Early human follicle ultrastructure comparison after slow cryopreservation in two different cryoprotectants
Date
2008-03
Authors
Els, Cecilia Lydia
Journal Title
Journal ISSN
Volume Title
Publisher
Stellenbosch : Stellenbosch University
Abstract
BACKGROUND: The cryopreservation and transplantation of ovarian tissue have
been shown to restore ovarian function temporarily and may also preserve the fertility
of young female cancer patients until after their sterilizing cancer treatment. Since
tissue samples are large and morphologically complex, the cryopreservation
methodology is difficult to optimize and standardise. Ovarian tissue cryopreservation
is therefore, still in its experimental stages and is not a routine option offered to
cancer patients.
OBJECTIVES: Our main aim was to initiate, develop and implement a practical
ovarian tissue cryopreservation and re-transplantation protocol which would restore
ovarian function, and possibly fertility, in young female cancer patients undergoing
sterilizing cancer therapies in South Africa. The objective of this study was to
improve the slow cryopreservation protocol for human ovarian tissue. The
ultrastructural effects after cryopreservation with two well-known cryoprotectants,
dimethyl sulfoxide (DMSO) and 1,2-propylene glycol (PROH), on early human
follicles were investigated and compared to identify and the better cryoprotectant.
MATERIALS AND METHODS: A single group experimental study design was used.
The participants consisted cancer patients of the Gynaecological Oncology Unit of
Tygerberg Hospital who entered on a basis of voluntary informed consent. Ovarian
tissue was obtained by laparoscopic oophorectomy. After dissection of the
ovary(ies), some fresh cortical tissue was sent for metastatic analysis and a few
strips taken as fresh control. Remaining dissected ovarian cortical tissue sections of
each patient were equally divided into the two cryoprotectant groups. Five resulting
groups could be compared: i) fresh tissue (control group); tissue equilibrated in ii)
DMSO; or iii) PROH and tissue equilibrated and cryopreserved in iv) DMSO or v)
PROH. Five tissue samples per patient were therefore fixed for standard histological
haematoxylin and eosin (HE) staining and transmission electron microscopy (TEM).
Tissue samples showing early follicles on HE slides were sent for TEM processing.
Ultrastructural studies on micrographs of primordial and primary follicles were
assessed according to a scoring system which gave an indication of follicular health.
Appropriate statistical tests were applied to analyse the mean scores where P≤0.05
was considered as statistically significant. RESULTS: No significant overall cryopreservation treatment effect was evident in
any of the follicular ultrastructures evaluated. This result indicated that the
cryopreservation protocol used did not induce significant damage to the cortex tissue
compared to the fresh control group. Comparison of the effect of PROH and DMSO
on the follicular ultrastructures showed that PROH tend to induce more extensive
damage, especially after cryopreservation. Correlation studies showed significant
positive relationships between the majority of the evaluated ultrastructures, especially
between the oocyte and granulosa cell layer and basal membrane. The stromal cells
and extracellular matrix did not correlate well with other structures. Correlations
indicated that the granulosa cells, oocyte and basal lamina are affected similarly and
that the damage in one of these structures may be representative of the damage in
the other structures.
CONCLUSION: The main aim of the study was achieved since results showed that
no significant damage was induced by the cryopreservation protocols. Ovarian tissue
cryopreserved in this study has shown to restore endocrine function temporarily after
heterotopic autotransplantation in menopausal patients. From the electron
microscopy evaluations, DMSO showed better cryopreservation results. The DMSO
cryopreservation protocol was also more time efficient and has shown the most
successful outcomes in the literature. The stromal tissue seemed to be affected
differently by cryopreservation compared to the primordial follicle ultrastructures.
Younger patients are needed for future studies since a larger initial follicular reserve
may allow for larger follicle sample sizes.
Description
Thesis (MScMedSc (Obstetrics and Gynaecology))--Stellenbosch University, 2008.
Keywords
Cryopreservation of organs, tissues, etc., Cryoprotectants, Ovarian tissue, Fertility, Cancer -- Treatment, Cancer -- Treatment -- Complications, Tissues -- Preservation, Dissertations -- Medicine, Theses -- Medicine, Dissertations -- Obstetrics and gynaecology, Theses -- Obstetrics and gynaecology