Impact of low- or moderate-risk gonadotoxic chemotherapy prior to testicular tissue freezing on spermatogonia quantity in human (pre)pubertal testicular tissue.

STUDY QUESTION: What is the impact of low- or moderate-risk gonadotoxic chemotherapy received prior to testicular tissue freezing (TTF), and of the cancer itself, on spermatogonia quantity in testicular tissue from (pre)pubertal boys? SUMMARY ANSWER: Vincristine, when associated with alkylating agents, has an additional adverse effect on spermatogonia quantity, while carboplatin has no individual contribution to spermatogonia quantity, in testicular tissue of (pre)pubertal boys, when compared to patients who have received non-alkylating chemotherapy. WHAT IS KNOWN ALREADY: The improved survival rates after cancer treatment necessitate the inclusion of fertility preservation procedures as part of the comprehensive care for patients, taking into consideration their age. Sperm cryopreservation is an established procedure in post-pubertal males while the TTF proposed for (pre)pubertal boys remains experimental. Several studies exploring testicular tissue of (pre)pubertal boys after TTF have examined the tubular fertility index (TFI, percentage of seminiferous tubule cross-sections containing spermatogonia) and the number of spermatogonia per seminiferous tubule cross-section (S/T). All studies have demonstrated that TFI and S/T always decrease after the introduction of chemotherapeutic agents, especially those which carry high gonadotoxic risks such as alkylating agents. STUDY DESIGN, SIZE, DURATION: Testicular tissue samples from 79 (pre)pubertal boys diagnosed with cancer (from 6 months to 16 years of age) were cryopreserved between May 2009 and June 2014. Their medical diagnoses and previous chemotherapy exposures were recorded. We examined histological sections of (pre)pubertal testicular tissue to elucidate whether the chemotherapy or the primary diagnosis affects mainly TFI and S/T. PARTICIPANTS/MATERIALS, SETTING, METHODS: (Pre)pubertal boys with cancer diagnosis who had been offered TTF prior to conditioning treatment for hematopoietic stem cell transplantation were included in the study. All the patients had previously received chemotherapy with low- or moderate-risk for future fertility. We have selected patients for whom the information on the chemotherapy received was complete. The quantity of spermatogonia and quality of testicular tissue were assessed by both morphological and immunohistochemical analyses. MAIN RESULTS AND THE ROLE OF CHANCE: A significant reduction in the number of spermatogonia was observed in boys treated with alkylating agents. The mean S/T values in boys exposed to alkylating agents were significantly lower compared to boys exposed to non-alkylating agents (P = 0.018). In contrast, no difference was observed for patients treated with carboplatin as the sole administered alkylating agent compared to the group of patients exposed to non-alkylating agents. We observed an increase of S/T with age in the group of patients who did not receive any alkylating agent and a decrease of S/T with age when patients received alkylating agents included in the cyclophosphamide equivalent dose (CED) formula (r = 0.6166, P = 0.0434; r = -0.3759, P = 0.0036, respectively). The TFI and S/T decreased further in the group of patients who received vincristine in combination with alkylating agents (decrease of 22.4%, P = 0.0049 and P < 0.0001, respectively), but in this group the CED was also increased significantly (P < 0.0001). Multivariate analysis, after CED adjustment, showed the persistence of a decrease in TFI correlated with vincristine administration (P = 0.02). LIMITATIONS, REASONS FOR CAUTION: This is a descriptive study of testicular tissues obtained from (pre)pubertal boys who were at risk of infertility. The study population is quite heterogeneous, with a small number of patients in each sub-group. Our results are based on comparisons between patients receiving alkylating agents compared to patients receiving non-alkylating agents rather than chemotherapy-naive patients. The French national guidelines for fertility preservation in cancer patients recommend TTF before highly gonadotoxic treatment. Therefore, all the patients had received low- or moderate-risk gonadotoxic chemotherapy before TTF. Access to testicular tissue samples from chemotherapy-naive patients with comparable histological types of cancer was not possible. The functionality of spermatogonia and somatic cells could not be tested by transplantation or in vitro maturation due to limited sample sizes. WIDER IMPLICATIONS OF THE FINDINGS: This study summarizes the spermatogonial quantity of (pre)pubertal boys prior to TTF. We confirmed a negative correlation between the cumulative exposure to alkylating agents and spermatogonial quantity. In addition, the synergistic use of vincristine in combination with alkylating agents showed a cumulative deleterious effect on the TFI. For patients for whom fertility preservation is indicated, TTF should be proposed for chemotherapy with a predicted CED above 4000 mg/m2. However, the data obtained from vincristine and carboplatin use should be confirmed in a subsequent study including more patients. STUDY FUNDING/COMPETING INTEREST(S): This study had financial support from a French national research grant PHRC No. 2008/071/HP obtained by the French Institute of Cancer and the French Healthcare Organization. The sponsors played no role in the study. The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Minimal residual disease detection by multicolor flow cytometry in cryopreserved ovarian tissue from leukemia patients.

