Cortex Fragments Research Articles

P-343 Assessing the effect of adipose tissue-derived stem cell-conditioned medium on follicles and stromal cells in cultured ovarian tissue

Abstract Study question Does addition of adipose tissue-derived stem cell-conditioned medium (ASC-CM) enhance follicle survival and development and stromal cell viability in ovarian tissue culture? Summary answer Adding ASC-CM to ovarian tissue in vitro culture (IVC) promotes follicle survival, development, estradiol (E2) production, and stromal cell viability. What is known already Despite extensive efforts to optimize ovarian tissue IVC conditions, outcomes of follicle in vitro development remain suboptimal, mainly showing poor follicle survival and growth. IVC conditions that best resemble the in vivo environment are likely to generate less cellular distress and yield better outcomes. ASCs are known for promoting cell proliferation and survival through secretion of growth factors and cytokines. CM obtained from ASC culture contains these beneficial biomolecules and could potentially be a valuable supplement for ovarian tissue IVC. Study design, size, duration This prospective experimental study used 8 fragments of bovine ovarian cortex, which were cultured for 8 days in two different groups: (i) standard ovarian tissue culture (OT Ctrl D8); and (ii) ovarian tissue cultured with ASC-CM supplementation (OT+CM D8). One fragment per sample served as a non-cultured control (D0). Half of the culture medium was replaced every other day, and medium from days 2 and 8 was stored for further analyses. Participants/materials, setting, methods E2 production was evaluated on days 2 and 8 by ELISA. Follicle classification was established using hematoxylin and eosin (H&E) staining. Follicle and stromal cell apoptosis rates were assessed by TUNEL assays, while growth differentiation factor-9 (GDF-9) labeling served as a follicle quality marker. ASC-CM was analyzed to identify and measure soluble factors like vascular endothelial growth factor (VEGF), interleukin-6 (IL-6) and transforming growth factor beta 1 (TGF-β1), associated with protective effects. Main results and the role of chance The OT+CM D8 group showed significantly lower primordial follicle rates (p = 0.03) and higher secondary follicle rates (p = 0.02) than the OT Ctrl D8 group. Concerning apoptosis, the OT+CM D8 group demonstrated significantly lower follicle (p = 0.014) and stromal cell (p = 0.001) rates than the OT Ctrl D8 group. Furthermore, follicles in the OT+CM D8 group exhibited a significant increase (p = 0.002) in GDF-9 expression compared to those in the OT Ctrl D8 group. E2 production was also significantly higher (p = 0.04) after 8 days of culture in the OT+CM D8 group. All studied paracrine factors (VEGF, IL-6 and TGF-b1) were found to be present in the ASC-CM. Limitations, reasons for caution This study focuses only on improving follicle outcomes during the first step of ovarian tissue IVC, where follicles remain in situ within the tissue, and not on subsequent steps. Wider implications of the findings Our study demonstrates that adding ASC-CM to ovarian tissue IVC medium enhances follicle survival, development and E2 secretion, and promotes stromal cell viability. Moreover, our data suggest that ASC-derived factors VEGF, IL-6 and TGF-β1 could be possible paracrine mediators underlying the beneficial effects. Trial registration number not applicable

P-786 Mesenchymal stem cells as a potential source of ovarian theca interna cell differentiation

