Treatment with trypLE before freezing improves thawing integrity and functionality of sheep ovarian tissue

doi:10.21203/rs.3.rs-4991603/v1

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Research Article

Treatment with trypLE before freezing improves thawing integrity and functionality of sheep ovarian tissue

https://doi.org/10.21203/rs.3.rs-4991603/v1

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Objective: To study innovative approaches to ovarian tissue cryopreservation, a critical issue for fertility preservation in pediatric cancer patients. Despite historical attempts, recent advances in cancer treatment have underscored the urgent need for more effective and reliable ovarian tissue cryopreservation methods. Our research aims to evaluate if decreasing the rigidity of stroma before cryopreservation by investigating pre-treatments with enzymes can enhance the quality of ovarian tissue post-thawing.

Design: Our research evaluated the use of five commonly used enzymes to disaggregate tissue (trypLE, collagenase, dispase, accutase and hyaluronidase) before freezing ovarian tissue to decrease rigidity and facilitate cryopreservation. Sheep ovaries, with high similarity to human ovaries, were used as an animal model. Tissue structure, cell proliferation, apoptosis and viability were assessed before and after thawing.

Results: Our findings showed that enzymatic treatment with trypLE before freezing offered immediate benefits post-thawing with the highest viability values and percentage of intact follicles. However, 2 and 7 days after thawing and culture, dispase showed the highest viability but with loci of necrosis at the edges and more damaged follicles compared to trypLE. A decrease in viability was observed after thawing and culturing the samples. The pretreatment with accutase damaged the tissue severely with also the lowest viability values. Ki67-positive follicles and stromal cells were observed in fresh samples, but only trypLE and hyaluronidase maintained Ki67-positive antral follicles after 2 days culture. Besides, only trypLE maintained all follicles negative to caspase-3 after thawing, and 7 days after culture primordial follicles were apoptotic in all treatments apart from trypLE.

Conclusion: our findings suggest that trypLE pretreatment could provide a beneficial approach for maintaining the functions and viability of cryopreserved ovaries after thawing. Further research is needed to fully understand their impact and optimize cryopreservation protocols in this important clinical context.

cryopreservation

ovarian tissue

follicles

fertility

enzymes

cancer

prepubertal

Fertility preservation in girls with pediatric cancer is a crucial and challenging issue in modern medicine (1). Often, cancer treatment in childhood, such as chemotherapy and radiotherapy, can have devastating effects on these patients' future reproductive capacity. Since procreation capability is considered to be valuable in the quality of life, offering fertility preservation options to the patients seems not only ethical but also necessary.

While fertility can be preserved by freezing oocytes or embryos in post-pubertal woman, these modalities are not an option for younger patients as they do not respond to hyperstimulation ovarian treatment due to lack of maturation of the hypothalamic-pituitary axis (2). The primary fertility preservation option that exists for pre-pubertal girls or oncologic patients undergoing immediate gonadotoxic cancer treatment is ovarian tissue cryopreservation (OTC) and its subsequent transplantation (OTT) (3). Currently, it is estimated that more than 10,000 girls and woman worldwide have undergone OTC (4) and more than 200 children have been conceived from OTT, with an estimated birth rate of around 47% (5). Human OTC is performed through two general approaches: slow freezing and vitrification. The slow-cooling method is the current gold standard for ovarian tissue cryopreservation since it was employed in most of the cases when a live birth has been achieved after cryopreservation and transplantation (3). However, this process presents significant challenges. Transplanted ovarian tissue seems to undergo an initial over-recruitment of follicles and a dysfunctional folliculogenesis, which cause an acute impairment of the follicular pool in grafted tissue. There is loss of approximately 50% of follicles because of damage during preservation, ischemia and oxidative stress damage (6, 7).

Physical junction of granulosa cells to the oocyte is very important in early stages of follicular development. It has been demonstrated that rigidity of stroma impacts the ability of follicles to accommodate and survive the current cryopreservation process. Limited information about rigidity modulation and ovarian tissue preservation is available. Only one study conducted on mouse ovaries using collagenase as a facilitating enzyme has demonstrated that pretreatment of ovaries with collagenase before vitrification maintained ovarian reserve (8). The underlying basis for the improved preservation is related to accommodating high osmotic solutions. Authors observed that brief treatment with collagenase before exposure to the hyperosmotic solution maintained the cadherin adhesions between the oocyte and granulosa cell layers. Consequently, secondary follicles collected from frozen-thawed ovaries that were pretreated with collagenase showed normal follicular development.

