Oocytes recovered after ovarian tissue slow freezing have impaired H2AX phosphorylation and functional competence

2015 ◽  
Vol 27 (8) ◽  
pp. 1242
Author(s):  
Sam Sudhakaran ◽  
Shubhashree Uppangala ◽  
Sujith Raj Salian ◽  
Sachin D. Honguntikar ◽  
Ramya Nair ◽  
...  
Keyword(s):  
Ovarian Tissue ◽  
Germinal Vesicle ◽  
Slow Freezing ◽  
Ovarian Tissue Cryopreservation ◽  
Strontium Chloride ◽  
H2ax Phosphorylation ◽  
Germinal Vesicle Breakdown ◽  
Functional Competence ◽  
Dna Strand

It has been shown that oocytes isolated from ovarian tissue cryopreservation acquire DNA damage during the process of freeze–thawing. Using a mouse model, here we have investigated the functional competence and phosphorylation of H2AX (γ-H2AX) in germinal vesicle (GV) and parthenogenetically activated oocytes derived from conventional ovarian tissue slow freezing and vitrification techniques. The number of GV-stage oocytes with γ-H2AX foci was not significantly different between the slow-freezing and vitrification groups. Although the in vitro maturation (IVM) potential of GV oocytes in the slow-freezing group showed a significant delay (P < 0.0001) in the process of germinal vesicle breakdown, no difference in the maturation rate was observed between the two protocols. Nevertheless, parthenogenetic activation of IVM oocytes using strontium chloride showed a significantly lower activation rate in the slow-freezing group compared with the vitrification (P < 0.05) and control (P < 0.01) groups. Importantly, H2AX phosphorylation was significantly perturbed in the slow-freezing group in comparison to the control (P < 0.05). Therefore, we conclude that impaired sensing of DNA strand breaks and repair processes are associated with the reduced functional competence of the oocytes recovered from the slow-freezing group, which may have a significant impact on the reproductive outcome.

scholarly journals Graft site and gonadotrophin stimulation influences the number and quality of oocytes from murine ovarian tissue grafts

Reproduction ◽  
2006 ◽  
Vol 131 (5) ◽  
pp. 851-859 ◽  
Author(s):  
Hsiao Yun Yang ◽  
Shae-Lee Cox ◽  
Graham Jenkin ◽  
Jock Findlay ◽  
Alan Trounson ◽  
...  
Keyword(s):  
Ovarian Tissue ◽  
Germinal Vesicle ◽  
Fertilization Rate ◽  
Ovarian Tissue Cryopreservation ◽  
Control Group ◽  
Developmental Potential ◽  
Oocyte Yield ◽  
Tissue Grafts

Ovarian tissue cryopreservation and subsequent transplantation can restore fertility in cancer patients. This study used a mouse ovarian grafting model to investigate whether the graft site (bursal cavity, the kidney capsule or subcutaneous) influences the number, fertilization rate and developmental potential of oocytes recovered from grafts and whether using a standard gonadotrophin stimulation protocol would increase oocyte yield from the grafts. Mouse ovarian tissue was grafted into four week old mice and collected three weeks later. Graft recipients were treated either with or without exogenous gonadotrophin stimulation prior to graft collection. Grafted ovaries yielded oocytes that were either at the germinal vesicle (GV) stage or mature metaphase II (MII) stage at collection. These GV oocytes were matured beforein vitrofertilization (IVF), while the MII oocytes underwent IVF immediately. Oocytes collected from the oviducts of non-grafted superovulated mice of the same age served as controls. Two-cell embryos were transferred to pseudopregnant recipients and recovered at day 15 of gestation or left to go to term. Graft retrieval and the number of oocytes from each graft were lowest from the subcutaneous graft site. The number of two-cell embryos produced was significantly higher for oocytes from the grafts to the bursa as compared with the other sites. All graft sites gave rise to embryos with comparable implantation rates and developmental potential to fetuses and offspring following transfer. However, the oocytes from grafted ovaries had a significantly lower developmental potential when compared with the control group. Stimulation with exogenous gonadotrophins did not significantly increase oocyte yield from grafted ovaries but did enhance oocyte maturation and development. In conclusion, graft site affects the number and quality of oocytes produced from ovarian grafts.

scholarly journals Slow Freezing Versus Vitrification of Mouse Ovaries: from Ex Vivo Analyses to Successful Pregnancies after Auto-Transplantation

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Carmen Terren ◽  
Maïté Fransolet ◽  
Marie Ancion ◽  
Michelle Nisolle ◽  
Carine Munaut
Keyword(s):  
Ex Vivo ◽  
Fertility Restoration ◽  
Ovarian Tissue ◽  
Reference Method ◽  
Slow Freezing ◽  
Ovarian Tissue Cryopreservation ◽  
Freezing Damage ◽  
Specific Proteins

