• Toxicological Research
• /
• v.33 no.4
• /
• pp.315-323
• /
• 2017

Preconceptual sex selection is still a highly debatable process whereby X- and Y-chromosome-bearing spermatozoa are isolated prior to fertilization of the oocyte. Although various separation techniques are available, none can guarantee 100% accuracy. The aim of this study was to separate X- and Y-chromosome-bearing spermatozoa using methods based on the viability difference between the X- and Y-chromosome-bearing spermatozoa. A total of 18 experimental semen samples were used, written consent was obtained from all donors and results were analysed in a blinded fashion. Spermatozoa were exposed to different pH values (5.5, 6.5, 7.5, 8.5, and 9.5), increased temperatures ($37^{\circ}C$, $41^{\circ}C$, and $45^{\circ}C$) and ROS level ($50{\mu}M$, $750{\mu}M$, and $1,000{\mu}M$). The live and dead cell separation was done through a modified swim-up technique. Changes in the sex-chromosome ratio of samples were established by double-label fluorescent in situ hybridization (FISH) before and after processing. The results indicated successful enrichment of X-chromosome-bearing spermatozoa upon incubation in acidic media, increased temperatures, and elevated $H_2O_2$. This study demonstrated the potential role for exploring the physiological differences between X-and Y-chromosome-bearing spermatozoa in the development of preconceptual gender selection.

• Clinical and Experimental Reproductive Medicine
• /
• v.46 no.1
• /
• pp.8-13
• /
• 2019

Objective: Density gradient centrifugation (DGC) is frequently used to isolate high-motility fractions of spermatozoa. We compared the efficacy of four DGC media in terms of the percentage of morphologically normal spermatozoa, DNA fragmentation level, and hyaluronic acid (HA) binding ability. Methods: Thirty men with a total motile spermatozoa count > 80 million participated. Semen samples were divided into four aliquots, which were processed using PureSperm, PureCeption, Sidney, and SpermGrad media, respectively. The DNA fragmentation level was measured using the Halosperm assay kit and HA binding ability was measured using the HBA assay kit. Results: The mean percentage of morphologically normal spermatozoa was significantly enhanced after DGC using all four media (10.3%, 9.9%, 9.8%, and 10.7%, respectively; p< 0.05 for each when compared with 6.9% in raw semen). The DNA fragmentation level was significantly reduced after DGC using PureSperm, PureCeption, and SpermGrad media (6.0%, 6.5%, and 4.9%, respectively; p< 0.05 for each when compared with 11.2% in raw semen), but not after DGC using Sidney media (8.5%, p> 0.05). HA binding ability did not change after DGC using any of the four media. Conclusion: The four media were equally effective for obtaining a sperm fraction with highly motile, morphologically normal sperm. PureSperm, PureCeption, and SpermGrad media were equally effective for acquiring a sperm fraction with less DNA fragmentation.

• Development and Reproduction
• /
• v.4 no.2
• /
• pp.203-213
• /
• 2000

To investigate the effects of reactive oxygen species (ROS) on capacitation, acrosome reaction in human spermatozoa. Human spermatozoa were incubated with xanthine-xanthine oxidase (X-XO), $H_2O$$_2$, sodium nitroprusside (SNP) or lymphocyte. Otherwise, spermatozoa were incubated under low $O_2$ (5 ％) condition. Chlortetracycline (CTC) staining was conducted to assess capacitation and acrosome reaction. Analysis of lipid peroxidation was done by spectrophotometric determination of malondialdehyde (MDA) production in spermatozoa. $H_2O$$_2$, X-XO, SNP and lymphocyte treatment significantly increased capacitated spermatozoa within 1 h of incubation. There was no significant difference in capacitation between low- and high $O_2$ groups. In the presence of low concentration of $H_2O$$_2$, lipid peroxidation decreased significantly. However, under the high concentration of $H_2O$$_2$, lipid peroxidation significantly increased at the end of incubation compared to control. In the presence of high concentration of lymphocytes, lipid peroxidation significantly increased compared to control at 1hr of incubation. There was no significant difference in lipid peroxidation according to $O_2$ concentration examined. Acrosome reaction (AR) was evaluated by CTC staining after the progesterone challenge. In all ROS groups, AR increased compared to control. The X(100 $\mu$M) - XO (100mIU) system was the most potent to induce AR. Taken together, it suggested positive control of AR by ROS and the positive relationship between the lipid peroxidation and AR. The early onset of capacitation in the presence of ROS suggest that ROS might be a positive regulator of sperm capacitation and hyperactivation.

