Cortical Strips Research Articles

Responsive deep brain stimulation for the treatment of Tourette syndrome.

To report the results of 'responsive' deep brain stimulation (DBS) for Tourette syndrome (TS) in a National Institutes of Health funded experimental cohort. The use of 'brain derived physiology' as a method to trigger DBS devices to deliver trains of electrical stimulation is a proposed approach to address the paroxysmal motor and vocal tic symptoms which appear as part of TS. Ten subjects underwent bilateral staged DBS surgery and each was implanted with bilateral centromedian thalamic (CM) region DBS leads and bilateral M1 region cortical strips. A series of identical experiments and data collections were conducted on three groups of consecutively recruited subjects. Group 1 (n = 2) underwent acute responsive DBS using deep and superficial leads. Group 2 (n = 4) underwent chronic responsive DBS using deep and superficial leads. Group 3 (n = 4) underwent responsive DBS using only the deep leads. The primary outcome measure for each of the 8 subjects with chronic responsive DBS was calculated as the pre-operative baseline Yale Global Tic Severity Scale (YGTSS) motor subscore compared to the 6month embedded responsive DBS setting. A responder for the study was defined as any subject manifesting a ≥ 30 points improvement on the YGTSS motor subscale. The videotaped Modified Rush Tic Rating Scale (MRVTRS) was a secondary outcome. Outcomes were collected at 6months across three different device states: no stimulation, conventional open-loop stimulation, and embedded responsive stimulation. The experience programming each of the groups and the methods applied for programming were captured. There were 10 medication refractory TS subjects enrolled in the study (5 male and 5 female) and 4/8 (50%) in the chronic responsive eligible cohort met the primary outcome manifesting a reduction of the YGTSS motor scale of ≥ 30% when on responsive DBS settings. Proof of concept for the use of responsive stimulation was observed in all three groups (acute responsive, cortically triggered and deep DBS leads only). The responsive approach was safe and well tolerated. TS power spectral changes associated with tics occurred consistently in the low frequency 2-10Hz delta-theta-low alpha oscillation range. The study highlighted the variety of programming strategies which were employed to achieve responsive DBS and those used to overcome stimulation induced artifacts. Proof of concept was also established for a single DBS lead triggering bi-hemispheric delivery of therapeutic stimulation. Responsive DBS was applied to treat TS related motor and vocal tics through the application of three different experimental paradigms. The approach was safe and effective in a subset of individuals. The use of different devices in this study was not aimed at making between device comparisons, but rather, the study was adapted to the current state of the art in technology. Overall, four of the chronic responsive eligible subjects met the primary outcome variable for clinical effectiveness. Cortical physiology was used to trigger responsive DBS when therapy was limited by stimulation induced artifacts.

Intraoperative ECoG in bottom-of-the-sulcus syndrome using a novel flexible strip electrode.

The recording of epileptiform discharges from bottom-of-sulcus focal cortical dysplasia (BOSD) is often difficult during intraoperative electrocorticography (ECoG) due to the deep localization. We describe the use in this scenario of a new-generation electrode strip with high flexibility, easily adapted to cortical gyri and sulci. A right-handed 20-year-old male with drug-resistant focal epilepsy due to BOSD of the inferior frontal gyrus and daily focal aware seizures was evaluated for epilepsy surgery. Based on electroclinical and neuroimaging results, a focal cortectomy guided by ECoG was proposed. ECoG recordings were performed with new-generation cortical strips (Wise Cortical Strip; WCS®) and standard cortical strips. ECoG, performed on the convexity of the frontal cortical surface, recorded only sporadic spikes with both types of strips. Then, after microsurgical trans-sulcal dissection, WCS was molded along the sulcal surface of the suspected BOSD based on 3D-imaging reconstruction, showing continuous/subcontinuous 3-4-Hz rhythmic spike activity from the deepest electrode. Registration after resection of the BOSD did not show any epileptiform activity. Pathology showed dysmorphic neurons and gliosis. No surgical complications occurred. The patient is seizure-free after 12 months. This single case experience shows that highly flexible electrode strips with adaptability to cortical gyrations can identify IEDs originating from deep location and could therefore be useful in cases of bottom of the sulcus dysplasia.

Safety of ovarian cryopreservation and transplantation in patients with acute leukemia: a case series

