The first comparative study on the effectiveness of slow freezing and vitrification of human ovarian tissue by xenotransplantation model in Vietnam

Abstract

Purpose

To compare the effectiveness of slow freezing (SF) and vitrification (VT) for ovarian tissue cryopreservation using a xenograft model.

Methods

From September 2020 to August 2023, ovarian tissues from patients aged 18 to 37 undergoing benign ovarian surgery were divided into three groups. Group 1: fresh tissues (FC) were immediately fixed for analysis. Group 2 (SF/VT): tissues were cryopreserved by SF or VT without transplantation. Group 3 (SF-T/VT-T): tissues were cryopreserved using SF or VT, followed by transplantation into NOD-SCID mice for one week, after which the tissues were collected and analyzed.

Results

A total of 49 ovarian tissue fragments were analyzed. Regardless of the cryopreservation technique, follicle survival, development, function, and vascularization were significantly reduced compared to the FC group, particularly after transplantation (p < 0.001). The survival rate of follicles in the SF-T group was notably higher (90.9%) than in the VT-T group (82.6%) (p < 0.001). For cell proliferation, indicated by Ki-67 positivity, the SF-T group showed a higher median number of positive follicles (2.5; range: 0–18) compared to the VT-T group (2; range: 0–11) (p = 0.04). Neo-vascularization, assessed via CD31 positivity, was significantly greater in the SF-T group (61%) than in the VT-T group (47%) (p = 0.016). Additionally, the median number of AMH-positive follicles was significantly higher in the SF-T group (3; range: 0–23) compared to the VT-T group (2; range: 0–25) (p = 0.03).

Conclusion

Both SF and VT are feasible methods for ovarian tissue cryopreservation. SF may be the preferred cryopreservation technique for fertility preservation in cases tissue transplantation is anticipated.

Introduction

As of December 2019, the American Society for Reproductive Medicine officially recognized ovarian tissue cryopreservation (OTC) as an established method for preserving female fertility [1]. OTC was started in late 1990s with slow freezing, until year 2005 with vitrification. Comparative data between slow freezing and vitrification has also become abundant [2, 3]. Since having been approved, OTC’s indications have broadened. OTC is now available not only to patients undergoing gonadotoxic treatments for cancer or benign conditions but also to those with non-malignant ovarian diseases, genetic or metabolic disorders that place them at high risk for premature ovarian insufficiency. Additionally, individuals choosing to preserve their fertility for age-related social reasons can also benefit from OTC [2–4]. Furthermore, OTC remains the most suitable option for safeguarding reproductive function in prepubertal girls, for whom other fertility preservation techniques are not viable [5].

There are two common techniques for cryopreserving ovarian tissue: slow freezing and vitrification. Slow freezing, widely adopted as the standard technique in most centers for OTC [6], involves the use of low concentrations of cryoprotectants such as dimethyl sulfoxide (DMSO), propanediol, or ethylene glycol (EG). The process entails gradually cooling the tissue to approximately –140 °C, over several hours using a programmable freezer, followed by immersion in liquid nitrogen at –196 °C for long-term storage [7, 8]. However, this method can have detrimental effects on cells due to the formation of ice crystals during the freezing process [9].

Conversely, vitrification employs an ultrafast cooling rate combined with a high concentration of cryoprotectants, resulting in the rapid solidification of cells and their surrounding environment into a glass-like state [10]. Vitrification is well-established as a routine clinical procedure for oocyte and embryo freezing and offers several advantages for OTC, including a reduced risk of ice crystal formation, cost-effectiveness in terms of equipment, and less time consumption [8]. Consequently, if vitrification proves to be as effective as slow freezing, it may represent a more economically efficient solution for fertility preservation [8].

Supporting evidence in the literature remains mixed, with some studies favoring slow freezing [11], others advocating for vitrification [12, 13], and some reporting comparable outcomes between the two techniques [14–16]. However, by 2020, the number of babies born following vitrification was reported to be four [8, 17], whereas more than 200 babies had been born following slow freezing [18, 19]. The substantial methodological variability across studies highlights the need for further research to definitively identify the superior technique.