• Clinical and Experimental Reproductive Medicine
• /
• v.24 no.2
• /
• pp.187-192
• /
• 1997

The survival rate and motility recovered after cryopreservation of testicular spermatozoa in testicular sperm extraction (TESE)-ICSI program is low. The purpose of this study was to assess the availability and efficiency of mouse empty zona pellucida in cryopreserving human TESE spermatozoa. Mouse empty zonae pellucidae were obtained by extraction of cytoplasm with or without cytochalasin B treatment. Motile sperm from proven-fertile donor and two azoospermic patients after TESE were individually inserted into empty zona pellucida and cryopreserved. Two to five days after cyropreservation, the frozen sperm were thawed and the rates of recovery and motility were observed. The ooplasmic extraction rates of control (N=80) and cytochalasin B treated oocytes (N=80) were 94.0% and 96.2%, respectively (p>0.05). The post-thaw recovery rates of spermatozoa and rates of motility recovery of ejaculate (N=70) and testicular (N=70) sperm were 97.1%, 97.1% and 95.7%, 94.3%, respectively (p>0.05). The results of this study showed that the mouse zone pellucida is useful for cryostorage of single testicular spermatozoa.

• Clinical and Experimental Reproductive Medicine
• /
• v.49 no.4
• /
• pp.270-276
• /
• 2022

Objective: The present study assessed the biological characteristics of human spermatozoa at different time intervals (0, 1, 1.5, and 2 hours) after incubation at 37℃. Methods: Twenty-five normozoospermic semen samples were incubated at 37℃. Incubation was performed at four time intervals of 0 (after liquefaction), 1, 1.5, and 2 hours. The samples were evaluated for sperm parameters at each time interval. Results: The rate of sperm progressive motility decreased at 1.5 hours compared to 0 hours as well as 2 hours compared to 1 hour and 0 hours. The rate of non-motile spermatozoa also decreased after 2 hours compared to after 0 hours. No significant changes were observed in sperm viability (p=0.98) and non- progressive motility (p=0.48) at any time intervals. Abnormal sperm morphology increased at 1.5 hours of incubation time (p<0.001). No significant changes were observed in DNA fragmentation at 1 hour compared to 0 hours (median [interquartile range]: 19.5 [4] vs. 19 [4]), as well as at 1.5 hours compared to 1 hour (20 [5]). However, a significant increase in DNA fragmentation was observed at 1.5 hours compared to 0 hours. The mitochondrial membrane potential decreased remarkably after 1 hour of incubation time. No significant differences were observed in the acrosome reaction or malonaldehyde levels at any time point (p=0.34 and p=0.98, respectively). Conclusion: The incubation of normozoospermic samples before use in assisted reproductive technology should be less than 1.5 hours to minimize the destructive effects of prolonged incubation time on general and specific sperm parameters.

• Clinical and Experimental Reproductive Medicine
• /
• v.16 no.2
• /
• pp.173-177
• /
• 1989

Defective or inadequate semen quality, usually presenting as low sperm count or poor sperm motility , is recognizable by semen analysis. However, the ability of spermatozoa to fertilize an ovum is not determined used in various experiments. In this study, hamster oocyte sperm penetration assay was used to determine the fertilizing capacity of sperms in 20 subjects which divided into two groups, group A with 10 normal fertile men, and group B with 10 infertile men. The % penetration in group A and group B were 61% and 35% respectively, which showed statistically not significant but fertilization index was significantly different between group A(FI=2.24) and group B(FI=O.05). Additionally it seemed that the percentage of sperm penetraton was influenced more by the motility of spermatozoa than by the number.

• Clinical and Experimental Reproductive Medicine
• /
• v.27 no.4
• /
• pp.393-403
• /
• 2000

Objective: To investigate the effects of ROS on kinematic parameters in human spermatozoa. To verify the changes in above parameters, human spermatozoa were incubated with xanthine-xanthine oxidase (X-XO), $H_2O_2$, sodium nitroprusside (SNP) or lymphocyte. Otherwise, spemlatozoa were incubated under low $O_2 (5%) condition. Methods: CASA was employed to analyze sperm motion parameters. Results: Under $H_2O_2 treatment, all kinematic parameters of spermatozoa were dramatically increased during 30 min, but gradually decreased thereafter. Under the low concentration of $H_2O_2 (125 ${\mu}M$ and 250 ${\mu}M$), the movement velocity (VAP, VCL, VSL) decreased, but forward movement increased. Under the 1mM $H_2O_2, sperm showed reduced kinematic parameters except during first 30 min of incubation. In the cases of X-XO and SNP treatment, the movement velocity increased but the forward movement reduced. After incubation for 3 hr treatment, the kinematic parameters gradually decreased in high concentration of X-XO. However these parameters maintained or increased in low concentration of X-XO. There was no obvious changes in the above parameters in the high concentration of SNP. In the presence of high concentration of lymphocytes, all parameters decreased. Under the 5% $O_2 condition, the parameters of the movement velocity and movement pattern increased, but forward movement decreased. Taken togethers, it suggested that ROS increased the movement velocity but decreased the forward movement and lateral head replacement. $H_2O_2, X-XO, SNP and lymphocyte treatment significantly increased capacitated spermatozoa within I h of incubation. There was no significant difference in capacitation between low- and high $O_2 group. Conclusion: The early onset of capacitation in the presence of ROS suggest that ROS might be a positive regulator of sperm capacitation and hyperactivation. These results demonstrate that low concentration ROS facilitates the movement velocity but high concentration ROS was inhibitory.

