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Pôle de Recherche en Gynécologie, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain, Brussels, BelgiumGynecology Department, Cliniques Universitaires Saint-Luc, Brussels, Belgium
In recent years, some countries and fertility preservation networks have started adopting 24 h transportation for ovarian tissue, a practice that has the potential to spread very quickly due to the high costs and bureaucracy involved in the establishment of ovarian tissue cryobanks. While pregnancies and live births have been reported after such long periods of transportation, this, however, remains an empirical procedure. This review aims to prompt reflection on ovarian tissue transport, highlighting the lack of knowledge in humans by providing a counterpoint looking into more than 40 studies published in different animal models. By discussing these studies in animals, the findings of various models can be deciphered, and light shed on the patterns identified. Like the development of different assisted reproductive technology procedures, this is an important step in creating guidelines for future studies on human ovarian tissue transportation.
Assisted reproductive technology (ART) is vital for the preservation of animal and human gametes. In animals, techniques have been developed for the selection of species/breeds of economic interest and also the conservation of endangered species (
One of the most recent ART strategies, ovarian tissue cryopreservation and transplantation, is now considered an established procedure in a growing number of countries, yielding more than 130 live births to date (
), it is important to stress that there are still many challenges. Tissue cryopreservation requires specialized equipment, trained staff as well as designated time and space to prepare and freeze the ovarian cortex. Due to strict European rules, specific protocols and high costs, only large hospitals can maintain an ovarian tissue cryobank. One way of increasing access to this strategy is improving ovarian tissue transportation and creating a network for fertility preservation, which would allow small clinics and less developed countries to extend the availability of ovarian tissue cryopreservation and transplantation (
Fertiprotekt, oncofertility consortium and the danish fertility-preservation networks - what can we learn from their experiences? Clinical medicine insights.
Organ preservation: Current concepts and new strategies for the next decade.
Transfusion medicine and hemotherapy: offizielles Organ der Deutschen Gesellschaft fur Transfusionsmedizin und Immunhamatologie.2011; 38: 125-142https://doi.org/10.1159/000327033
), with adapted medium composition, temperature and time for transport, the same cannot be said for ovarian tissue. Indeed, there are still no standard procedures for ovarian tissue transportation (
), when the blood supply is re-established, but it does not only occur after grafting. In fact, ischaemia begins when ovarian tissue is taken from the patient and continues until its cryopreservation and/or transplantation (
). Since the ovary is a complex organ, with a diversity of cells and structures of great importance for ensuring follicle survival and development, its transportation is not straightforward. Media used for human ovarian tissue transportation at present, such as alpha-minimum essential medium (α-MEM) (
), the aim here is to systematically review studies on ovarian tissue transportation in both animals and humans. Species, media, timepoints, temperatures, analyses and optimal transport conditions were addressed for each study to better understand practices that are well established, as well as possible gaps in knowledge and future strategies that can be implemented to prepare guidelines for ovarian tissue transport.
Materials and methods
This review was carried out according to the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA-P) (
), and registered on PROSPERO on 6 January 2020 (CRD42020155900).
Search strategy
A PubMed search was conducted of the literature in English, from 1990 to 2020, focusing on transportation of human and animal ovarian tissue, using the following keywords: ‘ovary and transportation’, ‘ovarian tissue and transportation’, ‘ovary and transport’, ‘ovarian tissue and transport’, ‘ovary and storage’ and ‘ovarian tissue and storage’. Results were downloaded to EndNote software (Thomson Reuters, Canada), references were merged and duplicates were removed. Any risk of bias was avoided.
Screening and eligibility
Screening was carried out for article titles and abstracts containing the keywords by J.M.V.V. and C.A.A. Only full-text English reports that specifically compared different conditions for ovarian tissue transportation were included. Scientific articles reporting successful outcomes of cryopreservation were excluded, unless the main goal of the research was to analyse tissue transport.
A data-charting table was developed by the authors to extract the main variables. This table was continually updated while writing this review. Disagreements were resolved through discussion and consensus. Charting was implemented using Airtable (USA), a customizable online platform.
Data items
Data were extracted on article characteristics (such as authors, year, country and institution), tested temperatures, timepoints and media, and analyses and best conditions were scrutinized for each study. Data were cross-checked by the authors.