Ethical barriers and the limited availability of human ovarian tissue preclude the widespread use of human ovaries for research. Consequently, a robust animal model comparable with humans in needed. The sheep ovary is an adequate candidate for research due to the similar ovarian architecture, limited number of developed follicles, single ovulation with primordial follicles distributed superficially in the cortex and a comparable collagen-dense cortical layer (9, 10, 11, 12).

In this study, we aimed to improve the ovarian tissue cryopreservation protocol by treating tissue briefly with 5 different enzymes (TrypLE, collagenase, dispase, accutase, hyaluronidase) before exposure to freezing media to determine the treatment that maintained the functions and viability of ovarian tissue after thawing.

2.1. Experimental design: A strategy for optimizing the cryopreservation of ovarian tissue by decreasing the rigidity of the tissue by treatment before freezing with 5 different enzymes (trypLE, collagenase, dispase, accutase, hyaluronidase) was studied, comparing with an untreated control. Sheep ovaries show similarities to human ovaries in terms of fibrous cortical tissue and were used as an animal model in this project (13). Ovaries from adult sheep, 3–4 years old (approximately 40 ovaries), with small or no dominant follicles, were obtained from the abattoir and transported in PBS (Phosphate Buffer Solution) with antibiotics. Upon arrival at the laboratory, the medullary layer from the ovaries was removed to isolate the cortical area, i.e. the layer containing follicles. Samples of approximately 1 mm thick were cut into quadrilaterals of 1 cm x 1 cm. This study was approved by the Ethics Committee of Animal Experimentation from the University of Zaragoza, Zaragoza, Spain with the code of PI06/22NE.

2.2. Enzymatic pre-freezing treatment of samples

The effect of 5 enzymatic pre-freezing treatments was analyzed, including a control without enzymes. Four replicates were carried out per treatment and assessment time. The samples were evaluated before freezing, at 0 h of thawing and after 2 and 7 days of culture. The enzymatic treatments used were: 1) TrypLE (1X; 9 ml PBS and 1 ml of the commercial solution of the TrypLE® enzymatic cocktail at 10X concentration); 2) Collagenase Type I (0.83 mg/ml); 3) Dispase (2.5 mg/ml); 4) Accutase (Acumax®); 5) Hyaluronidase (25 mg/ml). Samples of 1 cm x 1 cm x 1 mm dimensions were subjected to the different enzymatic treatments for 10 minutes at 37°C and then immediately washed three times with abundant PBS supplemented with 20% of FCS for enzyme inactivation before initiating the freezing process. Untreated sample with enzymes before freezing were considered as the control.

2.3. Freezing/thawing

All samples were frozen following the modified slow freezing protocol proposed by Herraiz et al. (14). Briefly, 3 solutions were used: Solution 1: DMEM (Dulbecco's Modified Eagle Medium) + 5% FBS (fetal bovine serum); Solution 2: Solution 1 + 10% DMSO (dimethyl sulfoxide); and Solution 3: Solution 1 + 12% DMSO. The samples for each treatment were deposited in tubes with 8 ml of Solution 1. Then, 8 ml of Solution 2 (final concentration of DMSO 5%) were added and incubated for 20 minutes. After incubation, 8 ml of liquid was removed, adding 8 ml of Solution 3 (final concentration of DMSO 8.5%) and incubating for 15 minutes. Then samples were transferred to the cryotube containing the final freezing solution (DMSO 8.5%) using forceps. This process was carried out at room temperature, next transferring the tubes to a cell freezer, where the temperature was decreased at a 1°C/minute rate until reaching − 80°C (approximately 24 hours). A cell freezer was used in this study as it is a cost-effective alternative to a programable freezer as demonstrated in a previous study (15). Finally, the samples were stored in a liquid nitrogen tank at a temperature of -196°C. For thawing, cryovials were immersed in a water bath at 42°C and agitated until were completely thawed. Subsequently, three washes with PBS were performed to remove the cryoprotectants.