Abstract Study question Are mesenchymal stem cells (MSCs) the cell source of origin during the differentiation of ovarian stromal cells into theca interna cells (TICs)? Summary answer MSC-like cells were observed during in vitro TIC differentiation, particularly on day 2, suggesting their potential role in the origin of TICs. What is known already Folliculogenesis is the process during which a follicle develops from its primordial towards its antral stage. Follicles in the antral stage consist of an oocyte, granulosa and theca cells. While the oocyte is surrounded by granulosa cells in the primordial stage, theca cells differentiate from their precursor cell from the secondary stage onwards. Recent studies identified the existence of theca stem cells (TSCs) in both humans and animals. These TSCs express both platelet-derived growth factor receptor (PDGFR) and nerve growth factor receptor (NGFR), and demonstrate the ability to differentiate into mesenchymal cell lineages, resembling characteristics of MSCs. Study design, size, duration Ovarian stromal cells were isolated from cryopreserved cortex fragments, and cultured in vitro for 13 days, using cell media enriched with various growth factors and hormones, as previously established by our group. Flow cytometry, immunocytochemistry, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and enzyme-linked immunosorbent assay (ELISA) were employed to analyze the cells and their media at days 0, 2, 8 and 13. Participants/materials, setting, methods Whole ovaries from three postmenopausal organ donors were obtained (50, 54, and 64 years) and the cortex was cryopreserved in small 5x5 fragments prior to the isolation and differentiation into TICs. Main results and the role of chance Immunocytochemistry results showed an increase in the proportion of cells positive for TIC markers from day 0 to day 8 (12.13 ± 2.11% vs. 57.73 ± 49.99%), while the proportion of MSC-like cell markers was increased significantly from day 0 to day 2 (4.54 ± 1.71% vs. 57.34 ± 18.05%). This TIC proportion aligns with the rising androstenedione levels from day 2 to day 13 (81.31 ± 25.09 pg/ml vs. 384.29 ± 245.82 pg/ml). Flow cytometry analysis identified a CD73+ CD90+ CD105+ CD34- CD45- HLA-DR- cell population, expanding from less than 1% on day 0, to 15.30 ± 7.9% on day 2. A CD13+ cell population and its corresponding gene expression increased significantly from the beginning towards the end of the culture (13.52 ± 7.9% vs. 84.13 ± 11.08, and 1.38 ± 0.97 vs. 285.80 ± 27.43, flow cytometry and gene expression analysis respectively). Moreover, NGFR and PDGFRA expression was induced on day 0 and day 2, respectively, compared to day 13 (1.12 ± 0.60 vs. 0.08 ± 0.08, and 2.03 ± 1.11 vs. 0.24 ± 0.14). In conclusion, our study provides valuable insights into the differentiation potential of TICs, suggesting a possible origin from MSCs. Limitations, reasons for caution Due to the limited accessibility of a large quantity of human ovarian tissue from postmenopausal women, this study is restricted to three biological replicates. When examining statistical differences, it is crucial to bear in mind the considerable biological variabilities that exist among humans. Wider implications of the findings Characterizing the origin of TICs will allow for a comprehensive study of TICs, leading to a broader knowledge of these cells. The in vitro differentiation to TICs from easily accessible cells, like MSCs, holds potential significance in toxicity studies and investigating TIC-related disorders, such as polycystic ovary syndrome (PCOS). Trial registration number not applicable

Heterotopic ovarian allotransplantation in a caprine model: Effects of implant site on morphological parameters

This study aimed to investigate a new implantation site (intra-auricular subcutaneous - IA) compared to intramuscular (IM) in the cervical portion (cervical splenius muscle) of the neck for ovarian transplantation in goats. Morphological aspects of the implant, follicular activation and morphology, and type I and III collagen deposits of the transplanted tissue were evaluated. Four fragments of the ovarian cortex were allotransplanted at the IA and IM sites in all goat recipients and recovered 7 (IA-7; IM-7) or 15 (IA-15; IM-15) days later and submitted to histological analysis. Two fragments/animal were separated for the fresh control (FC) group. There was a higher percentage of normal and developing primordial follicles at the IA-7 site (P < 0.05) compared to the other treatments, with similar values to the fresh control. Type I and III collagen fibers differed between the groups (P < 0.05), showing a considerable decrease in type I collagen fibers at the IA-7 site compared to the FC. However, the IM-7 and IA-15 sites showed higher values of type I collagen fibers, showing similarity to the FC. Therefore, we conclude that the IA site in goats is an effective site for ovarian tissue transplantation, as it is easily accessible, low invasive and has presented satisfactory rates of morphology and follicular activation.

A petavoxel fragment of human cerebral cortex reconstructed at nanoscale resolution.

To fully understand how the human brain works, knowledge of its structure at high resolution is needed. Presented here is a computationally intensive reconstruction of the ultrastructure of a cubic millimeter of human temporal cortex that was surgically removed to gain access to an underlying epileptic focus. It contains about 57,000 cells, about 230 millimeters of blood vessels, and about 150 million synapses and comprises 1.4 petabytes. Our analysis showed that glia outnumber neurons 2:1, oligodendrocytes were the most common cell, deep layer excitatory neurons could be classified on the basis of dendritic orientation, and among thousands of weak connections to each neuron, there exist rare powerful axonal inputs of up to 50 synapses. Further studies using this resource may bring valuable insights into the mysteries of the human brain.

