Royan Institute First Attempts: Autotransplantation of Vitrified Human Ovarian Tissue in Cancer Patients.

Today, timely diagnosis and therapeutic progress open a road of hope for survival in cancerous patients. Increased knowledge about the various cytotoxic treatment's impacts on ovarian function and fertility has resulted in a surge in the number of patients seeking to preserve their fertility before starting the anti-cancer treatment process. In this regard, embryo cryopreservation can be recommended for fertility preservation when the woman is married and has adequate time for ovarian stimulation. If patients are prepubertal girls or not married women, oocytes or ovarian tissue can be frozen instead to be used in the future. In this regard, the first attempts for ovarian tissue transplantations were conducted in 2016 and in 2019 for two cancerous patients whose ovarian tissue was cryopreserved in the Royan Human Ovarian Tissue Bank (Tehran, Iran). Unfortunately, the transplantations did not result in a live birth.

A self-gelling hydrogel based on thiolated hyaluronic acid for three-dimensional culture of ovine preantral follicles.

Three-dimensional (3D) ovarian follicle culture offers a promising option for fertility preservation in patients who cannot receive ovarian tissue transplantation. Our research evaluated the potential of a hydrogel composed of thiolated hyaluronic acid (HA-SH) for ovine preantral follicle development compared to routinely used alginate hydrogel (ALG). Synthesized via a carbodiimide reaction, HA-SH facilitated a self-crosslinking hydrogel through disulfide bond formation. Ovine preantral follicles (200-300 µm) retrieved through mechanical and enzymatic methods were encapsulated individually in either ALG or HA-SH hydrogels. Although both hydrogels adequately supported follicle survival, 3D integrity, and antrum formation over a 17-day in vitro culture, follicle growth was significantly higher within the HA-SH hydrogel. Gene expression analysis underscored that some folliculogenesis-related genes (ZP3, BMP7, and GJA1) and a steroidogenic gene (CYP19A1) demonstrated higher expression levels in HA-SH encapsulated follicles versus ALG. Collectively, our findings advocate for HA-SH hydrogel as a potent biomaterial for in vitro follicle cultures, attributing its efficacy to facile gelation, bio-responsiveness, and superior support for follicle growth.

The effect of mTOR activation and PTEN inhibition on human primordial follicle activation in ovarian tissue culture.

PURPOSE: The effect of PTEN inhibitor (Bpv(HOpic); Bpv) and mTOR activators (phosphatidic acid (PA) and propranolol (PP)), were evaluated on the activation and subsequent development of human primordial follicles in ovarian tissue culture. METHODS: Slow frozen-thawed human ovarian cortical strips were incubated for 24 h in different groups: (1) Control (base medium), (2) Bpv (100 µM), (3) PA (200 µM), (4) PA + PP (50 µm), and (5) Bpv + PA + PP. Afterward, the medium was exchanged, and all groups were cultured without stimulators for 6 additional days. The proportion of normal and degenerated follicles, estradiol secretion, and expression of RPS6, FOXO3a, and AKT proteins was evaluated and compared between groups. RESULTS: After 24 h of culture, there was no significant difference between the proportion of primordial and growing follicles in either of the experimental groups. This non-significant change was also observed for the phosphorylated protein to total protein ratios of RPS6, FOXO3a, and AKT proteins. After 7 days of culture, the proportion of the transitional follicles was significantly higher in comparison to the primordial follicles for the PA, PA + PP, and Bpv + PA + PP groups. The estradiol level was significantly higher on the last day compared to the first day, in PA, PA + PP, and Bpv + PA + PP groups. Hormonal secretion was significantly higher in the PA and PA + PP groups and lower in the Bpv and Bpv + PA + PP groups compared to the control on day 7 of culture. CONCLUSION: Temporary in vitro treatment of human ovarian tissue with mTOR activators enhances the initiation of primordial follicle development and positively influences steroidogenesis after short-term culture.

Effects of N-Acetyl-L-Cystein Antioxidant on Ex Vivo Culture of Vitrified Premature Mouse Ovarian Tissue.