With increased success, ovarian tissue cryopreservation has recently become a standard technique for fertility preservation. However, malignant cell introduction through ovarian tissue transplantation remains a major concern for patients with acute leukemias. This study aimed to investigate the safety of performing autologous ovarian tissue transplantation in survivors of acute leukemia. Clinical, histopathological, and molecular data of 4 women with acute myeloid leukemia and 2 women with acute lymphoblastic leukemia who underwent ovarian tissue cryopreservation and transplantation were analyzed in this case series. Following cryopreservation of 66% to 100% of an ovarian cortex with a slow freezing method, all women received high-dose multiagent alkylating preconditioning chemotherapy for allogeneic hematopoietic stem cell transplantation. Before the ovarian tissue transplantation, (1) antral follicle counts, serum antimüllerian hormone and follicle-stimulating hormone levels were assessed to confirm primary ovarian insufficiency; (2) all recipients were cleared by their hematologist-oncologists; (3) representative cortical strips were screened for leukemia infiltration by histologic (hematoxylin and eosin staining), immunohistochemical (CD3, CD20, CD34, CD68, CD117, CD163, PAX-5, Tdt, lysozyme, and MPO), and molecular marker evaluation (BCR/ABL p190 and AML1/ETO) where appropriate. The median age was 20 years (interquartile range, 15-32) at ovarian tissue cryopreservation. Before undergoing hematopoietic stem cell transplantation, all patients received induction or consolidation chemotherapy that included cytarabine+ daunorubicin or Berlin-Frankfurt-Munich-95 protocol and were in remission. The mean serum antimüllerian hormone was 1.9±1.7 ng/mL before ovarian tissue cryopreservation. In all cases, ovarian tissue screening for leukemic cells was negative. Ovarian transplantation was performed laparoscopically with or without robotic assistance, after a median of 74.5 months (interquartile range, 41-120) after ovarian tissue cryopreservation. Ovarian function resumed in all patients after a median of 3.0 months (range, 2.5-4.0), and 2 women had 1 live birth each. The median graft longevity was 35.5 months (interquartile range, 18-57) after ovarian tissue transplantation. After a median follow-up of 51 months (interquartile range, 20-74), all patients remained relapse-free. In 1 patient, the graft was removed during cesarean delivery and was negative for immunochemical leukemia markers. Our long-term follow-up demonstrated no evidence of disease relapse after ovarian tissue transplantation in patients with acute leukemia who received allogeneic hematopoietic stem cell transplantation. This safety profile may be explained by the fact that these patients are induced into remission by nongonadotoxic induction chemotherapy before undergoing ovarian tissue cryopreservation. We propose that ovarian tissue cryopreservation should not be excluded as a fertility preservation option for young women with leukemia who are due to receive preconditioning chemotherapy before allogeneic hematopoietic stem cell transplantation.

Relationship between Amino Acid Metabolism and Bovine In Vitro Follicle Activation and Growth

The amino acid metabolism of bovine follicles during in vitro growth (IVG) was evaluated to identify potential indicators of health during culture. The bovine ovarian cortex was sliced, prepared as strips, and cultured for 6 days. Tissue samples were examined histologically before and after 6 days of culture, and the degree of follicle activation was classified as either high or low based on the number of growing secondary follicles present (high: 7~11; low: 0~1). In a separate experiment, secondary follicles (diameter range: 100~200 μm) were manually isolated and cultured, and their growth was monitored for 6 days. Cultured follicles were classified as growth or degenerate based on diameter change during culture (growth: +60.5~74.1 μm; degenerate: -28~15.2 μm). Free amino acids and their metabolites were measured in the spent culture medium from each group. In cultured ovarian cortical strips, the concentration of α-aminoadipic acid was significantly higher in the low activation group than in the high group (p < 0.05), while those of methionine, lysine, and arginine were higher in the high activation group. In cultured isolated secondary follicles, concentrations of methionine, tyrosine, histidine, and hydroxyproline were higher in the degenerate group (p ≤ 0.05). In conclusion, amino acid metabolism has the potential to serve as an indicator of primordial follicle activation and subsequent growth rate during bovine IVG.

Transplantation of Refrozen Ovarian Cortical Strips Retrieved from a Cryopreserved Whole Ovary: Proof of Feasibility.

We report successful clinical outcomes after transplantation of refrozen-rethawed cortical strips from a cryopreserved whole ovary in a patient diagnosed with stage IIIb rectal adenocarcinoma. Whole ovary cryopreservation was proposed as a fertility preservation strategy in 2006 prior to radiotherapy, chemotherapy and oncological surgery. To allow for minimal residual disease screening before ovarian reimplantation, the whole ovary was thawed and dissected into cortical strips. While awaiting the results, the majority of the cortical strips were refrozen. These refrozen-rethawed cortical strips were laparoscopically grafted to 2 sites: the previously irradiated pelvic cavity and the non-irradiated extrapelvic cavity. Ovarian function resumption was assessed by recovery of menses, hormone levels, ultrasound and oocyte pick-up following controlled ovarian stimulation (COS). Restoration of ovarian function occurred 6 months after reimplantation, with recovery of menses and estradiol secretion. A total of 12 cycles were followed by the IVF department. A second reimplantation was performed 1.5 years later, since the grafts were found to have stopped functioning for >3 consecutive months. Overall, 3 fertilizable oocytes were retrieved transabdominally from the extrapelvic graft following COS, yielding 2 embryos and culminating in one fresh embryo transfer, but no pregnancy. Concerning the reimplantation site, no ovarian activity was detected in the graft placed in the previously irradiated pelvic cavity. Indeed, only fibrotic-looking tissue was observed in the pelvic site at second laparoscopy 1.5 years later, while ovarian activity was noted in the extrapelvic graft, showing a large antral follicle. All in all, transplantation of refrozen-rethawed cortical strips from a cryopreserved whole ovary can lead to ovarian function resumption and embryo development if grafted to a non-irradiated field.

The effect of mTOR activation and PTEN inhibition on human primordial follicle activation in ovarian tissue culture.

