Effects of AavLEA1 Protein on Mouse Ovarian Tissue Cryopreservation by Vitrification

Abstract

Conventional ovarian tissue cryopreservation often destroys the structural, functional, and DNA integrity of the ovarian tissue. How to effectively retain the ultrastructure and subsequent function of ovarian tissue during cryopreservation has long been an issue of concern. Late embryogenesis abundant (LEA) proteins are a class of highly hydrophilic proteins and have been reported to protect various cells from water stress. However, whether LEA proteins exert protective effects on ovarian tissue cryopreservation remains unknown. To investigate the benefit of LEA proteins in ovarian tissue cryopreservation, we purified the recombinant AavLEA1 protein, a member of Group 3 LEA proteins, then cryopreserved the mouse ovaries with this protein by vitrification, and obtained the ovarian follicle structure, cellular proliferation, apoptosis, and GAPDH gene expression of postcryopreservation ovaries. We found that recombinant AavLEA1 protein protected the ovarian follicles from cryoinjury, improved the proliferative ability of follicles, decreased the apoptosis, and promoted the GAPDH gene expression. These results indicated that the LEA protein enhanced the antiapoptosis ability of ovarian cells and retained DNA/RNA integrity against cryoinjury during ovarian tissue vitrification. LEA proteins exert beneficial effects on ovarian tissue cryopreservation, and maybe provide a novel cryoprotective agent for ovarian tissue cryopreservation.

Introduction

Fertility preservation (including egg, embryo, and ovarian tissue cryopreservation) is one of the most critical issues addressed in female cancer patients at the reproductive age.^1,2 As a promising option that could help women reserve their ovaries, ovarian tissue cryopreservation provides a prospective approach for female fertility restoration.^3,4 Ovarian tissue cryopreservation avoids ovarian stimulation and does not require a male partner or sperm donor; thus it becomes the only available option for prepubertal girls. In addition to safeguarding fertility, autotransplanting frozen-thawed ovarian tissue grafts is also conducive to restore long-term hormonal function.⁵ However, conventional ovarian tissue cryopreservation by slow freezing is time-consuming and requires expensive equipment. Besides, it has the potential to form ice crystals during freezing, which may mechanically and physically damage ovarian cells.⁶ Conventional ovarian tissue cryopreservation is reported to impair follicular morphology and gene expression⁷ and cause DNA fragmentation and follicular pool exhaustion.^8,9 Thereby it limits the developmental potential of follicles and shortens the grafts expectancy.^10,11 How to effectively retain the ultrastructure and subsequent function of ovarian tissue during cryopreservation has long been an issue of concern.

Slow freezing and vitrification are the two most commonly used methods for ovarian tissue cryopreservation. Vitrification effectively inhibits the ice crystal formation and thus prevents cryoinjury and maintains the integrity of ovarian stroma and the morphology of blood vessels.^12,13 Nevertheless, ovarian tissue vitrification needs a higher concentration of cryoprotective agents (CPAs) and a faster cooling rate.¹⁴ High concentrations of CPAs such as dimethyl sulfoxide (DMSO), ethylene glycol (EG), glycerol, and so on, exhibit severe osmotic or chemical toxicity to cells in conventional vitrification.¹⁵ Cryoinjury, which results from the cytotoxicity of CPAs and the mechanical damage owing to osmotic shock,^15,16 plays a vital role in optimization of the vitrification process during ovarian tissues cryopreservation. Therefore, attempts have been made to explore the nontoxic CPAs to improve ovarian tissues cryopreservation.¹⁷

Late embryogenesis abundant (LEA) proteins are mainly a family of highly hydrophilic and generally unstructured proteins produced by nonaquatic plants and lower animals. These proteins allow their survival from stressful environments, such as freezing, desiccation, and high salinity.^18–20 Studies have reported that LEA proteins considerably enhance the tolerance of mammalian cells to water stress.¹⁹ In our previous study, we found that recombinant AavLEA1 protein, a Group 3 LEA protein from the nematode Aphelenchus avenae, effectively protected human umbilical cord matrix mesenchymal stem cells (hUCM-MSCs) against cryogenic damage.²¹

Despite the nontoxic and protective properties of LEA proteins, the effects of LEA proteins on ovarian tissue cryopreservation are uncertain. Therefore, the aim of this study was to determine whether LEA proteins protect mouse ovaries from cryoinjury during vitrification, thus optimizing the ovarian tissue cryopreservation protocols for fertility preservation.

