Ovarian Tissue Fragments Research Articles

Metallic-based phthalocyanine nanoemulsions for photodynamic purging of ovarian tissue in leukemia patients

For cancer patients with a high risk of ovarian tissue metastasis, ovarian autotransplantation is not advised due to the potential spread of malignant cells. Ex vivo purging of ovarian fragments may offer a more suitable alternative for fertility restoration. Eradicating malignant cells should be done selectively without affecting follicles or ovarian stromal cells (SCs). As a clinically licensed method, photodynamic therapy (PDT) is a minimally invasive treatment to destroy cancer cells. This study evaluates the effectiveness of nanoemulsions (NE) containing two phthalocyanine photosensitizers; aluminum (III) phthalocyanine (AlPc) and zinc (II) phthalocyanine (ZnPc) in eliminating cancer cells. Human leukemic malignant (HL-60) and ovarian stromal cells (SCs) were treated with AlPc/ZnPc loaded NEs with or without diode laser irradiation. HL-60 leukemia cells in 2D culture were eliminated by treatment with 10 nM AlPc-NE or 0.1 µM ZnPc-NE, while no toxicity was observed in SCs. In 3D culture models, although the cells showed more resistance to the NEs as a result of limited oxygen and photosensitizer penetration, the treatment remained selective for cancer cells. These approaches have the potential to eliminate malignant cells from ovarian tissue fragments.

Vitrification of feline ovarian tissue: Comparison of protocols based on equilibration time and temperature

Global contraction of biodiversity pushed most members of Felidae into threatened or endangered list except the domestic cat (Felis catus) thence preferred as the best model for conservation studies. One of the emerging conservation strategies is vitrification of ovarian tissue which is field-friendly but not yet standardized. Thus, our main goal was to establish a suitable vitrification protocol for feline ovarian tissue in field condition. Feline ovarian tissue fragments were punched with biopsy punch (1.5 mm diameter) and divided into 4 groups. Group 1 was fresh control (Fr), while the other three were exposed to 3 vitrification protocols (VIT_CT, VIT_RT1 and VIT_RT2). VIT_CT involved two step equilibrations in solutions containing dimethyl sulfoxide (DMSO) and ethylene glycol (EG) for 10 min each at 4 °C. VIT_RT1 involved three step equilibration in solutions containing DMSO, EG, polyvinylpyrrolidone and sucrose for 14 min in total at room temperature, while in VIT_RT2 all conditions remained the same as in VIT_RT1 except equilibration timing which was reduced by half. After vitrification and warming, fragments were morphologically evaluated and then cultured for six days. Subsequently, follicular morphology, cellular proliferation (expression of Ki-67, MCM-7) and apoptosis (expression of caspase-3) were evaluated, and data obtained were analysed using generalised linear mixed model and chi square tests. Proportions of intact follicles were higher in Fr (P = 0.0001) and VIT_RT2 (P = 0.0383) in comparison to the other protocols both post warming and after the six-day culture. Generally, most follicles remained at primordial state which was confirmed by the low expression of Ki-67, MCM-7 markers. In conclusion, VIT_RT2 protocol, which has lower equilibration time at room temperature has proven superior thus recommended for vitrification of feline ovarian tissue.

Histopathological evaluation of the viability of auto-transplanted ovary following vitrification-thawing process in rabbit as an animal model.

Cryopreservation and re-transplantation of ovarian tissue is a relatively new technique to preserve fertility. This study evaluated the preservation of ovarian follicles after the vitrification-thawing procedure by implanting ovarian fragments into the gluteus muscle of rabbit. Bilateral ovariectomy was performed on each rabbit. The cortices were separated from the medullae of ovaries and divided into four fragments. One fresh cortical fragment was used as a control, fixed in 10.00% formalin. Three fragments underwent vitrification-thawing for two weeks. Two of these vitrified-thawed fragments were auto-transplanted into separate locations within the opened gluteus muscle, while the final fragment was fixed in 10.00% formalin. Eight weeks after re-implantation, biopsies were collected from the ovarian fragments and fixed in 10.00% formalin. The numbers and areas (µm2) of morphologically normal follicles were evaluated on sections stained with Hematoxylin and Eosin through light microscopy. The follicular morphology in the ovarian fragments immediately after vitrification-thawing remained similar to that of the fresh ovary. The number of morphologically intact follicles was significantly lower in the ovarian fragments eight weeks after re-implantation than fresh ovary and ovarian tissue fragments after vitrification-thawing procedure. However, follicular development to the antral stage was observed in all samples eight weeks after re-implantation. There was no statistical difference in the areas (µm2) of primordial, primary, pre-antral, and antral follicles in the ovarian fragments before cryopreservation, immediately after vitrification-thawing, and following re-implantation procedures. Auto-transplantation of ovarian tissue into the gluteus muscle of rabbits could be a viable approach for preserving ovarian follicles after vitrification-thawing procedure.

