Effects of first and second division modes on euploidy acquisition in human embryo.

The aim of this study was to non-invasively investigate euploid embryos using methods other than pre-implantation genetic testing for aneuploidy. The study focused on direct cleavage (DC) observed during early embryo development. We also investigated the relationship between the mode of early embryo division and embryo ploidy. Embryos were divided into the normal cleavage (NC) and DC groups, and the DC group was further subdivided into the DC-First (DC-F) and DC-Second (DC-S) groups, depending on whether DC was observed at the first or second cleavage, respectively. The acquisition rates of euploid embryos and embryos appropriate for transfer were compared between the groups. Our results revealed that the timing of the first division did not differ between blastocyst grades or in embryos with varying degrees of ploidy. Further, the timing of the first cleavage did not affect the acquisition rate of embryos appropriate for transfer and euploid embryo formation rate did not significantly differ between the DC and NC groups. We also noted that for embryos appropriate for transfer, euploidy acquisition rate did not differ significantly between the DC and NC groups. Further, the euploidy acquisition rate of embryos did not differ between the DC-F and DC-S groups. However, the acquisition rate of embryos appropriate for transfer, including those with low mosaicism, was significantly higher in the DC-S group than in the DC-F group. These findings indicated that the number of good-quality blastocysts formed was significantly higher in the NC group than in the DC group and the acquisition rate of embryos appropriate for transfer, including those with low mosaicism, was significantly higher in the DC-S group than in the DC-F group.

Original delayed-start ovarian stimulation protocol with a gonadotropin-releasing hormone antagonist, medroxyprogesterone acetate, and high-dose gonadotropin for poor responders and patients with poor-quality embryos.

Introduction: The delayed-start gonadotropin-releasing hormone antagonist protocol seems effective for patients who are poor ovarian responders, but there are insufficient data on whether it is also effective for patients with poor-quality embryos and low rates of good blastocyst formation. Specifically, the effectiveness of delayed-start gonadotropin-releasing hormone antagonists with progesterone has not been adequately investigated. Therefore, we compared the efficacy of the original delayed-start gonadotropin-releasing hormone antagonist protocol using medroxyprogesterone acetate (MPA) and high-dose gonadotropin in patients with poor ovarian response. Methods: Overall, 156 patients with recurrent assisted reproductive technology failure who underwent the original protocol were included. They received cetrorelix acetate (3 mg) and MPA (10 mg) on cycle day 3, and high-dose gonadotropin was initiated on day 11. When the leading follicle reached 14 mm, ganirelix acetate (0.25 mg) was administered until the trigger day. The number of oocytes retrieved, metaphase II (MII) oocytes, two pronuclear (2PN) zygotes, and good blastocysts and live birth rates were compared between the previous (Cycle A) and original (Cycle B) cycles in three groups (Group A, all patients; Group B, poor responders; and Group C, patients with poor-quality embryos). Results: In Group A (n=156), the number of MII oocytes (3.6 ± 3.3 versus 4.5 ± 3.6), 2PN zygotes (2.8 ± 2.9 versus 3.8 ± 3.1), good blastocysts (0.5 ± 0.9 versus 1.2 ± 1.6), and live birth rates (0.6 versus 24.4) significantly increased in Cycle B. Similar results were obtained in Group B (n=83; 2PN zygotes [1.7 ± 1.7 versus 2.3 ± 1.8], good blastocysts [0.4 ± 0.7 versus 0.9 ± 1.3], live birth rates [0 versus 18.1]) and Group C (n=73; MII oocytes [5.1 ± 3.8 versus 6.6 ± 4.0], 2PN zygotes [4.0 ± 3.4 versus 5.4 ± 3.4], good blastocysts [0.7 ± 1.1 versus 1.6 ± 1.9], and live birth rates [1.4 versus 31.5]). Conclusion: This original protocol increased the number of MII oocytes retrieved, 2PN zygotes, good blastocysts, and live birth rates in both poor responders and in patients with poor-quality embryos.

Smooth endoplasmic reticulum cluster presence does not affect embryo ploidy.

