Cryopreservation of ovarian tissue

Cryopreservation is the process of freezing and storing biological material at ultra-low temperatures
(such as -196⁰C) in liquid nitrogen (LN2) for unlimited periods. Thanks to the application
of this biotechnology, all biological activities including the biochemical reactions
leading to cell death and DNA degradation discontinue. From this point of view, cryopreservation
biotechnology focuses on preservation of cells that have many applications in the
fields of human and veterinary medicine, agriculture and aquaculture. In addition, this form
of biotechnology also has many applications in biomedical research, specifically in the areas
of immunology, virology, neurobiology, toxicology and the pharmaceutical industry.
Cryopreservation Biotechnology in Biomedical and Biological Sciences describes principles and applications
of cryopreservation biotechnology in different research areas and includes seven
chapters that have been written by experts in their research fields. The book chapters are
divided into four sections.
Section I, "Mechanism of Cryopreservation" contains one chapter entitled "Cryoprotectants
and their usage in cryopreservation process", which describes the importance and mechanism
of the cryopreservation process and also its comparison to vitrification. In addition, the
functions and physical and chemical properties of cryoprotectants are discussed.
Section II, "Application of Cryopreservation in Human Medicine Researches" is divided into
four chapters. The first chapter, "Clinical outcomes of assisted reproductive techniques using
cryopreserved gametes and embryos in human medicine", provides valuable information
regarding fertility treatments in human reproductive medicine, focusing on
cryopreservation of spermatozoa, oocytes and embryos. The second chapter, "Cryopreservation
of platelets: advances and current practices" focuses on the long-term preservation of
platelets, which is necessary for the coagulation of blood and stopping bleeding. The third
chapter, "Cryopreservation of preantral follicles", provides valuable information regarding
the structure and development of ovarian follicles. Beside this, advantages and necessities of
using oocytes in preantral follicles for the aim of cryopreservation are explained in humans
and mammals. The fourth chapter, "Vitrification: Fundamental principles and its application
for cryopreservation of human reproductive cells", is related to mechanisms of cryopreservation
in terms of application of this technique in human medicine with emphasis on
reproductive cells. In this way, mechanisms underlying the problem of the intra- and extracellular
ice formation, the role of cryoprotectants, stages of the warming and cooling process
and use of this biotechnology in the field of IVF industry are explained.
Section III, "Application of Cryopreservation in Veterinary Medicine Research", contains one
chapter entitled "Biological signals of sperm membrane resistance to cryoinjury in boars",
discusses biological mechanisms before the cryopreservation/vitrification process causing
cryoinjuries in sperm cells in boars.
Section IV, "Application of Cryopreservation in Agricultural Research", contains one chapter
entitled "Cryopreservation protocols for grapevine shoot tips", which presents valuable information
regarding cryopreservation of shoot tips of grapevines, which is an economically
important agricultural product.
This book covers different applications of cryopreservation biotechnology. I hope that this
book will be helpful for researchers studying cryobiology and related issues. I would like to
thank all the authors for their distinguished contributions, IntechOpen Publishing Company,
and its Author Service Manager Ms. Dolores Kuzelj for her help in publishing this book.

Cryopreservation of ovarian tissue may be a potential alternative for the conservation of genetically superior animals, including high milk- and meat-producing goat breeds. However, until now, no information was available concerning the cryopreservation of preantral follicles (PF) enclosed in caprine ovarian tissue. The objective of the present study was to evaluate the structural and ultrastructural characteristics of caprine PF after exposure to and cryopreservation of ovarian tissue in 1.5 and 3 M glycerol (GLY) and ethylene glycol (EG). At the slaughterhouse, each ovarian pair from five adult mixed breed goats was divided into nine fragments and randomly distributed into treatment groups. One fragment was immediately fixed for histological examination and ultrastructural analysis, after slaughter (control). Four of the ovarian fragments were equilibrated at 20 °C for 20 min in 1.8 ml of MEM containing 1.5 or 3 M GLY or EG for a toxicity test and the final four fragments were slowly frozen using these cryoprotectants at the concentrations above. After toxicity testing and freezing/thawing, the ovarian fragments were fixed for histological examination. Histological analysis showed that after toxicity testing and cryopreservation of the ovarian tissue in GLY or EG at both concentrations, the percentage of normal PF was significantly lower than controls. Ultrastructural analysis of PF frozen in 1.5 and 3 M GLY, as well as 3 M EG demonstrated that these follicles remained morphologically normal. In conclusion, we demonstrated cryopreservation of caprine PF in ovarian tissue.