2.4. Assessment and Culturing

The samples were evaluated before freezing, at 0h post-thaw, and after 2 and 7 days of culture. DMEM medium supplemented with L-glutamine (1%) and fetal bovine serum (20%) was used as culture medium. Samples were covered with 2 ml of culture medium and placed in an incubator at 37°C and 5% CO2 to meet the specified observation times. The next parameters were assessed: viability, tissue integrity and cell proliferation and apoptosis.

2.4.1. Viability

To assess tissue viability, calcein (4µg/ml) and propidium iodide (6µg/ml) staining was used. The samples were incubated in calcein and propidium iodine for 30 minutes at 37ºC and washed three times with PBS before clearing. Samples were cleared to eliminate the excess of pigments and improve visibility of tissue viability, following the protocol proposed by Lempereur et al. (16). Briefly, 400 µl of 50% clearing solution (clearing solution stock MD+: PBS, 1:1) were added to the samples, incubating for 2 hours. Next the samples were incubated for 45 minutes in 100% clearing solution. The clearing solution MD + contains a combination of 45% sucrose (w/v), 20% nicotinamide (w/v), 20% triethanolamine (w/v) and 0.1% TritonX-100 (v/v). Then the samples were visualized under a confocal microscope (Nikon TI-E-c1 Confocal modular), at 590/50 nm wavelength for calcein (green, labeling live cells) and 650LP nm for propidium iodide (red, labeling dead cells), with a fixed gain of 95 and 97 for red respectively. The samples were observed in frames of z-stack adjusted from the bottom to the top of the tissue slices. Images were exported from the EZ-C1 software into ImageJ/Fiji software. In the figures shown in this manuscript, maximum orthogonal projections, no image manipulation was performed prior to export into ImageJ/Fiji software to generate the figures and quantifying green and red fluorescence intensity in arbitrary units (AU). These data were used to calculate the tissue viability percentage (green AU/Sum and red AU; %).

2.4.2. Morphological analysis

Morphological analyses of ovarian tissue were evaluated from hematoxylin and eosin staining. For both morphological analysis and immunohistochemistry, tissue samples were fixed in 4% PFA for 24 hours at 4°C, after which they were processed in mesh cassettes (Mesh Biopsy Cassette) using a standard protocol (automated tissue processor; Tissue-Tek Xpress x50). The blocks were made with the block making unit (Leica EG1150) after which they were solidified on a cold plate. The paraffin-embedded samples were cut into 3 µm thick sections (Leica RM2255 Rotating Microtome), which were placed in a tempered bath from where they were collected with Adhesive Plus Microscope Slides (Bio-Optica; Ref. 09-3000) for hematoxylin-eosin slides and with Flex IHC Microscope Slides (Dako Agilent; Ref. K8020) for immunohistochemistry. The slides were placed on vertical racks and left to dry overnight at 37°C, after which a deparaffinization and rehydration process was carried out. For hematoxylin and eosin slides, the nuclei of the cells in tissue sections were stained by immersing in Carazzi's Hematoxylin (Bio-Optica; Ref. 05-06012/L) for 40 minutes. Sections were rinsed in tap water for 5 min. Cell cytoplasm was stained by immersing in 3% of eosin yellowish aqueous solution (C.I. 45380) for 10 min and dipped in de-ionised water for 1 minute. After eosin staining, sections were dehydrated by immersion in ascending alcohol solutions (70%, 96% and absolute alcohol 100%) for 15 s/each. Finally, all sections were cleaned by xylene for 15 s. and mounted with a cover slipping reagent (Leica CV Mount) using glass coverslips. The staining process was made in a Stainer Integrated Workstation (Leica ST5020-CV5030). Hematoxylin and eosin-stained preparations were evaluated under a light field optical microscope for morphological study of ovarian follicles and stroma. The number of follicles were counted. Intact follicles were considered in follicles maintaining the integrity of membranes and zones (theca area, basal lamina, granulosa area, zona pellucida, plasma membrane, nuclear membrane). Intact stroma was considered when the connective and interstitial tissue did not exhibit white spaces, maintained good cellularization, and did not show focal areas of necrosis or apoptosis.

