Cryopreservation Of Human Spermatozoa Research Articles

The evaluation effect of nanoliposome-loaded Mito-Tempo on sperm parameters during human sperm cryopreservation.

The aim of this study is the evaluation effect of nanoliposome-loaded Mito-Tempo on sperm parameters during human sperm cryopreservation. Semen samples of 50 Asthenoteratozoospermia men (random) were collected. Sperm parameters were analyzed based on World Health Organization (WHO, 2010) criteria (2021) and each sample was divided into 5 groups (E1-E5). E1 (control group): the sperm was cryopreserved without nanoliposome, and Mito-Tempo. E2: sperm cryopreservation with Mito-Tempo-loaded nanoliposome (Mito-Tempo 0.1 mM) + freezing medium. E3: sperm cryopreservation with Mito-Tempo-loaded nanoliposome (Mito-Tempo 0.2 mM) + freezing medium. E4: in this group, the cryopreservation sperm with Mito-Tempo 0.3 mM + freezing medium. E5: the cryopreservation sperm with Mito-Tempo 0.2 mM + freezing medium. The result of this study indicated that sperm parameters and total antioxidant capacity (TAC) significantly increase in E3 and E4 groups, compared to E1, E2, and E5 groups respectively (P < 0.05). The percentage of abnormal morphology, DNA fragmentation index (DFI), malondialdehyde (MDA), and the levels of ROS significantly decrease in E3 and E4 groups, compared to E1, E2, and E5 groups (P < 0.05). In addition, the sperm parameters and stress oxidative factors significantly improve in E3 group compared to other groups (P < 0.05). In conclusion, the combination of Mito-Tempo with nanoliposome due to its ability to cooperate with lipid layers may lead to significant performance in reducing oxidative stress damage and increasing the quality of sperm parameters.

Addition of Vitamin C Mitigates the Loss of Antioxidant Capacity, Vitality and DNA Integrity in Cryopreserved Human Semen Samples.

Cryopreservation of human spermatozoa is a necessity for males suffering from infertility who cannot produce fresh semen for insemination. However, current ART cryopreservation protocols are associated with losses of sperm motility, vitality and DNA integrity, which are thought to be linked to the induction of oxidative damage and the toxic properties of commercial cryoprotectants (CPAs). Preventing or mitigating these losses would be hugely beneficial to sperm survival during ART. Therefore, in this in vitro investigation, lipid peroxidation, production of reactive oxygen species, movement characteristics, antioxidant capacity, vitality, and DNA integrity were examined in semen samples both pre- and post-cryopreservation with CPA supplementation. The findings revealed a 50% reduction in antioxidant capacity with CPA addition, which was accompanied by significant increases in generation of reactive oxygen species and formation of lipid aldehydes. These changes were, in turn, correlated with reductions in sperm viability, motility and DNA integrity. Antioxidant supplementation generated bell-shaped dose-response curves with both resveratrol and vitamin C, emphasising the vulnerability of these cells to both oxidative and reductive stress. At the optimal dose, vitamin C was able to significantly enhance vitality and reduce DNA damage recorded in cryopreserved human spermatozoa. An improvement in sperm motility did not reach statistical significance, possibly because additional pathophysiological mechanisms limit the potential effectiveness of antioxidants in rescuing this aspect of sperm function. The vulnerability of human spermatozoa to reductive stress and the complex nature of sperm cryoinjury will present major challenges in creating the next generation of cryoprotective media.

Evaluation of the effects of hydroxytyrosol on human sperm parameters during cryopreservation

Human sperm cryopreservation is a routine procedure in assisted reproductive technology, but it has detrimental effects on different sperm parameters due to oxidative stress. Our objective was to assess the impacts of hydroxytyrosol (HT), as an antioxidant, on human sperm parameters following cryopreservation. In the first phase, 20 normal human semen samples were cryopreserved using the rapid freezing method with different concentrations of HT including 0, 50, 100, 150, and 200 μg/mL. In the second phase, 20 normal semen samples were collected and cryopreserved with 50 and 100 μg/mL HT. The beneficial effects of HT were determined by evaluation of motility (computer-assisted sperm analysis; CASA), viability (Eosin-nigrosine stain), DNA integrity (sperm chromatic dispersion test, SCD), reactive oxygen species (DCF and DHE staining by flowcytometry) lipid peroxidation (malondialdehyde, MDA test) and mitochondrial membrane potential (JC1 staining by flowcytometry) of sperm after cryopreservation. After thawing, sperm motility had an increasing trend in 50 and 100 μg/mL HT groups in comparison with other groups, althought the difference was not significant. However, sperm viability was significantly increased at 50 and 100 μg/mL HT. Our data also showed that sperm DNA fragmentation was significantly decreased after thawing at 100 μg/mL in comparison with 0 and 50 μg/mL HT. However, the level of intracellular reactive oxygen species, lipid peroxidation and mitochondrial membrane potential were not significantly different between groups. Our results showed that HT may have protective effects on the viability and DNA integrity of human sperm during the freezing-thawing process.