Tu Du Hospital, the largest Obstetrics and Gynecology hospital in Vietnam and home to the country’s first IVF center, aims to establish the most effective OTC method tailored to the specific needs of IVF centers in Vietnam. To expand fertility preservation options for patients, we conducted the first study in Vietnam to compare the effectiveness of slow freezing and vitrification using a xenograft model, with approval from the Department of Science and Technology of Ho Chi Minh City. Our assessment includes a detailed analysis of histological and immunohistochemical results, marking a significant advance in reproductive technology in the region.

Materials and methods

The study was conducted from September 2020 to August 2023, with approval from the Department of Science and Technology of Ho Chi Minh City and the Ethics Committee of Tu Du Hospital (reference number 1103/BVTD-HĐĐĐ). After obtaining informed consent from patients, biopsies of the ovarian cortex were collected. The study included Vietnamese women aged 18 to 37 who underwent laparoscopy or laparotomy for benign ovarian surgery at Tu Du Hospital. Exclusion criteria included ovarian tumors suspected of malignancy during surgery, crushed or torn tissues, histologically confirmed malignant tumors, deaths of mice during the study, and instances where grafted tissues detached from the mice before collection.

Ovarian tissue preparation

After removing the remaining medullary tissue, ovarian samples were cut into 5 × 5x1 mm pieces. Biopsies from each patient were divided into three groups. Group 1 consisted of fresh tissue for control (FC group), which was fixed in 10% neutral buffered formalin for histological and immunohistochemical evaluation after collection. In Group 2, tissue fragments were equally divided between slow freezing and vitrification (SF and VT groups) without transplantation. After freezing/vitrification and subsequent thawing/warming, the fragments were evaluated by histology and immunohistochemistry. In Group 3, following freezing/vitrification and thawing/warming, the samples were transplanted into NOD-SCID mice for one week (SF-T and VT-T groups). For comparative analysis, each mouse received two ovarian grafts on the back, with the left side designated for slow freezing and the right side for vitrification. The tissues were then evaluated similarly to those in Groups 1 and 2 after collection.

Slow freezing and thawing procedures

Slow freezing was performed using a previously described method [20]. The freezing solution consisted of 10% dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) and 20% human serum albumin (HSA; Grifols Biologicals LLC, Los Angeles, CA, USA), both diluted in L-15 medium (Sigma-Aldrich). First, the ovarian tissue pieces were washed in the freezing solution and then transferred to sterile cryovials containing 0.8 mL of the freezing solution. The cryovials were loaded into a programmable freezer (Planner PLC, Sunbury-On-Thames, UK) at 0 °C for 15 min. The cooling protocol included several steps: from 0 °C to −8.0 °C at a rate of −2.0 °C/min, followed by manual seeding and cooling to −40.0 °C at a rate of −0.3 °C/min, and then to −140 °C at a rate of −30 °C/min. Finally, the vials were stored in liquid nitrogen at −196 °C.

For the thawing procedure, the cryovials were removed from the liquid nitrogen container and shaken in the air for 2 min, then immersed in a 37 °C water bath for 2 min until completely thawed. The ovarian tissue pieces were then washed in L-15 medium through three steps, with each step lasting 5 min. After the final wash, one piece was kept in a tube containing L-15 medium and promptly xenografted into a NOD-SCID mouse. Simultaneously, the other piece was fixed in 10% neutral buffered formalin for histological and immunohistochemical evaluation.

Vitrification and warming procedures

The tissue pieces were first incubated in an equilibrium medium containing L-15 medium, 10% ethylene glycol (EG; Sigma-Aldrich), 10% DMSO, 0.1 M sucrose (Sigma-Aldrich), 0.1 M trehalose (Sigma-Aldrich), and 6% HSA for 15 min. They were then transferred to a vitrification solution (VS) containing 20% EG, 20% DMSO, 0.3 M sucrose, 0.1 M trehalose, and 6% HSA in L-15 medium for up to 10 min. Finally, the tissue pieces were placed on a sterilized gauze device [21], which was rapidly plunged into fresh liquid nitrogen. The gauze devices were then placed into cryovials and stored in a liquid nitrogen tank.

