• Journal of Life Science
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• v.23 no.9
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• pp.1079-1087
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• 2013

Speed germination success and robust vegetative growth of citrus rootstock through improved sowing methods and fertilizer inputs offer the usage of root system for the citrus. The current study evaluated the influence of seed coat removal and different fertilizer concentrations on plant germination and plant growth of spontaneous rootstock siblings. Decoated and coated seeds of diploid and tetraploid plants were sown in tubes. Commercial fertilizer concentrations of 0, 2, 4, 6, 8 and $10g{\cdot}l^{-1}$ were added. The experimental layout followed a randomized block $2{\times}6$ factorial design (seed coat removal ${\times}$ fertilizer concentration) for each rootstock. Fertilizer concentrations were 0, 10, 20 and $30g{\cdot}l^{-1}$ of the fertilizer for the resistance of the strength on the salt level. The germination rate of seeds without testa sown in vitro was improved (67-80%) compared to that of nontreated seeds. The eventual tree height of the seeds without testa in the diploid group was increased due to higher fertilization compared to that in the nontreated group. The removal of seed testa promoted the seed germination of both diploid and tetraploid trifoliate orange and resulted in greater height. Their vegetative development was also increased due to the increased fertilization of the rootstock. The Fv/Fm value for the diploid plants was 0.4 and 0.8 for the tetraploid ones under salt stress after 11 days of treatment. The removal of seed testa may improve the seed germination of trifoliate orange. Tetraploid trifoliate orange appears to possess resistance to salt stress compared to the diploid variety.

• Clinical and Experimental Reproductive Medicine
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• v.28 no.4
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• pp.307-315
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• 2001

Objective: To evaluate the clinical outcomes and influencing factors of in vitro fertilization and embryo transfer (IVF-ET) in patients with failed pregnancy after microsurgical reversal of tubal sterilization. Materials and Methods : From January, 1997 to December, 2000, IVF-ET was performed in two groups; the study TR (tubal reanastomosis) group consisted of 147 cycles in 66 patients with failed microsurgical reversal of tubal sterilization, and the control group of 115 cycles in 67 patients with bilateral tubal occlusion (BTO). The two groups were evaluated and compared for clinical characteristics, clinical pregnancy rates, and factors influencing the outcomes of IVF-ET. Results: Compared with the control BTO group, age and the previous parity were significantly higher ($36.3{\pm}2.7$ vs. $33.6{\pm}2.0$ years, p<0.05; $1.6{\pm}0.7$ vs. $0.2{\pm}0.4$, p<0.05), and the clinical pregnancy rate per cycle was significantly lower (23.8% (35/147) vs. 29.3% (34/115), p<0.05) in the TR group. Difference in the clinical pregnancy rates was age-related, since there was no significant difference between the two groups, except for the previous parity ($1.6{\pm}0.7$ vs. $0.1{\pm}0.3$, p<0.05), when the patients aged 37 years or older were excluded. No difference was found in terms of the following: the proportion of controlled ovarian hyperstimulation (COH) cycles with GnRH agonist ultrashort protocol, the duration of COH, the dosage of gonadotropins used, and the numbers of oocytes retrieved and of embryos transferred, irrespective of age correction. Conclusions: The outcomes of IVF-ET following the failed microsurgical reversal of tubal sterilization depend upon patient age. The previous fertility of patients does not seem to be a factor of better IVF-ET prognosis.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.2
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• pp.207-216
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• 1998

