Low Level of Cystic Fibrosis Transmembrane Conductance Regulator Is Associated with Human Sperm Autophagy and Vitality

Low sperm motility is one of the main causes of male infertility. Cystic fibrosis transmembrane conductance regulator (CFTR, an anion channel protein) is related to the progressive motility of sperm. CFTR disruptor CFTRinh-172 or forskolin (FSK) in this study were used to treat human sperm separately, and the rates of sperm autophagy and progressive motility, mitochondrial membrane potential (MMP) and ATP concentration, and the expression levels of related factors were detected to explore their relationship. It was showed that sperms treated with CFTRinh-172 or FSK reduced the levels of cAMP, CFTR and PKA, but increased sperm autophagy rate, expression levels of AMPK and LC3B. However, reactive oxygen species content had no significant difference. It was indicated that low level of CFTR performed with cAMP and its downstream effectors such as PKA and AMPK to regulate mitochondrial structure and function, leading to increased autophagy rate and reduced vitality of sperm.

Characterization of nucleohistone and nucleoprotamine components in the mature human sperm nucleus

Aim： To simultaneously determine the localization of histones and protamines within human sperm nuclei. Methods： Immunofluorescence of the core histones and protamines and fluorescence in situ hybridization of the telomere region of chromosome 16 was assessed in decondensed human sperm nuclei. Results： Immunofluorescent localization of histones, protamine 1 （PRM1） and protamine 2 （PRM2） along with fluorescence in situ hybridization localization of chromosome 16 telomeric sequences revealed a discrete distribution in sperm nuclei. Histones localized to the posterior ring region （i.e. the sperm nuclear annulus）, whereas PRM1 and PRM2 appeared to be dispersed throughout the entire nucleus. Conclusion： The co-localization of the human core sperm histones with the telomeric regions of chromosome 16 is consistent with the reorganization of specific non-protamine regions into a less compacted state.

Can Helicobacter pylori infection influence human reproduction?

Helicobacter pylori (H. pylori) infection could be associated with extra-digestive diseases. Here, we report the evidences concerning the decrease in reproductive potential occurring in individuals infected by H. pylori, especially by strains expressing CagA. This infection is more prevalent in individuals with fertility disorders. Infected women have anti-H. pylori antibodies in cervical mucus and follicular fluid that may decrease sperm motility and cross react immunologically with spermatozoa, conceivably hampering the oocyte/sperm fusion. Infection by CagA positive organisms enhances the risk of preeclampsia, which is a main cause of foetus death. These findings are supported by the results of experimental infections of pregnant mice, which may cause reabsorption of a high number of foetuses and alter the balance between Th1 and Th2 cell response. Infected men have decreased sperm motility, viability and numbers of normally shaped sperm and augmented systemic levels of inflammatory cytokines, such as tumor necrosis factor-α, which may damage spermatozoa. In countries where parasitic infestation is endemic, detrimental effects of infection upon spermatozoa may not occur, because the immune response to parasites could determine a switch from a predominant Th1 type to Th2 type lymphocytes, with production of anti-inflammatory cytokines. In conclusion, the evidences gathered until now should be taken into consideration for future studies aiming to explore the possible role of H. pylori infection on human reproduction.

Human sperm immobilization effect of Carica papaya seed extracts: an in vitro study

Aim: To examine if the seed extracts of Carica papaya, which showed anfispermatogenic/sperm immobilizationproperties in animal models, could cause human sperm immobilization in vitro. Methods: Chloroform extract, ben-zene chromatographic fraction of the chloroform extract, its methanol and ethyl acetate sub-fractions and the isolatedcompounds from the sub-fractions i. e., ECP 1 & 2 and MCP 1 & 2, of the seeds of Carica papaya were used at con-centrations of 0.1%, 0.5%, 1% and 2%. Sperm motility was assessed immediately after addition of extracts and ev-ery 5 minutes thereafter for 30 minutes. Results: There were dose-dependent spermicidal effects showing an instantfall in the sperm motility to less than 20% at 2% concentration. Isolated compounds ECP 1 & 2 were more effective in-ducing a motility of less than 10%. Many of the spermatozoa became vibratory on the spot. Total inhibition of motilitywas observed within 20-25 rain at all concentrations of all products. Scanning and transmission electron microscopyrevealed deleterious changes in the plasma membrane of the head and mid-piece of spermatozoa. Sperm viability testand the number of abnormal spermatozoa after completion of incubation suggested that the spermatozoa were infertile.The effects were spermicidal but not spermiostatic as revealed by the sperm revival test. Conclusion: The results re-veal spermicidal activity in vitro of the seed extracts of Carica papaya.

