Genotoxicity assessment of mouse oocytes by comet assay before vitrification and after warming with three vitrification protocols.

OBJECTIVE: To assess the genotoxicity of three oocyte vitrification protocols. DESIGN: Murine assay. SETTING: Biogenotoxicology research laboratory. ANIMAL(S): CD1 female mice. INTERVENTION(S): Three mouse oocyte groups were exposed to three commercialized human oocyte vitrification protocols. Protocols 1 and 2 contained dimethyl sulfoxide and ethylene glycol (EG), and protocol 3 contained EG and 1,2-propanediol (PrOH). DNA damage was first evaluated by comet assay after oocyte exposure to the three different equilibration and vitrification solutions. Comet assay was also performed after full vitrification and warming procedure and compared with a negative control group (oocytes stored in medium culture only) and a positive control group (oocytes exposed to hydrogen peroxide just before comet assay). MAIN OUTCOME MEASURE(S): DNA damage was quantified as Olive tail moment (OTM). Statistical analysis consisted of a Shapiro-Wilk test. Then, median protocol OTM was compared with the negative control group with the Mann-Whitney U test. The difference was considered to be statistically significant if the P value was <.05. RESULT(S): In both parts of our study, protocols 1 and 2 did not induce significant DNA damage, whereas protocol 3 induced statistically higher DNA damage compared with the negative control group. CONCLUSION(S): Vitrification protocols containing PrOH induced significant DNA damage on mouse oocytes, both before cooling and after warming. Therefore, for the moment, we prefer vitrification techniques without PrOH while we await more studies on PrOH toxicity and long-term evaluation.

Respiratory distress syndrome after elective caesarean section in near term infants: a 5-year cohort study.

OBJECTIVE: to assess the incidence of respiratory distress syndrome (RDS) in late preterm (34(0/7)-36(6/7)) and just term (37(0/7)-37(6/7)) infants born via elective caesarean section (CS) in a tertiary care maternity facility. METHODS: retrospective cohort study between 2005 and 2009. Hundred and eighty-eight near term infants, divided in two groups: group A: 125 late preterm (34(0/7)-36(6/7)) and group B: 63 just term (37(0/7)-37(6/7)), from elective CS (except CS after pre-mature rupture of membranes and foetuses presenting congenital malformation) were included. RESULTS: In group A the overall incidence of RDS (RDS at or shortly after birth, requiring respiratory support or oxygen therapy) was 44% (n = 55) vs. 15.9% (n = 10) in group B (p < 0.01). The incidence of SRDS (requiring admission in the neonatal intensive care unit (NICU)) in group A was 13.6% (n = 17) and 3.2% (n = 2) group B (p < 0.01). The risk decreased significantly as gestational age (GA) increased: for RDS, 50.9% at 34 weeks of gestation (WG), 52.5% at 35 WG, 21.5% at 36 WG, and 15.9% at 37 WG; for admission, 30.2% at 34 WG, 25% at 35 WG, 9.4% at 36 WG, and 6.3% at 37 WG. Among late preterm infants with RDS, 30.9% (n = 17) developed severe RDS (SRDS). CONCLUSIONS: Late preterm infants born via elective CS are at high risk for RDS and NICU admission. The risk is influenced by each additional week spent in utero. As the incidence of CS is increasing within this population, new preventative strategies must be sought.

Evaluating methods of mouse euthanasia on the oocyte quality: cervical dislocation versus isoflurane inhalation.

Cervical dislocation is a commonly used method of mouse euthanasia. Euthanasia by isoflurane inhalation is an alternative method which allows the sacrifice of several mice at the same time with an anaesthesia, in the aim to decrease pain and animal distress. The objective of our study was to assess the impact of these two methods of euthanasia on the quality of mouse oocytes. By administering gonadotropins, we induced a superovulation in CD1 female mice. Mice were randomly assigned to euthanasia with cervical dislocation and isoflurane inhalation. Oviducts were collected and excised to retrieve metaphase II oocytes. After microscopic examination, oocytes were classified into three groups: intact, fragmented/cleaved and atretic. Intact metaphase II oocytes were employed for biomedical research. A total of 1442 oocytes in the cervical dislocation group were compared with 1230 oocytes in the isoflurane group. In the cervical dislocation group, 93.1% of the oocytes were intact, versus 65.8% in the isoflurane group (P ≤ 0.001). In light of these results, we conclude that cervical dislocation is the best method of mouse euthanasia for obtaining intact oocytes for biomedical research.

