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Validating Reference Gene Expression Stability in Human Ovarian Follicles, Oocytes, Cumulus Cells, Ovarian Medulla, and Ovarian Cortex Tissue.

ABSTRACT: Human ovarian cells are phenotypically very different and are often only available in limited amounts. Despite the fact that reference gene (RG) expression stability has been validated in oocytes and other ovarian cells from several animal species, the suitability of a single universal RG in the different human ovarian cells and tissues has not been determined. The present study aimed to validate the expression stability of five of the most used RGs in human oocytes, cumulus cells, preantral follicles, ovarian medulla, and ovarian cortex tissue. The selected genes were glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta-2-microglobulin (B2M), large ribosomal protein P0 (RPLP0), beta-actin (ACTB), and peptidylprolyl isomerase A (PPIA). Overall, the stability of all RGs differed among ovarian cell types and tissues. NormFinder identified ACTB as the best RG for oocytes and cumulus cells, and B2M for medulla tissue and isolated follicles. The combination of two RGs only marginally increased the stability, indicating that using a single validated RG would be sufficient when the available testing material is limited. For the ovarian cortex, depending on culture conditions, GAPDH or ACTB were found to be the most stable genes. Our results highlight the importance of assessing RGs for each cell type or tissue when performing RT-qPCR analysis.

SUBMITTER: Cadenas J

PROVIDER: S-EPMC8778884 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature

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Validating Reference Gene Expression Stability in Human Ovarian Follicles, Oocytes, Cumulus Cells, Ovarian Medulla, and Ovarian Cortex Tissue.

Cadenas Jesús J Pors Susanne Elisabeth SE Nikiforov Dmitry D Zheng Mengxue M Subiran Cristina C Bøtkjær Jane Alrø JA Mamsen Linn Salto LS Kristensen Stine Gry SG Andersen Claus Yding CY

International journal of molecular sciences 20220114 2

Human ovarian cells are phenotypically very different and are often only available in limited amounts. Despite the fact that reference gene (RG) expression stability has been validated in oocytes and other ovarian cells from several animal species, the suitability of a single universal RG in the different human ovarian cells and tissues has not been determined. The present study aimed to validate the expression stability of five of the most used RGs in human oocytes, cumulus cells, preantral fol ...[more]

PMID: 35055072

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Project description:Study questionWhat is the X chromosomal content of oocytes and granulosa cells of primordial/primary (small) follicles and stromal cells in ovaries of young patients with Turner's syndrome (TS)?Summary answerSmall ovarian follicles were detected in one-half of the patients studied, and X chromosome analysis revealed that most oocytes were normal, granulosa cells were largely monosomic, while stromal cells showed a high level of mosaicism.What is known alreadyMost women with TS experience a premature reduction or complete loss of fertility due to an accelerated loss of gametes. To determine whether fertility preservation in this group of patients is feasible, there is a strong need for information on the X chromosomal content of ovarian follicular and stromal cells.Study design, size, durationSmall follicles (<50 μm) and stromal cells were isolated from ovarian tissue of young TS patients and analysed for their X chromosomal content. In addition to ovarian cells, several other cell types from the same patients were analysed.Participants/materials, setting, methodsAfter unilateral ovariectomy, ovarian cortex tissue was obtained from 10 TS patients (aged 2-18 years) with numerical abnormalities of the X chromosome. Ovarian cortex fragments were prepared and cryopreserved. One fragment from each patient was thawed and enzymatically digested to obtain stromal cells and primordial/primary follicles. Stromal cells, granulosa cells and oocytes were analysed by FISH using an X chromosome-specific probe. Extra-ovarian cells (lymphocytes, buccal cells and urine cells) of the same patients were also analysed by FISH. Ovarian tissue used as control was obtained from individuals undergoing oophorectomy as part of their gender affirming surgery.Main results and the role of chanceOvarian follicles were detected in 5 of the 10 patients studied. A method was developed to determine the X chromosomal content of meiosis I arrested oocytes from small follicles. This revealed that 42 of the 46 oocytes (91%) that were analysed had a normal X chromosomal content. Granulosa cells were largely 45,X but showed different levels of X chromosome mosaicism between patients and between follicles of the same patient. Despite the presence of a low percentage (10-45%) of 46,XX ovarian cortex stromal cells, normal macroscopic ovarian morphology was observed. The level of mosaicism in lymphocytes, buccal cells or urine-derived cells was not predictive for mosaicism in ovarian cells.Limitations, reasons for cautionThe results are based on a small number (n = 5) of TS patient samples but provide evidence that the majority of oocytes have a normal X chromosomal content and that follicles from the same patient can differ with respect to the level of mosaicism of their granulosa cells. The functional consequences of these observations require further investigation.Wider implications of the findingsThe results indicate that despite normal ovarian and follicular morphology, stromal cells and granulosa cells of small follicles in patients with TS may display a high level of mosaicism. Furthermore, the level of mosaicism in ovarian cells cannot be predicted from the analysis of extra-ovarian tissue. These findings should be considered by physicians when offering cryopreservation of ovarian tissue as an option for fertility preservation in young TS patients.Study funding/competing interest(s)Unconditional funding was received from Merck B.V. The Netherlands (Number A16-1395) and the foundation 'Radboud Oncologie Fonds' (Number KUN 00007682). The authors have no conflicts of interest.Trial registration numberNCT03381300.

Project description:ObjectiveOvarian tissue cryopreservation is one of the crucial options for fertility preservation. Transplantation of cryopreserved ovarian tissue was proven to restore ovarian endocrine function in patients with premature ovarian insufficiency. Ovaries from deceased donors potentially serve as an excellent and readily available tissue for the translational and basic research. In this study, we used ovaries obtained from 5 deceased donors aged 18-26 years, to evaluate the number and quality of ovarian follicles isolated before and after cryopreservation.DesignPreclinical.SettingAcademic biomedical research laboratory.PatientsDe-identified deceased human donors.InterventionsSlow-freeze cryopreservation and thawing.Main outcome measuresFollicle count, follicle density, follicle viability using immunohistochemical staining (TUNEL).ResultsThe follicle density negatively correlated with age in both cryopreserved/thawed and fresh group. A total of 2803 follicles from fresh and 1608 follicles from cryopreserved tissues were classified and analyzed using Hematoxylin and eosin staining. There was no significant difference in the percent of morphologically normal follicles between two groups. TUNEL assay indicated no higher DNA damage in the follicles and the stroma cells after cryopreservation. Morphologically normal preantral follicles were enzymatically isolated from both fresh and cryopreserved tissue with 88.51 ± 5.93% (mean ± SD) of the isolated follicles confirmed viable using LIVE/DEAD evaluation.ConclusionsOur results indicate the ovarian tissue from deceased donors maintain high quality after long time extracorporeal circulation and transportation from the hospital to the laboratory. High survival rate of follicles at different developmental stages suggested tolerance to the cryopreservation process. Human ovarian tissues obtained from deceased donors is an ample source tissue and can be applied to promoting research and future clinical applications.

Dataset Information

Validating Reference Gene Expression Stability in Human Ovarian Follicles, Oocytes, Cumulus Cells, Ovarian Medulla, and Ovarian Cortex Tissue.

Publications

Validating Reference Gene Expression Stability in Human Ovarian Follicles, Oocytes, Cumulus Cells, Ovarian Medulla, and Ovarian Cortex Tissue.

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