The effect of PTEN inhibitor (Bpv(HOpic); Bpv) and mTOR activators (phosphatidic acid (PA) and propranolol (PP)), were evaluated on the activation and subsequent development of human primordial follicles in ovarian tissue culture. Slow frozen-thawed human ovarian cortical strips were incubated for 24h in different groups: (1) Control (base medium), (2) Bpv (100µM), (3) PA (200µM), (4) PA + PP (50µm), and (5) Bpv + PA + PP. Afterward, the medium was exchanged, and all groups were cultured without stimulators for 6 additional days. The proportion of normal and degenerated follicles, estradiol secretion, and expression of RPS6, FOXO3a, and AKT proteins was evaluated and compared between groups. After 24h of culture, there was no significant difference between the proportion of primordial and growing follicles in either of the experimental groups. This non-significant change was also observed for the phosphorylated protein to total protein ratios of RPS6, FOXO3a, and AKT proteins. After 7days of culture, the proportion of the transitional follicles was significantly higher in comparison to the primordial follicles for the PA, PA + PP, and Bpv + PA + PP groups. The estradiol level was significantly higher on the last day compared to the first day, in PA, PA + PP, and Bpv + PA + PP groups. Hormonal secretion was significantly higher in the PA and PA + PP groups and lower in the Bpv and Bpv + PA + PP groups compared to the control on day 7 of culture. Temporary in vitro treatment of human ovarian tissue with mTOR activators enhances the initiation of primordial follicle development and positively influences steroidogenesis after short-term culture.

P-446 Influence of strip thickness and FSH supplementation on follicle growth in the in vitro culture of strips of bovine ovarian cortical tissue in gas-permeable dishes

Abstract Study question Do tissue thickness and FSH supplementation affect follicle growth and health in the vitro culture of bovine ovarian cortical strips (BOCS) in gas-permeable dishes? Summary answer Culture of 0.5mm thin BOCS with 5ng/ml FSH does improve follicle health and yield to secondary follicles compared with 1mm thick BOCS What is known already Oxygen availability inside tissue has been demonstrated to represent a key factor in follicle health and growth during in vitro culture of bovine and human ovarian cortical strips (HOCS). Although, strip thickness can limit nutrients and gases diffusion in and out of the innermost tissue zone, the presence of the outer medulla in thick strips could positively affect follicle growth. The role of FSH on the progression of primordial to secondary follicles in ovarian organ culture is still debated. Study design, size, duration Bovine ovaries from animals aged 8-24 months were collected at a slaughterhouse. In each experiment (n = 3), BOCS of varying thickness collected from the same ovary were cultured with or without FSH in gas-permeable dishes for 10 and 15 days. Participants/materials, setting, methods Slices either 0.5mm or 1mm thick were cut with a tissue-slicer and were chopped into 1x1mm2 strips. BOCS were cultured for 10 or 15 days at the same tissue/medium volume ratio (groups of either five 1mm strips or ten 0.5mm strips in 5ml medium) with 0, 1, or 5ng/ml FSH. Follicle stages were assessed by histology. Follicle viability was estimated by labeling with live-dead far red and propidium iodide at the confocal laser scanning microscope. Main results and the role of chance Overall, 2314 follicles were analyzed (histology, 998; viability, 1316). At day 0 most follicles were primordial (primordial, 89.4%; primary, 8.7%; secondary, 1.9%), and had a high viability (94.69%). The best follicle growth and viability was observed in 0.5mm thin BOCS cultured with 5ng/ml FSH. In particular, when compared to 1mm thick BOCS cultured with 5ng/ml FSH, 0.5mm thin BOCS cultured with 5ng/ml FSH showed a higher and significant proportion of secondary follicles at day 10 (0.5 vs 1mm, % secondary follicles: 26.5 vs 10, P &amp;lt; 0.05) and a significantly higher proportion of viable follicles at day 15 (0.5 vs 1mm, % viable follicles: 89.4 vs 60.7, P &amp;lt; 0.01). These results demonstrate that smaller BOCS thickness and 5ng/ml FSH supplementation significantly improve the growth and health of secondary follicles. Limitations, reasons for caution Although the bovine is considered a reliable model for human folliculogenesis, the study should be validated on human ovarian tissue. Wider implications of the findings A limiting step in the production of mature oocytes starting from primordial follicles is the low yield to secondary follicles at the end of organ culture. Given the similarities between bovine and human folliculogenesis, the best culture conditions herein identified could contribute to the refinement of human in vitro folliculogenesis. Trial registration number Not applicable

O-265 Luteinizing hormone is able to protect reproductive health in cancer patients

