انجماد

Developing culture and cryopreservation methods for spermatogonial stem cells (SSCs) is essential for genetic studies, regenerative medicine, transgenic animal production and recombinant drug development. Establishing an optimal culture system for these cells is crucial. This study aimed to investigate the in vitro effects of different concentrations of L-arginine on the proliferation of frozen-thawed caprine SSCs.

This experimental study was repeated five times. Isolated spermatogonial cells were frozen- thawed and cultured in six groups with a basal culture medium (DMEM containing 1% antibiotics and 5% FBS) supplemented with different concentrations of L-arginine (0, 50, 100, 200, 500 and 1000 μmol/L). All experimental groups were cultured for 10 days. The number and surface area of colonies formed were assessed on days 4, 7, and 10. Expression of spermatogonial genes (ID4, BCL6, PGP9.5, VASA, PLZF and OCT4) was determined by RT-PCR.

The viability rates of fresh cells before and after the addition of a cryoprotectant agent were 87.14 ± 2.6% and 80.72 ± 1.21%, respectively, which decreased to 65.38 ± 1.26% after thawing. The number and surface area of spermatogonial colonies in the fourth treatment group (200 μmol/L L-arginine) were significantly higher compared to other experimental groups (P < 0.05). Finally, the isolated cells expressed SSC surface markers.

The addition of 200 μmol/L L-arginine likely enhances the colonization of frozen-thawed goat SSCs in short- term culture.

Ovarian tissue freezing is the most effective method to maintain fertility for immature girls and women diagnosed with cancer. Nonetheless, because of the chance that malignant cells might reappearing following tissue transplantation, it is crucial to isolate the follicles from the frozen-thawed ovarian tissue of these individuals and employ them in the process of in vitro maturation process or artificial ovarian framework. This study aimed to assess the application of neutral red (NR) vital dye alongside collagenase IA for effectively isolating viable follicles from the vitrified human ovarian tissue samples.

Two categories existed: the category with NR and the group without NR. Chopped (0.5×0.5 mm) strips of vitrified-warmed ovarian tissue from 10 transsexual individuals were placed into two falcon tubes with HTCM and Collagenase IA (1mg/ml). Neutral red (NR) was introduced to one of the falcons. Follicles were then isolated mechanically. The morphology, size, and viability of the follicles were assessed. The condition of the follicles was evaluated using fluorescent staining methods involving Calcein-AM and Ethidium homodimer-I. The t-test method was used to evaluate the data.

The number of isolated follicles with Neutral Red (46.50±25/02) exceeded those without NR (6.6±5.58; P < 0/0001). Additionally, according to the morphological studies, a majority of the isolated follicles from the transsexual ovarian cortex were primordial (77.4%), and primary (21.12%) follicles, with only a small number of secondary follicles (1.4%) identified in these tissues. Live/dead staining verified the viability of isolated follicles by displaying a green hue.

The finding indicates that combining collagenase I with the vital dye Neutral red significantly facilitates the of follicles from dense human ovarian tissue.

To assist save endangered species from extinction and to aid in their care, it is crucial to sustaining their reproductive capacity. The fetus's life and growth processes begin at conception and proceed in accordance with a biological clock's timing. With today's understanding through cryobiology, observing scientific ethics problems and interfering with the clock's operation by stopping its biological time is possible. Practically, it is accomplished by storing the cell at -196 °C, or the temperature of liquid nitrogen, where all metabolic activity ceases. Since 200 years ago, germ cell preservation has been a common practice. Since then, there have been many improvements, particularly for at-risk women. Since then, significant progress has been achieved, and several freezing techniques are being used to preserve ovarian tissue, follicles, and oocytes in women who are at risk of infertility. Different approaches have different levels of success. Among preservation techniques, vitrification performs better and is used more frequently. The cellular configuration of the mammalian oocyte is intricate. This cell's constituent parts are particularly sensitive to osmosis and variations in temperature. For instance, the alterations to the cell membrane that occur during maturation, in vitro fertilization, and the differential between the permeability of water and cryoprotectants can all be mentioned. Oocyte freezing results in a variety of impairments, including a reduction in the quality and viability of cells after thawing.

Recent studies are looking for ways to enhance freezing procedures and raise the caliber of frozen oocytes. The favorable or negative effects of freezing on the oocyte and its potential, or embryo development, are the subject of this review article.

 Cryopreservation by various mechanisms, such as the induction of oxidative, osmotic, and temperature stress, causes a reduction in sperm quality after the freeze-thaw process. This study aimed to investigate the effect of the simultaneous addition of chlorogenic acid and lipoic acid as an antioxidant supplement to sperm freezing medium in patients with oligoasthenospermia.

 Sperm samples from patients with oligoasthenozoospermia were divided into five groups as follows: fresh sample (pre-frozen semen), control (sperm freezing medium without supplements), CGA group (sperm freezing medium with 100 µM chlorogenic acid), ALA group (sperm freezing medium with 200 µM lipoic acid) and CGA+ALA group (sperm freezing medium with chlorogenic acid and lipoic acid). Total mobility, viability rate (eosin-nigrosin staining) and DNA fragmentation index (Halo test) were assessed before and after freeze-thawing.