2.4.3. Immunohistochemistry

After rehydration, antigen retrieval was performed by means of the PT Link (Dako) at 95ºC for 20 min in a low pH buffer (Dako antiger retrieval, low pH). After retrieval, automated immunostaining was performed with a previously optimized protocol (automatic immunostaining equipment; Dako Autostainer Plus Link).

To assess cell apoptosis, the primary antibody used was Human/Mouse Active Caspase-3 (Rabbit Polyclonal, R&D System Biotechne), dilution 1/125, detected with ImmPRESS® HRP Horse Anti-Rabbit IgG Polymer Detection Kit Peroxidase from Vector Laboratories. Follicles at all stages were evaluated and categorized as apoptotic when the oocyte and/or > 50% of granulose were positive. To assess cell proliferation, the primary antibody used was Ki 67 Human (Mouse monoclonal, Clone MIB-1, Agilent DAKO), and Envision Flex HRP from Dako was used as the visualization system. Follicles were classified into 2 categories: resting, when all granulosa cells were negative for Ki67 or proliferating, with at least 1 granulose cell was positive for Ki67.

2.5. Statistical Analysis of Viability

Viability data were analyzed using SPSS (Statistical Package for the Social Sciences) version 19.0 (SPSS, Chicago, IL). Normality of distribution was assessed using the Kolmogorov-Smirnov test, revealing a non-normal distribution in the data. Significant differences were considered at p < 0.05. Differences between treatments were compared using the Kruskal-Wallis test, followed by post-hoc analysis. Only descriptive statistics were used for follicular statistics because of the low numbers per category in each treatment.

3.1. Viability

Ovarian tissue clearing permits better visualization and analysis of tissue viability. In samples before freezing (fresh), the highest viability percentages were observed with dispase treatment, while the lowest were observed with accutase. Immediately after thawing, the highest viability was observed in samples treated with trypLE, while accutase showed the lowest viability values. When culturing tissue samples after thawing for 2 and 7 days, the dispase treatment continued to exhibit the highest viability values. In general, viability improvement compared to the control before freezing was observed in treatments with trypLE, collagenase, and dispase. However, immediately after thawing, which is typically when transplantation occurs, only trypLE treatment showed better viability results than control. After 2 days of culture, control exhibited the worst viability results, and after 7 days of culture, only dispase treatment showed improvements in terms of viability compared to the control. It is worth noting that in all treatments, a decrease in viability was observed after thawing and culturing of the samples (Table 1). Regardless of the treatment, viability decline could be observed by comparing the total viability values over time of assessment (Fig. 1).

Table 1

Tissue viability values (%) before freezing and at thawing (0h. 2 days. and 7 days of culture). Values expressed as mean ± standard error. Different letters within the same column indicate significant differences between treatments (p < 0.05).
	Fresh	0h	2d	7d
TrypLE	76.89 ± 0.21a	77.85 ± 0.9a	67.24 ± 0.08abd	63.26 ± 0.42adf
Collagenase	81.76 ± 0.5b	66.71 ± 0.25b	66.76 ± 0.2be	58.32 ± 0.23b
Dispase	87.58 ± 0.05c	65.27 ± 0.2c	72.04 ± 0.3c	70.57 ± 0.26c
Accutase	71.08 ± 0.44d	47.79 ± 0.54d	66.03 ± 0.35de	60.06 ± 0.16dg
Hyaluronidase	71.49 ± 0.2d	70.25 ± 0.3e	61.81 ± 0.46f	59.69 ± 0.21ed
Control	74.01 ± 034f	70.32 ± 0.33e	61.52 ± 0.22g	60.06 ± 0.25fg
P-value	< 0.001	< 0.001	< 0.001	< 0.001

Figure 1. Percentage of tissue viability measured as a percentage of the total treatments over time (Mean ± SEM).

3.2. Follicular and stromal integrity

In samples before freezing, most of the follicles were found intact (219 total follicles with only 1 damaged with dispase treatment and 1 in control) and the stroma was intact.

