Razi Journal of Medical Sciences
مجله علوم پزشکی رازی
RJMS
Medical Sciences
http://rjms.iums.ac.ir
39
journal39
2228-7043
2228-7051
en
jalali
1400
12
1
gregorian
2022
3
1
29
1
online
1
fulltext
fa
بررسی اثرپارامترهای تاثیرگذار بر اسپرم های انسانی کپسوله شده در هیدروژل آلجینات در طی روند انجماد
Evaluation of Parameters Affecting Encapsulated Human Spermatozoa in Alginate Hydrogel during Cryopreservation
علوم تشریحی
Anatomy
پژوهشي
Research
<span style="font-size:11pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="tab-stops:10.5pt"><span style="direction:rtl"><span style="unicode-bidi:embed"><span style="line-height:115%"><span style="font-family:Calibri,"sans-serif""><b><span lang="FA" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"B Mitra""><span style="color:#0070c0">زمینه و هدف: </span></span></span></span></b><span lang="FA" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"B Mitra""><span style="color:black">انجماد اسپرم فرایند اجتناب ناپذیری است که در مراکز درمان ناباروری انجام میشود و میتواند منجر به ایجاد آسیبهایی در اسپرم گردد و هنوز روش بهینهای برای انجماد اسپرم معرفی نشده است. راهکارهای مختلفی در جهت حفظ سلول اسپرم برای مقابله با صدمات ناشی از انجماد وجود دارد که یکی از آنها استفاده از کپسوله کردن سلول اسپرم با کمک آلجینات میباشد. هدف اصلی این مطالعه یافتن یک روش بهینه برای کپسوله کردن اسپرم برای استفاده در انجماد اسپرم میباشد. </span></span></span></span></span></span></span></span></span></span></span></span><br>
<span style="font-size:11pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="tab-stops:10.5pt"><span style="direction:rtl"><span style="unicode-bidi:embed"><span style="line-height:115%"><span style="font-family:Calibri,"sans-serif""><b><span lang="FA" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"B Mitra""><span style="color:#0070c0">روش کار:</span></span></span></span></b><span lang="FA" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"B Mitra""><span style="color:black"> در این مطالعه در چهار مرحله اثر غلظتهای مختلف آلجینات، کلراید کلسیم، و نحوه افزودن مواد محافظ انجماد در روند کپسوله کردن اسپرم توسط آلجینات بررسی شد. در هر مرحله اسپرمها بعد از کپسوله شدن با روش انجماد سریع منجمد شدند. بعد از ذوب و باز شدن آلجینات، حرکت و زندهمانی اسپرم بین گروههای مختلف بررسی شد. از روش رنگ آمیزی ائوزین-نیگروزین برای بررسی سلامت غشاء و زندهمانی اسپرم استفاده شد. فراساختار هیدروژل آلجینات توسط میکروسکوپ الکترونی روبشی بررسی گردید. </span></span></span></span></span></span></span></span></span></span></span></span><br>
<span style="font-size:11pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="tab-stops:10.5pt"><span style="direction:rtl"><span style="unicode-bidi:embed"><span style="line-height:115%"><span style="font-family:Calibri,"sans-serif""><b><span lang="FA" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"B Mitra""><span style="color:#0070c0">یافتهها:</span></span></span></span></b><span lang="FA" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"B Mitra""><span style="color:black"> <a name="_Hlk72310023">حرکت و زندهمانی اسپرم در گروه آلجینات 5/1% در مقایسه با آلجینات 1% کاهش داشت. </a></span></span></span></span><span lang="AR-SA" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"B Mitra""><span style="color:black">حرکت و زندهمانی</span></span></span></span><span lang="FA" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"B Mitra""><span style="color:black"> اسپرم در گروه 150 میکرولیتر نسبت به گر</span></span></span></span><span lang="AR-SA" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"B Mitra""><span style="color:black">وه 100 میکرولیتر کلراید کلسیم </span></span></span></span><span lang="FA" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"B Mitra""><span style="color:black">روند کاهشی </span></span></span></span><span lang="AR-SA" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"B Mitra""><span style="color:black">نشان داد. </span></span></span></span><span lang="FA" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"B Mitra""><span style="color:black">حرکت پیشرونده و تام اسپرم بین گروه افزودن محیط محافظ انجماد قبل از کپسوله کردن و بعد از کپسوله کردن اختلاف معنیداری وجود نداشت. گروهی که مواد محافظ انجماد را قبل از کپسوله کردن دریافت نکرده بود نسبت به همه گروهها کاهش معنیداری را در میزان زندهمانی اسپرم نشان داد. </span></span></span></span><span lang="AR-SA" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"B Mitra""><span style="color:black">میزان حرکت تام و زندهمانی در گروهی که قبل از کپسوله کردن زمان داشت در مقایسه با گروهی که زمان نداشت به طور معنیداری بیشتر بود. ساختار متخلخل هیدروژل آلجینات توسط </span></span></span></span><span lang="FA" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"B Mitra""><span style="color:black">میکروسکوپ الکترونی روبشی تایید شد.</span></span></span></span> <span lang="FA" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"B Mitra""><span style="color:black"></span></span></span></span></span></span></span></span></span></span></span></span><br>
