scholarly journals 115DIMETHYLSULPHOXIDE AND 1,2-PROPANEDIOL DIFFERENTIALLY CRYOPROTECT BOVINE AND PORCINE PRIMORDIAL, PRIMARY AND SECONDARY FOLLICLES

2004 ◽  
Vol 16 (2) ◽  
pp. 180
Author(s):  
E. Papasso Brambilla ◽  
A. Paffoni ◽  
T.A.L. Brevini ◽  
M. De Eguileor ◽  
G. Ragni ◽  
...  
Keyword(s):  
Structural Integrity ◽  
Developmental Stages ◽  
Ovarian Tissue ◽  
Normal Structure ◽  
Ovarian Tissue Cryopreservation ◽  
Protective Effects ◽  
Chi Square ◽  
Primordial Follicles ◽  
Chi Square Test ◽  
Tissue Cryopreservation

Ovarian tissue cryopreservation is of interest for many areas of assisted reproduction. Normal structure and intra- as well as intercellular organization have to be maintained in order to preserve follicle viability. In the present work we studied the effects of two largely used cryoprotectants, namely, dimethylsulphoxide (DMSO) and 1,2-propanediol (PROH), on follicle morphology. Experiments were carried out on bovine and porcine ovaries and the two cryoprotectants were assessed for their ability to preserve structural integrity of primordial, primary and secondary follicles, in order to investigate the protective effects of these molecules on specific developmental stages. Fragments from each ovary were divided in three groups: a) immediately fixed (control); b) cryopreserved in 1.5M DMSO; c) cryopreserved in 1.5M PROH. To allow equilibration with the cryoprotectant, samples were held for 30min at 4°C in order to minimize toxic effects. Cryopreservation was carried out in a controlled rate freezer (Planer Planerple, Sunbury, Middlesex, UK), with the following protocol: precooling (4°C); cooling at −2°C/min to 9°C; seeding at 9°C and standby for 10 minutes; cooling at −0.3°C/min to 40°C and 10°C/min to 140°C; plunging into liquid nitrogen. Samples were rapidly thawed. Dilution of cryoprotectants was carried out in 3 steps of ten minutes each at 4°C (1M; 0.5M; 0M). Samples were then fixed, paraffin embedded, serially sectioned and evaluated with a Nikon TE200 inverted microscope. Follicles of each developemental stage were scored in three categories using the criteria previously described by Paynter et al. (1999, Cryobiology 38, 301–309) and presented in the table below as I=Intact; SA=Small Abnormalities; GA=Great Abnormalities. Statistical differences were assessed by the chi-square test (P<0.05). The results obtained showed that both cryoprotectants are effective for the preservation of secondary follicles. The use of PROH was unable to protect primary and primordial follicles in both species. Conversely, DMSO showed a satisfying cryoprotecting effect for pig primary and bovine primordial follicles but had a poor protecting capability for pig primordial and bovine primary follicles. Altogether the present results suggest that the choice of the cryoprotectant needs to be carefully targeted in relation to the follicular stage and the species of interest. This work was funded by Industria Farmaceutica Serono SpA. Table 1

P-461 Pre-selected for an award: A 16-year bicentric retrospective analysis of ovarian tissue cryopreservation (OTC) in paediatric patients: indications, results and outcome

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
M Grellet-Grün ◽  
B Delepine ◽  
P Le Van Quyen ◽  
A Durlach ◽  
C Greze ◽  
...  
Keyword(s):  
Survival Rate ◽  
Ovarian Tissue ◽  
Adult Population ◽  
Ovarian Tissue Cryopreservation ◽  
Primordial Follicles ◽  
Paediatric Patients ◽  
The Mean ◽  
Tissue Cryopreservation