PURPOSE: We examined the impacts of the smooth endoplasmic reticulum cluster (sERC) presence on embryonic development and blastocyst ploidy. METHODS: Patients who underwent oocyte retrieval from January 2019 to November 2021 were included in the study. We classified the oocytes into three groups: normal oocytes in the sERC ( -) cycle, normal oocytes in the sERC ( +) cycle, and sERC ( +) oocytes. Next, the levels of serum estradiol, progesterone, anti-Mullerian hormone, follicle-stimulating hormone, and human menopausal gonadotropin were compared between the groups. Moreover, fertilization, degeneration, and abnormal fertilization rates were compared between groups. To investigate developmental outcomes, the blastocyst and good-quality blastocyst rates after intracytoplasmic sperm injection were compared. The quality of the transferred blastocysts was evaluated at follow-up. Additionally, embryos were submitted for next-generation sequencing analysis to examine the effect of sERC presence on ploidy. RESULTS: The sERC ( +) group had significantly higher serum estradiol, serum progesterone, and serum anti-Mullerian hormone concentrations compared to those in the sERC ( -) group (P < 0.01). The abnormal fertilization rate was higher in the sERC ( +) cycle-sERC ( +) oocyte group (16.1%; 37/230) than in the sERC ( +) cycle-normal oocyte (6.2%; 63/971) and sERC ( -) cycle-normal oocyte groups (7.1%; 174/2467) (P < 0.01). After embryo transfer, nine women gave birth, and no confirmed congenital anomalies were observed. There was no significant difference in ploidy between the sERC ( +) and sERC ( -) groups. CONCLUSION: The occurrence rates of embryos with euploidy were similar between the sERC ( +) and sERC ( -) groups.

A novel trophectoderm biopsy technique for all blastocyst stages.

Purpose: This study was conducted to assess the effectiveness of a new trophectoderm (TE) biopsy method that does not require prior opening of the zona pellucida at the blastocyst stage. Methods: TE biopsy was conducted using a modified extrusion method for embryos during the cleavage stage. In this method, culture medium was injected into the perivitelline space to help extrude TE cells from the zona pellucida before TE biopsy. Results: Our extrusion method preserves the embryo culture environment until immediately before biopsy because it does not require opening of the zona pellucida prior to TE biopsy. Furthermore, this method does not require a waiting time for blastocyst hatching after laser irradiation, thereby minimizing damage to the embryos and maintaining the time schedule of culture operations. Conclusions: TE biopsy using this novel extrusion method may be useful in various applications, including the collection of TE cells for next-generation sequencing analysis.

The effects of differences in trophectoderm biopsy techniques and the number of cells collected for biopsy on next-generation sequencing results.

Purpose: To examine how differences in trophectoderm biopsy techniques affect the frequency of mosaic embryos and sequencing results. Methods: We examined differences in next-generation sequencing (NGS) analysis results among operators or according to biopsy technique. Additionally, we determined the cut-off for the number of collected cells to predict the occurrence of mosaicism. We collected cells according to the cut-off value and examined whether there was a difference in the NGS analysis results between the pulling and flicking methods. Results: There was no difference in the NGS analysis results among the operators. Regarding re-biopsy, changes in the mosaic were observed in all specimens. The cut-off value for the number of collected cells was five, and when more than five cells were collected, there was no difference in the NGS analysis results between the two methods. Conclusions: We demonstrated that if trophectoderm biopsy techniques and NGS are stable, the cell collection location has a greater effect on NGS results than the biopsy technique.

The effect of repeated cryopreservation and thawing using cryotip on the clinical outcomes of embryos.