Carboxylated Poly-L-lysine Potentially Reduces Human Sperm DNA Fragmentation after Freeze-Thawing, and Its Function Is Enhanced by Low-Dose Resveratrol.

Sperm DNA fragmentation (SDF) that occurs during the freezing-thawing of sperm may negatively impact the treatment outcomes of assisted reproductive technologies (ART). In a previous study, we developed a human sperm cryopreservation reagent containing carboxylated poly-L-lysine (CPLL) that reduced SDF after freeze-thawing compared with clinically popular cryopreservation reagents containing human serum albumin. However, it is unclear whether CPLL reduces SDF, as it differed from the constituents of the commercial cryopreservation reagents used for comparison. Therefore, here, we examined whether CPLL reduces the SDF of human sperm and evaluated reactive oxygen species (ROS) levels and lipid peroxidation (LPO), which are the causes of SDF; mitochondrial injury, ROS production; and impaired sperm motility. Furthermore, optimal antioxidants and their concentrations that could further enhance the reduction in SDF were determined for future clinical application in ART and underwent the same functional evaluations. CPLL can reduce SDF via inhibition of intracytoplasmic ROS and LPO. Furthermore, the addition of 0.1 mM resveratrol avoided the enhancement of SDF, which potentially affects mitochondrial and cytoplasmic ROS and LPO. This novel human sperm cryopreservation reagent containing CPLL and resveratrol has the potential to improve treatment outcomes in ART using frozen sperm.

Problems of human spermatozoa cryopreservation: research methods, solutions.

Cryopreservation of male gametes is one of the most important methods of assisted reproductive technologies, which allows preserving gametes for research or further use. However, the fertilizing ability of spermatozoa after cryopreservation decreases by 30-70%, which makes it urgent to search for new substances with cryoprotective properties. The review considers the main causes of cell damage during cryopreservation. The relevance of methods for assessing the formation of crystals and the physicochemical properties of cryoprotective media depending on various compositions is discussed. The problem of stabilization of the spermatozoa membrane during cryopreservation is considered. A possible solution to the problem of membrane integrity may consist in modification of the basic cryoprotective media with yolk emulsion or development of methods for saturation of the membrane phospholipid layer with cholesterol.

Omega-3 Fatty Acids as Supplements and Cryoprotectants and Positive Outcomes for Human Seminal Cryopreservation: A Study in the Iraqi Context

Background: With the increasing deterioration in male fertility rates over the past few decades, assisted reproductive therapy via sperm banking has gained considerable attention from different health authorities worldwide. The process of sperm cryopreservation exerts certain harmful effects on sperm quality parameters. The aim of the current study was to examine the effect of omega-3 on human sperm cryopreservation as a dietary supplement and as a cryoprotectant stimulant.&#x0D; Methods: From healthy men, 120 seminal fluid samples were randomly collected for cryopreservation (for 30 days). All samples were divided into three groups (40 in each). The supplement group (SG) (first group) had been given dietary supplements of omega-3 (30% EPA: 20% DHA) for eight weeks before sample collection. The samples of the cryoprotectant group (CG) (second group) were treated with omega-3 additive in the diluent at the time of being collected and before we cryopreserved them. The samples from the control group (third group) were cryopreserved without any dietary or cryoprotectant supplementation. Initial seminal analysis was recorded and post-thawing assessment of sperm motility, vitality (using eosin test) and oxidative stress (using a nitro blue tetrazolium (NBT) test) were compared.&#x0D; Results and Conclusion: SG samples had greater initial seminal fluid volumes, as well as better sperm motility and vitality, but the oxidative stress assessment did not differ significantly pre-cryo. Post-thawing assessment revealed that the CG group had the best parameters of motility, vitality and oxidative stress. These results may be related to the positive effects of omega-3 fatty acids on reproductive glands, hormonal milieu, sperm function and structure, making it a suitable biostimulant for improving the outcome of human sperm banking. Results were comparable to multiple previous animal studies.

P-098 Cryopreservation process deregulates the expression of pathways important for fertility in human spermatozoa