P-426 In vitro and in vivo toxicity of carboplatin and paclitaxel regimen on BRCA-mutated ovarian tissue fragments

Abstract Study question Does carboplatin and paclitaxel regimen have a toxic cumulative impact on the ovarian reserve of young breast cancer patients with germline Breast Cancer (BRCA) mutation? Summary answer Based on follicle classification and survival through apoptosis and DNA repair mechanism recruitment analyses, gonadotoxic but no cumulative effect was observed in BRCA-mutated ovarian tissue. What is known already Ovarian function recovery and fertility issues remain major concerns for young patients diagnosed with cancer. Breast cancer is the first cancer diagnosed among women under 40 years and around 10% of them are BRCA germline mutation carriers. Beside their predisposition to breast and ovarian cancers, BRCA carriers might be more sensitive to chemotherapy-induced gonadotoxicity due to the role of BRCA in DNA repair mechanism. Current therapies for BRCA-mutated breast cancer patients often include carboplatin and paclitaxel, drugs that are both considered as moderately gonadotoxic. However, the effect of combined therapy in BRCA-mutated ovarian tissue is unknown. Study design, size, duration Ovarian tissue cryopreserved for fertility preservation before chemotherapy and donated for research by breast cancer patients with or without BRCA1/2 mutation (< 35 years at diagnosis) were used in this study. Two models were investigated: 3 days in vitro culture with carboplatin (10 µg/mL) and/or paclitaxel (1 µM) and ovarian tissue xenograft into mice followed by 3 weeks injections of carboplatin (50mg/kg/week) and paclitaxel (10mg/kg every 3 days). Control conditions were performed for each model. Participants/materials, setting, methods Ovarian tissue from 3 BRCA-mutated and 3 non-mutated patients were thawed and exposed in vitro and in vivo to chemotherapy. Follicle density (number of follicles/mm²) as well as ratios of quiescent/activated and healthy/atretic follicles were evaluated following haematoxylin-eosin staining. Apoptosis and DNA repair mechanisms were analysed using TUNEL/GDF9 co-staining and phosphorylated H2A histone family member X (γH2AX)/ZP-3 co-staining, respectively. Genes expression level will be evaluated on isolated follicles to assess the activation process. Main results and the role of chance First, the impact of in vitro culture and in vivo xenotransplantation was evaluated by follicle counting and classification. A decrease of follicular density (5 to 95% irrespective of patient, model, nor condition) and an increase of activated and atretic follicles was observed after both in vitro and in vivo experiments in all conditions. However, a slight increase of quiescent follicle pool was observed in the in vitro control condition, with no difference between mutated and non-mutated fragments. Then, immunofluorescence staining was performed to evaluate follicle damage. For quantification, 30 to 150 follicles per patient were counted within each condition and classified as positive if at least one cell was stained. Apoptosis was assessed with TUNEL/GDF9 co-staining. In both models, the ratio of positive follicles was higher in fragments treated with chemotherapy (40-60%) compared to control conditions (15-20%). Recruitment of DNA repair complex was analysed using γH2AX/ZP3 co-staining. In both models, an increase in positive follicles ratio was observed in treated fragments (15-40%) compared to control conditions (5-10%). Interestingly, the positive follicles ratio was lower in BRCA-mutated (15-18%) than in non-mutated fragments (25-40%) after 3-days in vitro culture, irrespective of the treatment used. Limitations, reasons for caution Reasons of caution include the high inter-variations between patients as well as intra-variations in one patient regarding follicular density within ovarian tissue. Moreover, BRCA-mutated patients included (27.67 ± 2.08) are younger than control ones (33.67 ± 1.53). Ongoing experiments with additional patients should support the results already obtained. Wider implications of the findings Carboplatin and paclitaxel combined exposure in vitro or in vivo does not seem to have a cumulative deleterious impact on ovarian reserve. Ovarian tissue with BRCA-mutation did not appear to be more sensitive to chemotherapy exposure. Further investigations focusing on DNA repair mechanisms will be performed in both models. Trial registration number Not applicable

Enhanced expression of certain biomarkers as a predictive factor for malignant transformation of atypic lesions in ovarian endometriosis

BackgroundPatients with ovarian endometriosis (OE) are known to have a 2.5-fold increase in the risk of malignant neoplasms. OE is polymorphic condition histologically subdivided on two forms: “without atypia” and “with atypia”. First form is considered benign, while second one is precursor of ovarian malignant transformation. AimAssess of the morphological and immunohistochemical characteristics indicative of the risk of malignant neoplastic transformation of OE atypical lesions. Materials and methodsSeventy-eight fragments of ovarian tissue (resected regarding ovarian endometrioid cists) were studied histologically for determination of cystic ovarian endometriosis foci with and without atypia. Thirty-five histological slides with OE and 8 slides with adenocarcinomas of different types were studied immunohistochemically. Monoclonal antibodies to Ki-67, Всl-2, р53 proteins and polyclonal antibodies to HNF-1β were used. Results were evaluated by means of Histological score (H-score) method. ResultsThirty-five foci of cystic OE without atypia and 32 foci with epithelial atypia were observed. In foci with epithelial atypia, the expression of Ki-67, Bcl-2 and HNF-1β was significantly higher relatively to the foci without atypia, while expression of р53 was the same in foci with and without atypia. From 4 subtypes of studied ovarian adenocarcinomas the expression of HNF-1β was only observed in clear cell adenocarcinoma. ConclusionEnhanced expression of biomarkers Ki-67, Bcl-2 and HNF-1β in cystic OE with atypia indicates on the risk of malignant transformation of lesions with atypia. Using of these biomarkers can be useful for differential diagnostics of OE with and without atypia and for decision about surgical treatment.

