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.

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.

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.

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.

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.

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.

An Introduction to The Royan Human Ovarian Tissue Bank.

From December 2000 until 2010, the researchers at Royan Institute conducted a wide range of investigations on ovarian tissue cryopreservation with the intent to provide fertility pres- ervation to cancer patients that were considered to be candidates for these services. In 2010, Royan Institute established the Royan Human Ovarian Tissue Bank as a subgroup of the Embryology Department. Since its inception, approximately 180 patients between the ages of 747 years have undergone consultations. Ovarian samples were cryopreserved from 47 patients (age: 7-35 years) diagnosed with cervical adenocarcinoma (n=9); breast carcinoma (n=7), Ewing's sarcoma (n=7), opposite side ovarian tumor (n=7), endometrial adenocarci- noma (n=4), malignant colon tumors (n=3), as well as Hodgkin's lymphoma, major thalas- semia and acute lymphoblastic leukemia (n=1-2 patients for each disease). Additionally, two patients requested ovarian tissue transplantation after completion of their treatments.

Vitrification of mouse preantral follicles versus slow freezing: Morphological and apoptosis evaluation.

The aim of this study was evaluation of survivability, maturation rate and apoptotic gene expression of preantral follicles after vitrification and slow freezing technique. Normal mouse preantral follicles were randomly divided into three experimental groups. In the control group, follicles were cultured immediately; in the vitrification and slow freezing groups, follicles were cultured after vitrification-warming and slow freezing-thawing procedures. Follicular viability was assessed by using 0.4% trypan blue, and molecular evaluation of messenger RNA levels of apoptosis-related genes was performed by the semi-quantitative RT-PCR method after 3 h of culture. Oocyte maturation rates were also evaluated on day 14 of culture. Survival and maturation rate in the slow freezing group were significantly lower than those in control and vitrification groups (P ≤ 0.05). Although there was no difference in Survivin expression among the three experimental groups, Bcl-2 expression was significantly lower in the slow freezing group compared to the other groups (P ≤ 0.05). The expression of Bax, P53, Fas and Bax/Bcl-2 ratio in the slow freezing group was significantly higher than control and vitrification groups (P ≤ 0.05). Preantral follicle vitrification seems to be better than slow freezing as seen in the survival, maturation and expression rates of apoptotic gene variants.