Limits...
Different chromatin and energy/redox responses of mouse morulae and blastocysts to slow freezing and vitrification.

Somoskoi B, Martino NA, Cardone RA, Lacalandra GM, Dell'Aquila ME, Cseh S - Reprod. Biol. Endocrinol. (2015)

Bottom Line: After warming, the chromatin integrity, mitochondrial distribution pattern and energy/oxidative status were compared among groups.Cryopreservation altered the quantitative bioenergy/redox parameters at a greater extent in the morulae than in the blastocysts.However, effects induced by vitrification were related to mitochondrial pattern, as only embryos with homogeneous mitochondrial pattern in small aggregates had reduced energy status.

View Article: PubMed Central - PubMed

Affiliation: Department and Clinic of Obstetrics and Reproduction, Szent Istvan University, Budapest, Hungary. somoskoibence@gmail.com.

ABSTRACT

Background: The ability to cryopreserve mammalian embryos has become an integral part of assisted reproduction, both in human and veterinary medicine. Despite differences in the size and physiological characteristics of embryos from various species, the embryos have been frozen by either of two procedures: slow freezing or vitrification. The aim of our study was to compare the effect of slow freezing and vitrification to the chromatin structure, energy status and reactive oxygen species production of mouse morulae and blastocysts.

Methods: Mouse morulae and blastocysts were randomly allocated into vitrification, slow freezing and control groups. For slow freezing, Dulbecco phosphate buffered saline based 10% glicerol solution was used. For vitrification, G-MOPS™ based solution supplemented with 16% ethylene glycol, 16% propylene glycol, Ficoll (10 mg/ml) and sucrose (0.65 mol/l) was used. After warming, the chromatin integrity, mitochondrial distribution pattern and energy/oxidative status were compared among groups.

Results: Cryopreservation affected chromatin integrity at a greater extent at the morula than the blastocyst stage. Chromatin damage induced by slow freezing was more relevant compared to vitrification. Slow freezing and vitrification similarly affected mitochondrial distribution pattern. Greater damage was observed at the morula stage and it was associated with embryo grade. Cryopreservation altered the quantitative bioenergy/redox parameters at a greater extent in the morulae than in the blastocysts. Effects induced by slow freezing were not related to embryo grade or mitochondrial pattern, as affected embryos were of all grades and with both mitochondrial patterns. However, effects induced by vitrification were related to mitochondrial pattern, as only embryos with homogeneous mitochondrial pattern in small aggregates had reduced energy status.

Conclusions: This study shows for the first time the joint assessment of chromatin damage and mitochondrial energy/redox potential in fresh and frozen mouse embryos at the morula and blastocyst stage, allowing the comparison of the effects of the two most commonly used cryopreservation procedures.

No MeSH data available.


Related in: MedlinePlus

Effects of slow freezing and vitrification on chromatin integrity of mouse embryos at the morula and blastocyst stage. Panel A: percentages of embryos graded according to chromatin damage (for details and criteria see M&M) as grade A (no damage, white segments), grade B (slight damage, gray segments) or grade C (severe damage, black segments). Embryos were grouped according to their developmental stage, observed after slow freezing/thawing or vitrification/warming and compared with controls. Numbers of analyzed embryos per group are indicated on each histogram and segment. Chi square test with the Yates correction: comparisons slow freezing vs control and vitrification vs control: *P < 0.05; **P < 0.0001; comparisons slow freezing vs vitrification: # P < 0.001; ## P < 0.0001. Panel B: Representative photomicrographs of control grade A morula (A1) and control grade A blastocyst (A2), slow frozen grade C morula (B1) and slow frozen grade C blastocyst (B2), vitrified grade B morula (C1) and vitrified grade B blastocyst (C2) are shown. The nuclei of embryos were stained with Hoechst 33258. For each embryo, UV light images are shown. Arrows indicate signs of chromatin damage: white thin arrows indicate micronuclei and white thick arrows indicate lobulated nuclei. Scale bar represents 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4419566&req=5

