The use of fresh versus frozen spermatozoa in men with nonobstructive azoospermia (NOA) undergoingin vitro fertilization (IVF)has been a debated hot topic among reproductive specialists. Each approach presents distinc...The use of fresh versus frozen spermatozoa in men with nonobstructive azoospermia (NOA) undergoingin vitro fertilization (IVF)has been a debated hot topic among reproductive specialists. Each approach presents distinct advantages and disadvantages,with fresh sperm typically showing superior sperm quality, while frozen sperm offers logistical flexibility and a reliable backup forrepeated cycles. This review summarizes the latest advancements in sperm retrieval and cryopreservation techniques, providingpractitioners with a comprehensive analysis of each option’s strengths and limitations. Comparative studies indicate that, althoughfresh sperm often has better quality metrics, cryopreservation methods such as vitrification have significantly improved postthawoutcomes, making frozen sperm a viable choice in assisted reproductive technologies (ART). The findings show comparablerates for fertilization, implantation, clinical pregnancy, and live birth between fresh and frozen microdissection testicular spermextraction (micro-TESE) sperm in many cases, although patient-specific factors such as timing, cost-effectiveness, and proceduralconvenience should guide the final decision. Ultimately, the choice of using fresh or frozen sperm should align with the individualneeds and conditions of patients. This tailored approach, supported by the latest advancements, can optimize ART outcomes andprovide personalized reproductive care.展开更多
Azoospermia is characterized by the absence of sperm in the ejaculate and is categorized into obstructive azoospermia(OA)and nonobstructive azoospermia(NOA).For men with NOA,testicular sperm extraction(TESE)is the onl...Azoospermia is characterized by the absence of sperm in the ejaculate and is categorized into obstructive azoospermia(OA)and nonobstructive azoospermia(NOA).For men with NOA,testicular sperm extraction(TESE)is the only method to obtain sperm for assisted reproductive technology(ART).Given the rarity of these sperm and the unpredictable success of subsequent retrieval attempts,cryopreservation of microdissection-TESE-obtained sperm is essential.Effective cryopreservation prevents the need for repeated surgical procedures and supports future ART attempts.After first delving into the physiological and molecular aspects of sperm cryopreservation,this review aims to examine the current methods and devices for preserving small numbers of sperm.It presents conventional freezing and vitrification techniques,evaluating their respective strengths and limitations in effectively preserving rare sperm,and compares the efficacy of using fresh versus cryopreserved testicular sperm.展开更多
Oocyte cryopreservation is an essential procedure in assisted reproductive technologies,aimed at preserving fertility,particularly for women undergoing IVF treatment or at risk of ovarian damage due to radiation,chemo...Oocyte cryopreservation is an essential procedure in assisted reproductive technologies,aimed at preserving fertility,particularly for women undergoing IVF treatment or at risk of ovarian damage due to radiation,chemotherapy,or surgery.Despite its growing use,the survival and fertilization rates of cryopreserved oocytes remain suboptimal,largely due to cryo-induced oxidative stress.The generation of Reactive Oxygen Species(ROS)during freezing and thawing causes considerable damage to key cellular components,including proteins,lipids,DNA,and mitochondria.This oxidative stress compromises oocyte quality and reduces developmental potential.To address these challenges,the use of additives-especially antioxidants-has shown significant promise in mitigating oxidative damage.Enzymatic antioxidants such as Superoxide Dismutase(SOD)and Catalase(CAT),along with non-enzymatic antioxidants like glutathione,melatonin,and resveratrol,have demonstrated the ability to neutralize ROS and improve oocyte viability and developmental outcomes.Recent studies highlight the potential of Mitoquinone(MitoQ),a mitochondria-targeted antioxidant,to effectively counteract mitochondrial ROS and enhance cellular defense mechanisms during cryopreservation.This review explores the cellular mechanisms of cryodamage,the role of oxidative stress in oocyte cryopreservation,and the potential of various antioxidant strategies to enhance oocyte survival and function.Developing effective antioxidant supplementation approaches may significantly improve the outcomes of cryopreservation in reproductive medicine.展开更多
Chuan Huang,Yu-Lin Tang,Jian-Ling Hu,Wen-Jun Zhou,Zeng-Hui Huang,Xue-Feng Luo,Zheng Li,Wen-Bing Zhu.Update on techniques for cryopreservation of human spermatozoa.Asian Journal of Andrology 2022;24:563-9.Yang Xiong,Fu...Chuan Huang,Yu-Lin Tang,Jian-Ling Hu,Wen-Jun Zhou,Zeng-Hui Huang,Xue-Feng Luo,Zheng Li,Wen-Bing Zhu.Update on techniques for cryopreservation of human spermatozoa.Asian Journal of Andrology 2022;24:563-9.Yang Xiong,Fu-Xun Zhang,Yang-Chang Zhang,Chang-Jing Wu,Feng Qin,Jiu-Hong Yuan.Genetically predicted insomnia causally increases the risk of erectile dysfunction.Asian Journal of Andrology 2023;25:421-5.展开更多
Cryopreservation is a fundamental technology in biomedical research,regenerative medicine,and tissue engineering,enabling the long-term storage of cells,tissues,and organs.However,its effectiveness is limited by chall...Cryopreservation is a fundamental technology in biomedical research,regenerative medicine,and tissue engineering,enabling the long-term storage of cells,tissues,and organs.However,its effectiveness is limited by challenges such as intracellular ice formation,cryoprotectant toxicity,and reduced post-thaw viability.This review explores the crucial role of encapsulation in enhancing cryopreservation efficiency,with a focus on recent advances in materials science,bioengineering,and cryobiology.Emerging technologies,such as nanotechnology and stimuli-responsive polymers,are transforming encapsulation strategies.Innovations such as microfluidic systems offer precise control over cooling rates and cryoprotectant distribution,thereby mitigating conventional limitations.The review also addresses current obstacles related to scaling up encapsulation processes and ensuring the long-term biocompatibility and stability of preserved specimens.By synthesizing recent findings,this work provides a comprehensive resource for researchers and clinicians seeking to enhance biopreservation techniques and their applications in contemporary medicine and biotechnology.Finally,the review identifies critical knowledge gaps that must be addressed to improve the efficacy of cryopreservation strategies and advance their clinical translation.展开更多