 In the control group, there was a decrease in total sperm motility and viability and an increase in DFI. Freezing sperm in the CGA, ALA, and CGA+ALA groups was associated with a significant increase in viability and total sperm motility after thawing. The results of the Halo test also showed that the increase in sperm DFI caused by the freeze-thaw process was less in all three experimental groups than in the control group, and this decrease was statistically significant (P<0.05).

 The simultaneous addition of chlorogenic acid and lipoic acid to the sperm freezing medium can improve the quality of sperm samples after the freeze-thaw process in terms of mobility, viability, and DNA fragmentation, thus increasing the efficiency of this technique in the treatment of male infertility.

Cryonics or cryopreservation is one of the newest topics in biomedicine and has been able to keep incurable patients with some hope of recovery. Considering that in cryobiology, the heart and brain and all body mechanisms are stopped and bringing people back to life is currently not a practical possibility, we are faced with the question of whether it is permissible to freeze incurable patients from a jurisprudential point of view or not? In this research, we intend to discuss the permissibility or impermissibility of cryobiology by analyzing the nature of freezing.

This research has been carried out by descriptive and analytical method and using library tools. First the nature of freezing and then the jurisprudential validity or invalidity of freezing has been discussed.

The examination of Shari'i evidence shows that, considering that the freezing of a patient is considered a type of death that can be proven in the theory of unstable death, and currently there is no practical possibility to revive such people, the freezing of incurable patients is not allowed from the Shari'a point of view.

Sperm cryopreservation is an important assisted reproductive technique. However, due to increased production of reactive oxygen species and oxidative stress, it can impair sperm function and reduce quality of fertility. The aim of this study was to investigate the effects of nicotinic acid and folic acid on maintenance of sperm function during cryopreservation by evaluation of sperm parameters and chromatin concentration.

Thirty samples were collected from normozoospermic men and examined for chromatin quality, viability, membrane integrity, morphology and motility. The samples were frozen and placed in 4 groups: control, folic acid (50 nM), nicotinic acid (10 mM) and a combination of both. After cryopreservation, the four groups were compared with one another in regard to the sperm parameters. Also the sperm parameters were compared before and after freezing in every group. We assessed chromatin quality by TB staining, viability by eosin-nigrosin staining, membrane integrity by hypo osmotic swelling test, and morphology and movement by CASA software.

Before cryopreservation chromatin quality, membrane integrity, normal morphology, sperm motility were lower and immotile sperms were higher in all groups compared to those after cryopreservation (p <0.001). The highest chromatin quality was detected in the folic acid, folic acid + nicotinic acid groups and the lowest chromatin quality was observed in the control group (p</05). The rates of sperm viability and normal morphology were lowest in the control group and highest in the folic acid and other groups (p <0.05). percentage of membrane integrity was highest in the folic acid group followed by nicotinic acid + folic acid, nicotinic acid and control groups, respectively (p <0.05). Folic acid played an important role in maintaining sperm parameters.

Nicotinic acid and folic acid have a positive effect on maintaining sperm function during cryopreservation

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.

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).

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.

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.

One of the problems after cryopreservation and ovarian tissue transplantation is ischemia followed by follicles mortality. In the present study, hyaluronic acid hydrogel was used to improve angiogenesis in vitrified ovarian tissue transplantation in rat.

In this experimental study, 22 adult female rats (~8 weeks old) with normal estrous cycles were ovariectomized, then their right ovaries were vitrified and warmed into two groups without hyaluronic acid hydrogel (VT) and encapsulated with hyaluronic acid hydrogel (VT+HA) was autotransplanted into the dorsal muscle. Daily vaginal monitoring was performed until re-initiation of first full oestrus cycles. The ovaries were removed at the end of the first estrus cycle and angiogenesis genes VEGF, CD31 and CD34 evaluated by real time PCR. Data were analyzed by Mann-Whitney test and the significance level was considered P<0.05.

All transplants were completely successful (100% successful transplant). There was no statistically significant difference in CD34 and CD31 genes between two groups. But VEGF gene expression was significantly lower in VT+HA group (0.25±0.06) than VT group (1.00±0.05) (P<0.05). Thus, the VT group appeared more successful than the VT+HA group in expressing angiogenic genes at the end of the first estrus cycle.

Hyaluronic acid hydrogel does not play any effective role in the growth and improvement of angiogenesis after transplantation in the short term. But due to the angiogenic property of hyaluronic acid, increasing the expression of angiogenic genes in the VT + HA group may need more time.

Reactive oxygen species (ROS) are of the most important detrimental factors on sperm quality during freezing process, which increases the lipid peroxidation (LPO) of cell membranes. In this study, the concentration of malondialdehyde (MDA) levels in semen of men with oligospermia before and after cryopreservation process was measured to evaluate the effect of folic acid and nicotinic acid on the MDA concentration after freezing.

