Human Ovarian Cortical Tissue Research Articles

In vitro growth of secondary follicles from cryopreserved-thawed ovarian cortex.

Can secondary follicles be obtained from cultured cryopreserved-thawed human ovarian cortical tissue? We obtained high-quality secondary follicles from cultured cryopreserved-thawed human ovarian cortical tissue from cis female donors (cOVA), but not from trans masculine donors (tOVA) in the same culture conditions. The in vitro growth of oocytes present in unilaminar follicles into metaphase II stage (MII) oocytes has been previously achieved starting from freshly obtained ovarian cortical tissue from adult cis female donors. This involved a multi-step culture protocol and the first step included the transition from unilaminar follicles to multilayered secondary follicles. Given that the ovarian cortex (from both cis female and trans masculine donors) used for fertility preservation is cryopreserved, it is crucial to investigate the potential of unilaminar follicles from cryopreserved-thawed ovarian cortex to grow in culture. Cryopreserved-thawed ovarian cortical tissue from adult trans masculine donors (n = 3) and adult cis female donors (n = 3) was used for in vitro culture following the first culture step described in two published culture protocols (7-8 days and 21 days) and compared to freshly isolated ovarian cortex from trans masculine donors (n = 3) and to ovarian cortex prior to culture. Ovarian cortical tissue was obtained from adult trans masculine donors undergoing gender-affirming surgery while using testosterone, and from adult cis female donors undergoing oophorectomy for fertility preservation purposes before chemotherapy. The ovarian cortex was fixed either prior (day 0) or after the culture period. Follicular survival, growth, and morphology were assessed through histology and immunofluorescence. We quantified the different stages of follicular development (primordial, primary, secondary, and atretic) after culture and observed an increase in the percentage of secondary follicles as well as an increase in COLIV deposition in the stromal compartment regardless of the culture media used. The quality of the secondary follicles obtained from cOVA was comparable to those prior to culture. However, in the same culture conditions, the secondary follicles from tOVA (fresh and cryo) showed low-quality secondary follicles, containing oocytes with small diameter, granulosa cells that expressed abnormal levels of KRT19 and steroidogenic-marker STAR and lacked ACTA2+ theca cells, when compared to tOVA secondary follicles prior to culture. The number of different donors used was limited. Our study revealed that cryopreserved-thawed cOVA can be used to generate high-quality secondary follicles after culture and those can now be further tested to evaluate their potential to generate functional MII oocytes that could be used in the clinic. However, using the same culture protocol on tOVA (fresh and cryo) did not yield high-quality secondary follicles, suggesting that either the testosterone treatment affects follicular quality or adapted culture protocols are necessary to obtain high-quality secondary follicles from tOVA. Importantly, caution must be taken when using tOVA to optimize folliculogenesis in vitro. This research was funded by the European Research Council Consolidator Grant OVOGROWTH (ERC-CoG-2016-725722 to J.S.D.V. and S.M.C.D.S.L.), the Novo Nordisk Foundation (reNEW NNF21CC0073729 to H.C., F.W., J.S.D.V., S.M.C.D.S.L.), and China Scholarship Council (CSC 202008320362 and CSC 202008450034 to H.C. and F.W.), respectively. The authors have no conflicts of interest to declare. N/A.

P-665 High FSH level impairs VEGF secretion by thawed human ovarian tissue

Abstract Study question How does a high FSH level as related to POI (premature ovarian insufficiency) affect VEGF secretion by thawed human ovarian tissue? Summary answer After thawing, the high FSH concentration of 175mIU/mL (10ng/mL) significantly decreased VEGF secretion by human ovarian tissue by 25.6%. 17.5mIU/mL did not change VEGF secretion. What is known already Cryopreservation of ovarian cortical tissue has become an important method in fertility preservation. However, 70% of the follicles do not survive the reimplantation process due to an insufficient blood supply and oxidative stress. Human ovarian tissue secretes angiogenic factors, including VEGF, spontaneously. Its regulation by FSH is not known although 50% of the reimplantation patients show increased FSH levels (>25mIU/mL) due to POI. Study design, size, duration Provided a signed consent, biopsy punches of ovarian cortical tissue from 8 patients undergoing fertility preservation were studied. Slow freezing and thawing was applied as in the intern routine instructions. Participants/materials, setting, methods Human ovarian cortical tissue was cultured in full medium at 37 °C for 48h after thawing in presence or absence of 17.5mIU/mL (1ng/mL) or 175mIU/mL (10ng/mL) recombinant human FSH. VEGF-A secretion was measured by ELISA. The expression of VEGF-A, HIF-1α and FSHR was assessed by immunohistofluorescence per follicle. The effect of FSH was assessed by one-way-ANOVA with Tukey’s post test. The effect of culture or follicular maturation stage was analyzed by t test. Main results and the role of chance After thawing, VEGF secretion recovers and rises over time. The addition of 175mIU/mL FSH significantly impaired VEGF-A expression compared to untreated ovarian tissue (p = 0.016). The analysis of 481 follicles showed similar results of FSH affecting VEGF-A expression by immunostaining. In particular, primordial follicles expressed significantly less VEGF-A compared to growing follicles (primary+secondary; p < 0.0001). 48h tissue culture significantly increased VEGF-A expression in primordial follicles (p = 0.0041). The addition of the high FSH level significantly decreased VEGF-A expression in growing follicles (p = 0.0005). Limitations, reasons for caution Although VEGF-A is a key factor in angiogenesis, more factors are known to affect angiogenesis. The direct effect on angiogenesis should be evaluated. Moreover, the conditions in tissue culture may not represent those after reimplantation. Wider implications of the findings High FSH levels, as seen in patients with POI, might impair ovarian VEGF expression and angiogenesis. Hormone replacement therapy (HRT) might be endorsed before and during ovarian tissue reimplantation. More clinical and biological data are required to understand the regulators of angiogenesis after ovarian tissue reimplantation adequately. Trial registration number not applicable