Optimization of practical ways to obtain mature follicles from cryopreserved ovarian tissues, especially in patients suffering from ovarian dysfunction, is very important. In vitro ovarian tissue culture allows faster screening of follicle development and reduces follicle isolation damage. During ovarian tissue culture, controlling oxidative stress is critical to support better follicular development and less damage. Immature Naval Medical Research Institute (NMRI) mouse ovaries (8-days-old) were randomly distributed into four cultured groups; non-vitrified, vitrified, non-vitrified N-acetyl-L-cysteine (NAC)+, and vitrified NAC+. Ovaries of vitrified groups along with non-vitrified ovaries were cultured on agar gel in the presence or absence of NAC for 5 days. Afterward, morphological evaluations, mRNA expressions of Gdf9, Bmp6, Lif, Amh, Bax, and Bcl2 genes, malondialdehyde, and total antioxidant capacities were compared between four groups at the first and last day of culture. Good preservation of tissue integrity and an increase of follicular development were observed in all groups. In addition, the expression of Gdf9, Lif, Bax, and Bcl2 genes were increased and Amh was decreased in groups cultured in the presence of NAC compared to groups cultured without NAC. Although total antioxidant capacity was not significantly different between the experimental groups, the lipid peroxidation and apoptotic index were significantly reduced in the presence of NAC. Thus, it appears that NAC antioxidant acts as a contributory factor for the ex vivo culture of ovarian tissue and reduces oxidative stress, apoptotic index, and improves follicular development, especially in non-vitrified groups.

Human ovarian tissue in-vitro culture: primordial follicle activation as a new strategy for female fertility preservation.

Cryopreservation and transplantation of ovarian tissue is the only fertility preservation option used for prepubertal girls and women who don't have a chance for embryo or oocyte vitrification. For women with aggressive cancer, hormone-responsive malignancies, autoimmune diseases, etc. ovary transplantation cannot be performed so an alternative technology called in-vitro follicle activation is thinkable. In this method, dormant primordial follicles are activated from the resting primordial pool by in-vitro culture and enter their growth phase. Different in-vitro culture media and supplements in addition to various culturing methods have been conducted for activating these dormant follicles. Furthermore, several signaling pathways such as Hippo, phosphatidylinositol-3-kinase, and mTOR influence follicle activation. Therefore, the addition of different activators of these signaling pathways can beneficially regulate this culture system. This review summarizes the findings on different aspects of human ovarian tissue culture strategies for in-vitro follicular activation, their medium, and different factors involved in this activation. Afterward, signaling pathways important for follicle activation and their clinical applications towards improving activation in culture are also reviewed.

Two-Decade Experience of Royan Institute in Obtaining Mature Oocyte from Cryopreserved Ovarian Tissue: In Vitro and In Vivo Approaches.

Ovarian tissue cryopreservation (OTC) holds promise for preservation of fertility among women who have lost their fertility due to diseases such as cancer. OTC has significantly assisted such cases by helping them maintain normal hormonal levels and menstrual cycles. Appropriate strategies to develop and extract mature oocytes from OTC could overcome a range of complications that are associated with ovarian dysfunction, caused by aging, and primary or secondary ovarian insufficiency. Scientists from different departments at The Royan Institute (Tehran, Iran) have been conducting studies to find the best way to extract mature oocytes from animal and human cryopreserved ovarian tissues. The various studies conducted in this area in the past 20 years, by researchers of the Royan Institute, are collated and provided in this review, in order to provide an idea of where we are now in the area of fertility preservation.

Female Reproductive Health in SARS-CoV-2 Pandemic Era.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic struck global health systems with overgrowing demands in many fields of health care; yet, reproductive care, particularly pregnancy care remains a special focus of interest. Pregnancy is a major physiologic change that alters temporarily normal function of many organs, and specifically the immune system. Therefore, pregnant women are more susceptible to respiratory pathogens compared to the others. The current pandemic may have serious consequences on pregnancy whether directly or indirectly. In the present review, direct and indirect possible adverse effects of SARS-CoV-2 infection on female reproductive system by focusing on pregnancy and delivery has been discussed in details. In addition, the pregnancy consequences and whether maternal infection can affect infants were deliberated. The adverse impact of luck down and related psychological complications and obesity on pregnant women were discussed as well. Finally, the effects of SARS-CoV-2 vaccination on maternal health and pregnancy outcome was analyzed.