For warming, the cryovials were removed from the storage tank, and the gauze devices containing the ovarian tissue pieces were taken out of the cryovials while still in liquid nitrogen. The devices were immediately immersed in a thawing solution (0.5 M sucrose, 0.2 M trehalose, and 6% HSA in L-15 medium) for 3 min. The pieces were allowed to fully thaw before being transferred to diluting solution 1 (0.5 M sucrose and 6% HSA in L-15 medium) and then to diluting solution 2 (0.25 M sucrose in L-15 medium), each for 3 min at room temperature. After the thawing procedure, the pieces were washed twice in washing solution (L-15 medium) for 3 min per step.

Following the final wash, one tissue piece was kept in a tube containing L-15 medium and promptly xenografted into a NOD-SCID mouse. Simultaneously, the other piece was fixed in 10% neutral buffered formalin for histological and immunohistochemical evaluation.

Xenotransplantation into NOD/SCID mice

The procedures for animal care and transplantation adhered to the animal welfare guidelines approved by the Animal Care and Use Committee of the Stem Cell Institute, University of Science, VNU-HCM (reference code 220,102/SCI-AEC). Twenty female NOD-SCID mice (BioLASCO, Taiwan Co., Ltd, Taiwan), aged ten weeks, were used in the study. The mice were housed in groups of three to four per cage under HEPA-filtered hoods in a positive airflow ventilated rack. The housing conditions were carefully controlled, with a temperature of 22 ± 2 °C, humidity levels of 45% to 65%, and a 12-h light/dark cycle. The mice were provided with laboratory food and water ad libitum. All bedding, cages, food, and water were autoclaved before use.

Before xenografting, the mice were anesthetized via intraperitoneal injection with a combination of 20 mg/kg body weight tiletamine + zolazepam (Zoletil, Virbac, France) and 5 mg/kg body weight xylazine (Xyla, Interchemie, Netherlands). This was followed by an intramuscular injection of 8 mg/kg gentamicin (Gentamicin Kabi, Fresenius Kabi, Germany) in 3 days.

To graft the ovarian tissue, a small incision was made on the back of each mouse, the tissue was inserted, and the skin was closed using Caresilk-Silk sutures. Two fragments from the same patient were grafted in the same mouse, the slow freezing one was put on the left and the vitrified one on the right. After one week of transplantation, the mice were sacrificed, the borders of skin around the tissues were cut, the skin was opened to take the fragments out. The grafted tissue fragments were fixed for histological and immunohistochemical assessment.

Histologic analysis

After fixation and paraffin embedding, ovarian tissue samples were sectioned into 5-µm-thick slices. Each slide contained three consecutive sections and was sequentially numbered: the 1st slide for Ki-67 staining, the 2nd for CD31 staining, the 3rd for AMH staining, and the 4th for hematoxylin and eosin (H-E) evaluation. The slides were deparaffinized using xylene and rehydrated through a series of ethanol solutions (100%, 90%, and 70%) before staining.

The H-E slides were sent to the Histology-Embryology-Genetics Department at Pham Ngoc Thach University of Medicine for microscopic evaluation of follicle count. Two experts independently evaluated each slide, with the final result determined by consensus. Only follicles containing an oocyte with a visible nucleus were counted to prevent duplication of the same follicle in different sections.

Follicles were counted and classified according to their developmental stage based on the protocol described by Gougeon [22]. A primordial follicle is characterized by an oocyte surrounded by a single layer of flattened granulosa cells, while a primary follicle contains an oocyte encircled by a single layer of cuboidal granulosa cells. A secondary follicle features an oocyte surrounded by at least two layers of cuboidal granulosa cells. The antral follicle stage is identified by the presence of an oocyte, multiple layers of granulosa cells, and a fluid-filled cavity.

