• Clinical and Experimental Reproductive Medicine
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• v.42 no.1
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• pp.14-21
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• 2015

Objective: It has previously been suggested that embryos developing from intracytoplasmic sperm-injected (ICSI) zygotes with three pronuclei (3PN) are endowed with a mechanism for self-correction of triploidy to diploidy. 3PN are also observed in zygotes after conventional in vitro fertilization (IVF). The parental origin, however, differs between the two fertilization methods. Whereas the vast majority of 3PN IVF zygotes are of dispermic origin and thus more likely to have two centrioles, the 3PN ICSI zygotes are digynic in origin and therefore, more likely to have one centriole. In the present study, we examine whether the parental origin of 3PN embryos correlates with the karyotype. Methods: The karyotype of each nucleus was estimated using four sequential fluorescence in situ hybridizations-each with two probes-resulting in quantitative information of 8 different chromosomes. The karyotypes were then compared and correlated to the parental origin. Results: 3PN ICSI embryos displayed a significantly larger and more coordinated reduction from the assumed initial 3 sets of chromosomes than 3PN IVF embryos. Conclusion: The differences in the parental origin-and hence the number of centrioles-between the 3PN IVF and the 3PN ICSI zygotes are likely to be the cause of the differences in karyotypes.

• Clinical and Experimental Reproductive Medicine
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• v.36 no.3
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• pp.209-217
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• 2009

Objective: To analyze current issues and to propose alternatives for "the cryopreserved embryos generated before 2005". Methods: The differences in attitude among the stakeholders such as sperm donors, oocyte donors, and IVF clinics were presupposed. We want to forecast the impediments which occur inevitably in the process of "getting the informed consent" and "discarding the cryopreserved embryos generated before 2005". Results: Even though there is a specific guideline for "the cryopreserved embryos generated before 2005" at November 23, 2006, no consensus about the process related to "getting the informed consent" has been made. Conclusion: Unavoidably, it seems to be entering a period of massive discard of "the cryopreserved embryos generated before 2005". This is actually opposed to the intent of the Bioethics and Safety Act, which is to protect human dignity and prevent harm to human beings. We have to make reasonable due process to determine the destiny of "the cryopreserved embryos generated before 2005".

• Clinical and Experimental Reproductive Medicine
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• v.49 no.2
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• pp.149-158
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• 2022

Objective: Optimizing culture media for the incubation of immature oocytes is a vital strategy to increase the oocyte maturation rate during in vitro maturation (IVM) programs. This study evaluated the IVM and fertilization rates of human germinal vesicle (GV) and metaphase I (MI) oocytes using two different maturation media (commercial and homemade) with or without growth differentiation factor 9-β (GDF9-β). supplementation. Methods: Immature oocytes from intracytoplasmic sperm injection (ICSI) cycles were collected and assigned to one of two IVM culture media (commercial or homemade; cleavage-stage base). After maturation, MII oocytes were examined under an inverted microscope for the presence of the polar body, zona pellucida (ZP) birefringence, and meiotic spindle (MS) visualization after maturation in four conditions (commercial or homemade medium, with or without GDF9-β. ICSI was done for matured oocytes, and fertilization was confirmed by the visualization of two distinct pronuclei and two polar bodies. Results: No significant differences were found between the two culture media in terms of the time and rate of oocyte maturation or the rate of fertilization (p>0.05). Growth factor supplementation increased the 24-hour maturation rate for both GV and MI oocytes only in homemade medium. The maturation rate after 24 hours was higher for MI oocytes (p<0.05). Similar results were observed for MS visualization and ZP structure in both types of media (p>0.05). Conclusion: Higher rates of oocyte maturation and fertilization were observed after application of homemade medium supplemented with GDF9-β. Therefore, this combination may be recommended as an alternative for clinical IVM programs.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.4
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• pp.339-344
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• 2000

Objective: To evaluate silane-coated silica particles (Sil-select) as an alternative to polyvinylpyrrolidone-coated particles (Percoll) for gradient separation of spermatozoa, for use in assisted reproduction. Methods: 20 normal semen based on WHO criteria were included in this study. Recovery of motile and morphologically normal spermatozoa after using two-layer Percoll and Sil-select gradient respectively was recorded. Motility, HOST (hypoosmotic swelling test) and the detection of malondialdehyde for LPO (lipid peroxidation) after 24 h of incubation at $37^{\circ}C$ in a 5% $CO_2$ incubator were compared. Results: Percoll (78.5%) and Sil-select (79.1%) showed a significant increase in the motility compared to ejaculate (60.9%) but no difference between Percoll and Sil-select. Normal sperm morphology significantly increased after Percoll (57.6%) and Sil-select (53.7%) compared to ejaculate (35.8%) but no difference between Percoll and Sil-select. No differences in the recovery of motile spermatozoa and motility, HOST and the production of malondialdehyde after 24 h incubation were found when comparing the use of Percoll and Sil-select. Conclusion: Sil-select seems to be an attractive alternative to Percoll for sperm separation in assisted reproduction.

• Clinical and Experimental Reproductive Medicine
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• v.19 no.2
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• pp.143-152
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• 1992

The present experiments were focussed to modify a short slow-cooling protocol used for freezing of early stage embryo(Testart et al., 1986) and also to apply the modified method for the cryopreservation of hamster oocytes with Zona or without. The protocol was modified by changing the 4-step equilibration into 1-step and the 1-step thawing into 2-step. The oocytes were added in 1.5M PROH and 0.1M Sucrose, seeded at $-7^{\circ}C$, slow cooled($0.3^{\circ}C$/min) to $-30^{\circ}C$ before plunging to $-196^{\circ}C$. The oocytes were thawed at $23-25^{\circ}C$ air(20sec/150sec) and/or 33-35 water(10sec). The survival of the frozen-thawed oocytes was determined by morphologic criteria and their fertilizing ability was also estimated by Sperm Penetration Assay(SPA) system(Chang et al, 1990) using fertile men semen sample. One-step equilibration showed slightly higher survival rate(83.9% vs. 71.0%) and fertilization rate(83.9% vs. 71.0%) compared with four-step(p>0.05). And two-step thawing(air & water exposing) of oocytes frozen after 1-step equilibration showed significantly higher survival rate(96.3%) than one-step thawing at air(85.2%) or water(65.0%) only(p<0.05). Therefore, by the modified method(l-step equilibration & 2-step thawing), Zona-intact(ZI) and Zona-free(ZF) oocytes were frozen and thawed. ZI-oocytes showed significantly higher survival rate(95.4%, 308/323 vs. 67.6%, 240/355) than ZF-oocytes(P<0.01). But the survival of ZF-oocytes was as high as ZI-oocytes in fourteen of twenty-four replicates. ZI-oocytes was also significantly higher fertilization rate($92.4{\pm}8.9%$ vs. $63.7{\pm}18.5%$) and higher mean number of penetrated sperm($6.2{\pm}4.2$ vs. $3.9{\\pm}3.3$) than ZF-oocytes, but not higher than control(fresh oocytes;$99.3{\pm}2.4%$, $8.4{\pm}4.2$)(P<0.001). Conclusively, this modified method will contribute to freeze effectively the hamster oocytes for simplifing of the logical consideration of performing SPA and also to freeze the human and other animal oocytes.

• 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.