High FSH levels impair VEGF secretion of human, frozen-thawed ovarian cortical tissue in vitro

Cryopreservation and reimplantation of human ovarian tissue restore the ovarian hormonal function and fertility due to the preservation of follicles. As the success depends on proper angiogenesis, different approaches aim to support this process. In mice, pretreatment of ovarian tissue with FSH shows increased follicular numbers probably due to the supported angiogenesis by an increased vascular endothelial factor (VEGF) expression. However, in human tissue it remains completely unclear, which effect the hormonal status of the patient has at the time point of reimplantation. Frozen-thawed human ovarian cortical tissue was cultured for 48 h with 0, 1 or 10 ng/mL recombinant human FSH. VEGF-A expression was assessed by ELISA and immunohistofluorescence (IHF) analysis. By IHF, HIF-1α and FSHR expression dependency on culture and FSH concentration was analyzed. Follicles at all stages expressed VEGF-A, which increases during folliculogenesis. Frozen-thawed human ovarian cortical tissue secreted a not statistically different amount of VEGF-A, when cultured in presence of 1 ng/mL FSH (17.5 mIU/mL). However, the presence of 10 ng/mL FSH (175 mIU/mL) significantly decreased VEGF-A expression and secretion. The high FSH concentration increased especially the VEGF-A expression of already growing follicles. The presence of pre-menopausal concentrations of FSH had no significant effect on VEGF-A expression, whereas the presence of elevated FSH levels decreased cortical VEGF-A expression. A hormonal pre-treatment of women with elevated FSH concentrations prior to reimplantation might be considered to support angiogenesis. Here, we show that VEGF-A expression by follicles is affected by FSH dependent on the concentration.

Dynamic in vitro culture of bovine and human ovarian tissue enhances follicle progression and health

In vitro ovarian cortical tissue culture, followed by culture of isolated secondary follicles, is a promising future option for production of mature oocytes. Although efforts have been made to improve the culture outcome by changing the medium composition, so far, most studies used static culture systems. Here we describe the outcome of 7 days cultures of bovine and human ovarian cortical tissue in a dynamic system using a novel perifusion bioreactor in comparison to static culture in conventional and/or gas permeable dishes. Findings show that dynamic culture significantly improves follicle quality and viability, percentage and health of secondary follicles, overall tissue health, and steroid secretion in both species. Model predictions suggest that such amelioration can be mediated by an enhanced oxygen availability and/or by fluid-mechanical shear stresses and solid compressive strains exerted on the tissue.

P-430 Characterization of secondary follicles from cultured cryopreserved-thawed human ovarian cortical tissue