BACKGROUND: Cryopreservation of ovarian tissue is a fertility-preservation option for women before gonadotoxic treatments. However, cryopreserved ovarian tissue transplantation must be performed with caution in women with malignancies that may metastasize to the ovaries. For this purpose, detecting minimal residual disease (MRD) in the ovarian cortex using sensitive methods is a crucial step. We developed an automated ovarian tissue dissociation method to obtain ovarian cell suspensions. RESULTS: We assessed MRD by multicolor flow cytometry (MFC) in cryopreserved ovarian cortex of 15 leukemia patients: 6 with B-cell acute lymphoblastic leukemia (B-ALL), 2 with T-cell acute lymphoblastic leukemia (T-ALL) and 7 with acute myeloid leukemia (AML). Ovarian MRD was positive in 5 of the 15 leukemia patients (one T-ALL and 4 AML). No B-ALL patient was positive by MFC. Quantitative reverse-transcribed polymerase chain reaction was performed when a molecular marker was available, and confirmed the MFC results for 3 patients tested. Xenografts into immunodeficient mice were also performed with ovarian cortical tissue from 10 leukemia patients, with no evidence of leukemic cells after the 6-month grafting period. CONCLUSIONS: In conclusion, this is the first study using MFC to detect MRD in ovarian cortical tissue from acute leukemia patients. MFC has been accepted in clinical practice for its ease of use, the large number of parameters available simultaneously, and high throughput analysis. We demonstrate here that MFC is a reliable method to detect MRD in cryopreserved ovarian tissue, with a view to controlling the oncological risk before ovarian tissue transplantation in leukemia patients.

Assessment of the architecture and integrity of frozen-thawed testicular tissue from (pre)pubertal boys with cancer.

BACKGROUND: Testicular tissue freezing is proposed for fertility preservation to (pre)pubertal boys with cancer before highly gonadotoxic treatment. Studies accurately comparing human (pre)pubertal testicular tissue quality before freezing and after thawing are exceptional. No study has reported this approach in a systematic manner and routine care. OBJECTIVES: To assess the impact of a control slow freezing protocol on testicular tissue architecture and integrity of (pre)pubertal boys after thawing. MATERIALS AND METHODS: (Pre)pubertal boys (n = 87) with cancer from 8 Reproductive Biology Laboratories of the French CECOS network benefited from testicular tissue freezing before hematopoietic stem cell transplantation. Seminiferous tubule cryodamage was determined histologically by scoring morphological alterations and by quantifying intratubular spermatogonia and the expression of DNA replication and repair marker in frozen-thawed testicular fragments. RESULTS: A significant increase in nuclear and epithelial score alterations was observed after thawing (p < 0.0001). The global lesional score remained lower than 1.5 and comparable to fresh testicular tissue. The number of intratubular spermatogonia and the expression of DNA replication and repair marker in spermatogonia and Sertoli cells did not vary significantly after thawing. These data showed the good preservation of the seminiferous tubule integrity and architecture after thawing, as previously reported in our studies performed in prepubertal mice and rats. DISCUSSION: The current study reports, for the first time, the development of a semi-quantitative analysis of cryodamage in human (pre)pubertal testicular tissue, using a rapid and useful tool that can be proposed in routine care to develop an internal and external quality control for testicular tissue freezing. This tool can also be used when changing one or several parameters of the freezing-thawing procedure. CONCLUSION: Control slow freezing protocol without seeding maintains the seminiferous tubule architecture and integrity, the concentration of spermatogonia and the expression of DNA replication and repair marker in spermatogonia and Sertoli cells after thawing.

Development of Ovarian Tissue Autograft to Restore Ovarian Function: Protocol for a French Multicenter Cohort Study.

