Objective:The primary objective of this study was to develop an artificial ovary made of polylactic acid (PLA) material, which could potentially be used for fertility preservation. This scaffold was used to carry umbilical cord mesenchymal stem cells (UCMSCs) for xenotransplantation to study follicular growth, viability, and angiogenesis. Materials and Methods:The experimental study involved transplanting human ovarian cortical pieces into 3Dprinted baskets, with or without human UCMSCs. Four groups were considered as control: vitrified-warmed ovarian tissue (OT), OT transplantation without 3D-printed baskets without UCMSCs, OT+D; OT transplantation with 3D-printed baskets and without UCMSCs, and OT+D+C; OT transplantation with 3D-printed baskets and UCMSCs. Eighteen rats were divided into three transplantation groups, with grafts implanted into their back muscles. After one week, comparisons were made between OT, OT+D, and OT+D+C with control groups. Histological evaluations and gene expression analyses, including CX37, CX43, KL, GDF9, and VEGF, were conducted to evaluate factors such as angiogenesis, cell proliferation, and follicle development. Results:The results revealed that OT+D had the highest follicular growth (99.3%) and transition rate (74.08%). OT+D+C indicated increased follicular survival, reduced mortality (P<0.0001), and a higher percentage of primordial follicles (59.1%) compared to OT and OT+D groups. UCMSCs' paracrine secretions likely preserved the follicular reserve. Additionally, OT+D+C exhibited significantly increased blood vessel number and diameter (P<0.0001) compared to other groups. Conclusion:This study underscores the significance of utilizing a three-dimensional basket scaffold to enhance the development of ovarian follicles and facilitate angiogenesis. The integration of stem cells with the scaffold demonstrated a noteworthy synergistic effect, resulting in improved preservation and viability of the follicles and Reduced fibrosis. These findings illuminate the promising potential of scaffold-based strategies in advancing reproductive therapies and tissue engineering, thereby offering prospects for innovative treatments in the future.

This study aims to conduct a comparative study of two strains of laboratory rats, hooded and Wistar, to select a suitable alternative to nude rats for ovarian tissue xenotransplantation. The study investigated the effects of ovarian tissue transplantation using three experimental groups: (1) the control group receiving vitrified-warmed human ovarian tissue, (2) the OTW (ovarian tissue transplantation in Wistar) group with human ovarian tissues in Wistar rats, and (3) the OTH (ovarian tissue transplantation in hooded) group with ovarian tissues transplanted into hooded rats. A total of 12 rats (6 each from the two transplantation groups) were used, with tissue samples implanted in the dorsal neck muscles. Outcomes were evaluated using histological analyses, including hematoxylin and eosin and Masson's trichrome staining, as well as immunohistochemical assessments of CD31 for angiogenesis, fibrosis, and necrosis. The OTH group exhibited more blood vessel numbers and the Vegf gene expression than both the OTW and control groups, with significantly lower levels of fibrosis and necrosis compared to the OTW and control groups. The study indicates that hooded rats could serve as a valuable alternative for specific research when nude mice are not available. Their immune systems are more compatible with human ovarian tissue transplants than Wistar rats, and they exhibit calmer behaviors, making them better suited for laboratory work.

BACKGROUND:We investigated the applicability of human decellularized ovarian scaffold (HDOS) to create an artificial ovary for preserving germ cells and forming follicular-like structures in a mouse chemotherapy-induced premature ovarian failure (chemo-POF) model after seeding stromal cells and transplantation. METHODS:The present study was conducted in three steps. (1) Creation of a mouse POF model, (2) Production of an artificial ovary (AO), (3) Transplantation of AO to ovariectomized mice. For the first step, female NMRI mice received chemotherapy drugs to induce POF. For AO production, mouse ovarian cell suspension (OCS) from the POF model and the control group were isolated separately and seeded into human decellularized ovarian scaffolds (HDOSs). AO was transplanted to ovariectomized intact and POF mice, which were followed up for 2 months (AOII). After transplantation, histological assessments, hormonal evaluation (FSH/E2), and gene expression analysis were conducted (Blimp1). RESULTS:A follicle-like structure formed two months after the transplantation. No significant increase was observed in the FSH level in both the Intact/AOII and POF/AOII groups. The gene expression of Blimp1 showed a significant increase in the Intact/AOII group compared to the other groups. CONCLUSIONS:The bioengineered ovary is capable of forming a follicle-like structure in the POF model after cell seeding and transplantation. We also demonstrated the efficacy of these extracellular matrices (ECMs) in preserving germ cells.

