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1
Cheon, Yong Pil, Myung Chan Gye, Chung-hoon Kim, Byung Moon Kang, Yoon Seok Chang, Sung Rye Kim, and Moon Kyoo Kim. "Role of actin filaments in the hatching process of mouse blastocyst." Zygote 7, no. 2 (May 1999): 123–29. http://dx.doi.org/10.1017/s0967199499000477.
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Hatching has been suggested to occur as a result of protease-mediated lysis and the blastocoele tension. However, even if rupturing is initiated at multiple sites, interestingly only a single site is used for escape. This implies that there are several mechanisms involved in hatching. In this study, the involvement of actin filaments in mouse embryo hatching was examined. We treated mouse embryos with cytochalasin B for 12 h or 24 h at the morula, middle blastocyst, expanded blastocyst, lobe-formed blastocyst and hatching blastocyst stages, and measured the amount and distribution of actin filaments using a confocal microscope. At morula, middle blastocyst, lobe-formed blastocyst and hatching blastocyst stages embryonic development was completely arrested by cytochalasin B. However, when transferred to cytochalasin-B-free medium, the embryos resumed development and escaped the zona pellucida. In the expanded blastocysts development was almost completely inhibited by cytochalasin B, but rupturing occurred in some embryos. However, development stopped completely at the ruptured stage. Distribution of actin filaments was prominent at rupturing and hatching sites regardless of cytochalasin B treatment. The amount of actin filaments was prominent at hatching embryos compared with other developmental stages of embryos. These actin filaments were distributed intensively between the trophectodermal cells, and formed locomotion patterns. Taken together, these results suggest that not only tension and lytic enzymes are required to rupture, but the activity of actin filaments may have a crucial role in the process of hatching.
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Kim, Y. J., K. S. Ahn, S. M. Park, B. C. Lee, H. Shim, and C. Ahn. "28 IMPROVED HATCH RATE AFTER PARTIAL DISSECTION OF ZONA PELLUCIDA IN CLONED PIG EMBRYO." Reproduction, Fertility and Development 29, no. 1 (2017): 121. http://dx.doi.org/10.1071/rdv29n1ab28.
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For last 20 years, the efficiency of animal cloning has remained extremely low, despite many attempts to improve it. Although nuclear transfer experiments have been almost optimized, artificial holes are inevitably made in the zona pellucida (ZP) during nuclear transfer experiments, such as enucleation of maternal genome or injection of nuclear donor. Hatching from the ZP is a prerequisite for mammalian embryo implantation, and the condition of the ZP has a lot of influence on hatching. The present studies were performed to investigate the effects of artificial holes in the ZP, because of the nuclear transfer procedure, on hatching of clone embryos in pigs. All experiments were done in triplicate. Statistical analysis was performed using SPSS statistical software (SPSS Inc., Chicago, IL, USA). First, we made a slit in the ZP of porcine parthenote that was identical to the artificial holes of nuclear transfer experiment and compared in vitro development of Day 7 embryos with control group with intact ZP. Of slit blastocysts, 89.9% (80/89) were trapped at a slit, which looked like typical figure-eight shape, and did not complete the hatching process until Day 8, though 68.8% (64/93) of control blastocysts completed the hatching at Day 7. Then, to solve these abnormal hatchings caused by a slit in the ZP, we applied partial zona dissection (PZD) to porcine clone embryos and compared the hatching process with that of conventional clone embryos. Contrary to conventional clone blastocysts that were trapped at slit in the ZP (91.4%; 43/47), 89.5% (60/67) of clone blastocysts in PZD group were preferentially hatched through dissected hole at Day 7. These results suggest that trapping of conventional clone blastocyst in a slit of the ZP could be avoided by PZD. Through this study, we demonstrated that a slit in the ZP would hinder a blastocyst from hatching from the ZP and that partial dissection at the ZP could help clone blastocyst to hatch without trapping at a slit in the ZP. This assisted hatching in clone embryos would be useful for the successful hatching of clone blastocysts with a capacity of full-term development, so that the efficiency of animal cloning might be improved.
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Eswari, S., G. Sai Kumar, and G. Taru Sharma. "Expression of mRNA encoding leukaemia inhibitory factor (LIF) and its receptor (LIFRβ) in buffalo preimplantation embryos produced in vitro: markers of successful embryo implantation." Zygote 21, no. 2 (August 14, 2012): 203–13. http://dx.doi.org/10.1017/s0967199412000172.
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SummaryThe objective of this study was to evaluate the effect of supplementation of recombinant leukaemia inhibitory factor (LIF) in culture media on blastocyst development, total cell number and blastocyst hatching rates and the reverse transcription-polymerase chain reaction analysis of preimplantation buffalo embryos to determine whether they contain the LIF-encoding mRNA and its beta receptor (LIFRβ) genes in different stages of preimplantation buffalo embryos. Cumulus–oocyte complexes retrieved from slaughterhouse buffalo ovaries were matured in vitro and fertilized using frozen buffalo semen. After 18 h of co-incubation with sperm, the presumptive zygotes were cultured in modified synthetic oviductal fluid without (control) or with rhLIF (100 ng/ml). There was no significant difference in the overall cleavage rate up to morula stage however the development of blastocysts, hatching rate and total cell numbers were significantly higher in the LIF-treated group than control. Transcripts for LIFRβ were detected from immature, in vitro-matured oocytes and in the embryos up to blastocyst stage, while transcripts for the LIF were detected from 8–16-cell stage up to blastocyst, which indicated that embryo-derived LIF can act in an autocrine manner on differentiation process and blastocyst formation. This study indicated that the addition of LIF to the embryo culture medium improved development of blastocysts, functional (hatching) and morphological (number of cells) quality of the blastocysts produced in vitro. The stage-specific expression pattern of LIF and LIFRβ mRNA transcripts in buffalo embryos indicated that LIF might play an important role in the preimplantation development and subsequent implantation of buffalo embryos.
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Leonavicius, Karolis, Christophe Royer, Chris Preece, Benjamin Davies, John S. Biggins, and Shankar Srinivas. "Mechanics of mouse blastocyst hatching revealed by a hydrogel-based microdeformation assay." Proceedings of the National Academy of Sciences 115, no. 41 (September 19, 2018): 10375–80. http://dx.doi.org/10.1073/pnas.1719930115.
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Mammalian embryos are surrounded by an acellular shell, the zona pellucida. Hatching out of the zona is crucial for implantation and continued development of the embryo. Clinically, problems in hatching can contribute to failure in assisted reproductive intervention. Although hatching is fundamentally a mechanical process, due to limitations in methodology most studies focus on its biochemical properties. To understand the role of mechanical forces in hatching, we developed a hydrogel deformation-based method and analytical approach for measuring pressure in cyst-like tissues. Using this approach, we found that, in cultured blastocysts, pressure increased linearly, with intermittent falls. Inhibition of Na/K-ATPase led to a dosage-dependent reduction in blastocyst cavity pressure, consistent with its requirement for cavity formation. Reducing blastocyst pressure reduced the probability of hatching, highlighting the importance of mechanical forces in hatching. These measurements allowed us to infer details of microphysiology such as osmolarity, ion and water transport kinetics across the trophectoderm, and zona stiffness, allowing us to model the embryo as a thin-shell pressure vessel. We applied this technique to test whether cryopreservation, a process commonly used in assisted reproductive technology (ART), leads to alteration of the embryo and found that thawed embryos generated significantly lower pressure than fresh embryos, a previously unknown effect of cryopreservation. We show that reduced pressure is linked to delayed hatching. Our approach can be used to optimize in vitro fertilization (IVF) using precise measurement of embryo microphysiology. It is also applicable to other biological systems involving cavity formation, providing an approach for measuring forces in diverse contexts.
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Morató, Roser, Míriam Castillo-Martín, Marc Yeste, and Sergi Bonet. "Cryotolerance of porcine in vitro-produced blastocysts relies on blastocyst stage and length of in vitro culture prior to vitrification." Reproduction, Fertility and Development 28, no. 7 (2016): 886. http://dx.doi.org/10.1071/rd14203.
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The aim of our study was to assess whether the cryotolerance of in vitro-produced embryos could be influenced by the length of in vitro culture and size of blastocoel cavity before vitrification, using the pig as a model. For this purpose we analysed the cryoresistance and apoptosis rate of blastocysts at different stages of development as derived on Day 5 and 6 of in vitro culture. Blastocysts were subsequently vitrified, warmed and cultured for 24 h. Re-expansion rates were recorded at 3 and 24 h and total cell number and apoptotic cells were determined at 24 h. Day-6 blastocysts showed the highest rates of survival after warming, which indicates higher quality compared with Day-5 blastocysts. Higher re-expansion rates were observed for expanded blastocysts and those in the process of hatching when compared with early blastocysts. Total cell number and apoptotic cells were affected by blastocyst stage, vitrification–warming procedures and length of in vitro culture, as expanding and hatching–hatched blastocysts from Day 6 presented higher percentages of apoptotic cells than fresh blastocysts and blastocysts vitrified at Day 5. Our findings suggest that the cryotop vitrification method is useful for the cryopreservation of porcine blastocysts presenting a high degree of expansion, particularly when vitrification is performed after 6 days of in vitro culture. Furthermore, these results show that faster embryo development underlies higher blastocyst cryotolerance and provide evidence that blastocoel cavity expansion before vitrification is a reliable index of in vitro-produced embryo quality and developmental potential.
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Pasquariello, R., J. R. Herrick, Y. Yuan, A. F. Ermisch, J. Becker, L. Yao, C. Broeckling, W. B. Schoolcraft, J. P. Barfield, and R. L. Krisher. "73 Fatty Acid Supplementation in Culture Medium with Reduced Nutrient Concentrations Improves Bovine Blastocyst Development Compared with Standard Culture Medium." Reproduction, Fertility and Development 30, no. 1 (2018): 175. http://dx.doi.org/10.1071/rdv30n1ab73.
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Lipids are a potent source of cellular energy and are metabolized within mitochondria via fatty acid β-oxidation, a process that also requires carnitine. Embryos metabolize lipids during pre-implantation development, but relatively little is known about the effect of fatty acid supplementation for early bovine embryogenesis in culture. The objective of this study was to evaluate the effect of lipid supplementation (via albumin) and l-carnitine (C; 5 mM) during embryo culture in a novel medium with reduced concentrations of nutrients, compared with our standard culture medium (control). Following in vitro maturation and IVF, zygotes were cultured using a serum-free sequential media system (0-72 h step 1; 72-168 h step 2). Concentrations of salts, bicarbonate, and protein [2.5 mg mL−1 fatty acid-free (FAF) or fraction V (FrV) BSA] were the same in all treatments to maintain consistent osmolarity and pH. Nutrients (glucose/fructose, citrate, lactate, pyruvate, amino acids, vitamins, and EDTA) were diluted to 6.25% of control. In addition to the control medium (100%+FAF; n = 587), experimental treatments included 6.25%+FAF+C (essentially lipid free; n = 573) and 6.25%+FrV+C (lipid rich; n = 585). Following in vitro culture (7 reps), hatching blastocysts were stained to determine inner cell mass (ICM; SOX2+) and trophectoderm (TE; CDX2+) cell numbers. Lipid content of single expanded blastocysts was determined using gas chromatography coupled to an ISQ-LT MS/MS (GC-MS). Data (mean ± SEM) were analysed by ANOVA. Embryo cleavage did not differ between treatments. Blastocyst development (per cleaved embryo) was higher (P < 0.05) after culture in lipid rich (38.3 ± 1.5%) compared with control (29.6 ± 2.2%) and lipid free (28.1 ± 3.6%). Blastocyst hatching was reduced (P < 0.05) in lipid free (1.4 ± 0.7%) but not in lipid rich (5.2 ± 1.7) compared with control (9.8 ± 2.1). However, blastocysts developed in lipid rich and lipid free had reduced cell numbers compared with control: TE, 98.7 ± 5.9 and 98.8 ± 9.1 v. 160.3 ± 9.0; ICM, 19.2 ± 2.9 and 25.2 ± 6.1 v. 43.3 ± 4.0; and total cell number, 117.9 ± 7.3 and 124.0 ± 8.7 v. 203.6 ± 10.2, respectively. Analysis by GC-MS identified 40 annotated lipids (i.e. triacylglycerols and phosphatidyl cholines) that were significantly reduced in blastocysts cultured in lipid rich compared with control. In summary, blastocyst development was significantly improved after supplementation of fatty acids and l-carnitine to a medium with reduced nutrient concentrations. The mechanism underlying this phenomenon may be related to increased lipid metabolism in the low nutrient environment. Although more embryos developed in this novel medium, these blastocysts had reduced cell numbers even though blastocyst expansion and hatching were not affected. This reduced nutrient medium may provide an experimental model in which to independently study pathways controlling cell proliferation and blastocyst development. Future studies will investigate whether embryo cell number can be rescued while maintaining improved blastocyst development.