Paternal trans-fatty acid and vitamin E diet affects rat offspring's semen quality and PPARs expression.

Trans-fatty acids (TFAs) consumption has created concerns regarding male/female reproductive system. However, the effects of TFA in paternal diet on offspring's reproduction have not been addressed. The purpose of this study is to investigate the effects of rat paternal TFAs and vitamin E consumption on offspring's sperm quality and expression pattern of peroxisome proliferator-activated receptors (PPARs) in testis tissues. Forty adult male rats were randomly divided into four groups: Control diet (C); Control diet plus TFA (CTH); diet supplemented with vitamin E (E) and a diet containing vitamin E and TFA (ETH). Mother rats had normal diet during gestation period. Three offspring from each group were chosen randomly and their testicular samples were collected, and sperm parameters were measured by CASA. Our results indicate that feeding fathers with TFA can negatively affect offspring's sperm concentration and motility, while consumption of vitamin E can improve these parameters (p < .05). The paternal diet containing TFA down-regulated the expression of PPARß and PPARγ genes, whereas vitamin E-containing diet up-regulated the transcription of PPAR genes. In conclusion, TFA intake in paternal diet may have negative effects on reproductive system of the offspring while vitamin E may not diminish these effects.

The effect of agar substrate on growth and development of cryopreserved-thawed human ovarian cortical follicles in organ culture.

OBJECTIVE: To preserve human ovarian tissue structure and improve follicular growth and survival during in-situ culture, various biomaterials are used. In this study we aimed to compare agar as a cultivation substrate with matrigel-coated insert in order to achieve an optimum system for in-situ human follicle culture. STUDY DESIGN: Frozen-thawed human ovarian cortical tissues were cultured on either matrigel-coated inserts or agar-soaked substrates. The proportion of morphologically viable and degenerated follicles at different developmental stages, secreted hormonal levels, and apoptotic and proliferation gene expressions were compared between the cultured groups after 7-days of culture. RESULTS: The follicular growth was not significantly different between the two cultured groups, although showing higher percentage of growing follicles in agar cultured group. The secreted hormonal levels didn't have any difference between two cultured groups. Although the apoptotic gene expressions didn't show any difference between the cultured groups, the apoptotic index was lower in agar cultured group. In addition, Ki67 gene expression, a proliferative marker, showed a significantly higher expression in agar cultured group. CONCLUSION: Based on the results, agar is as suitable as matrigel-coated inserts for the survival and growth of follicles during culture. Therefore, agar can be an inexpensive alternative substrate for culturing frozen-thawed human ovarian cortical strips.

The combination of basic fibroblast growth factor and kit ligand promotes the proliferation, activity and steroidogenesis of granulosa cells during human ovarian cortical culture.

Different factors, such as basic fibroblast growth factor (bFGF) and kit ligand (KL), are used in ovarian cortical culture to promote activation of primordial follicles. In the present study, the effects of bFGF and KL, alone and in combination, were evaluated on human follicular activation and growth during in-situ cortical culture. Slow frozen-thawed human ovarian cortical tissues (n = 6) were cultured in 4 different groups: 1) control (base medium), 2) KL (base medium; BM + 100 ng/ml KL), 3) bFGF (BM + 100 ng/ml bFGF) and 4) bFGF + KL (BM + 100 ng/ml KL + 100 ng/ml bFGF) for a week. The proportion of morphologically normal and degenerated follicles at different developmental stages, secreted hormonal levels and specific gene expressions were compared. Although the proportion of growing follicles was higher than primordial counterpart in all cultured groups, no significant differences were observed among the cultured groups. In all cultured groups, anti-Müllerian hormone (AMH), progesterone and estradiol hormones levels increased after 7 days of culture; however, this increase was only significant for estradiol in the bFGF + KL group. The expression of Ki67 gene indicated an increase in ovarian cell proliferation in the three experimental groups compared to the control group, however this increment was only significant for the bFGF + KL group. It can be concluded that KL and bFGF factors individually have no beneficial effects on in-situ follicular growth, but their combination positively influences steroidogenesis of granulosa cells without significantly increasing the number of growing follicles.