Immunohistochemical staining and evaluation

Staining with Ki-67, AMH, and CD31 was performed to evaluate cell proliferation, follicular function, and angiogenesis, respectively. The slides were placed in preheated 80 °C citrate buffer (Sigma-Aldrich) at pH 6.0 for 10 min in a water bath to facilitate heat-induced epitope retrieval. The samples were then blocked with 5% goat serum (Sigma-Aldrich) for one hour. After blocking, the sections were incubated with primary antibodies for 12 h at 4 °C: Ki-67 (1 mg/ml, Ki-67 Monoclonal Antibody, Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA), AMH (1:1000 dilution, AMH Polyclonal Antibody, Invitrogen, Thermo Fisher Scientific), or CD31 (1 mg/ml, CD31 Monoclonal Antibody, Invitrogen, Thermo Fisher Scientific). After two washes with washing buffer (PBS with 0.5% Triton X-100), the slides were incubated with secondary antibodies (Goat anti-Rabbit IgG-594 or Goat anti-Mouse IgG-488, Thermo Fisher Scientific) for 2 h at room temperature in the dark. After washing twice, the slides were counterstained with DAPI (Thermo Fisher Scientific) to detect nuclei.

Using the Axio Observer A1 microscope (Zeiss), the number of Ki-67 or AMH-positive follicles, as well as CD31-positive vessels, were counted. Fluorescence images were captured using an LSM 800 microscope (Zeiss). Follicles were classified as developing if at least one granulosa cell stained positive for Ki-67. Follicles were considered AMH-positive if at least one granulosa cell showed a positive signal for AMH. A tissue section was deemed CD31-positive if it contained at least one CD31-positive blood vessel.

Ovarian follicle viability was assessed based on the DAPI staining of granulosa and oocyte nuclei. Live follicles displayed clear, uniform nuclei, while dead follicles had dark, translucent nuclei or no visible nuclei.

Statistical analyses

Continuous data are presented as mean ± standard deviation (SD) or median with interquartile range (IQR), while categorical data are described using frequency and percentage. The 95% confidence intervals for percentages were estimated using the Wilson score interval. To account for multiple statistical tests, p-values were adjusted using the Benjamini–Hochberg correction.

Comparisons of viable follicles, H-E-stained follicle types, and CD31-positive tissues between ovarian tissue preservation methods were performed using the Chi-squared test. Comparisons of the total number of observed HE-stained tissues, AMH-positive, and Ki67-positive follicles between preservation methods were conducted using the Kruskal–Wallis test. Results were considered statistically significant when the two-sided p-value was less than 0.05. All statistical analyses were performed using R version 4.3.0.

Results

Graft recovery and macroscopic findings

A total of 61 ovarian tissue pieces were obtained from 7 patients (5 to 9 pieces per patient). Most patients were under 35 years old, with a mean age of 27.00 ± 3.55 years. The tissue pieces were randomly allocated into three groups: 7 for fresh ovarian tissues (control group), 14 for non-xenotransplantation, and 40 for xenotransplantation. In the latter two groups, the tissue pieces were equally divided between the two cryopreservation methods. In the xenotransplantation group, 40 tissue pieces were grafted into 20 NOD-SCID mice (Fig. 1). After one week, out of the 20 mice, 14 mice yielded two viable tissue pieces per mouse (Fig. 2), while 6 mice did not and were subsequently excluded from the analysis. Therefore, data from the tissue pieces of 14 mice were analyzed.

Fig. 1.

Study design diagram

Fig. 2.

Retrieval of the grafted ovarian tissue fragments. Back of NOD-SCID mice after one week of xenotransplantation (left: slow freezing; right: vitrification) (a); Vessels presenting on the frozen-thawed (b) and vitrified-warmed ovarian fragments (c)

Follicle survival and development

Morphologically normal follicles were observed in all groups (Fig. 3). The median number of follicles stained with H-E per slide was as follows: 11.8 (range: 1–24) in the control group, 2.82 (range: 0–8.37) in the SF group, 0 (range: 0–4.95) in the VT group, 1.66 (range: 0–9) in the SF-T group, and 0 (range: 0–5.67) in the VT-T group. There was a statistically significant difference in the median number of follicles across the five groups (p < 0.001). The median number of follicles gradually decreased from the fresh sample group to the slow freezing and vitrification groups and further decreased in the groups with transplantation compared to those without.

Fig. 3.