Cryopreservation of living cells and tissues plays a vital role in biomedical research,clinical applications,biotechnology innovation,the development of new vaccines and drugs,and the conservation of endangered specie...Cryopreservation of living cells and tissues plays a vital role in biomedical research,clinical applications,biotechnology innovation,the development of new vaccines and drugs,and the conservation of endangered species.While significant technological breakthroughs have been achieved in cooling methods-particularly through vitrification for large tissue and organs-the lack of optimal rewarming technology remains a key obstacle to successful cryopreservation,especially for larger samples such as tissues and organs.The primary challenges during the warming process include non-uniformity heating and insufficient rewarming rates,which can lead to thermal stress-induced structural damage and lethal ice recrystallization,ultimately compromising the integrity and functionality of biological materials.In recent years,various advanced warming techniques have emerged,employing different energy conversion approaches to achieve volumetric heating while minimizing the risk of overheating.These techniques involve thermal,mechanical-thermal,and electromagnetic-thermal energy conversions.However,each method presents its own limitation.This review aims to summarize recent advancements in rewarming technologies for cryopreservation,with a focus on their mechanisms,applications,and the key challenges that must be addressed to enable broader adoption in medical and commercial contexts.展开更多
Organoids are three-dimensional structures derived from stem cells that recapitulate the gene expression profiles and functional characteristics of their tissue of origin,rendering them invaluable tools for disease mo...Organoids are three-dimensional structures derived from stem cells that recapitulate the gene expression profiles and functional characteristics of their tissue of origin,rendering them invaluable tools for disease modeling,drug screening,and precision medicine.Despite their promise,the widespread application of organoids is limited by extended culture durations and technical complexity.Cryopreservation has emerged as a critical strategy to overcome challenges related to the long-term storage and application of organoids,offering a range of preservation approaches tailored to organoid development.Nevertheless,conventional cryopreservation techniques encounter significant limitations when applied to organoids.To address these issues,the development of naturally derived,low-toxicity Cryoprotectants(CPAs),along with the optimization of CPA loading methods and refinement of cooling and warming protocols,is essential to mitigate cryoinjury.Looking forward,the comprehensive enhancement of cryopreservation technologies may facilitate the transformation of organoids into“off-the-shelf”products,enabling scalable production,batch standardization,and centralized distribution.Such advancements will lay the foundation for the establishment of Next-Generation Living Biobanks(NGLB).展开更多
Fertility preservation and pregnancymanagement are critical considerations for patients undergoing organtransplantation.Innovations in assisted reproductive technologies,hormonalmodulation,and personalized medicine ha...Fertility preservation and pregnancymanagement are critical considerations for patients undergoing organtransplantation.Innovations in assisted reproductive technologies,hormonalmodulation,and personalized medicine have expanded options for these patients,who face unique challenges due to immunosuppressive therapy and organ functionconcerns.This mini-review explores advancements in cryopreservationtechniques,pre-conception counseling,and multidisciplinary strategies forsafe pregnancies post-transplantation.Emphasis is placed on balancing maternalhealth,graft function,and fetal outcomes.The integration of reproductive andtransplant medicine is paving the way for improved quality of life andreproductive autonomy for this patient population.展开更多
Germplasm resources are essential for the sustainable development of biodiversity and husbandry of local chickens, as well as for the breeding and industry of superior quality chickens. Unfortunately, many local and i...Germplasm resources are essential for the sustainable development of biodiversity and husbandry of local chickens, as well as for the breeding and industry of superior quality chickens. Unfortunately, many local and indigenous chicken breeds are at risk of declining numbers, emphasizing the need to conserve breed resources for endangered chickens. Primordial germ cells(PGCs) are crucial for preserving germplasm resources by inheriting genetic information from parents to offspring and ensuring stability of genetic material between germlines. In this study,PGCs were isolated from chicken embryos' gonads and cultured in FAcs medium without feeder cells. Over a period of approximately 40 d, the cells proliferated to a number of up to 10^(6), establishing various cell lines. Particularly, 18 PGC lines were created from Rugao Yellow chicken and Shouguang chicken, with an efficiency ranging from 39.1 to 45%. Furthermore, PGCs that had been cultured for 40 passages exhibited typical PGC characteristics, suchas glycogen staining reaction, and expression of pluripotency and reproductive markers. These results confirmthat PGCs maintain stem cell properties even after long-term in vitro culture. Additionally, PGCs cryopreserved for up to 120 d remained viable, maintained typical PGC morphologies, and possessed stable cell proliferation ability. Through intravascular injection into chicken embryos, green fluorescent protein(GFP)-PGCs were found in the recipient embryos' gonads and could develop into gametes to produce offspring, indicating that even after extended culture, PGCs retain their migratory and lineage-transmitting capabilities. This research offers valuable insights into the in vitro cultivation and preservation of PGCs of Chinese indigenous chickens. The findings of this study can be applied in transgenic chicken production and the preservation of genetic resources of indigenous chicken breeds.展开更多
Dear Editor,We are much obliged that Hadziselimovic1 has used our data2 to highlight the substantial proportion of boys with cryptorchidism where gonadotropin insufficiency is an important factor related to the pathog...Dear Editor,We are much obliged that Hadziselimovic1 has used our data2 to highlight the substantial proportion of boys with cryptorchidism where gonadotropin insufficiency is an important factor related to the pathogenesis.We have recently presented a study on a series of 453 consecutive boys with bilateral nonsyndromic cryptorchidism,in which we conducted hormonal evaluations and assessed germ cell numbers in testicular biopsies.3 In this series,45%of the boys were classified as having gonadotropin insufficiency.3 Identifying these patients at the time of surgery is important.A recent follow-up study of 208 boys with nonsyndromic bilateral cryptorchidism from our department showed that the boys with gonadotropin insufficiency had an impaired fertility potential after surgery compared to boys with an intact hypothalamus–pituitary–gonadal feedback mechanism.4 In a review from 2022,Hadziselimovic5 suggested that infertility in patients diagnosed with cryptorchid testes is a consequence of a hormonal deficiency rather than temperature-induced cellular damage.展开更多
Sperm cryopreservation is an essential technique for male fertility preservation,especially in men who are undergoing medical treatment.Conventional cryopreservation methods face limitations such as oxidative stress,D...Sperm cryopreservation is an essential technique for male fertility preservation,especially in men who are undergoing medical treatment.Conventional cryopreservation methods face limitations such as oxidative stress,DNA fragmentation,and cytotoxicity associated with traditional cryoprotectants like dimethyl sulfoxide(DMSO).Recent breakthroughs have focused on improving post-thaw sperm viability with novel cryoprotectants and innovative freezing strategies.Prospective approaches include the use of amino acid-based cryoprotectants,deep eutectic solvents,and antioxidants that have been described to prevent oxidative damage and maintain DNA integrity.Vitrification,a high-speed freezing technique that prevents ice crystal formation,has demonstrated superior outcomes compared to conventional slow freezing.Moreover,the Direct Dropping Method,a cryoprotectant-free approach,has been introduced as a contamination-minimizing technique that preserves sperm functionality.Multiomics tools are also utilized to determine biomarkers for protocol optimization.Despite these advancements,cryoprotectant toxicity is a central challenge,emphasizing the necessity for safer agents.Future research must focus on long-term sperm functionality and individualized cryopreservation strategies to maximize reproductive outcomes.The current review highlights the challenges associated with sperm cryopreservation,explores innovative strategies and novel cryoprotectants,underscores the significance of maintaining DNA integrity,and proposes future research directions to improve fertility preservation outcomes.展开更多