Noninvasive sexing of human preimplantation embryos using RT-PCR in the spent culture media: A proof-of-concept study.

Preimplantation genetic testing (PGT) routinely requires biopsy which is an invasive approach. The aim of this study was to examine a noninvasive approach for sexing of preimplantation embryos using polymerase chain reaction (PCR)/reverse transcriptase-PCR (RT-PCR) based on the presence of SRY DNA/RNA in the spent culture medium. Two groups were evaluated: in group 1, 40 embryos of routine PGT volunteers were cultured individually after biopsy and in group 2, 31 embryos were cultured individually until Day-5 post-fertilization. Each group was further divided into three subgroups: RNA extraction (RE), nucleic acid (NA) and DNase treated (DT). In the NA and DT subgroups, cDNA synthesis was performed directly on culture medium with or without DNase treatment in DT and NA subgroups, respectively. The results of sexing based on the PCR/RT-PCR in the culture medium, were compared with the results of sexing by fluorescence in situ hybridization (FISH) technique. In group 1, all samples were correctly diagnosed. In group 2, five female samples were misdiagnosed. Test's sensitivity, specificity and accuracy were 100 %, 94.44 % and 96.88 %, in RE, 100 %, 81.82 % and 93.55 % in DT and 100 %, 71.43 % and 85.71 % in NA, respectively. Preimplantation sexing without embryo biopsy, in the spent embryo culture media using RNA amplification based methods including RE and DT seem to be more reliable while nucleic acid based method, NA, led to the highest misdiagnoses probably due to DNA contamination. Since all male samples were correctly diagnosed in all subgroups of this preliminary study, preimplantation noninvasive sexing on culture medium seems feasible, however all sources of nucleic acid contamination must be carefully avoided.

A novel approach for human sperm cryopreservation with AFPIII.

Sperm cryopreservation causes different stresses including thermal shock, osmotic damage, and ice crystal formation, thereby reducing sperm quality. Few studies have evaluated the application of AFPs in cryopreservation. The effects of antifreeze protein III (AFP III) on human sperm cryopreservation is not fully understood therefore, we conducted this study to investigate the effects of AFPIII treatment on human sperm parameters following cryopreservation. First, for 20 semen samples the effects of various concentrations of AFPIII (0, 0.01, 0.1, 1, 5, 10 µg/ml) were evaluated. Sperm parameters, such as motility and viability were assessed in order to identify an optimal dose. Next, liquefied 20 semen samples were divided into three aliquots and diluted in glycerol-egg-yolk-citrate (GEYC) cryopreserved without AFPIII (control), with optimal dose of AFPIII, as well as fresh groups. After thawing, samples were evaluated for plasma membrane integrity (PMI), DNA fragmentation index (DFI), reactive oxygen species (ROS), and total antioxidant capacity (TAC) levels. Spermatozoa treatment with 0.01, 0.1 and 1 µg/ml AFPIII increased the sperm motility and viability compared to the control group, but the highest concentrations were ineffective. In conclusion, the results showed that the addition of AFPIII to GEYC at 1 µg/ml improved motility, PMI, viability and TAC, and decreased ROS and DNA fragmentation of cryopreserved human semen compared to the control group.

Chitosan Hydrogel Supports Integrity of Ovarian Follicles during In Vitro Culture: A Preliminary of A Novel Biomaterial for Three Dimensional Culture of Ovarian Follicles.