Abstract Study question Is luteinizing hormone (LH) able to protect ovarian follicles against cyclophosphamide (CPM)-induced damage in women? Summary answer LH significantly reduces primordial follicle (PMF) loss in ovarian cortical strips cultured in vitro with phosphoramide mustard (PM), the active metabolite of CPM. What is known already Cancer therapies are cause of premature ovarian insufficiency (POI) in female patients, since they induce a severe reduction of PMFs. For this reason, several compounds have been analyzed as adjuvant therapies to protect the ovarian reserve without interfering with cancer treatment on tumor cells. We recently demonstrated the protective effect of LH against ovotoxicity induced by PM and cisplatin on the ovary of prepuberal and adult mice. These results suggest the possibility to use LH prior or in concomitance with anticancer drugs in order to preserve oocytes in human patients, therefore preventing the early onset of menopause and/or infertility. Study design, size, duration Ovarian cortical tissues were collected from nine patients (age ± SD: 15.33 ± 4.50) who have cryopreserved their tissue before receiving anticancer treatment. For each patient, ovarian cortical strips were thawed and randomly assigned to the experimental conditions: Control (CTRL), PM and PM+LH. LH was added 1 hour before the treatment with PM. Samples were analyzed after 8, 16, 24 and 48 hrs of treatment. Participants/materials, setting, methods Ovarian cortial strips were placed onto culture plate insert and cultured for a maximum of 48 hours, in αMEM with 10% Serum Substitute Supplement with/out 200mIU/ml LH and/or 10μM PM. The samples were processed for: - Histology, for PMFs and primary follicles (PFs) analysis; - Immunohistochemistry, for the expression of protein involved in DNA damage, apoptosis and follicle activation; - Real-Time PCR, for the expression of apoptotic and inflammation related genes. Main results and the role of chance The follicle density in the untreated group varied from 233.03 to 3420.27 PMFs/mm3 between the nine patients analysed. Relative follicular density (%) was performed to analyse statistical differences between the groups. Relative PMFs density (%) was significantly reduced in PM vs CTRL either after 24 and 48 hrs, while this reduction was significantly counteracted by LH (24 hrs: CTRL=76.19±2.12; PM = 44.95±5.06; PM+LH=73.97±11.64. 48 hrs: CTRL=66.47±3.96; PM = 36.76±3.96; PM+LH=55.74±8.72). To investigate the mechanism underlying the observed effects, the expression of markers involved in DNA damage (gH2AX), apoptosis (Cleaved caspase 3, NOXA, PUMA), follicle activation (p-AKT, FOXO3a), cell cycle arrest (p21, Ki67), and inflammation (IL1β, TNFα) were analysed. The results showed that LH did not prevent DNA damage induced by PM, since gH2AX positive oocytes were seen both in PM and PH+LH group at 16h; at 24hrs however we observed a downregulation of the gH2AX expression in the PH+LH group (PM@ 100% vs PM+LH@35%). LH also counteracts the activation of chemotherapy-induced apoptotic processes, by reducing the levels of pro-apoptotic factors such as NOXA, PUMA and CC3, and follicles activation lowering AKT-FOXO3a signaling axis. Moreover, PM treatment creates a proinflammatory microenvironment, as shown by increased IL1β and TNFα gene expression, partially counteracted by LH pretreatment. Limitations, reasons for caution Experiments are carried out in vitro; the functionality of the cortex in xenografts should be evaluated. Careful evaluation of which patients might use this protector is needed. Wider implications of the findings These findings demonstrate that LH is able to reduce PMFs loss in human ovarian cortex exposed to PM. These results encourage thinking about the use of the hormone as a ferto-protector agent in clinical trials to prevent the premature onset of menopause and/or infertility in women undergoing anticancer treatments. Trial registration number not applicable

Protective effects of a SIRT1 inhibitor on primordial follicle activation and growth induced by cyclophosphamide: insights from a bovine in vitro folliculogenesis system

PurposeAlthough oncological advances have improved survival rates of female cancer patients, they often suffer a reduced fertility due to treatment side effects. In the present study, we evaluated the potential fertoprotective effects of the specific inhibitor of SIRT1, EX-527, on the gonadotoxic action exerted by cyclophosphamide (CPM) on loss of primordial follicles (PFs).MethodsThe effects of the CPM metabolite phosphoramide mustard (PM) on follicle activation, growth and viability and the protective action of EX-527 against PM effects were evaluated on bovine ovarian cortical strips in vitro cultured for 1 or 6 days. To understand whether PFs exposed to PM plus EX-527 were able to activate and grow to the secondary stage after suspension of the treatment, strips cultured for 3 days in PM plus EX-527 for 3 days were transferred to plain medium until day 6. Follicle growth and health were evaluated through histology and viability assay at a confocal microscope. In order to investigate the molecular pathways underlying the ovarian response to PM in the presence of EX-527, we analysed the protein level of SIRT1, HuR, PARP1 and SOD2 after 1 day of in vitro culture.ResultsWe found that (1) PM, the main CPM active metabolite, promotes PF activation; (2) the ovarian stress response induced by PM includes a SIRT1-dependent pathway; and (3) EX-527 reduces PF activation and growth induced by PM.ConclusionSIRT1 can represent a candidate molecule to be targeted to protect ovarian follicles from alkylating agents and EX-527 could represent a potential fertoprotective agent for cancer patients.

The effect of agar substrate on growth and development of cryopreserved-thawed human ovarian cortical follicles in organ culture

ObjectiveTo preserve human ovarian tissue structure and improve follicular growth and survival during in-situ culture, various biomaterials are used. In this study we aimed to compare agar as a cultivation substrate with matrigel-coated insert in order to achieve an optimum system for in-situ human follicle culture. Study designFrozen-thawed human ovarian cortical tissues were cultured on either matrigel-coated inserts or agar-soaked substrates. The proportion of morphologically viable and degenerated follicles at different developmental stages, secreted hormonal levels, and apoptotic and proliferation gene expressions were compared between the cultured groups after 7-days of culture. ResultsThe follicular growth was not significantly different between the two cultured groups, although showing higher percentage of growing follicles in agar cultured group. The secreted hormonal levels didn’t have any difference between two cultured groups. Although the apoptotic gene expressions didn’t show any difference between the cultured groups, the apoptotic index was lower in agar cultured group. In addition, Ki67 gene expression, a proliferative marker, showed a significantly higher expression in agar cultured group. ConclusionBased on the results, agar is as suitable as matrigel-coated inserts for the survival and growth of follicles during culture. Therefore, agar can be an inexpensive alternative substrate for culturing frozen-thawed human ovarian cortical strips.