Follicular and stromal damage was observed after thawing, with separation of the granulosa area from the oocyte, as well as loss of shape of the basal plasma and nuclear membranes in the follicles, with appearance of decellularization in the stromal part (Fig. 2). TrypLE and hyaluronidase treatments maintained the integrity of the tissue better at 0h, with 10% of damage. The accutase treatment presented the worst results with multiple foci of pyknosis and necrosis coinciding with the lowest viabilities measured at 0h. Around 50% of the follicles showed disintegration of the granulosa areas, vacuoles, and loss of shape in the treatments with accutase. Treatment with collagenase and dispase produced stromal damage mainly at the edges of the tissue with more intact stroma in the central part and a percentage of affected follicles of around 30%. The control presented similar results of follicular alteration (30%) and some foci of decellularization and necrosis at the stromal level.

Figure 2. HE preparations of ovarian tissue at 0h post-thawing. a) TrypLE. b) Collagenase. c) Dispase. d) Accutase. e) Hyaluronidase. f) Control. Blue arrows indicate healthy follicles. Yellow arrows indicate damaged follicles. Red arrows indicate areas of necrosis or pyknosis. Purple arrows indicate areas of decellularization.

At 2 days of culture post-thawing, more damage in follicles and stroma was observed in all treatments, with more foci of necrosis and decellularization (Fig. 3). TrypLE treatment again maintained the tissue integrity better with few foci of necrosis and 20% of the follicles altered. The hyaluronidase treatment also maintained stromal integrity in the inner part of the tissue, showing more decellularization at the edges; and similar percentages of follicular viability at the follicular level (30%). In collagenase treatment and in control, 50% of the follicles were damaged with more cellular disorganization at the stromal level. The dispase treatment showed significant tissue disorganization at the edges with some foci of necrosis, maintaining better integrity in the central area and with follicles positioned in the peripheral area altered (50%). The accutase treatment exhibited the greatest damage at the follicular level (70%) with alterations mainly in the granulosa area of the follicles, multiple foci of pyknosis and higher decellularization.

Figure 3. HE preparations of ovarian tissue at 2 days post-thawing. a) TrypLE. b) Collagenase. c) Dispase. d) Accutase. e) Hyaluronidase. f) Control. Blue arrows indicate healthy follicles. Yellow arrows indicate damaged follicles. Red arrows indicate areas of necrosis or pyknosis. Orange rectangles indicate damaged edges.

At 7 days of culture post-thawing, an increase in foci of pyknosis and necrosis, with more decellularization and damage at the follicular level were observed in all treatments (Fig. 4). The trypLE treatment was the treatment that better maintained tissue integrity (follicular damage 50%), while in the hyaluronidase treatment the edges of the tissue and the follicles were more damaged (60%). In the dispase treatment and in the control, follicular damage increased to 70% with severe stromal alterations, primarily at the edges of the tissue in the case of dispase. The most damage results at 7 days were again found in the accutase and collagenase treatments, where most of the tissue was necrotic and the follicles were altered (80%).

Figure 4. HE preparations of ovarian tissue at 7 days post-thawing. a) TrypLE. b) Collagenase. c) Dispase. d) Accutase. e) Hyaluronidase. f) Control. Blue arrows indicate healthy follicles. Yellow arrows indicate damaged follicles. Red arrows indicate areas of necrosis or pyknosis. Purple arrows indicate areas of decellularization. Orange rectangles indicate damaged edges.

3.3. Immunohistochemistry

Primordial and primary follicles were negative to Ki67 in all treatments and times. However, antral follicles in fresh samples were mostly Ki67 positive in all treatments (34 follicles positive to Ki67; 3 follicles negative to Ki67). After thawing, antral follicles positive to Ki67 were observed in all treatments except in accutase. Only trypLE (5 Ki67 positive and 5 Ki67 negative) and hyaluronidase (5 Ki67 positive and 4 Ki67 negative) maintained positive antral follicles to Ki67 after thawing and 2 days culture. However, no positive Ki67 follicles were observed after thawing and 7 days culture in any treatment (44 follicles negative to Ki67).

