The effects of platelet lysate on maturation,fertilization and embryo development of NMRI mouse oocytes at germinal vesicle stage

Purpose

Improving in vitro maturation could increase the rate of pregnancy from oocytes matured in vitro. Consequently, patients will be prevented from using gonadotropin with its related side effects. In this study, the maturation medium was enriched by platelet lysate (PL), then maturation and subsequent developments were monitored.

Methods

Oocytes at germinal vesicle stage with cumulus cells (cumulus–oocyte complex) and without cumulus cells (denuded oocytes) were obtained from mature female mice. The maturation medium was enriched by 5 and 10 % PL and 5 % PL + 5 % fetal bovine serum (FBS) as experimental groups; the control groups’ media consisted of 5 and 10 % FBS. After 18 h, the matured oocytes were collected and, after fertilization, subsequent development was monitored.

Results

The rates of maturation, fertilization and 2-cell embryo development for the denuded oocyte groups in experimental media 5 % PL and 5 % PL + 5 % FBS were significantly higher than those of the control groups (P < 0.05), while the results for the cumulus–oocyte complex groups were similar between the experimental groups and control groups.

Conclusions

The results of this study indicated that platelet lysate could improve the maturation rate in the absence of granulosa cells compared to media with FBS. This extract also had positive effects on fertilization and embryo development.

     

Effects of differing oocyte-secreted factors during mouse in vitro maturation on subsequent embryo and fetal development

Purpose

We hypothesised that varying native oocyte-secreted factor (OSF) exposure or using different recombinant OSF peptides would have differential effects on post-in vitro maturation (IVM) embryo and fetal development.

Methods

Mouse cumulus oocyte complexes (COCs) were treated with the purified mature domain of GDF9 and/or BMP15 or were co-cultured with denuded oocytes (DOs) from 0 h or 3 h of IVM. DOs were matured for 3 h as either intact COCs+/-FSH before denuding, or as DOs + FSH. COCs were fertilised and blastocyst development was assessed on days 5 and 6, and either differentially stained for ICM numbers or vitrified/warmed embryos were transferred to recipients to assess implantation and fetal rates.

Results

No improvement in embryo development was observed with the addition of GDF9 and/or BMP15 to IVM. In contrast, embryos derived from COCs co-cultured with DOs had significantly improved blastocyst rates and ICM numbers compared to controls (P < 0.05). The highest response was obtained when DOs were first added to COCs at 3 h of IVM, after being pre-treated (0–3 h) as COCs + FSH. Compared to control, co-culture with DOs from 3 h did not affect implantation rates but more than doubled fetal yield (21 % vs 48 %; P < 0.05). GDF9 Western blot analysis was unable to detect any differences in quantity or form of GDF9 (17 and 65 kDa) in extracts of DO at 0 h or 3 h.

Conclusions

This study provides new knowledge on means to improve oocyte quality in vitro which has the potential to significantly aid human infertility treatment and animal embryo production technologies.     

Influential effect of age on oocyte morphometry,fertilization rate and embryo development following IVF in mice

Changes of oocyte quality and decreasing of ovarian follicle reserve in advanced age are the major challenges in infertility treatments. One factor associated with aging is the thickness of zona pellucida, which has adverse relation with embryo score. Although, there is no correlation between perivitelline space and granulation with embryo quality, an inversely correlation is observed between these two factors with subsequent embryo quality. The aim was to investigate the influence of age on the oocyte quality, fertilization rate and embryo development following in vitro fertilization setting in mice. NMRI mice (N = 21) were categorized into 3 groups regarding to their ages (groups I–III; 25, 30, 35 weeks old, respectively). Standard in vitro fertilization protocol was conducted for each group. After collection of the oocytes, three points of zona pellucida and perivitelline space diameters were measured in each group and mean values were calculated. Also, the number of oocytes, fertilization rate, the number of cleavage embryos and blastocyst formation were compared among the groups. All the changes were insignificancy age related, as the mean value of zona pellucida and perivitelline space diameters as well as the number of oocytes, rate of fertilization and 2 cells embryos were higher in group I compared to other groups. Also, there was significant difference between some evaluated parameters, such as the number of generated 4 cells cleavage embryos and blastocysts as well as oocytes degeneration rates. The advanced maternal age influenced negatively on the oocyte morphometry and cleavage and blastocyst embryo formation in animal model.     

