• Title, Summary, Keyword: Blastocyst

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A retrospective study of single frozen-thawed blastocyst transfer

  • Hur, Yong Soo;Ryu, Eun Kyung;Song, Seung Hyun;Yoon, San Hyun;Lim, Kyung Sil;Lee, Won Don;Lim, Jin Ho
    • Clinical and Experimental Reproductive Medicine
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    • v.43 no.2
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    • pp.106-111
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    • 2016
  • Objective: To study the clinical outcomes of single frozen-thawed blastocyst transfer cycles according to the hatching status of frozen-thawed blastocysts. Methods: Frozen-thawed blastocysts were divided into three groups according to their hatching status as follows: less-than-expanded blastocyst (${\leq}EdB$), hatching blastocyst (HgB), and hatched blastocyst (HdB). The female age and infertility factors of each group were evaluated. The quality of the single frozen-thawed blastocyst was also graded as grade A, tightly packed inner cell mass (ICM) and many cells organized in the trophectoderm epithelium (TE); grade B, several and loose ICM and TE; and grade C, very few ICM and a few cells in the TE. The clinical pregnancy and implantation rate were compared between each group. The data were analyzed by either t-test or chi-square analysis. Results: There were no statistically significant differences in average female ages, infertility factors, or the distribution of blastocyst grades A, B, and C in each group. There was no significant difference in the clinical pregnancy and implantation rate of each group according to their blastocyst grade. However, there was a significant difference in the clinical pregnancy and implantation rate between each group. In the HdB group, the clinical pregnancy and implantation rate were similar regardless of the blastocyst quality. Conclusion: There was an effect on the clinical outcomes depending on whether the blastocyst hatched during single frozen-thawed blastocyst transfer. When performing single frozen-thawed blastocyst transfer, the hatching status of the frozen-thawed blastocyst may be a more important parameter for clinical outcomes than the quality of the frozen-thawed blastocyst.

Associations of post-warming embryo or blastocyst development with clinical pregnancy in vitrified embryo or blastocyst transfer cycles

  • Hong, Yeon Hee;Lee, Jang Mi;Kim, Seul Ki;Youm, Hye Won;Jee, Byung Chul
    • Clinical and Experimental Reproductive Medicine
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    • v.47 no.2
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    • pp.140-146
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    • 2020
  • Objective: To investigate whether the degree of post-warming embryo or blastocyst development is associated with clinical pregnancy in vitrified embryo or blastocyst transfer cycles. Methods: Ninety-six vitrified cleavage-stage embryos and 58 vitrified blastocyst transfer cycles were selected. All transfer cycles were performed from February 2011 to March 2019, and all vitrified embryos or blastocysts were warmed from 4 PM to 6 PM and then transferred the next morning from 9 AM to 10 AM. The scores of the cleavage-stage embryos and blastocysts were assessed at warming and at transfer using the modified Steer method and the Gardner method, respectively. The mean embryo or blastocyst score, score of the single top-quality embryo or blastocyst, and the difference in the score between warming and transfer were compared between nonpregnant and pregnant women. Results: In the cleavage-stage embryo transfer cycles, both the top-quality embryo score at transfer and the difference in the score between warming and transfer were significantly associated with clinical pregnancy. A top-quality embryo score at transfer of ≥ 60.0 (area under the curve [AUC], 0.673; 95% confidence interval [CI], 0.531-0.815) and a difference in the score between warming and transfer of ≥ 23.0 (AUC, 0.675; 95% CI, 0.514-0.835) were significant predictors of clinical pregnancy. In blastocyst transfer cycles, the top-quality blastocyst score at transfer was the only significant factor associated with clinical pregnancy. A top-quality blastocyst score at transfer of ≥ 38.3 was a significant predictor of clinical pregnancy (AUC, 0.666; 95% CI, 0.525-0.807). Conclusion: The top-quality embryo score at transfer and the degree of post-warming embryo development were associated with clinical pregnancy in vitrified cleavage-stage embryo transfer cycles. In vitrified blastocyst transfer cycles, the top-quality blastocyst score at transfer was the only significant factor affecting clinical pregnancy.