Abstract Study question Does cryopreservation impact on transcriptome of conventional frozen human spermatozoa? Summary answer The cryopreservation of human sperm alters pathways important for fertility, although the upregulation of some genes can compensate for the harmful effects of freezing. What is known already Sperm cryopreservation is a widely used procedure for storing gametes for later use, for preserving fertility in patients before gonadotoxic treatments or surgery, and for sperm donation programs. The cryopreservation technique can cause damage to sperm through induction of DNA alterations, fragmentation and oxidation, and altered motility, mitochondrial activity and morphology. Transcriptomic alterations have been reported in cryopreserved sperm from various animal species, but no experimental data are available on the effect of cryopreservation on the transcriptome-wide profile of human sperm. Study design, size, duration Semen samples were obtained from 20 normospermic men from April to May 2022. Each sample was divided in two aliquots. From one aliquot total RNA was immediately extracted. The second aliquot was slowly cryopreserved and after a week of storage in liquid nitrogen total RNA was extracted. A total of 13 paired RNA samples passed quality control and were analyzed after randomization into 4 pools, each of 6 patients. Participants/materials, setting, methods The men who participated in this study had a median age of 35.0 years (range: 29.0-46.0). RNA was extracted by miRNeasy Micro Kit (Qiagen), analyzed by High Sensitivity RNA on 2200 Tape Station system (Agilent), amplified by Ovation Pico WTA SystemV2 (Nugen), labeled and hybridized on GE 4x44K v2 microarrays (Agilent). The paired Significance Analysis of Microarray (SAM) was performed using the limma hgug4112a.db and samr packages in R/BioConductor. Main results and the role of chance The expression profiles of cryopreserved sperm significantly differed from those of fresh sperm in 219 down-regulated and 28 up-regulated unique transcripts. The gene ontology analysis disclosed that cryopreservation downregulates genes involved in sperm motility by the mitochondria function (CABS1, SPATA19) and the correct organization of the sperm midpiece and flagellum cytoskeleton (ACTL7A, AKAP4, C9ORF24, CAPZB, CCIN, IQCG, ODF2, SPATA6, SPATA6L, TBC1D21), in fertilization (ACTL7A, AKAP4, PLCZ1, PRSS37, TUBGCP3), early embryo development (AGT, CLMN, DCC, DHRS3, EGFLAM, FAM20A, FREM1, GPI, HEMGN, KRTDAP, IFT172, MICAL2, MLF1, NF2, PGRMC2, PHACTR1, POPDC3, RO60, ROBO1, RORA, SGCA, SPRR2D, TCF4, TNC, TNNI3), oxidant detoxification (TXNDC29) and DNA repair (BRIP1, ERCC6, TRIP12), calcium ion binding and homeostasis (AMY1C, ANO1, ANO2, ASGR1, CABS1, CPNE9, FREM1, KCNIP2, ITPR3, PKD2L1, PLCZ1, SELENOK, SYN3, TNNI3). Upregulated genes were enriched in pathways related to the negative regulation of DNA damage response (ALOX15B, CD74, RPL10, SNAI1, THBS1), cellular response to calcium ion (ALOX15B, FOSB, HLA-DQB1, S100A4, THBS1), regulation of transcription (CD74, FOSB, HLA-DRB1, MAMLD1, RASD1, RPL10, SNAI1, TCF15, TLE1), protein stabilization (CD74), protein ubiquitination (BAG1, DCAF6, ERCC6, HERPUD2, KLHL7, MARCHF8, NF2, PSMA6, RNF133, SERGEF, TRIP12, UBE2DNL, UBL3, UBQLN3, ZNRF3) and ubiquitin protein ligase binding (GPI, HSPA1L, PRKAR2A, STX8). Limitations, reasons for caution Data validation in a larger sample cohort and by quantitative PCR would be useful, although we have applied stringent criteria for gene selection (FDR = 0). Further research should be undertaken to experimentally test whether the length of cryostorage can have any effect on the gene expression profile of sperm. Wider implications of the findings Specific pathways are deregulated by cryopreservation, in accordance with altered sperm morphology, motility and molecular integrity reported in literature. Functional enrichment analyses also revealed gene expression changes to compensate for the sperm cryoinjury. This finding is noteworthy for safety issue of sperm banking i.e., fertility preservation, gamete donation. Trial registration number not applicable

P-048 Vitrification of human semen impairs the mobility of spermatozoa when compared to the conventional slow-freezing

