Ovarian Cortex Freezing

AbstractEndothelial cells (ECs), the primary component of the vasculature, play a crucial role in neovascularization. However, the number of endogenous ECs is inadequate for both experimental purposes and clinical applications. Porcine ovarian putative stem cells (poPSCs), although not pluripotent, are characterized by great plasticity. Therefore, this study aimed to investigate whether poPSCs have the potential to differentiate into cells of endothelial lineage. poPSCs were immunomagnetically isolated from postnatal pig ovaries based on the presence of SSEA-4 protein. Expression of mesenchymal stem cells (MSCs) markers after pre-culture, both at the level of mRNA: ITGB1, THY, and ENG and corresponding protein: CD29, CD90, and CD105 were significantly higher compared to the control ovarian cortex cells. To differentiate poPSCs into ECs, inducing medium containing vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF), epidermal growth factor (EGF), ascorbic acid, and heparin was applied. After 14 days, poPSC differentiation into ECs was confirmed by immunofluorescence staining for vascular endothelial cadherin (VECad) and vascular endothelial growth factor receptor-2 (VEGFR-2). Semi-quantitative WB analysis of these proteins confirmed their high abundance. Additionally, qRT-PCR showed that mRNA expression of corresponding marker genes: CDH5, KDR was significantly higher compared with undifferentiated poPSCs. Finally, EC functional status was confirmed by the migration test that revealed that they were capable of positive chemotaxis, while tube formation assay demonstrated their ability to develop capillary networks. In conclusion, our results provided evidence that poPSCs may constitute the MSC population in the ovary and confirmed that they might be a potential source of ECs for tissue engineering.

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Superficial ovarian cortex vascularization is inversely related to the follicle reserve in normal cycling ovaries and is increased in polycystic ovary syndrome

Human Reproduction ◽

10.1093/humrep/dep008 ◽

2009 ◽

Vol 24 (5) ◽

pp. 1142-1151 ◽

Cited By ~ 28

Author(s):

F. Delgado-Rosas ◽

M. Gaytan ◽

C. Morales ◽

R. Gomez ◽

F. Gaytan

Keyword(s):

Polycystic Ovary Syndrome ◽

Polycystic Ovary ◽

Ovary Syndrome ◽

Ovarian Cortex

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Isolation, purification, and culture of oogonial stem cells from adult human and bovine ovarian cortex

The Lancet ◽

10.1016/s0140-6736(14)60308-1 ◽

2014 ◽

Vol 383 ◽

pp. S45 ◽

Cited By ~ 11

Author(s):

Cheryl E Dunlop ◽

Rosemary A Bayne ◽

Marie McLaughlin ◽

Evelyn E Telfer ◽

Richard A Anderson

Keyword(s):

Stem Cells ◽

Ovarian Cortex ◽

Adult Human

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O-071 The ovarian endometriotic cyst

Human Reproduction ◽

10.1093/humrep/deab126.017 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

M Nisolle

Keyword(s):

Ovarian Reserve ◽

Cyst Wall ◽

Ovarian Tissue ◽

Young Patients ◽

Childbearing Age ◽

Excessive Bleeding ◽

Ovarian Cortex ◽

Ablative Surgery ◽

Endometriotic Cyst ◽

Combined Technique

Abstract text "The ovarian endometriotic cyst” Ovarian endometriomas affect 17 to 44% of women with endometriosis, and are often associated with pelvic pain and infertility. Patients suffering from endometriosis frequently present an already reduced ovarian reserve, assessed by AMH dosage or by antral follicular count during TVS. Pain and infertility are the main indications for endometrioma surgery which is a complex procedure as endometriosis leads to inflammation around the lesions, causing fibrosis. Three main surgical procedures have been described: the ovarian cystectomy, the endometrioma ablation or the combined technique. During the cystectomy, after ovarian mobilization and adhesions lifting, an incision of the cortex is realized to find a cleavage plane between the cyst wall and the ovarian cortex. Traction and countertraction movements are performed to carefuly dissociate the cyst from the ovarian cortex. It is crucial to handle the ovarian tissue as atraumatically as possible. With this technique, the cyst wall as well as the surrounding fibrosis are excised with the risk of oocytes removal responsible for decreased ovarian reserve. The ablative surgery is defined by the fenestration and vaporization of the endometrioma cyst. The ablation is carried out using a laser or plasma energy or electrocoagulation. Once the endometrial cyst has been emptied of its contents, the entire internal surface of the endometrioma must be sprayed or evaporated using the different chosen techniques. Where feasible, the cyst may be turned inside out to facilitate further treatment. The combined technique associates partial cystectomy (80-90%) and ablation of the 10-20% remaining endometrioma. This method is especially useful while operating large endometriomas. It prevents excessive bleeding or damage to the ovarian tissue. In cases of large ovarian endometrioma, the three-step approach has been proposed, consisting on an opening and drainage of the cyst followed by a 3 months’ administration of Gn-RH agonists in order to reduce its diameter and vascularization. A second surgical procedure is then scheduled to ablate the remaining cyst wall. In conclusion, it is crucial to keep in mind that endometriosis and especially the presence of endometrioma reduce fertility whereas in the majority of cases, the ovarian reserve is already diminished in relation to the patient's age. Ovarian preservation must be one of our priorities in young patients of childbearing age and it is therefore really important to carry out surgeries that are as atraumatic as possible. Trial registration number: Study funding: Funding source:

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O-191 Assessing the use of tumour-specific DARPin-toxin fusion proteins for ex vivo purging of cancer metastases from human ovarian cortex tissue fragments before autotransplantation

Human Reproduction ◽

10.1093/humrep/deab127.092 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

L Eijkenboom ◽

V Palacio-Castañeda ◽

F A Groenman ◽

D D M Braat ◽

C C M Beerendonk ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Fusion Proteins ◽

Breast Cancer Cells ◽

Ex Vivo ◽

Ovarian Tissue ◽

In Vitro Growth ◽

Ovarian Cortex ◽

Positive Breast Cancer

Abstract Study question Is it possible to eradicate cancer cells from ovarian cortex by using tumour-specific designed ankyrin repeat protein (DARPin)-toxin fusion proteins, without compromising the ovarian tissue? Summary answer Purging ovarian cortex ex vivo from experimentally induced breast cancer tumour foci is possible by tumour-targeted DARPin-toxin fusion proteins trough inhibition of protein synthesis. What is known already Ovarian tissue cryopreservation and autotransplantation is a successful technique for fertility restoration in cancer patients. The procedure is not without risk since malignant cells may still be present in the graft. Procedures to detect cancer cells render the tissue fragment useless for autotransplantation. Strategies to circumvent this problem such as in vitro maturation of follicles or the construction of artificial ovaries are pursued but are still experimental. Alternatively, we have shown ex vivo purging of ovarian cortex is possible by elimination of rhabdomyosarcoma after treatment with verteporfin. This allows treatment of cortex fragments before autotransplantation without compromising ovarian tissue integrity. Study design, size, duration Human ovarian cortex fragments harbouring breast cancer tumour foci were exposed for 24 h to DARPins fused to the translocation and catalytic domain of Pseudomonas aeruginosa exotoxin A (DARPin-toxin fusion proteins) targeting EpCAM or HER2. After treatment with the DARPin-toxin fusion proteins the tissue was cultured for an additional 6 days to allow any remaining tumour cells to form foci. In addition, the functional integrity of the ovarian tissue was analysed after purging. Participants/materials, setting, methods Breast cancer cell lines expressing different levels of EpCAM and HER2 were introduced in human ovarian tissue to form tumour foci. After purging with DARPin-toxin fusion proteins, the presence of any remaining cancer cells in the tissue was analysed with (immuno)histochemistry and RT-qPCR. Possible detrimental effects on the viability of ovarian cortex and follicles were determined by (immuno)histology, a follicular viability assay and an assay to determine the in vitro growth capacity of small follicles. Main results and the role of chance Ovarian cortex harbouring EpCAM-positive breast cancer cells showed a significant decrease in the number of tumour foci after treatment with the EpCAM-targeted DARPin-toxin fusion proteins. Although exposure to the EpCAM-specific DARPin had no effect on morphology or viability of follicles, a decrease in oocyte viability after in vitro growth experiments was observed, presumably due to low level expression of EpCAM on oocytes. In contrast to the EpCAM-specific DARPin-toxin fusion protein, the DARPin-toxin fusion protein targeting HER2 had no detrimental effects on morphology, viability or in vitro growth of follicles while foci of HER2-positive breast cancer cells were severely affected as indicated by the presence of apoptotic bodies, tumour cell remnants and the absence of viable tumour cells. The histological results after purging with the HER2-specific DARPin-toxin fusions proteins were confirmed by RT-qPCR, showing a decrease to basal levels of HER2 mRNA in the ovarian cortex tissue. Limitations, reasons for caution The effect of DARPin-toxin fusion proteins depends heavily on the expression of their target on the cancer cell. The target protein should not be expressed by ovarian cortex as this may lead to tissue damage. The functional integrity of ovarian cortex after the treatment requires further investigation in vivo. Wider implications of the findings Purging metastases from ovarian cortex without harming ovarian tissue is possible by targeting tumour specific surface expressed antigens with DARPin-toxin fusion proteins. Purging ovarian cortex tissue with DARPin-toxin fusion proteins provides a feasible therapeutic strategy to prevent reintroduction of cancer by autotransplantation in case of malignancies expressing tumour-specific surface markers. Trial registration number not applicable

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‘Spontaneous’ sex reversal in organ cultures of the embryonic male gonad of the bird

Development ◽

10.1242/dev.31.3.611 ◽

1974 ◽

Vol 31 (3) ◽

pp. 611-620

Author(s):

Gregory F. Erickson

Keyword(s):

Germ Cells ◽

Primordial Germ Cells ◽

Calf Serum ◽

Sex Reversal ◽

Biologically Active ◽

Foetal Calf Serum ◽

Developmental Sequence ◽

Organ Cultures ◽

Male Gonad ◽

Ovarian Cortex

The left embryonic testis of the bird (4–8 days of incubation) was organ cultured in medium that contained 10% foetal calf serum. Under these conditions, the germinal epithelium (GE) of the 4-day gonad differentiates into an ovarian cortex and the male primordial germ cells (PGCs) complete a developmental sequence similar to normal oocytes, i.e. they divide mitotically, develop a Balbiani body, divide synchronously in groups of two, four, and eight germ cells, and some enter pre-leptotene. No medullary tissue develops in the 4-day explants. The pieces of 6- and 8-day gonad differentiate into true ovotestes in which the GE develops into a cortex and the medulla develops into seminiferous cords. The PGCs in the cortex differentiate as oocytes and those in the seminiferous cords differentiate as spermatogonia. The possibility that biologically active oestrogens are present in the growth medium is discussed.

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