Possibilities of reproductive function realization in patients with premature ovarian insufficiency

Premature ovarian insufficiency (POI) directly affects the reproductive health of women all over the world. Currently, the study of this pathology is becoming an increasingly important issue due to the widespread prevalence of premature menopause in the female population. In addition to genetic predisposition, an important role in the development of premature menopause is played by the increasing number of ovarian surgeries due to the development of surgery, as well as radiation and chemotherapy for cancer, leading to premature depletion of the follicular reserve in the ovaries. An important component of treatment for women with POI is not only the management of undesirable symptoms of menopause, prevention of cardiovascular complications and fractures due to decreased bone mineral density, but also the possibility of realizing their reproductive function in the future. Therefore, other treatment options may be offered to women with POI in the absence of spontaneous pregnancy in the natural cycle. Oocyte donation may be recommended for such women, but this treatment option may not be suitable if the patient wishes to have genetically related children. In such a case, oogonal stem cell transplantation, ovarian cortex fragmentation or PRP therapy are possible. In women who will undergo chemotherapy and radiation therapy for cancer, reimplantation of ovarian cortical tissue is recommended to preserve fertility. If there is a risk of malignant cell transfer with this treatment modality, primordial follicles can be isolated from ovarian tissue, cultured and activated in vitro, fertilized, and transferred into the uterine cavity. IVF with surrogate embryo transfer may be an alternative choice for a woman with NPT, which allows her to realize her reproductive plans.

Qualitative and quantitative characteristics of structures of cerebral cortex structure in young people under conditions of chronic alcohol intoxication

To study morphological changes of cerebral cortex in young people under the conditions of chronic alcohol intoxication (CAI). Morphometric examination of cerebral cortex fragments obtained from 28 persons who died with a CAI diagnosis (average age was 38 years), and 25 subjects who died from other causes, which are not associated with alcohol consumption (average age was 39 years), was carried out. It was shown that neurons of pathological shapes, including hypo- and hyperchromic, pyknotic and «shadow-like», were dominant in group of CAI. There was an increase in the glial index and a greater intensity of perivascular and pericellular edema compared to the control group. Morphological changes of cerebral cortex under the conditions of CAI are non-specific and largely similar to neurodegenerative alterations in other pathological conditions, senile dementia. Clearer histological criteria for alcoholic encephalopathy are needed, including with the use of immunohistochemical methods.

Accurate reduction of medial arch fracture fragments in intertrochanteric fractures: A novel technical note.

Intramedullary fixation is currently used to stabilize intertrochanteric fractures. Surgical reduction of the medial arch cortex is crucial to achieve stabilization of the internal fixation system, however, it is challenging to perform. To ensure anatomical reduction, we developed a novel surgical technique to assist in achieving accurate and convenient reduction. In this technique, right-angle forceps were used to pry and reset medial arch cortex fragments via a mini-helical blade incision. Noteworthily, all patients who underwent this technique achieved anatomical reduction with reduced operation times and bleeding. Our article illustrates intraoperative reduction techniques and summarizes tips and tricks that may be beneficial and educative for orthopedists.

Effect of alfacalcidol on bone regeneration in senile rats

Background. Among the drugs that have a pleiotropic effect on bone, attention is drawn to alfacalcidol – the precursor of the active form of D-hormone. There are studies that have revealed distinctive features of the effect of alfacalcidol on bone regeneration in osteoporosis and in young animals. The purpose was to study bone regeneration after treatment with alfacalcidol in senile animals. Materials and methods. A transcortical defect from the lateral to the medial femur was modeled in rats aged 24 months in the area of the metadiaphysis. A treatment with alfacalcidol was carried out from the 2-nd day and for 10 and 30 days before the animals was withdrawn from the experiment. Histological study of bone regeneration with morphometric analysis of tissues in the areas of callus had been performed. Results. On the 10-th day of the treatment with alfacalcidol in senile animals, connective tissue, osteoid and coarse-fibrous trabecular bone with a high density of osteoblasts were located in the defect area. On the 30-th day in treated animals, the bone tissue of the callus was mature; its area was larger than in untreated animals. In the area of cancellous bone there is a network of newly formed bone trabeculae. Destructive manifestations around the defect were reduced in animals treated with alfacalcidol. In untreated animals there was no dense bone fusion with cortex fragments; trabeculae of cancellous bone were intermittent. In the lateral part of the callus, the area of bone tissue was reduced compared to the medial part. Conclusions. Treatment of animals with alfacalcidol accelerates the formation of bone tissue in the defect and reduces destructive manifestations around the defect compared to untreated animals.