PAF Regulate Blastocyst Development to Hatching Stage through PKC Activity in the Mouse

  • Cheon Yong-Pil
    • Reproductive and Developmental Biology
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    • v.30 no.2
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    • pp.75-79
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    • 2006
  • The developmental regulation of the preimplantation mammalian embryos is a fundamental step for preparing the implantation and it may be regulated by several autocrine and paracrine factors including platelet-activating factor. PAF improved the embryonic survival and implantation but its role during blastocyst development is still largely unknown. In this study, the effects and the possible pathway of PAF on developmental regulation of blastocyst to hatching stage were investigated. Developmental pattern in hatching embryo was a concentration-response curve showing maximal activity at 1 nM PAF, with decreasing activity at higher concentrations. $50{\mu}M$ 1-(5-isoquinolimnesulfonyl)-2-methylpiperazinme dihydrochloride (H-7), a PKC inhibitor, inhibited the progression of blastocyst to hatching embryo. In addition H-7 blocked the PAF effects on the blastocyst development. Besides tetradecanoylphorbol acetate (TPA), a PKC activator stimulated development of blastocyst to the hatching stage. These finding revealed that PAF support the blastocyst development to the hatching embryo. Also it is suggested that PAF action pathways in hatching supporting include the PKC signaling pathway.

Concanavalin A Mediated Calcium Changes on Expansion and Hatching of the Mouse Blastocyst (Concanavalin A를 매개로한 세포내 Calcium의 변화와 생쥐 포배의 팽창과 부화)

  • Cheon, Yong Pil
    • Clinical and Experimental Reproductive Medicine
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    • v.32 no.4
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    • pp.337-346
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    • 2005
  • Objective: The oligosaccharide moieties of glycoproteins and proteoglycans have a vital function in blastocyst differentiation. Concanavalin (ConA), a lectin, is known to bind on the preimplantation embryos, especially on blastocyst. In this study, we investigated whether ConA can modulate the trophoblast development and about the regulating mediator. Also, we investigated whether expansion is enough for hatching procession of the mouse blastocyst. Method: Embryos were collected at 72 h post hCG injection and chemicals were treated after 24 h (96 hr post hCG injection). ConA or calcium ionophore A23187 were exposed to blastocyst and than analysis the developmental process for 48 hr. Intracellular free-$Ca^{2+}$ concentration in trophectoderm was measured with confocal laser microscope after exposing to ConA or calcium ionophore A23187. ConA-pretreated blastocyst exposed to the calcium ionophore A23187 and then analyzed the developmental process. Otherwise ouabain was treated to the blastocyst to block the $Na^+/K^+$-ATPase activity. Results: In contrast to the control blastocyst, the ConA-exposed blastocysts developed beyond the expansion stage with significantly high rate (90.4%) at 12 h post administration. ConA induced an increase the intracellular $Ca^{2+}$ concentration in trophectoderm. Calcium ionophore A23187 also stimulated expansion of blastocyst. Most of the control blastocysts developed to the hatching stage at 144 h post hCG injection. However, strongly 65% of the ConA-exposed embryos were arrested at expanded stage at same time point. The developmental progression rates to hatching stage of both ConA- and calcium ionophore A23187-expose blastocysts were significantly lower than that of the control. However ConA-pretreated embryos developed to the hatching stage like control embryos. Ouabain showed a tendency to delayed the progress to expansion stage but did not inhibit the development to the hatching stage. Conclusion: ConA-mediated expansion is the result of the increase of intracellular free-calcium in blastocyst stage embryo. It is suspected that expansion of the blasocyst is a essential indirect factor in hatching and the calcium may triggering the cellular mechanisms for the both expansion and hatching progression.