Results
A total of 324 records were found through database searches and a further 28 were identified from studies’ references. Of those, 293 were excluded and 58 were screened, based on the title and abstract that matched the study's criteria. From those, 52 full-text articles were considered as potentially eligible, of which 46 were included in the qualitative synthesis (Figure 1). Selected studies dated from 1992 to 2020 (Supplementary Figure 1). The studies were grouped by country, research group and species, outlined tested media, temperatures and timepoints, and considered applied analyses, as well as the main findings and best conditions in each study.
Figure 1PRISMA flow diagram for ovarian transportation studies.
Results from the 46 studies selected for systematic review are summarized in Table 1 (humans) and Table 2 (animals). They involved 13 countries, namely Italy (n = 2), Brazil (n = 15), Germany (n = 4), Iran (n = 1), the USA (n = 7), Japan (n = 7), Belgium (n = 2), China (n = 1), Turkey (n = 3), South Korea (n = 1), the UK (n = 1), Spain (n = 1) and Argentina (n = 1), and 28 different research groups, the majority being from Brazil (six groups) and Japan (six groups) (Supplementary Table 1).
Table 1Studies on ovarian tissue transportation in humans
Best conditions within the parameters observed in each study. Not applicable = the conclusion of this study was that transport duration should be limited to a minimum to avoid damage.
Increasing follicular and stromal cell proliferation in cryopreserved human ovarian tissue after long-term precooling prior to freezing: In vitro versus chorioallantoic membrane (cam) xenotransplantation.
a Best conditions within the parameters observed in each study. Not applicable = the conclusion of this study was that transport duration should be limited to a minimum to avoid damage.
Best conditions within the parameters observed in each study. Not applicable = the study aimed to verify metabolic activity in different media. The authors did not report best conditions ACP, powdered coconut water; BSA, bovine serum albumin; DPBS, Dulbecco's PBS; EGCG, epigallocatechin gallate; GSH, glutathione; ITS, insulin–transferrin–selenium; MEM, minimum essential medium; m-PBS, modified PBS; PBS, phosphate-buffered saline; SOD, superoxide dismutase; UW, University of Wisconsin
Lowering storage temperature during ovary transport is beneficial to the developmental competence of bovine oocytes used for somatic cell nuclear transfer.
Effect of low-temperature bovine ovary storage on the maturation rate and developmental potential of follicular oocytes after in vitro fertilization, parthenogenetic activation, or somatic cell nucleus transfer.
Structure of preantral follicles, oxidative status and developmental competence of in vitro matured oocytes after ovary storage at 4 degrees c in the domestic cat model.
Santos, R.R.D., Silva, J.R.V., Costa, S.H.F., Rodrigues, A.P.R., Lôbo, R.N.B., Figueiredo, J.R.D., 2002. Effect of 0.9% saline solution and phosphate buffer saline at different temperatures and incubation times on the morphology of goat preantral follicles %j brazilian journal of veterinary research and animal science. 39, 254-259.
Effect of braun-collins and saline solutions at different temperatures and incubation times on the quality of goat preantral follicles preserved in situ.
Effect of coconut water and braun-collins solutions at different temperatures and incubation times on the morphology of goat preantral follicles preserved in vitro.
Effect of melatonin supplementation in the long-term preservation of the sheep ovaries at different temperatures and subsequent in vitro embryo production.
a Best conditions within the parameters observed in each study. Not applicable = the study aimed to verify metabolic activity in different media. The authors did not report best conditionsACP, powdered coconut water; BSA, bovine serum albumin; DPBS, Dulbecco's PBS; EGCG, epigallocatechin gallate; GSH, glutathione; ITS, insulin–transferrin–selenium; MEM, minimum essential medium; m-PBS, modified PBS; PBS, phosphate-buffered saline; SOD, superoxide dismutase; UW, University of Wisconsin
Increasing follicular and stromal cell proliferation in cryopreserved human ovarian tissue after long-term precooling prior to freezing: In vitro versus chorioallantoic membrane (cam) xenotransplantation.
Effect of low-temperature bovine ovary storage on the maturation rate and developmental potential of follicular oocytes after in vitro fertilization, parthenogenetic activation, or somatic cell nucleus transfer.
Lowering storage temperature during ovary transport is beneficial to the developmental competence of bovine oocytes used for somatic cell nuclear transfer.