Background: The parallel and continued improvements in both infertility treatment and the management of malignancycases have brought to the forefront the potential for fertility preservation. Using ovarian follicular resourcescan effectively improve reproductive capacity and prevent infertility. The primary aim of this research was to try togenerate an appropriate in vivo environment for the growth of the mouse follicles. Hence, the possible effects of theovarian parenchyma cell suspension were explored on the growth and maturation of preantral follicles in vitro.Materials and Methods: In this experimental study, ovarian parenchymal cells were mechanically dissociated frompreantral follicles of 12-14 days-old NMRI mice and then divided into 5 experimental groups (G1: Control, G2: Freshfollicle with fresh parenchyma cell suspension, G3: Vitrified-warmed follicle with fresh parenchyma cell suspension,G4: Fresh follicle with frozen-thawed parenchyma cell suspension, and G5: Vitrified-warmed follicle with frozen-thawedparenchyma cell suspension). The diameter of the follicles and immature oocytes, viability, antrum formation,resumption of meiosis, in vitro fertilization (IVF), and Gdf9, Bmp6, and Bmp15 gene expression were examined ondifferent periods.Results: The diameter of the follicles and the oocytes on days 4 and 8, as well as the survival rate of the follicles upto day 12, were significantly higher in G2 and G4 compared to the Ctrl group (G1: 73.66%, G2:87.99%, G3: 82.70%,G4: 94.37%, and G5: 78.59%). Expression of growth marker genes for G3, and G5 groups was significantly higherthan other groups, which indicated the protective effects of parenchyma cell suspension on follicles damaged by vitrificationsolutions.Conclusion: The growth, survival, and maturation of preantral follicles could be enhanced by co-culturing them withovarian parenchyma cells. Further studies are needed to optimize the conditions for a successful parenchyma cellsuspension-induced in vitro maturation (IVM) to occur in infertility clinics.

Abstract Purpose The primary objective was to develop an artificial ovary, using a 3D printed scaffold made of polylactic acid (PLA) material which could potentially be used for fertility preservation. In ovarian tissue (OT) transplantation, it was essential to prioritize follicle viability by promoting ideal angiogenesis at the back of neck muscle rats to improve the chances of successful follicle development. Methods The study involved transplanting human frozen-thawed ovarian cortical pieces into 3D-printed PLA polymer baskets, with or without human umbilical cord mesenchymal stem cells (HUC-MSCs). Four groups were formed: control, ovarian transplantation), OT in 3D-printed baskets without stem cells (OT + D), and OT in 3D-printed baskets with HUC-MSCs (OT + D + C). Eighteen rats were divided into three transplantation groups, and grafts were implanted into their back muscles. After one week, comparisons were made between OT, OT + D, and OT + D + C with control groups. Histological and gene expression analyses, including CX37, CX43, KL, GDF9, and VEGF, were conducted to evaluate factors like angiogenesis, cell proliferation, and follicle development. Results The result showed that OT + D had the highest follicular growth (99.5%) and transition rate (74.95%). OT + D + C showed increased follicular survival, reduced mortality (p < 0.0001), and a higher percentage of primordial follicles (59.1%). UCMSCs' paracrine secretions likely preserved the follicular reserve. Additionally, OT + D + C exhibited significantly increased blood vessel number and diameter (p < 0.0001) compared to other groups. Conclusions The study conducted a comprehensive evaluation, analyzing factors such as graft recovery, follicular density, stromal integrity, angiogenesis, and gene expression to understand transplantation outcomes fully. It found that a 3D basket scaffold facilitates ovarian follicle development by promoting angiogenesis and aiding maturation. Furthermore, when combined with stem cells, the scaffold enhances follicular preservation, preventing cell death and maintaining viability through paracrine secretions.