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Ribeiro, E. S., M. C. Gonçalves, M. C. Pedrotti, L. T. Martins, R. P. C. Gerger, F. K. Vieira, K. C. S. Tavares, M. Bertolini, and A. Mezzalira. "74 EFFECT OF BETA-MERCAPTOETHANOL ON THE VITRIFICATION CRYOTOLERANCE OF BOVINE IN VITRO-PRODUCED EMBRYOS." Reproduction, Fertility and Development 21, no. 1 (2009): 137. http://dx.doi.org/10.1071/rdv21n1ab74.
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The control of oxidative processes in in vitro production (IVP) systems by the use of additives may be an alternative approach to improve embryo cryotolerance. The aim of this study was to verify the effect of β-mercaptoethanol (βME) on the cryotolerance of bovine IVP embryos. In 7 replications, and following IVM-IVF, presumptive zygotes (n = 3735) were in vitro-cultured in SOF medium supplemented or not with 100 μm βME (IVC treatment), at 38.5°C and high humidity. The initial 24 h of IVC was performed in 5% CO2 in air, with the remaining 6 days of IVC carried out in 5% CO2, 5% O2, and 90% N2. On Day 7, resulting blastocysts and expanded blastocysts were vitrified in glass micropipettes in a solution with 20% ethylene glycol + 20% propylene glycol. After warming, embryos were randomly allocated to 1 of 2 sub-groups for an additional 72 h of IVC to the hatching blastocyst (HBL) stage, in fresh SOF medium supplemented or not with 100 μm βME (PVC treatment), at 38.5°C, high humidity and 5% CO2. Experimental groups were as follows: G1 (βME-free medium during IVC and PVC); G2 (βME only during PVC); G3 (βME only during IVC); and G4 (βME during IVC and PVC). Cleavage (Day 2) and blastocyst (Day 7) rates in the IVC treatment and hatching rates (Days 7 to 9) for the PVC treatment were analyzed by the chi-square test, for P < 0.05. Total cell number (TCN) estimated by fluorescence staining in HBL derived from vitrified and nonvitrified embryos was analyzed by ANOVA. The use of βME during IVC did not affect cleavage rates (βME-free, 1491/1858, 80.2% v. βME, 1522/1877, 81.1%), but negatively affected development to the blastocyst stage (βME-free, 813/1858, 43.8% v. βME, 525/1877, 28.0%). Following vitrification, however, βME supplementation during PVC improved hatching rates (G2, 58.1% and G4, 63.8%) compared with groups without the additive (G1, 36.6% and G3, 42.0%). In addition, the presence of βME either during IVC or PVC, or during both culture periods, increased TCN in HBL from vitrified embryos (Table 1). The use of βME during IVC, irrespective of the presence of βME during the PCV period, caused an increase in TCN in HBL in G3 + G4, with no effects on hatching rates (Table 1b), whereas the addition of βME during PVC, irrespective of the presence of βME during the IVC period, resulted in greater hatching rates and TCN in HBL in G2 + G4 than in G1 + G3 (Table 1). In conclusion, the addition of βME during the IVC period did not affect cleavage, but reduced blastocyst yield. Despite that, βME supplementation during the IVC period appeared to have increased the cryotolerance of the resulting blastocysts, expressed by greater TCN in HBL, whereas βME supplementation during the PVC period also improved embryo survival to the vitrification process, manifested by greater hatching rates and TCN in HBL. Table 1.Effect of βME on the cryotolerance of bovine IVP embryos This study was supported by a grant from CNPq/Brazil.
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Silva, Thiago Velasco Guimarães, Priscila Di Paula Bessa Santana, Eduardo Baia de Souza, Ana Júlia Mota de Lima, Caroline de Araújo Santos, Nathália Nogueira da Costa Almeida, Vanessa Cunha de Brito, et al. "Sperm chromatin protamination influences embryo development in unsexed and sexed bull semen." Zygote 29, no. 4 (January 15, 2021): 264–69. http://dx.doi.org/10.1017/s0967199420000775.
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SummarySex selection through sperm sorting offers advantages in regards selection pressure in high-producing livestock. However, the sex-sorting process results in sperm membrane and DNA damage that ultimately decrease fertility. We hypothesized that given the role of protamines in DNA packaging, protamine deficiency could account, at least partially, for the DNA damage observed following sperm sex sorting. To test this, we compared protamine status between unsexed and sexed spermatozoa from two bulls using the fluorochrome chromomycin A3 (CMA3) and flow cytometry. Then, we assessed embryo development following in vitro fertilization (IVF) using the same sperm treatments. Overall, sperm protamination was not different between sexed and unsexed semen. However, one of the two bulls displayed higher rates of protamine deficiency for both unsexed and sexed semen (P < 0.05). Moreover, unsexed semen from this bull yielded lower blastocyst (P < 0.05) and blastocyst hatching rates than unsexed sperm from the other bull. CMA3-positive staining was negatively correlated with cleavage (R2 85.1, P = 0.003) and blastocyst hatching (R2 87.6, P = 0.006) rates in unsexed semen. In conclusion, while the sex-sorting process had no effect on sperm protamine content, we observed a bull effect for sperm protamination, which correlated to embryo development rates following IVF.
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Rascado, T. S., M. D. Guastali, D. M. Paschoal, M. J. Sudano, L. E. Vergara, R. R. Mazieiro, F. C. Landim, and J. P. Araujo. "208 COMPARISON OF THE EXPRESSION OF SOX2 AND Stat3 IN BOVINE BLASTOCYST AND HATCHED BLASTOCYST." Reproduction, Fertility and Development 25, no. 1 (2013): 252. http://dx.doi.org/10.1071/rdv25n1ab208.
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The investigation of the transcription factors involved in the regulation of pluripotency in pre-implantation embryos of cattle provides important information regarding the early embryonic development and derivation of embryonic stem cells. The present experiment aimed to compare the level of expression of SOX 2 and Stat3, pluripotency markers, in bovine blastocysts (D7) and hatched blastocysts (D10), because it is known that around hatching, the inner cell mass (ICM) differentiates into hypoblast and epiblast. Cumulus–oocyte complexes were matured in TCM 199 for 24 h and fertilized with frozen–thawed sperm. Presumptive zygotes were cultured in SOFaaci for 7 days (group 1) and for 10 days (group 2). All embryos were washed 3 times in PBS, pooled, and frozen at –80°C until RNA extraction. For quantification of SOX2 and Stat3 mRNA levels, total RNA was isolated from pools of 7 embryos per replicate (n = 3) and for each examined developmental stage using RNeasy Micro Kit (Qiagen). The reverse transcriptase Superscript III (Invitrogen) was used for the synthesis of cDNA (cDNA), and the qPCR was performed with the Gotaq qPCR Master Mix (Promega, Madison, WI, USA). As negative control, cDNA was replaced by nuclease-free water in the qPCR reaction. Quantification of expression was determined by the relative standard curve method and normalized to the housekeeping gene YWHAZ. Standard curves for SOX2, Stat 3, and YWHAZ were derived from 10-fold serial dilutions of bovine DNA and gave correlation coefficients greater than 0.99 and efficiencies greater than 94%. The data from 3 replicates were analysed by ANOVA, and it was found that the level of SOX2 abundance is 9.8-fold higher in D7 blastocyst cells compared with D10 blastocyst cells, and the level of Stat 3 transcription is 1.5-fold higher in blastocyst cells than in hatched blastocysts. The expression of pluripotency markers SOX2 and Stat3 was significantly higher in embryos with 7 days of development because at this stage the MCI had not yet begun the process of differentiation. Support of FAPESP is acknowledged.
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Das, S. K., X. N. Wang, B. C. Paria, D. Damm, J. A. Abraham, M. Klagsbrun, G. K. Andrews, and S. K. Dey. "Heparin-binding EGF-like growth factor gene is induced in the mouse uterus temporally by the blastocyst solely at the site of its apposition: a possible ligand for interaction with blastocyst EGF-receptor in implantation." Development 120, no. 5 (May 1, 1994): 1071–83. http://dx.doi.org/10.1242/dev.120.5.1071.
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Heparin-binding EGF-like growth factor (HB-EGF) is a newly discovered member of the EGF family of growth factors. HB-EGF can bind to two loci on cell surfaces, heparan sulphate proteoglycans and EGF-receptor (EGF-R), and either one or both of these interactions could play a role in cell-cell interactions. In the rodent, increased endometrial vascular permeability at the site of blastocyst apposition is considered to be an earliest discernible prerequisite event in the process of implantation and this event coincides with the initial attachment reaction between the blastocyst trophectoderm and uterine luminal epithelium. This investigation demonstrates that the HB-EGF gene is expressed in the mouse uterine luminal epithelium surrounding the blastocyst 6–7 hours before the attachment reaction that occurs at 2200–2300 hours on day 4 of pregnancy. It was further demonstrated that this gene is not expressed in the luminal epithelium at the site of the blastocyst apposition during the progesterone-maintained delayed implantation, but is readily induced in the luminal epithelium surrounding an activated blastocyst after termination of the delay by an estrogen injection. In vitro studies showed that HB-EGF induced blastocyst EGF-R autophosphorylation, and promoted blastocyst growth, zona-hatching and trophoblast outgrowth. These results suggest possible interactions between the uterine HB-EGF and blastocyst EGF-R very early in the process of implantation, earlier than any other embryo-uterine interactions defined to date at the molecular level.
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Sidrat, Tabinda, Abdul Aziz Khan, Muhammad Idrees, Myeong-Don Joo, Lianguang Xu, Kyeong-Lim Lee, and Il-Keun Kong. "Role of Wnt Signaling During In-Vitro Bovine Blastocyst Development and Maturation in Synergism with PPARδ Signaling." Cells 9, no. 4 (April 9, 2020): 923. http://dx.doi.org/10.3390/cells9040923.
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Wnt/β-catenin signaling plays vital role in the regulation of cellular proliferation, migration, stem cells cell renewal and genetic stability. This pathway is crucial during the early developmental process; however, the distinct role of Wnt/β-catenin signaling during pre-implantation period of bovine embryonic development is obscure. Here, we evaluated the critical role of Wnt/β-catenin pathway in the regulation of bovine blastocyst (BL) development and hatching. 6 bromoindurbin-3’oxime (6-Bio) was used to stimulate the Wnt signaling. Treatment with 6-Bio induced the expression of peroxisome proliferator-activated receptor-delta (PPARδ). Interestingly, the PPARδ co-localized with β-catenin and form a complex with TCF/LEF transcription factor. This complex potentiated the expression of several Wnt directed genes, which regulate early embryonic development. Inhibition of PPARδ with selective inhibitor 4-chloro-N-(2-{[5-trifluoromethyl]-2-pyridyl]sulfonyl}ethyl)benzamide (Gsk3787) severely perturbed the BL formation and hatching. The addition of Wnt agonist successfully rescued the BL formation and hatching ability. Importantly, the activation of PPARδ expression by Wnt stimulation enhanced cell proliferation and fatty acid oxidation (FAO) metabolism to improve BL development and hatching. In conclusion, our study provides the evidence that Wnt induced PPARδ expression co-localizes with β-catenin and is a likely candidate of canonical Wnt pathway for the regulation of bovine embryonic development.