Co-culture of human cryopreserved fragmented ovarian tissue with theca progenitor cells derived from theca stem cells.

Despite the significant advances in the in vitro development of human primordial follicles, it is still a challenging approach with great potential for improvements. Therefore, the present study aimed to investigate the effect of a feeder layer of human theca progenitor cells (hTPCs) on the development of primordial follicles embedded in human ovarian tissue. Fragments of frozen-thawed ovarian tissue were activated using the vanadate-derivative dipotassium bisperoxo (5-hydroxy-pyridine-2-carboxylic) oxovanadate (V) and kit ligand for 24h. Then, the specimens were divided into the co-culture and mono-culture groups and were cultured with and without a hTPC feeder layer for 6days, respectively. Afterward, the follicles were counted and classified, and the hormone levels and expression levels of apoptosis- and folliculogenesis-related genes were assessed. Both culture groups showed significant follicle growth (P < 0.05). However, the co-culture group had a significantly higher number of growing follicles compared to the other group (P < 0.05). Moreover, the expression levels of ZP1, ZP2, ZP3, BMP-7, AMH, and GDF9 were significantly higher in the co-culture group compared to the other group (P < 0.05), while the expression levels of P53 and CASP3 were significantly lower (P < 0.05). Also, the concentrations of estradiol, progesterone, testosterone, and androstenedione were significantly higher in the co-culture group compared to the other group (P < 0.05). The present study results provided novel evidence on the direct role of hTPCs in the growth and development of human primordial follicles. However, there is a need for future studies to illustrate the underlying mechanisms. Schematic summary of the results. According to our results, the expression of ZP1, ZP2, ZP3, and GDF9 in the oocytes, AMH in the granulosa cells, and BMP4 in the theca cells of the co-culture group were significantly higher than those of the mono-culture and non-culture groups, while the expression of apoptotic genes (BAX, CASP3, and P53) was significantly lower. Moreover, the co-culture group showed significantly increased levels of estradiol, progesterone, testosterone, and androstenedione in its culture media compared to the mono-culture groups.

Human ovarian gross morphology and subanatomy across puberty: insights from tissue donated during fertility preservation

To study ovarian gross morphologic and subanatomic features across pubertal development. Prospective cohort study. An academic medical center with specimens collected from 2018-2022. Tissue was obtained from prepubertal and postpubertal participants (0.19-22.96 years) undergoing ovarian tissue cryopreservation before treatment that put them at a significantly or high increased risk of developing premature ovarian insufficiency. Most participants (64%) had not received chemotherapy at tissue collection. None. Ovaries procured for fertility preservation were weighed and measured. Ovarian tissue fragments released during processing, biopsies used for pathology, and hormone panels were analyzed for gross morphology, subanatomic features, and reproductive hormones. Graphical analysis of best-fit lines determined age at maximum growth velocity. Prepubertal ovaries were significantly (1.4-fold and 2.4-fold) smaller than postpubertal ovaries by length and width and 5.7-fold lighter on average. Length, width, and weight grew in a sigmoidal pattern with age. Prepubertal ovaries were less likely to display a defined corticomedullary junction (53% vs. 77% in postpubertal specimens), less likely to have a tunica albuginea (22% vs. 93% in postpubertal specimens), contained significantly more (9.8-fold) primordial follicles, and contained primordial follicles at significantly deeper depths (2.9-fold) when compared with postpubertal ovaries. Ovarian tissue cryopreservation is a resource to study human ovarian biology and pubertal development. Maximum growth velocity occurs late within the pubertal transition (Tanner 3+) after changes in subanatomic features. This ovarian morphology model adds to foundational knowledge of human ovarian development and supports ongoing transcriptomics research.

Ex vivo purging of cancer cells from ovarian tissue using photodynamic therapy: a novel strategy to restore fertility in leukemia patients.