BACKGROUND: Sterility is a major late effect of radiotherapy and chemotherapy treatments. Iatrogenic sterility is often permanent and greatly impacts long-term quality of life. Ovarian tissue cryopreservation (OTC) performed before gonadotoxic treatments with subsequent autograft is a method of fertility preservation available for girls and women. Its application in prepubertal girls is of particular value as it is the only possible approach in this patient group. In addition, it does not require a delay in cancer therapy and no ovarian stimulation is needed. OBJECTIVE: The primary aim of this protocol is to help increase the implementation of ovarian tissue autografting in France. Knowledge is still lacking regarding the efficacy of ovarian transplantation in restoring ovarian function and regarding the safety of this procedure, especially the risk of cancer cell reseeding in certain types of cancer. A secondary aim of this study is to generate data to improve our understanding of these two essential aspects. METHODS: The DATOR (Development of Ovarian Tissue Autograft in Order to Restore Ovarian Function) study is ongoing in 17 university hospitals. The DATOR protocol includes the autograft of ovarian cortex fragments. Candidates are identified from an observational prospective cohort (called the Prospective Cohort of Patients Candidates for Ovarian Tissue Autograft [PERIDATOR]) of patients who have undergone OTC. Enrollment in the study is initiated at the patient's request and must be validated by the center's multidisciplinary team and by the study steering committee. The DATOR study begins with a total medical checkup. Ovarian tissue qualification and residual disease detection, if required, are performed. RESULTS: The study is ongoing. Currently, 38 patients have provided informed consent and have been entered into the DATOR study. Graft has been performed for 34 of these patients. An interim analysis was conducted on the first 25 patients for whom the period of at least 1 year posttransplantation was achieved. Out of these 25 patients, 11 women succeeded in becoming pregnant (pregnancy rate=44% [11/25]; delivery rate=40% [10/25]). Among these, 6 women conceived twice, and 1 pregnancy led to a miscarriage. CONCLUSIONS: Our preliminary analysis appears to be coherent with the accumulating body of evidence indicating the potential utility of ovarian tissue autograft for patients with premature ovarian failure. All these elements justify the pursuit of our study. TRIAL REGISTRATION: ClinicalTrials.gov NCT02846064; https://clinicaltrials.gov/ct2/show/NCT02846064. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/12944.

A protocol to isolate and qualify purified human preantral follicles in cases of acute leukemia, for future clinical applications.

BACKGROUND: Autotransplantation of cryopreserved ovarian cortex can be associated with a risk of cancer cell reseeding. This issue could be eliminated by grafting isolated preantral follicles. Collagenase NB6 is an enzyme produced under good manufacturing practices (GMP) in compliance with requirements for tissue engineering and transplantation in humans and thus can be used to isolate preantral follicles from ovarian tissue in the framework of further clinical applications. Multicolor flow cytometry is an effective tool to evaluate the potential contamination of follicular suspensions by leukemic cells. METHODS: The efficiency of collagenase NB6 was evaluated in comparison to collagenase type IA and Liberase DH, in terms of yield, morphology and viability. A short-term in vitro culture of follicles isolated with collagenase NB6 was conducted for 3 days in a fibrin matrix. A modelization procedure was carried out to detect the presence of leukemic cells in follicular suspensions using multicolor flow cytometry (MFC). RESULTS: No statistical differences were found between collagenase NB6, Liberase DH (p = 0.386) and collagenase type IA (p = 0.171) regarding the number of human preantral follicles isolated. The mean diameter of isolated follicles was significantly lower with collagenase NB6 (p < 0.0001). The survival rate of isolated follicles was 93.4% (n = 272) using collagenase NB6 versus 94.9% (n = 198) with Liberase DH and 92.6% (n = 298) using collagenase type IA. Even after 3 days of in vitro culture in a fibrin scaffold, most of the isolated follicles were still alive after using collagenase NB6 (90.7% of viable follicles; n = 339). The rate of isolated Ki67-positive follicles was 29 ± 9.19% before culture and 45 ± 1.41% after 3 days. In 23 out of 24 follicular suspensions analyzed, the detection of leukemic cells by MFC was negative. The purification had no significant impact on follicle viability. CONCLUSION: The isolation and purification of human preantral follicles were performed following good manufacturing practices for cell therapy. Multicolor flow cytometry was able to confirm that final follicular suspensions were free from leukemic cells. This safe isolation technique using collagenase NB6 can be considered for future clinical applications.