Can frozen-thawed human ovary withstand refreezing-rethawing in the form of cortical strips?

The aim of this study was to elucidate whether ovarian tissue is able to withstand a double freezing-thawing procedure. Human ovarian cortical biopsies from 4 thawed whole ovaries were divided into 4 experimental subgroups: (a) frozen-thawed non-grafted group, (b) frozen-thawed xenografted group, (c) refrozen-rethawed non-grafted group, and (d) refrozen-rethawed xenografted group. Xenografting was performed using 8 severe combined immunodeficient mice for a total duration of 21days. The following analyses were conducted: classic hematoxylin and eosin staining, Ki67 immunolabeling, transmission electron microscopy, Masson's green trichrome, and double CD34 immunostaining. Morphologically normal preantral follicles were detected in all groups. We observed a dramatic decline of more than 65% in early preantral follicle survival rates after grafting of both frozen-thawed (p< 0.0001) and refrozen-rethawed (p< 0.0001) ovarian tissue. However, mean follicle densities remained comparable between the frozen-thawed and refrozen-rethawed non-grafted groups, as well as both grafted groups. Equivalent proportions of proliferating early preantral follicles were identified in frozen-thawed and refrozen-rethawed samples, whether the tissue was grafted or not. Furthermore, we did not observe any significant difference in atretic follicle rates between any of the four groups, and the ultrastructural quality of follicles appeared unaffected by the refreezing procedure. Similar proportions of fibrosis were noted in the frozen-thawed and refrozen-rethawed groups, irrespective of grafting. Finally, no significant differences were witnessed in terms of vascularization. We were able to demonstrate, for the first time, that refrozen-rethawed ovarian tissue has the same functional characteristics as frozen-thawed ovarian tissue.

In-vitro fragmentation of ovarian tissue activates primordial follicles through the Hippo pathway.

STUDY QUESTIONWhat is the role of the Hippo and PI3K/Akt pathway in follicles during ovarian tissue culture in tissue derived from oncological patients and transgender men?SUMMARY ANSWERResults highlight a Hippo pathway driven primordial follicle activation in vitro, predominantly from Day 0 to Day 4.WHAT IS KNOWN ALREADY In-vitro ovarian tissue culture aims at activating and maturing primordial follicles for fertility restoration in patients with a threatened ovarian reserve. Not all patients are eligible for ovarian cortex transplantation and therefore several groups are attempting to culture ovarian tissue in-vitro. Cortex fragmentation disrupts the Hippo pathway, leading to increased expression of downstream growth factors and follicle growth. The PI3K/Akt pathway is considered the intracellular pathway to where different extracellular factors involved in primordial follicle activation in-vivo converge. In order to optimise current ovarian tissue culture models, information on progression of these pathways during tissue culture is mandatory.STUDY DESIGN, SIZE, DURATIONThe first step of a multistep cortex culture system was performed using 144 ovarian cortex pieces from a total of six patients. Per patient, 24 cortical strips were cultured for 6 days and six pieces per patient were collected for downstream analysis of follicle development and Hippo and PI3K/Akt pathway targets every second day.PARTICIPANTS/MATERIALS, SETTING, METHODSOvarian tissue was obtained from oncological (N = 3; 28.67 ± 4.51 years) and transgender (N = 3; 23.33 ± 1.53 years) patients. Follicles were analysed using haematoxylin-eosin staining and pathways were studied using immunohistochemistry and precise follicle excision by laser capture micro-dissection for RT-qPCR analysis. MIQE guidelines for RT-qPCR were pursued. Reference gene selection (GAPDH, RPL3A, 18s rRNA) was performed using GeNorm Reference Gene Selection Kit. Statistical analysis was conducted with IBM SPSS Statistics 23 (Poisson regression, negative binomial regression, ANOVA and paired t-test).MAIN RESULTS AND THE ROLE OF CHANCEImmunohistochemical analysis confirmed a Hippo pathway driven primordial follicle activation due to mechanical manipulation of the cortical strips. Ovarian tissue preparation and culture induced the inhibitory phosphorylated Yes-associated protein (pYAP) to disappear in granulosa cells of primordial follicles on Day 2. The stimulatory YAP on the contrary appeared in primordial granulosa cells over increasing culture days. Looking at the YAP target connective tissue growth factor (CTGF), a significantly up-regulated CTGF was noted in primordial follicles when comparing Day 2 and Day 4 (ratio Day 2/4 = 0.082; P < 0.05), clearly showing an effect on the Hippo pathway in primordial follicles during tissue culture. Follicle classification showed a significant drop in estimated primordial follicle counts in the oncological cohort (−78%; P = 0.021) on Day 2 and in the transgender cohort on Day 4 (−634%; P = 0.008). Intermediate follicle counts showed a non-significant increasing trend to during culture and this follicle recruitment and growth resulted in a significant rise in estimated primary follicle counts on Day 6 in oncological patients (170%; P = 0.025) and, although limited in absolute numbers, a significant increase in secondary follicles on Day 4 (367%; P = 0.021) in the transgender cohort. Subsequent antral follicle development could not be observed.LIMITATIONS, REASONS FOR CAUTIONA limitation is the small sample size, inherent to this study subject, especially as a large amount of tissue was needed per patient to reduce inter-patient variation in different downstream analysis techniques. A particular and specific weakness of this study is the inability to include an age-matched control group.WIDER IMPLICATIONS OF THE FINDINGSThese findings support an adapted tissue preparation for Hippo pathway disruption and a shorter first phase of tissue culture. This work may also have implications for transplantation of cryopreserved tissue as larger strips (and thus slower burnout due to less Hippo pathway disruption) could be a benefit.STUDY FUNDING/COMPETING INTEREST(S)This research was financially supported by the Foundation Against Cancer (Stichting tegen Kanker, TBMT001816N), the Flemish Foundation of Scientific Research (FWO Vlaanderen, FWO G0.065.11N10) and the Gender Identity Research and Education Society (GIRES) foundation. The authors declare no competing interests.TRIAL REGISTRATION NUMBERN/A.