Structure of preantral follicles, oxidative status and developmental competence of in vitro matured oocytes after ovary storage at 4 degrees c in the domestic cat model.
Effect of coconut water and braun-collins solutions at different temperatures and incubation times on the morphology of goat preantral follicles preserved in vitro.
Effect of braun-collins and saline solutions at different temperatures and incubation times on the quality of goat preantral follicles preserved in situ.
Santos, R.R.D., Silva, J.R.V., Costa, S.H.F., Rodrigues, A.P.R., Lôbo, R.N.B., Figueiredo, J.R.D., 2002. Effect of 0.9% saline solution and phosphate buffer saline at different temperatures and incubation times on the morphology of goat preantral follicles %j brazilian journal of veterinary research and animal science. 39, 254-259.
Effect of melatonin supplementation in the long-term preservation of the sheep ovaries at different temperatures and subsequent in vitro embryo production.
) (Supplementary Table 2). A total of 26 studies evaluated transportation of whole ovaries, one of half-ovaries and 16 of ovarian fragments, and one compared whole ovaries and fragments.
Tested parameters and assessment
Of the 46 publications analysed, 28 evaluated different temperatures, 34 monitored the effect of time, 13 investigated different media, six assessed the impact of additives in media and only two considered the influence of tissue size (Table 3). Tested temperatures ranged from 0 to 39°C, 4°C (n = 34) and 20°C (n = 13) being the most frequently evaluated. Timepoints varied from 1 to 96 h, 4 (n = 16), 12 (n = 20) and 24 h (n = 24) being the periods most commonly investigated. In total, 20 different media were tested over all the studies, with saline solution (n = 14) and phosphate-buffered saline (PBS; n = 12) emerging as the most prevalent, followed by MEM (n = 8) (Figure 2). The best study conditions for each species are summarized in Table 4.
Table 3Analysed parameters in ovarian tissue transport
Most studies focused their analysis on oocytes (43.7%, 20 studies) and ovarian tissue (50%, 23 studies) (Supplementary Figure 2). Regarding DNA/RNA analyses, one study applied the polymerase chain reaction (
). DNA fragmentation (using TdT (terminal deoxynucleotidyl transferase)-mediated dUTP nick-end labelling [TUNEL], in-situ end-labelling [ISEL] or other techniques) was performed in 10 studies (
Structure of preantral follicles, oxidative status and developmental competence of in vitro matured oocytes after ovary storage at 4 degrees c in the domestic cat model.
Lowering storage temperature during ovary transport is beneficial to the developmental competence of bovine oocytes used for somatic cell nuclear transfer.
) evaluated glucose, pyruvate and lactate concentrations, as well as pH and lactate dehydrogenase activity in media used for transportation. Two other studies analysed follicular fluid; Tellado and colleagues (
) calculated the pH of follicular fluid in antral follicles after storage of pig ovarian tissue.
In ovarian tissue, follicle morphology and ultrastructure were the focus of analyses, conducted using haematoxylin and eosin staining (23 studies) or transmission electron microscopy (TEM; 7 studies). For assessment of the transportation protocol, ovarian tissue fragments were cultured after storage in nine studies, and transplanted in one study (
). In oocytes, investigations were largely related to oocyte development after in-vitro maturation (IVM; 16 studies), nine of which involved IVF (results summarized in Table 2).
Effects on ovarian tissue
Analyses of ovarian tissue were performed in humans (
Increasing follicular and stromal cell proliferation in cryopreserved human ovarian tissue after long-term precooling prior to freezing: In vitro versus chorioallantoic membrane (cam) xenotransplantation.
Current achievements and future research directions in ovarian tissue culture, in vitro follicle development and transplantation: Implications for fertility preservation.
Effect of coconut water and braun-collins solutions at different temperatures and incubation times on the morphology of goat preantral follicles preserved in vitro.
Effect of braun-collins and saline solutions at different temperatures and incubation times on the quality of goat preantral follicles preserved in situ.
Santos, R.R.D., Silva, J.R.V., Costa, S.H.F., Rodrigues, A.P.R., Lôbo, R.N.B., Figueiredo, J.R.D., 2002. Effect of 0.9% saline solution and phosphate buffer saline at different temperatures and incubation times on the morphology of goat preantral follicles %j brazilian journal of veterinary research and animal science. 39, 254-259.