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Ramos-Ibeas, Priscila, Ismael Lamas-Toranzo, Álvaro Martínez-Moro, Celia de Frutos, Alejandra C. Quiroga, Esther Zurita, and Pablo Bermejo-Álvarez. "Embryonic disc formation following post-hatching bovine embryo development in vitro." Reproduction 160, no. 4 (October 2020): 579–89. http://dx.doi.org/10.1530/rep-20-0243.
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Failures during conceptus elongation are a major cause of pregnancy losses in ungulates, exerting a relevant economic impact on farming. The developmental events occurring during this period are poorly understood, mainly because this process cannot be recapitulated in vitro. Previous studies have established an in vitro post-hatching development (PHD) system that supports bovine embryo development beyond the blastocyst stage, based on agarose gel tunnels and serum- and glucose-enriched medium. Unfortunately, under this system embryonic disc formation is not achieved and embryos show notorious signs of apoptosis and necrosis. The objective of this study has been to develop an in vitro system able to support embryonic disc formation. We first compared post-hatching development inside agarose tunnels or free-floating over an agarose-coated dish in serum- and glucose-enriched medium (PHD medium). Culture inside agarose tunnels shaped embryo morphology by physical constriction, but it restricted embryo growth and did not provide any significant advantage in terms of development of hypoblast and epiblast lineages. In contrast to PHD medium, a chemically defined and enriched medium (N2B27) supported complete hypoblast migration and epiblast survival in vitro, even in the absence of agarose coating. Cells expressing the pluripotency marker SOX2 were observed in ~56% of the embryos and ~25% developed embryonic disc-like structures formed by SOX2+ cells. In summary, here we provide a culture system that supports trophectoderm proliferation, hypoblast migration and epiblast survival after the blastocyst stage.
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Almiñana, C., E. Corbin, G. Harichaux, V. Labas, G. Tsikis, C. Soleilhavoup, K. Reynaud, X. Druart, and P. Mermillod. "78 INTERCEPTION OF EXOSOMAL MESSAGES BETWEEN THE OVIDUCT AND THE EMBRYO: WHAT ARE THEY TWEETING ABOUT?" Reproduction, Fertility and Development 28, no. 2 (2016): 168. http://dx.doi.org/10.1071/rdv28n2ab78.
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Successful pregnancy requires an appropriate communication between the mother and the embryo(s). Recent studies indicate that exosomes, small (30–100 nm) membrane-bound vesicles of endocytotic origin, could act as intercellular vehicles in this unique communication system in the uterus. However, little is known about the role of these vesicles in the oviduct. Our study aimed at (1) demonstrating the existence of oviducal-embryo communication via exosomes, (2) deciphering the exosomal dialogue between them at the proteomic level, and (3) comparing the exosomal proteomic content to the oviducal fluid proteomic content in order to highlight the key role of exosomes in this dialogue. Cow oviducts (pool of 6 oviducts at different stages of the cycle in 4 replicates) were flushed, and exosomes were isolated by serial ultracentrifugation. Exosomes were measured by dynamic light scattering analysis, resulting in exosomes (63.25–97.03 nm) and microvesicle observations (>100 nm). Bovine embryos were produced in vitro up to the blastocyst and hatching/hatched blastocyst stages. To demonstrate the existence of the oviducal-embryo communication via exosomes, oviducal exosomes were labelled with green fluorescent dye (PKH67), filtered (0.22 µm) to remove microvesicles, and co-incubated with blastocysts and hatching/hatched (H) blastocysts for 20 h, under 5% CO2 and 5% O2 conditions. Subsequently, embryos were washed in exosome-free medium, fixed in 4% paraformaldehyde, and labelled with Hoechst 33342 and Actin Red Phallodin. Confocal microscopy observations confirmed that exosomes were internalized by blastocysts and H-blastocysts and located around the nucleus, demonstrating the existence of an oviducal-embryo communication via exosomes. Moreover, our results showed that the zona pellucida does not represent a barrier for exosomes and they act as natural nanoshuttles bringing oviducal signals into the embryo. Then, proteomic analysis by LC1D-nanoESI-LTQ-Orbitrap was used to decipher oviducal exosomal content, identifying 480 proteins. Gene ontology analysis revealed that a high number of these proteins were involved in metabolism (24.9%), cellular process (19.3%), and 0.8% reproductive processes. Further analysis revealed that more than 56% of exosomal proteins involved in cellular process were associated with cell-to-cell communication. Finally, exosomal proteins were compared with proteins present in oviducal fluid from a pool of samples from cows at Day 0 and Day 10 of the oestrous cycle. Comparative analysis showed that from a total of 607 proteins identified in both oviducal exosomes and fluid sources, 105 were specific to exosomes, 127 were specific to fluid, whereas 375 were common to both sources. Our findings provide the first evidence of oviducal-embryo communication via exosomes, an important first step in furthering the understanding of the oviducal environment and the role of exosomes as early mediators of embryo-maternal cross talk. This research was supported by the EU AgreenSkills fellowship n° 267196 and EU FECUND Project no 312097.
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Forell, F., C. Feltrin, A. D. Vieira, U. M. Costa, M. Hoelker, and J. L. Rodrigues. "52 ESTABLISHMENT OF A PREGNANCY WITH IDENTICAL TWINS AFTER THE TRANSFER OF A VITRIFIED CLONED BOVINE EMBRYO." Reproduction, Fertility and Development 22, no. 1 (2010): 184. http://dx.doi.org/10.1071/rdv22n1ab52.
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Identical twin pregnancies occur naturally in some species because of spontaneous embryo division during initial stages in development. Bovine embryo splitting usually does not compromise subsequent in vivo development, being a method commonly used in embryo transfer programs to enhance overall pregnancy and parity rates. The goal of this study was to establish pregnancies from vitrified bovine cloned embryos. A spontaneous twin pregnancy was obtained after the transfer of a vitrified cloned hatching blastocyst produced by nuclear transfer procedures. Briefly, selected COCs obtained via slicing from bovine ovaries collected in a local slaughterhouse, and in vitro-matured for 17 h, were denuded by pipetting in HEPES-SOF (HSOF)-BSA and screened for the presence of the first polar body. Selected oocytes were enucleated by micromanipulation in HSOF + cytochalasin B and Hoechst 33 342 under UV fluorescence control. Frozen-thawed cumulus cells obtained by ovum pickup (OPU) from a Holstein cow were used as nucleus donors at passage 2 and high confluence (>90%). A single cell was inserted into the perivitelline space by micromanipulation. Fusion was induced electrically between two 200-μm-wide electrodes, 150 μm apart, using a single 20-V electrical DC pulse for 45 μs. Fusion outcome was observed 30 min after the electrical stimulus, with the immediate activation of the reconstructed embryos in ionomycin for 5 min, followed by incubation in cycloheximide and cytochalasin D for 5 h. Then, activated embryos were cultured in vitro for 7 days in SOF with amino acids (SOFaa) +10% estrous cow serum (ECS), at 39°C, high humidity, and 5% CO2, 5% O2 and 90% N2. Day-7 blastocysts were vitrified in hand-pulled glass micropipettes (GMP) using super-cooled LN2 (Vieira et al. 2007 Anim. Reprod. Sci. 99, 377-383), with the subsequent transfer of the GMP content into a straw upon warming, for the direct transfer of 4 embryos to female recipients, 1 per recipient, without the embryo evaluation under a stereomicroscope (Vieira et al. 2008 Reprod. Domest. Anim. 43, 314-318). A pregnancy was diagnosed on Day 35; on Day 260 of pregnancy, the female recipient aborted identical 20-kg twin fetuses showing no macro- or microscopic alterations following the necropsy. Samples of donor cells and tissue samples from the female recipient and cloned twins subjected to microsatellite DNA analysis confirmed that the twins were indeed identical clones and derived from the donor cells. The embryo from which the twin pregnancy derived was a hatching blastocyst, vitrified when about half of the embryo, including half of the inner cell mass, was projected outside the zona pellucida. Most likely, the hatching blastocyst was physically and unintentionally bisected during the process of vitrification, warming, or embryo transfer, producing hemi-embryos that later developed into identical twins. This work was supported by the Brazilian National Council for Scientific and Technological Development (CNPq).
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Callesen, H., and P. Holm. "57 DEVELOPMENTAL CHARACTERISTICS OF LATER-STAGE PORCINE EMBRYOS PRODUCED IN VIVO OR IN VITRO." Reproduction, Fertility and Development 28, no. 2 (2016): 158. http://dx.doi.org/10.1071/rdv28n2ab57.
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Kinetics and morphological characteristics during pre-implantation embryo development are well established in most mammals. In porcine, such studies are few because of limited use of in vitro culture, but this has changed in recent years due to increasing use of in vitro production and cloning. Therefore, additional characterisation of especially later stage porcine embryos is needed. Here we studied in vitro development of porcine in vivo- and in vitro-derived zygotes collected from weaned, inseminated sows (slaughtered 2 days after first insemination; in vivo group, n = 112) or produced from immature oocytes from sow ovaries (matured and fertilized: Theriogenology 63, 2040–2052; in vitro group, n = 210). Both types were cultured for 7 days in vitro (Theriogenology 63, 2040–2052) in a time-lapse system (Theriogenology 50, 1285–1299) with images recorded every 0.5 h. Individual embryos were followed and characterised for stage and quality from first cleavage. The following was recorded: (i) zygote: inner (i.e. ooplasma) and outer diameter [i.e. including zona pellucida (ZP)], ZP thickness; (ii) compact morula: areas of compacted inner cells and of cellular debris; (iii) blastocyst: partial or total collapse of blastocoelic cavity and diameter at maximal expansion immediately before hatching. At the 1-cell stage, no difference was found between in vivo v. in vitro zygotes in ZP diameter (approximately 150 µm) and thickness (approximately 15.6 µm). In both groups, cleavage rate was around 65%, but more in vivo (85%) than in vitro (28%) zygotes developed beyond the morula stage. Embryos of both types that did not develop to this stage (n = 212) blocked predominantly at the 1st (50%) or 2nd (21%) cell cycle. Cell cycles were generally shorter in in vivo v. in vitro zygotes from compact morula until the hatched blastocyst stage (mean 128 v. 139 h from the 2-cell stage for in vivo v. in vitro; P < 0.05). Compacted in vivo morulae were 25% larger than in vitro, and the debris area was more than twice as large in in vitro. Hatching occurred after approximately two collapses in both in vivo and in vitro but at a larger ZP diameter in vitro. See Table 1 for further details. This study illustrates differences and similarities between morphology and developmental kinetics of in vivo- and in vitro-derived porcine zygotes, but also how various morphological characteristics indicate some of the possible causes of the reduced developmental ability of in vitro embryos. The in vitro period seems to result in more stressful conditions for the embryos, both during early development, but also during the later stages leading to the hatching process. Thus, further optimization of in vitro culture conditions is still needed in porcine. Table 1.Compact morula (CM) viable cell mass and debris, hatched blastocyst (HB) diameter, and early-hatched blastocyst (EB-XB) and expanded-hatched blastocyst (XB-HB) collapses for in vivo- and in vitro-derived zygotes
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Nims, S., D. Melican, T. Jellerette, R. Butler, and W. Gavin. "114EFFECT OF CYTOCHALASIN B TREATMENT ON VITRIFICATION OF CAPRINE PARTHENOGENIC BLASTOCYSTS." Reproduction, Fertility and Development 16, no. 2 (2004): 179. http://dx.doi.org/10.1071/rdv16n1ab114.