Dear Editor,I would like to congratulate Mamsen et al.i on their extensive and scientifically valuable work.I analyzed their raw data presented in Table 1 of the original article from a different perspective and disco...Dear Editor,I would like to congratulate Mamsen et al.i on their extensive and scientifically valuable work.I analyzed their raw data presented in Table 1 of the original article from a different perspective and discovered an effect not mentioned in the article.My analysis showed that luteinizing hormone(LH)levels are significantly lower in patients at high infertility risk(HIR),whose testes lack A dark(Ad)spermatogonia and display an abnormal ratio of germ cells per crosssectional tubule(G/T).展开更多
BACKGROUND As living biodrugs,mesenchymal stem cells(MSCs)have progressed to phase 3 clinical trials for cardiovascular applications.However,their limited immediate availability hampers their routine clinical use.AIM ...BACKGROUND As living biodrugs,mesenchymal stem cells(MSCs)have progressed to phase 3 clinical trials for cardiovascular applications.However,their limited immediate availability hampers their routine clinical use.AIM To validate our hypothesis that cryopreserved MSCs(CryoMSCs)are as safe and effective as freshly cultured MSC counterparts but carry logistical advantages.METHODS Four databases were systematically reviewed for relevant randomized controlled trials(RCTs)evaluating the safety and efficacy of CryoMSCs from various tissue sources in treating patients with heart disease.A subgroup analysis was performed based on MSC source and post-thaw cell viability to determine treatment effects across different CryoMSCs sources and viability status.Weighted mean differences(WMDs)and odds ratios were calculated to measure changes in the estimated treatment effects.All statistical analyses were performed using RevMan version 5.4.1 software.RESULTS Seven RCTs(285 patients)met the eligibility criteria for inclusion in the metaanalysis.During short-term follow-up,^(Cryo)MSCs demonstrated a significant 2.11%improvement in left ventricular ejection fraction(LVEF)[WMD(95%CI)=2.11(0.66-3.56),P=0.004,I2=1%],with umbilical cord-derived MSCs being the most effective cell type.However,the significant effect on LVEF was not sustained over the 12 months of follow-up.Subgroup analysis demonstrated a substantial 3.44%improvement in LVEF[WMD(95%CI)=3.44(1.46-5.43),P=0.0007,I2=0%]when using MSCs with post-thaw viability exceeding 80%.There was no statistically significant difference in the frequency of major cardiac adverse events observed in rehospitalization or mortality in patients treated with ^(Cryo)MSCs vs the control group.CONCLUSION ^(Cryo)MSCs are a promising option for heart failure patients,particularly considering the current treatment options for cardiovascular diseases.Our data suggest that ^(Cryo)MSCs could be a viable alternative or complementary treatment to the current options,potentially improving patient outcomes.展开更多
[Objective] The aim of this study was to establish the in vitro culture system of chicken fibroblasts.[Method] Tissue explant method and enzymatic digestion method were used to separate and culture chicken skin fibrob...[Objective] The aim of this study was to establish the in vitro culture system of chicken fibroblasts.[Method] Tissue explant method and enzymatic digestion method were used to separate and culture chicken skin fibroblasts respectively.The rate of cell growth,cryopreservation and recovery were compared.[Result] The primary chicken fibroblasts prepared by enzymatic digestion grew faster and converged together to form monolayer on 5 d post preparation;the passage cells prepared by these 2 methods grew at similar speed and formed monolayer within 2-3 d;homogeneous fibroblasts could be obtained by trypsin digestion and repeated attachment for 3-4 passages;there were 75%-80% of cells survived after cryopreservation and recovery;the growth curves of embryonic fibroblasts and skin fibroblasts were all normal and the two kind of cells still retained the normal number of chromosomes even at the twelfth passage.[Conclusion] The feeder layer cells needed for establishing ES cell lines could be obtained by culturing chicken fibroblasts through both tissue explant method and enzymatic digestion method.This study provided a basis for the successful establishment of ES cell lines.展开更多
[Objective] This study aimed to investigate the effects of cryopreservation on the quality and ultrastructure of Tibetan Mastiff sperm. [Method] The effects of cryopreservation on the quality of Tibetan Mastiff sperm ...[Objective] This study aimed to investigate the effects of cryopreservation on the quality and ultrastructure of Tibetan Mastiff sperm. [Method] The effects of cryopreservation on the quality of Tibetan Mastiff sperm were evaluated via motility, membrane integrity rate and acrosome intact rate. On that basis, the effects of cryopreservation on ultrastructure of sperm were observed under SEM and TEM. [Result] In Experiment 1, EG gave the best results not only in post-thaw motility rate （36.3%）, but also in low membrane integrity rate （38.0%） and acrosome intact rate （42.0% ）, but there was no significant difference between EG group and Glycerol group （P0.05）. In Experiment 2, the 5 cm freezing height obtained the best freezing-thawing results, but there was no significant difference between 2 and 5 cm height （P 0.05）, besides in acrosome intact rate. In Experiment 3, SDS and Vc added separately or together into extenders could improve freezing-thawing results, but there was not obvious difference between SDS group and Vc group （P0.05）, besides the lower motility of Vc group （P0.05）. Addition of SDS and Vc obtained the best results in post-thaw motility rate （44.1%）, and also in membrane integrity rate （48.0%） and acrosome intact rate （48.2%）. The ultrastructure of frozen-thawed sperm was also evaluated under SEM and TEM, results showed that cryopreservation caused various degrees of damage to Tibetan Mastiff sperm, more serious damages were observed in the acrosome such as swelling, vesiculation and even disappearance. [Conclusion] This study confirms that EG, horizontal height of 0.25 ml straw above LN 2 surface and additives SDS and Vc together can improve freezing effect. However, cryopreservation has certain damage to ultrastructure of sperm.展开更多