Fig1: Effects of slow freezing and vitrification on chromatin integrity of mouse embryos at the morula and blastocyst stage. Panel A: percentages of embryos graded according to chromatin damage (for details and criteria see M&M) as grade A (no damage, white segments), grade B (slight damage, gray segments) or grade C (severe damage, black segments). Embryos were grouped according to their developmental stage, observed after slow freezing/thawing or vitrification/warming and compared with controls. Numbers of analyzed embryos per group are indicated on each histogram and segment. Chi square test with the Yates correction: comparisons slow freezing vs control and vitrification vs control: *P < 0.05; **P < 0.0001; comparisons slow freezing vs vitrification: # P < 0.001; ## P < 0.0001. Panel B: Representative photomicrographs of control grade A morula (A1) and control grade A blastocyst (A2), slow frozen grade C morula (B1) and slow frozen grade C blastocyst (B2), vitrified grade B morula (C1) and vitrified grade B blastocyst (C2) are shown. The nuclei of embryos were stained with Hoechst 33258. For each embryo, UV light images are shown. Arrows indicate signs of chromatin damage: white thin arrows indicate micronuclei and white thick arrows indicate lobulated nuclei. Scale bar represents 20 μm.

Mentions: After both CP procedures, either SF or VF, chromatin damage was observed as formation of micronuclei or lobulated nuclei. In Figure 1 (Panel A), a percentage bar graph is reported in which embryos were graded as A, B and C, according to the described criteria (see Materials and Methods): grade A, embryos having all blastomeres with intact chromatin (Figure 1, Panel A, white segments), grade B, embryos having less than 20% blastomeres with damaged chromatin (gray segments) and grade C, embryos having more than 20% blastomeres with chromatin damage (black segments). In Figure 1 (Panel B), representative photomicrographs of embryos at the morula and blastocyst stage, and classified as grade A, B or C, are shown.Figure 1


Different chromatin and energy/redox responses of mouse morulae and blastocysts to slow freezing and vitrification.

Somoskoi B, Martino NA, Cardone RA, Lacalandra GM, Dell'Aquila ME, Cseh S - Reprod. Biol. Endocrinol. (2015)

Effects of slow freezing and vitrification on chromatin integrity of mouse embryos at the morula and blastocyst stage. Panel A: percentages of embryos graded according to chromatin damage (for details and criteria see M&M) as grade A (no damage, white segments), grade B (slight damage, gray segments) or grade C (severe damage, black segments). Embryos were grouped according to their developmental stage, observed after slow freezing/thawing or vitrification/warming and compared with controls. Numbers of analyzed embryos per group are indicated on each histogram and segment. Chi square test with the Yates correction: comparisons slow freezing vs control and vitrification vs control: *P < 0.05; **P < 0.0001; comparisons slow freezing vs vitrification: # P < 0.001; ## P < 0.0001. Panel B: Representative photomicrographs of control grade A morula (A1) and control grade A blastocyst (A2), slow frozen grade C morula (B1) and slow frozen grade C blastocyst (B2), vitrified grade B morula (C1) and vitrified grade B blastocyst (C2) are shown. The nuclei of embryos were stained with Hoechst 33258. For each embryo, UV light images are shown. Arrows indicate signs of chromatin damage: white thin arrows indicate micronuclei and white thick arrows indicate lobulated nuclei. Scale bar represents 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4419566&req=5