Synthetic Talc and Talc-Like Structures: Preparation, Features and Applications.

This contribution gives a comprehensive review about the progress in preparation methods, properties and applications of the different synthetic talc types: i) crystalline nanotalc synthesized by hydrothermal treatment; ii) amorphous and/or short-range order nanotalc obtained by precipitation, and iii) organic-inorganic hybrid talc-like structures obtained through a sol-gel process or a chemical grafting. Several advantages of nanotalc such as high chemical purity, high surface area, tunable submicronic size, high thermal stability, and hydrophilic character (leading to be the first fluid mineral) are emphasized. Synthetic nanotalc applications are also considered including its use as nanofiller in composite materials, as absorbers of organic compounds, as anticorrosion coatings and as agents for cosmetic applications. Regarding their high industrial application potential, intensive research has been carried out to better understand their behavior and develop processes to produce them. To facilitate further research and development, scientific and technical challenges are discussed in this Review article.

Validation of an automated technique for ovarian cortex dissociation: isolation of viable ovarian cells and their qualification by multicolor flow cytometry.

BACKGROUND: Ovarian tissue cryopreservation is a technique for fertility preservation addressed to prepubertal girls or to patients for whom no ovarian stimulation is possible before initiation of gonadotoxic treatments. Autotransplantation of frozen-thawed ovarian tissue is the only available option for reuse but presents some limitations: ischemic tissue damages post-transplant and reintroduction of malignant cells in cases of cancer. It is therefore essential to qualify ovarian tissue before autograft on a functional and oncological point of view. Here, we aimed to isolate viable cells from human ovarian cortex in order to obtain an ovarian cell suspension analyzable by multicolor flow cytometry. METHODS: Ovarian tissue (fresh or frozen-thawed), from patients with polycystic ovarian syndrome (reference tissue) and from patients who underwent ovarian tissue cryopreservation, was used for dissociation with an automated device. Ovarian tissue-dissociated cells were analyzed by multicolor flow cytometry; the cell dissociation yield and viability were assessed. Two automated dissociation protocols (named laboratory and commercial protocols) were compared. RESULTS: The effectiveness of the dissociation was not significantly different between reference ovarian tissue (1.58 × 106 ± 0.94 × 106 viable ovarian cells per 100 mg of ovarian cortex, n = 60) and tissue from ovarian tissue cryopreservation (1.70 × 106 ± 1.35 × 106 viable ovarian cells, n = 18). However, the viability was slightly different for fresh ovarian cortex compared to frozen-thawed ovarian cortex whether we used reference tissue (p = 0.022) or tissue from ovarian cryopreservation (p = 0.018). Comparing laboratory and commercial protocols, it appeared that cell yield was similar but cell viability was significantly improved when using the commercial protocol (81.3% ± 12.3% vs 23.9% ± 12.5%). CONCLUSION: Both dissociation protocols allow us to isolate more than one million viable cells per 100 mg of ovarian cortex, but the viability is higher when using the commercial dissociation kit. Ovarian cortex dissociation is a promising tool for human ovarian cell qualification and for ovarian residual disease detection by multicolor flow cytometry.

A new method for evaluating the risk of transferring leukemic cells with transplanted cryopreserved ovarian tissue.

PURPOSE: This study aimed to develop a method to detect ovarian residual disease by multicolor flow cytometry in acute leukemia patients. METHODS: We designed an experimental model consisting in adding acute leukemia cells to a cell suspension obtained from healthy ovarian cortex. Leukemic cell detection within the ovarian cell suspension required the development of a specific myeloid antibody panel different from that commonly used for minimal residual disease (MRD) monitoring in bone marrow. The method was then used to detect ovarian residual disease in 11 acute leukemia patients. RESULTS: Multicolor flow cytometry is able to evaluate the presence of viable leukemic cells in the ovarian cortex with good specificity and robust sensitivity of 10-4. We observed a good correlation between multicolor flow cytometry and quantitative polymerase chain reaction results. Ovarian residual disease detection by multicolor flow cytometry was positive in 3 out of 11 acute leukemia patients. CONCLUSION: Multicolor flow cytometry can potentially be applied to ovarian tissue from all acute leukemia patients and is essential to evaluate the risk of cancer re-seeding before autograft of ovarian tissue in case of acute leukemia.