The Studies on the Development of Human Blastocyst Embryos in IVF -ET Program - II. The Development of Human Blastocyst Embryos by co-culture with Cumulus Cells (IVF-ET Program에서 Blastocyst 배아의 발생에 관한 연구 - II. 난구세포 공동배양에 의한 Blastocyst 배아의 발생)

  • Lee, Suk-Won;Yoon, San-Hyun;Yoon, Hye-Gyun;Cho, Hyon-Jin;Heo, Yong-Soo;Yoon, Hye-Jin;Park, Se-Pill;Lee, Won-Don;Lim, Jin-Ho
    • Clinical and Experimental Reproductive Medicine
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    • v.25 no.1
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    • pp.35-41
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    • 1998
  • This study was carried out to investigate the development rates of human embryos co-cultured with cumulus cells to each blastocyst stage. Human zygotes were co-cultured on cumulus cell monolayer in YS medium supplemented with 20% hFF. On day 2, if patient had four or more "good" embryos (regular blastomeres without fragmentation), embryos were further cultured for 72hrs. Blastocysts on day 5 were classified into early blastocyst (ErB), early expanding blastocyst (EEB), middle expanding Blastocyst (MEB), and expanded blastocyst (EdB) on the basis of their morphological aspects of trophectoderm cells and blastocoele. Subsequently, maximum 3 of best blastocysts were transferred in 486 cycles. The results in this study were as follows: Patients who had four or more "good" embryos on day 2 were 498 persons, but patients whose embryos could not be transferred due to failure in development to the blastocyst stage on day 5 were 12 persons (2.4%). The development rate of embryos to the blastocyst stage was 58.2% (2,885/4,957) on day 5, and the rates that developed to the ErB, EEB, MEB, and EdB stage were 15.0% (743/4,957), 14.9% (739/4,957), 14.4% (714/ 4,957), and 13.9% (689/4,957), respectively. Total 1366 blastocysts were transferred in 486 cycles (mean number=2.81). The implantation rate and the ongoing implantation rate obtained by observing the number of G-sac and FHB were 29.9% (409/1,366) and 22.5% (308/1,366), respectively. The clinical pregnancy rate was 51.2% (249/486), and the ongoing pregnancy rate' was 39.1% (190/486). Among women showing ongoing pregnancy, women with singleton were 50% (95/190), women with twin were 37.9% (72/190), and women with triplet were 12.1% (23/190). Although triplet pregnancy rate in this study was high such as 12.1%, because many blastocysts with high viability were produced in our co-culture system using cumulus cells on day 5, we really believe that a multiple pregnancy except twin should not occur by selecting good embryos for maximum two blastocyst transfer. These results demonstrate that autologous cumulus cells may be used for the production of blastocysts with high developmental competence, and the use of autologous cumulus cells to be collected easily, and to be treated conveniently at OPU must be an effective means for obtaining high implantation and pregnancy rate.

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Effect of Conditioned Medium of Human Endothelial Cell Line(tHUE-2 cell) on In Vitro Development of Mouse 1-cell Embryos In Vitro Fertilized (체내 수정된 Mouse 1-세포배의 체외발생에 미치는 혈관내피세포주(tHUE-2세포) 배양액의 영향)

  • ;;;Y. Mitsui
    • Korean Journal of Animal Reproduction
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    • v.19 no.2
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    • pp.81-88
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    • 1995
  • Culture medium (ASF-301) of tHUE-2 cell, human endothelial cell line, and culture medium of these cells (conditioned medium : CM) which affect embryonic development of in vivo fertilized 1-cell embryos of mouse were examined. Two-cell stage block of mouse embryos was overicomed in ASF-301 and CM without EDTA, which usually added in basic medium (modified Whitten Medium: MWM, control) to overcome the 2-cell stage block. The developmental rates of embryos to the blastocyst stage were significantly increased in MWM containing 12.5% of growth factors added to ASF-301 (10mg/ $\ell$ transferrin, 1mg/$\ell$ insulin, 0.01mg/$\ell$ EGF) than those of 100% addition and control, 78.0% vs 20.8 and 52.3%, respectively (P<0.05), but the growth factors was not affected the hatching rate of blastocyst. Using ASF-301 or CM which was not treated, embryonic development into the blastocyst and hatched blastocyst stages were not affected. However, proportions of embryonic development into the blastocyst and hatched blastocyst stages were significantly higher in dilution (ASF-301 1:10; CM 1:3~1:6) than those in control (P,0.05). In ASF-301 dialyzed M.W.<10000 dialysis membrane, the developmental rate upto the hatched blastocyst stage was significantly increased, compared to ASF-301 which was not dialyzed (P<0.05), and hatching rate of blastocyst of these group was singnificantly increased than those in MWM (P<0.05). Compared to CM which was not dialyzed, however, in dialyzed CM was significantly decreased, compared to untreated CM (P<0.05), especially any hatched blastocyst was not appeared. As a result of these experiments indicated that a kind or porper treatment such as a dilution of complex synthetic cell culture medium and conditioned medium, and that a optimal concentration of growth factors are usuful for embryo cultrue in vitro.