PURPOSE: To compare the clinical outcomes of embryo transfers that were cryopreserved and thawed two or three times with those cryopreserved and thawed once by CryoTip. METHODS: Data for 388 single cryopreserved-thawed blastocyst transfer cycles, performed from April 2012 to March 2014, were assessed. The blastocysts were classified into three groups: blastocysts (A) cryopreserved once, (B) cryopreserved twice, and (C) cryopreserved three times. RESULTS: The pregnancy rate was 43.8% (134/306) in group A and 46.3% (38/82) in group B, while the miscarriage rate was 29.1% (39/134) in group A and 23.7% (9/38) in group B. The rate of improvement/maintenance of blastocyst grade was 84.0% (257/306) in group A and 80.5% (66/82) in group B. The pregnancy and miscarriage rates of the blastocysts that showed improvement/maintenance in the grade were 45.9% (118/257) and 29.7% (35/118) in group A and 48.5% (32/66) and 21.9% (7/32) in group B, respectively. The pregnancy rate was 33.3% (2/6), while the miscarriage rate was 0.0% (0/2) in group C. CONCLUSIONS: Pregnancy rates achieved with re-cryopreserved and rethawed blastocyst transfer were comparable to those achieved with single cryopreserved-thawed blastocyst transfer.

Synchrony of the first division as an index of the blastocyst formation rate during embryonic development.

Purpose: To devise an uninvasive selection system for human embryos with high developmental potential after a single oocyte retrieval cycle by comparing the in vitro and in vivo effectiveness of first division synchrony against subsequent embryonic developmental stages. Methods: The effects of using assisted reproductive technology on 948 embryos that were produced in 137 cycles were examined by dividing the embryos into "early cleavage" (first division within 25.90 hours) and "late cleavage" (first division at or after 25.90 hours) groups and comparing the blastocysts and good-quality blastocyst formation rates between the two groups. These two groups were each divided further into "high synchrony" (first division synchrony within 3.96 hours) and "low synchrony" (first division synchrony at or after 3.96 hours) groups. The blastocysts, good-quality blastocyst formation rates, and pregnancy rates were compared among these four groups. Results: Both the blastocysts and good-quality blastocyst formation rates were significantly higher in the early-cleavage groups than in the late-cleavage groups. The blastocyst formation rate of the latter was also significantly increased in the high-synchrony, compared with the low-synchrony, group. Conclusion: First division synchrony in a single oocyte retrieval cycle could be a useful assessment of the blastocyst formation rate that enables the selection of viable embryos at an early stage of culture.

Selection of human blastocysts with a high implantation potential based on timely compaction.

PURPOSE: Recently, we established a noninvasive system for selecting human blastocysts with a high pre-transfer implantation potential based on first and second division patterns. The present study was carried out to improve the selection system. METHODS: Embryos that completed first and second divisions within 25.90 and 37.88 h after culture, respectively, were selected using a time-lapse incubator. We examined the effects of compaction and blastocyst formation times on pregnancy rates after transferring these embryos at the blastocyst stage. RESULTS: The completion of compaction and blastocyst formation times (79.93 and 97.47 h after culture, respectively) of embryos resulting in pregnancies after transfer were significantly (P < 0.01) shorter than those (86.46 and 100.34 h after culture, respectively) of embryos that failed to induce pregnancies. Embryo selection based on completion of compaction time improved pregnancy rates (40.9 vs. 74.6%, P < 0.01). CONCLUSIONS: Of the embryos that formed two cells during the first division within 25.90 h after culture and four cells during the second division within 37.88 h after culture, those that completed compaction within 79.93 h after culture before reaching the blastocyst stage had a high implantation potential.

Developmental ability of embryos produced from oocytes with fragile oolemma by intracytoplasmic sperm injection.

PURPOSE: In intracytoplasmic sperm injection (ICSI) of oocytes with a fragile oolemma (fragile oocytes), breakage can occur at injection. In this study, we produced embryos from oocytes with a fragile and normal oolemma (normal oocytes) by ICSI and compared their ability to be fertilized and develop in vitro. We also investigated whether fragile oocyte-derived embryos could implant after blastocyst transfer to determine whether fragile oocytes should be used for assisted reproductive technology treatment. METHODS: Oocytes were divided into three groups-normal oocytes from cycles containing no fragile oocytes (group A), normal oocytes from cycles containing at least one fragile oocyte (group B), and fragile oocytes (group C), and their fertilization abilities after ICSI and the developmental abilities of resultant embryos were compared. RESULTS: The fertilization rate in group C (65.3 %) was significantly (P < 0.01) lower than those in groups A (84.6 %) and B (86.9 %), and the degeneration rate in group C (24.2 %) was significantly (P < 0.01) higher than those in groups A (0.71 %) and B (0.28 %). However, there were no significant differences in the blastocyst formation rates (59.7-67.5 %) of embryos among the different groups. In addition, the pregnancy rate after transfer of blastocysts in group C (50.0 %) was not significantly different from those in groups A (35.6 %) and B (45.8 %). CONCLUSIONS: The fertilization ability after ICSI of fragile oocytes is lower than that of normal oocytes but the resultant embryos have the same developmental ability as those of normal oocyte-derived embryos.

