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. 2022 Jan 18;27(2):97–106. doi: 10.1007/s12192-022-01252-6

Comparison between two cryopreservation techniques of human ovarian cortex: morphological aspects and the heat shock response (HSR)

Sérgio Galbinski 1,2, Lucas Stahlhöfer Kowalewski 3, Gisele Bettú Grigolo 3, Larissa Ramos da Silva 4, Mirela Foresti Jiménez 1,2,5, Mauricio Krause 3,✉, Nilo Frantz 6, Adriana Bös-Mikich 4
PMCID: PMC8943117  PMID: 35043289

Abstract

This study was tailored to compare the cryopreservation of the human ovarian cortex using closed metal container vitrification or the slow-freezing technique. Superficial ovarian cortical tissue biopsies were collected from 12 participants who underwent gynaecological videolaparoscopy. The fragmented samples were allocated to three experimental conditions: (a) fresh ovarian tissue, (b) slow-freezing, and (c) vitrification with a metal closed container. After thawing or rewarming, cellular morphological analyses were performed to determine tissue viability. The cellular response to thermal stress was measured by a putative increase in the immune quantification of the heat shock protein 70 kDa (heat shock protein 70 kDa response — HSR) after a heat challenge (2 h exposure at 42 °C). Both the total number of intact follicles and the frequency of primordial follicles were higher in fresh ovarian tissue than in the preserved samples, regardless of the technique employed. There was a trend towards an increase in the absolute number of intact follicles in the tissue preserved by vitrification. After cryopreservation, a higher HSR was obtained after slow-freezing. These results indicate that both cryopreservation techniques present advantages and may be used as alternatives to ovarian tissue cryopreservation.

Keywords: Vitrification, Slow-freezing, Human ovarian, Heat shock response

Introduction

Cryopreservation of human ovarian tissue was proven feasible in 1996, thus offering a new alternative method for fertility preservation (Rivas Leonel et al. 2019). Since the first infant was born following the transplantation of frozen/thawed ovarian tissue in 2004 (Donnez et al. 2004), over 130 live births have occurred worldwide after orthotopic auto-transplantation (Andersen et al. 2018; Donnez and Dolmans 2017; Meirow et al. 2005; Muraro et al. 2017; Tammiste et al. 2019; Xiao et al. 2017).

More recently, infertility specialists and oncologists have focused on cryopreservation techniques because they benefit oncologic female patients at risk of treatment-induced sterilisation and/or premature menopause (Demeestere et al. 2007; ESHRE Working Group on Oocyte Cryopreservation in Europe et al. 2017; Takae et al. 2019; von Wolff et al. 2019). Notably, cryopreservation not only allows the patient to generate a biological child but also re-establishes the post-transplantation ovarian physiological functions (Donnez and Dolmans 2010; Takae and Suzuki 2019; Xiao et al. 2017). The most employed techniques for preserving fertility involve the cryopreservation of embryos and oocytes. However, these techniques may present some implementation flaws.

While a number of studies have analysed ovarian tissue cryopreservation using the slow-freezing technique (Dalman et al. 2017; de Lemos Muller et al. 2018; Donnez and Dolmans 2017; Jensen et al. 2017), vitrification has also raised the interest of researchers (Almodin et al. 2004; Xiao et al. 2017). In Japan, for example, vitrification is the most employed technique for cryopreserving tissues. It is performed in 93% (25) of healthcare institutions in Brazil (Sanada et al. 2019). However, there is a lack of data evaluating which cryopreservation technique (slow-freezing or vitrification) leads to higher rates of follicular survival, ovarian stroma conservation, and post-transplantation live births (Chibelean et al. 2020).