Abstract Study question Can we obtain secondary follicles from cultured cryopreserved-thawed human cortical tissue? Summary answer We obtained a comparable percentage of secondary follicles after culture of cryopreserved-thawed human cortical tissue to that reported after culture of fresh human cortical tissue. What is known already The complete in vitro maturation of oocytes starting from primary oocytes (present in unilaminar follicles) until mature MII oocytes has been achieved previously by two different multi-step culture protocols. These protocols were applied to culture fresh ovarian cortical tissue from adult cisgender donors. However, the efficiency of these two protocols for growing unilaminar follicles to secondary follicles starting from cryopreserved cortical tissue from cisgender patients has not been investigated. As the ovarian tissue available for fertility preservation is cryopreserved, it is important to understand the potential of cryopreserved unilaminar follicles to mature in vitro. Study design, size, duration Cryopreserved ovarian cortical tissue from 4 cisgender adult donors was used for in vitro culture. According to the existing culture protocols, cortical ovarian tissue was cultured either for 8 days using the first step medium (Telfer’s medium) reported by M.McLaughlin 2018 (doi: 10.1093/molehr/gay002) or for 7 and 21 days using the first step medium (Xu’s medium) reported by Xu 2021 (doi: 10.1093/humrep/deab003). Participants/materials, setting, methods Ovarian cortical tissue obtained from adult cisgender donors undergoing oophorectomy for fertility preservation purposes was cryopreserved before chemotherapy. The cryopreserved ovarian cortical tissue was thawed and cut into small pieces. Several pieces were fixed immediately (day 0), the others were either cultured in Telfer’s medium or Xu’s medium. After culture, follicle survival, growth and morphology were assessed by histology and immunofluorescence. Main results and the role of chance We performed quantification of the different follicular stages (primordial, primary, secondary and atretic) after culture and observed that the percentage of secondary follicles increased independently of the culture media used. However, the ovarian cortical tissue cultured using Telfer’s protocol resulted in a higher percentage of secondary follicles and lower percentage of atretic follicles compared to that using Xu’s protocol. After culture, the ovarian cortical tissue was further immunostained for TUNEL, PCNA, COLLIV, STAR, AMH and KRT19. We observed that secondary follicles present in ovarian cortical tissue cultured in Telfer’s medium showed more proliferative (PCNA+) FOXL2+ granulosa cells and less apoptotic (TUNEL+) stromal cells. By contrast, ovarian cortical tissue cultured in Xu’s medium showed less proliferative (PCNA+) FOXL2+ granulosa cells and more apoptotic (TUNEL+) stromal cells. Moreover, the expression of AMH was high in granulosa cells of secondary follicles present in ovarian cortical tissue cultured in Telfer’s medium and low in Xu’s medium. Finally, in both Telfer’s and Xu’s medium cultures the expression of KRT19 was low in granulosa cells of secondary follicles present in ovarian cortical tissue. Limitations, reasons for caution The number of donors was limited and the study lacked fresh ovarian cortical tissue as control. Only the first step in Telfers’ and Xu’s protocol was performed, hence further culture is required to determine whether complete maturation of oocytes in vitro is possible starting from cryopreserved human cortical tissue. Wider implications of the findings Our study showed evidence of follicular growth in cryopreserved-thawed human ovarian cortical tissue after a period of in vitro culture. This is an important first step to achieve in vitro maturation of oocytes from cryopreserved-thawed human ovarian cortical tissue that could be used for clinical applications. Trial registration number NOT APPLICABLE

Identification of biomarkers and outcomes of endocrine disruption in human ovarian cortex using In Vitro Models

Endocrine disrupting chemicals (EDCs) are raising concerns about adverse effects on fertility in women. However, there is a lack of information regarding mechanisms and effects in humans. Our study aims to identify mechanisms of endocrine disruption using two EDCs, diethylstilbestrol (DES) and ketoconazole (KTZ)11Ketoconazole: KTZ.. Human ovarian cortical tissue obtained from Caesarean section patients was exposed to 10−9 M - 10−5 M KTZ and 10−10 M - 10−6 M DES in vitro for 6 days. Follicle survival and growth were studied via histology analysis and liquid-chromatography-mass spectrometry-based steroid quantification. RNA-sequencing was performed on COV434, KGN, and primary ovarian cells that were exposed for 24 h. Significantly lower unilaminar follicle densities were observed in DES 10−10 M group, whereas low KTZ exposure reduced secondary follicle density. KTZ 10−5 M reduced levels of pregnenolone and progesterone. RNA-sequencing revealed that 445 and 233 differentially expressed genes (false discovery rate < 0.1) altogether in DES and KTZ exposed groups. Gene set variation analysis showed that both chemicals modulated pathways that are important for folliculogenesis and steroidogenesis. We selected stearoyl-CoA desaturase (SCD) and 7-dehydrocholesterol reductase (DHCR7) for further validation. Up-regulation of both genes in response to KTZ was confirmed by qPCR and in situ RNA hybridization. Further validation with immunofluorescence focused on the expression of SCD in growing follicles in exposed ovarian tissue. In conclusion, SCD may serve as a potential novel human-relevant biomarker of EDC exposure and effects on ovaries.

Xenograft model of heterotopic transplantation of human ovarian cortical tissue and its clinical relevance.