Assessment of 1,2-propanediol (PrOH) genotoxicity on mouse oocytes by comet assay.

OBJECTIVE: To assess the genotoxicity of 1,2-propanediol (PrOH) on mouse oocytes by comet assay. DESIGN: In vitro assay using murine model. SETTING: Biogenotoxicology research laboratory. ANIMAL(S): CD1 female mice. INTERVENTION(S): Three 40-oocyte groups were exposed to different PrOH concentrations (5%, 7.5%, and 15%). Each concentration was tested during both long and short exposures (1-2 hours and 1-5 minutes) in comparison with control groups. DNA damage was evaluated by a single-cell gel electrophoresis assay, also called "comet assay," and analyzed with Komet software. MAIN OUTCOME MEASURE(S): DNA damage was quantified as Olive tail moment (OTM). Interpretation was done on OTM with the use of χ(2). RESULT(S): High PrOH concentrations (7.5% and 15%) induced significant DNA damage on mouse oocytes. The OTM χ(2) values were 4.16 ± 0.40 and 6.80 ± 0.4 with 7.5% PrOH at 1 and 2 hours, respectively, 24.35 ± 1.60 with 15% at 1 hour, and for 2h at 15% the DNA damage was too drastic to calculate OTM χ(2). After 1 and 5 minutes, the OTM χ(2) values were, respectively, 5.19 ± 0.26 and 6.06 ± 0.42 with 7.5%, and 7.53 ± 0.33 and 16.81 ± 0.67 with 15%. CONCLUSION(S): High concentrations of PrOH (7.5% and 15%) induced significant DNA damage on mouse oocytes, whatever the exposure duration. These results should be interpreted with caution, because additional data are needed to evaluate PrOH genotoxicity and DNA oocyte reparation after exposure to high PrOH concentrations.

Comet assay on mouse oocytes: an improved technique to evaluate genotoxic risk on female germ cells.

OBJECTIVE: To develop and validate an efficient comet assay on mouse oocytes without depellucidation. DESIGN: In vitro experiments using a murine model. SETTING: Biogenotoxicology research laboratory in Aix-Marseille II University, France. ANIMAL(S): CD1 prepubescent female mice. INTERVENTION(S): DNA lesions in oocytes were evaluated by the alkaline comet assay. After oocyte retrieval, we first studied the effect of zona pellucida (ZP) on comet morphology. For this study, we applied the comet assay to mature oocytes with and without ZP after exposure to simulated sunlight irradiation (SSI) compared with negative controls. Next, nondepellucidated mouse oocytes were exposed to three well-known genotoxic agents (SSI, methylmethanesulfonate [MMS], and hydrogen peroxide [H(2)O(2)]) and compared with negative controls. Images of oocytes were analyzed with Komet software. MAIN OUTCOME MEASURE(S): DNA damages were quantified and expressed as olive tail moment (OTM), defined as the product of the tail length and the fraction of total DNA in the tail. OTMχ(2) were calculated from OTM; they corresponded to the degrees of freedom (n) of each OTM distribution obtained from at least 50 oocytes. OTMχ(2) is an indicator of DNA lesions. The test was considered positive and statistically significant when OTMχ(2) increased in oocytes compared with the medium-only control cells. RESULT(S): There was no difference in comet aspect between oocyte groups with and without ZP. The three genotoxic agents significantly increased DNA damages as compared with the control groups. The OTMχ(2) values were (mean ± SD): 2.1 ± 0.07, 7.73 ± 0.35, 3.35 ± 0.15, and 12.4 ± 0.51 in control, SSI, MMS, and H(2)O(2) groups, respectively. CONCLUSION(S): Comet assay on non depellucidated mouse oocytes is a rapid and easy test. This assay would be useful to assess the genotoxicity on female germ cells of chemicals, drugs, or environmental pollutants and the efficiency of antioxidant molecules.