Effects of short-term in vitro heat stress on bovine preantral follicles

Heat stress occurs when heat dissipation mechanisms are overwhelmed by external and internal heat production. Ovarian reserve is an important contributor to reproductive success in cattle. The effects of heat stress on antral follicle development have been well studied; the consequences on preantral follicles are still largely unknown. This study aimed to investigate and characterize the effects of a short in vitro heat stress exposure on bovine preantral follicles, with a special focus on primordial follicles. Bovine ovarian cortex fragments (3 × 3 × 1 mm) were isolated and incubated at different temperatures for 2 h. The incubation methodology was validated using a non-incubated group. Incubation groups consisted in physiological temperature (38.5 °C) or heat stress (41 °C). Data on follicle count, diameter of the primordial follicles, number of granulosa cells, and markers for follicle proliferation and degeneration were recorded. Heat stress caused a significant decrease in the number of primordial follicles (p < 0.0001) compared to control and non-incubated groups. The diameter of these follicles also decreased under heat stress (p < 0.0001), as did the number of granulosa cells. In addition, an increase in the number of degenerated follicles (p < 0.0001) and a decrease in granulosa cell proliferation (p < 0.0001) were found after exposure to heat stress. The results of this in vitro study show that a brief exposure to heat stress can affect the follicular reserve in the ovarian cortex and therefore, provide relevant information of its effects on the reproductive performance of cattle.

A MODIFY SAGITTAL SPLIT OSTEOTOMY FOR ODONTOGENIC KERATOCYST SURGICAL TREATMENT

A 21-year-old male patient, on medication for schizophrenia, was forwarded from another service after an incisional biopsy that resulted in diagnosis of an odontogenic cyst. The first physical exam presented facial symmetry, satisfactory mouth opening, pain during palpation on the left lingual floor, and intraoral swelling. Orthopantogram and computed tomography revealed an osteolytic intraosseous well-defined lesion measuring about 45 × 16 × 12 mm and bone expansion located on the mandibular left side, surrounding the crown of an unerupted third molar. Due to the noncollaborative behavior, an excisional biopsy was programmed. The surgery was performed by a mandibular intraoral approach, modified sagittal split osteotomy, enucleation of the lesion, peripheral ostectomy in the surgical site, and buccal bone cortex fragment before lower left third molar extraction. The buccal cortical fragment was fixed in the original position by 2 titanium plates and 10 screws. After 32 months of follow-up there are no signs of recurrence.

P-485 The TurnerFertility study: data on fertility preservation (FP) by ovarian tissue cryopreservation (OTC) in young girls with Turner syndrome (TS)