Whole Ovary Cryopreservation and Transplantation: A Systematic Review of Challenges and Research Developments in Animal Experiments and Humans.

Ovarian tissue cryopreservation and transplantation is the only fertility preservation option that enables both restoration of fertility and resumption of ovarian endocrine function, avoiding the morbidity associated with premature menopause. It is also the only technique available to prepubertal patients and those whose treatment cannot be delayed for life-threatening reasons. Ovarian tissue cryopreservation can be carried out in two different ways, either as ovarian cortical fragments or as a whole organ with its vascular pedicle. Although use of cortical strips is the only procedure that has been approved by the American Society for Reproductive Medicine, it is fraught with drawbacks, the major one being serious follicle loss occurring after avascular transplantation due to prolonged warm ischemia. Whole ovary cryopreservation involves vascular transplantation, which could theoretically counteract the latter phenomenon and markedly improve follicle survival. In theory, this technique should maintain endocrine and reproductive functions much longer than grafting of ovarian cortical fragments. However, this procedure includes a number of critical steps related to (A) the level of surgical expertise required to accomplish retrieval of a whole ovary with its vascular pedicle, (B) the choice of cryopreservation technique for freezing of the intact organ, and (C) successful execution of functional vascular reanastomosis upon thawing. The aim of this systematic review is to shed light on these challenges and summarize solutions that have been proposed so far in animal experiments and humans in the field of whole ovary cryopreservation and transplantation.

Ovarian preantral follicles are responsive to FSH as early as the primary stage of development.

Follicle-stimulating hormone (FSH) is required for ovarian antral folliculogenesis and steroidogenesis, and there is increasing evidence that it may play critical roles in preantral follicle development. We hypothesized that preantral follicles begin responding to FSH as early as the primary stage of development. Our objectives were to establish whether the FSH receptor (FSHR) was expressed in bovine preantral follicles and to determine the effects of FSH in these follicles and the surrounding ovarian tissue. Preantral follicles were isolated from bovine ovaries and subjected to immunolocalization of FSHR. Ovarian cortical strips were cultured with FSH or vehicle for 2 or 4 days and subjected to RNA sequencing, hematoxylin/eosin staining and immunostaining for p42/44 MAPK. Finally, cortical strips were cultured for 4 days with FSH before Western blot analysis of total and phosphorylated p42/44 MAPK and total aromatase. We found greater FSHR labeling intensity per cell in preantral follicles at the primary stage compared to other stages (P < 0.05). FSH upregulated genes involved in energy metabolism and MAPK signaling and downregulated genes related to phagosome and allograft rejection in the ovarian cortex. Preantral follicles cultured in situ with FSH had greater expression of total p42/44 MAPK (P < 0.05), but no difference was detected in whole tissue Western blot for phosphorylated p42/44 MAPK or aromatase. We conclude that the FSHR is expressed in preantral follicles as early as the primary stage of development, and that FSH upregulates cell metabolism and activates MAPK signaling pathways in preantral follicles.

