In vitro growth (IVG) of human ovarian follicles in frozen thawed ovarian cortex tissue culture supplemented with follicular fluid under hypoxic conditions

Schallmoser, Andreas; Einenkel, Rebekka; Färber, Cara; Sänger, Nicole

doi:10.1007/s00404-022-06672-4

Cited by 7 publications

(4 citation statements)

References 102 publications

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“…In recent years, numerous studies have explored various culture conditions aimed at fostering follicle growth in vitro , predominantly relying on ovarian tissue culture ( Laronda et al , 2014 ; Xiao et al , 2015 ; Younis et al , 2017 ; Schallmoser et al , 2022 ). The achievement of our study is the demonstrated development of human ovarian primordial/intermediate follicles into secondary follicles within a remarkably short span of one week in a three-dimensional in vitro co-culture setting.…”

Section: Discussionmentioning

confidence: 99%

Influence of ovarian stromal cells on human ovarian follicle growth in a 3D environment

Grubliauskaitė,

Vlieghe,

Moghassemi

et al. 2023

Human Reproduction Open

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STUDY QUESTION Do ovarian stromal cells (OSCs) influence the viability and growth of human pre-antral follicles in vitro? SUMMARY ANSWER A feeder layer of oSCs promotes the growth and transition of low developmental stage follicles to the primary/secondary stage while maintaining a high proportion of viable follicles. WHAT IS KNOWN ALREADY In the ovary, follicles rely on the support of ovarian cells, which secrete essential factors for their survival and development. This phenomenon has also been demonstrated in vitro through the 3D culture of isolated mouse primary and secondary follicles on a feeder layer of ovarian stromal cells. This co-culture notably enhances follicle survival and growth. STUDY DESIGN, SIZE, DURATION Pre-antral follicles were isolated from human frozen-thawed ovarian tissue (OT) biopsies and then encapsulated in 1% alginate scaffolds. These embedded pre-antral follicles were either placed directly on the OSCs feeder layer or at the bottom of a culture dish for a 7-day in vitro culture (control). The study compared follicle viability, growth and hormone production between the different groups. PARTICIPANTS/MATERIALS, SETTING, METHODS Primordial/intermediate and primary follicles were isolated from frozen-thawed OT of cancer patients (n = 6). Ovarian stromal cells were then isolated from OT of post-menopausal women and cultured as a feeder layer. Follicle diameter was measured on days 0 and 7 using an inverted microscope to assess their development based on the increase in diameter. Viability was evaluated by staining a subset of follicles (n = 87) with calcein AM and ethidium homodimer-I, followed by classification into healthy/minimally damaged and damaged/dead follicles using confocal fluorescence microscopy. Additionally, estradiol levels were measured using ELISA. MAIN RESULTS AND THE ROLE OF CHANCE A total of 382 human preantral follicles (370 primordial/intermediate and 12 primary) with a mean diameter of 40.8 ± 9.9 µm (mean±SD) were isolated, embedded in 1% alginate hydrogel, and placed either on a monolayer of oSCs or directly on the plastic. By Day 7, the preantral follicles showed a significant size increase under both culture conditions (p < 0.0001 for D0 vs D7). The mean diameter of follicles (quiescent and growing) cultured on the feeder layer was 80.6 ± 11.0 μm compared to 67.3 ± 7.2 μm without it (p = 0.07). During the 7-day in vitro culture, the viability of the follicles significantly decreased only in the group without an OSCs monolayer compared to the D0 viability (p < 0.05). Additionally, more follicles transitioned to a higher developmental stage in the presence of OSCs (D0 primordial/intermediate: 184, primary: 7 vs D7 primordial/intermediate: 51, primary/secondary: 93) compared to those cultured without OSCs (D0 primordial/intermediate: 186, primary: 5 vs D7 primordial/intermediate: 84, primary/secondary: 65; p < 0.001). Specifically, 66 and 44 follicles reached the secondary stage (75 < x < 200 μm) in the presence and absence of OSCs, respectively. Moreover, the estradiol level was significantly higher (p = 0.006) in the alginate beads containing primordial and growing follicles cultured on the OSCs (54.1 ± 14.2 pg/ml) compared to those cultured without oSCs (29.9 ± 4.0 pg/ml). LARGE SCALE DATA N/A LIMITATIONS, REASONS FOR CAUTION This study was conducted using a short-term culture, and none of the primordial/intermediate/primary follicles reached the antral stage. Further in vitro studies are required to investigate follicular developmental capacity, physiology and steroidogenesis in alginate scaffolds with human ovarian stromal cells. WIDER IMPLICATIONS OF THE FINDINGS Activating and growing human primordial/intermediate follicles to a secondary stage in in vitro short-term culture has posed a longstanding challenge. However, co-culturing with human ovarian stromal cells has shown the potential to overcome this limitation. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS-PDR Convention grant number T.0004.20 awarded to C.A.A., Ph.D. scholarship awarded to H.V.), Fondation Louvain (awarded to C.A.A.; Ph.D. scholarship awarded to S.M., as part of a legacy from Mr Frans Heyes, and Ph.D. scholarship awarded to A.D. as part of a legacy from Mrs. Ilse Schirmer), Foundation Against Cancer (grant 2018-042 awarded to A.C.), and the European Community Structural Funds and Lithuanian Research Council (Agreement registration No. D-19-0874). The authors have no conflicts of interest to declare.