Abstract Study question What is the outcome of ovarian tissue cryopreservation (OTC) in paediatric patients from the beginning of its setting in two different French centres? Summary answer In our cohort of 75 paediatric patients who underwent OTC, the mean age, malignancy rate and survival rate were 9.7 years, 70.7% and 77.3% respectively. What is known already Cancer treatments of last decades improve the survival rate of children and adolescents; however chemo- and radiotherapy result in gonadal damage leading to acute ovarian failure and sterility. The preservation of fertility is now an integral part of care of children requiring gonadotoxic treatments. Currently OTC represents the only possibility of preserving the potential fertility in prepubertal girls. OTC is an effective fertility preservation option which allows long-term storage of primordial follicles, subsequent transplantation restores endocrine function and fertility. The efficacy of these techniques is well-demonstrated within adult population but the data are poor for paediatric patients. Study design, size, duration This is a retrospective study of OTC practice of two French centres from January 2004 to May 2020. Participants/materials, setting, methods A total of 75 patients from paediatrics units underwent cryopreservation of ovarian tissue before gonadotoxic therapy for malignant or benign diseases. The ovarian cortex was cut into fragments and the number of follicles per square millimeter was evaluated histologically. The long-term follow-up includes survival rate, hormonal and fertility status. Main results and the role of chance The mean age at OTC of 75 patients was 9.7 years [0.2 – 20], 32% were postpubertal. 53 had malignant disease and 22 had non-malignant disease. The most frequent diagnoses in this cohort included acute leukemia, hemoglobinopathies and neuroblastoma. Indication for OTC was stem cell transplantation for 78.7% (n = 59) girls. A third of each ovary was collected for 62,7% (n = 47) patients, a whole ovary for 33,3% (n = 25) patients and a third of one ovary alone for 4,0% (n = 3) patients. An average of 17 fragments [5-35] per patient was cryoconserved. A correlation was found between age and the number of fragments (p < 0.001). More fragments were obtained from partial bilateral harvesting than from whole ovary harvesting (p < 0.05). Histological analysis of ovarian tissue showed a median of 6.0 primordial follicles/mm2 [0.0–106.5] and no malignant cells were identified. A negative correlation was found between age and follicular density (p < 0.001). Median post-harvest follow-up was 92 months [1–188] 17 girls had died, 12 were still treated for their pathology and 46 were in complete remission. Of all patients, 29 have been subject to hormonal status evaluation and 26 were diagnosed with premature ovarian insufficiency (p < 0.001). One patient had undergone thawed ovarian tissue transplantation. Limitations, reasons for caution This study is a retrospective analysis. The cohort was not compared with a control group who did not undergo OTC or with an adult population. Furthermore, many of these girls are still young and do not intend to use the transplantation of thawed ovarian tissue yet. Wider implications of the findings OTC should be proposed to all girls with high risk of developing premature ovarian insufficiency following gonadotoxic therapies in order to give them the possibility of fertility and endocrine restoration. Trial registration number Not applicable

scholarly journals The Effectiveness of Anti-Apoptotic Agents to Preserve Primordial Follicles and Prevent Tissue Damage during Ovarian Tissue Cryopreservation and Xenotransplantation

2021 ◽  
Vol 22 (5) ◽  
pp. 2534
Author(s):  
Sanghoon Lee ◽  
Hyun-Woong Cho ◽  
Boram Kim ◽  
Jae Kwan Lee ◽  
Tak Kim
Keyword(s):  
Tissue Damage ◽  
Ovarian Function ◽  
Ovarian Tissue ◽  
Slow Freezing ◽  
Ovarian Tissue Cryopreservation ◽  
Second Step ◽  
Control Group ◽  
Dna Breaks ◽  
Primordial Follicles ◽  
Tissue Cryopreservation

The purpose of this study is to investigate the effectiveness of sphingosine-1-phosphate (S1P) and Z-VAD-FMK (Z-VAD) as anti-apoptotic agents to preserve ovarian function and prevent tissue damage during ovarian tissue cryopreservation and transplantation. This study consisted of two steps, in vitro and in vivo. In the first step, human ovarian tissues were cryopreserved using slow-freezing media alone, S1P, or Z-VAD (control, S1P, Z-VAD group); based on the outcomes in these groups, Z-VAD was selected for subsequent xenotransplantation. In the second step, human frozen/thawed ovarian tissues were grafted into fifty mice divided into three groups: slow-freezing/thawing and transplantation without an anti-apoptotic agent (Trans-control) and xenotransplantation with or without Z-VAD injection (Trans-Z-VAD-positive and Trams-Z-VAD-negative groups, respectively). In the first step, the Z-VAD group had a significantly higher primordial follicular count than the S1P (p = 0.005) and control groups (p = 0.04). Transplanted ovarian tissues were obtained 4 weeks after transplantation (second step). Angiogenesis was significantly increased in the Z-VAD-negative (p = 0.03) and -positive (p = 0.04) groups compared to the control group. This study demonstrated that slow-freezing and transplantation with Z-VAD is an effective method for preserving primordial follicle counts, decreasing double-strand DNA breaks, and increasing angiogenesis in a mouse model. Further molecular and clinical studies are needed to confirm these results.