Abstract Study question Does vitrification provide a better method for human sperm cryopreservation than slow freezing in terms of survival, mobility, maturity, and DNA fragmentation? Summary answer Vitrification of human semen yields similar outcome as slow-freezing considering survival, mobility, DNA fragmentation, and maturity but the mobility after thawing is lower. What is known already Slow freezing is the most common technique for cryopreservation of human sperm. Unfortunately, many sperm cells die during the process or lose their mobility and are thereafter unable of fertilization. Meanwhile, vitrification of animal sperm showed good outcomes in terms of sperm survival and motility while being easier to perform than slow freezing. So far, many studies focused on survival and mobility of human sperm after vitrification compared to slow freezing, with results still not unified. They also neglected the importance of sperm maturity and DNA fragmentation caused by the process. DNA damage can affect embryo development or cause abortion. Study design, size, duration The study was performed from March January to July 2022. It included 69 male participants who underwent a diagnostic spermiogram at the Division of gynaecology and obstetrics of University Medical Centre Ljubljana. All participants gave written consent of using the remaining of sample, after performing the spermiogram, for our study. Sperm samples were divided in three aliquots, one saved as native sample, one intended for vitrification, and one intended for slow freezing. Participants/materials, setting, methods Participants in the study had normal sperm quality parameters according to 5th edition of WHO manual. Slow freezing was performed using Sperm Freezing medium (Origio). For vitrification sperm was diluted in solution consisting of Sperm preparation medium (Origio), sucrose, and HSA in 1:1 (v/v) ratio, incubated for five minutes and then vitrified by adding 30 microliter droplets directly into liquid nitrogen. After thawing sperm survival, mobility, maturity, and DNA fragmentation were assessed. Main results and the role of chance The data are presented as mean values (± standard deviation) when normally distributed and as medians (IQR) when not normally distributed. The patient’s age was 35.0±5.1 years. In native sperm sample, sperm concentration was 69.6±59.0x106 cells per millilitre and total motility was assessed to be 60.0% (50.0%-70.0%). After thawing we noticed significantly lower mobility (p &amp;lt; 0.001), while also the mobility was significantly lower after vitrification when compared to slow-freezing (17.6% (12.4%-25.3%) and 14.5% (9.0%-22.6%), p = 0.007). Interestingly, there was no difference in survival after thawing the samples (20.5±10.7 % after slow freezing and 23.7±13.7 % after vitrification). Also there was no difference in maturity of sperm when checked with protamine staining (native vs. slow-freezing vs. vitrification; 53.6±15.2 % vs. 53.9±16.0 % vs. 52.4±15.2 %, p = 0.905) and also when checked with hyaluronan binding assay (78.0% (67.5%-82.4%) vs. 79.4% (71.7%-82.95%) vs. 75.3% (67.5%-81.1%), p = 0.607) . While sperm maturity was not affected with cryopreservation, DNA fragmentation was significantly higher after cryopreservation (native vs. slow-freezing vs. vitrification; 19.7±9.5%, vs. 24.7±11.6 vs. 27.1±15. %, p = 0.049). Limitations, reasons for caution We believe our study would be better by obtaining higher number of participants and by being performed over longer time period. We would also like to further optimise vitrification protocol. More knowledge is also needed in poor sperm samples, since we only included sperm samples of good quality. Wider implications of the findings We believe our finding are of great importance for the field of andrology. Our results show vitrification of human sperm can be comparable to slow freezing in most of parameters analysed, but lower mobility after vitrification is reason for caution. Trial registration number 20210024

The effects of coenzyme Q10 and curcumin supplementation in freezing medium for human sperm cryopreservation

BackgroundDespite its routine and frequent application, cryopreservation of human sperm is far from the desired efficacy, as freezing and thawing impair motility, viability, acrosomal unity, and DNA integrity. ObjectivesIn this study, the authors aimed to investigate whether adding antioxidants, coenzyme Q10, and curcumin into the freezing medium provide better efficacy in the cryopreservation of human sperm. MethodsThe semen samples from 40 healthy men aged 18–45 were collected in sterile containers by masturbation. Samples within normal reference values for sperm concentration (≥15 million/mL) and motility (progressive motile ≥ 32% and total motility ≥ 40%) were included in the study. Semen samples were equally divided into five groups and evaluated; i) pre-freezing sperm suspension, ii) frozen-thawed control (Ctrl) without any supplementation in freezing medium, iii) frozen-thawed with curcumin supplementation of 0.25 mM (Cur), iv) frozen-thawed coenzyme Q10 supplementation of 25 µM (CoQ10) and v) frozen-thawed curcumin (0.25 mM) plus coenzyme Q10 (25 µM) supplementation (CurCoQ10) into the freezing medium. Liquid nitrogen vapour freezing and rapid thawing were performed in each group (ii-v). Sperm motility, viability, acrosome integrity, and DNA fragmentation rates were compared and ultrastructural evaluations by transmission electron microscopy were undertaken between the groups. Additionally, the total antioxidant capacity/total oxidant capacity values were measured. ResultsAccording to CASA results, progressive motility was significantly higher in the CoQ10 group (9.4 ± 7.6) when compared with the Ctrl (7.1 ± 6.3), Cur (6.4 ± 4.8) and CurCoQ10 (8.1 ± 7.7) groups (p < 0.05). Flow cytometry results showed no difference in the viability and acrosome integrity values after thawing, but DNA fragmentation was significantly increased in the curcumin-added groups (p < 0.05). Acrosomal changes and sub-acrosomal defects were seen in all groups after thawing at the ultrastructural level. Mitochondrial membrane structure was preserved in CoQ10 and CurCoQ10 groups. ConclusionsOur results suggested that sperm ultrastructural morphology and motility were better preserved in the CoQ10 group during cryopreservation. In curcumin groups, DNA fragmentation and head defects were increased.