Antioxidative Protective Effect of Icariin on the FeSO_4/H_2O_2-damaged Human Sperm Based on Confocal Raman Micro-spectroscopy

Oxidative stress is implicated in male infertility and significantly higher reactive oxygen species are detected in 25% of infertile males. Although different agents of various alternative medicines, including traditional Chinese medicine, have been tried with varying success, evidence remains limited on whether and how much herbs or supplements might help increase the anti-oxidant ability of the sperm. This study examined the anti-oxidative effects of icariin, a flavonoid isolated from Herba Epimedii, on the human sperm. We prepared the FeSO4/H202-damaged human sperms, which were co-cultured with icariin in vitro, and then observed the changes of the sperm by employing Raman mi- cro-spectroscopy. The results showed that Raman mapping with a 514 nm excitation laser allowed clear differentiation of the nucleus, neck, and, in particular, the mitochondria-rich middle piece of a human sperm cell. The effect oficariin on different organelles of the sperm was quantified by localized spectral Raman signatures obtained within milli-seconds, and icariin could keep the ＂Raman fingerprint＂ of the human sperm the same as the control groups, suggesting that icariin could protect the human sperm from being damaged by FeSO4/H202. Icariin may serve as a tonifying and replenishing agent of herbal origin for enhancing reproductive fimctions.

Evaluation on Sensitivity of the Human Sperm Motility Assay for Detecting Endotoxin in Culture Medium

Objective To investigate the sensitivity of the human sperm motility assay for detecting endotoxin in culture mediumMaterials &. Methods Motile sperm were separated and exposed to different concentrations of endotoxin (0.5 ng/mL, 1ng/mL, 10ng/mL, 1000ng/mL, 10 000ng/ mL, and 50 000ng/mL), and sperm motility was determined after incubation. Effects of endotoxin on sperm motility in media without albumin were also examined. In addition, at the same concentrations of endotoxin (0. 5ng/mL, 1 ng/mL, and 10 ng/ mL ) , the sensitivity of the human sperm motility assay was compared to those of 1-cell and 2-cell mouse embryo bioassays.Results At levels of 0. 5ng/mL-1000ng/mL endotoxin in media with 2mg/mL albumin, sperm did not show significant change in motility during 24 h of incubation when compared with the control (P>0. 05). However, the sperm motility was significantly inhibited at endotoxin dosages of 10 000 and 50 000 ng/mL. In the absence of albumin supplementation, at endotoxin levels of 50 000ng/mL, and 1 000ng/mL, there was a marked decrease in sperm motility compared with the control after 2 h or 8 h of incubation, respectively (P<0. 01). In media containing 0. 5 ng/mL and 1 ng/ mL endotoxin, 1-cell and 2-cell mouse embryos had significantly reduced developmental rates in all developmental stages, and at the level of 10ng/mL, the development of the embryos was arrested. Conclusion The human sperm motility assay could detect high levels of endotoxin in culture medium but its sensitivity to endotoxin would be inferior to that of the 1-cell or 2-cell mouse embryo bioassay. In the absence of albumin supplementation, the sensitivity of the sperm motility assay could be improved.

Comparison of Three Different Techniques of Human Sperm DNA Isolation for Methylation Assay

Summary： Human sperm DNA is an important genetic and epigenetic material, whose chromatin structure differs from that of somatic cells. As such, conventional methods for DNA extraction of somatic cells may not be suitable for obtaining sperm DNA. In this study, we evaluated and compared three sperm DNA extraction techniques, namely, modified guanidinium thiocyanate method （method A）, traditional phenol-chloroform method （method B）, and TianGen kit method （method C）. Spectrophotometry and agarose gel electrophoresis analyses showed that method A produced DNA with higher quantity and purity than those of methods B and C （P〈0.01）. PCR results revealed that method A was more reliable in amplifying DEAD-box polypeptide 4 （DDX4） and copy number variations （CNVs） than methods B and C, which generated false-positive errors. The results of sperm DNA methylation assay further indicated that methods A and B were effective, and the former yielded higher quantitative accuracy. In conclusion, the modified guanidinium thiocyanate method provided high quality and reli- able results and could be an optimal technique for extracting sperm DNA for methylation assay.