Certain types of somatic cells, as well as follicular cumulus cells associating with mammalian oocytes, are known to produce beneficial effects on in vitro fertilization and pre implantation development of mammalian eggs when they are present in oocyte culture medium. To investigate the nature of the effects of somatic cells, the resistance of mouse oocytes against chymotrypsin treatment was examined after culture within various cell conditioned media. When mouse oocytes matured for 17-18 hr in the presence of cumulus cells were treated with 1 % chymotrypsin, half of them remained still alive even after 240 min $(t_{50}>240.0)$. In contrast half of mouse oocytes cultured without cumulus cells underwent degeneration within 65.0 min $(t_{50}=65.0{\pm}13.2min)$ of the same treatment. To see if the effects were duc to the secretory products of cumulus cells, mouse cumulus cells were cultured for 20 hr in medium containing 0.4% BSA and the supernatant of culture medium (conditioned medium) was taken. After maturation in the cumulus cell conditioned medium, mouse oocytes exhibited $t_{50}=190.0{\pm}10.8$ min upon chymotrypsin treatment whereas half of oocytes cultured without conditioned medium degenerated within 25.5 min. Human granulosa cell conditioned medium gave similar effects such that oocytes matured in conditioned medium exhibited $t_{50}=183.3{\pm}19.1$ min while $t_50$ of control group oocytes was $60.0{\pm}6.8$ min, Oocytes matured in vero cell conditioned medium exhibited $t_{50}=196.7{\pm}8.8$ min. On the other hand, amniotic cell conditioned medium resulted in the chymotrypsin resistance of $t_{50}=80.0{\pm}8.4$ min which was not statistically different from the control value of $t_{50}=48.0{\pm}13.2$ min. Based upon these results, it is suggested that certain somatic cell types including cumulus cells might change the biochemical properties of mouse oocyte membrane during meiotic maturation as revealed by the enhanced resistance against chymotrypsin treatment. Such effects of somatic cells appear to be mediated via the secretory products rather than direct communication between somatic cells and oocytes.

• Clinical and Experimental Reproductive Medicine
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• v.45 no.3
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• pp.129-134
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• 2018

Objective: In frozen and thawed embryos, the zona pellucida (ZP) can be damaged due to hardening. Laser-assisted hatching (LAH) of embryos can increase the pregnancy rate. This study compared thinning and drilling of the ZP before frozen embryo transfer (FET). Methods: Patients were randomly allocated into two groups for LAH using thinning or drilling on day 2 after thawing. Twenty-five percent of the ZP circumference and 50% of the ZP thickness was removed in the thinning group, and a hole $40{\mu}m$ in diameter was made in the drilling group. Results: A total of 171 in vitro fertilization/intracytoplasmic sperm injection FET cycles, including 85 cycles with drilling LAH and 86 cycles with thinning LAH, were carried out. The thinning group had a similar ${\beta}$-human chorionic gonadotropin-positive rate (38.4% vs. 29.4%), implantation rate (16.5% vs. 14.4%), clinical pregnancy rate (36.0% vs. 25.9%), miscarriage rate (5.8% vs. 2.4%), ongoing pregnancy rate (30.2% vs. 23.5%), and multiple pregnancy rate (7.0% vs. 10.6%) to the drilling LAH group. There were no significant differences in pregnancy outcomes between subgroups defined based on age (older or younger than 35 years) or ZP thickness (greater or less than $17{\mu}m$) according to the LAH method. Conclusion: The present study demonstrated that partial ZP thinning or drilling resulted in similar outcomes in implantation and pregnancy rates using thawed embryos, irrespective of women's age or ZP thickness.

• Reproductive and Developmental Biology
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• v.30 no.4
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• pp.271-277
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• 2006

Transforming growth $factor-{\beta}\;(TGF-{\beta})$ has been shown to have a positive effect on in vitro fertilization (IVF) and has been reported to stimulate meiosis at follicular level in variety of species. The study was designed to determine the expression patterns of $TGF-{\beta}1,\;TGF-{\beta}$ receptors type I, II and Smads gene in bovine oocytes and embryos. $TGF-{\beta}1$ and their receptors were observed in the unfertilized oocytes. $TGF-{\beta}1$ and type II receptor were not expressed at the blastocyst stage, however, only type I receptor was exclusively observed at the same stage. The blastocyst stage, in particular, showed high levels of mRNA expression patterns containing a $TGF-{\beta}1$ type I receptor. The mRNA expression pattern of Smad 2 at all stages of embryonic development was similar in all respect with $TGF-{\beta}1$ type I receptor. On the contrary, Smad 3 and 4 were expressed with high and low level mRNA at the blastocyst stage. In conclusion. it is suggested that $TGF-{\beta}1$ signaling may be regarded as an important entity during the preimplantation embryo development.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.2
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• pp.123-128
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• 1998