In humans, ovarian tissue pieces cultured for 15 days after 26 h of transportation in Brama I medium at 4°C showed no evidence of morphological damage (
Increasing follicular and stromal cell proliferation in cryopreserved human ovarian tissue after long-term precooling prior to freezing: In vitro versus chorioallantoic membrane (cam) xenotransplantation.
) exhibited increased follicle viability after 24 h of transport at 5°C and culture of ovarian pieces in vitro and in the chorioallantoic membrane of chick embryos for 5 days. Kyoya and co-workers (
) transported ovarian tissue in L-15 for up to 18 h at 4°C and cultured vitrified fragments for 24 h. They analysed oxygen consumption rates by scanning electrochemical microscopy and found no difference in follicle morphology or oxygen consumption from fresh controls. After transport in Celsior medium at 4°C for 24 or 48 h, Klocke and colleagues (
) performed vitrification and tissue culture for 18 days, reporting that malondialdehyde concentrations increased in the medium, indicative of oxidative stress. Oestradiol was also measured and its concentrations progressively fell with storage time. Apoptosis was assessed with anti-active caspase-3 after vitrification and warming, and the proportion of apoptotic follicles was not found to be significantly different from that in non-transported (fresh) tissue.
Animal ovarian tissue
Cow ovaries were stored at 4°C or 20°C for up to 18 h in saline or coconut water. Conservation at 4°C for up to 18 h and 20°C for up to 6 h kept the percentage of morphologically normal follicles similar to fresh controls, but the solution had little effect on the results (
) compared concentrations of glucose, pyruvate and lactate after transporting cow ovaries in IVF medium, L-15 medium, saline solution and PBS for up to 24 h at 4°C, and reported no glucose consumption, low pyruvate consumption and comparable lactate release in all media. Viability assays were performed using TUNEL and fluorescence-activated cell sorting, and showed low apoptosis and necrosis rates, indicating anaerobic metabolism during ovarian tissue transportation.
) stored ovarian tissue for up to 48 h in Dulbecco's PBS at 4°C and analysed follicle atresia by histology and DNA fragmentation using ISEL. They detected significant DNA degradation in granulosa cells after 12 h of storage. Using the same media and temperature, Wood and colleagues (
) observed that cold storage inhibited taphonomic changes to tissue for 48 h after removal from the animal. The authors reported that atresia found in samples was natural and not a result of cold storage. In a recent study, Martins and co-workers (
) evaluated follicle morphology before and after storage followed by vitrification. They used MEM at 4°C for 24 or 72 h and encountered higher survival rates in the 24 h storage group, with no difference in pre-antral follicle survival rates between fresh tissue and ovaries stored for 24 h.
All studies using goat ovarian tissue were from the same research group in Brazil. They tested five different media at four temperatures (4, 20, 35 and 39°C). Using histology or TEM, they observed better preservation of follicles with coconut water at 4°C than with Braun-Collins solution (
Effect of coconut water and braun-collins solutions at different temperatures and incubation times on the morphology of goat preantral follicles preserved in vitro.
Effect of braun-collins and saline solutions at different temperatures and incubation times on the quality of goat preantral follicles preserved in situ.
Santos, R.R.D., Silva, J.R.V., Costa, S.H.F., Rodrigues, A.P.R., Lôbo, R.N.B., Figueiredo, J.R.D., 2002. Effect of 0.9% saline solution and phosphate buffer saline at different temperatures and incubation times on the morphology of goat preantral follicles %j brazilian journal of veterinary research and animal science. 39, 254-259.
Effect of coconut water and braun-collins solutions at different temperatures and incubation times on the morphology of goat preantral follicles preserved in vitro.