Is it possible to purge leukemia cells from ovarian tissue (OT) fragments before transplantation? Our photodynamic therapy (PDT) approach has been shown to efficiently destroy leukemia cells from tumor-infiltration mimicking models (TIMs), indicating the feasibility of this technique to purge OT samples. Autotransplantation of cryopreserved OT is the most suitable option to preserve fertility for prepubertal girls and women who require immediate cancer treatment. Up until now, more than 200 live births have already been reported after OT cryopreservation and transplantation. Leukemia is the 12th most common cancer in Europe among prepubertal girls and women of reproductive age and in 2020, the estimated number of new leukemia cases was higher than 33000 in girls between 0 and 19 years old. Unfortunately, once their health has been restored, autotransplantation of cryopreserved OT for leukemia patients is not advised due to the high risk of transferring malignant cells back to the patient leading to leukemia recurrence. To safely transplant the OT from leukemia patients and restore their fertility, our goal was to develop a PDT strategy to eliminate leukemia ex vivo. To this end, we designed OR141-loaded niosomes (ORN) to create the most effective formulation for ex vivo purging of acute myelogenous leukemia cells from OT fragments (n = 4). Moreover, to ensure that such treatments are not harmful to follicle survival and development so they can be deemed a potential fertility restoration alternative, the effect of the ORN-based PDT purging procedure on follicles was assessed after xenografting the photodynamic-treated OT in SCID mice (n = 5). The work was carried out between September 2020 and April 2022 at the Catholic University of Louvain. After establishing the best ORN formulation, our PDT approach was used to eradicate HL60 cells from ex vivo TIMs prepared by microinjection of a cancer cell suspension into OT fragments. The purging efficiency was analyzed by droplet digital polymerase chain reaction and immunohistochemical analyses. Additionally, we evaluated the effect of ORN-based PDT on follicle density, survival and development, and tissue quality in terms of fibrotic areas and vascularization after 7-day xenotransplantation to immunodeficient mice. The ex vivo purging of TIMs demonstrated that our PDT strategy could selectively eradicate the malignant cells from tissue fragments without affecting OT normal cells, as evidenced by PCR and immunohistochemical analysis. Regarding the effect of our PDT approach on follicle population and OT quality, our results after xenotransplantation revealed no significant difference between the follicle density of control (non-treated, grafted OT) and PDT-treated groups (2.38 ± 0.63 and 3.21 ± 1.94 morphologically normal follicles/mm2, respectively). In addition, our findings showed that the control and PDT-treated OT could be equally vascularized (7.65 ± 1.45% and 9.89 ± 2.21%, respectively). Similarly, the proportions of fibrotic area did not differ between the control (15.96 ± 5.94%) and PDT-treated groups (13.32 ± 3.05%). N/A. This study did not use OT fragments from leukemia patients, but TIMs created after injection of HL60 cells into OT from healthy patients. Therefore, while the results are promising, whether our PDT approach will be equally successful in eliminating malignant cells from leukemia patients remains to be assessed. Our results showed that the purging procedure causes no significant impairment effect on follicle development and tissue quality, suggesting that our novel PDT procedure could be a promising strategy to destroy leukemia cells in fragments of OT, allowing safe transplantation in cancer survivors. This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS-PDR Convention grant number T.0004.20 awarded to C.A.A.); Fondation Louvain (awarded to C.A.A.; a Ph.D. scholarship awarded to S.M., as part of a legacy from Mr Frans Heyes, and a Ph.D. scholarship awarded to A.D. as part of a legacy from Mrs. Ilse Schirmer); and Foundation Against Cancer (grant number 2018-042 awarded to A.C.). The authors declare no competing interests.

Examination of the Effects of Thoracic Ganglion and Brain Extracts on Vitellogenin Gene Expression in the Ovary of Whiteleg Shrimp, Litopenaeus vannamei, and Possible Vitellogenesis-Stimulating Activity

In order to examine the role of the central nervous system in regulating ovarian maturation in the whiteleg shrimp, Litopenaeus vannamei, we incubated ovarian explants taken from adult shrimp with either brain or thoracic ganglion extracts at the level of five tissue equivalents for 6 h. For assessment of the effects of each tissue extract, transcriptional expression of vitellogenin mRNA in incubated ovarian tissue fragments was analyzed by quantitative real-time PCR. Results showed that crude brain extracts and a 90% aqueous acetonitrile eluate upregulated expression levels in a significant manner. These findings suggest that factors originating from brain tissue are involved in ovarian maturation, but that the thoracic ganglia do not likely harbor significant quantities of stimulatory substances. In the future, this knowledge may be useful in developing technology for inducing maturation and spawning in captive female penaeid.

Feline ovarian tissue vitrification: The effect of fragment size and base medium on follicular viability and morphology

To achieve optimal vitrification, tissue structure and fragment size represent a challenge for obtaining sufficient cooling velocity. Theoretically, thin ovarian tissue fragments lead to higher surface contact, hence higher solute penetration. Another critical factor is the concentration of cryoprotectants (CPA): CPA toxicity may occur with high concentrations, and as such, this may induce local apoptosis. Therefore two experiments were conducted: In experiment I, we compared the effect of sucrose supplementation in vitrification solution along with ovarian fragments of different sizes on post-warming tissue viability and follicle architecture. Fragments of two different sizes, with a thickness and radius of 1.5 × 0.75 mm and 3 × 1.5 mm respectively were vitrified in vitrification solution without sucrose and with 0.5 M sucrose supplementation. Post-warming, fragments of ovarian tissue (fresh and vitrified) were evaluated for viability (Calcein AM/Propidium Iodide) and for morphology (hematoxylin-eosin). In experiment II, we aimed to reduce cryoprotectant toxicity by using lower CPA concentrations in combination with an optimized carrier medium (HypThermosol®; HTS). Ovarian tissue fragments were randomly allocated to five groups (A: fresh controls; B: vitrified in GLOBAL® TOTAL® LP w/HEPES with 15% ethylene glycol (EG) and 15% DMSO; C: vitrified in HTS with 5% EG and 5% DMSO; D: vitrified in HTS with 10% EG and 10% DMSO; E: vitrified in HTS with 15% EG and 15% DMSO). Fragments (fresh and vitrified) were evaluated for morphology (hematoxylin-eosin) and for apoptosis through the activity of caspase-3. Results showed that follicular morphology was affected by the size of the fragment; smaller sized fragments contained a greater proportion of intact follicles (53.8 ± 2.0%) compared to the larger fragments (40.3 ± 2.0%). Our results demonstrated that 1.5 × 0.75 mm sized pieces vitrified in a vitrification solution supplemented with 0.5 M sucrose had more intact follicles (54.8 ± 1.3%; P = 0.0002) after vitrification. In addition, HTS presented no additional protective effect as a base medium, neither for follicular morphology nor apoptotic rate.