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In Vitro Development and Conception Rate after Transfer of Mouse Embryos Bisected at Morula or Blastocyst Stage (상실배 및 포기배에 분할한 생쥐 수정란의 체외발달 속도 및 이식 후 수태율)

  • 박희성;박준규;정장용;박충생
    • Journal of Embryo Transfer
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    • v.8 no.1
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    • pp.13-19
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    • 1993
  • These experiments were carried out to determine the effect of pregnancy in bisected embryo. The embryos of ICR mouse were microsurgically bisected at morula and blastocyst stage using microsurgical blade attached a micromanipulator. These bisected embryos without zona pellucida were cultured up to blastocyst stage and cell count and diameter of stained blastomere, and transferred pseudopregnant mice. And the development of these bisected embryos was compared with the results of production of young of the corresponding intact embryos or cell stage. When the bisected mouse embryos were cultured in vitro for 20 to 24 hours in morula stage(77.2%) or 3 to 6 hours in blastocyst stage(84.1%), them were developed to the expanded blastocyst stage. There were no significant(P<0.05) differences in the development rate of bisected embryos between in morula and blastocyst stages. The embryo size of blastocyst developed in vitro from bisected embryo was small(P<0.05)than intact embryo. However, the number of blastomeres with bisected embryo (24.7+1.3and 21.5+1.2 respectively) were significantly(P<0.05) reduced, compared with that of intacted embryos(36.3+1.1 and 41.4+1.2 respectively). When compared with the result of pregnancy rate(63.6%) after surgical transfer of bisected morulae, a similar result(65.4%) was obtained with bisected blastocyst stage(P< 0.05). However, production of youngs (38.8%) after transfer of bisected morula, a similar result (38.1%) was obtained with bisected blastocyst stage (P<0.05).

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Retrospective study of single vitrified-warmed blastocyst transfer cycles according to the presence of morphokinetic variables

  • Hur, Yong Soo;Ryu, Eun Kyung;Hyun, Chang Seop;Yang, Seong Ho;Yoon, San Hyun;Lim, Kyung Sil;Lee, Won Don;Lim, Jin Ho
    • Clinical and Experimental Reproductive Medicine
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    • v.45 no.1
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    • pp.52-55
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    • 2018
  • This study retrospectively assessed whether time-lapse data relating to developmental timing and morphology were associated with clinical outcomes, with the eventual goal of using morphokinetic variables to select embryos prospectively for cryopreservation. In this study, we examined the clinical outcomes of single vitrified-warmed blastocyst transfer cycles that were cultured in a time-lapse incubation system. The morphokinetic variables included uneven pronuclei, an uneven blastomere, multinucleation, and direct, rapid, and irregular division. A total of 164 single vitrified-warmed blastocyst transfer cycles were analyzed (102 cycles of regularly developed blastocysts and 62 cycles of blastocysts with morphokinetic variables). No significant differences in the age of females or the standard blastocyst morphology were found between these two groups. The regularly developed blastocysts showed significantly higher implantation and clinical pregnancy rates than the blastocysts exhibiting morphokinetic variables (30.4% vs. 9.7% and 37.3% vs. 14.5%, respectively; p< 0.01). The blastocysts that exhibited morphokinetic variables showed different mean development times compared with the regularly developed blastocysts. Although morphokinetic variables are known to have fatal impacts on embryonic development, a considerable number of embryos developed to the blastocyst stage. Morphokinetic variables had negative effects on the implantation and clinical pregnancy rates in vitrified-warmed blastocyst transfer cycles. These findings suggest that blastocysts cultured in a time-lapse incubation system should be considered for selective cryopreservation according to morphokinetic variables.