In terms of apoptosis, caspase-3 positive antral follicles were observed in fresh samples in dispase, accutase, hyaluronidase and control. Only triple maintained all follicles negative to caspase-3 just after thawing. First primordial follicles positive to caspase-3 were observed after thawing and 2 days culture. Only trypLE maintained some negative caspase-3 antral follicles after 2 days culture (5 follicles positive to caspase-3; 4 follicles negative to caspase 3) and moderate apoptosis in stroma. After 7 days culture all antral follicles were positive to caspase-3, and only trypLE maintained some primordial follicles negative to caspase-3 (12 follicles positive to caspase-3 and 6 negatives to caspase-3) (Fig. 5).

Figure 5. Immunohistochemistry. a) Follicle Ki67 positive in fresh tissue after collagenase treatment. Blue arrows indicate cells in proliferation. b) Follicle Ki67 negative in 0h after thawing in accutase. c) Follicle caspase-3 positive in 2 days culture after thawing in accutase treatment. Blue arrows indicate apoptotic cells. d) Follicle caspase-3 negative in fresh sample after trypLE treatment.

Although the first experiments in ovarian tissue freezing date back to the 18th century, it is only recently that this technique has received more attention. The evolution of cancer treatments and the increase in patient survival has resulted in resorting to this method to preserve their fertility. Given the enormous potential but still inconsistent success with the use of ovarian tissue cryopreservation as an assisted reproduction technology, it is critical that better animal models, such as the sheep, be used to advance this field. Protocols from other types of tissue have been adapted and optimized through the study of various factors considered important to maintain tissue viability and functionality during freezing, such as CPAs and freezing rates. However, the low quality of follicles after grafting remains a problem (6, 7). Conventional freezing negatively affects cell viability due to the formation of intracellular ice crystals (17).

The ovarian ECM, predominantly made of collagen, provides scaffolding for stromal cells and follicles, impacts cell behavior via physical adhesion and mechanical tension, and sequesters important paracrine factors involved in cell-to-cell communication (18, 19). Rigidity of the stroma impacts the ability of follicles to accommodate and survive the current cryopreservation processes and recover once the tissues are transplanted in culture and in vivo, allowing follicular development to proceed, thus restoring fertility. Only one paper has documented the use of low doses of collagenase treatment to improve fertility outcomes in mice by reducing the rigidity while tissue is subjected to hypoosmotic solutions during cryopreservation (8). The study presented herein documents the effect of pre-treatments with enzymes usually used to disaggregate tissues, to see if tissue quality was improved after freezing with this modulation of rigidity.

Cryopreservation procedure damages ovarian stromal and granulosa cells, leading to significant fibrotic areas after transplantation, asynchrony between granulosa cells and oocyte development, and thinner theca layers (20, 21). In our results we have found a general decrease in viability in agreement with other authors (22, 23). We would like to emphasize that this is the first study where the clearing technique has been used to assess the viability of ovarian tissue with satisfactory results. Some articles have been published where ovarian tissue viability was measured before and after freezing but without prior clearing (24, 25). In the optimization of the ovarian tissue viability assessment protocol, we attempted to measure viability following standard protocols that do not include clearing, finding a lack of homogeneous distribution of fluorochromes and with many sections remaining unstained, complicating result assessment. The goal of clearing is to remove tissue pigments for better visibility under microscopy. In our case, it allowed us to assess viability satisfactorily by achieving a homogeneous distribution of calcein and propidium iodide fluorochromes, staining the entire sample. This clearing protocol has been used in other tissues (26, 27). Similar clearing protocols have been used to assess follicles in ovarian in mouse model but not in either sheep or human models (28, 29).

Furthermore, this study is the first work where viability was quantified. This is a significant advancement as it allows us to have quantitative data for comparison with a large and representative sample size, enabling the identification of the best treatments in terms of viability. Other studies have assessed viability as mentioned earlier but not quantitatively (22, 24), limiting result interpretation. Zver et al. (30) described a method to quantify ovarian tissue before autograft by isolation of viable cells from human ovarian cortex to obtain an ovarian cell suspension analyzable by multicolor flow cytometry for viability and ovarian residual disease detection. However, our aim was not to obtain a cell suspension as we wanted to maintain the integrity of the tissue.