Transplantation of small ovarian tissue fragments using pipelle device is effective: method evaluation and reproductive outcomes.

To assess the feasibility, effectiveness, and reproductive outcomes of transplantation of tiny cryopreserved ovarian pieces through a pipelle cannula during laparoscopic surgery. A retrospective study of patients who underwent ovarian tissue transplantation for fertility restoration between 2004 and 2022. The "pipelle group" had their ovarian cortex cut into tiny pieces of ~ 1-2 mm3 before cryopreservation. The pieces were too small to be handled and transplanted via standard laparoscopic tools. Transplantation was performed using a pipelle cannula during laparoscopic surgery. The "control group" underwent transplants of ovarian cortex pieces 1-2mm thick, measuring approximately 25-50 mm2 pieces, using standard procedures. The pipelle group consisted of 4 patients aged 19, 21, 27, and 28years old at ovarian tissue cryopreservation (OTC). The control group consisted of 14 patients aged 21-30years old. All pipelle patients restored their endocrine activity, and all of them conceived. FSH levels dropped during the first 3months following the pipelle transplant. IVF cycle outcomes were similar for both groups. All patients from the pipelle group conceived, resulting in 5 pregnancies and 4 live births (one patient had 2 deliveries, and one additional pregnancy is ongoing), compared to the control group, where 8 patients achieved a total of 20 pregnancies and 18 live births. Pipelle transplantation for tiny cryopreserved ovarian pieces is feasible and effective. This study opens a door for patients who had their ovaries cut into small pieces and may even simplify the procedure in some instances, making ovarian transplant more accessible. (#6531-19-SMC) [18/09/2019].

New hyaluronan-based biomatrix for 3-D follicle culture yields functionally competent oocytes

BackgroundEncapsulation of follicles within a biomatrix is one approach to maintaining 3-D follicle architecture during culture. Hyaluronan is one component of the natural extracellular matrix (ECM) that provides support to cells in vivo. This report describes the application of a novel tyramine-linked hyaluronan for 3-D in vitro follicle culture and the production of developmentally competent metaphase II oocytes.Materials and MethodsEnzymatically isolated mouse preantral follicles or follicle clusters (FL-C) from fresh or vitrified ovaries were encapsulated in 3 mg/ml of hyaluronan gel (HA). Follicle growth, antrum formation and meiotic maturation to metaphase II oocytes was monitored. Chromatin staining was used to assess GV oocyte progression towards meiotic competence. Functional competence of in vitro matured (IVM) oocytes was evaluated by in vitro fertilization and ability to develop to blastocyst. Modifying the HA gel by inclusion of laminin (HA-LM), mouse sarcoma extracellular matrix (Matrigel;HA-MG) or placental extracellular matrix (HA-PM) was also tested to see if this might further enhance IVM outcomes.ResultsA total of 402 preantral follicles were cultured in HA gel. After hCG trigger, 314 oocyte-cumulus complexes ovulated from the embedded follicles. Meiotic maturation rate to the metaphase II stage was 73% (228/314). After insemination 83% (188/228) of IVM oocytes fertilized with a subsequent blastulation rate of 46% (87/188). A pilot transfer study with 3 recipient mice resulted in the birth of a single pup. HA gel supported individually isolated follicles as well ovarian tissue fragments containing clusters of 6–8 preantral follicles. Meiotic maturation was lower with FL-clusters from vitrified versus fresh ovaries (34% and 55%, respectively; p < 0.007). Modification of the HA gel with ECMs or laminin affected antrum formation and follicle retention. Maturation rates to the metaphase II stage were however not significantly different: 74% for HA gel alone as compared to HA-LM (67%), HA-MG (56%) and HA-PM (58%).ConclusionHyaluronan gel is an effective and versatile extracellular matrix based biomaterial for 3-D culture of ovarian follicles. This culture model allowed ovulation of functionally competent metaphase II oocytes, capable of fertilization, genomic activation and blastulation. Future testing with human follicles that require longer in vitro culture times should be considered.

Photodynamic therapy using OR141-loaded nanovesicles for eradication of leukemic cells from ovarian tissue

In 2020, the estimated number of new leukemia cases was higher than 30,000 in girls between 0 and 19 years old. Due to cancer treatment, some of these patients may lose both endocrine and reproductive functions. Transplantation of cryopreserved ovarian tissue is not advised after cancer remission because it has a high risk of reintroducing malignant cells in the patient, potentially leading to leukemia recurrence. To safely transplant the ovarian tissue from these patients and restore their fertility, our goal was to develop a photodynamic therapy (PDT) strategy to eliminate leukemia ex vivo. To this end, we designed, optimized, and characterized OR141-loaded niosomes (ORN) to develop the most effective formulation for ex vivo purging ovarian fragments from chronic myelogenous leukemia cells. After establishing the best ORN formulation, the PDT efficiency of optimized ORN was determined for human ovarian stromal cells and acute myeloid leukemia cell line (HL60). Blank niosomes treatment on ovarian stromal cells causes no significant toxicity, showing that the composition of the nanoparticle is not toxic. On the other hand, the in vitro studies showed that while ovarian stromal cells were still viable (82.04 ± 2.79%) after the treatment by 0.5 µM ORN, the same treatment yielded 95.43 ± 3.89% toxicity and cell death in the cancer cells. In conclusion, our results showed that our novel PDT procedure could be a promising strategy to destroy leukemia cells in ovarian tissue fragments allowing safe transplantation in cancer survivors.