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Section: Discussionmentioning

confidence: 99%

Influence of ovarian stromal cells on human ovarian follicle growth in a 3D environment

Grubliauskaitė,

Vlieghe,

Moghassemi

et al. 2023

Human Reproduction Open

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“…One major key aspect to assess the quality of ovarian cortex tissue is follicular viability that can be evaluated with calcein, a well-described fluorescence-based live assay [42][43][44]. The incubation period of 24 h prior viability measurements of fresh, slow frozen/thawed and vitrified/rapid warmed tissue enables the tissue to express potential damage that could potentially occur with these procedures and may potentially not be observed when analyzed at an earlier stage.…”

Section: Discussionmentioning

confidence: 99%

The effect of high-throughput vitrification of human ovarian cortex tissue on follicular viability: a promising alternative to conventional slow freezing?

Schallmoser

Einenkel

Färber

et al. 2022

Arch Gynecol Obstet

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Background The standard procedure most frequently used for ovarian tissue cryopreservation (OTC) is slow freezing, while vitrification has been proposed as promising alternative and has built an impressive catalog of success in fertility laboratories regarding cryopreservation of oocytes and embryos. Methods We developed and evaluated a high-throughput protocol for vitrification of human ovarian tissue suitable for clinical processing. Follicular viability was assessed via calcein staining prior and after cryopreservation analyzing ovarian tissue of a cohort of 30 patients. Results We found no significant differences regarding follicular viability between slow frozen and vitrified cortex tissue samples 24 h after thawing and rapid warming. Follicular viability of thawed and rapid warmed samples was not significantly different in comparison to fresh samples, indicating high proportions of follicular survival rates with both methods. Conclusions High-throughput vitrification is a promising option in a clinical setting. More research is required to determine the status of other tissue-specific quality indicators potentially influencing on autotransplantation.

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“…Cortex samples were slow frozen and thawed according to published protocols with modifications 21 – 26 . In brief, tissue was equilibrated in L-15 Leibovitz’s medium (Gibco Life technologies, NY, U.S.A.) supplemented with 11% human serum albumin [HSA] (Irvine Scientific, Santa Ana, USA), 10% dimethyl sulfoxide [DMSO] (CryoSure DMSO, WAK Chemie, Steinbach, Germany) for 40 min prior to slow freezing procedure at a cooling rate of − 2 °C per min.…”

Section: Methodsmentioning

confidence: 99%

Comparison of angiogenic potential in vitrified vs. slow frozen human ovarian tissue

Schallmoser,

Einenkel,

Färber

et al. 2023

Sci Rep

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Vitrification of ovarian tissue is a promising alternative approach to the traditional slow freezing method. Few empirical investigations have been conducted to determine the angiogenic profiles of these two freezing methods. In this study we aimed to answer the question whether one of the cryopreservation methods should be preferred based on the secretion of angiogenic factors. Tissue culture with reduced oxygen (5%) was conducted for 48 h with samples of fresh, slow frozen/thawed and vitrified/rapid warmed ovarian cortex tissue from 20 patients. From each patient, tissue was used in all three treatment groups. Tissue culture supernatants were determined regarding cytokine expression profiles of angiogenin, angiopoietin-2, epidermal growth factor, basic fibroblast growth factor, heparin binding epidermal growth factor, hepatocyte growth factor, Leptin, Platelet-derived growth factor B, placental growth factor and vascular endothelial growth factor A via fluoroimmunoassay. Apoptotic changes were assessed by TUNEL staining of cryosections and supplemented by hematoxylin and eosin and proliferating cell nuclear antigen staining. Comparing the angiogenic expression profiles of vitrified/rapid warmed tissue with slow frozen/thawed tissue samples, no significant differences were observed. Detection of apoptotic DNA fragmentation via TUNEL indicated minor apoptotic profiles that were not significantly different comparing both cryopreservation methods. Vitrification of ovarian cortical tissue does not appear to impact negatively on the expression profile of angiogenic factors and may be regarded as an effective alternative approach to the traditional slow freezing method.

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In vitro growth (IVG) of human ovarian follicles in frozen thawed ovarian cortex tissue culture supplemented with follicular fluid under hypoxic conditions

Cited by 7 publications

References 102 publications

Influence of ovarian stromal cells on human ovarian follicle growth in a 3D environment

Influence of ovarian stromal cells on human ovarian follicle growth in a 3D environment

The effect of high-throughput vitrification of human ovarian cortex tissue on follicular viability: a promising alternative to conventional slow freezing?

Comparison of angiogenic potential in vitrified vs. slow frozen human ovarian tissue

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