Minimal residual disease detection of leukemic cells in ovarian cortex by eight-color flow cytometry.

STUDY QUESTION: How can leukemic cells be detected in cryopreserved ovarian tissue? SUMMARY ANSWER: Multicolor flow cytometry (FCM) is useful to evaluate the presence of viable leukemic cells in the ovarian cortex with a high specificity and a robust sensitivity. WHAT IS KNOWN ALREADY: Storing ovarian tissue is an option to preserve fertility before gonadotoxic radiotherapy or chemotherapy treatments. However, transplantation of cryopreserved ovarian cortex to women cured of leukemia is currently not possible due to the risk of cancer re-seeding. STUDY DESIGN, SIZE, DURATION: We developed an automated ovarian cortex dissociation technique and we used eight-color FCM to identify leukemic cells with a series of dilutions added to ovarian single cell suspensions obtained from healthy cortex. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Healthy ovarian cortex originated from women between 23 and 39 years of age undergoing laparoscopic ovarian drilling for polycystic ovary syndrome. Blood or bone marrow cells were collected in acute lymphoblastic leukemia (ALL) patients at diagnosis. MAIN RESULTS AND THE ROLE OF CHANCE: The tissue dissociation technique yield was 1.83 ± 1.49 × 10(6) viable nucleated cells per 100 mg of ovarian cortex. No cell exhibiting a leukemic phenotype was present in the normal ovarian cortex. Added leukemic cells were detected using their leukemia-associated phenotype up to a dilution of 10(-4). When specific gene rearrangements were present, they were detected by real-time quantitative PCR at the same dilution. The ovarian cortex from two leukemia patients was then used, respectively, as positive and negative controls. LIMITATIONS, REASONS FOR CAUTION: Making available minimal residual disease (MRD) detection techniques (multicolor FCM, PCR and xenograft), that can be used either alone or together, is essential to add a fail-safe oncological dimension to pre-autograft monitoring. WIDER IMPLICATIONS OF THE FINDINGS: This approach can be performed on fresh ovarian tissue during cryopreservation or on frozen/thawed tissue before reimplantation and it is currently the only available technique in cases of ALL where no molecular markers are identified. This new perspective should lead to studies on ovarian tissue from leukemia patients, for whom the presence of MRD should be established before autograft. STUDY FUNDINGS/COMPETING INTEREST(S): The study was supported by the BioMedicine Agency, the Committee of the League against Cancer, the Besançon University Hospital, DGOS/INSERM/INCa and the regional Council of Franche-Comté. There were no conflicts of interest to declare.

Child with Beckwith-Wiedemann syndrome born after assisted reproductive techniques to an human immunodeficiency virus serodiscordant couple.

OBJECTIVE: To report a child with Beckwith-Wiedemann syndrome (BWS) born after assisted reproductive technology (ART) to an HIV serodiscordant couple. DESIGN: Case report. SETTING: Academic medical center. PATIENT(S): A child with BWS born after ART to an HIV serodiscordant couple. INTERVENTION(S): Assisted reproductive techniques. MAIN OUTCOME MEASURE(S): ART and HIV. RESULT(S): Since 2003, it has been suggested that there is an association between ART and BWS, which is a congenital overgrowth syndrome characterized by macrosomia, macroglossia, visceromegaly, umbilical and abdominal wall abnormalities, and an increased risk of developing embryonal tumors in childhood. It is a multigenic disorder resulting from genetic or epigenetic alterations of genes on chromosome 11p15. Methylation errors account for 50%-60% of sporadic cases and almost 100% of cases born after ART. We report the birth of a child diagnosed with BWS arising from an ART cycle to an HIV serodiscordant couple with no history of infertility. This case cannot constitute conclusive evidence but it raises various questions. CONCLUSION(S): Assisted reproductive technology seems to be in the causal pathway but a male/female factor or an iatrogenic factor is also possible.

[Chemotherapy and women fertility preservation]. / Chimiothérapie et préservation de la fertilité féminine.

