Mouse ovarian tissue vitrification on copper electron microscope grids versus slow freezing: a comparative ultrastructural study

2015 ◽  
Vol 27 (7) ◽  
pp. 1020 ◽  
Author(s):  
Ferda Topal-Celikkan ◽  
Sinan Ozkavukcu ◽  
Deniz Balci ◽  
Sibel Serin-Kilicoglu ◽  
Esra Atabenli-Erdemli
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Cancer Therapy ◽  
Ovarian Tissue ◽  
Light And Electron Microscopy ◽  
Slow Freezing ◽  
Ovarian Tissue Cryopreservation ◽  
Control Group ◽  
Primordial Follicles ◽  
User Friendly

There are many reasons, including cancer therapy, for premature ovarian failure and infertility. Oocyte, embryo and ovarian cryopreservation are current options for fertility preservation. Ovarian tissue cryopreservation is essential in patients whose cancer therapy cannot be delayed, including prepubertal girls, and is mostly performed using slow freezing. In the present study, mouse ovarian tissues were vitrified on copper electron microscope grids (n = 18) or conventionally slow frozen (n = 18). Post-thaw tissues were examined histologically using light and electron microscopy and compared with the control group. According to light microscopy observations, antral follicles were found to be better preserved with the slow freezing technique rather than vitrification. Electron microscopy revealed swollen mitochondria in the oocyte cytoplasm, condensations in the zona pellucida, breakages in the junctions of granulosa cells and vacuolisation in the extracellular space in pathologic follicles, which were relatively more frequent, in the vitrification group after thawing. These results indicate that ovarian slow freezing is preferable than vitrification on copper electron microscope grids, especially for larger follicles. Conversely, vitrification of ovarian pieces using cooper grids is user-friendly and provided good protection for primordial follicles and stromal cells. There is a need for further studies into advanced tissue vitrification techniques and carriers.

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.

scholarly journals Female fertility preservation: past, present and future

Reproduction ◽  
2018 ◽  
Vol 156 (1) ◽  
pp. F11-F27 ◽  
Author(s):  
Benjamin Fisch ◽  
Ronit Abir
Keyword(s):  
Cancer Therapy ◽  
Fertility Preservation ◽  
Ovarian Tissue ◽  
Young Patients ◽  
Reproductive Technologies ◽  
Ovarian Tissue Cryopreservation ◽  
Embryo Cryopreservation ◽  
Primordial Follicles ◽  
Anti Cancer

Anti-cancer therapy, particularly chemotherapy, damages ovarian follicles and promotes ovarian failure. The only pharmacological means for protecting the ovaries from chemotherapy-induced injury is gonadotrophin-releasing hormone agonist, but its efficiency remains controversial; ovarian transposition is used to shield the ovary from radiation when indicated. Until the late 1990s, the only option for fertility preservation and restoration in women with cancer was embryo cryopreservation. The development of other assisted reproductive technologies such as mature oocyte cryopreservation andin vitromaturation of oocytes has contributed to fertility preservation. Treatment regimens to obtain mature oocytes/embryos have been modified to overcome various limitations of conventional ovarian stimulation protocols. In the last decades, several centres have begun cryopreserving ovarian samples containing primordial follicles from young patients before anti-cancer therapy. The first live birth following implantation of cryopreserved-thawed ovarian tissue was reported in 2004; since then, the number has risen to more than 130. Nowadays, ovarian tissue cryopreservation can be combined within vitromaturation and vitrification of oocytes. The use of cryopreserved oocytes eliminates the risk posed by ovarian implantation of reseeding the cancer. Novel methods for enhancing follicular survival after implantation are presently being studied. In addition, researchers are currently investigating agents for ovarian protection. It is expected that the risk of reimplantation of malignant cells with ovarian grafts will be overcome with the putative development of an artificial ovary and an efficient follicle class- and species-dependentin vitrosystem for culturing primordial follicles.

scholarly journals Establishment of the ovarian tissue cryopreservation protocol on bovine model