Materials and Methods

LEA proteins purification

AavLEA1 proteins were obtained and purified according to a previous study.²¹ In brief, recombinant AavLEA1 proteins were overexpressed in Escherichia coli Rosetta (DE3; Novagen) and induced by isopropyl-b-d-thiogalactoside (IPTG). The recombinant proteins were purified from a bacterial lysate using Ni²⁺-nitrilotriacetate affinity resin (Ni-NTA; Qiagen) and HiLoad 16/60 Superdex 200 (GE Healthcare). Finally, AavLEA1 proteins were stored at −80°C for cryopreservation trials.

Collection of mouse ovaries

Female BALB/c mice (aged 8–10 weeks with an average weight of 22.81 ± 1.5 g, purchased from Boyuan Laboratory Supplies Co., Ltd., Hefei, China) were killed by cervical dislocation. Whole ovaries were aseptically removed and temporarily rinsed in phosphate-buffered saline (PBS) at room temperature (RT). The size of each ovary was ∼1.5 × 1 × 1 mm³. All ovaries were randomly divided into three groups: (1) fresh group (n = 28), in which the ovaries were fixed or lysed immediately once removed; (2) control group (n = 28), in which the ovaries were exposed to the conventional vitrification solutions without LEA protein; and (3) LEA protein group (n = 28), in which the vitrification solutions contained 0.1 mg/mL LEA protein. The investigation was approved by the Animal Ethics Committee of Anhui Medical University Animal Center.

Cryopreservation of mouse ovarian tissues

Vitrification

Whole ovaries were cryopreserved according to a modified vitrification protocol.²² The freezing and warming solutions were all prepared in basic medium containing HEPES-buffered M199 solution (Sigma-Aldrich) with 100 U/mL penicillin G and streptomycin (Hyclone). Ovaries in the control group were initially equilibrated in basic medium for 10 minutes followed by a second equilibration in basic medium added with 5% EG (v/v) and 5% DMSO (v/v) for 10 minutes, then exposed to a vitrification solution containing basic medium, 10% EG (v/v), and 10% DMSO (v/v) for another 10 minutes. However, the ovaries in the LEA protein group were further exposed to the vitrification solution supplemented with LEA protein (0.1 mg/mL) for 5 minutes after rinsing in the above vitrification solution for 5 minutes. The entire procedure was conducted at RT. Excess vitrification solution was removed using sterile gauze, then groups of three to four ovaries were immediately plunged into liquid nitrogen for ∼30 seconds until the samples became translucent, then transferred into a 1.8 mL plastic standard cryovials (Sorfa, Zhejiang, China) and stored in liquid nitrogen for 2 weeks.

Thawing

The cryovials were removed from the liquid nitrogen and allowed to stand in air at RT for 30 seconds and then immersed into a 37°C water bath for 60 seconds. The ovarian tissues were suspended in basic medium supplemented with descending concentrations of sucrose (0.5, 0.25, and 0 M) for 5 minutes in each washing step.