125 Transcriptomic changes in bovine ovarian cortex in response to FSH signaling

Preantral follicles serve as a reservoir of female gametes that could be used in assisted reproductive technologies in humans, livestock, and endangered animals. Invitro culture of ovarian cortex is a widely used method to grow preantral follicles. Follicle-stimulating hormone (FSH) is often added to the culture medium as a folliculogenesis-promoting factor. The roles of FSH in antral follicles is well known; however, the effects of FSH in preantral follicles and indirectly in the ovarian cortical cells is largely unknown. The aim of this study was to determine the transcriptomic responses of the ovarian cortex containing preantral follicles to FSH signalling over time. In 3 biological replicates, small strips of bovine ovarian cortex (10×5mm) were dissected from the medulla and evaluated under a stereomicroscope for removal of all visible antral follicles. Resulting cortical strips were cultured in defined medium with human-recombinant FSH or vehicle for 2 or 4 days at 38.5°C and 5% CO2. The RNA was isolated and subjected to cDNA library preparation and 3′-Tag RNA sequencing. Sequencing data analysis was performed using the edgeR and maSigPro packages (Bioconductor-R). Using a time-course analysis, genes up- or downregulated 2-fold or more and associated with an FDR&amp;lt;0.05 were considered differentially expressed (DEG) and were further analysed with NetworkAnalyst software. We found 252 DEG over time in response to FSH. In FSH-treated samples, significantly enriched biological functions from upregulated genes were associated with glycolysis, gluconeogenesis, carbon metabolism, and biosynthesis of amino acids. In contrast, significantly enriched biological functions from downregulated genes found in FSH-treated samples included phagosome and necroptosis. The germ cell markers BMP15, DAZL, DDX4, GDF9, and ZP2/ZP3 were expressed but unchanged by FSH, suggesting the presence of similar numbers of oocytes between samples. The gene B4GALT2, previously reported as a granulosa cell marker, was upregulated in FSH-treated samples at Day 4. The follicular marker RAB23 was expressed in all samples and not changed by FSH. One interesting finding was upregulation of MAPK signalling (represented by the genes MAPKAPK3, ELK4, MKNK2, and TGFB3) in response to FSH signalling, with no change in expression of the cAMP-response element responsive genes CYP19A1 and INHA. Together, these data indicate that FSH stimulates energy metabolism in ovarian cortical cells and represses negative cell function activity. We conclude that these responses are mostly mediated by granulosa cells, because the FSH receptor is not appreciably expressed in the ovarian cortex stroma. Moreover, the data suggest that FSH may utilise alternative signalling pathways, such as MAPK, in early follicles. This information enhances our understanding of FSH signalling pathways in the ovarian cortex, mediated by preantral follicles to create a positive environment for folliculogenesis.

Texture profile analysis reveals a stiffer ovarian cortex after testosterone therapy: a pilot study

PurposeThe importance of the surrounding ovarian stromal cells and extracellular matrix in the development and maturation of follicles has recently gained attention. An aberrant extracellular matrix has been described in ovaries of patients with polycystic ovary syndrome where a more rigid structural environment, possibly induced by endogenous testosterone, impairs normal folliculogenesis. In this context, we describe the textural parameters of the ovarian cortex of transgender men after prolonged testosterone administration compared to the textural parameters of the non-exposed ovarian cortex originating from female oncological patients.MethodsTexture profile analysis (TPA) was performed on ovarian cortex (5 × 5 mm) of oncological and transgender patients in order to measure stiffness, hardness, cohesiveness, and springiness of the ovarian cortex (LRXplus universal testing system). Statistical analysis was performed using repeated measurements mixed models and the Spearman rank order correlation test (IBM SPSS Statistics 23).ResultsA total of 36 frozen-thawed cortical strips (5 × 5 mm) were subjected to TPA. The superficial part of cortex fragments originating from transgender persons (fragments < 1.4 mm; N = 10) appeared to be significantly stiffer compared to cortex derived from oncology patients (fragments < 1.4 mm; N = 7) (6.78 ± 1.38 N/mm versus 5.41 ± 0.9 N/mm respectively, p = 0.036).ConclusionsThis is the first application of TPA in ovarian cortex to study the physical properties. Comparing the physical properties, we objectively describe an increased cortical stiffness in the most outer part of the ovarian cortex following prolonged testosterone administration in transgender men compared to the ovarian cortex of oncological patients. This preliminary and novel approach could be the start of future research to understand the physical properties of ovarian tissue.

Early ovine preantral follicles have a potential to grow until antral stage in two-step culture system in the presence of aqueous extract of Justicia insularis.

The objective of this study was to determine whether preantral follicles cultured in vitro for 7days within ovine ovarian cortical strips could be isolated at the secondary follicles (SF) and grown until antral stage during an additional 6days period of in vitro culture in the presence of aqueous extract of Justicia insularis. Fresh ovarian fragments from 16 adult sheep were fixed for histological analysis (Control 1) or in vitro cultured individually in α-MEM+ supplemented with 0.3mg/ml J.insularis (Step 1) for 7days. Part of the fragments then were fixed for histological analysis (in vitro culture group). Remaining fragments were exposed stepwise to increasing trehalose concentrations before immediate isolation of SF and viability assessment (Control 2) or after 6days of culture in α-MEM++ supplemented with 0.3mg/ml J.insularis (Step 2). In Step 1, percentage of follicular activation was 80%. In Step 2, a significant increase (p<0.05) in follicular diameter and antrum formation within 6days in vitro culture of isolated follicles was achieved. The total antioxidant capacity from both steps significantly increase (p<0.05) from day 2 to day 6. Confocal analysis of oocytes showed 57.14% oocytes with homogeneous distribution and 42.86% with peri-cortical distribution. In conclusion, SF can be successfully isolated from sheep ovarian cortex after 7days of culture and are capable of surviving and forming an antral cavity if cultured in vitro for an additional 6days in the presence of 0.3mg/ml J.insularis.

The effect of bpV(HOpic) on in vitro activation of primordial follicles in cultured swine ovarian cortical strips.