AbstractSlow freezing (SF) is the reference method for ovarian tissue cryopreservation. Vitrification (VT) constitutes an alternative but controversial method. This study compares SF and VT (open [VTo] and closed [VTc] systems) in terms of freezing damage and fertility restoration ability. In vitro analyses of C57Bl/6 SF or VTo-ovaries, immediately after thawing/warming or after culture (cult), revealed that event though follicular density was similar between all groups, nuclear density was decreased in VTo-ovaries compared to CT-ovaries (CT = 0.50 ± 0.012, SF = 0.41 ± 0.03 and VTo = 0.29 ± 0.044, p < 0.01). Apoptosis was higher in VTo-cult ovaries compared to SF-cult ovaries (p < 0.001) whereas follicular Bmp15 and Amh gene expression levels were decreased in the ovaries after culture, mostly after VTo (p < 0.001). Natural mating after auto-transplantation of SF, VTo and VTc-ovaries revealed that most mice recovered their oestrous cycle. Fertility was only restored with SF and VTo ovaries (SF: 68%; VTo: 63%; VTc: 0%; p < 0.001). Mice auto-transplanted with SF and VTo-ovaries achieved the highest number of pregnancies. In conclusion, in vitro, no differences between SF and VTo were evident immediately after thawing/warming but VTo ovaries displayed alterations in apoptosis and follicular specific proteins after culture. In vivo, SF and VTo ovary auto-transplantation fully restored fertility whereas with VTc-ovary auto-transplantation no pregnancies were achieved.

scholarly journals Influence of hydrogel encapsulation during cryopreservation of ovarian tissues and impact of post-thawing in vitro culture systems in a research animal model

2021 ◽  
Author(s):  
Paweena Thuwanut ◽  
Pierre Comizzoli ◽  
Alongkorn Pimpin ◽  
Weerayut Srituravanich ◽  
Wisan Sereepapong ◽  
...  
Keyword(s):  
Animal Model ◽  
Protein Phosphorylation ◽  
Fertility Preservation ◽  
Ovarian Tissue ◽  
Slow Freezing ◽  
Ovarian Tissue Cryopreservation ◽  
Culture Systems ◽  
Hydrogel Matrix ◽  
Hydrogel Encapsulation

Objective: Using domestic cats as a biomedical research model for fertility preservation, the present study aimed to characterize the influences of ovarian tissue encapsulation in biodegradable hydrogel matrix (fibrinogen/thrombin) on resilience to cryopreservation, and static versus non-static culture systems following ovarian tissue encapsulation and cryopreservation on follicle quality.Methods: In experiment I, ovarian tissues (n=21 animals; 567 ovarian fragments) were assigned to controls or hydrogel encapsulation with 5 or 10 mg/mL fibrinogen (5 or 10 FG). Following cryopreservation (slow freezing or vitrification), follicle viability, morphology, density, and key protein phosphorylation were assessed. In experiment II (based on the findings from experiment I), ovarian tissues (n=10 animals; 270 ovarian fragments) were encapsulated with 10 FG, cryopreserved, and in vitro cultured under static or non-static systems for 7 days followed by similar follicle quality assessments. Results: In experiment I, the combination of 10 FG encapsulation/slow freezing led to greater post-thawed follicle quality than in the control group, as shown by follicle viability (66.9%±2.2% vs. 61.5%±3.1%), normal follicle morphology (62.2%±2.1% vs. 55.2%±3.5%), and the relative band intensity of vascular endothelial growth factor protein phosphorylation (0.58±0.06 vs. 0.42±0.09). Experiment II demonstrated that hydrogel encapsulation promoted follicle survival and maintenance of follicle development regardless of the culture system when compared to fresh controls.Conclusion: These results provide a better understanding of the role of hydrogel encapsulation and culture systems in ovarian tissue cryopreservation and follicle quality outcomes using an animal model, paving the way for optimized approaches to human fertility preservation.

scholarly journals Requirement of mosXe protein kinase for meiotic maturation of Xenopus oocytes induced by a cdc2 mutant lacking regulatory phosphorylation sites.

1992 ◽  
Vol 12 (7) ◽  
pp. 3192-3203 ◽  
Author(s):  
K M Pickham ◽  
A N Meyer ◽  
J Li ◽  
D J Donoghue
Keyword(s):  
Protein Kinase ◽  
Oocyte Maturation ◽  
Xenopus Oocytes ◽  
Germinal Vesicle ◽  
Cyclin B1 ◽  
Phosphorylation Sites ◽  
Germinal Vesicle Breakdown ◽  
Regulatory Phosphorylation