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Production of human recombinant proteins in the milk of transgenic animals has been shown to be a viable production system. Protection of the animal genetics involved is paramount. Vitrification of embryos is a simple, time-efficient way of preserving an animal’s genetics without the formation of damaging ice crystals during the freezing process. Cytochalasin B has been shown to increase the viability of porcine blastocysts by reducing damage to microfilaments and other cytoskeletal components. These experiments utilized caprine parthenogenic blastocysts as a model to compare the viability of parthenotes treated with or without cytochalasin B prior to and during vitrification. Abattoir oocytes were in vitro-matured in M199 with 10% goat serum containing FSH, LH and gentamycin for 18 to 21h. Parthenogenic blastocysts were produced by treating in vitro matured abattoir oocytes with ionomycin for 5min (5μM) and with 6-dMAP (3mM) for 3h followed by culturing in SOF+0.8% BSA for 7 to 8 days at 38°C with 6% O2, 5% CO2, and 89% N2 in a modular incubator chamber. The experimental group was treated with cytochalasin B (5μg/mL)in the culture media for 30 to 45min prior to and thereafter throughout the vitrification process. All blastocysts (both the experimental group and the control group) were washed through two ovum culture media (OCM) droplets for 5min each. The blastocysts were incubated in vitrification solutions 1 and 2 (10% glycerol in OCM and 10% glycerol+20% ethylene glycol in OCM, respectively) for 5min each, followed by vitrification solution 3 (25% glycerol+25% ethylene glycol in OCM). They were then aspirated immediately into a 0.25cc cryopreservation straw, followed by an air bubble, and then a 0.25M sucrose solution in OCM. The straws were immediately plunged into liquid nitrogen and stored at −196°C. One to four days later, straws were thawed in air for 5s at room temperature, then in 22°C water for 15s. After thawing, the contents of the straw were expelled, mixed, held for 5min, and finally placed in OCM for 5min. Recovered embryos were placed in SOF+20% FBS and incubated at 38°C with 5% CO2 in air overnight. Viability was determined by re-expanding and subsequent hatching of the blastocyst. As shown in Table 1, there were no significant differences between re-expansion and hatching of blastocysts with cytochalasin B treatment compared to blastocysts not treated with cytochalasin B. These results suggest that, unlike porcine embryos (Dobrinsky et al., 2000 Biol Reprod 62, 564–570), cytochalasin B treatment does not improve the post-thaw viability of vitrified caprine parthenogenic blastocysts. Table 1
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Amir, Anna Aryani, Jennifer M. Kelly, David O. Kleemann, Zoey Durmic, Dominique Blache, and Graeme B. Martin. "Extracts of forage plants affect the developmental competence of ovine oocytes in vitro." Animal Production Science 59, no. 10 (2019): 1814. http://dx.doi.org/10.1071/an18170.
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Forage plants may contain secondary compounds that disrupt reproduction in ruminants so, as ‘duty of care’, proposed new forage species need to be tested for harmful effects on reproduction before industrial release. We evaluated the effects of Bituminaria bituminosa, Medicago sativa, Chicorium intybus, Trifolium subterraneum, Trifolium pratense, Biserrula pelecinus and Eremophila glabra, on the in vitro developmental competence of ovine oocytes. Crude methanolic extracts of each plant were added to the medium (final concentrations: 0, 50 or 100 μg dry extract per mL) used for in vitro maturation of cumulus-oocyte complexes derived from abattoir-sourced adult ewe ovaries. After in vitro fertilisation, we quantified cleavage rate, blastocyst rate, hatching rate, blastocyst efficiency, and total blastocyst cell number (TCN). Extract from B. pelecinus, at 50 μg/mL concentration, increased cleavage rate at (P < 0.05), and at 100 μg/mL, increased blastocyst rate and efficiency (P < 0.05). The other plant extracts did not affect these measures. TCN was affected by stage of development and treatment, but not by the interaction between stage and treatment. Within treatments, TCN was increased by C. intybus (at both 50 and 100 μg/mL) but decreased by M. sativa (at both 50 and 100 μg/mL; P < 0.05). We conclude that methanolic extracts of forage plants, present during in vitro oocyte maturation, did not disrupt subsequent fertilisation and embryo development until the blastocyst stage. On the contrary, B. pelecinus appears to improve fertilisation and embryo development. Overall, these observations suggest that these plants will not disrupt in vivo oocyte maturation but further testing is still required, especially for the other stages of the reproductive process.
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Kuzmany, A., V. Havlicek, C. Wrenzycki, S. Wilkening, G. Brem, and U. Besenfelder. "76 EFFECT OF CULTURE METHOD ON THE mRNA EXPRESSION BEFORE AND AFTER CRYOPRESERVATION IN BOVINE BLASTOCYSTS." Reproduction, Fertility and Development 23, no. 1 (2011): 143. http://dx.doi.org/10.1071/rdv23n1ab76.
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Blastocyst mRNA expression and cryopreservability are thought to be suitable indicators of embryo quality and developmental competence and have been shown to be affected by production methods and culture systems. The aim of the present study was to assess cryosurvival and levels of mRNA expression of selected genes [occludin, desmocollin 2, solute carrier family 2 member 3 (formerly glucose transporter 3), BAX, BCL xL, heat shock protein A1A (formerly heat shock protein 70.1), aquaporin 3, and DNA methyltransferase 1a] of bovine blastocysts derived by 4 different, established culture methods [in vitro production (IVP); multiple-ovulation embryo transfer (MOET); transfer into the heifer oviducts of gametes (GIFT); or in vitro derived cleaved stage embryos (Days 2–7)]. Linear models were used for the comparison of the relative abundances of the blastocyst mRNA transcripts. Separate 1-way ANOVA were used. The production methods were used as factors, except for the comparisons between pre- and post-cryopreservation, where 2-way ANOVA were used. The level of significance was set at P ≤ 0.05. A significant difference in re-expansion rates was found only at 24 h post-thawing, with significantly higher rates in blastocysts produced in vitro compared to embryos of the Days 2–7 group. Levels of mRNA expression were assessed using RT-qPCR. Before cryopreservation of embryos, no significant inter-group differences were seen. However, significantly more desmocollin 2 mRNA expression was detected in embryos of the MOET group compared with blastocysts derived by the other production methods. Post-cryopreservation, blastocysts of 3 embryo production groups (IVP, MOET, Days 2–7) were available for analysis. Compared with levels of mRNA expression before cryopreservation, re-expanded blastocysts after cryopreservation showed a significant up-regulation of heat shock protein A1A transcripts in all groups, and of solute carrier family 2 member 3 transcripts only in the IVP-derived group. The BAX, BCL-xL, occludin, and desmocollin 2 were significantly up-regulated in embryos of the MOET and IVP groups after cryopreservation, as compared with their counterparts before cryopreservation. None of the culture groups showed any pre- v. post-cryopreservation differences in the aquaporin 3 and the DNA methyltransferase 1 mRNA levels. Blastocysts derived by transfer of in vitro derived cleaved stage embryos into the oviduct of synchronised heifers (Days 2–7) did not show any pre- v. post-cryopreservation differences in the mRNA levels of any of the assessed genes. These results merit further investigation. After the process of cryopreservation and thawing, re-expanded embryos of the MOET and IVP groups do increase their mRNA levels to prepare for hatching and further development.
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Popelková, M., P. Chrenek, J. Pivko, A. V. Makarevich, E. Kubovičová, and J. Kačmárik. "Survival and ultrastructure of gene-microinjected rabbit embryos after vitrification." Zygote 13, no. 4 (November 2005): 283–93. http://dx.doi.org/10.1017/s096719940500331x.
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Morphological signs of injury and subsequent regeneration following vitrification of either rabbit gene microinjected (Gene-Mi) or intact in vitro cultured embryos derived from in vivo fertilized eggs were evaluated by post-warming recovery in culture and analysed by transmission electron microscopy (TEM). The percentages of vitrified/warmed Gene-Mi embryos that reached the blastocyst stage (69%) and hatched (57%) did not differ significantly from those of intact embryos (78% and 56%, respectively). In contrast, in vitro development of embryos to the blastocyst stage among non-vitrified intact (96%) and Gene-Mi (90%) embryos compared with both the intact vitrified (78%) and Gene-Mi vitrified (69%) groups, as well as hatching rate (94%, 90% vs 56%, 57%, respectively) varied significantly (p<0.001). Observations by TEM showed that the vitrified/warmed intact or Gene-Mi embryos without post-culture displayed severe degenerative changes among their cells. During 24 h of culture a proportion of the embryos were able to regenerate and complete the compaction process. Nevertheless the signs of previous injury were retained, such as swollen cytoplasmic organelles and remaining cellular debris in the perivitelline space. These observations indicate that the procedure of gene Mi does not siginificantly compromise embryo tolerance to cryopreservation and post-warming developmental ability. Severe changes in embryo morphology, observed at the ultrastructural level, can be attributed to a direct influence of the vitrification process rather than to the Mi procedure itself.
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Campos-Chillon, L., T. Suh, E. Carnevale, and G. Seidel. "241 EFFECT OF MEIOTIC ARREST BY ROSCOVITINE AND SUBSEQUENT IVM TIME ON DEVELOPMENTAL COMPETENCE OF IMMATURE BOVINE OOCYTES." Reproduction, Fertility and Development 17, no. 2 (2005): 271. http://dx.doi.org/10.1071/rdv17n2ab241.
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Maintaining immature bovine oocytes at the germinal vesicle stage by inhibiting M-phase promoting factor (MPF) activity is a reversible process when using roscovitine, and this can improve cytoplasmic maturation in vitro. However, optimum meiotic arrest times and subsequent IVM times have not been determined, so we evaluated the developmental competence of oocytes in relation to these times. Two by two factorial treatments consisting of 2 arrest times (8 h, 16 h) and 2 subsequent IVM times (16 h, 22 h) plus a control were replicated 6 times in this study. Semen from two bulls was used three times. Chemically defined media (CDM) were used throughout (Olson and Seidel 2000 J. Anim. Sci. 78, 152–157). Slaughterhouse-derived oocytes were arrested in meiosis in 1 mL of CDM-M without any hormones, but containing 50 μM roscovitine and 0.5% fatty acid-free (FAF)-BSA under 5% CO2 in air at 38.5°C. After 8 or 16 h of meiotic arrest, oocytes were washed and matured in 1 mL of CDM-M containing 0.5% FAF-BSA, 2 mM glucose, 50 ng/mL EGF, 15 ng/mL NIDDK-oFSH-20, 1 μg/mL USDA-LH-B-5, 1 μg/mL E2, and 0.1 mM cysteamine for 16 or 22 h under 5% CO2 in air at 38.5°C. Oocytes for the control group were matured in 1 mL of the CDM-M with hormones for 22 h. Ten oocytes from each group were fixed after IVM, stained with orcein, and evaluated for maturation to MII. For fertilization, motile sperm recovered from frozen-thawed semen were co-incubated for 18–20 h with ∼20 oocytes/group at a final sperm concentration of 0.5 × 106 sperm/mL in F-CDM. Presumptive zygotes were cultured in 0.5 mL of CDM-1 for 2.5 days and then in CDM-2 for 5.5 days in 5% CO2, 5% O2, 90% N2 in a humidified incubator at 39°C. Cleavage rates were evaluated after culture in CDM-1. Blastocyst rate, blastocyst stage (5 = early, 6 = full, 6.5 = expanding, 7 = expanded, 7.5 = hatching, 8 = hatched), and embryo quality (1 = excellent, 2 = good, 3 = fair, 4 = poor) were evaluated after CDM-2. Data were subjected to ANOVA; the arc sin transformation was used for percentage data, and least-squares means are presented. There were no significant differences in % cleavage (Cle), cell stage, or blastocyst quality among treatments (P > 0.1). However, meiotic arrest of oocytes for 16 h and subsequent IVM for 16 h improved embryo development to blastocysts compared to other roscovitine treatments (Table 1, P < 0.05). A bull effect for % blastocysts was observed, 19.9% and 25.2% for bulls 1 and 2, respectively (P < 0.08). Blastocyst production was improved by shortening oocyte maturation time from 22 to 16 h, when meiotic progression was previously inhibited for 16 h with roscovitine. Table 1. Effect of meiotic arrest and IVM times on oocyte maturation and embryo development
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Fanti, T., N. M. Ortega, R. Garaguso, M. J. Franco, C. Herrera, and A. A. Mutto. "239 USE OF CYCLIC ADENOSINE MONOPHOSPHATE MODULATORS IN IN VITRO PRODUCTION OF BOVINE EMBRYOS." Reproduction, Fertility and Development 27, no. 1 (2015): 209. http://dx.doi.org/10.1071/rdv27n1ab239.