Effects of early cleavage patterns of human embryos on subsequent in vitro development and implantation.

OBJECTIVE: To establish a noninvasive selection system of human embryos with a high implantation potential before transferring. DESIGN: Cohort study. SETTING: Independent IVF clinics. PATIENT(S): One hundred sixty-four patients, with 791 embryos fertilized, checked, and recorded. INTERVENTION(S): Embryos were divided into nine groups according to the number of cells they contained and the extent of their fragmentation during the first and second divisions. We examined the effects of the cell division pattern and division time of embryos on subsequent development in vitro and in vivo using a time-lapse incubator. MAIN OUTCOME MEASURE(S): Rates of blastocyst formation, high-quality blastocyst formation, and pregnancy. RESULT(S): The rates of embryos developed into blastocysts and blastocysts in which both the inner cell mass and trophectoderm grades were scored B or higher (good-quality blastocysts) were high in groups that formed two cells during the first division and four cells during the second division, regardless of the presence or absence of fragmentation. In these groups the first and second division times (25.90 and 37.88 hours after culture, respectively) of embryos developed into good-quality blastocysts were significantly shorter than those of other embryos. Although embryo selection based on the first and second division times did not affect the pregnancy rates after transfer at day 2 or 3 of culture, it improved the pregnancy rates after blastocyst transfer at day 5. CONCLUSION(S): Transfer of blastocysts derived from embryos that completed first and second divisions within 25.90 and 37.88 hours after culture, respectively, brings about high pregnancy rates.

Production of Cloned Miniature Pigs Expressing High Levels of Human Apolipoprotein(a) in Plasma.

High lipoprotein(a) [Lp(a)] levels are a major risk factor for the development of atherosclerosis. However, because apolipoprotein(a) [apo(a)], the unique component of Lp(a), is found only in primates and humans, the study of human Lp(a) has been hampered due to the lack of appropriate animal models. Using somatic cell nuclear transfer (SCNT) techniques, we produced transgenic miniature pigs expressing human apo(a) in the plasma. First, we placed the hemagglutinin (HA)-tagged cDNA of human apo(a) under the control of the ß-actin promoter and cytomegalovirus enhancer, and then introduced this construct into kidney epithelial cells. Immunostaining of cells with anti-HA antibody allowed identification of cells stably expressing apo(a); one of the positive clones was used to provide donor cells for SCNT, yielding blastocysts that expressed apo(a). Immunohistochemical analysis of tissue sections and RT-PCR analysis of total RNA from organs of cloned piglet revealed that apo(a) is expressed in various tissues/organs including heart, liver, kidney, and intestine. More importantly, a transgenic line exhibited a high level (>400 mg/dL) of Lp(a) in plasma, and the transgenic apo(a) gene was transmitted to the offspring. Thus, we generated a human apo(a)-transgenic miniature pig that can be used as a model system to study advanced atherosclerosis related to human disease. The anatomical and physiological similarities between the swine and human cardiovascular systems will make this pig model a valuable source of information on the role of apo(a) in the formation of atherosclerosis, as well as the mechanisms underlying vascular health and disease.

Osmolarity- and stage-dependent effects of glycine on parthenogenetic development of pig oocytes.