OBJECTIVE: Testing novel biomaterials for the three dimensional (3D) culture of ovarian follicles may ultimately lead to a culture model which can support the integrity of follicles during in vitro culture (IVC). The present study reports the first application of a chitosan (CS) hydrogel in culturing mouse preantral follicles. MATERIALS AND METHODS: In this interventional experiment study, CS hydrogels with the concentrations of 0.5, 1, and 1.5% were first tested for fourier transform infrared spectroscopy (FT-IR), Compressive Strength, viscosity, degradation, swelling ratio, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity and live/dead assay. Thereafter, mouse ovarian follicles were encapsulated in optimum concentration of CS (1%) and compared with those in alginate hydrogel. The follicular morphology, quality of matured oocyte and steroid secretion in both CS and alginate were assessed by enzyme-linked immunosorbent assay (ELISA). The expression of folliculogenesis, endocrine, and apoptotic related genes was also evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) and compared with day that in 0. RESULTS: The rates of survival, and diameter of the follicles, secretion of estradiol, normal appearance of meiotic spindle and chromosome alignment were all higher in CS group compared with those in alginate group (P≤0.05). The expression of Cyp19a1 and Lhcgr in CS group was significantly higher than that of the alginate group (P≤0.05). CONCLUSION: The results showed that CS is a permissive hydrogel and has a beneficial effect on encapsulation of ovarian follicle and its further development during 3D culture.

Evaluating two ovarian decellularization methods in three species.

Since there is dearth of practical ways to obtain mature follicles from cryopreserved or native ovarian tissues, especially in patients suffering from ovarian dysfunction, tissue engineering may help in restoring ovarian function and/or fertility. In the present study, the effects of sodium dodecyl sulfate (SDS) and sodium hydroxide (NaOH) on the decellularization of ovarian tissues were studied in order to ascertain their suitability in creating suitable bioscaffolds. Cells were removed from the ovarian tissues of mouse, sheep and human. The samples were distributed among three groups, viz., control (not treated), SDS and NaOH treated. Qualitative histological evaluations, quantitative assessments (nuclear contents, collagen and glycosaminoglycan), immunohistochemistry staining (for laminin, fibronectin and Collagen I), cell viability and scanning electron microscopic (SEM) assays were performed for all experimental groups. Finally, suspensions of mouse ovarian cells were injected into human NaOH treated scaffolds and subsequently auto-transplanted to ovariectomized mice. H&E and IHC staining (GDF-9) were performed on human recellularized NaOH treated scaffolds 1 month after auto-transplantation. Although histological studies and quantitative evaluations confirmed the successful decellularization and presence of key factors in ovarian scaffolds under both treatment methods, NaOH showed more interesting outcomes. Cell metabolic activity in sheep and human ovaries treated with NaOH was statistically (p < 0.05) higher than for SDS treated samples after 72 h. Moreover, spherical associations with cuboidal cells in human NaOH treated scaffolds were observed and this follicular reconstruction was also confirmed by GDF-9. NaOH was found to be more suitable than SDS for the decellularization of ovarian tissues and it supports follicular reconstruction better than SDS. This is a valuable finding in tissue engineering research and can help in the creation of appropriate ovarian bioscaffolds.

An improved method for vitrification of in vitro matured ovine oocytes; beneficial effects of Ethylene Glycol Tetraacetic acid, an intracellular calcium chelator.

Vitrification affects fertilization ability and developmental competence of mammalian oocytes. This effect may be more closely associated with an intracellular calcium rise induced by cryoprotectants. The present study aimed to assess whether addition of Ethylene Glycol Tetraacetic acid (EGTA) to vitrification solution could improve quality and developmental competence of in vitro matured ovine oocytes. Vitrified groups were designed according to the presence or absence of EGTA and/or calcium in base media, including: mPB1+ (modified PBS with Ca2+), mPB1- (modified PBS without Ca2+), mPB1+/EGTA (mPB1+ containing EGTA), mPB1-/EGTA (mPB1- containing EGTA). In vitro development, numerical chromosome abnormalities, hardening of zona pellucida, mitochondrial distribution and function of viable oocytes were evaluated and compared between groups. Quality of blastocysts was assessed by differential and TUNEL staining. Also, mRNA expression levels of six candidate genes (KIF11, KIF2C, CENP-E, KIF20A, KIF4A and KIF2A), were quantitatively evaluated by RT-PCR. Our results showed that calcium-free vitrification and EGTA supplementation can significantly increase the percentage of normal haploid oocytes and maintain normal distribution and function of mitochondria in vitrified ovine oocytes, consequently improving developmental rate after in vitro fertilization. qRT-PCR analysis showed no significant difference in mRNA expression levels of kinesin genes between vitrified and fresh oocytes. Also, the presence of calcium in vitrification solution significantly increased zona hardening. In conclusion, we have shown for the first time that supplementation of vitrification solution with EGTA, as a calcium chelator, improved the ability of vitrified ovine oocytes to preserve mitochondrial distribution and function, as well as normal chromosome segregation.