While slow-freezing is a well-established technique that has been successfully used for tissue post-transplantation in non-oncologic patients (Andersen et al. 2018; Donnez et al. 2004; Donnez and Dolmans 2013; Jensen et al. 2017; Meirow et al. 2005; Oktay et al. 2016; Oktay and Karlikaya 2000; Rosendahl et al. 2011; Tammiste et al. 2019; von Wolff et al. 2019), its execution requires an appropriate and expensive equipment. Vitrification, on the other hand, is based on the ability of highly concentrated cryoprotectant solutions to become viscous at a sufficiently low temperature, such that they rapidly solidify without ice formation (Rall 1987). The disadvantages of vitrification include the risk of cell damage induced by liquid nitrogen (LN2) or high-concentration cryoprotectants (Bielanski et al. 2000, 2003).

Recent studies comparing the use of slow-freezing (Fabbri et al. 2016; Silber 2016) with vitrification of ovarian tissue have shown very similar results regarding follicular preservation and tissue functionality (Chen et al. 2006; Klocke et al. 2015; Sheikhi et al. 2011). However, the use of plastic containers in vitrification may lead to an inferior efficiency because plastic is not an appropriate heat conductor (Amorim et al. 2011; Aquino et al. 2014; Bos-Mikich et al. 2012; Fabbri et al. 2016; Massignam et al. 2018b; Sheikhi et al. 2011; Xiao et al. 2017; Zhou et al. 2010). To overcome this issue, our group has designed a metal capsule for use in this technique (Patent # BR202013019739-0) (Aquino et al. 2014; Bos-Mikich et al. 2012; Bös-Mikich et al. 2013; Massignam et al. 2018b). The implementation of the metal capsule allows for rapid cooling of the biological material and eliminates the contact between the stored tissue and LN2, thus avoiding possible contamination. Preclinical studies evaluating the feasibility of closed metal container vitrification have demonstrated that tissue morphology and physiological viability are preserved after devitrification (Aquino et al. 2014; Massignam et al. 2018a).

Heat shock proteins (HSPs) are mainly involved in renaturation of intracellular protein, which is critical for cell homeostasis, thus being known as “stress proteins” (Krause et al. 2007). Heat shock protein (HSP70) is the most abundant and highly conserved HSP (Noble et al. 2008). The role of HSP70 is to unfold the aggregated proteins after thermal damage and refold them, thereby acting as a chaperone (Madden et al. 2008). Other functions include protein translocation, antiapoptotic, and anti-inflammatory functions. More recently, HSPs have been implicated in cellular signalling control and immune responses (Krause et al. 2015). The quantification of HSP70 has already been evaluated in ovarian tissues that have been subjected to different cryopreservation techniques (Maffei et al. 2014; Nikishin et al. 2018). However, in the two aforementioned studies, the authors analysed only baseline HSP levels after defrosting the tissue. The comparison between baseline levels of HSP70 and those obtained after heat stress (called heat shock response, HSR) can represent the physiological ability of cells to respond to challenging stimuli, indicating their cellular homeostasis capability.

Within this context, this study was tailored to compare the effectiveness of cryopreservation of human ovarian tissue using closed metal capsule vitrification or slow-freezing techniques. Additionally, the morphological aspects and tissue HSRs were evaluated.

Materials and methods

Participants and ethics

Twelve female participants volunteered to participate in this study. Informed consent was obtained from all participants prior to the initiation of the study. The study was approved by the local ethics committee (No. 17135). The present work was conducted in accordance with international laws on procedures to deal with human tissue, as well as with the CONSORT guidelines. All researchers maintained the anonymity of the data according to the resolution of the National Health Council CNS 466/2012.

Videolaparoscopy technique

After general anaesthesia, a Verress needle was inserted, and pneumoperitoneum was induced with CO2 at 13 mmHg. Subsequently, a 10-mm umbilical trocar was inserted, and laparoscopy was performed. After the abdominal cavity inventory, two 5-mm pelvic punctures were performed in both iliac fossae. During the procedure, a superficial cortical biopsy of the ovarian tissue was performed in the operated area with delicate forceps and scissors without electricity. Each fragment measured approximately 1 cm (length) × 1 cm (width) × 4 mm (depth). After removal from the cavity, the sample was stored in a physiological solution at room temperature (22–24 °C) to allow for transportation.