Abstract Study question Which girls with TS could benefit from FP by OTC, based on the presence of follicles in relation to karyotype, clinical and hormonal data? Summary answer Girls with TS who have favourable predictive parameters (e.g 46,XX cell line, a measurable AMH or spontaneous puberty) could benefit from FP by OTC. What is known already Infertility due to premature ovarian insufficiency is a major concern for girls with TS and their parents. Physicians are often asked about possible options to preserve their fertility. However, evidence for successful FP by OTC in these girls is lacking. Without evidence on the effectiveness of OTC in TS girls, it should only be offered in a research setting. Study design, size, duration A national prospective exploratory intervention study. Ovarian cortex will be obtained after unilateral ovariectomy from 106 girls with TS aged 2-18 years. Patients will be included between 2017 and 2022. Participants/materials, setting, methods All girls with TS who have completed the diagnostic work up of TS were included. After unilateral ovariectomy, one fragment of the ovarian cortex was used to determine the number of follicles by serial sectioning and staining. Karyotyping of ovarian cells, lymphocytes, buccal cells and urine cells was performed by Fluorescence in situ hybridization (FISH). Blood samples obtained before oophorectomy and during the yearly clinical visit after oophorectomy will provide information on hormonal parameters. Main results and the role of chance Currently, we have received 106 informed consent forms and 86 TS patients (age 3-19) had a unilateral ovariectomy. Oocytes were found in 32,6% (n = 28; age 5-19) of which 11 were prepubertal, 16 had a spontaneous puberty, 22 had numerical chromosome X aberrations, of whom one 45,X monosomy and 6 had structural chromosome X aberrations. In 6 patients with structural aberrations we found a low follicle density. In 24/28 patients AMH was measurable (0.1 - 4.79µg/L) and 25/28 patients had a FSH below 15 E/L. FISH was used to karyotype the ovarian cortex cells of 12 patients with a numerical aberration and revealed that 112 of the 119 oocytes (94,1%) had a normal X-chromosomal content. Granulosa cells were largely 45,X, but showed different levels of X chromosome mosaicism, not only between patients but also between individual follicles of the same patient. Despite the level of aneuploidy (0–80%) of ovarian stromal cells, no obvious morphological/histological abnormalities were observed in the ovarian cortex tissue. Hormone values and the chromosome pattern were found to be predictive parameters for the presence of follicles. The chance of finding follicles in girls with a 46,XX cell line was three times higher than in girls without a 46,XX cell line. Limitations, reasons for caution The final analysis will be performed when the dataset of 106 TS girls is completed. Further research is necessary to determine the effects of ovarian mosaicism on folliculogenesis, i.e. are follicles capable to grow to antral stages, and to elucidate if OTC is an effective method for FP in TS. Wider implications of the findings A combination of clinical, hormonal and karyotypic data could provide predictive parameters to define which girls with TS might benefit from fertility preservation. These parameters could help physicians during FP counselling to determine if OTC is an option for a certain girl with TS. Trial registration number NCT03381300

P-453 No major changes in ovarian function after unilateral ovariectomy in the context of ovarian tissue cryopreservation in girls with Turner syndrome

Abstract Study question What is the impact of unilateral ovariectomy on the ovarian function in girls with Turner syndrome (TS) undergoing ovarian tissue cryopreservation (OTC)? Summary answer In most girls with TS, ovarian markers remained stable up to three years after unilateral ovariectomy. What is known already Girls with TS have a complete or partial loss of one of the sex-chromosomes causing premature ovarian insufficiency which reduces the chances of a spontaneous pregnancy. OTC may be an option to preserve fertility at an early age to increase the chance on genetic offspring. However, this requires a unilateral ovariectomy for young girls with TS who are already subjected to an accelerated loss of ovarian reserve. Previous research in women without TS undergoing a unilateral ovariectomy has shown that the remaining ovary compensate for the function of the removed ovary with no effect on pubertal development and fertility outcome. Study design, size, duration This is a descriptive study of 28 girls with TS (age 5-19 years). Girls were monitored for up to three years after unilateral ovariectomy on pubertal development, and level of follicle stimulating hormone (FSH), luteinizing hormone (LH), oestrogen, Anti-Mullerian hormone (AMH) and inhibin B. Participants/materials, setting, methods The cohort was recruited from girls with TS undergoing OTC as part of the ongoing TurnerFertility study at a university medical centre. Follicles were found in the ovarian cortex fragments in 28 of the 86 cases. These cases were considered to have functional ovaries and were included in the study. Main results and the role of chance Follow-up data are not yet available for three girls because they had surgery only three months ago. Of the other 25 girls, two girls had a monosomic karyotype (45,X), fourteen were mosaic (45,X/46,XX), four had a mosaic with triple X-chromosome (45,X/47,XXX) and five girls had structural aberrations of the X chromosome. Ten girls were prepubertal at time of unilateral ovariectomy, twelve girls had a spontaneous thelarche and three girl had induced puberty. After unilateral ovariectomy, one of the 10 prepubertal girls entered puberty spontaneously and one of the prepubertal girls needed puberty induction. It is still unknown what the impact of a unilateral ovariectomy will be on the pubertal development of the other girls, due to their young age but AMH levels remained stable. 8/12 girls with spontaneous thelarche maintained a measurable AMH and FSH &amp;lt;20 IE/L during the median follow-up of 20 months (IQR 13-30). 4/12 girls with spontaneous thelarche before OTC needed hormone supplementation because of signs of premature ovarian insufficiency, e.g. irregular menstruations (n = 2), flushes (n = 1) and high FSH (84 IE/L; n = 1). However, these girls had unfavourable hormonal parameters before unilateral ovariectomy (e.g. AMH &amp;lt; 0.5 ug/L or FSH &amp;gt; 20 IE/L). Limitations, reasons for caution These results should be interpreted with caution because of the highly heterogeneous population of girls with TS. Moreover, the results should ideally be compared with age- and karyotype-matched controls to determine the natural course of ovarian function of girls with TS. Wider implications of the findings This cohort will be monitored for a longer period of time to observe the consequences on pubertal development, need for hormone supplementation, and eventually fertility outcome. If OTC becomes part of routine care for girls with TS, our results will be of great value in the counselling for future parenthood. Trial registration number NCT03381300