Histological analysis and follicle counting

After thawing, the ovarian tissues were fixed overnight in 4% paraformaldehyde, dehydrated in a series of alcohol solutions of ascending concentration, clarified with xylene, embedded in paraffin wax, and serially sectioned at 5 μm thickness. Then the ovarian tissues were stained with hematoxylin and eosin (HE) and observed under a light microscope of × 400 in 10 fields for each sample. Follicles at different developmental stages were categorized and counted according to the criteria described by Candy et al.²³ Meanwhile, follicles were scored as either morphologically normal or damaged based on the same criteria as in Candy et al. The normal follicles had intact oocyte and regular granulosa cell layers, but damaged follicles were characterized by cytoplasmic vacuolization, detachment of oocyte and granulosa cells, and a large area of irregular granulosa cells with pyknotic nuclei.²³ The total number and proportion of normal follicles were recorded in primordial follicles, primary follicles, secondary follicles, and antral follicles, respectively. Primordial and primary follicles were classified in a group owing to the similar morphological characteristics in mouse ovaries; meanwhile, the secondary and antral follicles were included in a group for similar percentage of morphologically healthy follicles.

Immunohistochemistry

Antigen Ki-67 is a nuclear protein that is associated with cellular proliferation. The proliferation analysis of follicles was performed using the Ki-67 assay. Sections were dewaxed in xylene and rehydrated through decreasing concentrations of alcohol, and immersed in Tris-buffered saline with 0.1% (v/v) Tween 20 (TBST) for 30 minutes. The antibody retrieval was achieved by microwaving each section in 10 mM sodium citrate (pH 6.0) until almost boiling two times, followed by washing three times with PBS, and then sections were immersed in hydrogen peroxide for 10 minutes, to quench endogenous peroxidase activity. After 3 × 2 minutes washes in PBS, sections were incubated with appropriate blocking solution (donkey serum) for 1.5 hours and then washed 3 × 2 minutes. Subsequently, rabbit anti-Ki67 (1:400; Cell Signaling) was added and incubated at 4°C overnight. A secondary antibody (ZSGB-BIO, Beijing, China) was then added and incubated at 37°C for 30 minutes. Color was developed with DAB (Sigma Co., St. Louis, MO) and counterstained with hematoxylin. The sections were observed under an optical microscope (Eclipse E200; Nikon Co., Tokyo, Japan) and photographed. The protein expressions were analyzed with the Image-Pro Plus image analysis software (Media Cybernetics, MD).

Immunofluorescence

The apoptosis of follicles and stroma were evaluated by the TUNEL assay kit (Elabscience, China). Ovarian sections were deparaffinized, rehydrated, and washed in PBS for 5 minutes. Then, ovarian sections were incubated with 50 μL of TUNEL reaction mixture at 37°C for 60 minutes in a humidified dark chamber. After washing, 50 μL streptavidin-FITC mixture was added. The fluorescence intensity of sections was obtained by a confocal scanning laser microscopy (LSM800; Zeiss, Germany), and analyzed by Zen software.

Culture in vitro and western blotting

To allow apoptotic pathway activation, the ovaries were cultured in vitro, and the according to the following specific steps. Thawed ovaries were placed in a transwell chamber containing a 150 μL mixture of Matrigel (Corning, Sweden) and DMEM/F-12 (Gibco) medium (1:1). Then the transwell chamber, as the upper chamber, was put into the lower chamber, the 24-well culture plate including 800 μL DMEM/F-12 medium. Then the ovaries were incubated in humidified air with 5% CO₂ for 4 days, with half the medium in the lower chamber removed and replaced with fresh DMEM/F-12 medium on alternate day. After in vitro culture, ovaries were suspended in radioimmunoprecipitation assay buffer (Beyotime, Shanghai, China) supplemented with 1% phenylmethanesulfonyl fluoride (Beyotime) and phosphatase inhibitor (Beyotime). The ovaries were cut with scissors and the protein concentration was determined by the BCA method. Proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred from the gels to polyvinylidene difluoride transfer membranes. The membranes were washed briefly in PBS-Tween and incubated with β-actin (1:2000; Sino Biological, Beijing, China), B cell lymphoma 2 protein (Bcl-2, 1:2000; Abcam, Cambridge, United Kingdom), and Bcl-2-associated X protein (Bax, 1:2000; Abcam) antibody at 4°C overnight. The membranes were next washed three times in PBS-Tween using a rotary shaker. The washed membranes were incubated with horseradish peroxidase-conjugated anti-rabbit for 1 hour. The membranes were washed again and processed with an ECL detection kit (Biosharp) according to the manufacturer's instructions, to visualize the proteins recognized by the antibodies. Western blots were digitally scanned and analyzed using ImageJ. All analysis was standardized to β-actin.