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In vitro embryo production systems (IVP) try to emulate and enhance molecular events that occur in in vivo reproductive systems in order to increase, not only the number of embryos generated, but also their quality. Despite advances, IVP processes are still inefficient compared with in vivo systems. Several studies have attributed this deficiency to a lack of oocyte competence due to spontaneous premature resumption of meiotic maturation in the oocyte following the removal from its follicular environment. Therefore, our objective was to increase oocyte competence avoiding premature resumption of meiosis by using cyclic adenosine monophosphate modulators. Cumulus-oocyte complexes (COC) were obtained from ovaries of slaughterhouses, washed, and randomly allocated in 2 culture systems. Oocytes in the control group (IVM) were cultured for a period of 24 h in basal medium TCM-199 with EGF (1 µg mL–1) supplemented with rhFSH (25 mIU mL–1). Oocytes in the biphasic in vitro maturation (b-IVM) group were cultured for 2 h in a basal medium supplemented with a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX, 500 µM), and an activator of adenylate cyclase (forskolin, 100 µM). Subsequently, COC were washed and cultured in basal medium supplemented with cilostamide (20 µM) and rhFSH (25 mIU mL–1) for 24 h. Maturation rates were analysed and IVF was performed with a dose of 1 × 106 sperm cells mL–1 in IVF-SOF medium. The presumptive zygotes were cultured in continuous-single-culture medium (Irvine) supplemented with 8 mg mL–1 of BSA until they reached the blastocyst stage. No significant differences in maturation, cleavage, and cryotolerance were observed between b-IVM and IVM groups (P > 0.05; Table 1). This study showed that b-IVM produced a significant increase in IVP compared with the control (IVM) at Days 7 and 8 (P < 0.01). Blastocyst hatching rate was significant (P < 0.05) for both treatment and day of analysis. The b-IVM group yielded an increase of 10 and 7.5% at Days 7 and 8, respectively, of IVP. The biphasic maturation showed an improvement in quality regarding the control group, in the timing analysis of production, and hatching percentages, and these results show that the use of cyclic adenosine monophosphate modulators in the oocyte maturation process enhances oocyte competence, which is reflected in increased productivity and embryo quality. We propose this treatment as an alternative to the standard protocols currently used in IVP of bovine embryos. Table 1.Effect of treatment on maturation, cleavage, and cryotolerance
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Zhang, Yan, Jing Guo, Xiao Wei Nie, Zi Yue Li, Yu Meng Wang, Shuang Liang, and Suo Li. "Rosmarinic acid treatment during porcine oocyte maturation attenuates oxidative stress and improves subsequent embryo development in vitro." PeerJ 7 (June 18, 2019): e6930. http://dx.doi.org/10.7717/peerj.6930.
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Background In vitro maturation (IVM) of oocytes has been widely used in the field of assisted reproductive technology. However, oocytes can be injured by oxidative stress during the process of IVM. Methods The present study was designed to evaluate the influences of rosmarinic acid (RA) on the IVM of porcine oocytes and the subsequent development of early-stage embryos as well as its underlying mechanisms. Various concentrations of RA (5 µM, 10 µM, and 25 µM) were treated with porcine oocyte maturation medium during the period of IVM. Results and Discussion The results showed that 5 µM RA treatment during the period of porcine oocyte IVM improves blastocyst quality and hatching ability after parthenogenetic activation. Furthermore, the presence of RA during the period of IVM dramatically improved the total number of cells after somatic cell nuclear transfer compared to the number of cells in the control group. Notably, RA treatment during the period of porcine oocyte IVM decreased intracellular reactive oxygen species generation not only in oocytes but also in cumulus cells. Further analysis showed that the intracellular free thiols levels in the oocytes were enhanced by treatment with RA during the period of porcine oocyte IVM compared to the free thiols levels in the control groups. These results indicate that RA improves the developmental competence of porcine oocytes during the IVM period by attenuating oxidative stress.
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Nguyen, Khanh Van, Thi Kim Yen Pham, Thi Au Hoang, Quang Minh Luu, and Doan Lan Pham. "Influence of fetal calf serum on the production of bovine embryos in vitro." Ministry of Science and Technology, Vietnam 65, no. 1 (March 15, 2023): 76–80. http://dx.doi.org/10.31276/vjste.65(1).76-80.
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The aim of the present study was to evaluate the effect of foetal calf serum (FCS) on the production of bovine embryo in vitro. We conducted two experiments: (1) to evaluate the effect of FCS on the production of bovine embryo in vitroand (2) to evaluate the effect of duration of FCS supplementation on the production of bovine embryo in vitro. In experiment 1, we divided mature bovine oocytes after in vitro fertilization into four embryo culture media: CR1aa, CR1aa+FCS, SOFaa, and SOFaa+FCS. Embryos cultured in CR1aa and SOFaa media supplemented with FCS resulted in a higher cleavage and blastocyst formation rates than when cultured in CR1aa and SOFaa without FCS (74.22 and 82.96% vs. 71.6 and 70.62%; 4.11 and 22.31% vs. 16.98 and 34.51%; respectively, p<0.05). The percentage of hatching blastocysts was highest when embryos were cultured in SOFaa medium supplemented with FCS (10.54%; p<0.05). Embryos cultured in the medium with FCS had a higher the average number of cells/blastocyst than the medium without FCS. The SOFaa+FCS group had a higher average number of cells/embryos than the other groups (94.32;p<0.05). In experiment 2, we added FCS to bovine embryo culture in vitro at the following times: 0 h, 48 h, and 120 h after fertilization. The rate of oocyte cleavage, blastocyst formation, and hatching blastocyst rate of the 0-h group were higher than that of 48-h and 120-h groups (82.96 vs 70.5 and 70.24%; 34.51 vs 24.21 and 23.18%; 10.5 vs 6.12 and 4.13%; respectively, p<0.05). However, there was no difference in the average number of cells/blastocysts between the groups. In conclusion, the addition of FCS to embryo culture media improved the efficiency of bovine embryo production in vitro and in vitro bovine embryo quality. The appropriate time to add FCS to bovine embryo culture in vitro was 0 h after fertilization.
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García-Jiménez, Maria, Klaus Rink, Enric Mestres, Ivette Vanrell, Gloria Calderón, and Nuno Costa-Borges. "Evaluation of the impact of laser-assisted hatching techniques on the hatching process of mouse blastocysts using time-lapse microscopy." F&S Science 2, no. 1 (February 2021): 43–49. http://dx.doi.org/10.1016/j.xfss.2020.12.004.
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Yamazaki, Kimie, Rika Suzuki, Eriko Hojo, Shunzo Kondo, Yoshihiro Kato, Ken Kamioka, Motonori Hoshi, and Hitoshi Sawada. "Trypsin-like Hatching Enzyme of Mouse Blastocysts: Evidence for Its Participation in Hatching Process before Zona Shedding of Embryos6. (embryo/hatching enzyme/protease/trypsin/strypsin)." Development, Growth and Differentiation 36, no. 2 (April 1994): 149–54. http://dx.doi.org/10.1111/j.1440-169x.1994.00149.x.
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McMillan, W. H., A. J. Peterson, S. F. Cox, S. J. Pearson, and M. J. Donnison. "Reproductive performance during early pregnancy in cattle with contrasting early pregnancy rates." BSAP Occasional Publication 26, no. 2 (September 2001): 283–88. http://dx.doi.org/10.1017/s0263967x00033796.
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AbstractCauses of variation amongst cattle within a herd in their ability to initiate and maintain pregnancy are largely unknown. An experimental animal resource has recently been established to understand the biology of early reproductive performance. This paper summarises the results achieved during the establishment phase and from several experiments aimed at determining the physiological basis of the difference between sub-herds of contrasting pregnancy rates on Day 60. Each of 155 contemporary yearling heifers received 2 in vitro-produced embryos on 6 separate occasions during a 26-month period. Sixty days after transfer, pregnancy and twinning rates were determined ultrasonically, pregnancies terminated and the process repeated. The interval between successive transfers was greater than 100 days. Heifers were ranked on their aggregate pregnancy rate performance after 6 rounds of transfer, and the highest (High) and lowest (Low) 25 were retained. Differences in reproductive performance during the establishment phase of the herd are reported. In addition, several subsequent experiments examined ovarian follicle turnover and progesterone levels during an oestrous cycle, early embryo development after either AI or embryo transfer, and protein, interferon tau and ubiquitin-cross-reactive protein levels in uterine luminal flushings.Pregnancy rates were 7-folder higher in the High sub-herd (76 vs. 11%), with much of this difference apparent by Day 25. The proportion of heifers observed in standing oestrus prior to embryo transfer and the interval from the end of synchronisation treatment to the onset of oestrus were similar in the sub-herds. Oestrous cycle length, ovarian follicular dynamics and progesterone profiles during the oestrous cycle were also similar. More conceptuses had elongated by Day 14 in the High sub-herd (67 vs. 14%, P<0.05), which also tended to have a higher pregnancy rate after artificial insemination (52 vs. 29, P<0.1). Total protein in flushings from the uterus was similar in the sub-herds on Day 14 and Day 17. Conceptuses in the High sub-herd were longer on Day 17 following embryo transfer (6.5 vs. 4.8, P<0.05). Interferon-tau levels were higher in the High sub-herd (25.9 vs. 16.1, P<0.01), although ubiquitin cross-reactive protein levels were also higher in the High sub-herd, but this difference just failed to reach significance. We conclude that: 1. Most of the difference in sub-herd pregnancy rate occurs within 3 weeks of ET; 2. Ovarian factors are unlikely to contribute to the difference; 3. Major differences occur after blastocyst hatching and probably depend upon a differing endometrial environment before Day 14; 4. Differences in the ability of the uterine milieu to stimulate the expression of interferon-tau may be responsible for the differences in pregnancy rate; 5. The two sub-herds are a unique experimental resource for understanding early pregnancy in cattle following either AI or ET.
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Rogers, K. D., B. A. Foster, E. J. Guiterrez, F. A. Diaz, and K. R. Bondioli. "41 Effects of Dimethyl Sulfoxide- or Glycerol-Based Vitrification Protocols on Zona Pellucida Hardening in Mature Bovine Oocytes." Reproduction, Fertility and Development 30, no. 1 (2018): 160. http://dx.doi.org/10.1071/rdv30n1ab41.