Implantation rates remain low following human in vitro fertilization (IVF). Suboptimal culture conditions may limit the ability of embryos to hatch as blastocysts, and artificial opening of the zona pellucida has been proposed as a means to promote subsequent hatching (assisted hatching). In this study, assisted hatching (AH) by zona drilling using acidic Tyrode's solution was performed in 320 patients, due to their age of more than 38 years (group A), the thick zona pellucida (group Z; $ZP\geq0.18{\mu}m$), and failures in implantation more than 3 times in previous IVF-ET trial (group P). This study was designed firstly, to study the effects of AH on the outcomes of IVF-ET according to the indications and secondly, to verify the appropriate application of AH. The results were as follows; 1. There was no difference in pregnancy rate between AH group (26.6%) and non-AH group (26.5%). 2. Assisted hatching (AH) showed significantly higher pregnancy rate of the patients with thick zona pellucid a than those of the patients with age factor and with the history of repeated implantation failure. But in the patients with age factor only, AH resulted in higher pregnancy rate. 3. Interestingly, the patients with complex factors including zona factor (Z: 33.9%; ZA: 30.4%; ZP: 31.6%; ZAP: 21.4%) showed higher pregnancy rates than other complex factors excluding zona factor (A: 24.4%, P: 0%; AP: 10.8%). From these results, AH is more helpful to the patients with thick zona pellucida rather than patients with older age and/or previous repeated implantation failure.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.2
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• pp.153-165
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• 1997

In male reproducible health, fertility and IVF (in-vitro fertilization), semen analysis has been most important. Semen analysis can be divided into concentration, motional and morphological analysis of sperm. The existing method which was developed earlier to analyze semen concentrated on the sperm motility analysis. To provide more useful and precise solutions for clinical problems such as infertility, semen analysis must include sperm morphological analysis. But the traditional tools for semen analysis are subjective, imprecise, inaccurate, difficult to standardize, and difficult to reproduce. Therefore, with the help of development of microcomputers and image processing techniques, we developed a new sperm morphology analyzer to overcome these problems. In this study the agreement on percent normal morphology was studied between different observers and a computerized sperm morphology analyzer on a slide-by-slide basis using strict criteria. Slides from 30 different patients from the SNUH andrology laboratory were selected randomly. Microscopic fields and sperm cells were chosen randomly and percent normal morphology was recorded. The ability of sperm morphology analyzer to repeat the same reading for normal and abnormal cells was studied. The results showed that there was no significant bias between two experienced observers. The limits of agreement were 4.1%${\sim}$-3.8%. The Pearson correlation coefficient between readers was 0.79. Between the manual and sperm morphology analyzer, the same findings were reported. In this experiments the slides were stained by two different methods, PAP and Diff-Quik staining methods. The limits of agreement were 7.2%${\sim}$-5.7% and 6.0%${\sim}$-6.3%, respectively. The Pearson correlation coefficients ware 0.76 and 0.91, respectively. The limits of agreement was tighter below 20% normal forms. In the experiments of repeatability, 52 cells stained by PAP and Diff-Quik staining methods were analyzed three times in succession. Estimating pairwise agreement, the kappa statistic for the pairs were 0.76, 0.81, 0.86, and 0.75, 0.88, 0.88 respectively. In this study it was shown that there was good agreement between manual and computerized assessment of normal and abnormal cells. The repeatability and agreement per slide of computerized sperm morphology analyzer was excellent. The computer's ability to classify normal morphology per slide is promising. Based on results obtained, this system can be of clinical value both in andrology laboratories and IVF units.