O-035 Ex vivo photodynamic therapy approach using a nanovesicular photosensitizer to allow safe autotransplantation of ovarian tissue from leukemia patients

Abstract Study question Is it possible to purge leukemia cells from ovarian tissue (OT) fragments before transplantation? Summary answer Our photodynamic therapy (PDT) approach showed to efficiently destroy leukemia cells from a tumor model, indicating the feasibility of this technique to purge OT samples. What is known already Autotransplantation of the cryopreserved OT is the most suitable option to preserve fertility for prepubertal girls and women that require immediate cancer treatment [1, 2]. Up to now, more than 200 live births have already been reported after OT cryopreservation and transplantation [3]. Leukemia is the 12th most common cancer in Europe among prepubertal girls and women of reproductive age [4]. Unfortunately, once their health has been restored, autotransplantation of cryopreserved OT for leukemia patients is not advised due to the high risk of transferring malignant cells back to the patient, leading to leukemia recurrence [3]. Study design, size, duration To safely transplant the OT from these patients and restore their fertility, our goal here was to develop a PDT strategy to eliminate leukemia ex vivo. To this end, we designed OR141-loaded niosomes (ORN) to create the most effective formulation for ex vivo purging OT fragments from myelogenous leukemia cells. Participants/materials, setting, methods After establishing the best ORN formulation, our PDT approach was used to eradicate HL60 from ex vivo tumor models prepared by microinjection of cancer cell suspension in ovarian fragments [5]. Additionally, we evaluated the effect of ORN-based PDT on follicle density, survival, and tissue quality was evaluated after 7-day xenotransplantation to SCID mice. Main results and the role of chance The ex vivo purging of tumor models demonstrated that the PDT strategy with ORN could selectively eradicate the malignant cells from tissue fragments without affecting ovarian tissue normal cells, as evidenced by PCR and immunohistochemical analysis. Regarding the effect of our PDT approach on follicle population and OT quality, our results after xenotransplantation revealed no significant difference between the follicle density of control (non-treated, grafted OT) and ORN-treated groups (2.38 ± 0.63 and 3.21 ± 1.94 morphologically normal follicles/mm2, respectively). In addition, the results showed that the control and ORN-treated ovarian tissue can be equally vascularized (7.65±1.45% and 9.89±2.21%, respectively). Moreover, the proportion of fibrotic area in control and ORN-treated groups were 15.96±5.94%, and 13.32±3.05%, which is not significantly different from the pre-graft fragments (14.22±6.65%). Limitations, reasons for caution This study did not use OT fragments from leukemia patients, but tumor models created after injection of HL60 cells in OT from healthy patients. Therefore, while the results are promising, it still remains to assess if our PDT approach will be equally successful to eliminate malignant cells from leukemia patients. Wider implications of the findings Our results showed that the purging procedure causes no significant impairing effect on follicles development and tissue quality, suggesting our novel PDT procedure could be a promising strategy to destroy leukemia cells in fragments of ovarian tissue allowing its safe transplantation in cancer survivors. Trial registration number not applicable

O-266 Encapsulation of human preantral follicles in an optimized hydrogel with similar mechanical strength of reproductive-age ovarian tissue

Abstract Study question Could a hydrogel with similar mechanical properties of reproductive age ovarian cortex support the growth of human preantral follicles? Summary answer Yes, our optimized PEGylated fibrin hydrogel with tailored mechanical strength provides a suitable substrate for follicle survival and growth. What is known already The most challenging part of assembling an engineered ovary is developing an appropriate scaffold that can maintain follicle spatial architecture and allow them to have radial growth and development1-3. Fibrin is a promising biomaterial due to its biocompatibility and bioactivity. Although fibrin has been used as a scaffold in fabricating different tissues such as the ovary, it has a fast degradation rate resulting in loss of physical support for the isolated follicles4-6. Therefore, various approaches such as physical or chemical modifications have been investigated to overcome the fibrin limitation7-10. Study design, size, duration PEGylation as a chemical reaction was used to control fibrin degradation. Moreover, to create an appropriate scaffold for encapsulating human preantral follicles, the Response Surface Methodology was used to find a hydrogel formulation with similar mechanical properties to the reproductive-age ovarian cortex. The chosen hydrogel was used to in vitro culture isolated human preantral follicles for 7 days. Participants/materials, setting, methods Design-Expert® software was employed to optimize PEGylated fibrin formulation to match mechanical properties of human ovarian tissues. Preantral follicles isolated from reproductive age patients were embedded in the hydrogel and in vitro cultured for 7 days. Follicle viability and diameter were evaluated on days 1 and 7. Moreover, confocal microscopy analysis was performed on whole follicles to confirm their growth by Ki67 staining of granulosa cells. Main results and the role of chance In this study, the biomechanically optimized PEGylated fibrin formulation was obtained by mathematical modeling. The hydrogel formulation was established by setting the specific goal of targeting 3178 ± 245 Pa, Young’s modulus of ovarian tissue fragments in reproductive-age women11, in order to obtain the optimized concentration of PEGylated fibrinogen and thrombin. Our results showed that the optimum condition with the desirability of 97.5% occurred by the PEGylated fibrin hydrogel containing 39.06 mg/ml of PEGylated fibrinogen and 50.36 IU/ml of thrombin with Young’s modulus of 3512.31±656.97 pa. Isolated follicles cultured in our hydrogel significantly increased in their diameter from 48.5±10.8µm (day 1) to 222.5±84.5µm (day 7), indicating the suitability of the optimized PEGylated fibrin formulation to support radial follicle growth. Moreover, follicles survival on days 1 and 7 did not significantly differ (86.9±8.9% and 83.4±6.4%, respectively). Follicle growth was confirmed by the presence of Ki67-positive granulosa cells from follicles at day 7. Limitations, reasons for caution In this study, the optimized hydrogel was first evaluated only in vitro, which is different from the actual physiological condition. Therefore, it is essential to perform a study analyzing the follicles after their encapsulation in the optimized hydrogel and transplantation, which will be the next step of our study. Wider implications of the findings Our results introduced an appropriate biomaterial, which resembles Young’s modulus of ovarian tissues in reproductive age, for encapsulating human preantral follicles. The optimal concentration of PEGylated fibrin allowed follicles to maintain their viability and have radial growth. Moreover, PEGylation enhanced the fibrin stability and physical support of follicles. Trial registration number not applicable