Real-time polymerase chain reaction procedure

Total RNA was extracted from each ovary using Trizol Reagent (Ambion, Carlsbad, CA) according to manufacturer's instructions. One microgram of total RNA was subjected to reverse transcription of mRNA using oligo-dT as a primer and a reverse transcription kit (Transgen Biotech, Beijing, China) to generate total cDNA. The expression levels of GAPDH mRNA were assessed by real-time polymerase chain reaction (RT-PCR) with SYBR Premix Ex Tap™ (TaKaRa, China) and the ABI 7500 real-time PCR system. The reaction conditions for gene amplification were 95°C for 10 minutes, 95°C for 15 seconds, and then 60°C for 1 minute. All procedures were performed following the manufacturer's instructions. The specific PCR primers were designed according to DNA sequences in the NCBI database. The β-actin was used as an internal standard. The primer sequences are given in Table 1. All data were processed using the 2^ΔΔCt method.

Table 1.

Primer Sequences

Genes	Primer sequence (5′-3′)
GAPDH	Forward: 5′-GACTTCAACAGCAACTCCCAC-3′
GAPDH	Reverse: 5′-TCCACCACCCTGTTGCTGTA-3′
β-actin	Forward: 5′-CTACCTCATGAAGATCCTGACC-3′
β-actin	Reverse: 5′-CACAGCTTCTCTTTGATGTCAC-3′

Statistical analyses

The results are given as mean ± standard deviation for at least three repeated trials. The statistical analyses were performed using SPSS 18.0 software. The individual comparisons were performed using Student's two-tail paired t-test. The percentage of normal follicles in each group was compared by the chi-square test. p < 0.05 was considered significant.

Results

Histological examination and follicle counting

To evaluate the influence of LEA proteins on ovary structure, we observed the follicular morphology of cryopreserved ovary after HE staining. A total of 30 ovaries randomly divided into three groups (10 sections in each group) were observed and analyzed. Morphologically normal and abnormal follicles in different growing stages are given in Figure 1A. The primordial follicles, characterized by one layer of flattened granulosa cells, constitute the ovarian reserve, which can be recruited for folliculogenesis and even the selection for ovulation. The primary and secondary follicles, characterized by one and two/more layers of cuboidal granulosa cells respectively, are advanced growing stages after the dormant primordial follicles are activated. As the follicles grow, a fluid-filled cavity appears and they are termed antral follicles, which continue to increase in size and eventually ovulate. Degenerated follicles in different stages were characterized by abnormal morphology, such as a shrunken cytoplasm or disorganized granulose cells. As given in Figure 1B, 73.76% ± 5.14% of total follicles in the fresh group were intact. The proportion of the total normal follicles is 62.72% ± 3.93% and 71.63% ± 2.83% in the control group and LEA protein group, respectively, and there was statistically significant difference between the control group and LEA protein group (p < 0.05). The proportion of normal follicles in the primordial and primary follicles in the LEA group was higher than that in the control group (p < 0.05) (Fig. 1C). Nevertheless, no significant difference was observed in the normal follicles proportion of secondary and antral follicles between the control group and the LEA protein group (Fig. 1D). The results confirmed that the LEA protein could protect the follicles of cryopreserved ovarian tissue by vitrification, especially for the primordial and primary follicles.

FIG. 1.

Histology and the proportion of morphologically normal follicles of mouse ovaries after cryopreservation. (A) Histological images by hematoxylin and eosin staining and classification of follicles in each stage after thawing. (a–d) Morphologically normal follicles: primordial follicles, primary follicles, secondary follicles, and antral follicles; (e–h) Morphologically abnormal follicles: primordial follicles, primary follicles, secondary follicles, and antral follicles. (B–D) The proportion of morphologically normal follicles at each stage of follicular development. (*p < 0.05). LEA, late embryogenesis abundant.