The osmolarities of media that are most effective for in vitro culture of mammalian oocytes and embryos are lower than that of oviductal fluid. Oocytes and embryos can survive the high physiological osmolarity in vivo perhaps owing to the presence of amino acids such as glycine, which serve as organic osmolytes in the female reproductive tract. In the present study, the effects of glycine on the parthenogenetic development of pig oocytes were examined in hypotonic or isotonic media. The results showed that culturing oocytes in isotonic media improved the cleavage rates (P<0.01) at 2 days in culture but inhibited any further development beyond cleavage when compared with the hypotonic media. However, addition of 4 mM glycine to the isotonic media resulted in improved blastocyst formation rates compared with that observed in the hypotonic media (P<0.01), and there was no inhibition of development beyond the cleavage stages in oocytes. The beneficial effects of glycine were observed only when oocytes were cultured in isotonic media and glycine was added at day 2 or 3 in culture. The results from the present study indicate that an isotonic medium with glycine is useful for in vitro culture of pig oocytes and that glycine may protect pig oocytes against the detrimental effects of increased osmolarity.

Effect of postactivation treatment with latrunculin A on in vitro and in vivo development of cloned embryos derived from kidney fibroblasts of an aged Clawn miniature boar.

The objective of this study was to examine the effect of postactivation treatment with latrunculin A (LatA), an actin polymerization inhibitor, on in vitro and in vivo development of somatic cell nuclear transfer (SCNT) embryos derived from kidney fibroblasts of an aged Clawn miniature boar (12 years old). After electric activation, SCNT embryos were treated with 0, 0.5 or 1 µM LatA and cultured in vitro. The rate of blastocyst formation was significantly higher (P<0.05) in SCNT embryos treated with 0.5 µM LatA (38%) than those in control (14%). When cloned embryos treated with 0.5 µM LatA were transferred into the oviducts of two recipient miniature gilts to assess their development in vivo, both recipients became pregnant; one maintained pregnancy to term, and a live piglet (weighing 220 g) was delivered by Caesarean section. The results of this study indicated that the postactivation treatment with LatA was effective in improving in vitro developmental capacity of SCNT miniature pig embryos derived from kidney fibroblasts of an aged animal and that miniature pig cloned embryos treated with LatA had the ability to develop to term.

Whole-genome amplification-based GenomiPhi for multiple genomic analysis of individual early porcine embryos.

The multiple displacement amplification (MDA) method, which relies on isothermal DNA amplification using the DNA polymerase of the bacteriophage phi29, was recently developed for high-performance, whole-genome amplification (WGA). The objective of the present study was to determine whether a target sequence could be successfully amplified by conventional PCR when the genomic DNA of a single Day-7 porcine blastocyst (derived from SCNT of a gene-engineered fibroblast) was amplified by the MDA method and used as a template. The yield of double-stranded DNA was 103.5 ± 16.0 ng/embryo (range, 75-125), as assessed by a PocoGreen assay. However, non-specific products (20 ± 5 ng/tube) were also generated, even in the negative control. Thus, ∼81% of the 103.5 ng (84 ng) of amplified DNA was estimated to be porcine sequences (2.2 × 10(3)-fold enrichment). In addition, PCR confirmed the presence of transgenes, as well as endogenous α-1,3-galactosyltransferase and homeobox Nanog genes in all embryos. Sequencing of the amplified products verified the fidelity of this system. In conclusion, the MDA-mediated WGA, which was simple, inexpensive, and did not require a thermal cycler, could be a powerful tool for multiple genomic analyses of individual early porcine embryos.

Latrunculin A dramatically improves the developmental capacity of nuclear transfer embryos derived from gene-modified clawn miniature pig cells.

This study was carried out to examine the effect of postactivation treatment with latrunculin A (LatA), an actin polymerisation inhibitor, on in vitro and in vivo development of somatic cell nuclear transfer (SCNT) embryos derived from gene-modified Clawn miniature pig cells. After the fusion and activation, SCNT embryos were treated with or without a cytoskeletal inhibitor [LatA or 10.4 microM cytochalasin B (CB) for 2 h]. The cleavage rate was significantly higher (p < 0.05) in embryos exposed to 0.5 microM LatA than those in embryos exposed to CB and without a cytoskeletal inhibitor. Moreover, the blastocyst formation rate was significantly higher (p < 0.05) in embryos exposed to 0.5 or 1 microM LatA than those in embryos exposed to CB and without a cytoskeletal inhibitor. In addition, five fetuses were obtained from recipient uteri after transfer of embryos treated with 0.5 muM LatA. The results of this study show for the first time that postactivation treatment with LatA is effective to improve in vitro developmental capacity of gene-modified cloned miniature pig embryos and embryos treated with LatA have the ability to develop into fetuses.