Complete Purging of Ewing Sarcoma Metastases from Human Ovarian Cortex Tissue Fragments by Inhibiting the mTORC1 Signaling Pathway.

Restoration of fertility by autologous transplantation of ovarian cortex tissue in former cancer patients may lead to the reintroduction of malignancy via the graft. Pharmacological ex vivo purging of ovarian cortex fragments prior to autotransplantation may reduce the risk of reseeding the cancer. In this study we have investigated the capacity of Everolimus (EVE), an inhibitor of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway, to eradicate Ewing’s sarcoma (ES) from ovarian tissue by a short-term ex vivo treatment. Exposure of experimentally induced ES tumor foci in ovarian tissue to EVE for 24 h completely eliminated the malignant cells without detrimental effects on follicle morphology, survival or early folliculogenesis. This indicates that effective purging of ovarian cortex tissue from contaminating ES tumor foci is possible by short-term exposure to EVE.

O-191 Assessing the use of tumour-specific DARPin-toxin fusion proteins for ex vivo purging of cancer metastases from human ovarian cortex tissue fragments before autotransplantation

Abstract Study question Is it possible to eradicate cancer cells from ovarian cortex by using tumour-specific designed ankyrin repeat protein (DARPin)-toxin fusion proteins, without compromising the ovarian tissue? Summary answer Purging ovarian cortex ex vivo from experimentally induced breast cancer tumour foci is possible by tumour-targeted DARPin-toxin fusion proteins trough inhibition of protein synthesis. What is known already Ovarian tissue cryopreservation and autotransplantation is a successful technique for fertility restoration in cancer patients. The procedure is not without risk since malignant cells may still be present in the graft. Procedures to detect cancer cells render the tissue fragment useless for autotransplantation. Strategies to circumvent this problem such as in vitro maturation of follicles or the construction of artificial ovaries are pursued but are still experimental. Alternatively, we have shown ex vivo purging of ovarian cortex is possible by elimination of rhabdomyosarcoma after treatment with verteporfin. This allows treatment of cortex fragments before autotransplantation without compromising ovarian tissue integrity. Study design, size, duration Human ovarian cortex fragments harbouring breast cancer tumour foci were exposed for 24 h to DARPins fused to the translocation and catalytic domain of Pseudomonas aeruginosa exotoxin A (DARPin-toxin fusion proteins) targeting EpCAM or HER2. After treatment with the DARPin-toxin fusion proteins the tissue was cultured for an additional 6 days to allow any remaining tumour cells to form foci. In addition, the functional integrity of the ovarian tissue was analysed after purging. Participants/materials, setting, methods Breast cancer cell lines expressing different levels of EpCAM and HER2 were introduced in human ovarian tissue to form tumour foci. After purging with DARPin-toxin fusion proteins, the presence of any remaining cancer cells in the tissue was analysed with (immuno)histochemistry and RT-qPCR. Possible detrimental effects on the viability of ovarian cortex and follicles were determined by (immuno)histology, a follicular viability assay and an assay to determine the in vitro growth capacity of small follicles. Main results and the role of chance Ovarian cortex harbouring EpCAM-positive breast cancer cells showed a significant decrease in the number of tumour foci after treatment with the EpCAM-targeted DARPin-toxin fusion proteins. Although exposure to the EpCAM-specific DARPin had no effect on morphology or viability of follicles, a decrease in oocyte viability after in vitro growth experiments was observed, presumably due to low level expression of EpCAM on oocytes. In contrast to the EpCAM-specific DARPin-toxin fusion protein, the DARPin-toxin fusion protein targeting HER2 had no detrimental effects on morphology, viability or in vitro growth of follicles while foci of HER2-positive breast cancer cells were severely affected as indicated by the presence of apoptotic bodies, tumour cell remnants and the absence of viable tumour cells. The histological results after purging with the HER2-specific DARPin-toxin fusions proteins were confirmed by RT-qPCR, showing a decrease to basal levels of HER2 mRNA in the ovarian cortex tissue. Limitations, reasons for caution The effect of DARPin-toxin fusion proteins depends heavily on the expression of their target on the cancer cell. The target protein should not be expressed by ovarian cortex as this may lead to tissue damage. The functional integrity of ovarian cortex after the treatment requires further investigation in vivo. Wider implications of the findings Purging metastases from ovarian cortex without harming ovarian tissue is possible by targeting tumour specific surface expressed antigens with DARPin-toxin fusion proteins. Purging ovarian cortex tissue with DARPin-toxin fusion proteins provides a feasible therapeutic strategy to prevent reintroduction of cancer by autotransplantation in case of malignancies expressing tumour-specific surface markers. Trial registration number not applicable