Evaluation on the Morphology and Membrane Integrity of Immotile Human Sperm

Objective To determine the membrane integrity in the head and tail regions of individual spermatozoon, and observe sperm morphology for samples with totally immotile sperm. Methods Ten infertile men with immotile sperm were enrolled into this study （group A）. The membrane integrity in the head and tail regions of individual spermatozoon of immotile sperm was examined by using the combined hypo-osmotic swelling-eosin Y exclusion test （HOS-EY test）. Sperm morphology was observed by light, scanning and transmission electron microscopy. Ten semen samples from normospermic donors were used as the control （group B）. Results The percentage of sperm with intact both head and tail membranes in group A was significantly lower than that in group B （P〈0.01）, whereas the value of sperm with defective head membrane but intact tail membrane in group A was significantly higher than that in group B （P〈0.01）. Abnormal sperm morphology in group A had a high incidence, and immotile sperm with viability and normal morphology could be observed in some cases. Most sperm had multiple ultrastructural defects. Conclussion Some immotile sperm had intact tail membrane but defective head membrane. Immotile sperm with viability and normal morphology could exist in some cases though abnormal sperm were in a great proportion. Carefully evaluating immotile sperm membrane integrity and morphology should benefit the treatment of patients with immotile sperm.

Inhibition of mouse acrosome reaction and sperm-zona pellucida binding by anti-human sperm membrane protein 1 antibody

Aim： To investigate the possible functions of human sperm membrane protein （hSMP-1） in the process of fertilization. Methods： A 576-bp cDNA fragment of HSD-1 gene coding for the extracellular domain of hSMP-1 was cloned and expressed. The localization of this protein on human and mouse sperm was determined by indirect immunofluorescent staining by using anti-recombinant hSMP-1 （anti-rhSMP-1） antibodies. Sperm acrosome reaction and spermzona pellucida （ZP） binding assay were carried out in 10-week-old BALB/c mice. Results： Recombinant hSMP-1 was successfully cloned and expressed. The expression of the native protein was limited on the acrosome of human and mouse sperm. Treatment of anti-rhSMP-1 antibodies significantly decreased the average number of sperms bound to each egg. Meanwhile, the percentage of acrosome reaction was decreased in comparison to pre-immune control after treatment with anti-rhSMP-1 （P 〈 0.05）. Conclusion： The results suggest that anti-rhSMP-1 antibody inhibited mouse acrosome reaction and sperm-ZP binding.

The opening of maitotoxin-sensitive calcium channels induces the acrosome reaction in human spermatozoa： differences from the zona pellucida

The acrosome reaction （AR）, an absolute requirement for spermatozoa and egg fusion, requires the influx of Ca2＋ into the spermatozoa through voltage-dependent Ca2＋ channels and store-operated channels. Maitotoxin （MTx）, a Ca2＋-mobilizing agent, has been shown to be a potent inducer of the mouse sperm AR, with a pharmacology similar to that of the zona pellucida （ZP）, possibly suggesting a common pathway for both inducers. Using recombinant human ZP3 （rhZP3）, mouse ZP and two MTx channel blockers （U73122 and U73343）, we investigated and compared the MTx- and ZP-induced ARs in human and mouse spermatozoa. Herein, we report that MTx induced AR and elevated intracellular Ca2＋ （[Ca2＋]~） in human spermatozoa, both of which were blocked by U73122 and U73343. These two compounds also inhibited the MTx-induced AR in mouse spermatozoa. In disagreement with our previous proposal, the AR triggered by rhZP3 or mouse ZP was not blocked by U73343, indicating that in human and mouse spermatozoa, the AR induction by the physiological ligands or by MTx occurred through distinct pathways. U73122, but not U73343 （inactive analogue）, can block phospholipase C （PLC）. Another PLC inhibitor, edelfosine, also blocked the rhZP3- and ZP-induced ARs. These findings confirmed the participation of a PLC-dependent signalling pathway in human and mouse zona protein-induced AR. Notably, edelfosine also inhibited the MTx-induced mouse sperm AR but not that of the human, suggesting that toxin-induced AR is PLC-dependent in mice and PLC-independent in humans.