Cryopreservation of few spermatozoa is still a major challenge for male fertility preservation. This study reports use a new micro-straw （LSL straw） for freezing few spermatozoa for intracytoplasmic sperm injection ...Cryopreservation of few spermatozoa is still a major challenge for male fertility preservation. This study reports use a new micro-straw （LSL straw） for freezing few spermatozoa for intracytoplasmic sperm injection （ICSI）. Semen samples from 22 fertile donors were collected, and each semen sample was diluted and mixed with cryoprotectant in a ratio of 1：1, and then frozen using three different straws such as LSL straw （50-100μl）, traditional 0.25 ml and 0.5 ml straws. For freezing, all straws were fumigated with liquid nitrogen, with temperature directly reducing to -130--140℃. Sperm concentration, progressive motility, morphology, acrosome integrity, and DNA fragmentation index were evaluated before and after freezing. After freezing-thawing, LSL straw group had significantly higher percentage of sperm motility than traditional 0.25 ml and 0.5 ml straw groups （38.5% vs 27.4% and 25.6%, P 〈 0.003）. Sperm motility and acrosomal integrity after freezing-thawing were significantly lower than that of before freezing. However, there was no significant difference in morphology, acrosome, and DNA integrity between the three types of straws （P 〉 0.05）. As LSL straws were thinner and hold very small volume, the freezing rate of LSL straw was obviously faster than 0.25 ml straw and 0.5 ml straws. In conclusion, LSL micro-straws may be useful to store few motile spermatozoa with good recovery of motility for patients undergoing ICSI treatment.展开更多
Sperm banking can preserve male fertility effectively, but the current conditions of sperm cryopreservation in China have not been investigated. This retrospective investigation was based on data collected at multiple...Sperm banking can preserve male fertility effectively, but the current conditions of sperm cryopreservation in China have not been investigated. This retrospective investigation was based on data collected at multiple centres in China from January 2003 to December 2008. The collected data included urogenital history, indication for cryopreservation, semen parameters, use rate, type of assisted reproductive technique （ART） treatment and pregnancy outcome. The study population included 1 548 males who had banked their semen during the study period at one of the clinics indicated above. Approximately 1.9% （30/1 548） of the cryopreserved semen samples were collected from cancer patients; about 88.8% （1 374/1 548） of the patients had banked their semen for ART and 8.6% （134/1 548） had a male infertility disease （such as anejaculation, severe oligozoospermia and obstructive azoospermia）. The total use rate of cryopreserved semen was 22.7% （352/1 548）, with 119 live births. The cancer group use rate was 6.7% （2/30）, with one live birth by intracytoplasmic single sperm injection （ICSI）. The ART group use rate was 23.2% （319/1 374）, with 106 live births. The reproductive disease group use rate was 23.1% （31/134）, with 12 live births. The semen parameters in each category varied; the cancer patient and infertility disease groups had poor semen quality. In vitro fertilization （IVF） and ICSI were the most common ART treatments for cryopreserved sperm. Semen cryopreservation as a salvage method is effective, but in many conditions it is underutilized, especially in cancer patients. Lack of awareness, urgency of cancer treatment and financial constraints are the main causes of the low access rate. The concept of fertility preservation should be popularized to make better use of this medical service in China.展开更多
Aim: To investigate the effect of cryopreservation on the plasma membrane integrity in the head and tail regions ofindividual sperm, and the relationship between intact cryopreserved sperm and its motility and zona-fr...Aim: To investigate the effect of cryopreservation on the plasma membrane integrity in the head and tail regions ofindividual sperm, and the relationship between intact cryopreserved sperm and its motility and zona-free hamster oocytepenetration rate. Methods: The eosin Y exclusion and the hypoosmotic swelling tests were combined to form a sin-gle test (HOS-EY test) to identify the spermatozoa with four types of membrane integrity. Results: After cryop-reservation, there was a marked decline in the percentage of spermatozoa with Type Ⅳ membrane integrity (head mem-brane intact/tail membrane intact), and a significant increase in those with Type Ⅰ (head membrane damaged/tail mem-brane damaged) and Type Ⅲ (head membrane damaged/tail membrane intact) membrane integrity (n = 50, P <0.01). The value of Type Ⅲ integrity had a wide range of variability, whereas Type Ⅱ (head membrane intact/tailmembrane damaged) was uncommon after thawing. A high correlation was observed between the percentage of Type Ⅳintegrity and sperm motility ( n = 50, r = 0.74, P < 0.01 ). However, the values of Type Ⅳ integrity were usuallylower than those of post-thaw motility in most cryopreserved samples. The value of Type Ⅳ integrity did not correlatewith the sperm penetration rate ( n = 25, r = 0.22, P > 0.05). Conclusion: (1) The HOS-EY test has the advan-tage of showing four patterns of membrane integrity in individual spermatozoon; (2) Cryopreservation causes a signifi-cant membrane rupture in the head and tail regions of spermatozoa; Type Ⅲ is the main transitional state of membranecryodamage; (3) Cryodamage to head and tail membrane may occur independently; the presence of an intact tail mem-brane does not necessarily indicate the intacmess of head membrane. (4) Intact membranes are closely related to post-thaw motility, but do not reflect the fertilizing potential.展开更多
Aim: To 1) compare post-wash and post-thaw parameters of sperm processed with PureSperm density gradient technique and swim-up method; and 2) test the efficacy of two commonly available density gradient media PureSper...Aim: To 1) compare post-wash and post-thaw parameters of sperm processed with PureSperm density gradient technique and swim-up method; and 2) test the efficacy of two commonly available density gradient media PureSperm and ISolate. Methods: This prospective study used semen specimens from 22 patients. Specimens from nine patients were processed by both PureSperm density gradient and swim-up method. These specimens were then cryopreserved. Thirteen specimens were processed by both PureSperm (40 % and 80 %) and Isolate (50 % and 90 %) double density gradient techniques. The two fractions processed by both PureSperm and swim-up were analyzed for post-wash sperm characteristics. Post-thaw analysis was done after 24 hours. Sperm fractions obtained after processing with PureSperm and ISolate were compared for post-wash sperm characteristics and ROS levels. Results: Specimens prepared with PureSperm had significantly higher median total motile sperm counts (TMSC) (32.2 x 10~6 vs. 17.6 x 10~6), recovery rates (69.2 % vs. 50.0 %), and longevity at 4 hours (83.0 % vs. 55.0 %) compared to specimen prepared by swim-up. Post-thaw specimens also had a higher recovery and longevity at 4 hours with PureSperm as compared to the swim-up. Semen specimens processed by PureSperm had significantly higher total sperm count, TMSC, and percentage recovery rates (30.0 % vs. 19.7 %) than ISolate. Conclusion: Semen quality is better preserved in fresh and cryopreserved semen prepared with PureSperm density gradient compared to swim-up. A significant enrichment of sperm is observed with PureSperm compared to ISolate. Higher recovery rates of mature motile sperm obtained after PureSperm sperm preparation may be beneficial for successful ART. i展开更多