МОРФОЛОГИЧЕСКИЕ ИЗМЕНЕНИЯ ТКАНЕЙ ПРИ ПРИМЕНЕНИИ БИПОЛЯРНОЙ КОАГУЛЯЦИИ И НЕОДИМОВОГО ЛАЗЕРА В ГИНЕКОЛОГИИ

The article is devoted to a comparative analysis of morphological changes in the tissues of the reproductive system of women under the influence of a neodymium laser and bipolar coagulation during laparoscopic operations. The results of assessing the reliability of differences in morphometric indicators of tissue damage (sections of the fallopian tubes, fragments of ovarian tissue, membranes of benign cystic formations of the ovaries, leiomyomatous nodes of the body of the uterus) under the influence of electro magnetic radiation of various wavelengths are presented. The recorded changes directly depend both on the applied heat or light energy, and on the histological structure of the tissue under study. Fundamental differences in artifactual changes in tissues of the reproductive system were revealed when using bipolar coagulation and laser radiation in surgical practice, and the dependence of morphological changes on the wavelength of a neodymium laser was established for the first time.

Dynamic Characterization of the Biomechanical Behaviour of Bovine Ovarian Cortical Tissue and Its Short-Term Effect on Ovarian Tissue and Follicles.

The ovary is a dynamic mechanoresponsive organ. In vitro, tissue biomechanics was reported to affect follicle activation mainly through the Hippo pathway. Only recently, ovary responsiveness to mechanical signals was exploited for reproductive purposes. Unfortunately, poor characterization of ovarian cortex biomechanics and of the mechanical challenge hampers reproducible and effective treatments, and prevention of tissue damages. In this study the biomechanical response of ovarian cortical tissue from abattoir bovines was characterized for the first time. Ovarian cortical tissue fragments were subjected to uniaxial dynamic testing at frequencies up to 30 Hz, and at increasing average stresses. Tissue structure prior to and after testing was characterized by histology, with established fixation and staining protocols, to assess follicle quality and stage. Tissue properties largely varied with the donor. Bovine ovarian cortical tissue consistently exhibited a nonlinear viscoelastic behavior, with dominant elastic characteristics, in the low range of other reproductive tissues, and significant creep. Strain rate was independent of the applied stress. Histological analysis prior to and after mechanical tests showed that the short-term dynamic mechanical test used for the study did not cause significant tissue tear, nor follicle expulsion or cell damage.

Transcriptomic Analysis of the Kuruma Prawn Marsupenaeus japonicus Reveals Possible Peripheral Regulation of the Ovary.

Crustacean reproduction has been hypothesized to be under complex endocrinological regulation by peptide hormones. To further improve our understanding of the mechanisms underlying this complex regulation, knowledge is needed regarding the hormones not only of the central nervous system (CNS) such as the X-organ/sinus gland (XOSG), brain, and thoracic ganglia, but also the peripheral gonadal tissues. For example, in vertebrates, some gonadal peptide hormones including activin, inhibin, follistatin, and relaxin are known to be involved in the reproductive physiology. Therefore, it is highly likely that some peptide factors from the ovary are serving as the signals among peripheral tissues and central nervous tissues in crustaceans. In this work, we sought to find gonadal peptide hormones and peptide hormone receptors by analyzing the transcriptome of the ovary of the kuruma prawn Marsupenaeus japonicus. The generated ovarian transcriptome data led to the identification of five possible peptide hormones, including bursicon-α and -β, the crustacean hyperglycemic hormone (CHH)-like peptide, insulin-like peptide (ILP), and neuroparsin-like peptide (NPLP). Dominant gene expressions for the bursicons were observed in the thoracic ganglia and the ovary, in the CNS for the CHH-like peptide, in the heart for NPLP, and in the ovary for ILP. Since the gene expressions of CHH-like peptide and NPLP were affected by a CHH (Penaeus japonicus sinus gland peptide-I) from XOSG, we produced recombinant peptides for CHH-like peptide and NPLP using Escherichia coli expression system to examine their possible peripheral regulation. As a result, we found that the recombinant NPLP increased vitellogenin gene expression in incubated ovarian tissue fragments. Moreover, contigs encoding putative receptors for insulin-like androgenic gland factor, insulin, neuroparsin, and neuropeptide Y/F, as well as several contigs encoding orphan G-protein coupled receptors and receptor-type guanylyl cyclases were also identified in the ovarian transcriptome. These results suggest that reproductive physiology in crustaceans is regulated by various gonadal peptide hormones, akin to vertebrates.