P-467 Optimizing in vitro growth of immature follicles from cryopreserved vs fresh human cortical ovarian tissue

Abstract Study question Can cryopreserved-thawed human ovarian cortex be cultured in vitro to produce growing follicles as efficiently as from fresh samples? Summary answer In contrast to fresh samples, cryopreserved-thawed ovarian cortex did not develop morphologically normal growing follicles, even when adding follicular growth stimulators. What is known already A multistep culture system has been shown to produce mature oocytes from immature follicles present in the human cortex, without the addition of follicular growth stimulators. However, these promising results were achieved using fresh human ovarian cortex tissue donated from cisgender women, which is not available for the purpose of fertility preservation. It remains unclear whether this multistep culture system can be used to mature follicles from cryopreserved-thawed human ovarian cortex. In addition, the applicability of the culture system to mature follicles in vitro has not been investigated using ovarian tissue from transmasculine people. Study design, size, duration Fresh and cryopreserved ovarian cortex tissue isolated from the same transmasculine people were cultured during 8 days (first step of the culture system) with or without the addition of a PI3K/Akt promotor, Sphingosine-1-Phosphate (S1P). The ovarian cortex fragments were collected a day 0 and day 8 for downstream morphological and quantitative analysis of the follicular population. This study also included data of in vitro culture of cryopreserved samples from oncological cisgender women. Participants/materials, setting, methods Ovarian cortex tissue was collected from six testosterone user transmasculine people (25,8 ± 4,9 years) and three oncological cisgender women (24,0 ± 6,0 years). Fresh cortex was either directly cultured or previously cryopreserved. All ovarian fragments were cultured in the same conditions for 8 days. After the culture period, follicular population was quantified on histological sections (haematoxylin-eosin) and compared to samples of day 0. Follicular cell types, cell proliferation and apoptosis were assessed by immunofluorescence. Main results and the role of chance Fresh ovarian cortex tissue isolated from transmasculine people showed morphologically normal primary follicles (PFs) and secondary follicles (SFs) after 8 days of culture and no antral follicles were observed. Moreover, the addition of the follicular growth stimulator, S1P, to the culture medium significantly increased the number of growing follicles (PFs, P &amp;lt; 0.05; SFs, P &amp;lt; 0.01). The addition of S1P stimulated the emergence of SFs that showed expression of anti-mullerian hormone (AMH), contained proliferative (PCNA+) granulosa cells and showed low levels of apoptotic cells (TUNEL assay). Structurally, these in vitro grown SFs showed an intact basement membrane assessed by the expression of collagen IV. By contrast, using the same culture protocol but starting from cryopreserved-thawed cortex tissue from the same patients resulted in a very low number of viable growing follicles (PFs and SFs) even in the presence of S1P. Lastly, when using the same culture protocol on cryopreserved-thawed samples from oncological cisgender women, we observed a low efficiency of follicular growth, comparable to that observed in cryopreserved ovarian tissue from (age-matched) transmasculine people. Limitations, reasons for caution Limitations of this study are: small sample size and lack of fresh cortex tissue from oncological cisgender women. Due to the high variability of follicular density among patients, the use of a larger number of samples is recommended. Wider implications of the findings Optimization of culture systems to grow and mature follicles in the ovarian cortex of cryopreserved samples (both from cis and trans persons) is urgently needed. Current culture protocols can be applied to fresh samples, but further characterization is needed to consider this as an alternative in fertility preservation treatment. Trial registration number Not applicable

Ovarian Stem Cells (OSCs) from the Cryopreserved Ovarian Cortex: A Potential for Neo-Oogenesis in Women with Cancer-Treatment Related Infertility: A Case Report and a Review of Literature.