A patch-clamp study on human sperm Cl^- channel reassembled into giant liposome

Aim: To record the single-channel currents and characterize the electrophysiological properties of the Cl^- channels inhuman sperm membrane. Methods: The membrane proteins extracted from the human sperm were reassembled intoliposome bilayer, and the liposomes were fused into giant liposomes with a diameter more than 10μm by dehydration-rehydration procedure. The giant liposomes were used to study the Cl^- channel activities by patch-clamp technique.Results: By patch clamping the giant liposome in an asymmetric NMDG (N-methyl-D-glucamine)-Cl (bath 100//pipette 200 mmol/L) solution system, three kinds of single-channel events with unit conductances of (74.1 ± 8.3) pS,(117.0±5.7) pS and (144.7±4.5) pS, respectively, were detected. Their activities were voltage-dependent and allwere blocked by SITS (4-acetamido-4'-isothiocyanato-stilbene-2', 2'-disulfonic acid) in a concentration-dependentmanner. By constructing the open and close dwell time distribution histograms and then fitting them with exponentialfunction, two time constants were obtained in both the open and the close states. The burst activity and conductancesubstate of the channels were observed. Conclusion; There exist three kinds of Cl^- channels with different conduc-tance in human sperm membrane at least. (Asian J Androl 2001 Sep; 3: 185 - 191)

Evaluation of Endotoxin in Culture Medium for Human in vitro Fertilization

To determine whether the presence of bacterial endotoxin in the commercial culture media utilized for human in vitro fertilization （IVF）, and evaluate the difference in detecting endotoxin in culture medium between the human sperm motility assay and the 2-cell mouse embryo assay. Methods Thirty-six batches of culture media commonly used in IVF laboratories from 3 manufacturers were determined for thepresence ofendotoxin before using the medium for the assisted reproductive programs （group A）. After being used, 25 specimens among above media were also tested （group B）. The chromogenic limulus amoebocyte lysate （LAL） test was used for quantification the content of endotoxin. In addition, the human sperm motility assay was compared with the 2-cell mouse embryo assay to evaluate the difference in detecting endotoxin in culture medium. Results Endotoxin was not detected in group A. However, 2 samples were positive in group B, Sperm did not show significant change in motility in group A during 24 h of incubation when compared with the control （P〉0. 05）. However, in group A the 2-cell embryo development to blastocyst was suppressed in 3 batches of media. Conclusions Regular screening of each batch of culture medium should be performed if possible although there was no evidence of endotoxin contamination in commercially prepared pre-tested media. Culture environment should be stringently controlled in case the medium is polluted. The sensitivity of the sperm motility assay was lower than that of the mouse embryo assay for detecting low levels of endotoxin or toxic compounds in the medium.

Sensitivity of sperm motility assay for detecting endotoxin effect on human sperm in vitro

Objective: To evaluate the sensitivity of sperm motility assay for detecting the endotoxin effect on human sperm in vitro. Methods: Motile human sperm were separately incubated for up to 24 hours with different concentrations of endotoxin (0.5, 1, 10, 1000, 10 000 and 50 000 ng/mL). Then the sperm motility was determined. The effect of endotoxin on the sperm motility in media without albumin was also determined. In addition, at the endotoxin concentrations of 0.5, 1 and 10 ng/mL, the sensitivity of the assay was compared to those of 1-cell and 2-cell mouse embryo bioassays. Results: At levels of 0.5-1 000 ng/mL endotoxin in media with 2 mg/mL albumin, sperm did not show significant change in motility after 24 h of incubation (P>0.05), while it was significantly inhibited at endotoxin levels of 10 000 and 50 000 ng/mL. In media without albumin, endotoxin levels of 50 000 and 1 000 ng/mL, markedly inhibited the sperm motility after 2 or 8 h of incubation (P<0.01). With media containing 0.5 and 1 ng/mL endotoxin, there was a significant reduction in the development rate at all developmental stages with 1-cell and 2-cell mouse embryo assays and at the level of 10 ng/mL, the embryo development was completely arrested. Conclusion: The sperm motility assay could detect high levels of endotoxin effect in vitro, but its sensitivity is low as compared with the 1-cell or 2-cell mouse embryo bioassay.