Xenografts of human ovarian cortical tissue provide a tractable model of heterotopic autotransplantation that is used for fertility preservation in patients undergoing ablative chemo/radiotherapy. This study describes the behavior of hundreds of xenografts to establish a framework for the clinical function of ovarian cortex following autotransplantation over short- and long-term intervals. More than 200 live births have been achieved using autotransplantation of cryopreserved ovarian cortical fragments, yet challenges remain to be addressed. Ischemia of grafted tissue undermines viability and longevity, typically requiring transplantation of multiple cortical pieces; and the dynamics of recruitment within a graft and the influence of parameters like size and patient age at the time of cryopreservation are not well-defined. Here, we describe results from a series of experiments in which we xenografted frozen/thawed human ovarian tissue (n = 440) from 28 girls and women (age range 32 weeks gestational age to 46 years, median 24.3 ± 4.6). Xenografts were recovered across a broad range of intervals (1-52 weeks post-transplantation) and examined histologically to quantify follicle density and distribution. The number of antral follicles in xenografted cortical fragments correlated positively with the total follicle number and was significantly reduced with increased patient age. Within xenografts, follicles were distributed in focal clusters, similar to the native ovary, but the presence of a leading antral follicle coincided with increased proliferation of surrounding follicles. These results underscore the importance of transplanting ovarian tissue with a high density of follicles and elucidate a potential paracrine influence of leading antral follicles on neighboring follicles of earlier stages. This temporal framework for interpreting the kinetics of follicle growth/mobilization may be useful in setting expectations and guiding the parameters of clinical autotransplantation.

Dynamic in vitro culture of cryopreserved-thawed human ovarian cortical tissue using a microfluidics platform does not improve early folliculogenesis.

Current strategies for fertility preservation include the cryopreservation of embryos, mature oocytes or ovarian cortical tissue for autologous transplantation. However, not all patients that could benefit from fertility preservation can use the currently available technology. In this regard, obtaining functional mature oocytes from ovarian cortical tissue in vitro would represent a major breakthrough in fertility preservation as well as in human medically assisted reproduction. In this study, we have used a microfluidics platform to culture cryopreserved-thawed human cortical tissue for a period of 8 days and evaluated the effect of two different flow rates in follicular activation and growth. The results showed that this dynamic system supported follicular development up to the secondary stage within 8 days, albeit with low efficiency. Surprisingly, the stromal cells in the ovarian cortical tissue were highly sensitive to flow and showed high levels of apoptosis when cultured under high flow rate. Moreover, after 8 days in culture, the stromal compartment showed increase levels of collagen deposition, in particular in static culture. Although microfluidics dynamic platforms have great potential to simulate tissue-level physiology, this system still needs optimization to meet the requirements for an efficient in vitro early follicular growth.

P-447 Enhancing oxygen availability in the dynamic culture of bovine ovarian cortical tissue improves the yield of secondary follicles

Abstract Study question Does enhancing oxygen availability during dynamic in vitro culture of bovine ovarian cortical tissue (BOCT) improve follicle growth and health? Summary answer Enhancing oxygen availability during dynamic in vitro culture of BOCT in perifusion bioreactors (PB) does improve follicle health and yield to secondary follicles What is known already Oxygen availability has been demonstrated to represent a key factor in follicle health and growth during in vitro culture of bovine and human ovarian cortical tissue (HOCT) under static culture conditions. Disruption of solutes gradients and application of physiological fluid mechanical stress, through in vitro dynamic culture of HOCT in a newly designed perifusion bioreactor have been shown to further enhance follicle growth and health. As it shows striking similarities with human, bovine folliculogenesis is considered a valuable model to study follicle growth in vitro Study design, size, duration Bovine ovaries from animals aged 8-24 months were collected at slaughterhouse. In each experiment (n = 3), BOCT strips from the same ovary were cultured for 6 days in perifusion bioreactors (PB, dynamic culture) and conventional dishes (CD, static culture). Culture outcome in static culture was analysed and compared to two bioreactor configurations in which medium oxygenation was kept low by using a standard tube reservoir (StPB) or was enhanced by using a gas-permeable dish reservoir (PB+O2). Participants/materials, setting, methods Slices of BOCT 0.5mm thick were cut with a tissue slicer and chopped into 1x1mm strips. In each experiment, fresh (D0) and cultured tissue (groups of ten strips) were analyzed. Follicle stages and health were assessed by histology (hematoxylin-eosin staining). Follicle viability was estimated by labelling with live-dead far-red and propidium iodide followed by clearing before analysis at the confocal laser scanning microscope. Main results and the role of chance Overall, 2417 follicles were analyzed (histology, 1476; viability, 941). At day 0 most follicles were primordial (primordial, 88.7%; primary, 10.6%; secondary, 0.7%), and had good quality (grade 1-2, 92.2%; grade 3, 7.8%), and high viability (91.8%). At day 6, follicle growth and health in StPB was superior than in CD (StPB vs CD - staging: primordial, 6.8 vs 16.3, P &amp;lt; 0.01; primary, 70.7 vs 74.1, NS; secondary, 22.5 vs 9.6%, P &amp;lt; 0.01; grading: grade 1 + 2, 71.4 vs 44.8, P &amp;lt; 0.01; grade 3, 28.6 vs 55.2%, P &amp;lt; 0.01). Dynamic culture in StPB better-preserved follicle viability compared to static culture in CD (StPB vs CD: 77.75 vs 64.9%, P &amp;lt; 0.01). Enhancing oxygen availability during dynamic culture increased follicle progression and viability (PB+O2 vs StPB - staging: primordial, 5.1 vs 6.8, NS; primary, 65.4 vs 70.7, NS; secondary, 29.5 vs 22.5%, P &amp;lt; 0.05; viability - 92 vs 77.75, P &amp;lt; 0.01). Overall, the obtained results demonstrate that i) disruption of stagnant layers of medium and application of shear stress to BOCT through dynamic culture improves follicle activation, growth and health; ii) enhancing oxygen availability by means of a gas-permeable medium reservoir further increases follicle progression and viability. 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 in vitro production of mature oocytes starting from primordial follicles is the low yield of secondary follicles after organ culture. The adoption of a newly designed dynamic bioreactor and modulation of oxygen availability could represent a valuable tool for multistep in vitro folliculogenesis. Trial registration number none