Developmental competence of in vitro matured ovine oocytes vitrified in solutions with different concentrations of trehalose.

This study aimed to determine the optimum concentration of trehalose in solutions used for vitrification of in vitro matured (IVM) ovine oocytes. IVM oocytes were randomly divided into four experimental (vitrified) and one control (fresh) groups. Experimental groups were treated with different concentrations (0.0, 0.25, 0.5 and 1.0 M) of trehalose. After warming, some viable oocytes were exposed to 0.25% pronase to test zona pellucida hardening, whereas the others were fertilized and cultured in vitro for 8 days to evaluate their developmental competence. Blastocysts quality was assessed by differential staining and TUNEL test. Survival and developmental rates of oocytes vitrified in the presence of 0.5 M trehalose were significantly higher than those of the other vitrified groups. Furthermore, there was a significant difference between fresh and vitrified groups in total blastocyst rate. Analysis of blastocysts quality also revealed a significant difference between the group treated with 0.5 M trehalose and other groups in terms of apoptotic index. Furthermore,zona pellucida digestion time period was longer in trehalose-free (0.0 M) group compared to other groups. In conclusion, we found that IVM ovine oocytes vitrified in solutions containing 0.5 M trehalose are fertilization-competent and are able to produce good-quality blastocysts with an apoptotic index comparable to that of the fresh oocytes. Therefore, 0.5 M may be considered the optimum concentration of trehalose to be used in solutions prepared for vitrification of oocytes.

Utilizing Fibrin-Alginate and Matrigel-Alginate for Mouse Follicle Development in Three-Dimensional Culture Systems.

In vitro culture of ovarian follicles is a new technique in reproductive technology, which helps in understanding the process of folliculogenesis. The in vitro culture of follicles could be carried out using three-dimensional (3D) natural scaffolds that mimic the ovarian tissue stroma. Selection of the right matrix and culture media in these scaffolds could increase the survival and maturation of the follicles. In this work, the applicability of matrigel-alginate (MA) and fibrin-alginate (FA) 3D scaffolds for folliculogenesis was assessed. The ovaries of 13-day-old Naval Medical Research Institute (NMRI) mice were isolated and distributed into control and vitrification groups. Preantral follicles (mean diameter: 120-140 µm) were mechanically isolated from control and vitrified-warmed ovaries, encapsulated in MA or FA scaffold and cultured for 12 days. Follicle survival, growth, maturation, and quantitative expression of oocyte maturation genes (Gdf9, Bmp15, Fgf8, KitL, Kit, and Amh) and proteins (GDF9 and BMP15) were assessed. Survival rate of culture preantral follicles in control groups was found to be significantly higher than vitrified follicles. Antrum formation was similar in all groups. Follicle diameters were significantly increased in all groups during culture period. A decreasing pattern of gene expression was seen for all genes in all groups. This trend was verified through evaluation of protein expression, during which there was strong staining in antral follicles from all groups in the last day of in vitro culture. The better survival and maturation rate of follicles in the MA compared to FA scaffold indicates that the MA matrix, being rich in extracellular matrix components, could mimic the ovarian condition better and presents a good environment for follicle development.

Slow freezing versus vitrification technique for human ovarian tissue cryopreservation: An evaluation of histological changes, WNT signaling pathway and apoptotic genes expression.

