• Clinical and Experimental Reproductive Medicine
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• v.43 no.4
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• pp.193-198
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• 2016

Objective: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzyme defect. G6PD plays a key role in the pentose phosphate pathway, which is a major source of nicotinamide adenine dinucleotide phosphate (NADPH). NADPH provides the reducing equivalents for oxidation-reduction reductions involved in protecting against the toxicity of reactive oxygen species such as $H_2O_2$. We hypothesized that G6PD deficiency may reduce the amount of NADPH in sperms, thereby inhibiting the detoxification of $H_2O_2$, which could potentially affect their motility and viability, resulting in an increased susceptibility to infertility. Methods: Semen samples were obtained from four males with G6PD deficiency and eight healthy males as a control. In both groups, motile sperms were isolated from the seminal fluid and incubated with 0, 10, 20, 40, 60, 80, and $120{\mu}M$ concentrations of $H_2O_2$. After 1 hour incubation at $37^{\circ}C$, sperms were evaluated for motility and viability. Results: Incubation of sperms with 10 and $20{\mu}M\;H_2O_2$ led to very little decrease in motility and viability, but motility decreased notably in both groups in 40, 60, and $80{\mu}M\;H_2O_2$, and viability decreased in both groups in 40, 60, 80, and $120{\mu}M\;H_2O_2$. However, no statistically significant differences were found between the G6PD-deficient group and controls. Conclusion: G6PD deficiency does not increase the susceptibility of sperm to oxidative stress induced by $H_2O_2$, and the reducing equivalents necessary for protection against $H_2O_2$ are most likely produced by other pathways. Therefore, G6PD deficiency cannot be considered as major risk factor for male infertility.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.3
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• pp.335-340
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• 1997

Fluorescence in situ hybridization (FISH) techniques allow the enumeration of chromosome abnormalities and from a great potential for many clinical applications. In order to produce quantitative and reproducible results, expensive tools such as a cooled CCD camera and a computer software are required. We have developed a Chromosome Image Processing System (Chips) using FISH that allows the detection and mapping of the genetic aberrations. The aim of our study, therefore, is to evaluate the capabilities of our original system using a black-and-white video camera. As a model system, three repetitive DNA probes (D18Z1, DXZ1, and DYZ3) were hybridized to variety different clinical samples such as human metaphase spreads and interphase nuclei obtained from uncultured peripheral blood lymphocytes, uncultured amniocytes, and germ cells. The visualization of the FISH signals was performed using our system for image acquisition and pseudocoloring. FISH images were obtained by combining images from each of probes and DAPI counterstain captured separately. Using our original system, the aberrations of single or multiple chromosomes in a single hybridization experiment using chromosomes and interphase nuclei from a variety of cell types, including lymphocytes, amniocytes, sperm, and biopsied blastomeres, were enabled to evaluate. There were no differences in the image quality in accordance with FISH method, fluorochrome types, or different clinical samples. Always bright signals were detected using our system. Our system also yielded constant results. Our Chips would permit a level of performance of FISH analysis on metaphase chromosomes and interphase nuclei with unparalleled capabilities. Thus, it would be useful for clinical purposes.

• Development and Reproduction
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• v.20 no.3
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• pp.219-225
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• 2016

Most of the commercial devices for vitrification are directly immersed into the warming solution (WS) for increasing of warming rate. However, the previous modified cut standard straw (MCS) which has reported is difficult to immerse into the WS. The aim of this study was to investigate whether the long cut straw (LCS) could be useful as a stable tool for vitrified-warmed human blastocysts. A total of 138 vitrified-warmed cycles were performed between November 2013 and November 2014 (exclusion criteria: women ${\geq}38$ years old, poor responder, surgical retrieval sperm, and severe male factor). The artificial shrinkage was conducted using 29-gauge needles. Ethylene glycol and dimethyl sulfoxide (7.5% and 15% (v/v)) were used as cryoprotectants. Freezing and warming were conducted using the LCS tool. The cap of LCS was removed using the forceps in the liquid nitrogen ($LN_2$) and then directly immersed into the first WS for 1 min at $37^{\circ}C$ (1 M sucrose). Only re-expanded blastocysts were transferred after it was cultured in sequential media for 18-20 h. A total of 294 blastocysts were warmed, and all were recovered (100%). Two hundred eighty-five embryos were survived (96.9%). The vitrified-warmed blastocysts of all patients were transferred without any cancellation. We were able to achieve a reasonable implantation (24.2%), following by clinical pregnancy (36.2%), which then continued to ongoing pregnancy (36.2%), and live birth (31.2%). Using LCS is achieved the acceptable rates of survival, pregnancy and live birth. Therefore, the LCS could be considered as a stable and simple tool for human embryo vitrificaton.