P-478 Deleterious influence of cyclophosphamide on primordial follicles derives from increased DNA damage and reduced proliferation in xenotransplanted human ovarian tissue

Abstract Study question What are the acute effects of cyclophosphamide (Cp) on primordial follicles (PrFs) in human ovarian tissue? Summary answer Administration of Cp damages PrFs via DNA fragmentation and results in reduced proliferation with no increase in PrF activation markers. What is known already Alkylating agents are highly gonadotoxic, and in cases where freezing oocytes, embryos, or ovarian tissue is impractical, a better understanding of the underlying damage mechanism could enable development of fertoprotective approaches (1). Studies from different groups suggest conflicting mechanisms of ovarian damage, with either PrF activation (2) or cell damage and apoptosis (3-4) proposed as drivers of PrF depletion. Few studies have examined this question using human tissue. We performed xenografts using ovarian tissue from a 17-month-old girl to ensure a graft with plentiful PrFs to test the acute effect of Cp. Study design, size, duration Cross-sectional study. We utilized a xenotransplantation model in which human ovarian tissue is co-transplanted with endothelial cells (ECs) into immunocompromised mice (NSG) (5). Three weeks after xenotransplantation, time 0, intraperitoneal (IP) injection of (saline/Cp) was followed by an IP injection of ethynyl-deoxyuridine (Edu), at 0 and 24 hours, followed by 5-chloro-2′-deoxyuridine (CldU), at 48 and 72 hours. Grafts were harvested at 96 hours. Participants/materials, setting, methods We co-xenotransplanted human ovarian cortical tissue from a 17-month-old girl, cryopreserved for fertility preservation, into immunocompromised mice. After 3 weeks, Cp (75mg/Kg)/saline was administered IP. Mice were then sequentially injected with EdU, followed by CldU (both at 100mg/kg for 2 days) 24 hours apart. Twenty-four hours later, mice were sacrificed and xenografts were harvested and sectioned. Slides were stained for EdU, CldU, VASA, γ-H2AX, FOXO3a, and 4',6-Diamidino-2-Phenylindole-Dilactate (DAPI) and imaged using a confocal microscope. Main results and the role of chance We used anti-VASA staining to evaluate oocyte morphology, confirming that chemotherapy was not sterilizing; we counted 76.5% of morphologically normal PrF in the control group (Ctrl) (130/170) and 35.1% (13/37) in the Cp group. In the Ctrl group, 11% stained positively for DNA fragmentation using anti-γ-H2AX, whereas 43% were positive in the Cp group (p = 0.0073). To evaluate activation, we stained for FOXO3a in the oocyte nucleus. No difference was found between the groups: 52.4% (74/144) versus 44.6% (37/83) in Ctrl versus Cp, respectively. Interestingly, when comparing EdU incorporation, the Ctrl group had higher incorporation at 72%, versus the Cp group with 40% of incorporation (p = 0.0485), and no difference was found with CldU incorporation: Ctrl 22% versus 27% in the Cp group, respectively (P = 0.6960). Limitations, reasons for caution We measured acute effects, DNA fragmentation, and proliferation, at five days post-Cp administration; however, this falls short of evaluating processes that appear later (fibrosis and neovascularization). Also, the results may partly stem from the patient’s young age. Repeating the experiment using adult-derived tissue might yield different results. Wider implications of the findings A better understanding of both the timeline and underlying mechanisms that contribute to chemotherapy-related gonadotoxicity could diminish the damage and depletion of the ovarian reserve (PrF). These results provide evidence that direct damage to PrFs, and not increased activation, contributes to gonadotoxicity in the acute phase following administration of Cp. Trial registration number NA

Workflow Optimization for Identification of Female Germline or Oogonial Stem Cells in Human Ovarian Cortex Using Single-Cell RNA Sequence Analysis.