). In another study, ovarian fragments cultured for up to 7 days after storage in MEM for 2 or 4 h exhibited follicle morphology similar to the fresh controls (
), by comparing the transportation of a whole ovary with fragments of equivalent to half, a quarter and an eighth its size. In that study, the smallest fragments showed better preservation of the follicle capacity to grow in vitro for up to 24 h. Gonçalves and colleagues (
) reported that sheep ovarian fragments can be stored for up to 6 h at 35°C in MEM supplemented with additives like insulin–transferrin–selenium, hypoxanthine, glutamine, bovine serum albumin and FSH (which they named MEM+). Other studies have described the positive impact of two anti-apoptotic drugs, Z-VAD-FMK (a pan-caspase inhibitor) and SP1 (a bioactive lipid in follicular fluid) (
In dogs, one study reported better preservation of follicle morphology when ovarian fragments were stored at 10°C in a coconut water-based medium compared with PBS. Indeed, while the percentage of morphologically normal follicles started to decrease after 24 h in PBS, it only started to fall after 36 h in coconut water-based medium (
). Although histology evidenced no significant changes after transport in Dulbecco's modified Eagle's medium, there was a significant reduction in implantation and live birth rates with prolonged tissue storage (>8 h).
Effects on oocyte maturation and fertilization, and embryo production
Studies on oocyte maturation and fertilization, as well as embryo production after ovarian tissue transportation, have only been conducted in animals. At the time of writing this review, no studies had been completed in humans. IVM of oocytes followed by IVF was performed after transport of bovine (
Effect of low-temperature bovine ovary storage on the maturation rate and developmental potential of follicular oocytes after in vitro fertilization, parthenogenetic activation, or somatic cell nucleus transfer.
Structure of preantral follicles, oxidative status and developmental competence of in vitro matured oocytes after ovary storage at 4 degrees c in the domestic cat model.
Effect of melatonin supplementation in the long-term preservation of the sheep ovaries at different temperatures and subsequent in vitro embryo production.
Effect of low-temperature bovine ovary storage on the maturation rate and developmental potential of follicular oocytes after in vitro fertilization, parthenogenetic activation, or somatic cell nucleus transfer.
) analysed the impact of time and temperature. Using PBS, saline or Ringer's solution, the studies suggest that bovine ovarian tissue is better preserved at 20°C for up to 5 h, but higher rates of maturation and blastocyst formation are obtained when storage was between 10 and 15°C when stored for 24 h. Moreover, Nagao and colleagues (
Structure of preantral follicles, oxidative status and developmental competence of in vitro matured oocytes after ovary storage at 4 degrees c in the domestic cat model.
) reported higher rates of meiosis resumption when storage was carried out at 4°C. At this temperature, these authors found decreased MII rates after 48 h of storage (
Structure of preantral follicles, oxidative status and developmental competence of in vitro matured oocytes after ovary storage at 4 degrees c in the domestic cat model.
), but they all agreed that with PBS or saline solution, cat ovarian tissue can be stored for up to 24 h at 4°C. Use of superoxide dismutase increased the viability of cumulus–oocyte complexes, embryo production and expression of the antiapoptotic gene Bcl-2, while reducing the expression of Bax, an apoptosis regulator (
In pigs, studies reported reduced oocyte viability and maturation rates at low temperatures in saline solution, suggesting that porcine ovaries should be stored for a short time (<6 h) at 25–35°C (
There is no consensus on the influence of transport temperatures on canine ovarian tissue. While some authors found this tissue to be more sensitive to low temperatures (
Ovaries from sheep were stored in PBS with different concentrations of melatonin, and there was an improvement in embryo quality with increasing concentrations of melatonin in the medium after storage at 4°C for 24 h (
Effect of melatonin supplementation in the long-term preservation of the sheep ovaries at different temperatures and subsequent in vitro embryo production.
). Goat oocytes showed higher meiotic resumption rates and a greater diameter of early antral follicles after 18 days of culture following ovarian tissue storage at 4°C for 4 h (
This is the first systematic review of the current state of ovarian tissue transportation. The authors identified 46 studies addressing optimal conditions for ovarian tissue preservation. Indeed, papers on this subject have been consistently published from 2004 to 2018 (an average of two or three per year), and continue to this day, testament to growing interest in this emerging topic of research.
While vital organ preservation has been developed and applied over the past 60 years (reviewed by
), preservation of ovarian tissue is relatively new. Hence, ovarian tissue transportation has only been recently discussed, with the concept of ‘the woman stays, the tissue moves’ established by the Danish network (
). This practice has the potential to spread extremely quickly due to the costs and bureaucracy involved in the establishment of ovarian tissue cryobanks. Pregnancies and live births have been reported after such long periods of transportation, but this systematic review demonstrates that this remains an empirical procedure.
