In vitro ovarian follicle growth: a comprehensive analysis of key protocol variables†

Simon, Leah E; Kumar, T. Rajendra; Duncan, Francesca E.

doi:10.1093/biolre/ioaa073

Cited by 49 publications

(36 citation statements)

References 166 publications

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“…Also, it is essential for the steroidogenesis and regulates the connection between oocyte and GCs (24). All the pervious researches increased the rate of follicle survival with addition of FSH to culture medium (11). The controversy is on the FSH dosages.…”

Section: Discussionmentioning

confidence: 99%

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How to Support the In Vitro Culture of Human Ovarian Cells and Follicles? A Preliminary Step for Assembling an Artificial Ovary

Moshrefi

Aflatoonian

Ghasemi-Esmailabad

et al. 2021

Preprint

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BackgroundThe first step in assembling an artificial ovary is supporting the in vitro growth of ovarian cells/follicles. Therefore, proliferation of granulosa or cumulus cells (CCs) is important and due to limited proliferation, improving the mediums for in vitro propagation of these cells is helpful. ObjectiveThis study aimed to characterized the appropriate media and supplements for in vitro culture of ovarian cells (OCs) and CCs. Material & MethodsCortical, medullar and hilar cells of ovary were cultured and their conditioned medium (CMs) collected. The expression of GDF9, as a key factor for ovarian follicular growth, was evaluated. CCs were collected from healthy women, who referred due to male factor infertility. To choose the optimum basal medium, a mixture of OCs was cultured with basal mediums, supplemented with two concentrations of fetal bovine serum (FBS) and human serum albumin (HSA). The cocktails were as follows: [Serum free mediums], [mediums+10%FBS], [mediums+20%FBS], [mediums+1%Alb)], [mediums+2%Alb], [mediums+10%FBS+1%Alb], [mediums+10%FBS+2%Alb], [mediums+20%FBS+1%Alb] & [mediums+20%FBS+2%Alb]. The same process was repeated for CCs. Also, the effect of various concentrations of L-Glutamine, bovine serum albumin (BSA), HSA, insulin transferrin selenium (ITS), Follitropin alfa® and Pregnyl® was evaluated on the growth of CCs. As well, CCs were treated with various concentrations of follicular fluids (FFs) and CMs. CMs were collected from ovarian, testicular, adipose & amniotic derived and ovarian carcinoma cells. FFs were collected from women with poor responders, endometriosis, advanced age, polycystic ovarian syndrome, male factor and unknown infertility. Then, CCs morphology, proliferation capacity and culture duration were evaluated. ResultsAll the ovarian cells expressed GDF9. αMEM+20%FBS and DMEMF12+20%FBS were the most suitable cocktails for OCs and CCs, respectively. 20%FBS was superior to 10% for both OCs and CCs. HSA could not support the growth of OCs and CCs, alone. The cocktail of mediums with 20%FBS superior to the others. The CMs of cortical and (hillar+medullar) cells and FFs from male factor and unknown infertility patients caused higher CCs proliferation. 17 mM/l L-Glutamine, 24 mg/ml BSA, 20 mg/ml HSA, 10 ng/ml ITS, 300 mIU/ml Follitropinα and 3.5 IU/ml Pregnyl led to higher proliferation of CCs. ConclusionCMs, serums and FFs can support the CCs growth alongside with basal mediums, supplemented with hormones, ITS and L-Glutamine, which are cheaper and more accessible.

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Section: Discussionmentioning

confidence: 99%

“…Beside the basal medium component, CCs growth needs some other ingredients. They are categorized to gonadotropins, serums as the protein source, survival factors like ITS and growth/paracrine factors (Table 4) (11). So, in the present study we compared different kinds of serum (FBS, HSA and BSA), ITS, L-Glutamine and hormones.…”

Section: Discussionmentioning

confidence: 99%

How to Support the In Vitro Culture of Human Ovarian Cells and Follicles? A Preliminary Step for Assembling an Artificial Ovary

Moshrefi

Aflatoonian

Ghasemi-Esmailabad

et al. 2021

Preprint

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“…Several culture systems are currently used to support in vitro folliculogenesis: Matrigel® (BD Bioscience, San Jose, CA), gelatin structure, alginate drop, or synthetic extracellular matrix like components. Most culture systems are currently based on enzymatic or mechanical isolation of follicles (3,4,5,6,7). An alternative approach is to work on ovarian fragments rather than on an isolated follicle, in order to maintain the architecture and interactions of the follicles.…”