This study is aimed at investigating the potentials of ex vivo expansion and pluri-differentiation of cryopreservation of adult human bone marrow mesenchymal stem cells （hMSCs） into chondrocytes, adipocytes and neur...This study is aimed at investigating the potentials of ex vivo expansion and pluri-differentiation of cryopreservation of adult human bone marrow mesenchymal stem cells （hMSCs） into chondrocytes, adipocytes and neurocytes. Cryopreserved hMSCs were resuscitated and cultured for 15 passages, and then induced into chondrocytes, adipocytes and neurocytes with corresponding induction medium. The induced cells were observed for morphological properties and detected for expressions of type II collagen, triglyceride or neuron-specific enolase and nestin. The result showed that the resuscitated cells could differentiate into chondrocytes after exposure to transforming growth factor 61 （TGF-~0, insulin-like growth factor I （IGF-I） and vitamin C （Vc）, and uniformly changed morphologically from a spindle-like fibroblastic appearance to a polygonal shape in three weeks. The induced cells were heterochromatic to safranin O and expressed cartilage matrix-procollagenal （If） mRNA. The resuscitated cells cultured in induction medium consisting of dexamethasone, 3-isobutyl-l-methylxanthine, indomethacin and IGF-I showed adipogenesis, and lipid vacuoles accumulation was detectable after 21 d. The resuscitated hMSCs were also induced into neurocytes and expressed nestin and neuron specific endolase （NSE） that were special surface markers associated with neural cells at different stage. This study suggested that the resuscitated hMSCs should be still a population ofpluripotential cells and that it could be used for establishing an abundant bMSC reservoir for further experiment and treatment of various clinical discases.展开更多
文摘The use of fresh versus frozen spermatozoa in men with nonobstructive azoospermia (NOA) undergoingin vitro fertilization (IVF)has been a debated hot topic among reproductive specialists. Each approach presents distinct advantages and disadvantages,with fresh sperm typically showing superior sperm quality, while frozen sperm offers logistical flexibility and a reliable backup forrepeated cycles. This review summarizes the latest advancements in sperm retrieval and cryopreservation techniques, providingpractitioners with a comprehensive analysis of each option’s strengths and limitations. Comparative studies indicate that, althoughfresh sperm often has better quality metrics, cryopreservation methods such as vitrification have significantly improved postthawoutcomes, making frozen sperm a viable choice in assisted reproductive technologies (ART). The findings show comparablerates for fertilization, implantation, clinical pregnancy, and live birth between fresh and frozen microdissection testicular spermextraction (micro-TESE) sperm in many cases, although patient-specific factors such as timing, cost-effectiveness, and proceduralconvenience should guide the final decision. Ultimately, the choice of using fresh or frozen sperm should align with the individualneeds and conditions of patients. This tailored approach, supported by the latest advancements, can optimize ART outcomes andprovide personalized reproductive care.
文摘Azoospermia is characterized by the absence of sperm in the ejaculate and is categorized into obstructive azoospermia(OA)and nonobstructive azoospermia(NOA).For men with NOA,testicular sperm extraction(TESE)is the only method to obtain sperm for assisted reproductive technology(ART).Given the rarity of these sperm and the unpredictable success of subsequent retrieval attempts,cryopreservation of microdissection-TESE-obtained sperm is essential.Effective cryopreservation prevents the need for repeated surgical procedures and supports future ART attempts.After first delving into the physiological and molecular aspects of sperm cryopreservation,this review aims to examine the current methods and devices for preserving small numbers of sperm.It presents conventional freezing and vitrification techniques,evaluating their respective strengths and limitations in effectively preserving rare sperm,and compares the efficacy of using fresh versus cryopreserved testicular sperm.
基金Anhui Province Clinical Medical Research Translation Special Program(No.2204295107020002).
文摘Oocyte cryopreservation is an essential procedure in assisted reproductive technologies,aimed at preserving fertility,particularly for women undergoing IVF treatment or at risk of ovarian damage due to radiation,chemotherapy,or surgery.Despite its growing use,the survival and fertilization rates of cryopreserved oocytes remain suboptimal,largely due to cryo-induced oxidative stress.The generation of Reactive Oxygen Species(ROS)during freezing and thawing causes considerable damage to key cellular components,including proteins,lipids,DNA,and mitochondria.This oxidative stress compromises oocyte quality and reduces developmental potential.To address these challenges,the use of additives-especially antioxidants-has shown significant promise in mitigating oxidative damage.Enzymatic antioxidants such as Superoxide Dismutase(SOD)and Catalase(CAT),along with non-enzymatic antioxidants like glutathione,melatonin,and resveratrol,have demonstrated the ability to neutralize ROS and improve oocyte viability and developmental outcomes.Recent studies highlight the potential of Mitoquinone(MitoQ),a mitochondria-targeted antioxidant,to effectively counteract mitochondrial ROS and enhance cellular defense mechanisms during cryopreservation.This review explores the cellular mechanisms of cryodamage,the role of oxidative stress in oocyte cryopreservation,and the potential of various antioxidant strategies to enhance oocyte survival and function.Developing effective antioxidant supplementation approaches may significantly improve the outcomes of cryopreservation in reproductive medicine.
文摘Chuan Huang,Yu-Lin Tang,Jian-Ling Hu,Wen-Jun Zhou,Zeng-Hui Huang,Xue-Feng Luo,Zheng Li,Wen-Bing Zhu.Update on techniques for cryopreservation of human spermatozoa.Asian Journal of Andrology 2022;24:563-9.Yang Xiong,Fu-Xun Zhang,Yang-Chang Zhang,Chang-Jing Wu,Feng Qin,Jiu-Hong Yuan.Genetically predicted insomnia causally increases the risk of erectile dysfunction.Asian Journal of Andrology 2023;25:421-5.
基金supported by the National Natural Science Foundation of China(82172114)the"Challenge and Response"project for key and common technology research of Hefei(GJ2022SH08).
文摘Cryopreservation is a fundamental technology in biomedical research,regenerative medicine,and tissue engineering,enabling the long-term storage of cells,tissues,and organs.However,its effectiveness is limited by challenges such as intracellular ice formation,cryoprotectant toxicity,and reduced post-thaw viability.This review explores the crucial role of encapsulation in enhancing cryopreservation efficiency,with a focus on recent advances in materials science,bioengineering,and cryobiology.Emerging technologies,such as nanotechnology and stimuli-responsive polymers,are transforming encapsulation strategies.Innovations such as microfluidic systems offer precise control over cooling rates and cryoprotectant distribution,thereby mitigating conventional limitations.The review also addresses current obstacles related to scaling up encapsulation processes and ensuring the long-term biocompatibility and stability of preserved specimens.By synthesizing recent findings,this work provides a comprehensive resource for researchers and clinicians seeking to enhance biopreservation techniques and their applications in contemporary medicine and biotechnology.Finally,the review identifies critical knowledge gaps that must be addressed to improve the efficacy of cryopreservation strategies and advance their clinical translation.
基金supported by National Natural Science Foundation of China(Award Number:2346842).