Effects of Donor Cell Passage, Size and Type on Development of Porcine Embryos Derived from Somatic Cell Nuclear Transfer

  • Zhang, Y.H.;Song, E.S.;Kim, E.S.;Cong, P.Q.;Lee, S.H.;Lee, J.W.;Yi, Y.J.;Park, Chang-Sik
    • Asian-Australasian Journal of Animal Sciences
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    • v.22 no.2
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    • pp.194-200
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    • 2009
  • The aim of this study was to investigate the effects of donor cell passage, size and type on the development of nuclear transfer embryos. Porcine cumulus cells, fetal fibroblasts and oviductal epithelial cells from 1-2, 3-6 and 7-10 passages were used for the nuclear transfer. In the oocytes with the cumulus donor cells, fusion and cleavage rates of oocytes and cell numbers per blastocyst among the three different passage groups did not show any differences, but the rates of blastocyst formation from 1-2 and 3-6 passage groups were higher than those from 7-10 passage group. The rates of fusion, cleavage and blastocyst formation, and the cell numbers per blastocyst were higher in the embryos with the sizes of <20 and 20 ${\mu}m$ cumulus donor cells compared to the >20 ${\mu}m$ cumulus donor cell. In the oocytes with the fetal fibroblast donor cells, the rate of blastocyst formation from the 3-6 passage group was higher than from 1-2 and 7-10 passage groups. The embryos with the size of 20 $\mu{m}$ fetal fibroblast donor cell showed higher rate of blastocyst formation compared to those with <20 and >20 ${\mu}m$ donor cells. In the oocytes with the oviductal epithelial cells, the rates of blastocyst formation from 1-2 and 3-6 passage groups were higher compared to those from 7-10 passage group. The embryos with the sizes of <20 and 20 ${\mu}m$ oviductal epithelial donor cells had a higher rate of blastocyst formation compared to those with >20 ${\mu}m$ donor cell. Fusion and cleavage rates of oocytes, and cell numbers per blastocyst among the three different donor cell types from the 3-6 passage did not show any differences. However, the rate of blastocyst formation of somatic cell nuclear transfer (SCNT) embryos with the fetal fibroblast donor cell was higher than that of blastocyst formation of SCNT embryos with the cumulus and oviductal epithelial donor cells.

Effects of Oviductal Fluid, Culture Media and Zona Pellucida Removal on the Development of Porcine Embryos by Nuclear Transfer

  • Zhang, Y.H.;Song, E.S.;Kim, E.S.;Cong, P.Q.;Lee, S.H.;Lee, J.W.;Yi, Y.J.;Park, Chang-Sik
    • Asian-Australasian Journal of Animal Sciences
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    • v.22 no.7
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    • pp.962-968
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    • 2009
  • The aim of this study was to compare the effects of oviductal fluid, porcine zygote medium (PZM)-3, PZM-4 and PZM-5, and modified PZM-5 culture media, and determine the effects of zona pellucida (ZP) removal on the development of nuclear transfer (NT) embryos. There were no significant differences in the rates of fusion and cleavage among the five different oviductal fluid concentrations. However, the rates of blastocyst formation and the cell numbers per blastocyst were high in the embryos at the 14 and 28 $\mu{g}$/ml concentrations of oviductal fluid compared to the 0, 56 and 100 $\mu{g}$/ml concentrations. The rates of cleavage and blastocyst formation, and the cell numbers per blastocyst were higher in the PZM-3, PZM-5 and modified PZM-5 media than in the PZM-4 medium. However, there were no significant differences in the fusion rates of oocytes among the four culture media. The cell numbers per blastocyst in the embryos without ZP were significantly greater than those with ZP. However, there were no significant differences in the rates of fusion, cleavage and blastocyst formation between the embryos with and without ZP. In conclusion, we improved blastocyst development and the quality of NT embryos by replacing PVA with 3 mg/ml of BSA in PZM-5 medium and supplementing the PZM-5 medium with 14 $\mu{g}$/ml oviductal fluid. The NT embryos produced by the zona-free NT method had a high rate of blastocyst formation in the modified PZM-5 medium.