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Zona pellucida hardening is a natural process that occurs after oocyte fertilization to prevent polyspermic fertilization and to protect embryonic development. Pre-fertilization hardening of the zona pellucida, however, decreases fertilization rates. Cryoprotectants have also been shown to negatively affect fertilization rates, one possible mechanism of which being through zona hardening. This experiment was conducted to determine the effect of different cryoprotectants on hardening of the zona pellucida of mature bovine oocytes. Oocytes were collected by ovum pick-up (OPU) by transvaginal ultrasound guided aspiration (TUGA) from mixed-breed cows. After collection, oocytes were randomly assigned to 3 cryoprotectant treatment groups: dimethyl sulfoxide (DMSO), glycerol, or PBS (control). Drops (50 µL) of each vitrification solution were placed under mineral oil. Vitrification solution 1 (VS1) contained 10% ethylene glycol (EG), either 10% DMSO or glycerol, and 0.5 M sucrose. Vitrification solution 2 (VS2) contained 20% EG, 20% DMSO or glycerol, and 0.5 M sucrose. All oocytes were held in VS1 for 5 min before being transferred to VS2 for 45 s. All oocytes were washed in a common dilution solution (80% PBS, 20% calf serum, 0.025 M sucrose) for 5 min. Next, oocytes were moved to 50-µL drops of protease solution (0.1% protease) under mineral oil. Control oocytes were held in PBS for ~10 min before entering the protease solution to represent the same period as the vitrification procedure. The oocytes were observed until the zonae pellucidae were completely digested and times were recorded for each oocyte. This experiment included 4 replicates with a total of 88 oocytes used, 32 each in DMSO and glycerol and 24 in PBS. The data were analysed using ANOVA. The DMSO group had the lower mean zona digestion time out of the 2 cryoprotectants at 15.75 min and glycerol had the highest mean digestion time at 19.3 min. The control group (PBS) had the lowest mean of the 3 treatments at 12.7 min. The differences between DMSO and glycerol, and between DMSO and PBS were not significant (P = 0.0654 and 0.1073, respectively). However, both glycerol versus PBS and the average of DMSO and glycerol versus PBS were significantly different (P-value = 0.0053 and 0.0119, respectively). These results suggest that glycerol hardens the zona pellucida more than DMSO or PBS; however, there is not enough evidence to determine whether DMSO hardens the zona pellucida compared with PBS. This would suggest that, in relation to zona hardening and ensuring proper fertilization, glycerol-based cryoprotectants may be a better option than DMSO-based ones. Further, these results may be important in embryo vitrification as zona hardening may prevent blastocyst hatching, suggesting that glycerol-based cryoprotectants should be investigated as the optimal cryoprotectant here also.
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Stinshoff, H., K. Brüning, A. Hanstedt, D. Müller, S. Wilkening, and C. Wrenzycki. "117 EFFECT OF DIFFERENT CRYOPRESERVATION METHODS ON THE QUALITY OF IN VITRO-PRODUCED BOVINE EMBRYOS." Reproduction, Fertility and Development 22, no. 1 (2010): 217. http://dx.doi.org/10.1071/rdv22n1ab117.
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In vitro production (IVP) of bovine embryos has been greatly improved over the last couple of years. However, only one-third of the total number of embryos transferred worldwide are of in vitro origin. The IVP embryos still show remarkable differences compared with their in vivo-derived counterparts (i.e. bovine embryos produced in vitro are more sensitive to cryopreservation). So far, vitrification seems to be the most promising method to cryopreserve in vitro-produced bovine embryos. The aim of this study was to determine the effect of 2 different cryopreservation methods on the quality of in vitro-produced bovine embryos at the molecular level using a sensitive RT-qPCR assay. Bovine blastocysts were produced using abattoir ovaries and a standard protocol for IVP (Wrenzycki et al. 2001). They were randomly vitrified employing PBS plus ethylene glycol and DMSO or cryopreserved using a programmable freezer and 1.5 M ethylene glycol. After thawing, embryos from both groups were cultured for 48 h. After 24 h of culture re-expansion rates were documented, and after 48 h hatching rates were documented. After hatching, blastocysts were stored at -80°C for subsequent RT-qPCR analysis. The following gene transcripts known to play important roles during preimplantation development were analyzed: HSP70, GLUT-1, GLUT-3, E-CAD, ZO-1, DNMT3a, IFNτ, DCII. Re-expansion rates were 74.7% (68/91) and 75.0% (87/116) for vitrified and conventionally cryopreserved blastocysts, and 57.1% (52/91) and 55.2% (64/116) of re-expanded embryos hatched. The relative abundances of HSP70, GLUT-1, and ZO-1 transcripts were significantly affected in both groups of cryopreservation compared with the control group (hatched blastocysts without cryopreservation). Conventional cryopreservation had a significant effect on the amount of GLUT-3, DNMT3a, and IFNτ mRNA, whereas vitrification significantly affected DCII transcripts. E-CAD mRNA expression was similar in all groups of embryos. These results suggest that not only the cryopreservation process itself but also the method used to freeze the embryos had a significant influence on the mRNA expression of developmentally important genes in hatched bovine blastocysts. The support of the H.W. Schaumann Stiftung (Germany) and Gynemed Medizinprodukte GmbH & Co. KG (Germany) is gratefully acknowledged.
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Oliveira, Clara Slade, Naiara Zoccal Saraiva, Maria Helena Coelho Cruz, Bruna Mazeti, Leticia Zoccolaro Oliveira, Flavia Lombardi Lopes, and Joaquim Mansano Garcia. "HDAC inhibition decreases XIST expression on female IVP bovine blastocysts." REPRODUCTION 145, no. 1 (January 2013): 9–17. http://dx.doi.org/10.1530/rep-11-0343.
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During initial development, both X chromosomes are active in females, and one of them must be silenced at the appropriate time in order to dosage compensate their gene expression levels to male counterparts. Silencing involves epigenetic mechanisms, including histone deacetylation. Major X chromosome inactivation (XCI) in bovine occurs between hatching and implantation, althoughin vitroculture conditions might disrupt the silencing process, increasing or decreasing X-linked gene expression. In this study, we aimed to address the roles of histone deacetylase inhibition by trichostatin A (TSA) on female preimplantation development. We tested the hypothesis that by enhancing histone acetylation, TSA would increase the percentage of embryos achieving 16-cell stage, reducing percentage of embryos blocked at 8-cell stage, and interfere with XCI in IVF embryos. We noticed that after TSA treatment, acetylation levels in individual blastomeres of 8–16 cell embryos were increased twofold on treated embryos, and the same was detected for blastocysts. Changes among blastomere levels within the same embryo were diminished on TSA group, as low-acetylated blastomeres were no longer detected. The percentage of embryos that reached the 5th cleavage cycle 118 h after IVF, analyzed by Hoechst staining, remained unaltered after TSA treatment. Then, we assessedXISTandG6PDexpression in individual female bovine blastocysts by quantitative real-time PCR. Even thoughG6PDexpression remained unaltered after TSA exposure,XISTexpression was eightfold decreased, and we also detected a major decrease in the percentage of blastocysts expressing detectableXISTlevels after TSA treatment. Based on these results, we conclude that HDAC is involved on XCI process in bovine embryos, and its inhibition might delay X chromosome silencing and attenuate aberrantXISTexpression described for IVF embryos.
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Sidrat, Tabinda, Abdul Aziz Khan, Myeong-Don Joo, Lianguang Xu, Marwa El-Sheikh, Jong-Hyuk Ko, and Il-Keun Kong. "Extracellular vesicles improve embryo cryotolerance by maintaining the tight junction integrity during blastocoel re-expansion." Reproduction 163, no. 4 (April 1, 2022): 219–32. http://dx.doi.org/10.1530/rep-21-0320.
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Cryopreservation is a process in which the intact living cells, tissues, or embryos are preserved at subzero temperatures for preservation. The cryopreservation process highly impacts the survival and quality of the in vitro-produced (IVP) embryos. Some studies have highlighted the use of oviduct extracellular vesicles (EVs) to improve the cryotolerance of IVP embryos but the mechanism has not been well studied. The present study unravels the role of in vitro cultured bovine oviduct epithelial cells-derived EVs in improving the re-expansion and hatching potential of thawed blastocysts (BLs). The comparison of cryotolerance between synthetic oviduct fluid (SOF) and SOF + EVs-supplemented day-7 cryopreserved BLs revealed that the embryo’s ability to re-expand critically depends on the intact paracellular sealing which facilitates increased fluid accumulation during cavity expansion after shrinkage. Our results demonstrated that BLs cultured in the SOF + EVs group had remarkably higher re-expansion (67.5 ± 4.2%) and hatching rate (84.8 ± 1.4%) compared to the SOF group (53.4 ± 3.4% and 63.9 ± 0.9%, respectively). Interestingly, EVs-supplemented BLs exhibited greater influence on the expression of core genes involved in trophectoderm (TE) maintenance, formation of tight junction (TJ) assembly, H2O channel proteins (aquaporins), and Na+/K+ ATPase alpha 1. The EVs improved the fluid flux and allowed the transport of H2O into an actively re-expanded cavity in EVs-cultured cryo-survived BLs relative to control BLs. Our findings explored the function of EVs in restoring the TE integrity, improved the cell junctional contacts and H2O movement which helps the blastocoel re-expansion after thawing the cryopreserved BLs.
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Herrera, E. Y., C. de Frutos, R. Laguna-Barraza, A. Gutierrez-Adan, and D. Rizos. "88 EFFECT OF VITRIFICATION PROCEDURE ON SURVIVAL RATE OF BOVINE EMBRYOS PRODUCED IN VITRO." Reproduction, Fertility and Development 23, no. 1 (2011): 149. http://dx.doi.org/10.1071/rdv23n1ab88.
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Vitrification as a cryopreservation method has many advantages compared with slow freezing. Many variables in the vitrification process exists that influence the survival rates of vitrified oocytes and embryos. These include the cryoprotectants (type, concentration, and duration of exposure), the temperature of the vitrification solution at exposure, the device used for vitrification, and the quality and developmental stage of embryos. It is worthwhile to mention that vitrification protocols successfully used in bovine oocytes and embryos have been used also with human oocytes and embryos. Vitrification is relatively simple, requires no freezing equipment, and relies on the placement of the embryos in a very small volume of vitrification medium that must be cooled at extreme rates not obtainable in regular enclosed straws. The aim of the present study was to evaluate the efficiency of 4 different vitrification protocols on the survival rate of in vitro produced (IVP) bovine embryos. Blastocysts were produced by a standard IVP procedure following in vitro maturation, fertilization, and culture in synthetic oviduct fluid supplemented with 5% fetal calf serum (FCS). On Day 7 (Day of IVF = Day 0), a total of 297 blastocysts were vitrified using (i) the open pulled straw (OPS) in 20% DMSO and 20% ethylene glycol (EG) in a basal medium of TCM-199 with HEPES supplemented with 20% FCS; (ii) the modified OPS, in 20% DMSO, 20% EG, and 0.5 M sucrose in a basal medium of phosphate buffer saline (PBS) supplemented with 20% FCS; (iii) the cryoloop, in 15% DMSO, 15% EG, 10 mg mL–1 Ficoll 70, and 0.65 M sucrose in a basal medium of PBS supplemented with 20% FCS; and (iv) in 0.25 straws in 20% glycerol, 20% EG, 0.3 M sucrose, 3% polyethylene glycol, and 0.3 M xylose in a basal medium of PBS. After warming, embryos were placed in culture for additional 24 h. Re-expansion and hatching rates were measured at 2 and 24 h after warming. Data were analysed by 1-way ANOVA. At 2 h post-warming, the re-expansion of blastocysts vitrified with cryoloop was significantly higher compared with OPS, modified OPS, and the 0.25 straw methods (54.08 ± 15.53 v. 10.40 ± 3.00, 22.67 ± 9.20, and 8.82 ± 2.15, respectively; P ≤ 0.028). At 24 h post-warming, only embryos from cryoloop and modified OPS were still alive with a survival rate of embryos vitrified with cryoloop significantly higher than that of those vitrified with modified OPS (48.45 ± 17.56 v. 3.75 ± 3.75, respectively; P ≤ 0.007). Hatching rates at 24 h post-warming were not different between cryoloop and modified OPS groups (5.63 ± 4.40 and 1.25 ± 1.25, respectively). These results clearly demonstrate that embryo cryotolerance is affected by the method used for cryopreservation. Moreover, cryoloop vitrification was found to be more effective than OPS and 0.25 straw methods for the cryopreservation of bovine embryos.
