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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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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 Ca 2+ ), mPB1 - (modified PBS without Ca 2+ ), 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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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 β-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, β-CATENIN, FZD-2 and GSK-3β 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 β-CATENIN staining, while control and vitrification groups only showed β-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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Our objective was to evaluate the apoptosis incidence in immature mouse testicular tissue after two different protocols of vitrification and short-term culture. Testes of 7-day-old Naval Medical Research Institute mice were isolated and distributed into control and vitrification groups. In vitrification 1 group, testes were vitrified using a combination of ethylene glycol and DMSO in three steps, and in vitrification 2 group, testes were vitrified using a combination of ethylene glycol and sucrose in five steps. Then, fresh and vitrified-warmed testis fragments were cultured for 20 hours. Morphology, cell viability, apoptosis incidence, and apoptosis gene expression (BAX, BCL2, Caspase 3, Fas, Fas ligand, p53) were evaluated at 0, 3, and 20 hours of culture by light microscopy, flow cytometry, and real-time polymerase chain reaction, respectively. Significant decrease of early apoptosis (annexin V+/PI- cells in vitrification 1 and 2 groups at 0 hours of culture, 37.34 ± 0.91 and 30.72 ± 2.2, and at 20 hours of culture, 1.46 ± 0.28 and 0.76 ± 0.11, respectively), increase of late apoptosis (annexin V+/PI+ cells in vitrification 1 group at 0 hours of culture, 14.46 ± 0.86, and at 20 hours of culture, 37.18 ± 2.34), and BAX/BCL-2 ratio (in vitrification 1 and 2 groups at 0 hours of culture, 7.31 ± 0.31 and 6.83 ± 1.38, and at 20 hours of culture, 24.08 ± 4.32 and 9.35 ± 1.91, respectively) were observed in vitrification groups during culture period. Caspase 3 expression was significantly decreased in all groups after 3 hours of culture (in control, vitrification 1, and vitrification 2 groups at 0 hours of culture, 1.00 ± 0.0, 1.56 ± 0.09, and 0.79 ± 0.06, and at 20 hours of culture, 0.37 ± 0.0, 0.96 ± 0.10, and 0.12 ± 0.03, respectively). Expression of p53 was significantly lower in vitrification 1 (0.32 ± 0.02) and control (0.50 ± 0.03) groups in 20 hours of culture as compared with vitrification 2 (0.88 ± 0.14) group. Fas (in vitrification 1 and 2 groups at 0 hours of culture, 2.29 ± 0.23 and 1.14 ± 0.15, and at 20 hours of culture, 12.43 ± 0.46 and 6.7 ± 0.48, respectively) and Fas Ligand (in vitrification 1 and 2 groups at 0 hours of culture, 1.2 ± 0.28 and 5.24 ± 0.32, and at 20 hours of culture, 21.75 ± 2.00 and 25.82 ± 2.15, respectively) expressions significantly increased in vitrification groups after 20 hours of culture. Although both vitrification protocols cause cell death via apoptotic and necrotic pathway, it seems that vitrification 1 protocol induces cell death more via apoptotic pathway than via necrosis. The apoptosis incidence after vitrification may have occurred independent of p53., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Our aim was to evaluate the oocyte maturation rate and follicular genes expression pattern during in-vitro culture of vitrified mouse pre-antral follicles. Middle sized pre-antral follicles were isolated mechanically from the ovaries of pre-pubertal mice and distributed in vitrification and control groups. In the vitrification group, follicles were washed in equilibration and vitrification solutions and then were immersed in liquid nitrogen after loading on cryotop tips. After warming in descending concentrations of sucrose solutions, fresh and vitrified-warmed follicles were cultured for 13 days. Follicles survival rate and follicular genes expression were assessed during in vitro culture. Finally, at the end of the culture period oocytes maturation rate were compared in both groups. In the vitrification group, follicles survival rate was lower significantly comparing to the control group (P < 0.05), whereas oocytes maturation rate were similar. Although at the beginning of the culture period, expression of some genes such as Gdf9, Bmp15, Tgfβ1 and BmprII were higher in the vitrification group (P < 0.05), during the rest of the culture period expression pattern of all follicular genes were similar in both groups. In conclusion, survival rate of cryotop vitrified pre-antral follicles reduced during culture period while oocytes maturation and follicular genes expression did not show any noticeable alteration., (Copyright © 2015 Elsevier B.V. All rights reserved.)

To evaluate the effects slow-freezing and vitrification on three dimensional in vitro culture of preantral follicles, ovaries of 12-14 days old female NMRI mice were isolated and randomly assigned to fresh control, slow-freezing and vitrification groups. Slow-freezing was performed using programmable freezer. Vitrification was carried out in a medium consisting of ethylene glycol (EG) and dimethyl sulphoxide (Me2SO) by needle immersion method. middle sized preantral follicles were mechanically isolated and cultured for 12 days in 0.7% sodium alginate gel. The follicles development and quantitative expression of oocyte specific genes (Bmp15, Gdf9, Fgf8) and the growth related genes (Igf1, Kit, Kit-l) were assessed after 1, 8 and 12 days of culture. Both cryopreserved groups showed reduction of follicular survival rates compared to the control group on days 8 and 12 of culture (P < 0.05). Antrum formation rates reduced in slow-freezing after 12 days of culture (P < 0.05). Evaluation of gene expression showed reduction of Bmp15, Gdf9, Fgf8, Kit and Kit-l during 12 days of culture (P < 0.05). Kit and Kit-l expression in slow-freezing group significantly reduced on day 8 of culture (p < 0.05). Igf1 expression was lower in slow-freezing group on 1st day of culture than vitrification and control groups (P < 0.05). Finally, intergroup comparison showed same expression pattern of genes after 12 days of culture. Thus, cryopreservation of mouse ovaries by both methods can preserve most developmental parameters and expression of maturation genes. However, vitrification is a better method for cryopreservation of mouse ovaries due to greater antrum formation and expression of growth related markers., (Copyright © 2015. Published by Elsevier Inc.)