Background: Transplantation of cryopreserved follicles can be regarded as a promising strategy for preserving fertility in cancer patients under chemotherapy and radiotherapy by reducing the risk of cancer recurrence. The present study aimed to evaluate whether fibrin hydrogel supplemented with platelet lysate (PL) could be applied to enhance follicular survival, growth, and angiogenesis in cryopreserved preantral follicle grafts. Materials and Methods: Preantral follicles were extracted from 15 four-week-old NMRI mice, cryopreserved by cryotop method, and encapsulated in fibrin-platelet lysate for subsequent heterotopic (subcutaneous) auto-transplantation into the neck. Transplants were assessed in three groups including fresh follicles in fibrin-15%PL, cryopreserved follicles in fibrin-15%PL, and cryopreserved follicles in fibrin-0% PL. Two weeks after transplantation, histological, and immunohistochemistry (CD31) analysis were applied to evaluate follicle morphology, survival rate, and vascular formation, respectively. Results: Based on the results, fibrin-15% PL significantly increased neovascularization and survival rate (SR) both in cryopreserved (SR=66.96%) and fresh follicle (SR=90.8%) grafts, compared to PL-less fibrin cryopreserved transplants (SR=28.46%). The grafts supplemented with PL included a significantly higher percentage of preantral and antral follicles. Also, no significant difference was observed in the percentage of preantral follicles between cryopreserved and fresh grafts of fibrin-15% PL. However, a significantly lower (P=0.03) percentage of follicles (23.37%) increased to the antral stage in cryopreserved grafts of fibrin-15%PL, compared to fresh grafts (35.01%). Conclusion: The findings demonstrated that fibrin-PL matrix could be a promising strategy to improve cryopreserved follicle transplantation and preserve fertility in cancer patients at the risk of ovarian failure.

Introduction: L-carnitine (LC) has yielded relevant results on ischaemic/ reperfusion (I/R) injuries in organs such as the intestine, liver, kidney, brain and heart, because of its antioxidant activity. We aimed to evaluate the potential of using L-carnitine to improve the quality of grafted ovarian tissues. Materials and methods: The ovaries from 4-6-week old mice were grafted into back muscle site. Transplanted ovaries were divided into four groups. 200 mg/kg LC was injected intraperitoneally one day before surgical operation RESEARCH 24(101), January February, 2020 Medical Science ISSN 2321–7359 EISSN 2321–7367

Chick embryo extract (CEE) contains a variety of growth factors which may improve in vitro follicle growth. Therefore, the effect of CEE on mouse pre-antral follicle culture was evaluated. Different percentages of CEE (0, 0.50%, 1.00%, 5.00% and 10.00%) were added to culture medium. Hence, the osmolarity of media was measured. Pre-antral follicles with diameter of 120-150 μm were isolated from 12-14 days old mouse ovary and cultured for 12 days. After culture, the maturation rate was assessed. Granulosa cells viability was evaluated using MTT test and estradiol levels were evaluated using related radio-immunoassay (RIA). Genes expression (BMP15 and ALK6) was also evaluated. The osmolarity of media and granulosa cells viability were the same in all groups. Estradiol level in group with 10.00% CEE was significantly decreased compared to the control group. After 12 days culture, the percentage of antral follicles development was significantly higher in the group with 5.00% CEE compared to control group. The percentage of metaphase II and germinal vesicle breakdown oocytes was significantly higher in group 5.00% CEE compared to control group. The expression of BMP15 gene in antral follicles in 5.00% CEE and control groups was significantly lower compared to pre-antral follicles. However, the expression of ALK6 gene in antral follicles in 5.00% CEE and control groups was not significantly different compared to pre-antral follicles. The increasing effect of CEE on follicle viability with keeping normal gene expression indicates that addition of proper percentage of CEE to culture media improves culture conditions, making it a possible choice to be used as a follicular growth enhancer in infertility clinics.