• Journal of Animal Reproduction and Biotechnology
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• v.37 no.4
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• pp.292-297
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• 2022

Direct injection of CRISPR/Cas9 into zygotes enables the production of genetically modified nonhuman primates (NHPs) essential for modeling specific human diseases, such as Usher syndrome, and for developing novel therapeutic strategies. Usher syndrome is a rare genetic disease that causes loss of hearing, retinal degeneration, and problems with balance, and is attributed to a mutation in MYO7A, a gene that encodes an uncommon myosin motor protein expressed in the inner ear and retinal photoreceptors. To produce an Usher syndrome type 1B (USH1B) rhesus macaque model, we disrupted the MYO7A gene in developing zygotes. Identification of appropriately edited MYO7A embryos for knockout embryo transfer requires sequence analysis of material recovered from a trophectoderm (TE) cell biopsy. However, the TE biopsy procedure is labor intensive and could adversely impact embryo development. Recent studies have reported using cell-free DNA (cfDNA) from embryo culture media to detect aneuploid embryos in human in vitro fertilization (IVF) clinics. The cfDNA is released from the embryo during cell division or cell death, suggesting that cfDNA may be a viable resource for sequence analysis. Moreover, cfDNA collection is not invasive to the embryo and does not require special tools or expertise. We hypothesized that selection of appropriate edited embryos could be performed by analyzing cfDNA for MYO7A editing in embryo culture medium, and that this method would be advantageous for the subsequent generation of genetically modified NHPs. The purpose of this experiment is to determine whether cfDNA can be used to identify the target gene mutation of CRISPR/Cas9 injected embryos. In this study, we were able to obtain and utilize cfDNA to confirm the mutagenesis of MYO7A, but the method will require further optimization to obtain better accuracy before it can replace the TE biopsy approach.

• Reproductive and Developmental Biology
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• v.29 no.3
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• pp.201-205
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• 2005

The objective of this study was to investigative the effects of amino acids supplementation on maturation, fertilization and embryo development of pig oocytes. Essential amino acids (EA), non-essential amino acids (NA) or both amino acids (EA + NA) were supple-mented to North Carolina State University (NCSU) 23 medium containing porcine follicular fluid (pFF). When the amino acids were supplemented to the maturation medium, the maturation rates were higher (p<0.05) in the NA group than control ($83.3{\pm}0.04\%\;versus\;70.0{\pm}0.05\%$, but the subsequent cleavage rates and development to morula and blstocyst stage between aminoacid supplement groups and control were not different. The developmental rates to morula and blastocysts stage were not significantly different regardless of amino acid supplementation to culture medium. In addition, supplementation of amino acids did not significantly affect the rate of fertilization and polyspermy. When the amino acids were supplement to culture medium, the number of trophectodermal (TE) cells was significantly (p<0.05) higher in amino acid supplement group than that of control ($18.6{\pm}0.5\;versus\;16.1{\pm}0.6$), whereas the numbers of inner cell mass (ICM) cells were not different among the treaonent groups and control ($29.0{\pm}0.9\~31.5{\pm}1.2$). Total cell number was also significantly (p<0.05) higher in EANA group ($50.0{\pm}1.0$) than that of control group ($44.2{\pm}1.1$). These results indicate that the amino acid supplementation to maturation and culture medium may not significantly stimulate early embryo development, but may improve the TE cell number of blastocyst stage in the pig.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.3
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• pp.301-309
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• 1997

The objective of this study was to determine the microtubule assembly and chromatin configuration during the first cell cycle in bovine oocytes following injection of spermatozoon, sperm head and tail. The microtubule and chromatin configuration was imaged with fluorescent labeled monoclonal ${\alpha}$-tubulin antibody and propidium iodide under laser scanning confocal microscope. Microtubule and chromatin dynamics in bovine oocytes following intracytoplasmic sperm injection (ICSI) were not different from those observed during in vitro fertilization (IVF). Following ICSI, the microtubular aster was observed around sperm midpiece. During pronuclear formation, the sperm aster was enlarged and seen around male and female pronuclei. At mitotic metaphase, the microtubular spindle assemble astral poles and chromosomes were aligned on the spindle equator. At mitosis, asters were concentrated to each spindle pole and they filled the cytoplasm. After injection of the isolated sperm head, the microtubular aster was not seen around sperm head in any cases (0/18). Instead, microtubules were organized from the cytoplasm, which filled the whole cytoplasm during pronuclear apposition. These microtubules seem to move male and female pronuclei. These results suggest that isolated sperm head can develop into normal pronucleus in mature bovine oocytes, and competent to participate syngamy with the ootid chromatin. The functional microtubules following isolated sperm head injection in bovine oocytes appeared to be organized solely from maternal stores.