Objective: Reactive oxygen species have effects on gamete quality and gamete interaction; they influence spermatozoa, oocytes, embryos, and their environment. In this study, we evaluated the antioxidant effect of different concentrations of saffron (Crocus sativus L.) aqueous extract (SAE) and its ingredient, crocin, on the improvement of in vitro maturation (IVM) and subsequent in vitro fertilization (IVF) and embryo development of mouse oocytes., Materials and Methods: Cumulus oocyte complexes were collected from ovaries, and germinal vesicle oocytes were cultured in the presence of SAE and crocin. SAE was added at dosages of 5 μg/mL, 10 μg/mL, and 40 μg/mL; dosages of crocin were 50 μg/mL, 100 μg/mL, and 400 μg/mL. All dosages were added to maturation medium and a group without SAE or crocin was considered as the control group. Following IVM, metaphase II oocytes were fertilized and cultured in vitro in order to observe embryo development., Results: Both SAE and crocin improved the rate of IVM, IVF, and in vitro culture. Addition of 40 μg/mL SAE to maturation medium significantly increased the rate of IVM, IVF, and in vitro culture (p < 0.05). Furthermore 100 μg/mL crocin significantly increased the IVM rate compared to the control group (p < 0.05)., Conclusion: Use of SAE during IVM can affect on IVM, IVF, and early embryo development in a dose-dependent manner. SAE appears to have a stronger effect than pure crocin., (Copyright © 2014. Published by Elsevier B.V.)

Vitrification is considered a viable method for cryopreservation of ovarian tissue and selection of methods that minimize follicular damage is important. The objective of the present study was to evaluate the effects of two vitrification methods on ovarian tissue morphology, preantral follicles survival rate during in vitro culture, and relative expression of genes associated with oocyte maturation and cumulus expansion. Ovaries from 12-day-old mice were vitrified in media containing ethylene glycol, dimethyl sulphoxide, and sucrose. Before plunging in liquid nitrogen, ovaries were first loaded into an acupuncture needle (needle immersion vitrification [NIV]) or placed on a cold steel surface for 10 to 20 seconds (solid surface vitrification [SSV]). The integrity of the ovarian tissue was well-preserved after vitrification and was similar controls. Follicle viability in the SSV group was lower (P < 0.05) than in the control group after 6 days of culture and the NIV group after 10 day of culture. Follicle viability after 12 day of culture was 92.8%, 82.1%, and 58.4% in control, NIV, and SSV groups, respectively. Bmp15, Gdf9, BmprII, Alk6, Alk5, Has2, and Ptgs2 gene expression patterns were similar among groups. However, the level of gene expression in the vitrification groups during Days 6 to 10 were higher compared with the control group. In conclusion, ovarian tissue morphologic integrity was well-preserved, regardless of the vitrification method. Vitrification using the needle immersion method resulted in greater follicular survival after 12 day of culture than the SSV method. Gene expression patterns during culture did not seem to explain the reduced survival rate observed in the solid surface group., (Copyright © 2014 Elsevier Inc. All rights reserved.)

This paper reports studies on the effects of re-vitrification by the CPS (Closed Pulled Straw) method on the development of 4-cell stage mouse embryos. The procedure involved culturing 2-cell mouse embryos in G-1 medium until the 4-cell stage followed by the division of the normal 4-cell stage embryos into a control group (non-vitrified) and two experimental subgroups (vitrified and re-vitrified). Embryos in the vitrified subgroup were cryopreserved by the CPS vitrification method. In the second experimental subgroup (re-vitrified), embryos that were already vitrified were warmed and cryopreserved again by the same method. There was no significant reduction in the rate of blastocyst formation after vitrification and re-vitrification. However, re-vitrification reduced the total cell number, ICM (inner cell mass) percent and blastocyst diameter (P<0.05). These results showed that vitrification and re-vitrification by the CPS method did not negatively affect the development of vitrified-warmed 4-cell mouse embryos, whereas re-vitrification significantly reduced both the cell number and diameter of blastocysts., (Copyright © 2011 Elsevier Inc. All rights reserved.)

To determine the optimal vitrification conditions for sheep cumulus-oocyte complexes (COC), good-quality isolated COC were randomly divided into non-vitrified control, conventional straw, cryotop and solid-surface vitrification groups. In the conventional and cryotop methods, the vitrified COC were respectively loaded by conventional straw and cryotop, whereas in the solid-surface group, the vitrified COC were loaded by cryotop and then cooled before plunging in liquid nitrogen. The results indicated that the mean percentage of viability of vitrified-warmed COC was higher in both cryotop groups than that of the conventional group (83.84+/-2.85 and 78.56+/-1.72 versus 63.43+/-1.48%, P<0.05). In the cryotop group, although the mean percentage of oocyte maturation was similar to that in the control group (48.81+/-3.09 versus 51.94+/-3.01%), it was significantly higher than the other vitrification groups (48.81+/-3.09 versus 36.60+/-1.69 and 6.09+/-2.51%, respectively, P<0.05). However, the expression of maturation genes (GDF9, BMP15) was retarded after vitrification. Among the vitrification groups, the cryotop group had better expression. BMPRII was also expressed highly in the control, whereas ALK5 was similar in all groups. In conclusion, direct cryotop, when compared with other vitrification methods, seemed to be safe and could increase the viability, post-warming maturation and maturation-gene expression rates of sheep COC., (Copyright (c) 2009 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)