The vanadate-derivative dipotassium bisperoxo (5-hydroxy-pyridine-2-carboxylic) oxovanadate (V) (bpV(HOpic)), a pharmacological inhibitor of phosphatase and tensin homolog (PTEN), has been used in ovarian follicle culture systems for activation of follicular growth in vitro and suggested to be responsible for primordial follicle survival through indirect Akt activation. For pig ovarian tissue, it is still not clear which culture medium needs to be used, as well as which factors and hormones could influence follicular development; this also applies to bpV(HOpic) exposure. Therefore, ovarian cortical strips from pigs were cultured in 1µM bpV(HOpic) (N=24) or control medium (N=24) for 48hr. Media were then replaced with control medium and all tissue pieces incubated for additional 4days. The strips were embedded in paraffin for histological determination of follicle proportions at the end of the culture period and compared to histological sections from tissue pieces without cultivation, which had been embedded right after preparation; comparison of healthy follicles for each developmental stage was performed to quantify follicle survival and activation. After 6-day culture, follicle activation occurred in tissue samples from both cultured groups but significantly more follicles showed progression of follicular development in the presence of 1µMbpV(HOpic). The amount of non-vital follicles was not significantly increased during cultivation. BpV(HOpic) affects pig ovarian follicle development by promoting the initiation of follicle growth and development, similar as in rodent species and humans.

Robot-assisted orthotopic and heterotopic ovarian tissue transplantation techniques: surgical advances since our first success in 2000

To demonstrate the technical advances since the time we reported the first successful case in 2000 and our modern approach to autologous transplantation of frozen-thawed human ovarian tissue. A step-by-step video demonstration of three surgical approaches was created by editing the surgical footage obtained during ovarian transplantation procedures. Academic. Three patients who previously underwent ovarian tissue harvesting and cryopreservation before gonadotoxic cancer treatments or radical cancer surgery are presented. The illustrated techniques include robot-assisted orthotopic (technique 1) and heterotopic (technique 2) approaches using the da Vinci Xi (Intuitive Surgical) robotic system and a decellularized human extracellular tissue matrix (Alloderm; LifeCell Corp.) as a tissue scaffold, as well as a percutaneous autotransplantation approach (technique 3). Successful completion of procedures without complications and ovarian graft function with demonstration of E2 production and follicle development. All cases were completed without complications. Ovarian graft function was confirmed by E2 production, follicle growth by 10-14weeks after transplantation, and later embryo development. Since our first report of successful restoration of ovarian function after orthotopic transplantation of frozen-banked ovarian tissue in 2000 (1), followed by our first reports of subcutaneous heterotopic transplantation techniques (2, 3), ovarian tissue cryopreservation followed by subsequent transplantation has become a promising fertility preservation option for young women with cancer who do not have sufficient time to undergo oocyte or embryo cryopreservation and for prepubertal girls (4, 5). The same approach also has the advantage of restoring ovarian endocrine function and fertility without a need for assisted reproduction (6, 7). In the very first successful procedure that we reported in 2000, we used conventional laparoscopy, and the tissues were reconstructed and mounted on a polycellulose scaffold (Surgicel) (1, 7). Since then, we have made significant modifications in our surgical approach with potential improvements in outcomes. Here we illustrate three main techniques of ovarian tissue transplantation resulting in the restoration of ovarian function in all cases. In the first two cases, we illustrated the robot-assisted orthotopic and heterotopic approaches using Alloderm. Robotic ovarian transplantation may increase precision, provide more delicate graft handling, and reduce the time from tissue thawing to transplantation (6, 8). Alloderm is regenerated de-epithelized human cadaver skin, which consists of several extracellular matrix components. It has been safely used in the surgery and dentistry fields for enhancing tissue regeneration and vascularization (9, 10). Furthermore, our earlier laboratory work indicated the critical role of extracellular matrix in primordial follicle growth initiation and preantral follicle growth (11, 12). Prior to our use of Alloderm as part of ovarian transplant procedures, we tested it in human ovarian xenograft models and found Alloderm to incorporate well with ovarian tissue (8). Only after that test did we adopt it for use in ovarian transplants. The utility of the extracellular tissue matrix may thus enhance our ovarian autotransplantation techniques by facilitating ovarian reconstruction and potentially improving neovascularization. In fact, we have seen improved follicle growth and response to ovarian stimulation with the use of Alloderm in our first cases (8). We use heterotopic ovarian transplantation when the pelvis is not suitable for autotransplantation due to past radiation or scarring or when there are other medical contraindications for transplantation in the pelvis. The third technique we illustrated was percutaneous heterotopic ovarian autotransplantation. This is a simple approach that can be used in surgically high-risk patients, as it is done with local anesthesia or IV sedation and without entering abdominal cavity. Additionally, same approach can be utilized when there is heightened concern that the ovarian tissue may harbor a disease that can recur, requiring close surveillance and easier removal of the ovarian graft. While ovarian endocrine function and follicle growth are restored with efficiency using the percutaneous ovarian transplants, our initial experience suggests that oocyte quality may be impaired in SC locations (2, 3, 13). Hence that technique may be more suitable when the only purpose is restoration of ovarian endocrine function. However, we have encountered recurrent live births from spontaneous conceptions following SC ovarian transplants, prompting the question of whether the grafted tissue can augment the function of in situ menopausal ovary (13, 14). While ovarian cryopreservation and transplantation may no longer be considered experimental, there are many exciting questions remaining to be answered on the full potential of this procedure.

Complex spike synchrony dependent modulation of rat deep cerebellar nuclear activity.

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