Pharmacological Investigation of Voltage-dependent Ca^(2+) Channels in Human Ejaculatory Sperm in vitro

The types of the voltage-dependent calcium channels （VDCCs） in human ejaculatory sperm and the effects of calcium channel blocker （CCB） on human sperm motility parameters in vitro were investigated. The human sperm motility parameters in vitro in response to the pharmacological agents nifedipine （NIF, inhibitor of L-type VDCC） and ω-conotoxin （GVIA, inhibitor of N-type VDCC） were compared and analyzed statistically. The results showed that NIF （1, 5, 10 μmol/L） could not only significantly affect human sperm＇s shape but also spermatozoa motility after incubated at least 10 rain in vitro （P〈0.001）. GVIA （0.1, 0.5 and 1 μmol/L） could just only significantly affect human sperm＇s progressive motility （a %＋b %） after incubated for 20 min in vitro （P〈0.01）, but they both could not significantly affect spermic abnormality rate. It is suggested that L-type VDCC, non L-type VDCCs and isoform of L-type VDCC exist in the cell membrane of human sperm solely or together, and they participate in the spermic physiological processes especially the spermic motility.

Measurement and significance of sperm morphology

The measurement or evaluation and clinical significance of human sperm morphology has always been and still is a controversial aspect of the semen analysis for the determination of a male＇s fertility potential. In this review the background of the development of the evaluation criteria for sperm morphology will be discussed. Aspects of criticism on the strict criteria definition and use of the criteria for sperm morphology evaluation will be discussed as well as possible reasons for the decline in normal sperm morphology values and how we can compromise for this phenomenon resulting in the very low normal reference value as published in the 2010 WHO manual for the Examination and Processing of Human Semen. One of the possible solutions may be to give more attention to a limited number of abnormal sperm morphology categories and the inclusion of sperm morphology patterns. It is concluded in this review that if done correctly and with care and with strict application of existing guidelines as outlined in the 2010 WHO manual, sperm morphology measurement still has a very important role to play in the clinical evaluation of male fertility potential.

Assessment of released acrosin activity as a measurement of the sperm acrosome reaction

Aim： To develop a method for assessing sperm function by measuring released acrosin activity during the acrosome reaction （AR）. Methods： Human semen samples were obtained from 24 healthy donors with proven fertility after 3-7 days of sexual abstinence. After collection, samples were liquefied for 30 min at room temperature. Standard semen parameters were evaluated according to World Health Organization （WHO） criteria. Calcium ionophore A23187 and progesterone （P4） were used to stimulate the sperm to undergo AR. After treatment, sperm were incubated with the supravital dye Hoechst33258, fixed in a glutaraldehyde-phosphate-buffered saline solution, and the acrosomal status was determined by fluorescence microscopy with fluorescein isothiocyanate-labeled Pisum sativum agglutinin （FITC-PSA）. The percentage of sperm undergoing AR （AR%） was compared to sperm acrosin activities as assessed by spectrocolorimetry. The correlation between AR% and acrosin activity was determined by statistical analysis. Results： The AR% and released acrosin activity were both markedly increased with A23187 and P4 stimulation. Sperm motility and viability were significantly higher after stimulation with P4 versus stimulation with A23187 （P 〈 0.001）. There was a significant positive correlation between released acrosin activity and AR% determined by FITC-PSA staining （r= 0.916, P 〈 0.001）. Conclusion： Spectrocolorimetric measurement of released acrosin activity might serve as a reasonable alternative method to evaluate AR. （Asian J Androl 2008 Mar, 10： 236-242）

Retrospective descriptive analysis of the physiological kinetics of prostate-specific antigen in men older than 75 years

Several studies have compared prostate-specific antigen （PSA） kinetics in men with and without cancer, but there has been no adequate analysis of the longitudinal variation in PSA. The aim of this study was to assess the fluctuations in PSA in a cohort of elderly men in an attempt to define a physiological pattern of PSA kinetics. We searched a specific cohort of patients aged 〉 75 years and with PSA value 〈 2.0 ng mL^-1. A history of all PSA values over the past 10 years was compiled for each patient to create a database of patients fitting the following criteria： （1） minimum of five PSA measurements, （2） over at least 5 years. Exclusion criteria were： （1） PSA 〈 0.2 ng mL^-1 at each measurement and （2） having had more than one PSA test per year. In all, 1 327 patients （mean age： 78.52 years） fit the inclusion criteria. The mean variation from the first to the last PSA test was 0.05 ± 0.43, with a mean follow-up of 6.79 ± 1.71 years. Over the same period, the mean fluctuation from the lowest to the highest PSA value was 0.04 ± 0.55 （P = 0.925）. The mean annual PSA velocity （PSAV） was calculated by dividing the mean variation from the first to the last PSA test by the number of years of observation for each patient and was set at 0.0104 ± 0.1050. Concluding, in a large-scale cohort of elderly individuals considered healthy and evaluated for a considerable follow-up, the average annual PSAV as well as the average fluctuation from the lowest to the highest PSA value are insignificant.