文摘Cryopreservation of living cells and tissues plays a vital role in biomedical research,clinical applications,biotechnology innovation,the development of new vaccines and drugs,and the conservation of endangered species.While significant technological breakthroughs have been achieved in cooling methods-particularly through vitrification for large tissue and organs-the lack of optimal rewarming technology remains a key obstacle to successful cryopreservation,especially for larger samples such as tissues and organs.The primary challenges during the warming process include non-uniformity heating and insufficient rewarming rates,which can lead to thermal stress-induced structural damage and lethal ice recrystallization,ultimately compromising the integrity and functionality of biological materials.In recent years,various advanced warming techniques have emerged,employing different energy conversion approaches to achieve volumetric heating while minimizing the risk of overheating.These techniques involve thermal,mechanical-thermal,and electromagnetic-thermal energy conversions.However,each method presents its own limitation.This review aims to summarize recent advancements in rewarming technologies for cryopreservation,with a focus on their mechanisms,applications,and the key challenges that must be addressed to enable broader adoption in medical and commercial contexts.
基金supported by the National Natural Science Foundation of China(52076140).
文摘Organoids are three-dimensional structures derived from stem cells that recapitulate the gene expression profiles and functional characteristics of their tissue of origin,rendering them invaluable tools for disease modeling,drug screening,and precision medicine.Despite their promise,the widespread application of organoids is limited by extended culture durations and technical complexity.Cryopreservation has emerged as a critical strategy to overcome challenges related to the long-term storage and application of organoids,offering a range of preservation approaches tailored to organoid development.Nevertheless,conventional cryopreservation techniques encounter significant limitations when applied to organoids.To address these issues,the development of naturally derived,low-toxicity Cryoprotectants(CPAs),along with the optimization of CPA loading methods and refinement of cooling and warming protocols,is essential to mitigate cryoinjury.Looking forward,the comprehensive enhancement of cryopreservation technologies may facilitate the transformation of organoids into“off-the-shelf”products,enabling scalable production,batch standardization,and centralized distribution.Such advancements will lay the foundation for the establishment of Next-Generation Living Biobanks(NGLB).
基金Supported by European Union-Next Generation EU,through the National Recovery and Resilience Plan of the Republic of Bulgaria,No.BG-RRP-2.004-0008.
文摘Fertility preservation and pregnancymanagement are critical considerations for patients undergoing organtransplantation.Innovations in assisted reproductive technologies,hormonalmodulation,and personalized medicine have expanded options for these patients,who face unique challenges due to immunosuppressive therapy and organ functionconcerns.This mini-review explores advancements in cryopreservationtechniques,pre-conception counseling,and multidisciplinary strategies forsafe pregnancies post-transplantation.Emphasis is placed on balancing maternalhealth,graft function,and fetal outcomes.The integration of reproductive andtransplant medicine is paving the way for improved quality of life andreproductive autonomy for this patient population.
基金supported by the National Key Research and Development Program of China (2021YFD1200301 and 2021YFD1200302)the Natural Science Foundation of Jiangsu Province, China (BK20210813)+1 种基金the National Natural Science Foundation of China (32102534)the Yangzhou International Science and Technology Cooperation Projects, China (YZ2021175)。
文摘Germplasm resources are essential for the sustainable development of biodiversity and husbandry of local chickens, as well as for the breeding and industry of superior quality chickens. Unfortunately, many local and indigenous chicken breeds are at risk of declining numbers, emphasizing the need to conserve breed resources for endangered chickens. Primordial germ cells(PGCs) are crucial for preserving germplasm resources by inheriting genetic information from parents to offspring and ensuring stability of genetic material between germlines. In this study,PGCs were isolated from chicken embryos' gonads and cultured in FAcs medium without feeder cells. Over a period of approximately 40 d, the cells proliferated to a number of up to 10^(6), establishing various cell lines. Particularly, 18 PGC lines were created from Rugao Yellow chicken and Shouguang chicken, with an efficiency ranging from 39.1 to 45%. Furthermore, PGCs that had been cultured for 40 passages exhibited typical PGC characteristics, suchas glycogen staining reaction, and expression of pluripotency and reproductive markers. These results confirmthat PGCs maintain stem cell properties even after long-term in vitro culture. Additionally, PGCs cryopreserved for up to 120 d remained viable, maintained typical PGC morphologies, and possessed stable cell proliferation ability. Through intravascular injection into chicken embryos, green fluorescent protein(GFP)-PGCs were found in the recipient embryos' gonads and could develop into gametes to produce offspring, indicating that even after extended culture, PGCs retain their migratory and lineage-transmitting capabilities. This research offers valuable insights into the in vitro cultivation and preservation of PGCs of Chinese indigenous chickens. The findings of this study can be applied in transgenic chicken production and the preservation of genetic resources of indigenous chicken breeds.
文摘Dear Editor,We are much obliged that Hadziselimovic1 has used our data2 to highlight the substantial proportion of boys with cryptorchidism where gonadotropin insufficiency is an important factor related to the pathogenesis.We have recently presented a study on a series of 453 consecutive boys with bilateral nonsyndromic cryptorchidism,in which we conducted hormonal evaluations and assessed germ cell numbers in testicular biopsies.3 In this series,45%of the boys were classified as having gonadotropin insufficiency.3 Identifying these patients at the time of surgery is important.A recent follow-up study of 208 boys with nonsyndromic bilateral cryptorchidism from our department showed that the boys with gonadotropin insufficiency had an impaired fertility potential after surgery compared to boys with an intact hypothalamus–pituitary–gonadal feedback mechanism.4 In a review from 2022,Hadziselimovic5 suggested that infertility in patients diagnosed with cryptorchid testes is a consequence of a hormonal deficiency rather than temperature-induced cellular damage.
文摘Sperm cryopreservation is an essential technique for male fertility preservation,especially in men who are undergoing medical treatment.Conventional cryopreservation methods face limitations such as oxidative stress,DNA fragmentation,and cytotoxicity associated with traditional cryoprotectants like dimethyl sulfoxide(DMSO).Recent breakthroughs have focused on improving post-thaw sperm viability with novel cryoprotectants and innovative freezing strategies.Prospective approaches include the use of amino acid-based cryoprotectants,deep eutectic solvents,and antioxidants that have been described to prevent oxidative damage and maintain DNA integrity.Vitrification,a high-speed freezing technique that prevents ice crystal formation,has demonstrated superior outcomes compared to conventional slow freezing.Moreover,the Direct Dropping Method,a cryoprotectant-free approach,has been introduced as a contamination-minimizing technique that preserves sperm functionality.Multiomics tools are also utilized to determine biomarkers for protocol optimization.Despite these advancements,cryoprotectant toxicity is a central challenge,emphasizing the necessity for safer agents.Future research must focus on long-term sperm functionality and individualized cryopreservation strategies to maximize reproductive outcomes.The current review highlights the challenges associated with sperm cryopreservation,explores innovative strategies and novel cryoprotectants,underscores the significance of maintaining DNA integrity,and proposes future research directions to improve fertility preservation outcomes.