Immunohistochemistry and Immunofluorescence analysis

The proliferation and apoptosis of cryopreserved ovarian tissues were assessed by Ki67 immunohistochemistry and TUNEL immunofluorescence. The expression of Ki67 in the fresh (n = 3), control (n = 3), and LEA protein (n = 3) groups are given in Figure 2A, and the proportion of Ki67(+) cells were measured and analyzed (Fig. 2B). A significant increase of the expression of Ki67 was observed in the LEA protein group compared with the control group, and there was no significant difference between the fresh group and LEA protein group (Fig. 2B). The apoptosis of ovarian tissues was detected depending on the intensity of TUNEL(+) signal in the follicles and stroma. The relative intensity of TUNEL(+) cells was significantly higher in the control group (n = 3), whereas no difference was detected in the fresh (n = 3) and LEA protein (n = 3) groups (Fig. 3B). These results indicated that LEA protein could improve the proliferation and inhibit the apoptosis of cryopreserved ovarian tissue by vitrification.

FIG. 2.

Expression of Ki67 in the fresh (n = 3), control (n = 3), and LEA protein (n = 3) group. (A) Ovarian cells expression of Ki67 after cryopreservation was determined by immunohistochemistry. The brown stain indicates Ki67(+) cells. (B) Comparison of the relative Ki67 expression in each group (*p < 0.05).

FIG. 3.

TUNEL assay of ovarian cell apoptosis postcryopreservation. (A) Ovarian cells expression of TUNEL in the fresh (n = 3), control (n = 3), and LEA protein (n = 3) group. The green stain indicates TUNEL(+) cells. (B) The relative intensity of TUNEL(+) signal per group (*p < 0.05).

Western blotting analysis

To further investigate the possible role of the LEA proteins on apoptosis of ovaries, we assessed the expression of the apoptosis-related proteins in both the control group (n = 6) and LEA protein group (n = 6). After thawing and cultivation in vitro for 4 days, the protein of ovarian cells was extracted and analyzed as given in Figure 4A. Western blotting results analysis showed significantly decreased expression of cleaved-caspase-3 in the LEA protein group compared with that in the control group (Fig. 4B). In addition, an increase in the ratio of Bcl-2 to Bax was observed in LEA protein group (Fig. 4C). These results indicated that the LEA protein enhanced the expression of antiapoptosis proteins and inhibited the expression of proapoptosis proteins in the cryopreservation of ovarian tissues.

FIG. 4.

Western blotting showing the expression of apoptosis-related proteins in control (n = 6) and LEA protein (n = 6) group. (A) Bcl-2, Bax, cleaved-caspase-3 expression in the control and LEA protein group, (B) quantification of cleaved-caspase-3 expression in the control and LEA protein group, (C) Bcl-2/Bax ratio in the control and LEA protein group (*p < 0.05).

Real-time PCR analysis

We further explored the DNA/RNA integrity of cryopreserved mouse ovarian tissue based on detecting the expression of the housekeeping gene, GAPDH. After thawing, the ovaries were used for RNA purification, and the expression of GAPDH was analyzed by quantitative real-time PCR (RT-qPCR) (Fig. 5). Figure 5A shows that the GAPDH gene expression level of the LEA protein group (n = 6) was higher than that of the control group (n = 6). To quantify this observation, we performed real-time RT-PCR. Figure 5B presents the large increase of GAPDH gene expression in the LEA protein group (n = 6) compared with the control group (n = 6). These results reflect that cryopreservation with LEA protein can effectively retain the integrity of DNA and RNA.

FIG. 5.

Expression of GAPDH in the control group (n = 6) and LEA protein group (n = 6). (A) Analysis of amplified cDNA in the control group and LEA protein group by electrophoresis in 2% agarose gel and ethidium bromide staining. (B) quantitative real-time PCR results for the GAPDH gene in ovarian tissues (*p < 0.05).