Agressive chemotherapy can lead to premature ovarian failure and loss of fertility in women and children. Embryo cryopreservation is an established clinical procedure of fertility preservation but with several limitations. Others options are available. Cryopreservation ovarian cortex tissu have to be suggested in case of high gonadotoxic treatment. It doesn't require puberty and delay in initiation of chemotherapy. The first birth in France after orthotopic graft of ovarian tissu thawed have been recently described with a promising process. Oocyte cryopreservation is available for women without partner but the experience is limited. Gonadotrophin-releasing hormone (GnRH) agonist therapy as ovarian protectants seem interesting. Follicular growth and maturation in vitro are still experimental.

Live birth after ovarian tissue autograft in a patient with sickle cell disease treated by allogeneic bone marrow transplantation.

OBJECTIVE: To report the first case of restoration of ovarian activity and live birth after cryopreserved ovarian tissue autograft in a patient without cancer treated by allogeneic bone marrow transplantation. DESIGN: Case report. SETTING: University hospital. PATIENT(S): One woman with homozygous sickle cell anemia. INTERVENTION(S): An orthotopic autotransplantation of ovarian cortical strips was performed after freeze-thawing. MAIN OUTCOME MEASURE(S): Cryopreservation of ovarian tissue, bone marrow transplantation, ovarian autograft, and restoration of ovarian function. RESULT(S): In autumn 2005, biopsy samples of ovarian tissue were cryopreserved before chemotherapy followed by bone marrow transplantation. In spring 2008, because the patient had been menopausal for 2.5 years as a result of the conditioning therapy, an orthotopic autotransplantation of thawed ovarian cortex was performed. The patient conceived spontaneously in a natural cycle in autumn 2008, and delivered a healthy female child in June 2009. CONCLUSION(S): Cryopreservation of ovarian tissue with subsequent autotransplantation is an emerging procedure for preserving the fertility of young patients with a high risk of premature ovarian failure (POF) resulting from gonadotoxic treatment. This case opens up new perspectives in cases of nonmalignant diseases.

GR24, a synthetic analog of strigolactones, stimulates the mitosis and growth of the arbuscular mycorrhizal fungus Gigaspora rosea by boosting its energy metabolism.

Arbuscular mycorrhizal (AM) fungi are obligate biotrophs that participate in a highly beneficial root symbiosis with 80% of land plants. Strigolactones are trace molecules in plant root exudates that are perceived by AM fungi at subnanomolar concentrations. Within just a few hours, they were shown to stimulate fungal mitochondria, spore germination, and branching of germinating hyphae. In this study we show that treatment of Gigaspora rosea with a strigolactone analog (GR24) causes a rapid increase in the NADH concentration, the NADH dehydrogenase activity, and the ATP content of the fungal cell. This fully and rapidly (within minutes) activated oxidative metabolism does not require new gene expression. Up-regulation of the genes involved in mitochondrial metabolism and hyphal growth, and stimulation of the fungal mitotic activity, take place several days after this initial boost to the cellular energy of the fungus. Such a rapid and powerful action of GR24 on G. rosea cells suggests that strigolactones are important plant signals involved in switching AM fungi toward full germination and a presymbiotic state.

Use of trypan blue staining to assess the quality of ovarian cryopreservation.

OBJECTIVE: To check the efficiency of the cryopreservation procedure by using trypan blue staining of ovarian test fragments to assess the quality of frozen-thawed ovarian tissue. DESIGN: Prospective study. SETTING: University hospital. PATIENT(S): Patients with polycystic ovary syndrome undergoing laparoscopic ovarian drilling. INTERVENTION(S): Ovarian cortical biopsies obtained from polycystic ovary syndrome patients were frozen using a slow freezing-rapid thawing protocol. MAIN OUTCOME MEASURE(S): Primordial and primary follicle viability was assessed with trypan blue staining. Unstained isolated follicles were studied by transmission electron microscopy. Histologic and immunohistochemical analysis of apoptosis was performed on tissue sections. RESULT(S): The percentage of unstained follicles considered live was lower (P=.015) after freezing/thawing (71.9%) than before cryopreservation (87.3%). Transmission electron microscopy on follicles considered live confirmed the lack of ultrastructural damage. After freezing/thawing, tissue morphology was preserved, but immunohistochemical analysis shows a significant increase in the apoptosis process. CONCLUSION(S): Ovarian cortical test fragments combined with trypan blue staining on enzymatically isolated follicles is a useful and quick method of assessing the initial quality and viability of follicles in cryopreserved ovarian tissue. This type of test should be used routinely as quality control in ovarian cryopreservation procedures.