One of the challenges that must be overcome during ovarian tissue transplantation is Ischaemia/Reperfusion Injury (IRI). The most important hypothesis explaining the cellular events in I/R processes are calcium overload and oxygen free radicals constitute. Here, we study the effect of verapamil on IRI, and consequently on follicle survival during ovarian transplants in an autograft model. Female mice were randomly assigned into three groups in order to ovarian autotransplantation as follow: Group 1 (Control group), Group 2 (Transplanted group) and Groups 3 (Transplanted + Verapamil group). The grafted ovaries were collected at 3, 7 and 14 days after transplantation for evaluation of follicle content and morphology, apoptosis and Malondialdehyde (MDA) concentration. The results showed that verapamil treatment significantly preserved primordial follicular reserve and reduced the number of degenerated follicles compared to the transplanted group (P < 0.05). MDA levels were significantly higher on the 14th day after transplantation, in group 2 than in group 3. In conclusion, verapamil treatment is effective for the preservation of the follicular pool and reducing tissue damage induced by transplantation of ovarian tissue.

Introdution: One of the major challengs in ovarian tissue transplantation is overcomeing ischemia/ reperfusion injuries. During ischemia–reperfusion processes, oxygen free radicals constitute the most important component that induces damage of the grafted tissues. The aim of this study was to investigate the effect of L-Carnitine (LC) as an antioxidant on heterotopic transplantation of mouse ovarian tissue. Methods: In this laboratory experimental study, 5- week old female NMRI mice were divided into four groups: control, transplanted without administration (autograft), sham group (autograft+ saline) and LC group (autograft+ L- carnitine). Left ovarian tissues were transplanted into the Gluteal muscle for 3 weeks. After this time, ovarian tissues from all groups were removed and fixed in formalin for histological studies. Furthermore, rate of Caspase- 3 was assessed by immunohistochemistry test. Lipid peroxidation was assessed by measuring malondialdehyde (MDA). One-way ANOVA and Tudey test was used to analyze the data using the spss 16 software. Significance was defined as P≤0.0. Results: The study results indicated that total follicular count in transplantedwithout administration and sham groups was significantly lower than the control group (p<0.05), but there was no significant difference between the control and LC groups. In addition, the rate of caspase-3 was decreased in the LC group, but no significant difference existed between all groups (p<0.05). A significant reduction in the concentration of MDA was observed in the LC group than that in the other transplanted groups (p<0.05). Conclusion: In this study, LC could improve the ovarian reserve to some extent, but its effect was not significant.

Objective The aim of this study was to investigate the effects of static magnetic field (SMF) during transplantation of the ovarian tissue into the testis. Materials and Methods In this experimental study, ovaries of 6- to 8-week-old female Naval Medical Research Institute (NMRI) mice were randomly divided into four groups: i. Fresh ovaries were immediately transplanted into the testicular tissue (FOT group), ii. Fresh ovaries were exposed to the SMF for 10 minutes and then transplanted into the testicular tissue (FOT+group), iii. Vitrified-warmed ovaries were transplanted into the testicular tissue (VOT group), and iv. Vitrified-warmed ovaries were transplanted into the testicular tissue and the transplantation site was then exposed to the SMF for 10 minutes (VOT+group). Results The lowest percentages of morphologically dead primordial follicles and the highest percentages of morphologically intact primordial follicles were seen in the FOT+ group (4.11% ± 2.88 and 41.26% ± 0.54, respectively). Although the lowest significant percentage of maturation, embryonic development and fertility was observed in the VOT group as compared to the other groups, the difference in the fertility rate was not significant between the VOT and VOT+groups. Estrogen and progesterone concentrations were significantly higher in the FOT+group than those of the control mice. Conclusion It is concluded that, exposure of the vitrified-warmed ovaries to SMF retains the structure of the graft similar to that of fresh ovaries.