Fig1: Effects of slow freezing and vitrification on chromatin integrity of mouse embryos at the morula and blastocyst stage. Panel A: percentages of embryos graded according to chromatin damage (for details and criteria see M&M) as grade A (no damage, white segments), grade B (slight damage, gray segments) or grade C (severe damage, black segments). Embryos were grouped according to their developmental stage, observed after slow freezing/thawing or vitrification/warming and compared with controls. Numbers of analyzed embryos per group are indicated on each histogram and segment. Chi square test with the Yates correction: comparisons slow freezing vs control and vitrification vs control: *P < 0.05; **P < 0.0001; comparisons slow freezing vs vitrification: # P < 0.001; ## P < 0.0001. Panel B: Representative photomicrographs of control grade A morula (A1) and control grade A blastocyst (A2), slow frozen grade C morula (B1) and slow frozen grade C blastocyst (B2), vitrified grade B morula (C1) and vitrified grade B blastocyst (C2) are shown. The nuclei of embryos were stained with Hoechst 33258. For each embryo, UV light images are shown. Arrows indicate signs of chromatin damage: white thin arrows indicate micronuclei and white thick arrows indicate lobulated nuclei. Scale bar represents 20 μm.
Mentions: After both CP procedures, either SF or VF, chromatin damage was observed as formation of micronuclei or lobulated nuclei. In Figure 1 (Panel A), a percentage bar graph is reported in which embryos were graded as A, B and C, according to the described criteria (see Materials and Methods): grade A, embryos having all blastomeres with intact chromatin (Figure 1, Panel A, white segments), grade B, embryos having less than 20% blastomeres with damaged chromatin (gray segments) and grade C, embryos having more than 20% blastomeres with chromatin damage (black segments). In Figure 1 (Panel B), representative photomicrographs of embryos at the morula and blastocyst stage, and classified as grade A, B or C, are shown.Figure 1

Bottom Line: After warming, the chromatin integrity, mitochondrial distribution pattern and energy/oxidative status were compared among groups.Cryopreservation altered the quantitative bioenergy/redox parameters at a greater extent in the morulae than in the blastocysts.However, effects induced by vitrification were related to mitochondrial pattern, as only embryos with homogeneous mitochondrial pattern in small aggregates had reduced energy status.

View Article: PubMed Central - PubMed

Affiliation: Department and Clinic of Obstetrics and Reproduction, Szent Istvan University, Budapest, Hungary. somoskoibence@gmail.com.

ABSTRACT

Background: The ability to cryopreserve mammalian embryos has become an integral part of assisted reproduction, both in human and veterinary medicine. Despite differences in the size and physiological characteristics of embryos from various species, the embryos have been frozen by either of two procedures: slow freezing or vitrification. The aim of our study was to compare the effect of slow freezing and vitrification to the chromatin structure, energy status and reactive oxygen species production of mouse morulae and blastocysts.

Methods: Mouse morulae and blastocysts were randomly allocated into vitrification, slow freezing and control groups. For slow freezing, Dulbecco phosphate buffered saline based 10% glicerol solution was used. For vitrification, G-MOPS™ based solution supplemented with 16% ethylene glycol, 16% propylene glycol, Ficoll (10 mg/ml) and sucrose (0.65 mol/l) was used. After warming, the chromatin integrity, mitochondrial distribution pattern and energy/oxidative status were compared among groups.

Results: Cryopreservation affected chromatin integrity at a greater extent at the morula than the blastocyst stage. Chromatin damage induced by slow freezing was more relevant compared to vitrification. Slow freezing and vitrification similarly affected mitochondrial distribution pattern. Greater damage was observed at the morula stage and it was associated with embryo grade. Cryopreservation altered the quantitative bioenergy/redox parameters at a greater extent in the morulae than in the blastocysts. Effects induced by slow freezing were not related to embryo grade or mitochondrial pattern, as affected embryos were of all grades and with both mitochondrial patterns. However, effects induced by vitrification were related to mitochondrial pattern, as only embryos with homogeneous mitochondrial pattern in small aggregates had reduced energy status.

Conclusions: This study shows for the first time the joint assessment of chromatin damage and mitochondrial energy/redox potential in fresh and frozen mouse embryos at the morula and blastocyst stage, allowing the comparison of the effects of the two most commonly used cryopreservation procedures.

No MeSH data available.


Related in: MedlinePlus