• Clinical and Experimental Reproductive Medicine
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• v.22 no.2
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• pp.203-210
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• 1995

Many oocytes fail to fertilize and cleave in vitro and many embryos transferred back to uterus fail to implant or maintain implantation. Chromosomal abnormalities in the male and female gametes may contribute to this loss. The higher incidence of meiotic chromosomal abnormalities bas been found in oocytes than in sperm. The wide range of incidence of chromosomal abnormalities in unfertilized oocytes has been reported in human IVF program (26-63%). However, factors affecting chromosomal abnormalities are not well understood. The present study has been conducted to investigate effects of the method for ovarian hyperstimulation, women's age, and the number of oocytes retrieved per patients on the incidence of numerical chromosomal abnormalities. Five hundred eighty four unfertilized metaphase II oocytes were subjected to chromosomal analysis. Included unfertilized oocytes were from 220 patients (mean $age=32.7{\pm}3.0$) and three hundred thirty oocytes were legible for analysis. Two hundred fourty five oocytes out of 330 (73.3%) were normal, while 38 (11.5%) were hyperploidy, 35 (10.6%) were hypoploidy, and 12 (3.6%) were diploidy. Significant difference in chromosomal abnormalities was not found between two patient groups stimulated by follicular stimulating hormone/human menopausal gonadotrophin (FSH/HMG) (25.9%) and gonadotrophin-releasing hormone agonist/follicular stimulating hormone/human menopausal gonadotrophin (GnRHa/FSH/HMG) (28%). There was a tendency of increasing chromosomal abnormalities in unfertilized oocytes from older patients (<30 yrs: 20.3%, 30-34yrs: 26.9%, >34 yrs: 35.3%). The number of oocytes retrieved per patient had no effect the incidence of chromosomal abnormalities (1-5: 31. 4%, 6-10: 29.8%, 11-15: 28.6%, > 15: 16.5%). These results from the present study suggest that the chromosomal abnormalities observed in the unfertilized oocytes has not affected by the stimulation methods, patient's age, and the number of oocytes retrieved per patients.

• Clinical and Experimental Reproductive Medicine
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• v.36 no.3
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• pp.219-224
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• 2009

Objective: Hypogonadotropic hypogonadism (HH) is an uncommon cause of male infertility. We investigated the outcome of gonadotropin therapy for restoring fertility and pregnancy outcomes in patients with HH. Methods: Medical charts of 10 infertile male patients with HH treated with gonadotropin were reviewed. Initial testicular volume were estimated. Semen analysis parameters (semen volume, sperm counts, motility), serum leutenizing hormone (LH), follicle stimulating hormone (FSH), total testosterone were determined before and after human chorionic gonadotropin/human menopausal gonadotropin (hCG/hMG) treatment. Differences were analyzed statistically. Results: Of 10 patients, 7 (70%) succeed at pregnancy (nature pregnancy in 4). Semen analysis parameters, serum FSH, and testosterone were increased significantly after treatment. The population was stratified according to initial testicular volume into a small testis subset (testicular volume less than 10 cc in 4) and a large testis subset (testicular volume 10 cc or greater in 6). Semen analysis parameters and serum testosterone were increased significantly after treatment in large testis subset. Conclusion: Infertile men with HH initiate and maintain spermatogenesis with gonadotropin (hCG/hMG alone or combined) therapy, thus gonadotropin therapy is good choice in infertile men with HH.