With the evolution of the treatment of malignant neoplasms, the survival rates of patients undergoing chemo- or radiotherapy are increasing. The continuous development of techniques of assisted human reproduction has led to important strategies in an attempt to maintain reproductive function in patients subjected to treatment of neoplastic diseases, among them cryopreservation of embryos, gametes, and ovarian cortical tissue. The freezing of ovarian tissue is currently being proposed with the primary purpose of preserving ovarian function in these patients. Currently, the major challenge of groups working with preservation of fertility is the use of cryopreserved ovarian tissue after disease remission. The main alternatives presented today are the implantation of hetero- or orthotopic tissue and isolation of immature follicles from ovarian tissue followed by in vitro maturation and assisted reproduction procedures.

Butylparaben is used as a preservative in some foods, cosmetics, personal care products, household products and drug formulations and is widely used by manufacturing companies for its low cost and efficiency as a microbial agent. Butylparaben is of growing concern as recent studies have revealed that it mimics estrogen activity in in vitro and in vivo studies and thereby acting as a potent xenoestrogen. Exposure to chemicals showing estrogenic property is found to affect follicular population in ovary. In the present study sensitivity of butylparaben exposure on ovarian follicular population of immature 28 days old female C3H mice and adult female C3H mice of 8 weeks of age was analysed for three different doses of Butylparaben of 10 mg/Kg body weight/day, 50 mg/Kg body weight/day and 100 mg/Kg body weight/day. In this study olive oil was considered as control and 17β Estradiol as positive control. The mice were subcutaneously exposed to the chemical for 30 days following which they are sacrificed and 6 μm thick section of each ovary was prepared stained in hematoxylin and eosin. Every 12th and 20th section was examined for counting smaller follicles and larger follicles respectively. The results of butylparaben exposure showed fewer primary follicles and atretic follicles similar to the results obtained following mice exposed to 17β Estradiol. Thus the data obtained from the present study reveals the estrogenic nature of butylparaben and increases concern over the wide use of the chemical in human.

Objective Pulegone (PGN) is a monoterpene ketone, whose metabolites exert several cytotoxic effects in various tissues. The present study was conducted in order to evaluate the (R)-(+) PGN-induced alterations in ovarian aromatization, proto-oncogenes and estrogen receptorα (ERα) and ERβ receptors expressions. Materials and Methods In this experimental study, mature albino mice were divided into experimental (received 25 mg/kg, 50 mg/kg and 100 mg/kg PGN, orally for 35 days) and control (received 2% solution of Tween 80 as a PGN solvent, orally) groups. The mRNA levels of Erα, Erβ, p53, Bcl-2, and cytochrome p450 (Cyp19) as well as ovarian angiogenesis were analyzed through reverse transcription polymerase chain reaction and immunohistochemical techniques, respectively. Moreover, apoptosis of follicular cells, serum estrogen and progesterone levels and mRNA damage were investigated via using terminal transferase and biotin-16-dUTP staining, electrochemilunescence and fluorescent micr...

Objective: The aim of the present study was to examine whether lysophosphatidic acid (LPA) could decrease cell death and improve in vitro culture (IVC) conditions in cultured vitrified mouse ovarian tissue. Materials and Methods: In this experimental study, we collected and randomly divided 7-day-old mouse ovarian tissues into vitrified and non-vitrified groups. The ovaries were cultured in the presence and absence of LPA for one week. Morphology and follicular development were evaluated by hematoxylin and eosin (H&E) and Masson's trichrome (MTC) staining. The incidence of cell death was assessed by flow cytometry using annexin V/propidium iodide (PI) and a caspase-3/7 assay in all studied groups. Results: The vitrified groups had a significantly decreased follicle developmental rate compared to the non-vitrified groups (P<0.05). Overall, qualitative and quantitative results showed prominent follicular degeneration in the vitrified groups compared with the respective non-vitrified groups. Both LPA treated groups had a significantly higher proportion of preantral follicles compared to the non-LPA treated groups (P<0.05). Flow cytometry analysis results showed significantly greater early and late apoptotic cells in all groups (17.83 ± 8.80%) compared to necrotic cells (7.97 ± 0.92%, P<0.05). The percentage of these cells significantly increased in the vitrified groups compared with non-vitrified groups. LPA treated groups had a lower percentage of these cells compared to non-LPA treated groups (P<0.05). The lower enzyme activity was observed in non-vitrified (especially in the LPA+ groups) cultured ovaries compared to the vitrified group (P<0.05). Conclusion: Both vitrification and IVC adversely affected cell survival and caused cell death. We postulated that LPA supplementation of culture medium could improve the developmental rate of follicles and act as an anti-cell death factor in non-vitrified and vitrified ovarian tissues. It could be used for in vitro maturation of ovarian tissue. Citation: Abedpour N, Salehnia M, Ghorbanmehr N. The effects of lysophosphatidic acid on the incidence of cell death in cultured vitrified and non-vitrified mouse ovarian tissue: separation of necrosis and apoptosis border. Cell J. 2018; 20(3): 403-411.