This study compared slow freezing and vitrification of ovarian tissue by evaluation of histological changes, WNT signaling pathway and apoptotic genes expression. Ovarian tissue was obtained from women aging 27-38 years old. Ovarian cortex from each patient was divided into three pieces and randomly grouped as slow freezing, vitrification and control groups for investigation of WNT signaling gene expression and ß-CATENIN presence as well as histological studies. The stromal structure of all ovaries were preserved. The number of secondary follicles decreased in vitrified group (P < 0.05). WNT-3, ß-CATENIN, FZD-2 and GSK-3ß expressions were significantly higher in slow frozen and vitrified groups, compared to control group (P < 0.05). On the contrary, AXIN1 expression in slow frozen samples were significantly lower than that of the vitrified and control group. The expression of apoptotic genes, excluding CASP3, was significantly decreased in slow-frozen samples (P < 0.05). Conversely, BAX:BCL-2 percentage significantly increased in vitrification versus slow freezing and control(P < 0.05). Follicles in slow frozen samples displayed nuclear and cytoplasmic ß-CATENIN staining, while control and vitrification groups only showed ß-CATENIN protein in the cytoplasm. The presented data show that slow freezing results in a better preservation regardless of the type of follicle. Therefore, it is concluded that slow freezing is still an ideal method for ovary cryopreservation.

Fertility Preservation in Cancer Patients: In Vivo and In Vitro Options.

Oocyte, embryo and ovarian tissue cryopreservation are being increasingly proposed for fertility preservation among cancer patients undergoing therapy to enable them to have babies after the cancer is cured. Embryo cryopreservation is not appropriate for single girls without any sperm partner and also because oocyte retrieval is an extended procedure, it is impossible in cases requiring immediate cancer cure. Thus ovarian tissue cryopreservation has been suggested for fertility preservation especial in cancer patients. The main goal of ovarian cryopreservation is re-implanting the tissue into the body to restore fertility and the hormonal cycle. Different cryopreservation protocols have been examined and established for vitrification of biological samples. We have used Cryopin to plunge ovarian tissue into the liquid nitrogen and promising results have been observed. Ovarian tissue re-implantation after cancer cure has one problem- the possibility of recurrence of malignancy in the reimplanted tissue is high. Xenografting-implantation of the preserved tissue in another species- also has its drawbacks such as molecular signaling from the recipient. In vitro follicle culturing is a safer method to obtain mature oocytes for fertilization and the various studies that have been carried out in this area are reviewed in this paper.

Oocyte maturation, embryo development and gene expression following two different methods of bovine cumulus-oocyte complexes vitrification.

OBJECTIVE: To examine the maturational competence, embryo development and expression of genes involved in oocyte maturation and cumulus expansion (GDF9, BMP15, HAS2, TNFAIP6, FGF17 and FSHr) following two standard methods of bovine COCs vitrification. METHODS: Bovine cumulus-oocyte complexes (COCs) were aspirated from slaughtered ovaries and then distributed into three groups: non-vitrified COCs (control), vitrification 1 group (V1); vitrification was performed by 15% ethylene glycol (EG) and 15% DMSO in holding media (TCM-199 with 20% FCS); and vitrification 2 group (V2); vitrification was performed by 40% EG in holding media. After vitrification, COCs were warmed in two steps and cultured and then evaluated for nuclear maturation, embryo development and gene expressions. RESULTS: The mean (±SD) percentages of nuclear maturation and blastocyst/cleaved were higher in control group (79.5 ± 8.0 and 31.0 ± 5.1%) than the V1 (34.8 ± 9.1 and 4.4 ± 5.1%) and V2 (47.8 ± 11.7 and 7.1 ± 5.8%) groups (P < 0.05), respectively. Further, COCs in V2 group showed higher mean (±SD) percentages of cleavage compared to V1 group (31.8 ± 1.0 vs 21.7 ± 2.8%; P < 0.05). GDF9 and BMP15 expression levels were higher in COCs in the control than of the vitrification groups (P < 0.05). In addition, expression level of GDF9 and BMP15 was higher in V2 group than in V1group (P < 0.05). The expression of HAS2 and FGF17 in V1 group was lower (P < 0.05) than that of the V2 groups. CONCLUSIONS: Expression of oocyte maturation genes was affected by vitrification procedure and conditions. Using EG alone for vitrification of bovine immature COCs, resulted in higher expression of GDF9, BMP15 and production of more in vitro matured and cleaved oocytes.