文摘Dear Editor,I would like to congratulate Mamsen et al.i on their extensive and scientifically valuable work.I analyzed their raw data presented in Table 1 of the original article from a different perspective and discovered an effect not mentioned in the article.My analysis showed that luteinizing hormone(LH)levels are significantly lower in patients at high infertility risk(HIR),whose testes lack A dark(Ad)spermatogonia and display an abnormal ratio of germ cells per crosssectional tubule(G/T).
文摘BACKGROUND As living biodrugs,mesenchymal stem cells(MSCs)have progressed to phase 3 clinical trials for cardiovascular applications.However,their limited immediate availability hampers their routine clinical use.AIM To validate our hypothesis that cryopreserved MSCs(CryoMSCs)are as safe and effective as freshly cultured MSC counterparts but carry logistical advantages.METHODS Four databases were systematically reviewed for relevant randomized controlled trials(RCTs)evaluating the safety and efficacy of CryoMSCs from various tissue sources in treating patients with heart disease.A subgroup analysis was performed based on MSC source and post-thaw cell viability to determine treatment effects across different CryoMSCs sources and viability status.Weighted mean differences(WMDs)and odds ratios were calculated to measure changes in the estimated treatment effects.All statistical analyses were performed using RevMan version 5.4.1 software.RESULTS Seven RCTs(285 patients)met the eligibility criteria for inclusion in the metaanalysis.During short-term follow-up,^(Cryo)MSCs demonstrated a significant 2.11%improvement in left ventricular ejection fraction(LVEF)[WMD(95%CI)=2.11(0.66-3.56),P=0.004,I2=1%],with umbilical cord-derived MSCs being the most effective cell type.However,the significant effect on LVEF was not sustained over the 12 months of follow-up.Subgroup analysis demonstrated a substantial 3.44%improvement in LVEF[WMD(95%CI)=3.44(1.46-5.43),P=0.0007,I2=0%]when using MSCs with post-thaw viability exceeding 80%.There was no statistically significant difference in the frequency of major cardiac adverse events observed in rehospitalization or mortality in patients treated with ^(Cryo)MSCs vs the control group.CONCLUSION ^(Cryo)MSCs are a promising option for heart failure patients,particularly considering the current treatment options for cardiovascular diseases.Our data suggest that ^(Cryo)MSCs could be a viable alternative or complementary treatment to the current options,potentially improving patient outcomes.
基金Supported by National Natural Science Foundation of China(30801353)Shandong Education Department Foundation Project(G08LG53)~~
文摘[Objective] The aim of this study was to establish the in vitro culture system of chicken fibroblasts.[Method] Tissue explant method and enzymatic digestion method were used to separate and culture chicken skin fibroblasts respectively.The rate of cell growth,cryopreservation and recovery were compared.[Result] The primary chicken fibroblasts prepared by enzymatic digestion grew faster and converged together to form monolayer on 5 d post preparation;the passage cells prepared by these 2 methods grew at similar speed and formed monolayer within 2-3 d;homogeneous fibroblasts could be obtained by trypsin digestion and repeated attachment for 3-4 passages;there were 75%-80% of cells survived after cryopreservation and recovery;the growth curves of embryonic fibroblasts and skin fibroblasts were all normal and the two kind of cells still retained the normal number of chromosomes even at the twelfth passage.[Conclusion] The feeder layer cells needed for establishing ES cell lines could be obtained by culturing chicken fibroblasts through both tissue explant method and enzymatic digestion method.This study provided a basis for the successful establishment of ES cell lines.
基金Natural Science Foundation of Jiangsu Province (BK2008589)Qing Lan Project of Jiangsu ProvinceCooperation Project of Agricultural Production, Study and Research in Wuxi City~~
文摘[Objective] This study aimed to investigate the effects of cryopreservation on the quality and ultrastructure of Tibetan Mastiff sperm. [Method] The effects of cryopreservation on the quality of Tibetan Mastiff sperm were evaluated via motility, membrane integrity rate and acrosome intact rate. On that basis, the effects of cryopreservation on ultrastructure of sperm were observed under SEM and TEM. [Result] In Experiment 1, EG gave the best results not only in post-thaw motility rate （36.3%）, but also in low membrane integrity rate （38.0%） and acrosome intact rate （42.0% ）, but there was no significant difference between EG group and Glycerol group （P0.05）. In Experiment 2, the 5 cm freezing height obtained the best freezing-thawing results, but there was no significant difference between 2 and 5 cm height （P 0.05）, besides in acrosome intact rate. In Experiment 3, SDS and Vc added separately or together into extenders could improve freezing-thawing results, but there was not obvious difference between SDS group and Vc group （P0.05）, besides the lower motility of Vc group （P0.05）. Addition of SDS and Vc obtained the best results in post-thaw motility rate （44.1%）, and also in membrane integrity rate （48.0%） and acrosome intact rate （48.2%）. The ultrastructure of frozen-thawed sperm was also evaluated under SEM and TEM, results showed that cryopreservation caused various degrees of damage to Tibetan Mastiff sperm, more serious damages were observed in the acrosome such as swelling, vesiculation and even disappearance. [Conclusion] This study confirms that EG, horizontal height of 0.25 ml straw above LN 2 surface and additives SDS and Vc together can improve freezing effect. However, cryopreservation has certain damage to ultrastructure of sperm.
文摘Cryopreservation of few spermatozoa is still a major challenge for male fertility preservation. This study reports use a new micro-straw （LSL straw） for freezing few spermatozoa for intracytoplasmic sperm injection （ICSI）. Semen samples from 22 fertile donors were collected, and each semen sample was diluted and mixed with cryoprotectant in a ratio of 1：1, and then frozen using three different straws such as LSL straw （50-100μl）, traditional 0.25 ml and 0.5 ml straws. For freezing, all straws were fumigated with liquid nitrogen, with temperature directly reducing to -130--140℃. Sperm concentration, progressive motility, morphology, acrosome integrity, and DNA fragmentation index were evaluated before and after freezing. After freezing-thawing, LSL straw group had significantly higher percentage of sperm motility than traditional 0.25 ml and 0.5 ml straw groups （38.5% vs 27.4% and 25.6%, P 〈 0.003）. Sperm motility and acrosomal integrity after freezing-thawing were significantly lower than that of before freezing. However, there was no significant difference in morphology, acrosome, and DNA integrity between the three types of straws （P 〉 0.05）. As LSL straws were thinner and hold very small volume, the freezing rate of LSL straw was obviously faster than 0.25 ml straw and 0.5 ml straws. In conclusion, LSL micro-straws may be useful to store few motile spermatozoa with good recovery of motility for patients undergoing ICSI treatment.