Toxic effects of polychlorinated biphenyls （Aroclor 1254） on human sperm motility

Polychlorinated biphenyls （PCBs） are common environmental contaminants that represent a considerable risk to reproductive toxicity in exposed human populations. Although some experimental studies have suggested an association between the levels of PCBs and semen quality, the direct effects of PCBs on human sperm parameters remain largely unexplored. To this aim, a short-term in vitro incubation experiment that better imitated the putative exposure of sperm to Aroclor 1254 （a commercial PCB mixture） in male reproduction tissue was conducted. Human sperm were incubated with various concentrations （0, 1, 5, or 25 mg ｜^-1） of Aroclor 1254 for different amounts of time （3 and 6 h） in vitro. Sperm motility parameters were analyzed with computer-assisted sperm analysis （CASA）. The proportion of sperm with high mitochondrial membrane potential （ΔΨm） and the levels of intracellular reactive oxygen species （ROS） were detected to explore the probable cause of sperm impairment. Human sperm exposed to continuous Aroclor 1254 exhibited： （i） reduced sperm motility and kinematic parameters, （ii） a proportion of sperm with high ΔΨm that decreased in a dose-dependent manner （P 〈 0.05）, and （iii） increased levels of ROS compared with controls （P 〈 0.05）. In conclusion, Aroclor 1254 can decrease sperm motility, which may culminate in increased ROS and general mitochondrial dysfunction, thus affecting the fertilization potential of sperm. Our findings suggest a broader understanding of the effect of Aroclor 1254 on human soerm.

Human sperm testicular angiotensin-converting enzyme helps determine human embryo quality

Angiotensin-converting enzyme functions in the male reproductive system, but the extent of its function in reproduction is not fully understood. The primary objective of this work was to investigate the relationship between the testicular isoform of angiotensin-converting enzyme present in human spermatozoa and semen parameters, human embryo quality, and assisted reproduction success. A total of 81 semen samples and 635 embryos from couples undergoing oocyte donation cycles at the IVI Bilbao Clinic were analyzed. Semen parameters, embryos quality, and blastocyst development were examined according to the World Health Organization standards and the Spanish Association of Reproduction Biology Studies criteria. The percentage of testicular angiotensin-converting enzyme-positive spermatozoa and the number of molecules per spermatozoon were analyzed by flow cytometry. Both parameters were inversely correlated with human sperm motility. Higher percentages of testicular angiotensin-converting enzyme-positive spermatozoa together with fewer enzyme molecules per spermatozoon were positively correlated with better embryo quality and development. Our results suggest that embryos with a higher implantation potential come from semen samples with higher percentages of testicular angiotensin-converting enzyme-positive cells and fewer enzyme molecules per spermatozoon. Based on these findings, we propose that testicular angiotensin-converting enzyme could be used to aid embryologists in selecting better semen samples for obtaining high-quality blastocysts during in vitro fertilization procedures.

Changes on membrane integrity and ultrastructure of human sperm after freeze-drying

Objective To observe changes on membrane integrity and ultrastructure of human sperm after freeze-drying.Methods Semen samples from both normospermic donors（group A, n=15） and infertile men with abnormal sperm parameters（group B, n=15） were enrolled into this study. These samples were freeze-dried by using a freeze-drying method. The membrane integrity in the head and tail regions of individual spermatozoon was examined by using the combined hypo-osmotic swelling-eosin Y exclusion test. Sperm ultrastructure in groups A（n=3） and B（n=3） was observed by scanning electron microscopy（SEM）.Results After freeze-drying, all spermatozoa were types I（damaged both head and tail membranes） and III（damaged head membrane and intact tail membrane） membrane integrity in groups A and B. Type III of group B had lower value than that of group A（P〈0.01）. Under SEM, intact freeze-dried spermatozoa including abnormal morphology and normal-looking morphology were observed in both groups A and B. A few freezedried sperm heads had unsmooth or fuzzy surface. Isolated sperm heads, bent tails,broken sperm tails or fragmentary tails were more frequently seen in group B than those in group A.Conclusion Freeze-dried human spermatozoa could have intact structural components. However, freeze-drying resulted in severe damage on membrane integrity and ultrastructure of sperm. Samples from infertile men would have less resistance to freeze-drying.