Discussion

CPAs are indispensable for the success of ovarian tissue vitrification, to maintain a stable glass state without crystallization.²⁴ However, high CPA concentration increases the possibility of osmotic damage and CPA-induced cytotoxicity, especially for the conventional permeable CPAs. Cryobiologists have been trying to develop nontoxic and effective CPAs, such as trehalose and anti-freezing proteins. LEA proteins have the properties of nontoxicity, hydrophilicity, resistance to dry environment, and tolerance of dehydration stress. In this study, we investigated the effects of LEA proteins on mouse ovary vitrification and indicated that supplementation of recombinant LEA proteins can improve the efficiency of mouse ovary cryopreservation.

Previous studies have revealed the cryoprotective effects of LEA proteins on mammal cells. It was demonstrated that supplementation of recombinant AavLEA1 proteins provides protection to hUCM-MSCs against cryoinjury.²¹ Czernik et al. transfected three different LEA proteins with different subcellular targeting into sheep fibroblasts, and showed significantly improved functionality of the targeted organelles and cell viability after air drying.²⁵ Despite there having been few studies investigating the effect of LEA proteins on ovarian tissue cryopreservation, the results of this study were in line with many studies on cryopreservation of other cells. In this study, we showed the LEA protein improved the proportion of normal follicles after freezing/thawing. The percentage of morphologically normal primordial/primary follicles in the LEA protein group was significantly higher than that in the control group, although there was no significant difference when it came to secondary/antral follicles. LEA protein, a large-molecular-weight nonpermeable CPA, can prevent the loss of intracellular water and protect organisms from water depletion.

In addition, our study showed LEA protein improved the cellular proliferation and inhibited the apoptosis of cryopreserved mouse ovarian tissues. The proliferating cell nuclear antigen Ki67 activity was higher, and the TUNEL fluorescence intensity was lower in the LEA protein group than those in the control group. Furthermore, we also determined the expression of apoptosis-related protein, including Bcl-2, Bax, and cleaved-caspase-3. The Bcl-2 family plays a major role in modulating the ovarian mitochondrial apoptosis pathway. Bax can promote the release of mitochondrial cytochrome C and subsequent activation of caspase-3, and thus induce cellular apoptosis, whereas Bcl-2 can prevent these by forming heterodimers with Bax.²⁶ Therefore, the ratio of Bcl-2 (antiapoptotic protein) to Bax (proapoptotic protein) and the quantification of downstream caspase-3 are generally regarded as important factors in determining susceptibility to apoptosis.²⁷ In our study, the western blotting demonstrated the downregulated expression of cleaved-caspase-3 and the upregulated Bcl-2/Bax ratio in LEA protein group. These results may be owing to the protective effects of LEA protein against hyperosmolarity and maintaining enzyme activity. These could partly be owing to the high stability of LEA proteins, which is illustrated by the increase of intraprotein hydrogen bonds along with the decrease of protein–water hydrogen bonds as dehydration proceeds.²⁸ Rodriguez-Salazar et al.²⁹ also found that vegetative cells of the LEA1 mutant and the Azotobacter vinelandii disrupted the LEA1 gene were more sensitive to osmotic stress, which demonstrated that the LEA1 protein is indispensable for protection against hyperosmolarity and survival in dry conditions.

Furthermore, we determined that the GAPDH mRNA expression by RT-qPCR in frozen/thawed ovaries. The specific contributions of cryopreservation to nucleic acid degradation are controversial, but detecting the expression level of widely expressed genes like GAPDH (a well-known housekeeping gene) is an effective approach to assess the quality of DNA/RNA.³⁰ In addition, nuclear GAPDH is involved in maintaining DNA integrity by interacting with DNA repair enzymes and regulating telomeric DNA length.^31,32 Our results demonstrated that the LEA protein promoted the GAPDH mRNA expression; thus, cryopreservation with LEA protein may contribute to retaining the quality and integrity of DNA/RNA.