P–461 A 16-year bicentric retrospective analysis of ovarian tissue cryopreservation (OTC) in paediatric patients: indications, results and outcome

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
M Grellet-Grün ◽  
B Delepine ◽  
P L Va. Quyen ◽  
A Durlach ◽  
C Greze ◽  
...  
Keyword(s):  
Survival Rate ◽  
Ovarian Tissue ◽  
Adult Population ◽  
Ovarian Tissue Cryopreservation ◽  
Primordial Follicles ◽  
Paediatric Patients ◽  
The Mean ◽  
Tissue Cryopreservation

Abstract Study question What is the outcome of ovarian tissue cryopreservation (OTC) in paediatric patients from the beginning of its setting in two different French centres? Summary answer In our cohort of 75 paediatric patients who underwent OTC, the mean age, malignancy rate and survival rate were 9.7 years, 70.7% and 77.3% respectively. What is known already Cancer treatments of last decades improve the survival rate of children and adolescents; however chemo- and radiotherapy result in gonadal damage leading to acute ovarian failure and sterility. The preservation of fertility is now an integral part of care of children requiring gonadotoxic treatments. Currently OTC represents the only possibility of preserving the potential fertility in prepubertal girls. OTC is an effective fertility preservation option which allows long-term storage of primordial follicles, subsequent transplantation restores endocrine function and fertility. The efficacy of these techniques is well-demonstrated within adult population but the data are poor for paediatric patients. Study design, size, duration This is a retrospective study of OTC practice of two French centres from January 2004 to May 2020. Participants/materials, setting, methods A total of 75 patients from paediatrics units underwent cryopreservation of ovarian tissue before gonadotoxic therapy for malignant or benign diseases. The ovarian cortex was cut into fragments and the number of follicles per square millimeter was evaluated histologically. The long-term follow-up includes survival rate, hormonal and fertility status. Main results and the role of chance The mean age at OTC of 75 patients was 9.7 years [0.2 – 20], 32% were postpubertal. 53 had malignant disease and 22 had non-malignant disease. The most frequent diagnoses in this cohort included acute leukemia, hemoglobinopathies and neuroblastoma. Indication for OTC was stem cell transplantation for 78.7% (n = 59) girls. A third of each ovary was collected for 62,7% (n = 47) patients, a whole ovary for 33,3% (n = 25) patients and a third of one ovary alone for 4,0% (n = 3) patients. An average of 17 fragments [5–35] per patient was cryoconserved. A correlation was found between age and the number of fragments (p < 0.001). More fragments were obtained from partial bilateral harvesting than from whole ovary harvesting (p < 0.05). Histological analysis of ovarian tissue showed a median of 6.0 primordial follicles/mm2 [0.0–106.5] and no malignant cells were identified. A negative correlation was found between age and follicular density (p < 0.001). Median post-harvest follow-up was 92 months [1–188]: 17 girls had died, 12 were still treated for their pathology and 46 were in complete remission. Of all patients, 29 have been subject to hormonal status evaluation and 26 were diagnosed with premature ovarian insufficiency (p < 0.001). One patient had undergone thawed ovarian tissue transplantation. Limitations, reasons for caution This study is a retrospective analysis. The cohort was not compared with a control group who did not undergo OTC or with an adult population. Furthermore, many of these girls are still young and do not intend to use the transplantation of thawed ovarian tissue yet. Wider implications of the findings: OTC should be proposed to all girls with high risk of developing premature ovarian insufficiency following gonadotoxic therapies in order to give them the possibility of fertility and endocrine restoration. Trial registration number Not applicable

325 VIABILITY AND GROWTH OF CATTLE PREANTRAL FOLLICLES AFTER IN VITRO CULTURE OF OVARIAN FRAGMENTS IN α-TOCOPHEROL

2010 ◽  
Vol 22 (1) ◽  
pp. 318 ◽  
Author(s):  
L. A. Lisboa ◽  
E. R. Andrade ◽  
M. F. Hertel ◽  
F. A. Melo-Sterza ◽  
K. Moreno ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Ovarian Tissue ◽  
Nuclear Antigen ◽  
Cortical Tissue ◽  
Chi Square ◽  
Primordial Follicles ◽  
Preantral Follicles ◽  
Chi Square Test ◽  
Proliferating Cell