In 2004, the identification of female germline or oogonial stem cells (OSCs) that can support post–natal oogenesis in ovaries of adult mice sparked a major paradigm shift in reproductive biology. Although these findings have been independently verified, and further extended to include identification of OSCs in adult ovaries of many species ranging from pigs and cows to non–human primates and humans, a recent study rooted in single–cell RNA sequence analysis (scRNA-seq) of adult human ovarian cortical tissue claimed that OSCs do not exist, and that other groups working with OSCs following isolation by magnetic-assisted or fluorescence-activated cell sorting have mistaken perivascular cells (PVCs) for germ cells. Here we report that rare germ lineage cells with a gene expression profile matched to OSCs but distinct from that of other cells, including oocytes and PVCs, can be identified in adult human ovarian cortical tissue by scRNA-seq after optimization of analytical workflow parameters. Deeper cell-by-cell expression profiling also uncovered evidence of germ cells undergoing meiosis-I in adult human ovaries. Lastly, we show that, if not properly controlled for, PVCs can be inadvertently isolated during flow cytometry protocols designed to sort OSCs because of inherently high cellular autofluorescence. However, human PVCs and human germ cells segregate into distinct clusters following scRNA-seq due to non–overlapping gene expression profiles, which would preclude the mistaken identification and use of PVCs as OSCs during functional characterization studies.

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.

The combination of basic fibroblast growth factor and kit ligand promotes the proliferation, activity and steroidogenesis of granulosa cells during human ovarian cortical culture

Different factors, such as basic fibroblast growth factor (bFGF) and kit ligand (KL), are used in ovarian cortical culture to promote activation of primordial follicles. In the present study, the effects of bFGF and KL, alone and in combination, were evaluated on human follicular activation and growth during in-situ cortical culture. Slow frozen-thawed human ovarian cortical tissues (n = 6) were cultured in 4 different groups: 1) control (base medium), 2) KL (base medium; BM + 100 ng/ml KL), 3) bFGF (BM + 100 ng/ml bFGF) and 4) bFGF + KL (BM + 100 ng/ml KL + 100 ng/ml bFGF) for a week. The proportion of morphologically normal and degenerated follicles at different developmental stages, secreted hormonal levels and specific gene expressions were compared. Although the proportion of growing follicles was higher than primordial counterpart in all cultured groups, no significant differences were observed among the cultured groups. In all cultured groups, anti-Müllerian hormone (AMH), progesterone and estradiol hormones levels increased after 7 days of culture; however, this increase was only significant for estradiol in the bFGF + KL group. The expression of Ki67 gene indicated an increase in ovarian cell proliferation in the three experimental groups compared to the control group, however this increment was only significant for the bFGF + KL group. It can be concluded that KL and bFGF factors individually have no beneficial effects on in-situ follicular growth, but their combination positively influences steroidogenesis of granulosa cells without significantly increasing the number of growing follicles.

Improvement of in situ Follicular Activation and Early Development in Cryopreserved Human Ovarian Cortical Tissue by Co-Culturing with Mesenchymal Stem Cells

Follicular loss and tissue degeneration are great challenges in ovarian tissue culture systems. Mesenchymal stem cells (MSC) secrete a cocktail of growth factors and cytokines which supports adjacent cells and tissues. The aim of the current study was to investigate the impact of human bone marrow (hBM)-MSC, as co-culture cells, on human follicular development in ovarian cortical tissue (OCT) culture. For this purpose, warmed OCT fragments were co-cultured with hBM-MSC for 8 days and compared to monocultured OCT. During the culture period, ovarian follicle survival and development in the OCT were evaluated using histological observation, follicular developmental-related genes expression, and estradiol production. Furthermore, cell proliferation and apoptosis were assessed. The results showed that there were no significant differences in conserved ovarian follicles with a normal morphology between the two groups. However, the percentage of developing follicles, as well as follicular developmental gene expression, significantly increased in the co-culture group compared to the monoculture group. On the other hand, compared with the monoculture group, the co-culture group demonstrated a significant increase in cell proliferation, indicated by Ki67 gene expression, as well as a dramatic decrease in apoptotic cell percentage, revealed by TUNEL assay. These findings indicated that co-culturing of hBM-MSC with OCT could improve follicular activation and early follicular development in human ovarian tissue culture systems.

Initial steps in reconstruction of the human ovary: survival of pre-antral stage follicles in a decellularized human ovarian scaffold.