The aim of this review was to prompt reflection on ovarian tissue transport, given the lack of knowledge there is in humans, by scrutinizing more than 40 studies published in different animal models. Among different domestic animal species, cows, sheep and goats have been used as models for humans thanks to their physiological ovarian similarities (
). While no animal models can replace studies in our own species, they nevertheless provide valuable information that can be used in human medicine. By discussing these results in animals, an attempt can be made to identify patterns within the studies. As in the development of different ART procedures, the authors believe this is an important step to create guidelines for future studies on human ovarian tissue transportation.
This review found that, when ovaries are stored for longer periods, lower temperatures can preserve follicle morphology and yield better oocyte maturation rates. Only two studies in pigs reported better IVM results after storage at over 25°C for 6 h (
In fact, vital organs exposed to normothermic ischaemia remain viable for less than 1 h. Warm ischaemia leads to rapid depletion of ATP, accumulation of lactic acid, decreased pH, proteolysis, lipolysis and lipid peroxidation (
). Cold ischaemia causes an energy crisis, leading to injuries that are normally expressed after graft reperfusion, such as metabolic disruptions, oedema, autolysis and oxidative stress (
In ovarian follicle metabolism, it is known that primordial germ cells consume twice as much pyruvate as glucose and primordial follicles release lactate, indicating glycolysis and mitochondrial pyruvate oxidation (
). While there is a lack of information about metabolism during ovarian tissue transport, it was shown that when pig ovaries were stored at 25°C for 6 h, follicular fluid exhibited lower pH values (
Structure of preantral follicles, oxidative status and developmental competence of in vitro matured oocytes after ovary storage at 4 degrees c in the domestic cat model.
) compared cow ovarian tissue storage in saline solution, IVF medium and L-15 at 4°C for 24 h and observed an increased lactate release in the media, even though there was low consumption of pyruvate and no consumption of glucose in IVF medium or L-15. However, these metabolic responses are associated with the assembly of different ovarian cell types (
). It is important to stress that there is a lack of data on stromal ovarian cell metabolism, which, although not directly responsible for fertility, plays a significant role in the activation of primordial follicles and development of growing follicles (
Overall, for most species, ovarian tissue should be transported at low temperatures to reduce metabolic activity and injuries during this period, allowing the tissue to be preserved for up to 24 h with minimal damage.
In terms of media, there is no consensus on the best medium for ovarian tissue transport and studies have been carried out with 20 different media, none of them specific for ovarian tissue. The main media used to transport ovarian tissue are PBS and saline solution. However, follicle morphology analyses in different species show that ovarian tissue requires nutrients that are not present in these solutions. Adenine nucleotide metabolism plays a key role in organ ischaemia, and preservation solutions that take this into account yield a better quality of preservation than saline solution (
). Indeed, when ovaries stored in PBS or saline solution were compared with those in more complex media, the latter were able to maintain normal morphology for longer periods (
Effect of coconut water and braun-collins solutions at different temperatures and incubation times on the morphology of goat preantral follicles preserved in vitro.
Effect of melatonin supplementation in the long-term preservation of the sheep ovaries at different temperatures and subsequent in vitro embryo production.
) drugs also appeared to improve the quality of ovarian tissue, pointing to important factors to be studied in future experiments. For kidney, heart, lung and pancreas preservation, many substances have been used to avoid damage caused by reperfusion, such as xanthine oxidase inhibitors (
). Extrapolating knowledge acquired for different organs might well provide valuable insights into ovarian tissue preservation during transportation.
Most studies in humans were not part this systematic review because they reported outcomes of ovarian tissue cryopreservation and transplantation in clinical settings. In this context, the FertiPROTEKT network validated Custodiol as a transportation medium for up to 22 h, even though there are no data on its efficacy for ovarian tissue (
). In Denmark, 12 women underwent 18 transplantations and three babies were born to two women whose ovarian fragments were transported in α-MEM for 4–5 h prior to cryopreservation (
Increasing follicular and stromal cell proliferation in cryopreserved human ovarian tissue after long-term precooling prior to freezing: In vitro versus chorioallantoic membrane (cam) xenotransplantation.
). However, none of them is used in clinical practice for ovarian tissue transport prior to cryopreservation, stressing the importance of this topic for future research.