The development of culture systems to support the initiation of growth of primordial follicles is important to the study of the factors that control the earliest stages of folliculogenesis. The aims of the present study were to investigate the effects of α-tocopherol on survival, activation, and growth of cattle preantral follicles using histological and immunohistochemistry proliferating cell nuclear antigen (PCNA) studies. The ovarian cortex was divided into small fragments; one fragment was immediately fixed in Bouin (control). The other fragments were cultured for 2, 4, 6, or 8 d in culture plates with Minimum Essential Medium supplemented with insulin-transferrin-selenium (ITS), pyruvate, glutamine, hypoxanthine, BSA, and antibiotics (MEM+); and MEM+ plus α-tocopherol (5, 25, 50, 100, or 200 ng mL-1). Preantral follicles were classified according to their developmental stage (primordial, intermediate, primary, or secondary) and on the basis of morphological features (normal or degenerated). Pair-wise comparisons were done using Tukey’s procedure. Chi-square test was used to compare the percentage of follicles with PCNA-positive granulosa cells. All analyses were done with the SAS software (SAS Institute, Cary, NC, USA), and P < 0.05 was considered significant. The results showed that, compared with non-cultured cortical tissue (Day 0), the culture of ovarian tissue significantly reduced (P < 0.05) the percentage of normal follicles in all media tested, except for tissue cultured in the presence of 200 ng mL-1 of a-tocopherol. Furthermore, in all media tested, the percentage of primordial follicles was significantly reduced (P < 0.05), with a concomitant increase in the percentage of developing follicles. The highest percentage of secondary follicles was observed after 6 days of culture in MEM plus 200 ng mL-1 of a-tocopherol. The PCNA analysis confirmed the viability of follicles cultured with 200 ng mL-1 of a-tocopherol after 6 d. After 8 days of in vitro culture, we observed severe follicular degeneration in all media tested, suggesting that other supplements are recommended for longer periods of culture. In conclusion, the results of the present study indicate that 200 ng mL-1 of a-tocopherol maintains the survival of cattle preantral follicles and promotes activation of primordial follicles after 6 days of in vitro culture. Financial support: L. A. Lisboa is a recipient of CAPES support; E. R. Andrade and A. A. Alfieri are recipients of PRODOC/CAPES fellowships.

scholarly journals Ovarian cryopreservation for fertility preservation: indications and outcomes

Reproduction ◽  
2008 ◽  
Vol 136 (6) ◽  
pp. 681-689 ◽  
Author(s):  
R A Anderson ◽  
W H B Wallace ◽  
D T Baird
Keyword(s):  
Fertility Preservation ◽  
Ovarian Tissue ◽  
Invasive Procedure ◽  
Potential Method ◽  
Ovarian Tissue Cryopreservation ◽  
Hormonal Stimulation ◽  
Primordial Follicles ◽  
Subsequent Fertility ◽  
Female Fertility Preservation ◽  
Tissue Cryopreservation

Female fertility preservation provides significantly different challenges to that for the male, with the only established method being cryopreservation of embryos thus necessitating the involvement of a male. Other, experimental, options include oocyte or ovarian tissue cryopreservation. The latter has been regarded as a potential method for more than a decade, but has resulted in the birth of only five babies. It is not possible to be certain how many women have had ovarian tissue cryopreserved. Oocyte cryopreservation also remains experimental, but ∼100-fold more babies have been born through this technique over the last two decades. Ovarian tissue cryopreservation has the potential advantages of preservation of a large number of oocytes within primordial follicles, it does not require hormonal stimulation when time is short and indeed may be appropriate for the pre-pubertal. Disadvantages include the need for an invasive procedure, and the uncertain risk of ovarian contamination in haematological and other malignancies. We here review this approach in the context of our own experience of 36 women, highlighting issues of patient selection especially in the young, and uncertainties over the effects of cancer treatments on subsequent fertility. Of these 36 women, 11 have died but 5 have had spontaneous pregnancies. So far, none have requested reimplantation of their stored ovarian tissue. Ovarian cryopreservation appears to be a potentially valuable method for fertility preservation, but the indications and approaches best used remain unclear.

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