• The World Journal of Men's Health
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• v.41 no.1
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• pp.215-226
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• 2023

Purpose To clarify (phospho-) glycogen synthase kinase-3 (GSK3) isoform variants in the germline and soma of human testes and spermatozoa. Materials and Methods GSK3 isoform variants in normospermatogenic and Sertoli cell-only (SCO) testicular biopsies and spermatozoa were examined. In normospermatogenic testes, GSK3α and GSK3β variants 1 and 2 different in low complexity region (LCR) were expressed and their levels were decreased in SCO testes. GSK3β variant 3 was only expressed in SCO testes. GSK3β as well as GSK3α, the dominant isoforms in testes were decreased in SCO testes. In normospermatogenic testes, GSK3β were found in spermatogonia and markedly decreased in meiotic germ cells in which GSK3α was dominant. p-GSK3α/β were marginal in spermatogonia and early spermatocytes. In SCO testes, GSK3α/β immunoreactivity in seminiferous epithelia was weaker than those of normospermatogenic testes whereas p-GSK3α/β(Ser) immunoreactivity was visibly increased in Sertoli cells. GSK3α was dominant in ejaculated spermatozoa in which GSK3α and p-GSK3α(Ser) were found in the head, midpiece, and tail. In acrosome-reacted spermatozoa, GSK3α was found in the equatorial region of head, midpiece, and tail, and p-GSK3α(Ser) was only found in midpiece. During sperm capacitation, p-GSK3α(Ser) was significantly increased together with phosphotyrosine proteins and motility. In human male germ cells, GSK3 isoforms different in LCRs switch from GSK3β to GSK3α during meiotic entry, suggesting the isoform-specific roles of GSK3α and GSK3β in meiosis and stemness or proliferation of spermatogonia, respectively. In dormant Sertoli cells of SCO testes kinase activity of GSK3 might be downregulated via inhibitory phosphorylation. In spermatozoa, inhibitory phosphorylation of GSK3α might be coupled with activation of motility during capacitation.

• Development and Reproduction
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• v.4 no.2
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• pp.203-213
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• 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
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• v.27 no.1
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• pp.23-29
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• 2000

Objective: Zona pellucida (ZP) has been thought to be the barrier of egg to sperm penetration before and after fertilization. The phenomenon of ZP hardening has been considered as a post-fertilization event until now, and it is generally accepted that it is caused by the secretory products of cortical granules released during the cortical reaction. Hardening of ZP could occur "spontaneously" in mammalian oocytes in standard culture conditions, and that it is probably not a consequence of cortical reaction. The purpose of our study was to investigate the effect of human amniotic fluid (HAF) on nuclear maturation (NM) and fertilization ability of mouse immature oocytes. Methods: HAF was obtained from patients undergoing amniocentesis at $16{\sim}20$ weeks of gestation. HAF from five to ten patients was centrifuged and the supernatants was pooled. Cumulusenclosed mouse immature oocytes were incubated in the medium containing HAF, and examined to confirm NM and fertilization. Female ICR mice (about 3 weeks old) were stimulated with 7.5 IU PMSG. Immature oocytes were isolated at $48{\sim}52$ hrs post PMSG injection and cultured in TCM-199 supplemented with 20% HAF for 18 hrs. FBS was used as a control for the examination. Matured oocytes (MII) were fertilized with sperms collected from the epididymis of male mice (over 10 weeks old). Fertilization was in conducted T6 medium containing 15 mg/ml BSA, and confirmed at 6 hrs post-insemination. Fertilization rate was assessed in zona-intact or zona-free oocytes (denuded by trypsin). Evaluation of NM and fertilization was carried out by rapid staining method. ZP hardening was evaluated by incubating cumulus cell-free mature oocytes in 0.001% chymotrypsin at $37^{\circ}C$ for 10 min. Results: There was no significant difference between the effects of HAF (86.6%) and FBS (87.7%) supplements on NM of immature oocytes. When maturation medium was supplemented with HAF, total fertilization rates (7%) were significantly lower (p<0.01) than that of FBS (85.1%). In HAF group, fertilization rate was increased (p<0.01) in zona-free oocytes (7% versus 100%). The resistance of mouse oocyte ZP to digestion by chymotrypsin after maturation in vitro was significantly higher (p<0.01) in HAF group (86.7%) than in FBS (6.7%). To culture oocytes in FBS were very effective in preventing ZP hardening. However cultured oocytes in HAF showed high rate of ZP hardening (p<0.01). Conclusions: These results suggest that HAF can be used as a supplement for the NM of mouse immature oocytes in vitro. However, HAF induces spontaneous hardening of ZP of mouse immaure oocytes during maturation in vitro.