Cancer treatment related infertility (CTRI) affects more than one third of young women undergoing anti-cancer protocols, inducing a premature exhaustion of the ovarian reserve. In addition to ovarian suppression by GnRHa, oocyte and cortex cryopreservation has gained interest in patients with estrogen-sensitive tumors for whom the hormonal burst to prompt the multiple follicular growth could provide a further pro-life tumor pulsing. On the other hand, cortex reimplantation implies a few drawbacks due to the unknown consistency of the follicles to be reimplanted or the risk of reintroducing malignant cells. The capability of ovarian stem cells (OCSs) from fresh ovarian cortex fragments to differentiate in vitro to mature oocytes provides a tool to overcome these drawbacks. In fact, since ovarian cortex sampling and cryopreservation is practicable before gonadotoxic treatments, the recruitment of OSCs from defrosted fragments could provide a novel opportunity to verify their suitability to be expanded in vitro as oocyte like cells (OLCs). Here, we describe in very preliminary experiments the consistency of an OSC population from a single cryopreserved ovarian cortex after thawing as well as both their viability and their suitability to be further explored in their property to differentiate in OLCs, thus reinforcing interest in stemness studies in the treatment of female CTRI.

Transcriptional downregulation of ABC transporters is related to follicular degeneration after vitrification and in vitro culture of ovine ovarian tissue

ATP-binding cassette (ABC) transporters perform multiple functions in reproductive tissues. During ovarian tissue vitrification, the plasma membrane has important functions in the influx or efflux of water, and substances such as cryoprotectants and channel proteins that are required in this process. Thus, the present study aimed to verify the relative abundance of mRNA transcript of ABC transporters ABCB1, ABCG2, and MRP2 after vitrification and in vitro culture (IVC) of ovine ovarian tissue. For this study, the ovarian cortex fragments were proportioned into four groups as fresh control, vitrified control, fresh culture, and vitrified culture groups. After vitrification and in vitro culture, the ovarian tissue was evaluated using morphological procedures. Further, relative abundance of ABCB1, ABCG2, and MRP2 transporter mRNA transcripts in the ovarian cortex subjected to aforementioned treatment conditions were evaluated using qPCR. Our results showed a negative association between degenerated follicles and mRNA transcript abundances of ABCB1 and ABCG2. In addition, the percentage of growing follicles in the ovine ovarian cortex after vitrification was similar to that of the fresh control tissue without in vitro culture. The in vitro culture of fresh and vitrified tissue however, showed a significant decrease in the percentage of growing follicles. To the best of our knowledge, we believe that our data for the first time has studied the relative abundances of ABCB1 and ABCG2 mRNA transcripts in the ovine ovarian cortex. In addition, alterations of these protein channels may be indicative of a deleterious effect of osmotic stress on follicular survival during vitrification. Furthermore, these effects were detectable only after the IVC of the ovarian tissues. Nonetheless, further studies are required to investigate the functions of ABC transporters in ovine folliculogenesis, especially after in vitro culture of ovarian tissue.

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

Purging human ovarian cortex of contaminating leukaemic cells by targeting the mitotic catastrophe signalling pathway