This study was carried out to assess the different ovarian transplantation sites after short-time autografting. Female rats were randomized into five groups, with six rats in each group, including control (intact), cervical subcutaneous transplanted (CST), back subcutaneous transplanted (BST), subfascial transplanted (SFT) and intramuscular transplanted (IMT) groups. In all experimental groups, the right ovary was removed and transplanted into different sites. After three weeks, ovaries were removed for morphology assessment, follicular counting and the rates of corpus luteum (CL) and cyst formation. Transplanted ovaries in BST and SFT groups were full of cysts and did not have sufficient numbers of intact follicles and were excluded from experiments. In IMT and CST groups, re-anastomosis, follicular development and good homogeneity of the stromal tissue were seen. However, the difference in intact antral follicles between CST (7.92 ± 0.02%) and CST-Op (opposite ovary of CST group) (30.99 ± 0.03%) was significant as well as the difference between CST (7.92 ± 0.02%) and control (10.08 ± 0.01%) groups. In addition, the number of intact primordial follicles in the CST-Op (16.58 ± 0.02%) group was significantly less than that of the control (40.40 ± 0.03%) group. Interestingly, the number of CL was significantly increased in the CST-Op (11.71 ± 0.01%) and IMT-Op (9.16 ± 0.02%) groups compared to the control and experimental groups. Although both intramuscular and subcutaneous sites effectively preserved ovarian follicles after three weeks, cervical subcutaneous site was better suited for auto-transplantation in rat.

This study was designed to investigate the effects of applying 1 mT static magnetic field (SMF) during the vitrification process, on the viability of ovarian follicles after vitrification-warming and autotransplantation. The study was conducted in two phases. In the first phase, ovaries of female NMRI mice (6 to 8 weeks old) were randomly divided into three groups: 1- Freshly isolated ovaries fixed in Bouin solution (control group), 2- Ovaries vitrified-warmed without exposure to magnetic field (V1 group) and 3- Ovaries exposed to magnetic field during equilibration step of the vitrification process (V2 group). In the second phase, the vitrified (V1 and V2 groups) and fresh ovarian tissues were autografted into the back muscles of the mice from which the ovaries were extracted. In both phases, morphological aspects and molecular characteristics of active-apoptotic caspase-3 antibody were evaluated. Results indicated the lower percentages of morphologically intact primordial, primary and antral follicles in the V1 group (67.6, 49.5 and 17.6%, respectively) than those of control (97.3, 85.4 and 42.1%, respectively) and V2 (94.1, 78.8 and 40.9%, respectively) groups. In addition, the mean percentages of morphologically intact follicles in the V1 group were statistically lower than those in other groups, after transplantation. The rate of apoptosis in preantral follicles of the V1 group was significantly higher than that in the other groups. It was concluded that exposure of mice ovaries to SMF during vitrification resulted in greater resistance to injuries.