Stage-specific effects of osmolarity of a culture medium on development of pig oocytes and miniature pig somatic cell nuclear transfer embryos activated by ultrasound treatment.

Whether high osmolarity of a culture medium at the early culture stage affects the development of pig oocytes and miniature pig somatic cell nuclear transfer (SCNT) embryos activated by ultrasound was examined. When oocytes were cultured in modified porcine zygote medium-3 (mPZM-3) with increased NaCl to 138 mmol/L (mPZM-3+NaCl; 326 mOsm) or 50 mmol/L sucrose (mPZM-3+sucrose; 318 mOsm) for the first 2 days and then cultured in normal mPZM-3 (273 mOsm) for 5 days, the cleavage and blastocyst formation rates were significantly (P < 0.05) higher than those of oocytes cultured in mPZM-3 for 7 days. The cleavage and blastocyst formation rates of SCNT embryos cultured in mPZM-3+NaCl for the first 2 days and then cultured in mPZM-3 for 5 days were also significantly (P < 0.05) higher than those of embryos cultured in mPZM-3 for 7 days. These results showed that the high osmolarity of a culture medium induced by increasing NaCl concentration during the first 2 days improves the development of pig oocytes and miniature pig SCNT embryos activated by ultrasound.

Valproic acid enhances in vitro development and Oct-3/4 expression of miniature pig somatic cell nuclear transfer embryos.

The present study was carried out to examine the effects of valproic acid (VPA), a histone deacetylase inhibitor, on in vitro development of miniature pig somatic cell nuclear transfer (SCNT) embryos and on expression of a mouse Oct-3/4 promoter-driven enhanced green fluorescent protein (EGFP) gene (EGFP expression only detected in Oct-3/4-expressing cells) introduced into donor cells for SCNT during their development. The addition of 4 mM VPA to embryo culture medium for 48 h after activation significantly (p < 0.01) increased the blastocyst formation rate of SCNT embryos compared with the control, whereas VPA did not affect their cleavage rate. The rate of SCNT embryos expressing EGFP at 5 days of culture was not affected by the presence or absence of VPA treatment. At 7 days of culture, however, the addition of 4 mM VPA to embryo culture medium for 48 h after activation significantly (p < 0.05) increased the rate of SCNT embryos expressing EGFP compared with the control. The results indicate that VPA enhances the ability of miniature pig SCNT embryos to develop into blastocysts and maintains the ability of them to express Oct-3/4 gene.

Beneficial effects of reversine on in vitro development of miniature pig somatic cell nuclear transfer embryos.

Reversine, a 2-(4-morpholinoanilino)-6-cyclohexylaminopurine analog, can induce dedifferentiation of myogenic lineage-committed cells into multipotent mesenchymal progenitor cells, from which osteoblasts and adipocytes redifferentiate under lineage-specific inducing conditions. Although the molecular mechanism of how reversine causes dedifferentiation of a differentiated cell has not been fully elucidated, we speculated that it would be involved in reprogramming. In the present study, we examined whether reversine can enhance the development of somatic cell nuclear transfer (SCNT) embryos by improving the reprogramming state of the somatic cell nuclei. As donor cells, we used miniature pig fetal fibroblasts transfected with a plasmid construct containing a mouse Oct-3/4 promoter and enhanced green fluorescent protein (EGFP) cDNA. When the nuclei of these transfected cells are reprogrammed to an undifferentiated state in the SCNT embryos, EGFP expression is expected to commence under the control of the Oct-3/4 promoter. After SCNT, the resulting embryos were treated with 5 muM reversine for different durations (0, 6, 12, 18 and 24 h) or at different concentrations (0, 1, 5 and 10 muM) of reversine for 12 h and then cultured in vitro. When embryos were treated with 5 muM reversine for 12 h, the blastocyst formation rate was significantly (P<0.01) higher than that of embryos without reversine treatment. However, the strength and pattern of EGFP expression in the embryos were not affected by the same treatment. A normal-looking fetus was obtained 21 days after transfer of embryos treated with 5 muM reversine for 12 h into recipients. The present findings indicate that treatment with reversine is beneficial for enhancement of the in vitro development of miniature pig SCNT embryos, although the underlying mechanism is still unclear.