Can a reconstructed ovary using decellularized human ovarian tissue (DCT) support survival of pre-antral stage follicles? We have demonstrated an effective protocol for decellularization of human ovarian tissues and successful recellularization with isolated human ovarian cells and pre-antral follicles. Survivors of leukemia or ovarian cancer run a risk of reintroducing malignancy when cryopreserved ovarian tissue is transplanted to restore fertility. A reconstructed ovary free of malignant cells could provide a safe alternative. Decellularization of ovarian tissue removes all cells from the extracellular matrix (ECM) including possible malignancies and leaves behind a physiological scaffold. The ECM offers the complex milieu that facilitates the necessary interaction between ovarian follicles and their surroundings to ensure their growth and development. Previous studies have shown that decellularized bovine ovarian scaffolds supported murine follicle growth and restoration of ovarian function in ovariectomized mice. Optimizing a decellularization protocol for human ovarian tissues and testing biofunctionality of the decellularized scaffolds in vitro and in vivo by reseeding with both murine and human pre-antral follicles and ovarian cells. Donated human ovarian tissue and isolated pre-antral follicles were obtained from women undergoing ovarian tissue cryopreservation for fertility preservation. Ovarian cortical and medullary tissues were decellularized using 0.1% sodium dodecyl sulfate (SDS) for 3, 6, 18 and 24hours followed by 24hours of 1mg/mL DNase treatment and washing. Decellularization of ovarian tissues and preservation of ECM were characterized by morphological evaluation using Periodic Acid-Schiff (PAS) staining, DNA quantification, histochemical quantification of collagen content and immunofluorescence analysis for collagen IA, laminin, fibronectin and DNA. Human ovarian stromal cells and isolated human pre-antral follicles were reseeded on the DCT and cultured in vitro. Isolated murine (N= 241) and human (N= 20) pre-antral follicles were reseeded on decellularized scaffolds and grafted subcutaneously to immunodeficient mice for 3 weeks. Incubation in 0.1% SDS for 18-24hours adequately decellularized both human ovarian medullary and cortical tissue by eliminating all cells and leaving the ECM intact. DNA content in DCT was decreased by >90% compared to native tissue samples. Histological examination using PAS staining confirmed that the cortical and medullary tissues were completely decellularized, and no visible nuclear material was found within the decellularized sections. DCT also stained positive for collagen I and collagen quantities in DCT constituted 88-98% of the individual baselines for native samples. Human ovarian stroma cells were able to recellularize the DCT and isolated human pre-antral follicles remained viable in co-culture. Xenotransplantation of DCT reseeded with human or murine pre-antral follicles showed, that the DCT was able to support survival of human follicles and growth of murine follicles, of which 39% grew to antral stages. The follicular recovery rates after three weeks grafting were low but similar for both human (25%) and murine follicles (21%). N/A. Further studies are needed to increase recovery and survival of the reseeded follicles. Longer grafting periods should be evaluated to determine the developmental potential of human follicles. Survival of the follicles might be impaired by the lack of stroma cells. This is the first time that isolated human follicles have survived in a decellularized human scaffold. Therefore, this proof-of-concept could be a potential new strategy to eliminate the risk of malignant cell re-occurrence in former cancer patients having cryopreserved ovarian tissue transplanted for fertility restoration. This study is part of the ReproUnion collaborative study, co-financed by the European Union, Interreg V ÖKS. Furthermore, Project ITN REP-BIOTECH 675526 funded by the European Union, European Joint Doctorate in Biology and Technology of the Reproductive Health, the Research Pools of Rigshospitalet, the Danish Cancer Foundation and Dagmar Marshalls Foundation are thanked for having funded this study. The funders had no role in the study design, data collection and interpretation, or in the decision to submit the work for publication.

In-vitro culture of freeze-thawed human ovarian cortical tissue on agar scaffold

In-vitro culture of freeze-thawed human ovarian cortical tissue on agar scaffold

Ovarian tissue cryopreservation and transplantation: scientific implications.

After fresh or frozen ovary transplantation, FSH levels return to normal, and menstrual cycles resume by 150 days, coincident with anti-Müllerian hormone rising to higher than normal levels. AMH then returns to well below normal levels by 240 days, remaining as such for many years with nonetheless normal ovulation and fertility. To date, 20 babies have been born in our program from 11 fresh and 13 cryopreserved ovary transplant recipients with a live baby rate of over 70 % (11 babies from fresh and 9 from frozen). Globally, over 70 live births have been reported for both fresh and frozen ovary transplants with an approximate 30 % live birth rate. Given the rapid rise of AMH after the fall of FSH, with a subsequent AMH decrease with retention of ovarian function, it is tempting to speculate the existence of a shared mechanism controlling primordial follicle recruitment, fetal oocyte meiotic arrest, and recruitment in the adult ovary. With the massive recruitment of primordial follicles observed after human ovarian cortical tissue transplantation, which subsides to an extremely low recruitment rate, we will discuss how this phenomenon suggests a unifying theory implicating ovarian cortical tissue rigidity in the regulation of both fetal oocyte arrest and recruitment of follicles in the adult ovary. As the paper by Winkler-Crepaz et al. in this issue demonstrates, our in vivo results are consistent with the in vitro demonstration that primordial follicles in the fetal cortex are “locked” in development, resulting in meiotic arrest, which spares the oocytes from being rapidly lost all at once (Winkler-Crepaz et al., J Assist Reprod Genet, 1). Winkler-Crepaz et al. demonstrate that follicle loss after ovarian cortex transplantation is unlikely due to ischemic apoptosis, but rather from a “burst” of primordial follicle recruitment. In vivo, primordial follicles are normally resistant to further development or activation to prevent oocyte depletion. The dense fibrous ovarian cortex, through as yet unresolved mechanisms, arrests the further continuation of meiosis and also prevents a sudden depletion of all resting follicles in the adult ovary. Intrinsic tissue pressure is released after cortical tissue transplantation, temporarily resulting in a rapid follicle depletion. These results are consistent with the observation that once the ovarian reserve is reduced in the graft, the rate of recruitment diminishes and the ovarian tissue exhibits a relatively long duration of function.