<span style="font-size:11pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="tab-stops:10.5pt"><span style="direction:rtl"><span style="unicode-bidi:embed"><span style="line-height:115%"><span style="font-family:Calibri,"sans-serif""><b><span lang="FA" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"B Mitra""><span style="color:#0070c0">نتیجهگیری:</span></span></span></span></b><span lang="FA" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"B Mitra""><span style="color:black"> آلجینات 1% به همراه 100 میکرولیتر کلراید کلسیم و استفاده از مواد محافظ انجماد قبل و بعد از کپسوله کردن روش مناسبی برای کپسوله کردن اسپرم انسان توسط آلجینات به منظور انجماد میباشد. این مطالعه نشان داد که آلجینات میتواند برای کپسوله کردن اسپرم انسان مورد استفاده قرار بگیرد و مطالعات آینده باید تأثیر آلجینات در انجماد اسپرم را مورد بررسی قرار دهند.</span></span></span></span></span></span></span></span></span></span></span></span><br>
<span style="font-size:11pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="tab-stops:10.5pt"><span style="line-height:115%"><span style="font-family:Calibri,"sans-serif""><b><span style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"Times New Roman","serif""><span style="color:#0070c0">Background & Aims:</span></span></span></span></b> <span style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"Times New Roman","serif""><span style="color:black">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. </span></span></span></span></span></span></span></span></span></span><br>
<span style="font-size:11pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="tab-stops:10.5pt"><span style="line-height:115%"><span style="font-family:Calibri,"sans-serif""><b><span style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"Times New Roman","serif""><span style="color:#0070c0">Methods:</span></span></span></span></b> <span style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"Times New Roman","serif""><span style="color:black">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). </span></span></span></span></span></span></span></span></span></span><br>
<span style="font-size:11pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="tab-stops:10.5pt"><span style="line-height:115%"><span style="font-family:Calibri,"sans-serif""><b><span style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"Times New Roman","serif""><span style="color:#0070c0">Results:</span></span></span></span></b> <span style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"Times New Roman","serif""><span style="color:black">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. </span></span></span></span></span></span></span></span></span></span><br>
<span style="font-size:11pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="tab-stops:10.5pt"><span style="line-height:115%"><span style="font-family:Calibri,"sans-serif""><b><span style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"Times New Roman","serif""><span style="color:#0070c0">Conclusion:</span></span></span></span></b> <span style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"Times New Roman","serif""><span style="color:black">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.</span></span></span></span><span style="font-size:9.5pt"><span style="line-height:115%"><span style="font-family:"Times New Roman","serif""><span style="color:black"></span></span></span></span></span></span></span></span></span></span>
اسپرم انسان, کپسولهای آلجینات, انجماد
Cryopreservation, Alginate Capsules, Human Spermatozoa
70
83
http://rjms.iums.ac.ir/browse.php?a_code=A-10-5252-2&slc_lang=fa&sid=1
Somayeh
Feyzmanesh
سمیه
فیض منش
3900319475328460064357
3900319475328460064357
No
MSc, Department of Anatomical Sciences, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
کارشناسی ارشد، گروه علوم تشریح، دانشکده علوم پزشکی، دانشگاه تربیت مدرس، تهران، ایران
Nafiseh
Baheiraei
نفیسه
بحیرایی
n.baheiraei@modares.ac.ir
3900319475328460064358
3900319475328460064358
No
PhD, Department of Anatomical Sciences, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
دانشیار، گروه علوم تشریح، دانشکده علوم پزشکی، دانشگاه تربیت مدرس، تهران، ایران
Iman
Halvaei
ایمان
حلوایی
ihalvaei@modares.ac.ir
3900319475328460064359
3900319475328460064359
Yes
PhD, Department of Anatomical Sciences, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
استادیار، گروه علوم تشریح، دانشکده علوم پزشکی، دانشگاه تربیت مدرس، تهران، ایران