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Luz, M. R., C. C. Holanda, J. J. Pereira, N. S. Teixeira, R. Vantini, P. M. C. Freitas, A. E. P. Salgado, S. B. Oliveira, C. R. F. Guaitolini, and M. C. Santos. "99 SURVIVAL RATE AND IN VITRO DEVELOPMENT OF IN VIVO-PRODUCED AND CRYOPRESERVED DOG EMBRYOS." Reproduction, Fertility and Development 22, no. 1 (2010): 208. http://dx.doi.org/10.1071/rdv22n1ab99.
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Dogs have been used as an experimental model for human genetic diseases and for research applied to endangered Canidae. Moreover, application of assisted reproductive technologies (ART) by dog breeders is increasing. The aim of this study was to evaluate the survival rate and in vitro development of in vivo-produced and cryopreserved dog embryos. Seven cross-bred bitches were submitted to ovariohysterectomy 12 days after first mating or artificial insemination, and embryos were recovered by uterine horn flushing with 30 mL of PBS/horn (Nutricell, Campinas, SP, Brazil). Grade 1 and 2 morulae (MO; n = 7; IETS) and blastocysts (BL; n = 14) were frozen. For freezing, embryos were immersed in glycerol 10% (GLY; Nutricell, Campinas, SP, Brazil) or ethylene glycol 3,0 M (EG; Nutricell, Campinas, SP, Brazil) for 10 min. Straws were placed in the machine at -7.0°C (TK 3000, Uberaba, MG, Brazil), and equilibrated for 2 min. Seeding was performed at -7.0°C, and another equilibrium period of 2 min was performed. A cooling rate of -0.5°C/min until -32.0°C was used. Embryos were stored in N2L until thawing. Prior to in vitro culture, embryos were removed from N2L, kept at room temperature for 10 s, and put in a water bath at 25°C for 20 s. Embryos frozen in GLY were washed for 5 min in each thawing solution for cryoprotectant removal (0.6 M sacarose + glycerol 5%; 0.6 M sacarose + glycerol 2.5% and 0.6 M sacarose; Nutricell, Campinas, SP, Brazil). After that, embryos were washed 10 times in holding solution (Holding Plus, Bioniche, Pullman, WA, USA). Embryos frozen in EG were kept at room temperature for 10 s, put in a water bath at 25°C for 20 s, and were directly washed 10 times in holding solution. Comparison among groups was performed by ANOVA. After thawing, 9/11 (81.8%) embryos frozen in EG had rupture of zona pelucidae and 2/11 (18.2%) were intact, whereas 9/10 (90.0%) embryos frozen in GLY were intact (P < 0.05). All intact embryos (n = 11) were morphologically normal and were transferred to SOF medium (Nutricell, Campinas, SP, Brazil), cultured for 168 h at 38.3°C, and evaluated at 24-h intervals. After the last evaluation, for both cryoprotectants, hatching rate was 0.0%, but all embryos were morphologically normal. The results of this study suggest that dog embryos have a high in vitro survival rate in standard protocols used for mammalian embryo cryopreservation when glycerol 10% is used as cryoprotectant and that a long period of culture (possibly more than 10 days) is required for dog embryo hatching. On the other hand, this long period for in vitro hatching may reflect the delayed hatching and implantation that occurs in vivo in this species. Financial by FAPES/MCT/CNPq/CT-INFRA n. 19/2006; Processo n. 36600737/2007, ES- Brasil.
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Pathak, Madhulika, Vani Venkatappa, Surendra Sharma, and Polani B. Seshagiri. "Expression of IL-1β and implantation serine proteases is required for mouse blastocyst hatching." Reproduction, November 2020. http://dx.doi.org/10.1530/rep-20-0376.
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Mammalian blastocyst hatching is critically an indispensable process for successful implantation. One of the major challenges in IVF clinics is to achieve superior embryonic development with intrinsically potent hatching-competent blastocyst. However, the molecular regulation of hatching phenomenon is poorly understood. In this study, we examined the expression and function of one of the cytokines, IL-1β during blastocyst hatching in the mouse. In particular, the expression of IL-1β (Interleukin-1β), IL-1ra (Interleukin-1 receptor antagonist) and their functional receptor IL-1rt1 (Interleukin 1 receptor type-1) in morulae, zona intact- and hatched- blastocysts was studied. Supplementation of IL-1β to cultured embryos accelerated blastocyst development with improved hatching (treated: 89.6 ± 3.6% vs untreated: 65.4 ± 4.1%). When embryos were treated with IL-1ra, blastocyst hatching was decreased (treated: 28.8 ± 3.1% vs untreated: 67.5 ± 3.8%). Moreover, IL-1β and IL-1ra influenced the expression of hatching enzymes viz., implantation serine proteases (ISP 1 and ISP 2). While IL-1β increased the embryonic mRNA expression of ISPs (ISP1: 2-4; ISP2: 9-11 fold), IL-1ra decreased expression. The protein localization studies revealed increased nuclear presence predominantly of ISP 2 in IL-1β treated blastocysts. This is the first report to show the functional significance of embryonic IL-1β in regulating hatching-associated proteases, particularly ISP2. These findings have implications in our understanding of molecular regulation of blastocyst hatching and implantation failure in other species including humans.
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Yang, Guangping, Jianhua Chen, Yanni He, Hui Luo, Hongxia Yuan, Liangliang Chen, Lingli Huang, et al. "Neddylation Inhibition Causes Impaired Mouse Embryo Quality and Blastocyst Hatching Failure Through Elevated Oxidative Stress and Reduced IL-1β." Frontiers in Immunology 13 (July 4, 2022). http://dx.doi.org/10.3389/fimmu.2022.925702.
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Mammalian blastocyst hatching is an essential prerequisite for successful embryo implantation. As the rate-limiting step of current assisted reproductive technology, understanding the key factors regulating blastocyst hatching would be significantly helpful to improve the performance of the assisted reproductive practice. In early embryo development, the fine-tuned elimination of maternal materials and the balanced protein turnover are inevitable for the competent to hatch and implant into endometrium. Neddylation, a ubiquitination-like protein modification, has been shown to be involved in oocyte maturation and early embryo development. In this study, aiming to discover an unknown role of neddylation in the blastocyst hatching process, we provided functional evidence of neddylation in mammalian embryo quality and blastocyst hatching. Treatment with MLN4924, a specific neddylation inhibitor, lowered the embryo quality and dramatically reduced the hatching rate in mouse blastocysts. The transcriptional profile showed the upregulation of oxidative stress-related genes and aberrant expression of immune-related genes. The elevated oxidative stress was validated by qPCR and markers of apoptosis, DNA damage, reactive oxygen species, and cytoskeleton. Moreover, we found the secreted IL-1β level was reduced in an NF-κB-independent manner, leading to the final poor embryo quality and blastocyst hatching failure. This is the first report of neddylation being of great importance in the mammalian blastocyst hatching process. Further investigations uncovering more detailed molecular mechanisms of neddylation regulation in blastocyst hatching would greatly promote not only the understanding of this crucial biological process but also the clinical application in reproductive centers.
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Liu, Y., C. Jones, and K. Coward. "P–182 The mechanism of mouse embryo hatching and the impact of laser drilling the zona pellucida: an RNA sequencing study." Human Reproduction 36, Supplement_1 (July 1, 2021). http://dx.doi.org/10.1093/humrep/deab130.181.
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Abstract Study question What is the mechanism of embryo hatching? Will laser-assisted zona pellucida (ZP) drilling alter the embryonic transcriptome? Summary answer Hatching is an ATP-dependent process. Hatching is also associated with Rho-mediated signaling. Laser-assisted ZP drilling might cause alternation in embryo metabolism. What is known already Embryo hatching is a vital process for early embryo development and implantation. Animal data suggests that hatching is the result of multiple factors, such as mechanical pressure, protease activation, and the regulation of maternal secretions. However, little is known about the regulatory signaling mechanisms and the molecules involved. In addition, despite the extensive use of laser-assisted ZP drilling in the clinic, the safety profile of this technique at molecular level is very sparse. The impact of this technique on the embryonic transcriptome has not been studied systematically. Study design, size, duration Eighty mouse embryos were randomly divided into a laser ZP drilling group (n = 40) and an untreated group (n = 40). After treatment, embryos were cultured in vitro for two days. Then, hatching blastocyst (n = 8) and pre-hatching blastocyst (n = 8) from the untreated group, and the hatching blastocyst from the treatment group (n = 8) were processed for RNA sequencing (RNA-seq). Participants/materials, setting, methods Cryopreserved 8-cell stage mouse embryos (B6C3F1 × B6D2F1) were thawed, and a laser was used to drill the embryo ZP in the treatment group. Next, the treated and untreated embryos were individually cultured in vitro to the E4.5 blastocyst stage. The resulting blastocysts were lysed individually and used for subsequent cDNA library preparation and RNA-seq. Following data quality control and alignment, the RNA-seq data were processed for differentially expressed gene analysis and downstream functional analysis. Main results and the role of chance According to the RNA-seq data, 275 differentially expressed genes (DEGs) (230 up-regulated and 45 down-regulated, adjusted P < 0.05) were identified when comparing hatching and pre-hatching blastocysts in the control groups. Analysis suggested that the trophectoderm is the primary cell type involved in hatching, and revealed the potential molecules causing increased blastocyst hydrostatic pressure (Aqp3 and Cldn4). Functional enrichment analysis suggested that ATP metabolism and protein synthesis were activated in hatching blastocysts. DEGs were found to be significantly enriched in several gene ontology terms, particularly in terms of the organization of the cytoskeleton and actin polymerisation (P < 0.0001). Furthermore, according to QIAGEN ingenuity pathway analysis results, Rho signaling was implicated in blastocyst hatching (Actb, Arpc2, Cfl1, Myl6, Pfn1, Rnd3, Septin9, z-score=2.65, P < 0.0001). Moreover, the potential role of hormones (estrogen (z-score=2.24) and prolactin (z-score=2.4)) and growth factors (AGT (z-score=2.41) and FGF2 (z-score=2.213)) were implicated in the hatching process as indicated by the upstream regulator analysis. By comparing the transcriptome between laser-treated and untreated hatching blastocysts, 47 DEGs were identified (adjusted P < 0.05) following laser-assisted ZP drilling. These genes were enriched in metabolism-related pathways (P < 0.05), including the lipid metabolism pathway (Mvd, Mvk, Aacs, Gsk3a, Pik3c2a, Aldh9a1) and the xenobiotic metabolism pathway (Aldh18a1, Aldh9a1, Keap1, and Pik3c2a). Limitations, reasons for caution Findings in mouse embryos may not be fully representative of human embryos. Furthermore, the mechanism of hatching revealed here might only reflect the hatching process of embryos in vitro. Further studies are now necessary to confirm these findings in different conditions and species to determine their clinical significance. Wider implications of the findings: Our study profiled the mouse embryo transcriptome during in vitro hatching, identified potential key genes and mechanisms for future study. In addition, for the first time, we revealed the impact of laser-assisted ZP drilling on the transcriptome, this may help us to assess and improve the existing technique. Trial registration number Not applicable
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Pravdyuk, O., M. Gryshchenko, L. Shatalova, and K. Borodai. "P–163 The presence of trophectodermal vesicles on expanded ICSI-derived blastocysts is a good predictor of implantation." Human Reproduction 36, Supplement_1 (July 1, 2021). http://dx.doi.org/10.1093/humrep/deab130.162.