Immunohistochemistry data suggested a decrease in proliferation and increase in apoptosis after thawing. Few studies have used Ki67 to assess ovarian tissue after thawing. In most of the studies, PCNA is used, but PCNA did not appear to be a reliable quantitative marker of cell proliferation as it is an auxiliary protein to DNA polymerase delta, which is involved in DNA synthesis and repair (31). Thus, it is possible that the majority of cells in the ovarian tissue fragments were undergoing DNA repair rather than proliferation. In a similar study with sheep ovarian tissue, a decrease in Ki67 expression was also observed after thawing (32). Choi reported that ovarian tissue cryopreservation suppresses granulosa cell proliferation, and that this impairment is recovered within 48 h after culture (33). Ayuandari et al. (34) observed an increase in Ki65 after 12 weeks of grafting in comparison to pre-graft controls. Henry et al. (35) observed that granulosa cells associated with healthy follicles were able to proliferate as soon as 2 days of culture; thereafter, proliferation seemed to decrease, as observed after 6 days. In a recent study by Hossay et al. (36) proportions of Ki67 positive cells were greater on day 6 compared to just after thawing. In contrast, in our study, we could not find positive Ki67 cells after thawing either 2 or 7 days after thawing and culture, confirming that these cells are not in a growing phase. In the same study (36), caspase-3 was also used to assess apoptosis. Similarly, authors found a considerable increase in caspase-positive cells after day 6 of culture compared to just after thawing. It was demonstrated that cryopreservation-induced apoptosis in granulosa cells is mediated by activation of caspase-8, -9, and − 3 dependent apoptotic pathways (37). Other authors have found an increase in the expression of caspase-3 in granulose cells and stroma after thawing (38–40). These results are in agreement with our study where we observed an increase in caspase-3 positive cells after thawing and culture at day 2 and 6.

Regarding pretreatment with enzymes, only one study describes the use of collagenase to partially disaggregate mouse ovarian tissue before freezing (8). This study showed that during the vitrification process, there is a separation between the oocyte and the granulosa cells due to a loss of adhesion molecules without altering the extracellular matrix. However, with collagenase pre-treatment, the ratio between oocyte diameter and follicle diameter was maintained, preserving the adhesions. This would also translate into lower follicle atresia and therefore greater ovarian reserve. This study emphasizes the need to adapt this collagenase pre-treatment to different species. In this study, very low doses of collagenase (1-100 micrograms/ml) were used compared to our study (0.83 mg/ml), but it must be considered that mouse and human ovaries have many differences in terms of stromal characteristics and extracellular matrix, as well as in thicknesses. Our study showed little tissue disaggregation without affecting follicle quality after treatment at our selected collagenase concentration. The results obtained in both studies are difficult to compare since different cryopreservation techniques were performed, slow freezing in our study versus vitrification in the murine ovary study, with different assessment techniques and different animal species. Although the use of collagenase did not improve the viability of stromal and follicular morphology compared to the control, unlike the murine study (8), treating the tissue with trypLE resulted in improvements in tissue viability immediately after thawing, and treatment with dispase improved viability at 2 and 7 days of culture. It is worth mentioning that viability measurements were taken from central sections of the tissue piece, avoiding the edges, to ensure similar sections. Severely damaged edges were observed after dispase treatment. This damage was not reflected in terms of viability but was evident in hematoxylin-eosin sections with completely necrotic edges. Based on these sections, an improvement in stromal and follicular integrity was observed in treatments with trypLE and hyaluronidase.