In this experimental study, semen fluid sample was collected from 25 men with oligospermia in age range of 25-45 years. Each sample was divided into 5 groups: Fresh group, Freeze group without antioxidants (control), Freeze group with nicotinic acid (10 mM), Freeze group with folic acid (50 nM), and Freeze group with a combination of nicotinic acid (10 mm) + folic acid (50 nM). The concentration of MDA in nmol/ml was measured in each group with spectrophotometer at 535 nm.

The mean concentration of MDA in semen increased after freezing (1.73 ± 0.12) compared to before freezing (0.45 ± 0.02) (P < 0.001). Mean concentration of MDA in the group of folic acid + nicotinic acid (0.68 ± 0.06) was lower compared to other groups after freezing (P ± 0.001).

The combination of folic acid and nicotinic acid antioxidants with sperm freezing medium reduces the level of MDA and LPO of sperm membrane during freezing process, and thereby maintains the fertility potential in men with oligospermia.

Sperm associated antigens (SPAGs) play an important role in the incidence of various cancers including breast, lung, liver, and bladder. SPAGs are also important in sperm functions, such as motility. However, it seems that sperm cryopreservation as one of the assisted reproductive techniques (ART), can affect the expression of these genes. This study aimed to evaluate the effect of freezing on the expression of human SPAG 5 and 9 in human spermatozoa.

In the present experimental study, twelve semen samples in terms of normozoospermic parameters were collected from individuals referred to the Royan Institute, and progressive motile sperms were isolated by density gradient centrifugation (DGC). Each sample was divided into two, non-frozen (control) and frozen groups. After rapid freezing and three-day storage in liquid nitrogen, samples were thawed in tap water and incubated for 2 hours of recovery-time in a CO2 incubator. RNA extraction in both groups was performed using TRIzol; and SPAG5 and 9 were evaluated by Real-time PCR technique.

Based on statistical analysis, the expression of SPAG5 decreased significantly in the frozen group compared to the control group (P≤0.05); In contrast, there was no significant difference in the expression of SPAG9 between the control and frozen groups.

Considering the cold shock in the future of cell, a significant reduction in SPAG5 expression in the frozen group may indicate probable influences in the derived fetus.

  Freezing human semen is considered an effective technique in assisted reproductive techniques (ART). During cryopreservation, sperm cells are exposed to chemical and physical stress, resulting in damage to the axonum, increased morphological deficits in the middle segment, and decreased sperm motility. In this study, the effects of trehalose and resveratrol antioxidants on sperm parameters during freezing and thawing processes were evaluated.

A cross-sectional study was performed on 40 normal semen samples divided into 7 groups. The effects of these antioxidants on sperm parameters were investigated by computer analysis (CASA) and sperm chromatin (SCD) evaluation. It should be noted that P<0.05 was considered statistically significant.

Group 3 (freezing and treating with 100 mM trehalose) showed the highest percentage of motility, acrosome membrane integrity, and plasma membrane integrity compared to the control group and had healthier DNA after thawing and fewer survival of sperm.

According to the results, it can be concluded that trehalose samples in general improve the quality of semen and even after thawing it is involved in maintaining sperm motility. Resveratrol causes toxicity at high concentrations and causes undesirable changes in osmotic pressure, but at low concentrations it has beneficial effects on sperm survival and inhibits ROS as a potent antioxidant.

Ovarian vitrification is a technique for assisted reproductive technologies, while ovaries are vulnerable to the damage caused by the freezing procedure. Pentoxifylline has a protective effect as an antioxidant in reducing the free radicals in the culture medium. This study aimed to determine the antioxidant effects of pentoxifylline on the changes stereological vitrified ovarian tissue of mice after the vitrification process.

The study was performed on 25 mice ovaries, which were randomly divided into five groups (five mice each group). Group 1 (control): No treatment was performed in this group. Groups 2 and 3 (sham 1,2): In Group 2, after the ovaries are removed, they are placed for half an hour in a CO2 incubator in the saline phosphate buffer + bovine serum albumin (PB1 + BSA) environment, And in Group 3, in addition to being placed in the incubator, 1.8 mM of Pentoxifylline was added to the buffer environment. Groups 4 and 5 (Experimental 1,2): In Group 4, after the ovaries are removed from the animalchr('39')s body, the ovaries are vitrified for two weeks, after which time they are melted and placed in a CO2 incubator for half an hour in the saline phosphate buffer + bovine serum albumin (PB1 + BSA) environment, and In Group 5, in addition to vitrified, melting, and incubation, the ovaries were exposed to buffers containing 1.8 mM pentoxifylline. after tissue processing, ovarian tissue sections were studied with stereological methods.

Stereological estimation showed that the ovarian tissue volume and cortex in the sham2, experiment1, and experiment2 were increased compared to control and sham1 groups, but this increase was not statistically significant (P>0/05). The volume of the medulla was reduced in the sham2, experiment1 and experiment2 groups compared to the control and sham1 groups, but the reduction was not statistically significant (P>0/05).

According to the results of this study, pentoxifylline and Vitrification increase the volume of ovarian tissue, and pentoxifylline, despite its antioxidant properties, will not affect protecting the ovaries against the negative effects of Vitrification.