Human GV oocytes generated by mitotically active germ cells obtained from follicular aspirates.

Human female germline stem cells (FGSCs) have opened new opportunities for understanding human oogenesis, delaying menopause, treating infertility, and providing a new strategy for preserving fertility. However, the shortage of adult human ovaries tissues available impedes their future investigations and clinical applications. Here, we have established FGSC lines from scarce ovarian cortical tissues that exist in follicular aspirates (faFGSCs), which are produced and discarded in in vitro fertilization centers worldwide. The faFGSCs have characteristics of germline stem cells involved in the gene expression profile, growth characteristics, and a normal karyotype consistent with that of FGSCs obtained from ovarian cortexes surgically removed from patients (srFGSCs). Furthermore, faFGSCs have developmental potentials including spontaneous differentiation into oocytes under feeder-free conditions, communicating with granulosa cells by gap junctions and paracrine factors, entering meiosis after RA induction, as well as forming follicles after injection into human ovarian cortical tissues xenografted into adult immunodeficient female mice. Lastly, we developed a strategy guiding FGSCs differentiated into germinal vesicle (GV) stage oocytes in vitro and revealed their developmental mechanisms. Our study not only provides a new approach to obtain human FGSCs for medical treatment, but also opens several avenues to investigate human oogenesis in vitro.

Assessment of the effect of different vitrification solutions on human ovarian tissue after short-term xenotransplantation onto the chick embryo chorioallantoic membrane.

Chemotherapy can lead to the loss of fertility and premature ovarian failure in young women who suffer from malignant diseases. Freezing ovarian tissue by vitrification allows for the preservation of a large number of follicles prior to treatment, yet no established protocols have been optimized with respect to the vitrification solution. The aim of the present study was to evaluate the early stage of human ovarian tissue xenotransplantated onto the chick embryo chorioallantoic membrane after vitrification, and to determine the effect of different vitrification solutions on ovarian tissue quality-as defined by morphology and viability of follicles, neovascularization, cell proliferation, and apoptosis. Each vitrification protocol had a different impact on ovarian tissue at the early transplantation stage; one process using the lowest concentrations of ethylene glycol and dimethylsulfoxide, plus sucrose, demonstrated a moderate advantage compared to the other protocols. We also demonstrated that the chorioallantoic membrane model can be a useful alternative for short-term xenotransplantation studies of angiogenesis into human ovarian cortical tissue.

Oxygen consumption rate of early pre-antral follicles from vitrified human ovarian cortical tissue.

The study of human ovarian tissue transplantation and cryopreservation has advanced significantly. Autotransplantation of human pre-antral follicles isolated from cryopreserved cortical tissue is a promising option for the preservation of fertility in young cancer patients. The purpose of the present study was to reveal the effect of vitrification after low-temperature transportation of human pre-antral follicles by using the oxygen consumption rate (OCR). Cortical tissues from 9 ovaries of female-to-male transsexuals were vitrified after transportation (6 or 18 h). The follicles were enzymatically isolated from nonvitrified tissue (group I, 18 h of transportation), vitrified-warmed tissue (group II, 6 and 18 h of transportation) and vitrified-warmed tissue that had been incubated for 24 h (group III, 6 and 18 h of transportation). OCR measurement and the LIVE/DEAD viability assay were performed. Despite the ischemic condition, the isolated pre-antral follicles in group I consumed oxygen, and the mean OCRs increased with developmental stage. Neither the transportation time nor patient age seemed to affect the OCR in this group. Meanwhile, the mean OCR was significantly lower (P < 0.05) in group II but was comparable to that of group I after 24 h of incubation. The integrity of vitrified-warmed primordial and primary follicles was clearly corroborated by the LIVE/DEAD viability assay. These results demonstrate that the OCR can be used to directly estimate the effect of vitrification on the viability of primordial and primary follicles and to select the viable primordial and primary follicles from vitrified-warmed follicles.