Although the cryoprotective mechanisms of LEA protein in this study are not exactly clear, the mechanisms suggested by previous studies may be applicable to ovarian tissue cryopreservation, including improvement in cell tolerability against hyperosmotic stress,²¹ protection of the plasma membrane,³³ and stabilization of the vitrified state by increasing the glass-transition temperature.²⁸ However, the exact cryoprotective mechanism of LEA protein remains to be further investigated.

In conclusion, this study proved the protective effects of LEA protein on mouse ovary vitrification in terms of tissue morphology, cellular proliferation, apoptosis, and GAPDH gene expression, which were consistent with previous studies. LEA protein may represent a promising supplementary agent for reducing cryoinjury during ovarian tissue vitrification. However, LEA protein, a nonpermeable protective agent, cannot penetrate the cells to better inhibit the formation of intracellular ice crystals. Therefore, the introduction of LEA protein into cells may be a direction for further research. In addition, further studies with human ovarian tissue to elucidate the exact mechanism underlying this protective effect are necessary for the application of LEA proteins in clinical practice.

Footnotes

Author Disclosure Statement

No conflicting financial interests exist.

Funding Information

This work was supported by grants from National Key Technology Research & Development Program of China (No. 2017YFC1002004), National Natural Science Foundation of China (No. 81901437), and Anhui Provincial Natural Science Foundation (Nos. 1908085MH244, 1908085QH314).

References

Filatov

, Khramova

, Kiseleva

, et al. Female fertility preservation strategies: Cryopreservation and ovarian tissue in vitro culture, current state of the art and future perspectives. Zygote, 2016; 24:635–653.

Mahajan

. Fertility preservation in female cancer patients: An overview. J Hum Reprod Sci, 2015; 8:3–13.

Kristensen

, Andersen

. Cryopreservation of ovarian tissue: Opportunities beyond fertility preservation and a positive view into the future. Front Endocrinol, 2018; 9:347.

Rajabi

, Aliakbari

, Yazdekhasti

. female fertility preservation, clinical and experimental options. J Reprod Infertil, 2018; 19:125–132.

Leonel

ECR

, Corral

, Risco

, et al. Stepped vitrification technique for human ovarian tissue cryopreservation. Sci Rep, 2019; 9:20008.

Amorim

, Curaba

, Van Langendonckt

, et al. Vitrification as an alternative means of cryopreserving ovarian tissue. Reprod Biomed Online, 2011; 23:160–186.

Labrune

, Jaeger

, Santamaria

, et al. Cellular and molecular impact of vitrification versus slow freezing on ovarian tissue. Tissue Eng Part C Methods, 2020; 26:276–285.

Mathias

, D'Souza

, Uppangala

, et al. Ovarian tissue vitrification is more efficient than slow freezing in protecting oocyte and granulosa cell DNA integrity. Syst Biol Reprod Med, 2014; 60:317–322.

Fabbri

, Pasquinelli

, Keane

, et al. Optimization of protocols for human ovarian tissue cryopreservation with sucrose, 1,2-propanediol and human serum. Reprod Biomed Online, 2010; 21:819–828.

10.

Barbato

, Gualtieri

, Capriglione

, et al. Slush nitrogen vitrification of human ovarian tissue does not alter gene expression and improves follicle health and progression in long-term in vitro culture. Fertil Steril, 2018; 110:1356–1366.

11.

Amorim

, David

, Dolmans

M-M

, et al. Impact of freezing and thawing of human ovarian tissue on follicular growth after long-term xenotransplantation. J Assist Reprod Genet, 2011; 28:1157–1165.

12.

Corral

, Clavero

, Gallardo

, et al. Ovarian tissue cryopreservation by stepped vitrification and monitored by X-ray computed tomography. Cryobiology, 2018; 81:17–26.

13.

Keros

, Xella

, Hultenby

, et al. Vitrification versus controlled-rate freezing in cryopreservation of human ovarian tissue. Hum Reprod, 2009; 24:1670–1683.