Effect of long-term caffeine administration to mice on in vitro fertilization and embryo development using oocytes

Purpose

To evaluate the effect of long-term caffeine administration to mice on in vitro fertilization (IVF) of oocytes.

Methods

Mice were injected with different dosages (0, 0.1, and 1.0 mg/mouse/converted day) of caffeine for one month. Subsequently, the fertilization rate and embryo development to blastocyst stage were evaluated in IVF using oocytes from the mice.

Results

The retrieved average oocyte rate was significantly lower (27.4) in mice injected with 1.0 mg caffeine than in the control group (36.5; P < 0.05); the fertilization rate was significantly different between the 0 mg (317/401; 79.1 %) and 1.0 mg group (199/301; 66.1 %) (P < 0.05). At 96 h after insemination, the blastocyst formation rate was significantly decreased in the 1.0 mg group (94/199; 47.2 %) compared with the control (0 mg) group (237/317; 74.8 %) and 0.1 mg group (226/323; 70 %) (P < 0.05). When 1.0 mg caffeine was administered for two weeks, embryo development was significantly impacted.

Conclusions

Our findings suggest that caffeine administration negatively impacts oocytogenesis and embryonic development after IVF.     

Extended in vitro maturation of immature oocytes from stimulated cycles: an analysis of fertilization potential,embryo development,and reproductive outcomes

Purpose  

To investigate 24 h in vitro maturation (IVM) of cumulus-stripped immature oocytes from stimulated cycles.     

Live birth after frozen-thawed oocytes matured in vitro in a PCOS patient: a model for improving implantation rates in IVM cycles and objectively assessing the real potential of development of frozen oocytes matured in vitro

Over the past 20 years, in vitro maturation (IVM) of oocytes has emerged in the strategy of infertility treatment, with the main indication being in patients suffering from polycystic ovarian syndrome (PCOS). More recently, IVM has been proposed as an option for fertility preservation in women having to undergo gonadotoxic treatments. However, despite the increasing application of IVM, the potential of development of in vitro matured oocytes after thawing remains ill-established and few pregnancies have been reported so far. We report herein a case of live birth after frozen-thawed oocytes matured in vitro and embryo transfer during an artificial cycle in a 29-year-old patient with primary infertility due to PCOS.

The present case demonstrates that the transfer of frozen-thawed IVM oocytes during an artificial cycle in PCOS patients is feasible and leads to pregnancy and live birth. This strategy may also be an interesting option to objectively assess the developmental potential of these oocytes after freezing and thawing, which is a major concern for physicians who include the IVM approach in their fertility preservation program.     

Combination of calcium ionophore A23187 with puromycin salvages human unfertilized oocytes after ICSI

OBJECTIVE: To determine whether oocyte activation using a combination of calcium ionophore A23187 (A23187) with puromycin could salvage human unfertilized oocytes after ICSI. STUDY DESIGN: One hundred and thirteen discarded unfertilized oocytes 20-68 h after ICSI were assigned to four groups: ICSI 20-h group, ICSI 44-h group, ICSI 68-h group and control. All unfertilized oocytes were exposed to A23187 (5 microM) for 5 min and subsequently were incubated with puromycin (10 microg/ml) for 4 h. Sex chromosomal analysis was performed by dual color fluorescence in situ hybridization (FISH). RESULTS: The combination of A23187 with puromycin could activate the unfertilized oocytes 20-68 h after ICSI. The best results were achieved in the ICSI 20-h group, which exhibited an activation rate of 91.2% (31/34), a cleavage rate of 64.7% (22/34) and 44.1% (15/34) high-quality embryos. The activation rate, cleavage rate and the number of high-quality embryos appeared to decrease with the cultured time of unfertilized oocytes after ICSI. FISH analysis showed six embryos with XX and seven embryos with XY in 16 embryos derived from 2PN2PB. CONCLUSIONS: The combination of calcium ionophore A23187 with puromycin could effectively salvage unfertilized oocytes within 20 h after ICSI.