Histological evaluation of the ovarian tissue fragments. Fresh tissue (a); SF group (b); SF-T group (c); VT group (d); VT-T group (e) (red arrow: viable primordial follicle)

The proportion of viable follicles in the control group was significantly higher than in each experimental group (p < 0.001) (Figs. 4, 5). However, no significant difference was observed in the proportion of viable follicles between the SF and VT groups (p = 0.966). Notably, the SF-T group showed a significantly higher proportion of viable follicles compared to the VT-T group (p < 0.001) (Fig. 5).

Fig. 4.

Immunofluorescence staining for viable follicles. Viable (a,b) and non-viable follicles (c,d). Images with white light (a,c) and cell nuclear stained with DAPI (b,d)

Fig. 5.

Proportion of viable follicles across the five groups: control, SF, VT, SF-T, and VT-T. *p < 0.001

Regarding follicle development, primordial follicles were the most prevalent, followed by primary and secondary follicles (Table 1). Interestingly, the proportion of primordial follicles in the cryopreserved groups, both with and without xenotransplantation, tended to be higher than in the fresh samples.

Table 1.

Distribution of follicle types (primordial, primary, and secondary) across the five groups: FC, SF, VT, SF-T, and VT-T

	FC	SF	VT	SF-T	VT-T
Primordial follicles	87.6 (1133/1293)	92.3 (686/743)	96.3% (975/1012)	95.1 (1689/1775)	97.9 (1067/1090)
Primary follicles	10.3%a(133/1293)	5.24 (39/743)	2.6%b(26.4/1012)	3.56 (63.3/1775)	1.8%b(19.6/1090)
Secondary follicles	2.09% (27/1293)	2.44 (18/743)	1.07% (https://doi.org/10.8/1012)	1.31 (23.26/1775)	0.29 (3.17/1090)

^a,b Indicate the statistical difference (p < 0.05) in only specific follicle classes for follicle proportion

Within the same follicle class, there were statistically significant differences in the proportion of primary follicles between the FC group and the VT group (p = 0.03) and between the FC group and the VT-T group (p = 0.01). However, no statistical differences were observed between groups for primordial or secondary follicles (Table 1).

Follicle development was confirmed by Ki-67 staining (Fig. 6). The proportion of Ki-67 positive follicles was significantly higher in the control group compared to the other four groups (p < 0.001) (Fig. 7). While no significant difference was observed in the proportion of Ki-67 positive follicles between the two cryopreservation methods, either before (p = 0.9) or after transplantation (p = 0.6), the proportion of Ki-67 positive follicles was significantly higher prior to xenografting than after xenografting for both methods (p < 0.001) (Fig. 7). The median of positive follicles in each slide between 5 groups were significantly different (p < 0.001) with 9 (2–36), 5 (0–10.2), 1 (0–5), 2.5 (0–18), 2 (0–11) in the control group, SF group, VT group, SF- T group, and VT- T group, respectively. The median of Ki67 positive follicles in SF- T group 2.5 (0–18) was higher than the VT -T group 2 (0–11) (p = 0.04).

Fig. 6.

Immunohistochemistry evaluation of the ovarian tissue fragments. Cell nuclei stained with DAPI (a, d, g); Ki-67 positive follicles (b, c); AMH-positive follicles (e, f) and with DAPI (f); CD31-positive staining surrounding follicles (h, i)

Fig. 7.

Proportion of Ki-67-positive follicles across the five groups: control, SF, VT, SF-T, and VT-T. *p < 0.001

Follicle function

Among the five groups, there was a significant difference in the percentage of AMH-positive follicles, with the highest proportion observed in the FC group compared to SF, SF-T, VT, and VT-T (p < 0.001) (Fig. 8). Additionally, a statistically significant difference was found in the percentage of AMH-positive follicles between the two cryopreservation methods, both with and without xenografting (p < 0.001) (Fig. 8). The proportion of AMH-positive follicles was significantly higher in the SF group (73.2%) compared to the VT group (64.8%). However, in the xenografting groups, the VT-T group (70.3%) had a significantly higher proportion of AMH-positive follicles than the SF-T group (63.2%) (p < 0.001). Despite this, the median number of AMH-positive follicles was significantly lower in the VT-T group (2, range: 0–25) compared to the SF-T group (3, range: 0–23) (p = 0.03).