Phosphatidylcholine and cholesterol-loaded-cyclodextrins can replace egg yolk in the cryopreservation of equine spermatozoa

In the horse breeding industry, cryopreservation of spermatozoa has become an integral aspect of modern-day protocols. To avoid sperm damage during the freezing process, freezing media containing egg yolk are used. Recently, urgency to find an alternative to egg yolk has been increased due to the prevalence of Avian Influenza Virus (AIV). In the UK, the World Organization for Animal Health reports that between July and December 2022, an AIV outbreak led to the death of 4.62 million birds (56.73 million globally), with substantial consequences for agriculture, environment and egg availability (WAHIS. 2023; https://wahis.woah.org/#/dashboard/qd-dashboard: Accessed: 12 January 2023). Recent research has also called into question the biosafety of egg yolk, with RNA from highly pathogenic and low pathogenic AIV found on and in eggs (Stephens.et.al. Avian Diseases. 2020; 64:143-148). The cryoprotectant potential of phosphatidylcholine, the main phospholipid in egg yolk, has been investigated in diluents used for cryopreservation of human spermatozoa with some success (Sicchieri.et.al. Translational Andrology and Urology. 2021; 10:397-407). The chemically defined origin and increased biosafety make phosphatidylcholine a desirable alternative to egg yolk for use in cryopreservation. Ejaculates from four horses (n=4) were frozen using four different freezing extenders: a reference medium containing egg yolk and 3% dimethylformamide (control) and media without egg yolk supplemented with 1.5mg/ml2-hydroxypropyl-β-cyclodextrin cholesterol complex, 3% dimethylformamide and either 2% (P1), 4% (P2) or 6% phosphatidylcholine (P3). Samples were diluted and centrifuged on a density gradient medium (1000xg’ for 20 minutes). The cell band was aspirated and resuspended (200 × 10⁶ cells/ml) with allotted treatment extender. Samples were packed into 0.25ml straws, cooled to 5˚C for 25 minutes, then frozen using a programmable freezing unit (IceCube14s, Minitube, Germany). Temperature of the straws was reduced from 4 ˚C to -143˚C in 9 minutes, after which they were plunged directly into liquid nitrogen (-196˚C). Viability was assessed using NucleoCounter® NC-100™. Total motility analyses were performed with computer assisted semen analysis (Hamilton Thorne, USA). No difference in post-thaw viability was found between any of the media tested (P>0.05). No difference in total motility was found between the control (36.75±1.93σM), P1 (29.25±3.82σM) or P2 media (19.5±6.35σM) (P>0.05). However, post-thaw total motility of sperm cryopreserved in P3 media (16±6.01σM) was lower than the control (P<0.05). Media supplemented with 2-4% phosphatidylcholine and 1.5mg/ml cholesterol was effective in limiting loss of total motility and membrane integrity of equine spermatozoa, establishing the need for further breeding trials into the use of phospholipids for egg yolk lipid replacement to improve biosafety. The authors wish to recognize the support of Tullis Matson and Stallion AI Services Ltd in this study.

Sperm Cryopreservation Today: Approaches, Efficiency, and Pitfalls.

The cryopreservation of human spermatozoa has been an option for patients undergoing chemo or radiotherapies since the late 1950s. Presently, there are different techniques for the cryopreservation of spermatozoa. The most commonly used techniques are programmable slow freezing and freezing on liquid nitrogen vapors, while the use of vitrification is still not accepted as clinically relevant. Although there have been many improvements, the ideal technique for achieving better post-thaw sperm quality continues to be a mystery. A major obstacle during cryopreservation is the formation of intracellular ice crystals. Cryodamage generated by cryopreservation causes structural and molecular alterations in spermatozoa. Injuries can happen because of oxidative stress, temperature stress, and osmotic stress, which then result in changes in the plasma membrane fluidity, motility, viability, and DNA integrity of the spermatozoa. To prevent cryodamage as much as possible, cryoprotectants are added, and in some clinical trial cases, even antioxidants that may improve post-thaw sperm quality are added. This review discusses cryopreservation techniques, cryodamage on molecular and structural levels, and cryoprotectants. It provides a comparison of cryopreservation techniques and describes recent advances in those techniques.

Beneficial effects of trehalose and gentiobiose on human sperm cryopreservation.

The protection of human sperm during cryopreservation is of great importance to infertility. Recent studies have shown that this area is still a long way from its ultimate aim of maintaining the maximum viability of sperm in cryopreservation. The present study used trehalose and gentiobiose to prepare the human sperm freezing medium during the freezing-thawing. The freezing medium of sperm was prepared with these sugars, and the sperm were then cryopreserved. The viable cells, sperm motility parameters, sperm morphology, membrane integrity, apoptosis, acrosome integrity, DNA fragmentation, mitochondrial membrane potential, reactive oxygen radicals, and malondialdehyde concentration was evaluated using standard protocols. A higher percentage of the total and progressive motility, rate of viable sperm, cell membrane integrity, DNA and acrosome integrity, and mitochondrial membrane potential were observed in the two frozen treatment groups compared to the frozen control. The cells had less abnormal morphology due to treatment with the new freezing medium than the frozen control. The higher malondialdehyde and DNA fragmentation were significantly observed in the two frozen treatment groups than in the frozen control. According to the results of this study, the use of trehalose and gentiobiose in the sperm freezing medium is a suitable strategy for sperm freezing to improve its motion and cellular parameters.