Cryopreservation of ovarian tissue may be a potential alternative for the conservation of genetically superior animals, including high milk-and meat-producing goat breeds. However, until now, no information was available concerning the cryopreservation of preantral follicles (PF) enclosed in caprine ovarian tissue. The objective of the present study was to evaluate the structural and ultrastructural characteristics of caprine PF after exposure to and cryopreservation of ovarian tissue in 1.5 and 3 M glycerol (GLY) and ethylene glycol (EG). At the slaughterhouse, each ovarian pair from five adult mixed breed goats was divided into nine fragments and randomly distributed into treatment groups. One fragment was immediately fixed for histological examination and ultrastructural analysis, after slaughter (control). Four of the ovarian fragments were equilibrated at 20 8C for 20 min in 1.8 ml of MEM containing 1.5 or 3 M GLY or EG for a toxicity test and the final four fragments were slowly frozen using these cryoprotectants at the concentrations above. After toxicity testing and freezing/thawing, the ovarian fragments were fixed for histological examination. Histological analysis showed that after toxicity testing and cryopreservation of the ovarian tissue in GLY or EG at both concentrations, the percentage of normal PF was significantly lower than controls. Ultrastructural analysis of PF frozen in 1.5 and 3 M GLY, as well as 3 M EG demonstrated that these follicles remained morphologically normal. In conclusion, we demonstrated cryopreservation of caprine PF in ovarian tissue. #

Cryopreservation of ovarian tissue may be a potential alternative for the conservation of genetically superior animals, including high milk-and meat-producing goat breeds. However, until now, no information was available concerning the cryopreservation of preantral follicles (PF) enclosed in caprine ovarian tissue. The objective of the present study was to evaluate the structural and ultrastructural characteristics of caprine PF after exposure to and cryopreservation of ovarian tissue in 1.5 and 3 M glycerol (GLY) and ethylene glycol (EG). At the slaughterhouse, each ovarian pair from five adult mixed breed goats was divided into nine fragments and randomly distributed into treatment groups. One fragment was immediately fixed for histological examination and ultrastructural analysis, after slaughter (control). Four of the ovarian fragments were equilibrated at 20 8C for 20 min in 1.8 ml of MEM containing 1.5 or 3 M GLY or EG for a toxicity test and the final four fragments were slowly frozen using these cryoprotectants at the concentrations above. After toxicity testing and freezing/thawing, the ovarian fragments were fixed for histological examination. Histological analysis showed that after toxicity testing and cryopreservation of the ovarian tissue in GLY or EG at both concentrations, the percentage of normal PF was significantly lower than controls. Ultrastructural analysis of PF frozen in 1.5 and 3 M GLY, as well as 3 M EG demonstrated that these follicles remained morphologically normal. In conclusion, we demonstrated cryopreservation of caprine PF in ovarian tissue. #

In this study we aimed testing the efficiency of a newly developed device for vitrification of ovaries without contact with liquid nitrogen, Ovarian Tissue Cryosystem (OTC). From each ovarian pair, fragments were recovered and immediately fixed for analysis (fresh control) or submitted to vitrification (fragments, hemi-ovary or whole ovary), either or not followed by in vitro culture for two days. Vitrification was performed using the OTC system. The OTC is a cylindrical structure made by stainless steel and composed by three pieces (basis, insert and cover), which can be hermetically closed avoiding contact of the tissue with liquid nitrogen during vitrification. Before and after culture, the ovarian tissue was histologically evaluated. Independently from the size of the ovarian tissue, it was observed a decrease (P<0.05) in the rates of normal preantral follicles when fragments (58.1%), hemi-ovary (54.4%) and whole ovary (54.3%) were vitrified, in comparison with fresh control (68.1%). These data were confirmed by ultrastructural analysis, which showed a great extension of degeneration in follicles vitrified in the whole ovary. Follicular survival after vitrification followed by culture was higher (P<0.05) when ovarian fragments were vitrified (36.1%) than in those enclosed in vitrified hemi-ovary (22.3%) or whole ovary (18.4%). In conclusion, the Ovarian Tissue Cryosystem (OTC) opens a new possibility for successful vitrification of caprine ovarian fragments.