Fig. 8.

Proportion of AMH-positive follicles across the five groups: control, SF, VT, SF-T, and VT-T. *p < 0.001

Vascularization

Overall, ovarian stromal tissue showed good vascularization in all groups, with visible vessels on the surface, at the periphery, and within the center of the tissue sections. Cells and tubular structures stained positive for CD31 throughout (Fig. 6). The percentage of CD31-positive slides differed significantly among the groups, with the VT-T group showing the lowest proportion (p < 0.001) (Fig. 9). The proportion of CD31-positive slides was significantly higher in the SF group (87%) compared to the VT group (73%) (p = 0.034). Similarly, the SF-T group (61%) had a significantly higher proportion of CD31-positive slides than the VT-T group (47%) (p = 0.016). Additionally, the control group (83%) exhibited a significantly higher proportion of CD31-positive slides than both the SF-T (61%) (p = 0.05) and VT-T (47%) groups (p < 0.001) (Fig. 9).

Fig. 9.

Proportion of CD31-positive slides across the five groups: control, SF, VT, SF-T, and VT-T. *p < 0.001

Discussion

This research represents the first study in Vietnam to investigate the efficacy of human ovarian tissue cryopreservation (OTC) and compare two cryopreservation methods, aiming to establish a standard protocol for clinical practice. As noted by Soleimani et al. [23], early events following transplantation are critical for the success of the procedure. Additionally, both Amorim et al. [24] and Soleiman et al. [23] indicated that follicle survival and early development can be assessed within the first week of transplantation. Therefore, for our initial study, we selected a one-week transplantation period to evaluate the outcomes.

Regarding the histological results, follicular distribution differed across the study groups (FC, SF, VT, SF-T, VT-T). After freezing and thawing, the median number of follicles significantly decreased, with the lowest numbers observed in the SF-T and VT-T groups. This reduction in follicle numbers after cryopreservation, particularly after transplantation, is consistent with findings from other studies. The loss may result from follicular cell detachment from the basal membrane due to cryodamage, hypoxia during the revascularization process, or premature follicle activation leading to uncontrolled growth [9, 11, 19, 25]. Notably, despite these challenges, the majority of primordial follicles, which constitute the ovarian reserve [26], were preserved in both cryopreservation techniques, with no significant difference in their proportion after freezing. This finding aligns with other reports [6, 14, 15], though it contrasts with Lee et al. [11], who compared the number of intact primordial follicles rather than their proportions.

In terms of follicle survival, the percentage of viable follicles after slow freezing was significantly higher than after vitrification (p < 0.001). Interestingly, Silber et al. [12] reported higher viability rates for vitrification (89.1%) compared to slow freezing (42%), indicating ongoing debate over the superiority of one technique over the other. For slow freezing, we use the standard protocol that has been used in many publications and shown the effectiveness. However, for the VT protocol, we have used trehalose and sucrose as external CPA with the expectation that would enhanced the VT results. Variability in protocols, devices, freezing methods, and chemicals used could explain these discrepancies. To date, there is no universally accepted vitrification protocol that outperforms others [10]. Evaluating the effectiveness of cryopreservation techniques is often done at different stages: immediately post-freezing [15, 27], after experimental culture [28, 29], or post-transplantation in mice [11, 24, 30, 31]. Studies suggest that while about 7% of follicles are lost after thawing, up to 60% may be lost after transplantation, primarily due to delayed vascularization [31]. Thus, our approach of assessing follicle viability post-xenotransplantation is crucial for determining clinical applicability. In our study, both techniques yielded over 80% follicle survival one week after xenotransplantation, though slow freezing demonstrated better results.