New Technique for Human Sperm Cryopreservation Using Emptied Sheep's Ovarian Follicles.

This study aimed to investigate the efficacy of emptied ovarian follicles of sheep as a container for cryopreservation of human spermatozoa to preserve the presence of low concentrations of spermatozoa at the post-thawing stage. This research was performed on 30 semen samples from oligozoospermic patients and 10 samples from normozoospermic males. They were diagnosed according to the standard criteria of the World Health Organization 2010. Semen samples were classified into four groups of G1-G4 according to sperm concentration: 3-5 million/mL, 6-10 million/mL, 11-15 million/mL, and 16-20 million/mL, respectively. Each sample was divided into two equal parts. One part was cryopreserved without cryoprotectant, while the other was diluted 1:1 with 10% glycerol-based cryosolution. The ovarian follicles of sheep were obtained from a local slaughterhouse and prepared by slicing the ovaries and evacuating the follicular fluid and oocyte. The emptied follicles were injected with the prepared semen samples. After cryopreservation and thawing, the semen mixture aspired outside the follicles, and sperm parameters were measured, namely concentration, progressive motility, total motility, and normal morphology. Sperm concentration and progressive and total sperm motility were significantly (P<0.01) decreased in all groups at the post-thawing stage, compared to the pre-freezing stage. The sperm concentration was significantly higher (P<0.01) in samples cryopreserved without cryoprotectant, compared to that in those cryopreserved with glycerol. However, progressive and total motility were significantly (P<0.01) higher in samples cryopreserved with glycerol, compared to that in the samples cryopreserved without cryoprotectant in all groups. Moreover, no significant difference was found between the pre-freezing and post-thawing stages in terms of normal morphology. Emptied ovarian follicles are an appropriate carrier for cryopreservation of human sperms, especially for patients with oligozoospermia. The best sperm survival rate in this technique was observed when using glycerol-based cryosolution.

Comparative Epigenetic Analysis of Imprinting Genes Involved in Fertility, in Cryopreserved Human Sperms with Rapid Freezing versus Vitrification Methods.

Choosing the optimal method for human sperm cryopreservation seems necessary to reduce cryoinjury. The aim of this study is to compare two cryopreservation methods including rapid-freezing and vitrification, in terms of cellular parameters, epigenetic patterns and expression of paternally imprinted genes (PAX8, PEG3 and RTL1) in human sperm which play a role in male fertility. In this experimental study, semen samples were collected from 20 normozoospermic men. After washing the sperms, cellular parameters were investigated. DNA methylation and expression of genes were investigated using methylation-specific polymerase chain reaction (PCR) and real-time PCR methods, respectively. The results showed a significant decrease in sperm motility and viability, while a significant increase was observed in DNA fragmentation index of cryopreserved groups in comparison with the fresh group. Moreover, a significant reduction in sperm total motility (TM, P<0.01) and viability (P<0.01) was determined, whereas a significant increase was observed in DNA fragmentation index (P<0.05) of the vitrification group compared to the rapid-freezing group. Our results also showed a significant decrease in expression of PAX8, PEG3 and RTL1 genes in the cryopreserved groups compared to the fresh group. However, expression of PEG3 (P<0.01) and RTL1 (P<0.05) genes were reduced in the vitrification compared to the rapid-freezing group. Moreover, a significant increase in the percentage of PAX8, PEG3 and RTL1 methylation was detected in the rapid-freezing group (P<0.01, P<0.0001 and P<0.001, respectively) and vitrification group (P<0.01, P<0.0001 and P<0.0001, respectively) compared to the fresh group. Additionally, percentage of PEG3 and RTL1 methylation in the vitrification group was significantly increased (P<0.05 and P<0.05, respectively) compared to the rapid-freezing group. Our findings showed that rapid-freezing is more suitable method for maintaining sperm cell quality. In addition, due to the role of these genes in fertility, changes in their expression and epigenetic modification may affect fertility.

A new cryotop vial device system provides an aseptic cryoprotectant-free and centrifuge-free cryopreservation of human spermatozoa (a closed system)