• Hwankyungkyoyuk
• /
• v.11 no.1
• /
• pp.275-290
• /
• 1998

Nowadays, mankind is suffering from serious pollutions. In proportion to industrial modernization, polltional problems have been increased and arised new one endlessly. Todays, we are cope with a newkind pollution materials threatening the life of mankind with root out. It is Environmental Hormon, that is, Endocrine Disrupting Chemical(EDs). EDs was made by dioxin, PCB, DDT, cadmium, lead, ether and even diethylstillbestrol(DES) that used to contraceptive pill etc. These EDs makes changing from normal human hormon to abnormal one and it kill off or fade out spermatozoa. Then the spermatozoa was infected with EDs makes man imposex, sexual malformations and infertility also. It is the more critical phenomenons that EDs invade into the field of wild-life; birds, invertebrate animals, fishes, mammalia especially. England TV(BBC) journalist Cadury, D. introduced a lot of case studies that many kind of male animals was changed feminization by EDs in her book (The Feminization of Nature;1997). Otherwise, Gibbs, P.E. and Bryan, G. W. reported about dog-whelk snail (a sort of sea shell) was changed gender from male to female by EDs in their study. Briefly speaking, Environmental Hormon (EDs) stem from polluted materials maloperate human and animal informational codes of hormon. It is a significant event to an individual ontogeny and ecological system. To overcome these problem WHO, OECD, UNEP held many work-shops, international seminars and researches but it is not enough yet. On May of 1997, the International Meeting of Environmental Administrators makes a $\ulcorner$Declaration on EDs Problems$\lrcorner$ for solving it fortunately and NHK(japan) reported Super Critical Fluid as a new materials for decrease toxicity of dioxin in recent. Key Words : Environmental Hormon, EDs, imposex feminization, gender, DES, spermatozoa. Super Critical Fluid.

• Clinical and Experimental Reproductive Medicine
• /
• v.25 no.3
• /
• pp.331-340
• /
• 1998

We determined the incidence of activation, male pronuclear formation and apposition of pronuclei in porcine oocytes following intracy-toplasmic injection of various porcine sperm components and foreign species spermatozoa, such as mouse, human or cattle. The porcine oocytes were activated by injection of a spermatozoon or an isolated sperm head. Neither isolated sperm tail nor perinuclear material removed sperm head activated oocytes. Because injection of mouse, bovine or human spermatozoon activated porcine oocytes, the sperm born activation factors is not strict species specific. Male pronuclear formation and pronuclear apposition were observed in the porcine oocytes following injection of porcine, bovine, mouse or human spermatozoa. The electrical stimulation following sperm cell injection did not enhance the incidence of male pronuclear formation nor pronuclear apposition comparent with sperm cell injection alone (p>0.1). Mitosis and two cell division in some oocytes were observed at 20 to 24 h after injection of porcine spermatozoon. However, none of oocytes following injection of mouse, bovine or human spermatozoa developed to the mitotic metaphase or normally divided to the two cell stage. These results suggested that the oocyte activating factor(s) presented in the perinuclear material and it is not species specific for the porcine oocyte.

• Clinical and Experimental Reproductive Medicine
• /
• v.19 no.1
• /
• pp.65-69
• /
• 1992

The sucess of intrauterine inseminations with washed human spermatozoa was evaluated in 92 patients. In according to indication of insemination, intrauterine inseminations of women with hostile cervical mucus yield a 35.3% and 12.8% with male factors and 27.3% with unexplained infertility. In ovulation induction group with variable agents including clomiphen, human menopausal gonadotropin (HMG) and GnRH analog, the pregnancy rate was 27.8% (22/79) and in natural cycle group, 15.4% (2/13). The fetal loss rate in insemination group was 12.5% (3/24). Multiple pregnancies were 7 cases and ovulation induction were performed in 6 cases among them. Intrauterine insemination with washed human spermatozoa therefore represents an effective and safe procedure selected infertile couples.