2016 ◽  
Vol 19 (2) ◽  
pp. 24-32
Author(s):  
Dung Thi Phuong Nguyen ◽  
Lan Thi Thu Nguyen ◽  
Quang Nhat Nguyen ◽  
Tuong Manh Ho ◽  
Loc Minh Tai Nguyen ◽  
...  
Keyword(s):  
Ovarian Function ◽  
Ovarian Tissue ◽  
Neutral Red ◽  
Slow Freezing ◽  
Ovarian Tissue Cryopreservation ◽  
Control Group ◽  
Ovarian Cortex ◽  
Group 2 ◽  
Tissue Cryopreservation ◽  
Cryopreservation Protocol

Ovarian tissue cryopreservation is a suitable method for fertility preservation on women receiving treatment that may threaten the ovarian function and subsequent fertility. The whole ovarian or a part of ovarian can be cryopreserved for future use. This study was aimed to establish ovarian tissue cryopreservation protocols on bovine model for human application in Vietnam. In this method, bovine ovarians were collected from a slaughterhouse and kept at 4 oC up to a maximum of 12 hours before doing experiments. The ovarian cortex was cut into pieces of 10x10x1 mm. These pieces were randomly divided into 3 groups: (1) fresh species (control group), (2) species were freezed by slow-freezing method and (3) pieces were freezed by vitrification. After thawing, ovarian cortex pieces were treated with Collagenase Ia for the follicle isolation. The isolated follicles then were stained with Neutral Red. The rate of viable follicles was used as the outcome measure to assess the efficiency of the cryopreservation protocol. In results, the rates of viable follicles were 72.46 ± 6.11 % and 59.09 ± 7.08 % after slow-freezing and vitrification comparing to the control group, respectively. This was the first study which successfully established a protocol of ovarian tissue cryopreservation on bovine model in Vietnam. The protocol should be improved for further application to human treatment in the near future.

126 EFFECTIVE METHOD FOR IN VITRO CULTURE OF CRYOPRESERVED OVINE OVARIAN TISSUE

2016 ◽  
Vol 28 (2) ◽  
pp. 193
Author(s):  
A. Seisenbayeva ◽  
Y. Toishibekov ◽  
U. Iglmanov ◽  
B. Valiyeva ◽  
B. Katubayeva
Keyword(s):  
In Vitro Culture ◽  
Room Temperature ◽  
Culture Media ◽  
Ovarian Tissue ◽  
Farm Animals ◽  
Penicillin G ◽  
Ovarian Tissue Cryopreservation ◽  
Control Group ◽  
Primordial Follicles