Single sperm cryopreservation is a new method to preserve small numbers of spermatozoa in small droplets. So far, several devices have been introduced for this technique, but more studies are needed for its optimization. The aim of this study was optimizing the previous device for low number of spermatozoa and low volume semen, which led to design of Cryotop Vial device. Normal semen samples from 25 patients were prepared by swim-up method and divided into four groups: Fresh (F), Rapid freezing (R) and ultra-rapid freezing with Cryotop Device (CD) and Cryotop Vial Device (CVD). In R group, the diluted sperm suspension with sperm freezing medium was cooled in the vapor phase and then immersed in liquid nitrogen. Ultra rapid freezing was performed with sucrose in small volume using the Cryotop Device (CD) or Cryotop Vial Device (CVD). Sperm viability, motility, fine morphology, mitochondrial activity and DNA fragmentation were assessed in all samples. All sperm parameters decreased significantly in all the cryo groups compared to the fresh group. The comparison between the cryo groups showed that progressive motility (69.28 ± 6.82 vs. 55.68 ± 9.04, and 54.76 ± 5.34, p < 0.001) and viability (77.36 ± 5.48 vs.68.84 ± 8.51, p < 0.001, and 70.04 ± 7.44, P = 0.002) were significantly higher in the CVD compared to the CD and R groups respectively. DNA fragmentation was also significantly lower in both ultra-rapid freezing groups (CD and CVD) compared to the R group. Fine morphology and mitochondrial activity were not different between the cryo groups. The CVD as a cryoprotectant and centrifuge-free technique preserved sperm motility, viability and DNA integrity after cryopreservation better than other groups.

The effects of purslane (Portulaca oleracea) and fennel (Foeniculum vulgare Mill) hydroalcoholic extracts on the functional parameters of human spermatozoa after vitrification.

Reactive oxygen species (ROS) are produced during cryopreservation of human sperm and impair sperm function. Antioxidant compounds, such as fennel and purslane, reduce the damaging effects of ROS. This study aimed to evaluate motility parameters, plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), intracellular ROS, and DNA damage to determine the optimum concentrations of hydroalcoholic extracts of fennel and purslane for human spermatozoa cryopreservation. Twenty human sperm samples were used and divided into seven equal groups consisting of fennel hydroalcoholic extract (5, 10, and 15 mg/L), purslane hydroalcoholic extract (25, 50, and 100 mg/L), and no additive. Supplementation of 25 mg/L and 50 mg/L purslane extract and 10 mg/L fennel extract in cryopreservation extender significantly increased the motility and PMI of sperm with a significant reduction in intracellular ROS compared to control groups (p<0.05). A 50 mg/L concentration of purslane extract elevated progressive motility and MMP compared to the control group (p<0.05). No significant differences were seen for motion patterns and DNA damage of frozen-thawed human sperm in extender containing these extracts. The results showed that supplementation of 50 mg/L purslane extract and 10 mg/L fennel extract in semen cryopreservation extender has the potential to decrease intracellular ROS and subsequently elevate the motility and PMI of human sperm.

The Effects of Silymarin Supplementation on Post-Thawed Human Sperm Functional Parameters.

This study investigated the effect of silymarin on human sperm quality during cryopreservation. Samples were collected from 20 normospermic individuals, and each sample was divided into different concentrations of silymarin comprising the following groups: (0, 20, 100, 500, and 1000 μg/mL silymarin). Sperm quality parameters, such as plasma membrane integrity, mitochondrial membrane potential, acrosomal membrane integrity, and caspase 3 were estimated. Silymarin concentrations of 100-500 μg/mL significantly increased motility, plasma membrane integrity, and mitochondrial activity compared with the frozen control group. Acrosomal integrity was increased in the 1000 μg/mL silymarin group. Moreover, 20 and 100 μg/mL concentrations significantly decreased the percentage of caspase 3. The addition of silymarin antioxidant to the frozen medium reduced damage in the sperm after freezing and thawing. This is the first study that showed silymarin can be useful in cryopreservation of human sperm.

Evaluation of supplementation of cryopreservation medium with gallic acid as an antioxidant in quality of post-thaw human spermatozoa.

Human sperm cryopreservation is a way to preserve sperm in the clinic. But, the production of reactive oxygen species (ROS) during cryopreservation, has certain negative impacts on the quality of sperm and its reproductive capacity. The goal of this study is to see how sperm cryopreserved in media enriched with gallic acid alter post-thaw morphology, motility, viability, DNA structure, and plasma membrane lipid peroxidation. Four groups were considered for performing this study: (1) Fresh sperm before cryopreservation; (2) cryopreserved control sperm without any supplementation; (3) cryopreserved sperm using freezing media supplemented with 50 μg/ml gallic acid and (4) cryopreserved sperm using freezing media supplemented with 100 μg/ml gallic acid. This study's results indicated that the addition of doses of 50 μg/ml gallic acid to cryopreservation medium significantly improved sperm morphology, motility, vitality, and DNA integrity and reduced DNA fragmentation and lipid peroxidation as compared to the medium without any gallic acid supplementation (p< 0.05); but, the concentration of 100 μg/ml gallic acid had no significant effects on the mentioned sperm parameters (p> 0.05). This study's findings represent the possibility of enhancing sperm characteristics and lowering detrimental effects of ROS on sperm DNA structure during cryopreservation by supplementing sperm freezing medium with gallic acid as a natural antioxidant. Therefore, Gallic acid may help improve the quality and subsequently fertilization potential of cryopreserved human sperm.