• Clinical and Experimental Reproductive Medicine
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• v.38 no.1
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• pp.31-36
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• 2011

Objective: To determine whether the serum ${\beta}$-human chorionic gonadotropin (hCG) profile following preimplantation genetic diagnosis (PGD) is lower than that of intracytoplasmic sperm injection (ICSI) cycles. Methods: A total of 129 PGD cycles and 1,161 age-matched ICSI cycles, which resulted in pregnancy (serum ${\beta}-hCG{\geq}5$ mIU/mL) on post-ovulation day (POD) 12 were included. We compared the mean serum ${\beta}$-hCG levels on POD 12, 14, 21, and 28, doubling time of serum hCG, and created a cut-off value for predicting a singleton pregnancy in each group. Results: The mean serum ${\beta}$-hCG concentration of the PGD group was significantly lower than that of the control group on POD 12, 14, and 21. The doubling time of serum ${\beta}$-hCG at each time interval showed no significant difference. The cut-off-value of serum ${\beta}$-hCG for predicting a single viable pregnancy was 32.5 mIU/mL on POD 12 and 113.5 mIU/mL on POD 14 for the PGD group, which was lower than that for the control group. Conclusion: Blastomere biopsy may decrease the ${\beta}$-hCG-producing activity of the trophoblasts, especially in early pregnancy. Setting a lower cut-off value of serum ${\beta}$-hCG for predicting pregnancy outcomes in PGD may be needed.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.3
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• pp.281-292
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• 1997

The objective of this study was to compare retrospectively the survival and pregnancy rates(PR) of cryopresered-thawed embryos obtained from intracytoplasmic sperm injection (ICSI) or conventional in vitro fertilization (IVF). Ninety-six cycles of cryopresered-thawed embryo transfer (ET) were performed in 79 patients from June, 1996 to September, 1997 and grouped as followings: 20 cycles (16 patients) inseminated by ICSI (ICSI Group) and 76 cycles (63 patients) by conventional IVF (IVF Group). Slow-freezing and rapid-thawing protocol was used with 1.5M propanediol (PROH) and 0.1M sucrose as cryoprotectant. All embryos were frozen-thawed at the two pronuclear (2 PN) stage excluding four cycles in which the early cleavage stage embryos were frozen, and allowed to cleave in vitro for one day before ET. The duration from freezing to thawing was comparable in both groups ($mean{\pm}SD$, $112.1{\pm}80.0$ vs. $124.8{\pm}140.1$ days). The age of female ($31.2{\pm}3.4$ vs. $32.6{\pm}3.3$ years) and the endometrial thickness prior to progesterone injection ($9.4{\pm}2.0$ vs. $9.3{\pm}1.8$ mm) were also comparable in both groups. There was no significant difference in the outcomes of cryopreserved-thawed ET between two groups: survival rate ($85.2{\pm}16.1%$ vs. $82.2{\pm}19.7%$), cleavage rate ($96.9{\pm}6.7%$ vs. $94.7{\pm}13.0%$), cumulative embryo score (CES, $54.5{\pm}31.1$ vs. $49.0{\pm}20.0$), preclinical loss rate (5.0% vs. 5.3%), clinical miscarriage rate (0% vs 29.4%), clinical PR per transfer (35.0% vs. 22.4%), implantation rate (9.9% vs. 5.6%), and multifetal PR (42.9% vs. 17.6%). In conclusion, human embryos resulting from ICSI can be cryopreserved-thawed and transferred successfully, and the survival rate and PR are comparable to conventional IVF.