Today, ovarian tissue cryopreservation is used for preserving the reproductive function of women, as well as the genetic material of rare and endangered species or domestic animals breeds. In the last 20 years genetic diversity of farm animals breeds suffered considerable losses in Kazakhstan; therefore, genetic preservation of valuable local breeds is desirable. The aim of this study was to compare the effectiveness of different in vitro culture media on morphology of ovine ovarian tissue cryopreserved by a slow-freezing protocol with 1.5 M dimethyl sulfoxide (DMSO). Ovaries were collected from indigenous Chuyi breed and immediately transported to the laboratory at 30°C within 1 h. Ovaries were rinsed several times in PBS supplemented with antibiotics (75 mg L–1 of penicillin-G, 50 mg L–1 of streptomycin sulfate). In Hepes-buffered medium 199, halved and the medulla removed with curved iris. Using a scalpel, the cortex was cut into 5- × 3- × 1-mm strips. Ovarian strips were equilibrated sequentially in freezing medium containing 0.25, 0.75, and 1.5 M DMSO with 0.5 M sucrose (5 min each). Then, ovarian strips were frozen in plastic straws using a programmable freezer Planer Kryo-360 3,3 (Planer, UK) and cooled as follows: stabilised at 20°C for 5 min, cooled from 20°C to –70°C at 5°C min–1, seeded to the temperature –7°C, cooled again to –30°C at 0.3°C min–1, cooled to –150°C at 35°C min–1, and finally plunged into liquid nitrogen and stored for 10 days. The straws were thawed at room temperature for 1 min, and then immersed in a water bath at 37°C for 2 min, warmed at room temperature with Dulbecco’s PBS (DPBS), supplemented with 10% FCS and 0.75 M sucrose (15 min), then DPBS + 10% FCS (30 min), and finally placed in the culture media for 10 min. Fresh and frozen tissue pieces were randomly distributed into 12 groups for further culture: 1) TCM 199 + 10% FBS; 2) TCM 199 + 10% native ovine serum (NOS); 3) TCM-Hepes + 10% FBS; 4) TCM-Hepes + 10% NOS; 5) DMEM + 10% FBS; 6) DMEM + 10% NOS; with and without 7.5 mg mL–1 of FSH. After 7 days of culture, the effects of different culture media on ovarian tissue morphology was evaluated by light microscopy after hematoxylin and eosin staining of tissue sections. The best result was observed when frozen ovarian tissue was cultured in the presence of FSH. The best result was observed in group 3 and 4 with FSH. The percentages of normal primordial, primary, and preantral follicles were: 1) TCM 199 + 10% FBS + FSH = 53.5 ± 3.1, 39.7 ± 3.8a, 28.5 ± 3.2; 2) TCM 199 + 10% NOS + FSH = 49.4 ± 2.3a, 36.7 ± 3.3a, 25.3 ± 4.1; 3) TCM-Hepes + 10% FBS + FSH = 66.3 ± 2.5, 45.7 ± 3.9, 35.1 ± 3.8; 4) TCM-Hepes + 10% NOS + FSH = 86.5 ± 3.8b, 75.4 ± 4.2b, 45.7 ± 3.5; 5) DMEM + 10% FBS + FSH = 42.1 ± 3.5a, 33.7 ± 2.9a, 21.3 ± 4.9, 20.7 ± 3.9; 6) DMEM + 10% NOS + FSH = 41.3 ± 3.9a, 32.9 ± 2.5a; control group = 98.2 ± 1.1, 93.7 ± 1.7, 90.3 ± 1.9 (ab, P < 0.01). The majority of follicles in groups without FSH were degenerated. In group 4) TCM-Hepes + 10% NOS without FSH, a damaged structure of primordial follicles was observed.

scholarly journals An Electron Microscope Study of Basophile Substances of Frozen-Dried Rat Liver

1958 ◽  
Vol 4 (3) ◽  
pp. 291-300 ◽  
Author(s):  
Henry Finck
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Rat Liver ◽  
Microscope Study ◽  
Electron Microscope Study ◽  
Freeze Drying ◽  
Light And Electron Microscopy ◽  
Enzyme Treatment ◽  
Dense Granules ◽  
Homogeneous Matrix

Small pieces of liver from rats subjected to different dietary regimes were fixed by freeze-drying, and postfixed by in vacuo heating and denaturation with alcohol. Specimens were digested with ribo- or deoxyribonuclease, and stained with gallocyanin-chromalum, azure II, the Feulgen procedure or alcoholic platinic tetrabromide. Some specimens were reserved as controls of the effects of enzyme treatment. Stained and unstained specimens were embedded in methacrylate and examined by light and electron microscopy. Basophilic and Feulgen-positive substances, after contact with watery reagents, were found by electron microscopy to exist as small dense granules embedded in a less dense homogeneous matrix, forming the walls of submicroscopic vacuoles. These granules were absent after digestion with nucleodepolymerases. In specimens (unstained, or stained with platinic tetrabromide) which had not passed through water, the dense (basophile) substances in nuclei and cytoplasm were found to exist, not as granules, but as ill defined submicroscopic concentrates which blended imperceptibly into the homogeneous matrix of the vacuolar walls. Objections to the use of stains for improving contrast conditions in electron microscopy of tissues are discussed, and it is concluded that the reagents do not necessarily produce the observed increases in contrast by selectively stabilizing certain structures. The concept of microsomes as pre-existing distinct morphological entities in intact (unhomogenized) cells is thought to be inconsistent with the distribution of basophile substances in frozen-dried liver.