This is the first study to identify the effect of enzymes for ovarian tissue freezing. These enzymes were selected because they are commonly used enzymes for tissue treatment for cell isolation. Hyaluronidase enzyme is used in the denudation of oocytes (removing them from the cumulus oophorus) in assisted reproduction laboratories to facilitate sperm penetration in the in vitro fertilization process. However. it is used in much lower concentrations than those used in this study as direct contact with the oocyte is very damaging while higher concentrations are required to penetrate tissue in this study (0.08 mg/ml for oocytes vs 2.5 mg/ml for tissue) (41). The trypLE enzyme complex is used to isolate cells such as mesenchymal stem cells for the analysis of their surface antigens and has not been used in ovarian tissue previously. The dispase enzyme has been used similarly to isolate different types of cells, such as lymphocytes labeled with certain antibodies or tumor cells. In ovarian tissue, it was used for follicular isolation for designing artificial ovaries (42). As for the accutase enzyme, although it has been proven to be an excellent cell isolator in other types of tissues (fibroblasts, keratinocytes, vascular endothelial cells), this is the first study where it has been used in ovarian tissue and the results are not as promising, showing the lowest viability values and alterations in follicular and stromal morphology.

Altogether, our results show that viability decreases over time after thawing as well as stromal and follicular integrity. This suggests that the current strategy of implanting the tissue immediately after thawing is appropriate. Further studies focused on other culture media and supplements should be studied in future research as a recent study conducted on human tissue showed better primordial follicle reserve in the tissue cultured with the Chorioallantoic Membrane (36).

The next steps of this research line will focus on assessing the effect of these enzymes in human adult tissue. Results will be compared and their effect on human pre-pubertal tissues will be evaluated, to determine conclusively whether these enzymatic pretreatments should be included in the prepubertal ovarian tissue freezing protocol to develop a more effective fertility preservation method for girls with cancer.

Treatment with trypLE before freezing has beneficial effects after thawing ovarian tissue samples. Accutase treatment is not advisable before ovarian tissue cryopreservation. As currently practiced, our results suggest grafting immediately after thawing is beneficial. Further experimental studies are required to optimize the treatment with tripLE (concentration and time of incubation) before ovarian tissue cryopreservation to maintain ovarian tissue integrity and functionality.

ACKNOWLEDGEMENTS

We would like to thank the slaughterhouse Mercazaragoza (Zaragoza, Spain) for providing the sheep ovaries. Besides we would like to thank Hector Castro and Sandra González for their help in confocal assessments and FIJI software, and Jane Morrell for helping with English grammar. We acknowledge the use of Servicios Científico-Técnicos del CIBA (IACS-Universidad de Zaragoza).

DECLARATION OF INTEREST

Authors declares no conflict of interest.

AUTHOR CONTRIBUTIONS

AM: investigation, methodology and writing original draft; MG: data analysis and writing original draft; JC: conceptualization, formal analysis and review and editing; MR and AI: methodology, resources and visualization; MF: methodology; CM: conceptualization, formal analysis, investigation, methodology, project administration, supervision review and editing.

Funding statement: The authors did not receive support from any organization for the submitted work.

Disclosure Statement: The authors have no conflict of interest to declare.

Attestation Statement: Data regarding any of the subjects in the study have not been previously published unless specified. Data will be made available to the editors of the journal for review or query upon request.

Capsule: Rigidity of stroma impacts on the ability of follicles to accommodate and survive the current cryopreservation process. Enzymatic treatment with trypLE before freezing maintained the highest viability and proliferation values, with the lowest apoptosis.

Author roles: AM: investigation, methodology and writing original draft; MG: data analysis and writing original draft; JC: conceptualization, formal analysis and review and editing; MRC and AID: methodology, resources and visualization; ARR: investigation, conceptualization and editing; MFC: methodology; CM: conceptualization, formal analysis, investigation, methodology, project administration, supervision review and editing.

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No competing interests reported.

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Treatment with trypLE before freezing improves thawing integrity and functionality of sheep ovarian tissue

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Abstract

Figures

1. INTRODUCTION

2. MATERIAL AND METHODS

2.2. Enzymatic pre-freezing treatment of samples

2.3. Freezing/thawing

2.4. Assessment and Culturing

2.4.1. Viability

2.4.2. Morphological analysis

2.4.3. Immunohistochemistry

2.5. Statistical Analysis of Viability

3. RESULTS

3.1. Viability

3.2. Follicular and stromal integrity

3.3. Immunohistochemistry

4. DISCUSSION

5. CONCLUSIONS

Declarations

References

Additional Declarations

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Version 1