Transportation and preparation of the samples

The ovarian tissue fragments were washed with a 0.9% physiological solution under sterile conditions and placed in 50-ml plastic tubes containing the physiological solution (pH 7.4). Transport from the surgery room to the laboratory (20–30 min) was performed at room temperature. Each fragment of ovarian tissue was sectioned with a 22-mm scalpel blade to avoid tissue injury (Bos-Mikich et al. 2012). First, 1-cm strips were made. Each strip was subsequently sectioned into several smaller fragments measuring 2 mm (width) × 3 mm (length) × 1–2 mm (depth) (Haino et al. 2018; Suzuki et al. 2015). A random sample was selected for storage in paraformaldehyde for morphological evaluation of primordial, primary, and secondary follicles.

The fragments were carefully transferred from one solution to another with the aid of sterile brushes to minimise the medium in each transfer.

Slow-freezing and thawing technique

The samples were placed in 50-ml plastic tubes containing 30 ml of equilibration solution [0.1 mol/L sucrose and 1.5 mol/L ethylene glycol (EG) in phosphate-buffered saline (PBS) solution] for 30 min. The tubes were placed on an oscillating stirring table at 1 °C. Subsequently, the samples were placed in 1.8 ml cryotubes (Nunc A/S, Denmark) containing 1 ml of cryoprotectant. The tubes were then cryopreserved using a programmable freezing device (K10, Planner, UK). The following cooling curve was used: 2 °C/min at − 9 °C, with a 5-min pause. The induction of nucleation was performed with a cooled calliper. Then, the temperature drop was 0.3 °C/min to − 40 °C and 10 °C/min to − 140 °C. The cryotubes were removed from the freezing device, directly immersed in LN2 at − 196 °C, and stored in an LN tank.

The samples were thawed in three 10-min stages. Cryotubes containing frozen tissue were placed in a water bath at 37 °C. When the solution became liquid, the tissue sample was withdrawn and placed in the first thawing medium (0.75 mol/L EG and 0.25 mol/L sucrose in PBS). The sample was then transferred to the second thawing medium (0.25 mol/L sucrose in PBS) at room temperature. After 10 min, the tissue was transferred to PBS for 10 min (Rosendahl et al. 2011).

Vitrification and rewarming technique

The previously prepared ovarian tissue fragments were transferred to the equilibration solution with 7.5% EG and dimethylsulfoxide (DMSO), followed by the vitrification solution with 15% EG and DMSO, both in HTF medium (Irvine), for 25 and 15 min, respectively. Tissue samples were placed at the base of the metal containers. Finally, the system was immersed in LN2 for storage. Each metal container received 10–12 fragments of a single ovary. The metal capsule (Patent #BR 202,013,019,739–0) was made of stainless steel, with a volume of 1 ml and a height of approximately 2 cm (Aquino et al. 2014).

For rewarming, the metal capsules were withdrawn from the LN2, exposed to tap water, and then the lid was removed. The base of the capsule containing the tissue samples was placed in a water bath at 37 °C for 1 min. The contents were transferred to the first rewarming solution containing 1 M sucrose in HTF for 1 min, immediately followed by the second solution containing sucrose (0.5 M) for 3 min, and then the final solution with 0.25 M sucrose for 5 min. All tissue manipulations were performed using sterile brushes. Finally, the tissue fragments were left in the HTF-HEPES medium. All solutions were maintained at room temperature (Aquino et al. 2014). The samples were fixed in paraformaldehyde solution for histology or cultured in an incubator with 5% CO2 atmosphere at 37 °C in HTF medium for 1 h before being subjected to the thermal stress test.

Ovarian HSR test

Considering the importance of HSRs for tissue/cell stress adaptation and proteostasis, we quantified the expression of HSP70 in ovarian cells under heat stress conditions (a normal and expected response in healthy cells). Briefly, the fresh, thawed, or rewarmed tissues were incubated at two different temperatures: 37 °C (control) and 42 °C (heat-stressed) for 2 h in a suitable incubator (5% CO2, Form Scientific, Marietta, OH, USA). After the heat shock, the samples were placed in an incubator for an additional 8 h (37 °C in 5% CO2) to recover from the HS and reach the peak of HSP70 expression (Krause et al. 2020; MULLER 2019). After incubation, the tissue fragments were homogenised and prepared for total protein quantification and western blot analysis for HSP70.

