15. Maternal plasma, placental plasma, placenta tissue, amniotic fluid and fetal tissue were collected for concentration analysis after all the animals were sacrificed following one repeated dose on GD19. The results indicated that Cmax, AUC(0₋t) and AUC(0₋∞) increased in a dose-dependent manner both on GD6 and GD15. At 7 mg/kg group, the total serum clearance value on GD15 was reduced to approximately 16.4% of that on GD6, and the volume of distribution was also significantly decreased (p<0.05). Curdione could be detected in the maternal plasma, placental plasma, placenta tissue, amniotic fluid and fetal tissue, and its concentration in the fetal tissue reached saturation at 21 mg/kg. In conclusion, curdione presents with the risk of accumulation in pregnant SD rats and may affect the fetus via transference through the placental barrier system.