Objective: Retrieval of high quality follicles and oocytes from transplanted ovaries is essential for higher fertility preservation efficiency. The effect of vascular endothelial growth factor (VEGF) was evaluated on the survival rate of preantral follicles following ovarian transplantation. Material and Methods: Prepubertal female mice were divided to 6 groups including: control (C), transplanted with no VEGF treatment (T) and transplanted with different dosages of VEGF [0.5 µg/mL (TV1), 1 µg/mL (TV2), 2 µg/mL (TV3), and 4 µg/mL (TV4)]. Twenty-one days later, the left ovaries were removed and transplanted on gluteal muscle. Each dose was injected directly into transplanted ovary. Twenty-one days after transplantation, the ovaries were taken, and follicles and cumulus-oocyte-complexes (COCs) were released using 26-gauge needles with a stereo microscope. The number of healthy COCs, matured oocytes, and in vitro developed embryos after fertilization in vitro were evaluated to determine the best dose of VEGF. Follicle number and follicular growth was evaluated relative to the dose of VEGF provided. Transplantation and VEGF treatment with the best dose was performed as mentioned above and in vitro follicle growth in transplanted ovaries was compared with opposite ovaries (OPP). Results: COC retrieval was significantly lower in the transplanted groups compared with the control group (p<0.05). The percentage of metaphase II oocytes was significantly lower in the group treated with 4 µg/mL VEGF compared with the controls (p<0.01). In the TV2 (1 µg/mL) and TV3 (2 µg/mL) groups, the percentages of morula and blastocysts were significantly improved compared with the T group (p<0.01). In the OPP group, the number of follicles was significantly higher compared with the transplanted groups (p<0.01). Conclusion: The improving effect of VEGF on in vitro maturation and in vitro development outcome indicates that VEGF administration may increase transplantation efficiency for fertility preservation.

BACKGROUND: Ovarian cryopreservation has emerged as an important method of fertility preservation. Magnetic field enhanced cryopreservation has been considered in recent times as a promising type of ovarian cryopreservation but the effectiveness of the process is still not clear. OBJECTIVE: The aim of the present study was to investigate the effect of applying 1-mT SMF (static magnetic field) on the vitrification of ovarian tissue and the follow-up investigation of the morphology and functions of vitrified- warmed ovarian tissue after transplantation. MATERIALS AND METHODS: Ovaries of 6-8 week-old female mice from the Naval Medical Research Institute (NMRI) were exposed of the static magnetic field during different steps of the vitrification process. Immunohistological studies were performed on the ovaries. RESULTS: The mean percentage of damaged primordial follicles was lowest in control group and the group with ovaries exposed to magnetic field during the equilibration step. The latter group also had the highest percentage of intact primordial follicles after transplantation. CONCLUSION: Exposure of mice ovaries to static magnetic field during first step of vitrification process (the equilibration step) resulted in greater resistance against injury.

Improving in vitro maturation could increase the rate of pregnancy from oocytes matured in vitro. Consequently, patients will be prevented from using gonadotropin with its related side effects. In this study, the maturation medium was enriched by platelet lysate (PL), then maturation and subsequent developments were monitored.

One of the most important factors affecting survival rate of ovarian follicles during transplantation period is proper vascular development. The objective of the study was to evaluate the effect of vascular endothelial growth factor (VEGF) on auto-transplanted ovarian tissue. Twenty-one-day-old female mice (n=30) transplanted as control group and 21-day-old female mice (n=40) were divided into 4 groups that were treated with 0.5, 1, 2 and 4 mu g/mL of VEGF directly injected to auto-transplanted ovarian tissue. Twenty-one days after transplantation, mice were treated with 7.5 IU pregnant mare serum gonadotropin and human chorionic gonadotropin. Transplanted ovaries were removed and sections were prepared from transplanted tissues for staining. The most effective dosage of VEGF on transplanted tissue was determined over H&E (hematoxylin and eosin) staining results. Slides were compared using TUNEL staining and CD31 assay for the most effective dosage. The percentages of preantral and antral follicles were not significantly different between transplanted group with 4 mu g/mL VEGF and non-transplanted group. Lower apoptotic areas and higher CD31 expression were observed in transplanted ovaries treated with 4 mu g/mL VEGF when compared to transplanted ovaries without VEGF treatment. VEGF positively affects the quality of ovarian tissue during transplantation. Survival rate of follicles and follicular development has improved with the effect of VEGF.