Protein quantification and western blot for HSP70

Ovarian tissue protein quantification was determined using a BCA Protein Assay Kit (Thermo Scientific, Massachusetts, USA), and samples (1 µg) were mixed with 5 × Laemmli loading buffer [50 mM Tris, 10% SDS, 10% glycerol, 10% 2-mercaptoethanol, and 2 mg/ml bromophenol blue] at a final concentration of 1:5, boiled for 5 min, and electrophoresed. For sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), equivalent amounts of protein (1 µg) were applied in a 10% polyacrylamide minigel for 2 h at 100 V. Proteins were then transferred onto nitrocellulose membranes (GE Healthcare, Chicago, USA) according to the manufacturer’s instructions (Bio-Rad, Hercules, USA) (2 h, 100 V). For immunoblotting, membranes were blocked in 2% BSA in wash buffer [50 mM Tris, 5 mM EDTA, 150 mM NaCl (TEN)-Tween 20 0.1% solution, pH 7.4] for 30 min and then incubated overnight at 1:1,000 dilution with monoclonal anti-HSP70 antibodies produced from mice (Sigma-Aldrich, St. Louis, USA). After appropriate washing, the membranes were probed with anti-mouse IgG and biotin antibodies at a 1:10,000 dilution (Sigma-Aldrich, St. Louis, USA) for 1 h. Membranes were then incubated with a final Streptavidin − Peroxidase Polymer, Ultrasensitive (Sigma-Aldrich, St. Louis, USA) at a 1:1,000 dilution for 1 h. Visualisation of the blots was performed using the chemiluminescence reagent, p-coumaric acid, and luminol in the ImageQuantTM LAS 4000 chemiluminescence system (GE Healthcare, Chicago, USA) and quantified in ImageJ (version 1.51f; NIH, Maryland City, USA). A standard molecular weight marker (RPN 800 rainbow full range Bio-Rad, CA, USA) was used as a reference to determine the molecular weights of the bands. The data were normalised using the Ponceau S method (Klein et al. 1995).

Histological analysis of ovaries

Ovarian morphology, particularly of the primordial, primary, and secondary follicles, was evaluated from 5-μm-thick histological sections that were stained with haematoxylin and eosin for analysis under a light microscope. For histological processing, the thawed or rewarmed fragments and controls were placed in a 4% paraformaldehyde solution before being dehydrated in increasing sets of ethanol at room temperature. After diaphanization, the ovary fragments were placed in paraffin for histological sections, and the slices were subsequently stained with haematoxylin and eosin.

The follicle development stages were classified according to the number of layers and the shape of the granulosa cells. The primordial follicles were classified as those containing a single layer of flattened cells, and the primary follicles were those with a single and complete layer of cuboid granulosa cells (Gougeon 1996). The criteria for considering a primordial or primary follicle as damaged included the presence of vacuoles in the ooplasm or pycnotic germinal vesicle, follicular cells detached from the follicular basement membrane, retraction of the oocyte, and incomplete layer of follicular cells surrounding the oocyte. Secondary and antral follicles were not the main focus of our analyses because they no longer belong to the ovarian reserve; however, these were described based on their morphology whenever present in the histological sections. All analyses were performed by the same investigator.

Statistical analysis and sample size calculation

The sample calculation was based on a previous model of vitrification of bovine ovarian tissue in which the rate of viable primordial follicles after rewarming was 93% (Aquino et al. 2014); data from the Denmark program of slow-freezing cryopreservation of human ovarian tissue (Rosendahl et al. 2011), in which the rate of viable primordial follicles after thawing was 68%, was also considered. Considering a power of 80% and a significance level of 5%, the WINPEPI program indicated the necessity of collecting 46 primordial follicles in each experimental group and 46 primordial follicles in the control group (fresh tissue), totalling 138 follicles. The average number of follicles per standard sample was 10 (Donnez and Dolmans 2013; Rosendahl et al. 2011). Thus, enrolment of 12 to 14 participants was required to accomplish this study.

