Islam, Nadia
(2020)
The effects of gonadal tissue/gamete cryopreservation on microRNAs.
PhD thesis, University of Nottingham.
Abstract
Advanced technologies have steadily been established for conserving the potential of biological parenthood in couples who are at risk of losing their fertility due to suffering from benign, malignant cancers as well as other genetic
diseases. Methods for fertility preservation differ significantly, depending mainly on the patient’s age and the disease they are suffering from. Cryopreservation of gametes has been suggested to be a putative novel approach
to preserving fertility in women and men who are at risk of losing fertility. However, the effects of the freezing/thawing process are not fully understood and ways to assess the tissue’s potential to perform normally after thawing, are an important research goal.
MicroRNAs are evolutionarily conserved, single-stranded, non-coding RNA molecules, consist of 18–22 nucleotides. They are important intercellular signalling molecules, known to have major roles in post-transcriptional gene regulation and protein synthesis. In many cells, specific microRNAs are
expressed differentially in disease states compared to normal tissue. Recent reports have identified microRNAs during the process of folliculogenesis and spermatogenesis which may be able to modulate reproduction. Over 200 microRNAs have been reported in human follicular fluid and using bioinformatics analysis, microRNA expression in granulosa cells and follicular fluid and its correlation with endocrine, reproductive and metabolic functions have been studied. During spermatogenesis they are also expressed in a precise manner, therefore taking part in every step of male germ cell development. The lack of tight regulation of these processes by microRNAs, can cause disruptions and prevent certain stages of development occurring within the correct time frame during folliculogenesis or spermatogenesis, and it can lead to
abnormalities in oocyte/sperm development.
In this thesis, we investigated the possible association between microRNA expression pattern and cryopreservation that could help in the assessment of ovarian tissue damage and oocyte/sperm quality post freeze-thaw cycles. We have selected five target microRNAs (24, 193b, 320A, Let7-b and 34C), as these microRNAs have been shown to play an important role in regulation of folliculogenesis and spermatogenesis in a variety of species.
For ovarian cortical tissue, we investigated the possible interplay between steroid production and microRNAs’ expression pattern that could help in the assessment of tissue damage post freeze-thaw cycles. Ovine ovarian cortical tissues were cultured in rotatory cell culture system with or without
cryopreservation, spent culture media from the system was analysed for steroid production (oestradiol and progesterone) and microRNA expression profile (24, 193b and 320A). The number of primordial, transitory, primary, secondary and antral follicles were analysed between fresh, fresh-cultured and cryopreserved cultured ovarian cortical tissues. The findings confirmed that, down-regulation of microRNA-193b and microRNA- 320A together with up-regulation of microRNA-24 could have a synergistic role in cell apoptosis, and consequently leading to reduced oestradiol and progesterone production, which can be further explored as novel non-invasive markers of cell damage following cryopreservation.
In terms of oocyte, we investigated the expression pattern of three microRNAs (24, 193b and 320A) in spent culture media supplemented with or without Insulin-Transferrin-Selenium as indicators of oocyte quality; post maturation and post vitrification. Ovine oocytes were matured in media supplemented with or without Insulin-Transferrin-Selenium. Following maturation, oocyte cumulus expansion was measured, and viable oocytes were then vitrified and tested again for viability after warming. The changes in expression pattern of microRNAs’ in spent culture media supplemented with or without Insulin-Transferrin-Selenium was investigated, both post- maturation and post- vitrification, and the differential expression was correlated with cumulus expansion, oocyte maturation and oocyte viability. The findings confirmed that, oocytes matured in media supplemented with Insulin-Transferrin-Selenium showed a significant increase in cumulus expansion and nuclear maturation, and viability following vitrification. Downregulated microRNA- 24 and 193B and upregulated 320A might be associated with improved oocyte maturation, viability and cumulus expansion, which could be boosted by the presence of Insulin-Transferrin-Selenium supplementation. These microRNAs can further be explored as markers of oocyte quality following maturation and vitrification.
In case of sperm, we studied the expression pattern of five microRNAs (24, 193b, 320A, Let-7b and 34C) in human semen samples as marker of sperm quality, pre and post density gradient centrifugation. The expression pattern of these microRNAs was correlated with semen parameters (concentration, progressive motility and velocity). The findings confirmed that, the effect of DGC in samples was a positive one based on the expression levels of target microRNAs. Also, negative correlation of microRNA-193b and positive correlation of microRNA-34C with velocity along with increased expression of microRNA-34C following DGC can possibly be further re-enforced utilised as a marker for fertility.
We also investigated the possible association between sperm cryopreservation and semen parameters (concentration, progressive motility and velocity)/microRNAs’ expression pattern that could help in the assessment of sperm quality post freeze-thaw cycles. Semen parameters and microRNAs’ (24, 193b, 320A, Let-7b and 34C) expression profile was evaluated in human spermatozoa subjected to cryopreservation (controlled rate freezing and vapour cooling) using 2 different types of cryoprotectants (Test-yolk buffer and Quinns advantage sperm freezing media). Both the freezing methods (controlled rate freezing and vapour cooling) as well as CPAs (Test-yolk buffer and Quinns advantage sperm freezing media) were comparable in maintaining sperm concentration, PM% and velocity. Nonetheless, based on the findings of microRNAs’, Quinns advantage sperm freezing media seems to be superior to Test-yolk buffer using both the freezing methods (controlled rate freezing and vapour cooling).
In conclusion, down-regulation of microRNA-193b and microRNA-320A along with up-regulation of microRNA-24 for cryopreserved then cultured ovarian cortical tissue might have a synergistic role in cell apoptosis and consequently reduced oestradiol and progesterone production. Moreover, downregulated microRNA-24 and microRNA-193B and upregulated microRNA-320A plays an important role in oocyte maturation, viability and cumulus expansion in in presence of ITS. These microRNAs’ (24, 193b and 320A) could be further explored as novel markers of cell injuries and/or damage following cryopreservation for ovarian cortical tissue and oocytes. As for the sperm, the negative and positive correlation of microRNA-193b and microRNA-34C respectively with velocity can possibly be further utilised as a marker for sperm quality following preparation and cryopreservation. Both tested sperm freezing methods were comparable in maintaining sperm concentration, progressive motility and microRNA expression, nonetheless, supplementation of egg yolk resulted in poorer outcomes.
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