PurposeIs it possible to eliminate metastasised chronic myeloid leukaemia (CML) and acute myeloid leukaemia (AML) cells from ovarian cortex fragments by inhibition of Aurora B/C kinases (AURKB/C) without compromising ovarian tissue or follicles?MethodsHuman ovarian cortex tissue with experimentally induced tumour foci of CML, AML and primary cells of AML patients were exposed to a 24h treatment with 1 μM GSK1070916, an AURKB/C inhibitor, to eliminate malignant cells by invoking mitotic catastrophe. After treatment, the inhibitor was removed, followed by an additional culture period of 6 days to allow any remaining tumour cells to form new foci. Ovarian tissue integrity after treatment was analysed by four different assays. Appropriate controls were included in all experiments.ResultsFoci of metastasised CML and AML cells in ovarian cortex tissue were severely affected by a 24h ex vivo treatment with an AURKB/C inhibitor, leading to the formation of multi-nuclear syncytia and large-scale apoptosis. Ovarian tissue morphology and viability was not compromised by the treatment, as no significant difference was observed regarding the percentage of morphologically normal follicles, follicular viability, glucose uptake or in vitro growth of small follicles between ovarian cortex treated with 1 μM GSK1070916 and the control.ConclusionPurging of CML/AML metastases in ovarian cortex is possible by targeting the Mitotic Catastrophe Signalling Pathway using GSK1070916 without affecting the ovarian tissue. This provides a therapeutic strategy to prevent reintroduction of leukaemia and enhances safety of autotransplantation in leukaemia patients currently considered at high risk for ovarian involvement.

In Vitro Activation and Development of Goat Preantral Follicles Enclosed in Ovarian Tissue Co-cultured with Mesenchymal Stem Cells.

The development of culture systems capable of maintaining follicular growth since the preantral stage has been the target of investigations. Mesenchymal stem cells (MSC) present potential for use in a wide range of applications, including research aimed at preserving fertility. Therefore, this study investigated the use of caprine Wharton's Jelly Mesenchymal Stem Cells (WJMSC) on the survival and in vitro development of goat preantral follicles enclosed in ovarian fragments cultured for 1 or 7days. Fragments of the ovarian cortex were immediately fixed (non-cultured control) or distributed in four treatments: ovarian tissue cultured in control medium (α-MEM+); ovarian tissue cultured in α-MEM+ supplemented with FBS (α-MEM+ + FBS); ovarian tissue co-cultured with stem cells in α-MEM+ (α-MEM+ + SC); and ovarian tissue co-cultured with stem cell in α-MEM+ + FBS (α-MEM+ + SC + FBS). The rates of cell proliferation, follicular survival, and activation, as well as follicular diameter, were evaluated. After 7days, the treatment co-cultured with stem cells showed a higher (P < 0.05) percentage of morphologically normal preantral follicles compared to the other treatments, as well as a higher (P < 0.05) activation rate compared to cultured control. Moreover, the follicular diameter was higher (P < 0.05) in the treatment co-cultured with stem cells compared to co-cultured with stem cells plus FBS. This study demonstrates for the first time that in vitro co-culture of caprine WJMSC with preantral follicles enclosed in goat ovarian tissue improves activation and early follicular development.

In Vitro Follicular Activation and Stem Cell Therapy as a Novel Treatment Strategies in Diminished Ovarian Reserve and Primary Ovarian Insufficiency

Usually poor ovarian response (POR) to gonadotropins reflects a diminished ovarian reserve (DOR) that gives place to few recruitable follicles despite aggressive stimulation. The reduction in the quantity and quality of the oocytes with advanced age is physiological. However, some women experience DOR much earlier and become prematurely infertile, producing an accelerated follicular depletion towards primary ovarian insufficiency (POI). Up to now, egg donation has been commonly used to treat their infertility. In the last thirty years, specialists in assisted reproduction have focused their attention on the final stages of folliculogenesis, those that depend on the action of gonadotrophins. Nevertheless, recently novel aspects have been known to act in the initial phases, with activating and inhibiting elements. In vitro activation (IVA) combining the in vitro stimulation of the ovarian Akt signaling pathway in ovarian cortex fragments with a method named Hippo-signaling disruption. Later, a simplification of the technique designated Drug-Free IVA have shown encouraging results in patients with POI. Another innovative therapeutic option in these patients is the infusion of bone marrow-derived stem cells (BMDSC) in order to supply an adequate ovarian niche to maintain and/or promote follicular rescue in patients with impaired or aged ovarian reserves. In this review, for the first time, both therapeutic options are addressed together in a common clinical setting. The aim of this review is to analyze the physiological aspects on which these innovative techniques are based; the preliminary results obtained up to now; and the possible therapeutic role that they may have in the future with DOR and POI patients.