Results were analysed using the Statistical Package for Social Sciences Program (SPSS®, version 20.0, IBM, New York, USA). Categorical variables were described as frequencies and percentages. The asymmetric quantitative variables are described as median, minimum, and maximum values. Symmetrical variables are described as the mean and standard deviation or using confidence intervals. The Friedman test was used to determine the statistical differences in the number of follicles and in the HSP delta among the groups. The Wilcoxon test followed by a Bonferroni correction was used for multiple comparisons. The comparison of HSP70 expression was performed using the Bonferroni correlation model of the generalised estimating equation model (GEE), as the measurements of the different techniques and temperatures were not independent variables. The Spearman correlation coefficient was used to analyse the correlation between the quantitative variables. For the association of categorical variables, the chi-square test was used. A significance level of 5% was considered for the established comparisons.

Results

Demographic data characteristics of the enrolled participants (n = 12), whose mean age was 34.6 years (± 3.2) and mean body mass index was 27.5 (± 3.1), are depicted in Table 1.

Table 1.

Demographic data of enrolled participants

Clinical characteristics Sample data
Age (years) 34.6 ± 3.2
Weight (kg) 73.7 ± 12.4
Height (cm) 163.3 ± 5.2
BMI (kg/m2) 27.5 ± 3.1
Polycystic ovary 8.3% (1)
Endometriosis 16.7% (2)
Arterial hypertension 25% (3)
Tabagism 8.3% (1)
Diabetes mellitus 16.7% (2)
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BMI, body mass index. Age, weight, and BMI were represented by mean ± standard deviation. The associate clinical characteristics are presented as frequency percentage and number of occurrences (n) in brackets

Regarding the morphological evaluation of the follicles (Table 2), all samples subjected to cryopreservation, irrespective of the method, displayed a decreased total number of intact follicles (p = 0.007). Although both techniques reduced the total number of intact primordial follicles, the magnitude of the decrease was higher in the samples subjected to slow-freezing (p = 0.004). Finally, both techniques also induced a decrease in the number of intact primary follicles compared to the control condition (fresh storage) (p = 0.035).

Table 2.

Morphological characterisation of the human ovarian follicles cryopreserved by two distinct techniques

Follicles morphology analyses Fresh condition Slow-freezing Closed metal container vitrification p
Total intact 21 (1–330) 4 (0–43)a 11 (0–42)a 0.007
Total damaged 2 (0–78) 1 (0–16) 5 (0–8) 0.273
Intact primordial 8 (0–298) 2 (0–23)a 4 (0–30)a,b 0.004
Damaged primordial 1 (0–59) 0 (0–11) 2 (0–6) 0.10
Intact primary 6 (0–64) 2 (0–15)a 4 (0–15)a 0.035
Damaged primary 1 (0–19) 1 (0–5) 1 (0–5) 0.690
Intact secondary 1 (0–8) 0 (0–5) 0 (0–8) 0.552
Damaged secondary 0 (0–0) 0 (0–0) 0 (0–0) Not performed
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Data presented as median (followed by the minimum and maximum values) and compared by the Friedman test. ap < 0.05 compared to the control condition (fresh condition); bp < 0.05compared to slow-freezing method

The percentage of intact primordial follicles decreased in the cryopreserved samples (p < 0.001) (Table 3).

Table 3.