14.

Wowk

. Thermodynamic aspects of vitrification. Cryobiology, 2010; 60:11–22.

15.

Sanfilippo

, Canis

, Smitz

, et al. Vitrification of human ovarian tissue: A practical and relevant alternative to slow freezing. Reprod Biol Endocrinol, 2015; 13:67.

16.

Lee

, Ryu

, Kim

, et al. Comparison between slow freezing and vitrification for human ovarian tissue cryopreservation and xenotransplantation. Int J Mol Sci, 2019; 20:3346.

17.

Lee

, Youm

, Lee

, et al. Effect of antifreeze protein on mouse ovarian tissue cryopreservation and transplantation. Yonsei Med J, 2015; 56:778–784.

18.

Bartels

. Desiccation tolerance studied in the resurrection plant Craterostigma plantagineum. Integrative and Comparative Biology, 2005; 45:696–701.

19.

, Chakraborty

, Borcar

, et al. Late embryogenesis abundant proteins protect human hepatoma cells during acute desiccation. Proc Natl Acad Sci U S A, 2012; 109:20859–20864.

20.

Tompa

, Kovacs

. Intrinsically disordered chaperones in plants and animals. Biochem Cell Biol, 2010; 88:167–174.

21.

Wang

, Zhang

, Zhu

, et al. Effects of recombinant AavLEA1 protein on human umbilical cord matrix mesenchymal stem cells survival during cryopreservation. Biopreserv Biobank, 2020; 18:290–296.

22.

Zhou

X-H

, Wu

Y-J

, Shi

, et al. Cryopreservation of human ovarian tissue: Comparison of novel direct cover vitrification and conventional vitrification. Cryobiology, 2010; 60:101–105.

23.

Candy

, Wood

, Whittingham

. Effect of cryoprotectants on the survival of follicles in frozen mouse ovaries. J Reprod Fertil, 1997; 110:11–19.

24.

Mukaida

, Oka

. Vitrification of oocytes, embryos and blastocysts. Best Pract Res Clin Obstet Gynaecol, 2012; 26:789–803.

25.

Czernik

, Fidanza

, Luongo

, et al. Late Embryogenesis Abundant (LEA) proteins confer water stress tolerance to mammalian somatic cells. Cryobiology, 2020; 92:189–196.

26.

Liu

, Qiu

, Xue

, et al. Small extracellular vesicles derived from embryonic stem cells restore ovarian function of premature ovarian failure through PI3K/AKT signaling pathway. Stem Cell Res Ther, 2020; 11:3.

27.

Zhang

, Deng

, Zhang

, et al. Effect of PGC-1α overexpression or silencing on mitochondrial apoptosis of goat luteinized granulosa cells. J Bioenerg Biomembr, 2016; 48:493–507.

28.

Hand

, Menze

, Toner

, et al. LEA proteins during water stress: Not just for plants anymore. Annu Rev Physiol, 2011; 73:115–134.

29.

Rodriguez-Salazar

, Moreno

, Espin

. LEA proteins are involved in cyst desiccation resistance and other abiotic stresses in Azotobacter vinelandii. Cell Stress Chaperones, 2017; 22:397–408.

30.

Kumar

, Singh

, Bhatia

, et al. Audit of quality and quantity of nucleic acid yield from pediatric acute leukemia cases following a bio-banking initiative. Indian J Hematol Blood Transfus, 2019; 35:77–82.

31.

Kosova

, Khodyreva

, Lavrik

. Role of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in DNA repair. Biochemistry (Mosc), 2017; 82:643–654.

32.

Sirover

. Pleiotropic effects of moonlighting glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in cancer progression, invasiveness, and metastases. Cancer Metastasis Rev, 2018; 37:665–676.

33.

Moore

, Hand

. Cryopreservation of lipid bilayers by LEA proteins from Artemia franciscana and trehalose. Cryobiology, 2016; 73:240–247.