The p34cdc2 protein kinase is a component of maturation-promoting factor, the master regulator of the cell cycle in all eukaryotes. The activity of p34cdc2 is itself tightly regulated by phosphorylation and dephosphorylation. Predicted regulatory phosphorylation sites of Xenopus p34cdc2 were mutated in vitro, and in vitro-transcribed RNAs were injected into Xenopus oocytes. The cdc2 single mutants Thr-14----Ala and Tyr-15----Phe did not induce germinal vesicle breakdown (BVBD) upon microinjection into oocytes. In contrast, the cdc2 double mutant Ala-14/Phe-15 did induce GVBD. Both the Ala-14 and Ala-14/Phe-15p34cdc2 mutants were shown to coimmunoprecipitate cyclin B1 and to phosphorylate histone H1 in immune complex kinase assays. Microinjection of antisense oligonucleotides to c-mosXe was used to demonstrate the role of mos protein synthesis in the induction of GVBD by the Ala-14/Phe-15 cdc2 mutant. Thr-161 was also mutated. p34cdc2 single mutants Ala-161 and Glu-161 and triple mutants Ala-14/Phe-15/Ala-161 and Ala-14/Phe-15/Glu-161 failed to induce GVBD in oocytes and showed a decreased binding to cyclin B1 in coimmunoprecipitations. Each of the cdc2 mutants was also assayed by coinjection with cyclin B1 or c-mosXe RNA into oocytes. Several of the cdc2 mutants were found to affect the kinetics of cyclin B1 and/or mos-induced GVBD upon coinjection, although none affected the rate of progesterone-induced maturation. We demonstrate here the significance of Thr-14, Tyr-15, and Thr-161 of p34cdc2 in Xenopus oocyte maturation. In addition, these results suggest a regulatory role for mosXe in induction of oocyte maturation by the cdc2 mutant Ala-14/Phe-15.

P–431 Human oocytes in-vitro maturation efficacy from infancy to adulthood – is there an optimal age?

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
G Karavani ◽  
P Wasserzug-Pash ◽  
T Mordechai-Daniel ◽  
M Klutstein ◽  
T Imbar
Keyword(s):  
Fertility Preservation ◽  
In Vitro Maturation ◽  
Ovarian Tissue ◽  
Age Groups ◽  
Ovarian Tissue Cryopreservation ◽  
Success Rates ◽  
Human Oocytes ◽  
Tertiary Center ◽  
Significant Difference

Abstract Study question Does human oocytes in-vitro maturation (IVM) effectiveness change throughout childhood, adolescence and adulthood in girls and women undergoing fertility preservation via ovarian tissue cryopreservation (OTC) prior to chemo-radiotherapy exposure? Summary answer The optimal age for IVM is from menarche to 25 years, while pre-menarche girls and women older than 30 years have extremely low maturation rates. What is known already In vitro maturation of oocytes from antral follicles seen during tissue harvesting is a fertility preservation technique with potential advantages over OTC, as mature frozen and later thawed oocyte used for fertilization poses decreased risk of malignant cells re-seeding, as compared to ovarian tissue implantation. We previously demonstrated that IVM performed following OTC in fertility preservation patients, even in pre-menarche girls, yields a fair amount of oocytes available for IVM and freezing for future use. Study design, size, duration A retrospective cohort study, evaluating IVM outcomes in chemotherapy naïve patients referred for fertility preservation by OTC that had oocyte collected from the medium with attempted IVM between 2003 and 2020 in a university affiliated tertiary center. Participants/materials, setting, methods A total of 133 chemotherapy naïve patients aged 1–35 years with attempted IVM were included in the study. The primary outcome was IVM rate in the different age groups – pre-menarche (1–5 years and ≥6 years), post-menarche (menarche–17 years), young adults (18–24 years) and adults (25–29 and 30–35 years). Comparison between paired groups for significant difference in the IVM rate parameter was done using the Tukey’s Studentized Range (HSD) Test. Main results and the role of chance A gradual increase in mean IVM rate was demonstrated in the age groups over 1 to 25 years (4.6% (1–5 years), 23.8% (6 years to menarche) and 28.4% (menarche to 17 years), with a peak of 38.3% in the 18–24 years group, followed by a decrease in the 25–29 years group (19.3%), down to a very low IVM rate (8.9%) in the 30–35 years group. A significant difference in IVM rates was noted between the age extremes – the very young (1–5 years) and the oldest (30–35 years) groups, as compared with the 18–24-year group (p &lt; 0.001). Number of oocytes matured, percent of patients with matured oocytes and overall maturation rate differed significantly (p &lt; 0.001). Limitations, reasons for caution Data regarding ovarian reserve evaluation was not available for most of the patients, due to our pre-op OTC procedures protocol. None of our patients have used their frozen in-vitro matured oocytes, as such further implications of age on in-vitro matured oocytes quality and implantation potential has yet to be evaluated. Wider implications of the findings: Our finding of extremely low success rates in those very young (under 6 years) and older (≥30 years) patients suggest that IVM of oocyte retrieved during OTC prior to chemotherapy should not be attempted in these age group. Trial registration number N/A

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