Number and frequency of follicles at distinct developmental stages obtained in human ovarian tissue samples submitted to cryopreservation

Primordial follicles Primary follicles Secondary follicles
Technique Sum Total Intact Total Intact Total Intact
Fresh condition 984

768

(78%)

695

(90.5%)

186

(18.9%)

154

(82.8%)

30

(3.1%)

30

(100%)
Slow-freezing 134

64

(47.8%)

45

(70.3%)*

58

(43.3%)

45

(77.6%)

12

(8.9%)

12

(100%)
Closed metal container vitrification 218

124

(56.9%)

98

(79%)*

 78 (35.8%) 58 (74.4%)

16

(7.3%)

16

(100%)
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Data are presented as total number and frequencies in brackets of each follicular developmental stage and the total number of obtained follicles. *p < 0.001 compared to fresh condition

Figure 1 shows examples of intact and damaged primordial and primary follicles from human ovarian tissue. Figure 2 shows the morphology of follicles at distinct developmental stages kept under fresh condition (A) or subjected to slow-freezing followed by thawing (B), and Fig. 3 shows the morphology of vitrified-rewarmed (A) and frozen-thawed (B) damaged ovarian follicles.

Fig. 1.

Fig. 1

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Morphology of primordial and primary follicles kept under fresh condition (A) or submitted to closed metal container vitrification followed by rewarming (B). Haematoxylin–eosin staining (× 200)

Fig. 2.

Fig. 2

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Morphology of follicles at distinct developmental stages kept under fresh condition (A) or submitted to slow-freezing followed by thawing (B). Haematoxylin–eosin staining (× 400)

Fig. 3.

Fig. 3

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Morphology of vitrified-rewarmed (A) or frozen-thawed (B) damaged ovarian follicles. Haematoxylin–eosin staining (× 400)

Intact primordial follicles present a single continuous layer of follicle cells surrounding the oocyte, no vacuoles in the ooplasm, and dispersed nuclear heterochromatin. Primary follicles present a single layer of cuboidal follicular cells surrounding the oocyte. Fig. 1 shows examples of normal primordial and primary follicles in fresh (A) and in vitrified-rewarmed ovarian tissue (B). Fig. 2 shows examples of normal primordial (A) and normal primary (B) follicles in fresh and frozen-thawed ovarian tissues, respectively. Finally, Fig. 3 depicts examples of primordial follicles in which the oocyte presents a large vacuole (A) and shrunken, picnotic nucleus (B) after vitrification/rewarming. Similarly, altered follicular and oocyte features were observed after freezing.

Regarding the thermal-induced HSR, the cryopreserved samples presented with decreased quantification in the expression of HSP70 at 42 °C compared to the samples kept in fresh conditions (p = 0.001) (Table 4). In addition, samples subjected to closed metal container vitrification also had a decreased HSR compared to the fresh condition. In line with these findings, when the HSR delta (difference between 37 °C and 42 °C) was analysed (Fig. 4), HSP70 expression was decreased in cryopreserved samples and there were no significant differences in the HSRs displayed by the samples subjected to cryopreservation.

Table 4.

Thermal stress induced HRS in previously cryopreserved ovarian follicles

Technique T Median 95% IC p
(temperature)		 p
(interaction)
Inferior limit Upper limit
Fresh condition 37 °C 2.193 1.487 2.899 0.153  < 0.001
42 °C 2.498 1.835 3.161
Slow-freezing 37 °C 1.665 0.996 2.334 0.070  < 0.001
42 °C 1.108a 0.825 1.391
Closed metal container vitrification 37 °C 0.970a,b 0.689 1.251 0.001  < 0.001
42 °C 0.449a,b,c 0.333 0.565
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HSR, heat shock response (expression of HSP70); T, temperature (°C); 95% IC, 95% confidence interval. ap < 0.001 compared to fresh condition; bp < 0.001 compared to slow-freezing technique; and cp < 0.001 compared to tissue samples submitted to the same condition but with HSR evaluated at different temperatures (generalised estimating equation model, GEE)

Fig. 4.