Enhancement of cytoplasmic maturation of in vitro-matured pig oocytes by mechanical vibration.

The effects of mechanical vibration during in vitro maturation and/or in vitro culture after artificial activation of pig oocytes on maturation and development were examined. In addition, the optimal conditions were applied to in vitro production of blastocysts derived from miniature pig somatic cell nuclear transfer (SCNT) embryos. Mechanical vibration during in vitro maturation did not affect the rates (60.5 +/- 1.9-69.5 +/- 2.2%) of oocytes reaching the metaphase-II stage. However, the blastocyst formation rates after activation of oocytes matured with mechanical vibration for 5 sec at intervals of 30-60 min or for 10 sec at intervals of 60 min were significantly (P<0.05) higher than those of oocytes matured without mechanical vibration (25.7 +/- 2.0-28.1 +/- 2.7% vs. 12.3 +/- 1.4% and 25.8 +/- 1.8% vs. 15.7 +/- 1.9%, respectively). In contrast, mechanical vibration during in vitro culture after activation did not affect the blastocyst formation (11.6 +/- 5.2-16.5 +/- 3.0%) of oocytes. Mechanical vibration for 5 sec at intervals of 60 min during in vitro maturation of oocytes did not affect fusion (66.8 +/- 3.5-72.1 +/- 3.1%) with miniature pig somatic cells after enucleation. However, the blastocyst formation rate of SCNT embryos was improved (P<0.05) by mechanically vibrating recipient oocytes for 5 sec at intervals of 60 min during in vitro maturation, regardless of the presence or absence of the same treatment during in vitro culture (17.6 +/- 2.5% vs. 9.4 +/- 0.9% and 13.0 +/- 0.3% vs. 7.4 +/- 0.9%, respectively). The results indicated that mechanical vibration enhances the cytoplasmic maturation of in vitro-matured pig oocytes, resulting in improvement of their parthenogenetic development. In addition, it was shown that in vitro maturation of oocytes with mechanical vibration can be applied to efficient production of blastocysts derived from miniature pig SCNT embryos.

Development of a noninvasive monitoring system for evaluation of Oct-3/4 promoter status in miniature pig somatic cell nuclear transfer embryos.

The present study was carried out to develop a noninvasive monitoring system for evaluation of Oct-3/4 promoter gene status in miniature pig somatic cell nuclear transfer (SCNT) embryos during in vitro development. Miniature pig fetal fibroblasts (MPFFs) were transfected with a gene construct consisting of two expression units, a mouse Oct-3/4 promoter-driven enhanced green fluorescent protein (EGFP) gene (EGFP expression only detected in Oct-3/4-expressing cells) and a neomycin resistance gene. After neomycin selection, MPFFs that did not express EGFP were fused with enucleated pig oocytes, cultured in vitro and assessed for EGFP expression. EGFP expression was detectable in all morulae (at 4-6 days of culture) and 50.0% of blastocysts (at 5-6 days of culture), whereas none of the 1-cell to 16-cell embryos at 1-5 days of culture expressed EGFP. On the other hand, EGFP expression was not maintained in all blastocysts at 7 days of culture. The reactivity with anti-Oct-3/4 antibodies also peaked from the morula to blastocyst stages at 5 days of culture. The results showed that reactivation of the Oct-3/4 promoter gene of donor nuclei occurs in the morula to blastocyst stages at 4-6 days after SCNT and that this noninvasive monitoring system using Oct-3/4 promoter-driven EGFP gene would be useful for evaluation of the reprogramming status of donor nuclei.