Volume 29, Issue 1 (3-2022)                   RJMS 2022, 29(1): 70-83 | Back to browse issues page

Research code: 1
Ethics code: IR.MODARES.REC.1397.198
Clinical trials code: 1

XML Persian Abstract Print


Download citation:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:

Feyzmanesh S, Baheiraei N, Halvaei I. Evaluation of Parameters Affecting Encapsulated Human Spermatozoa in Alginate Hydrogel during Cryopreservation. RJMS 2022; 29 (1) :70-83
URL: http://rjms.iums.ac.ir/article-1-6993-en.html
PhD, Department of Anatomical Sciences, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran , ihalvaei@modares.ac.ir
Abstract:   (753 Views)
Background & Aims: Today, sperm cell cryopreservation, as a suitable method, is widely used in infertility clinics to maintain sperm cell fertility potential. But sperm cryopreservation has deleterious effects on sperm parameters. For this purpose, there are various ways to protect sperm cells during cryopreservation like sperm cell encapsulation with alginate (ALG). Sodium ALG (C6H7O6Na) is sodium salt of alginic acid, which is a natural anionic and hydrophilic polysaccharide and is mainly extracted from the cell wall of brown seaweed (7). Due to its non-toxicity and high biocompatibility, ALG hydrogel can create semi-permeable membranes around the sperm cells (8). To the best of our knowledge, no study has been performed to optimize the method of encapsulation of sperm with ALG in human sperm for clinical use. The main purpose of this study was to find an optimal method for encapsulating human sperm for use in cryopreservation.
Methods: In this study, in four stages, the effect of different concentrations of ALG, calcium chloride, and how to add cryoprotectant agent (CPA) in the process of encapsulation of sperm by ALG were investigated. In this study, the direct swim-up method was used to prepare sperm samples. ALG hydrogels were prepared by dissolving sodium ALG powder in a water solvent (12). To evaluate the optimal concentration, the solutions were well homogenized at concentrations of 1 and 1.5% by volume (W/V) at room temperature. Calcium chloride was selected as a cross-linker for cross-linking and final hydrogel formation and was used with a concentration of 102 mM/L. For evaluation of the effects of ALG concentration, twelve normozoospermic samples were divided into the following groups after preparation and subjected to freezing and thawing: 1- ALG 1% + CPA, 2- ALG 1.5% + CPA, 3- control. To determine the best concentration of calcium chloride, which was used as a crosslinker to form ALG hydrogels, twelve normozoospermic samples after preparation were divided into the following groups and then subjected to freezing and thawing: 1- ALG + CPA + CaCL2 (100µL), 2- ALG + CPA + CaCL2 (150µL): 3- control. For evaluation of addition of CPA before encapsulation, twelve normozoospermic samples, were prepared and divided into the following groups and then subjected to freezing and thawing: 1- ALG + CPA, 2- ALG, 3- control. To investigate the effects of giving time before encapsulation twelve normozoospermic samples were divided into the following groups and then subjected to freezing and thawing: 1- ALG + (CPA + NaCl), 2- ALG + group (CPA + NaCl) 3 Min, 3- control. At each stage, the sperm samples were frozen by rapid freezing after encapsulation. To freeze the samples in the cryotube, they were first placed horizontally at a distance of three cm above the level of liquid nitrogen in the nitrogen vapor for thirty minutes and then immersed in the liquid nitrogen. During thawing, the cryotubes were placed at 35 °C for two minutes after leaving the liquid nitrogen. The samples were then transferred to a laminar hood and placed in the medium containing 150 μL of sodium citrate 119 mM/L solution at pH = 7.5. After 30 seconds, the pre-warmed Ham’s F10 with human serum albumin was added dropwise and after complete washing of the cryotube with the medium, the samples were centrifugated for 10 minutes at 300 g. After removing the supernatant, the final pellet was used for analysis. After thawing and dissolving ALG, sperm motility and viability were assessed for all groups. Eosin-nigrosin staining method was used to evaluate membrane integrity and sperm viability. The ultrastructure of ALG hydrogel was examined by scanning electron microscopy (SEM).
Results: SEM showed that the prepared ALG hydrogel had a porous structure with interconnected porosity that could act as a semi-permeable membrane. The progressive motility in the 1.5% ALG group was zero. There was also a significant difference between total (progressive + non-progressive) motility of sperm between the 1% and 1.5% ALG groups (P˂0.001). Total sperm motility in ALG groups showed a significant decrease compared to the control group. Sperm viability rate in the 1% ALG group was significantly higher than the 1.5% ALG group (P˂0.01) and both ALG groups showed a significant decrease in viability rate compared to the control group. Progressive and total motility of sperm in the 150 μL calcium chloride group compared to the 100 μL group showed a decreasing trend while total motility in the 150 μL group compared to the control group showed a significant decrease (P <0.05). The rate of sperm viability in the 150 μL group compared to the 100 μL group of calcium chloride and the control group showed a decreasing trend. Regarding the adding CPA before encapsulation on sperm parameters, progressive and total sperm motility in the different ALG groups showed a significant decrease compared to the control group and there was no significant difference between the groups of adding CPA before encapsulation and after encapsulation. The rate of sperm viability after thawing in the group that received CPA before encapsulation showed a significant increase compared to the group that received before cryopreservation, but there was no significant difference compared to the control group. The group that did not receive CPA before encapsulation showed a significant reduction in sperm viability compared to all groups (P <0.001). Regarding the effects of giving time before encapsulation, progressive motility in both groups that used ALG for cryopreservation showed a significant decrease compared to the control group. However, the total motility in the group that had time was significantly higher than the group that did not have time (P <0.05). Sperm viability rate in the group without time did not show a significant difference compared to the group that had three minutes, but there was a significant decrease compared to the control group.
Conclusion: Encapsulation of sperm with ALG is a promising method that can prevent the side effects of cryopreservation. It seems that 1% ALG with 100 µL of calcium chloride and the use of CPA before and after encapsulation is a good way to encapsulate human sperm by ALG for freezing.
Full-Text [PDF 943 kb]   (218 Downloads)    
Type of Study: Research | Subject: Anatomy

Add your comments about this article : Your username or Email:
CAPTCHA

Send email to the article author


Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

© 2024 CC BY-NC 4.0 | Razi Journal of Medical Sciences

Designed & Developed by : Yektaweb