Fig. 4

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HSP70 immunocontent of human ovarian cortex tissue samples. Fresh tissue (not submitted to cryopreservation), slow frozen tissues (tissues submitted to slow-freezing cryopreservation technique), and vitrified tissues (tissues submitted to the vitrification technique) were compared in terms of their baseline immunocontent of HSP70 (A), heat-induced HSP70 (B), and delta (variation from 37 to 42 °C) (C). Briefly, the fresh, thawed, and rewarmed tissues were incubated at two different temperatures: 37 °C (control) and 42 °C (heat-stressed) for 2 h in a suitable incubator (5% CO2). After incubation, the tissue fragments were homogenised and prepared for total protein quantification and western blot analysis for HSP70

When the correlation between the histological subtype and HSP70 expression at 37 °C or 42 °C was evaluated, there was a positive, statistically significant, direct, and strong correlation between the number of total and primordial follicles and HSP70 expression at 37 °C for the samples kept under fresh conditions (Table 5). No other correlation was found between the histological subtypes and HSP70 expression in cryopreserved samples.

Table 5.

Spearman’s correlation between the histological subtype and the HSP70 expression

HSP at 37 °C HSP at 42 °C
Fresh Primordial ( +) 0.722* 0.497
Primordial ( −) 0.733* 0.248
Primary ( +) 0.393 0.102
Primary ( −) 0.019  − 0.254
Secondary ( +) 0.269 0.055
Secondary ( −) - -
Total ( +) 0.658* 0.413
Total ( −) 0.471 0.032
Vitrification Primordial ( +) 0.127 0.021
Primordial ( −) 0.215  − 0.021
Primary ( +) 0.461  − 0.201
Primary ( −)  − 0.34  − 0.251
Secondary ( +) 0.482  − 0.46
Secondary ( −) - -
Total ( +) 0.245  − 0.144
Total ( −) 0.171  − 0.046
Slow-freezing Primordial ( +)  − 0.018  − 0.082
Primordial ( −) 0.195 0.094
Primary ( +) 0.521 0.169
Primary ( −) 0.050  − 0.270
Secondary ( +) 0.244  − 0.024
Secondary ( −) - -
Total ( +) 0.448 0.039
Total ( −) 0.190  − 0.095
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rs, Spearman’s correlation coefficient; *p < 0.05

The age of the enrolled participants was positively correlated with the number of secondary follicles in the samples subjected to closed metal container vitrification (rs =  − 0.724, p = 0.008). Patient age was also correlated with the total number of follicles (rs =  − 0.607, p = 0.036) and the number of primordial follicles in the samples that were cryopreserved by slow-freezing (rs =  − 0.591, p = 0.043).

Discussion

The current literature regarding cryopreservation methods for ovarian tissue has reported contradictory results when slow-freezing (Dalman et al. 2017) and vitrification techniques have been compared (Fabbri et al. 2016). Studies have indicated not only some advantages in using slow-freezing but also promising results when vitrification is employed.

In the vitrification technique, the container plays a critical role in preventing direct contact of the ovarian tissue sample with LN2 (thus avoiding tissue injury and contamination) and assisting in cooling the sample. Thus, the nature of the material used for its fabrication can consistently influence the cooling curves (Gosden, 2011). Considering that metal is a much faster heat conductor than plastic, our group developed a metallic container, initially made of aluminium foil and later of stainless steel (Bös-Mikich et al. 2013). Notably, it has been demonstrated that this device was effective in the vitrification of bovine ovarian tissue samples, with preservation of tissue morphology (Aquino et al. 2014) and post-rewarming biochemical aspects of the sample (Massignam et al. 2018b). In addition, the container was tested for sealing using a dry red powder dye (Sudan) (Aquino et al. 2014).

Other metallic devices have also been proposed for closed vitrification. The use of a titanium device allowed the resumption of hormonal cycles after heterotopic transplantation in a study carried out in monkeys (Suzuki et al. 2015). In humans, the use of a silver container allowed better preservation of the cells compared to a plastic container (Xiao et al. 2017).