文摘Sperm banking can preserve male fertility effectively, but the current conditions of sperm cryopreservation in China have not been investigated. This retrospective investigation was based on data collected at multiple centres in China from January 2003 to December 2008. The collected data included urogenital history, indication for cryopreservation, semen parameters, use rate, type of assisted reproductive technique （ART） treatment and pregnancy outcome. The study population included 1 548 males who had banked their semen during the study period at one of the clinics indicated above. Approximately 1.9% （30/1 548） of the cryopreserved semen samples were collected from cancer patients; about 88.8% （1 374/1 548） of the patients had banked their semen for ART and 8.6% （134/1 548） had a male infertility disease （such as anejaculation, severe oligozoospermia and obstructive azoospermia）. The total use rate of cryopreserved semen was 22.7% （352/1 548）, with 119 live births. The cancer group use rate was 6.7% （2/30）, with one live birth by intracytoplasmic single sperm injection （ICSI）. The ART group use rate was 23.2% （319/1 374）, with 106 live births. The reproductive disease group use rate was 23.1% （31/134）, with 12 live births. The semen parameters in each category varied; the cancer patient and infertility disease groups had poor semen quality. In vitro fertilization （IVF） and ICSI were the most common ART treatments for cryopreserved sperm. Semen cryopreservation as a salvage method is effective, but in many conditions it is underutilized, especially in cancer patients. Lack of awareness, urgency of cancer treatment and financial constraints are the main causes of the low access rate. The concept of fertility preservation should be popularized to make better use of this medical service in China.
文摘Aim: To investigate the effect of cryopreservation on the plasma membrane integrity in the head and tail regions ofindividual sperm, and the relationship between intact cryopreserved sperm and its motility and zona-free hamster oocytepenetration rate. Methods: The eosin Y exclusion and the hypoosmotic swelling tests were combined to form a sin-gle test (HOS-EY test) to identify the spermatozoa with four types of membrane integrity. Results: After cryop-reservation, there was a marked decline in the percentage of spermatozoa with Type Ⅳ membrane integrity (head mem-brane intact/tail membrane intact), and a significant increase in those with Type Ⅰ (head membrane damaged/tail mem-brane damaged) and Type Ⅲ (head membrane damaged/tail membrane intact) membrane integrity (n = 50, P <0.01). The value of Type Ⅲ integrity had a wide range of variability, whereas Type Ⅱ (head membrane intact/tailmembrane damaged) was uncommon after thawing. A high correlation was observed between the percentage of Type Ⅳintegrity and sperm motility ( n = 50, r = 0.74, P < 0.01 ). However, the values of Type Ⅳ integrity were usuallylower than those of post-thaw motility in most cryopreserved samples. The value of Type Ⅳ integrity did not correlatewith the sperm penetration rate ( n = 25, r = 0.22, P > 0.05). Conclusion: (1) The HOS-EY test has the advan-tage of showing four patterns of membrane integrity in individual spermatozoon; (2) Cryopreservation causes a signifi-cant membrane rupture in the head and tail regions of spermatozoa; Type Ⅲ is the main transitional state of membranecryodamage; (3) Cryodamage to head and tail membrane may occur independently; the presence of an intact tail mem-brane does not necessarily indicate the intacmess of head membrane. (4) Intact membranes are closely related to post-thaw motility, but do not reflect the fertilizing potential.
文摘Aim: To 1) compare post-wash and post-thaw parameters of sperm processed with PureSperm density gradient technique and swim-up method; and 2) test the efficacy of two commonly available density gradient media PureSperm and ISolate. Methods: This prospective study used semen specimens from 22 patients. Specimens from nine patients were processed by both PureSperm density gradient and swim-up method. These specimens were then cryopreserved. Thirteen specimens were processed by both PureSperm (40 % and 80 %) and Isolate (50 % and 90 %) double density gradient techniques. The two fractions processed by both PureSperm and swim-up were analyzed for post-wash sperm characteristics. Post-thaw analysis was done after 24 hours. Sperm fractions obtained after processing with PureSperm and ISolate were compared for post-wash sperm characteristics and ROS levels. Results: Specimens prepared with PureSperm had significantly higher median total motile sperm counts (TMSC) (32.2 x 10~6 vs. 17.6 x 10~6), recovery rates (69.2 % vs. 50.0 %), and longevity at 4 hours (83.0 % vs. 55.0 %) compared to specimen prepared by swim-up. Post-thaw specimens also had a higher recovery and longevity at 4 hours with PureSperm as compared to the swim-up. Semen specimens processed by PureSperm had significantly higher total sperm count, TMSC, and percentage recovery rates (30.0 % vs. 19.7 %) than ISolate. Conclusion: Semen quality is better preserved in fresh and cryopreserved semen prepared with PureSperm density gradient compared to swim-up. A significant enrichment of sperm is observed with PureSperm compared to ISolate. Higher recovery rates of mature motile sperm obtained after PureSperm sperm preparation may be beneficial for successful ART. i
基金Project supported by the Science Foundation (No. 2003C23015) and the Natural Science Foundation (No. Y204139) of Zhejiang Province, China
文摘This study is aimed at investigating the potentials of ex vivo expansion and pluri-differentiation of cryopreservation of adult human bone marrow mesenchymal stem cells （hMSCs） into chondrocytes, adipocytes and neurocytes. Cryopreserved hMSCs were resuscitated and cultured for 15 passages, and then induced into chondrocytes, adipocytes and neurocytes with corresponding induction medium. The induced cells were observed for morphological properties and detected for expressions of type II collagen, triglyceride or neuron-specific enolase and nestin. The result showed that the resuscitated cells could differentiate into chondrocytes after exposure to transforming growth factor 61 （TGF-~0, insulin-like growth factor I （IGF-I） and vitamin C （Vc）, and uniformly changed morphologically from a spindle-like fibroblastic appearance to a polygonal shape in three weeks. The induced cells were heterochromatic to safranin O and expressed cartilage matrix-procollagenal （If） mRNA. The resuscitated cells cultured in induction medium consisting of dexamethasone, 3-isobutyl-l-methylxanthine, indomethacin and IGF-I showed adipogenesis, and lipid vacuoles accumulation was detectable after 21 d. The resuscitated hMSCs were also induced into neurocytes and expressed nestin and neuron specific endolase （NSE） that were special surface markers associated with neural cells at different stage. This study suggested that the resuscitated hMSCs should be still a population ofpluripotential cells and that it could be used for establishing an abundant bMSC reservoir for further experiment and treatment of various clinical discases.