Section: Introductionmentioning

confidence: 99%

A New Bioreactor to Promote Follicular Growth With or Without Activin A

Jovet

Fraison

Lornage

et al. 2021

Preprint

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Background: The aim of the present study was to evaluate the effect of Activin A on the activation of in vitro folliculogenesis of human ovarian tissues with or without our new three-dimensional structure (3D-structure). Methods: Five fresh ovarian human tissues were cultured in vitro in 4 groups with 100ng/mL Activin A or without Activine A and within or without a 3D-structure for 20 or 22 days of in vitro culture. Follicular density and quality were evaluated, and follicular diameters were measured. Estradiol secretion was quantified. Proliferation and apoptosis through immunostaining were performed.Results: The proportion of primordial follicles was significantly reduced, and the proportion of primary and secondary follicles was significantly increased in all four groups (p<0.001). Antral cavities were observed in the four culture groups. Activin A supplementation did not significantly affect the follicular density, follicular quality, follicular growth, or estradiol secretion (p>0.05). The 3D-structure increased the density of primary follicles and decreased the estradiol secretion (p<0.001). Tissular proliferation was significantly lower in the 3D-structure group compared to the non-3D-structure group (p=0.008). Regarding tissular apoptosis, it was significantly higher in the Activin group compared to the non-Activin group (0.006). Conclusion: The presence of Activin A did not seem to play a key role in in vitro folliculogenesis activation. However, the results may indicate that the 3D-structure could be more physiological and could prevent a pejorative in vitro folliculogenesis flare-up.

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

Desai

Spangler

Nanavaty

et al. 2022

Preprint

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Background Within the ovary, bidirectional communication between oocytes and surrounding granulosa cells is required for appropriate nuclear and cytoplasmic maturation (1, 10–14). The 3-D architecture of the growing follicle is however difficult to maintain during in vitro maturation using conventional 2-D tissue culture systems. Encapsulation of follicles within a biomatrix is one approach to maintaining 3-D follicle architecture during culture. Hyaluronan is one component of natural extracellular matrices (ECM) that provide support to cells in vivo. This report describes the application of hyaluronan for 3-D in vitro follicle culture and the production of developmentally competent metaphase II oocytes. Materials and Methods A novel tyramine-linked hyaluronan (HA) hydrogel was used for follicle encapsulation. Enzymatically isolated mouse preantral follicles or follicle clusters (FL-C) from fresh or vitrified ovaries were encapsulated in 3 mg/ml HA gel for in vitro follicle maturation. Follicle growth, antrum formation and meiotic maturation to metaphase II oocytes was monitored. Chromatin staining was used to assess GV oocyte progression towards meiotic competence. Spindle morphology was evaluated in metaphase II oocytes using immunostaining and polarized light. Functional competence of in vitro matured oocytes was evaluated by in vitro fertilization and ability to develop to blastocyst. Modifying the HA gel by inclusion of laminin (HA-LM), mouse sarcoma extracellular matrix (Matrigel;HA-MG) or placental extracellular matrix (HA-PM) was also tested to see if this might further enhance IVM outcomes. Results A total of 402 pre-antral follicles were embedded in HA gel. Antrums were observed in 55% of follicles by day 8. Post-hCG trigger, 314 oocyte-cumulus complexes ovulated from the cultured follicles and 84% (264/314) underwent GVBD. The maturation rate to MII was 72.6%. (228/314). The fertilization rate was 82.5% (188/228). The subsequent blastulation rate with IVM oocytes was 46.3% (87/188). Blastocysts were vitrified for later use. Thawed blastocysts were transferred into three recipient and resulted in the birth of a single pup. HA gel supported individually isolated follicles as well ovarian tissue fragments containing clusters of 6–8 preantral follicles. Meiotic maturation to the metaphase II stage was significantly lower with FL-clusters from vitrified versus fresh ovaries (34% and 55%, respectively; p < 0.007). Modification of the HA gel with ECMs or laminin affected antrum formation and follicle retention within the gel during in vitro culture. Significantly lower antrum formation was noted amongst follicles in HA-LM (29%), HA-MG (18%) and HA-PM (26%) as compared to HA alone, 48%; p = 0.006). The HA-Matrigel encapsulated follicle treatment group had a lower rate of ovulation (37%) as compared to all three of the other treatment groups ( 65–67%; P = 0.001). The rate of maturation to metaphase II oocytes was 74% for HA gel alone as compared to HA-LM (67%), HA-MG (56%) and HA-PM (58%) but these differences were not statistically significant. HA alone proved to be entirely sufficient for supporting the development of mature competent metaphase II oocytes. Conclusion Tyramine-linked hyaluronan gel is an effective ECM based biomaterial for 3-D culture of ovarian follicles in clusters or individually. In vitro matured metaphase II oocytes are functionally competent, capable of being fertilized and developing to the blastocyst stage. The versatility, simplicity of use and ability to be used at various rigidities give this new biomatrix numerous advantages, making it particularly attractive for ovarian follicle culture.

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In vitro ovarian follicle growth: a comprehensive analysis of key protocol variables†

Cited by 49 publications

References 166 publications

How to Support the In Vitro Culture of Human Ovarian Cells and Follicles? A Preliminary Step for Assembling an Artificial Ovary

How to Support the In Vitro Culture of Human Ovarian Cells and Follicles? A Preliminary Step for Assembling an Artificial Ovary

A New Bioreactor to Promote Follicular Growth With or Without Activin A

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

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