Regarding cell proliferation, no significant difference was observed between slow freezing and vitrification in terms of Ki-67 positive follicles after transplantation. However, the median number of Ki-67 positive follicles in the SF-T group was significantly higher than in the VT-T group. This finding mirrors the results of Lee et al. [11] and may explain the higher proportion of growing follicles observed in the SF-T group compared to the VT-T group. Similarly, for AMH secretion, while the VT-T group showed a higher proportion of AMH-positive follicles, the SF-T group had a significantly higher median number of AMH-positive follicles (p = 0.03), likely due to the greater number of viable follicles in the SF-T group. The proportion of AMH-positive follicles is important; however, in the clinical aspect, the number of AMH-positive follicles is more important because the aim of OTC is to preserve as many viable follicles with good function as possible. So, this result may need to be considered while choosing which freezing technique will be applied for the future human transplantation.

In comparison, studies like Lee et al. [11], which quantified AMH production via Western blot, found reduced AMH expression in both SF and VT groups compared to fresh tissue, without noting differences between the two techniques. Oktem et al. [32] also reported significantly lower AMH production in vitrified ovarian tissues compared to fresh and SF samples, though their study used a 3-day culture model rather than xenografting. Our findings corroborate these reports, as AMH expression decreased after cryopreservation, regardless of the method used.

Finally, the proportion of CD31-positive slides, an indicator of vascularization, was significantly higher in the SF group compared to the VT group, both with and without transplantation. This is consistent with the findings of Lee et al. [11] after transplantation. However, Herraiz et al. [13], using a nude mouse model and a six-month transplantation period, reported only a slight increase in vascularized area in SF grafts compared to VT grafts. In our study, the one-week xenografting period may not have been long enough for full neovascularization to develop. It is well-established that increased vascularization following transplantation is crucial for enhancing primordial follicle survival, both in mice and in human ovarian tissues [33, 34].

Our study has some limitations. The results are assessed after one week of transplantation. Though some researches proved that this duration is enough to evaluate; however, the longer-term outcomes may produce more information. Additionally, here is a model of xenograft transplantation; human transplantation may show more practical data for each OTC technique. The human transplantation is prerequisite to confirm which OTC technique should be applied in the specific clinical condition. So, future research in the longer-term models and human application should be necessary.

Conclusion

In conclusion, our study demonstrates that both slow freezing and vitrification are viable methods for OTC. However, slow freezing showed superior outcomes after xenotransplantation, particularly in terms of follicle survival, development, and function. Both cryopreservation techniques performed effectively in our setting, and either method could be recommended for OTC in Vietnam. With these results, we are confident in our ability to offer this fertility preservation option to cancer patients, providing a valuable strategy to safeguard reproductive potential in our country. However, a long-term transplantation model should be conducted to confirm the results. Further research on the topic will be necessary.

Author’s contributions

Conceptualization was made by Thi Minh Chau Le. Methodology was written by Thi Minh Chau Le, Christiani Andrade Amorim. Recruitment was performed by Thi Minh Chau Le, Thi Hanh Dung Tran, Van Phuc Pham, Thanh Long Dang, Khue Tu Duong, Thi Ngoc Tuyen Huynh, Thanh Tan Hua, Thi Hong Nhung Nguyen, Ba My Nhi Nguyen. Formal analysis and statistics were performed by Thi Minh Chau Le, Phuc Thinh Ong, Nguyen The Nguyen Pham. The study was consulted by Quang Thanh Le, Ba My Nhi Nguyen, Minh Tuan Vo. The first draft of the manuscript was written by Thi Minh Chau Le, Christiani Andrade Amorim, Thi Hanh Dung Tran, Khue Tu Duong. Critical revision of the manuscript for important intellectual content and approved the version to be published: Thi Minh Chau Le, Christiani Andrade Amorim. All authors read and approved the final manuscript.

Funding

The study was funded by the Department of Science and Technology of Ho Chi Minh city.

Data availability

With the approval of Tu Du hospital, we will provide our data if requested.

Declarations

Ethics approval

Approval of this study was granted by the Department of Science and Technology of Ho Chi Minh City and the Ethics Committee of Tu Du Hospital (reference number 1103/BVTD-HĐĐĐ). The procedures involving NOD/SCID mice were approved by the Animal Care and Use Committee of the Stem Cell Institute, University of Science, VNU-HCM (reference code 220102/SCI-AEC).

Competing interest

All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript.

Consent to participate

Informed consent was obtained from all individual patients included in the study.

Consent to publish

Patients signed informed consent regarding publishing their data.