O-185 Cryopreservation of Human Spermatozoa: Utilization of L-Proline as a Novel Additive to Improve Sperm Quality Following Freezing-Thawing Process

Abstract Study question Does supplementation of human sperm freezing media with L-Proline improve sperm quality and DNA Integrity following the freezing-thawing process? Summary answer The inclusion of L-proline as a novel additive to human sperm cryopreservation media improves sperm parameters and DNA integrity via mitigating oxidative stress. What is known already Sperm cryopreservation is an essential aspect of assisted reproductive technique (ART) and male fertility preservation. Although frozen-thawed semen has great practical benefits for reproduction, it is widely reported that the cryopreservation process induces physical and chemical detrimental changes in sperm functions. Indeed, freezing induces nitro-oxidative stress, lipid peroxidation, DNA fragmentation, and apoptosis. L-proline plays versatile roles in osmotic protection and oxidative stress, cell signaling, programmed cell death, and nutrient adaptation. This multifunctional amino acid is a natural osmoprotectant and the protective effect of L-proline against freezing-thawing-induced damages in stallion and donkey spermatozoa have been reported. Study design, size, duration Thirty normozoospermic semen samples were collected by masturbation after 3–5 days of sexual abstinence from men who were referred to the IVF clinic of Kermanshah Motazedi Hospital from December 2020 to June 2021. Each prepared semen sample was aliquoted to 4. In aliquots 1 to 4, experimental concentrations of L-proline (0, 1, 2, and 4 mmol/L), were included in the freezing medium. Participants/materials, setting, methods 30 normozoospermic, healthy, non-smoker men were enrolled. Sperm parameters (progressive motility, viability, and morphology) were assessed. Sperm chromatin quality was measured by Aniline blue (AB), Toluidine blue (TB), and Chromomycin A3 (CMA3) staining. DNA integrity was evaluated by the Sperm chromatin dispersion (SCD) test. Furthermore, the levels of reactive oxygen species (ROS), lipid peroxidation (LPO), and total antioxidant capacity (TAC) were determined in sperm freezing media. All assessments were conducted before cryopreservation and after thawing. Main results and the role of chance Our findings showed that sperm progressive motility and viability were significantly higher in the 4 mmol/L L-proline treated groups compared to the control groups (p = 0.003, p = 0.0200) respectively. The percentage of normal morphology was improved in L-Proline-treated groups. However, this improvement was not considerable (p &amp;gt; 0.5). Moreover, the level of ROS production significantly diminished in the 4 mmol/L L-proline group compared to the control group (p &amp;lt; 0.001). Besides, the level of TAC was significantly enhanced in the 4 mmol/L L-proline group by comparison to the control group (p &amp;lt; 0.001). Furthermore, the results of AB and TB tests demonstrated that chromatin packaging after supplementation with 4 mmol/L L-proline significantly improved compared to the control group (p &amp;lt; .0001). In this regard, there was a significant improvement between the 4 mmol/L L-proline group and the control group in terms of CMA3 evaluation (p &amp;lt; .0001). Also, there was a difference between the 4 mmol/L L-proline group and the control group in reducing the level of fragmented DNA. Although 4 mmol/L of Praline could diminish the SCD level compared to the control group, this amelioration was not statically significant (p = 0.053). Limitations, reasons for caution We were not able to perform further studies to verify the effects of L-proline on mitochondrial activity and membrane potential, and sperm apoptosis as well as to confirm the efficiency and safety of L-proline in terms of more indicators such as the in vitro embryonic development and live birth rate. Wider implications of the findings These findings can draw attention to the potential role of L-proline as a novel additive and antioxidant to human freezing medium in preserving sperm quality and protecting spermatozoa against ROS attack during sperm cryopreservation in infertility clinics. Trial registration number N/A

Cyanidin-3-Ο-glucoside supplementation in cryopreservation medium improves human sperm quality.

Currently, the cryopreservation of human spermatozoa must overcome the adverse effects of excessive oxidation. In this study, we aimed to evaluate the effect of supplementation of cryopreservation medium with cyanidin-3-Ο-glucoside (C3G) on sperm quality. Semen samples were obtained from men with normozoospermia according to WHO criteria (n=39). The sperm parameter values were compared after cryopreservation in medium supplemented with and without C3G.Compared with the control group (without additive), low doses (50 μM and 100 μM) of C3G improved sperm viability and motility and decreased the reactive oxygen species (ROS) of spermatozoa, while high doses (200 μM) of C3G did not obviously enhance sperm quality. The amount of DNA fragmentation index (DFI) and high DNA stainability (HDS) after freezing were higher in the control group than in the C3G supplementation groups. Low-concentration C3G supplementation (50μM) was negatively correlated with sperm ROS levels (r= -0.2, p= 0.03). Collectively, our findings suggest that C3G could be an efficient semen cryoprotectant that ameliorates oxidative stress in human sperm during cryopreservation.