P–449 Evaluation of ovarian reserve in female children and adolescents with non-iatrogenic primary ovarian insufficiency to establish criteria for ovarian tissue cryopreservation

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
A Volodarsky-Perel ◽  
M Zajicek ◽  
D Shai ◽  
H Raanani ◽  
N Gruber ◽  
...  
Keyword(s):  
Fertility Preservation ◽  
Ovarian Reserve ◽  
Predictive Value ◽  
Ovarian Function ◽  
Ovarian Tissue ◽  
Antral Follicle ◽  
Ovarian Tissue Cryopreservation ◽  
Positive Parameter ◽  
Primordial Follicles

Abstract Study question What is the predictive value of ovarian reserve evaluation in patients with non-iatrogenic primary ovarian insufficiency (NIPOI) for follicle detection in ovarian tissue harvested for cryopreservation? Summary answer Ovarian tissue cryopreservation (OTCP) should be considered if patients present at least one of the following parameters: detectable AMH, FSH≤20mIU/ml, detection of ≥ 1 antral follicle. What is known already In pre-pubertal girls suffering from NIPOI, which majorly has a genetic etiology, fertility preservation using OTCP is commonly practiced. When OTCP was performed in an unselected group of children and adolescents with NIPOI, only 26% of them had follicles in ovarian tissue while 74% did not benefit from the surgery. The role of preoperative evaluation of anti-müllerian hormone (AMH) serum level, follicular stimulating hormone (FSH) serum level, and trans-abdominal ultrasound for the antral follicle count to predict the detection of primordial follicles in the harvested ovarian tissue is unclear. Study design, size, duration We conducted a retrospective analysis of all patients ≤ 18 years old who were referred for fertility preservation counseling due to NIPOI at a single tertiary hospital between 2010 and 2020. If initial evaluation suggested a diminished ovarian reserve and at least one positive parameter indicating a follicular activity (AMH &gt; 0.16ng/ml, FSH ≤ 20mIU/ml, detection of ≥ 1 antral follicle by transabdominal sonography), OTCP was offered. Patients with 46XY gonadal dysgenesis were excluded. Participants/materials, setting, methods OTCP was performed laparoscopically in all cases. A fresh sample of cortical tissue was fixed in buffered formaldehyde for histological analysis. The rest of the ovarian tissue was cut into small cuboidal slices 1–2 mm in thickness and cryopreserved. After the serial sections, the histological slides were evaluated for the presence of follicles by a certified pathologist. Follicles were counted and categorized as primordial, primary, and secondary. Main results and the role of chance During the study period, 39 patients with suspected NIPOI were referred to the fertility preservation center. Thirty-seven patients included in the study were diagnosed with Turner’s syndrome (n = 28), Galactosemia (n = 3), Blepharophimosis-Ptosis-Epicanthus Inversus syndrome (n = 1), and idiopathic NIPOI (n = 6). Of 28 patients with Turner’s syndrome, 6 had 45X monosomy, 15 had mosaicism and 7 had structural anomalies in X-chromosome. One patient with gonadal dysgenesis and one with the presence of Y-chromosome in 20% of somatic cells were excluded from the study. OTCP was conducted in 14 patients with at least one positive parameter suggesting ovarian function. No complications of the surgical procedure or the anesthesia were observed. Primordial follicles were found in all patients with two or three positive parameters (100%) and in three of six cases with one positive parameter (50%). In total, of the 14 patients who underwent OTCP with at least one positive parameter, 11 (79%) had primordial follicles at biopsy (mean 23.9, range 2–47). This study demonstrates a positive predictive value of 79% for the detection of primordial follicles in patients who had at least one positive parameter of ovarian reserve evaluation. If two or three parameters were positive, the positive predictive value increased to 100%. Limitations, reasons for caution This study did not examine the negative predictive value of our protocol as OTCP was not recommended in the absence of positive parameters. The future fertility potential of cryopreserved tissue in the population with NIPOI is unclear and should be discovered in further studies. Wider implications of the findings: We suggest the evaluation of ovarian reserve by antral follicles count, AMH, and FSH serum levels prior to OTCP in patients with NIPOI. By recommendation of OTCP only if ≥ 1 parameter suggesting the ovarian function is positive, unnecessary procedures can be avoided. Trial registration number Not applicable

scholarly journals Comparison between Slow Freezing and Vitrification for Human Ovarian Tissue Cryopreservation and Xenotransplantation