The complexity differences among oocytes, embryos, and human ovarian tissue are remarkable. In fact, the ovarian tissue consists of a variety of cellular subtypes (e.g. oocytes, granulosa cells, stromal cells, and blood cells) in addition to the fibrous stroma. Thus, maintaining the cryostability of ovarian tissue is more challenging. Primordial follicles are important biomarkers for assessing fertility potential. Since it has been demonstrated that primordial follicles are more resistant to cryoinjury (thus presenting with a preserved morphology and integrity), these cells are commonly used to assess the viability of the ovarian tissue after cryostorage (Hovatta et al. 1996; Zhou et al. 2010). In line with this, the morphology of primordial follicles was used to evaluate the efficacy of the cryopreservation techniques in this study. Our results demonstrated that the number of intact primordial follicles after rewarming was higher than that obtained after thawing, indicating better preservation of the tissue submitted to the closed metal container vitrification. Some studies have corroborated these findings (Herraiz et al. 2014; Huang et al. 2008; Shi et al. 2017), and some studies failed to find any significant difference between the two evaluated techniques (Fabbri et al. 2016; Oktem et al. 2011).

Although the follicles may be morphologically intact after cryopreservation, their ability to develop and produce an oocyte to be fertilised, with the ultimate goal of pregnancy and live birth, may be affected by cryopreservation, regardless of the method (Shi et al. 2017). While some studies compared the two techniques exclusively using morphological parameters (Dalman et al. 2017), we investigated the ability of the ovarian cortex tissue (including follicles and stroma) to physiologically respond to non-lethal stress by evaluating the expression of heat shock proteins. These proteins, particularly HSP70, are involved in the activation of a molecular pathway known as HSR (please see Krause et al. (2015) for review). Healthy cells and tissues are expected to respond to heat stress (exposure to high temperatures) via an increase in HSP70 expression (de Lemos Muller et al. 2018).

When the ovarian tissue was kept under fresh conditions, an increased HSR was observed. This expected response was hampered by rewarming or thawing, indicating a dramatic reduction in the proteostasis machinery. Despite the cryopreservation-induced reduction in HSP70 expression at 37 °C, the magnitude of the effect was found to be smaller for the slow-freezing technique although there were no statistically significant differences between the deltas of both groups. Together, these data suggest better cytoprotection in samples subjected to slow-freezing. These findings have been previously described in animal models (Maffei et al. 2014). However, we found a positive correlation between the number of intact primordial follicles and HSR.

With regard to HSP, our data showed that both cryopreservation techniques resulted in decreased numbers of viable follicles followed by a reduced heat-induced HSR, suggesting an inability to react to a stressful event. The levels of HSP70 were different from those previously reported by Nikishin and colleagues (Nikishin et al. 2018) in native or vitrified/rewarmed samples. These discrepancies may be related to the fact that our samples were subjected to heat-induced stress 1 h after rewarming or thawing. Different results may be obtained after a prolonged recovery time, or it may suggest that each technique may require a distinct recovery duration after the heat challenge. This hypothesis warrants further investigation and is currently being tested in our laboratory.

Collectively, our results indicate that each cryopreservation technique may have different advantages, such as a higher number of intact primordial follicles following closed metal container vitrification and a better-preserved cytoprotection effect in the case of slow-freezing. Our results also corroborate that heat-induced HSR tests efficiently evaluate the stress response of ovarian tissues kept under different conditions. Finally, our results demonstrated that the closed metal container is a promising device for use in vitrification, although further investigation is warranted.

Acknowledgements

The authors would like to thank all the patients enrolled in the study. We thank the Federal University of Rio Grande do Sul (UFRGS), Department of Physiology, for supporting this work.

Author contribution

SG, MK, MFJ, and ABM conceptualised and designed the study. SG performed the tissue collection. LSK, GBG, NF, and LRS conducted the histological and viability analyses. SG, MK, MFJ, and ABM analysed the results. SG and MK drafted the manuscript. MFJ and ABM critically revised the text. All authors read and approved the final manuscript.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. MK was supported by a research fellowship from CNPq (process# 302959/2020–3). This work was partially supported by FAPERGS. MK was responsible for the grant support from FAPERGS (Edital FAPERGS/Decit/SCTIE/MS/CNPq/SESRS n. 03/2017–PPSUS #17/2551–0001424-3).

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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