Ultimately, it is clear that different media compositions influence follicle viability and fertility outcomes, which could be related to cell metabolism. Although low temperatures reduce metabolism 12-fold, studies in animal models have shown lactate release in media (
), suggesting that cells might be using internal sources of energy instead of nutrients present in the media. These data indicate that ovarian tissue metabolism is affected by storage, even at low temperatures, and might impact fertility after transplantation. Moreover, it reinforces the importance of understanding whole tissue dynamics, beyond ovarian follicles.
The reliability of assessing transportation parameters was also considered in this review. Assessment of ovarian tissue transport differs according to application. In this sense, using IVM, IVF and in-vitro culture to analyse oocytes isolated from transported tissue provides important clues to how fertility is affected by storage time, temperature and medium. Results from species that rely on these techniques for fertility preservation offer direct answers on how maturation and embryo development rates are impacted by transportation. Unfortunately, findings of this type do not apply to follicles included in ovarian tissue.
Most studies on ovarian tissue evaluated only follicle morphology after storage. While morphology is an important criterion to observe, it does not translate to normal follicle activity, so it is necessary to allow a re-establishment of tissue activity to gauge the effects of the procedure. Some studies have used in-vitro culture of ovarian fragments, but this system is not very well established for many species, yielding low rates of follicle survival in both short- and long-term culture (
Current achievements and future research directions in ovarian tissue culture, in vitro follicle development and transplantation: Implications for fertility preservation.
), it might be a valuable technique to evaluate tissue transport. Indeed, transplantation of mouse ovarian tissue was used to assess ovarian tissue storage and revealed a significant reduction in both implantation and live birth rates, even though the morphology before transplantation did not show any difference in the percentage of morphologically normal follicles (
This systematic review was, however, limited by the fact it was based on full-text publications in English. Studies published in different languages were not included in the investigation.
This review highlights the limited knowledge there is on ovarian tissue transportation conditions for all species addressed. Results from studies in this systematic review, as well as reports from clinical settings, demonstrate that finding a solution that meets ovarian tissue needs is essential to improving ART techniques. While there is mostly a consensus on maintaining ovarian tissue at low temperatures (namely 4°C), it is vital to consider different types of ovarian cells, as well as their interactions and needs, to be able to establish a standard protocol for ovarian tissue transport according to each species. Elucidating stromal cell metabolism and whole ovary dynamics is essential to developing a specific medium to transport ovarian tissue destined for transplantation. Moreover, interdisciplinary studies will play a crucial role in the development of ovarian tissue transport media; much has been learned and developed for vital organs, and this knowledge can be used to study how to improve the quality of ovarian tissue after transport. Assessment of follicle development and function should also be addressed in future studies to understand how fertility is affected by transport protocols once tissue vascularization has been restored (Figure 3).
Figure 3Guidelines for developing a standard protocol for ovarian tissue transportation.
With increasing demand for ovarian tissue cryopreservation and transplantation around the world, creating standard protocols for efficient transport is imperative in order to reach more patients, who can then have biopsies taken in small clinics and hospitals, and frozen in national tissue bank centres. It is also important to stress that improving transport may also affect the quality of the grafts and reproductive outcomes, which in turn can have a positive impact on birth rates after ART.
Conclusion
Upon review of the current literature, it is clear that there are no set standards for ovarian tissue transportation and, to date, its evaluation has been largely empirical. Ovarian tissue should be transported at 4°C to minimize ischaemic damage, and it is vital to create guidelines for ovarian tissue transportation to enhance ART outcomes in all species. To be able to develop a medium that meets the needs of ovarian tissue, it is essential to study the metabolic activity of this tissue, and not only isolated follicles, to discern how whole-tissue dynamics affect fertility. Moreover, assessment of transported tissue should be conducted after its return to activity. Finally, to shed light on the effects of procuring ovarian tissue for grafting, xenotransplantation is still the best means of evaluating ovarian tissue transportation.
Acknowledgments
The authors would like to thank Mira Hryniuk, BA, for the English language revision of this manuscript. This study was supported by grants from Wallonia-Brussels International (WBI) (grant WBI-IN awarded to J. M. V. Vilela) and the Fonds National de la Recherche Scientifique de Belgique (FNRS) (C. A. Amorim is an FRS-FNRS Research Associate; grant 5/4/150/5 awarded to M. M. Dolmans).
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