2019 ◽  
Vol 20 (13) ◽  
pp. 3346 ◽  
Author(s):  
Sanghoon Lee ◽  
Ki-Jin Ryu ◽  
Boram Kim ◽  
Dahyeon Kang ◽  
Yoon Young Kim ◽  
...  
Keyword(s):  
Ovarian Tissue ◽  
Primordial Follicle ◽  
Research Unit ◽  
University Hospital ◽  
Slow Freezing ◽  
Ovarian Tissue Cryopreservation ◽  
Ki 67 ◽  
Immunodeficient Mice ◽  
Human Ovarian Tissue ◽  
Tissue Cryopreservation

Two methods for the cryopreservation of human ovarian tissue were compared using a xenotransplantation model to establish a safe and effective cryopreservation method. Ovarian tissues were obtained from women who underwent benign ovarian surgery in the gynecology research unit of a university hospital. The tissues were transplanted into 112 ovariectomized female severe combined immunodeficient mice 4 weeks after slow freezing or vitrification cryopreservation. Tissues were retrieved 4 weeks later. Primordial follicular counts decreased after cryopreservation and xenotransplantation, and were significantly higher in the slow freezing group than in the vitrification group (p < 0.001). Immunohistochemistry and TUNEL assay showed that the Ki-67 and CD31 markers of follicular proliferation and angiogenesis were higher in the slow freezing group (p < 0.001 and p = 0.006, respectively) and DNA damage was greater in the vitrification group (p < 0.001). Western blotting showed that vitrification increased cellular apoptosis. Anti-Müllerian hormone expression was low in transplanted samples subjected to both cryopreservation techniques. Electron microscopy revealed primordial follicle deformation in the vitrification group. Slow freezing for ovarian tissue cryopreservation is superior to vitrification in terms of follicle survival and growth after xenotransplantation. These results will be useful for fertility preservation in female cancer patients.

Protective Effect of Octreotide Against Liver Ischemic Reperfusion Injury in Rats

2020 ◽  
Vol 10 (8) ◽  
pp. 1115-1121
Author(s):  
Shuangfa Zou ◽  
Huiping Sun ◽  
Yanhua Peng ◽  
Shuo Yang ◽  
Jinfeng Yang
Keyword(s):  
Electron Microscopy ◽  
Reperfusion Injury ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Light And Electron Microscopy ◽  
Control Group ◽  
Sham Operation ◽  
Morphological Alterations ◽  
Saline Treatment ◽  
Octreotide Treatment

Liver ischemia-reperfusion injury (LIRI) is an inevitable complication during liver resection and liver transplantation. This study explored the effect of octreotide pretreatment on LIRI in rat model. Thirty male SD rats were included. They were divided into three groups: control group (sham operation plus saline treatment); ischemia/reperfusion group (IR group, ischemia/reperfusion operation plus saline treatment) and octreotide treatment group (IR + Oct group, ischemia/reperfusion operation plus octreotide treatment). The serum liver enzymes (ALT, AST) were tested to assess the liver damage in the rats. Light and electron microscopy was used to identify morphological alterations in each group. The expressions of HMGB1, RIP1 and RIP3 were measured by Immunohistochemistry and Western Blot. The levels of AST, ALT in IR group increased significantly (P < 0 05), and were significantly reduced by Octreotide pretreatment (P < 0 05). Morphology of control group remained grossly normal by transmission electron microscopy. While mitochondrial degeneration, cristae disruption, swelling, rupture was observed in IR group. The microscopic morphology of liver cells was basically normal and occasionally a small number of mitochondria were a little swelled in pretreatment with octreotide group. The expressions of HMGB1, RIP1 and RIP3 in pretreatment with octreotide were significantly down-regulated compared with those in pretreatment without octreotide (P < 0 001). The present study suggested that octreotide pretreatment play a protective role in LIRI, due to the decreased necrotizing apoptosis of hepatocytes. The mechanisms underlying these effects may be associated with the inhibition of HMGB1/RIP1/RIP3 necrotizing apoptosis signals.

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