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Abstract Study question Is the implantation rate (IR) higher in blastocysts with trophectodermal vesicles (TVs) compared to blastocysts without TVs or euploid blastocysts with unknown spontaneous hatching status? Summary answer The blastocysts with TVs demonstrate significantly higher IR in comparison to blastocysts without TVs or euploid blastocysts with unknown spontaneous hatching status. What is known already After ICSI spontaneous hatching mainly occurs by trophectoderm cell herniation via a small slit in the zona pellucida. At the beginning of this process, TVs are formed on the outside of the zona pellucida. It was previously shown that the clinical pregnancy rate was similar after the transfer of expanded blastocysts and expanded blastocysts with TVs. But another study showed that transfer of blastocysts of more advanced hatching stages yields better pregnancy rates than expanded blastocyst transfer. It remains unclear whether there is an association between the presence of TVs and IR in the transfers of single vitrified blastocysts. Study design, size, duration This retrospective cohort study was conducted from October 2018 to November 2019 and included 477 transfers of a single vitrified blastocyst. Cases were divided into 3 groups. Group 1 included transfers of blastocysts without TVs and with assisted hatching (AH). Group 2 contained the transfers of blastocysts at TVs stage of spontaneous hatching and without AH. Group 3 consisted of transfers of the euploid blastocysts with AH performed and unknown spontaneous hatching status. Participants/materials, setting, methods The age of women was between 21 and 39. Embryo transfers following oocyte donation programs were excluded from the study. This study included only transfers of the ICSI-derived fully expanded blastocysts with top-graded inner cell mass and trophectoderm. AH was performed using laser Saturn 3. The primary outcome was the implantation rate. Statistical analysis was performed using Pearson’s chi-square test and likelihood ratio test. Preimplantation genetic testing for aneuploidy (PGT-A) was performed by next-generation sequencing. Main results and the role of chance The number of cases in groups 1,2 and 3 was 133, 49, and 295, respectively. The average age in the groups was about 32.5 and did not differ between groups. The implantation rate in group 3 with PGT-A was 60% (177 out of 295), which was insignificantly higher compared to group 1 - 55% (73 out of 133) (p = 0.34). In group 2, the implantation rate was 76% (38 out of 49), which exceeded significantly the outcomes in groups 1 and 2 (p = 0.016). Thus the transfer of expanded blastocyst with TVs gives higher IR in comparison to expanded blastocyst. Therefore TVs could be utilized as a morphological marker for embryo selection. Furthermore, according to obtained results the presence of TVs on nontested blastocysts predicts implantation better than euploidy does in blastocysts with unknown spontaneous hatching status. Limitations, reasons for caution This is a retrospective nonrandomized study with its inherited limitations. Wider implications of the findings: Based on the results of the study embryo selection practice could be optimized. To maximize the outcomes of PGT-A programs embryo culture and biopsy workflow could be modified to allow collecting data on spontaneous hatching and TVs presence before performing the biopsy. Trial registration number Not applicable
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Adams, T., M. Markle, and C. Leisinger. "P-527 Comparison of implantation rate and preimplantation genetic testing between hatching and hatched blastocysts." Human Reproduction 37, Supplement_1 (June 29, 2022). http://dx.doi.org/10.1093/humrep/deac107.486.
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Abstract Study question Does embryo stage, hatching versus hatched blastocyst, affect ploidy status and implantation rates (IR)? Summary answer The embryo stage may influence ploidy status, but no correlation was made between embryo stage and IR. What is known already It is generally accepted that hatched embryos present increased vulnerability when handled and are often selected against. The literature on this varies with some reporting that the transfer of hatched blastocysts resulted in lower pregnancy rates compared to hatching or expanded blastocysts. Other studies have found no difference in clinical outcome with the transfer of hatched blastocysts compared to other stages. Reports defining the relationship between hatched or hatching blastocysts and ploidy rates are limited. Study design, size, duration Retrospective analysis of IR of 224 transferred euploid embryos and ploidy rate (P-Rt) of 1431 embryos produced in two IVF labs in the United States between 2019-2021. Embryos underwent assisted hatching on Day 3 and were subjected to preimplantation genetic testing (PGT) on Day 5 or Day 6 followed by vitrification. IR was defined as the presence of an intrauterine implantation sac with the detection of a fetal heartbeat on ultrasound. Participants/materials, setting, methods Trophectoderm biopsies were evaluated by NextGen Sequencing at the same genetics laboratory (Ovation Genetics, Nashville, TN). P-Rt included the following categories: Normal (N) or euploid, Abnormal (ABN) or aneuploid, Low-Mosaic (LM), and High-Mosaic (HM). Euploid embryos were transferred in subsequent frozen embryo transfer (FET) cycles and monitored by clinical ultrasound. Chi-square statistical analysis was performed to identify potential differences (p < 0.05). Main results and the role of chance No significant difference was observed in IR after transferring euploid hatched blastocysts compared to euploid hatching blastocysts. The IR of hatched blastocysts was 40.3% compared to 50.3% for hatching blastocysts (p = 0.21). A significant difference in ABN and HM embryos was observed according to embryo stage (p = 0.02). The ABN rate was 21.2% for hatched blastocysts compared to 28.0% for hatching blastocyst and the HM rate of hatched blastocysts was 10.3% versus hatching blastocyst at 7.5%. No significant difference in Normal and LM ploidy rates were observed according to embryo stage. The N ploidy rate of hatched blastocysts was 58.1% versus 54.4% in hatching blastocysts, and LM rate for hatched blastocysts was 10.3% compared to 10.1% in hatching blastocysts. Limitations, reasons for caution The limited data set did not include fresh embryo transfers or the transfer of untested embryos. The inclusion of increased embryo numbers could strengthen the power of the evaluation and allow for more robust statistical analysis. Wider implications of the findings Embryo selection is a multifactorial process consisting of a wide range of parameters. Numerous advances in embryo production and evaluation have resulted in the need for an algorithm or guide to rank embryos for transfer. Further investigation is warranted to elucidate comprehensive recommendations for embryo selection. Trial registration number not applicable
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Herrero Grassa, L., J. Ten Morro, Á. Linares Bernabéu, E. Álvarez Socias, L. Cascales Romero, S. García Cano, J. A. Ortiz Salcedo, A. Bernabéu, and R. Bernabéu. "P-178 Time to hatching: the most predictive morphokinetic parameter for embryo implantation in machine learning algorithms following time-lapse incubation and blastocyst transfer in oocyte-donation programs." Human Reproduction 37, Supplement_1 (June 29, 2022). http://dx.doi.org/10.1093/humrep/deac107.172.
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Abstract Study question Which is the most predictive parameter when machine learning (ML) is applied to a known implantation database (KID) of day 5 embryo transfer database in an egg donation program? Summary answer Time to hatching (tiHB) is the most predictive embryonic parameter when machine learning algorithms were used on reproductive data in an oocyte donation program. What is known already Artificial intelligence is becoming an encouraging tool in medicine, also in ART, where the amount of data generated in the IVF lab has dramatically increase, favored by time-lapse technology. Numerous embryo selections algorithms based on logistic regressions have been developed for predicting blastocyst formation and implantation potential, but with machine learning, we can train algorithms and connect different morphological and morphokinetic embryo parameters with implantation or even live birth embryo potential. The aim of this study was to test machine learning algorithms and to identify predictive embryonic morphokinetic parameters when comparing the different models generated after machine learning analysis. Study design, size, duration Retrospective analysis of 405 embryos in a KID obtained after 392 embryo-transfers (13 double and 379 single-ET) performed in an oocyte donation program in 4 fertility clinics (year 2021). Recipientś average age: 42.2±4.2 years. The embryos were cultured in Global® Total® culture medium in Geri® (Genea Biomedx) time-lapse incubators after ICSI until embryo transfer at blastocyst stage. Only sperm samples >1x106 spermatozoa/ml were included. All parameters were registered by one single trained senior embryologist. Participants/materials, setting, methods Thirty-five variables were initially analyzed: classic morphokinetic markers, time intervals (including total thinning time before hatching: tiHB-tFB and total blastulation time before hatching: tiHB-Tcav) and morphological measurements (blastocyst and inner cell mass diameter 110h post-injection). Eighty percent of the data was used for model training and 20% was reserved for model validation. Twelve supervised and unsupervised predictive machine learning models were developed. The software used to carry out the analysis was SPSS (v20.0) R (4.0.5). Main results and the role of chance The basic characteristics of the embryo population were similar. From the 405 embryos transferred, 216 blastocysts came from vitrified oocytes (53.3%). The implantation rate was 57.03% (231 gestational sacs) and the miscarriage rate was 16.8%. The classification-supervised algorithms applied included binary logistic regression, neural networks, support vector machines, neighborhood-based methods, classification trees, boosting and bagging methods. The algorithms were optimized by minimizing the AUC. Cluster analysis (unsupervised) was also performed. In the 6 best predictive models, the variables with the highest relevance were tiHB (hatching initiation time), tiHB-tFB and tiHB-tcav: variables related to hatching initiation. Furthermore, in the cluster analysis, these three variables appeared grouped in the same cluster. From the blastocysts population that implanted, 57.6% (133/231) were initiating hatching, while from those embryos that did not, only 42.3% (74/174) began the hatching process. Other variables such as the diameter of the transferred blastocyst, which we assumed to be valuable as an objective morphological parameter, did not show a high predictive capacity in the models obtained. Blastocyst average diameter of implanting blastocysts was 157.9±24.9 µm and non-implanting was 153.9±26.1 µm. Limitations, reasons for caution Morphology and morphokinetic parameters require subjective annotation and thus might have intrinsic intra-reader variability. Our findings need to be validated prospectively. Wider implications of the findings Time to blastocyst hatching appears to have significative impact in most ML predictive models. Hatching-related variables seems to have predictive power. Despite numerous variables influencing IVF outcome (intrinsic and extrinsic to embryo development) ML and AI approaches may improve the prioritization of the most viable embryo favoring single embryo transfer. Trial registration number 2021ibmad001
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Sanguinet, P., G. Regnier-Vigouroux, B. Keppi, A. Chiron, M. Montagut, G. Queré, and D. Nogueira. "P-286 Can laser assisted hatching help implantation of warmed blastocysts in presence of fragmented cells ?" Human Reproduction 37, Supplement_1 (June 29, 2022). http://dx.doi.org/10.1093/humrep/deac107.275.
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Abstract Study question Does laser assisted hatching (LAH) following warming of blastocysts containing fragmentated cells improves blastocyst expansion and implantation? Summary answer The application of LAH does not improve blastocyst expansion and implantation regardless the presence or absence of fragmented cells. What is known already The absence of natural hatching is one of the hypotheses of implantation failure after cryopreservation, a process that could lead to hardening of the zona pellucida. Another theory is the fact that cellular fragments inside the zona pellucida, surrounding the trophectoderm, could impairs the exit of the blastocyst through the zona decreasing its chances of implantation. Available data regarding the effect of LAH on blastocysts after vitrification are inconclusive and limited to small samples. Evaluating the effectiveness of LHA performed at the time of blastocyst warming when cellular fragments are present could elucidate its impact on hatching and implantation Study design, size, duration A bicentric prospective randomized study including 344 successive FET cycles from January 2020 until March 2021. Patients were enrolled only once in the study. Patients underwent a natural cycle or hormonal replacement treatment for FET. Blastocysts graded ≥ BL3BB (Gardner scoring) underwent artificial collapse and were vitrified on D5 or D6. Only blastocysts surviving post-warming were considered in the analysis. Primary end point was clinical pregnancy rate. Participants/materials, setting, methods Patients ≤42 years with ≤3 previous oocyte retrievals scheduled for the first elective single embryo transfer (eSET) with vitrified/warmed blastocysts. Survived blastocysts were randomized immediately after warming to LAH group (n = 172) or to control group (no-LAH, n = 172). Cellular fragmentation was annotated as a percentage of the total volume of the embryo (0%, ≤25%, ≤50%, >50%) and LHA was performed on the opposite side. Embryo expansion was annotated at time of transfer, at 3 hours post-warming. Main results and the role of chance Patients age were similar between LHA (33.1±9.3) and controls (34.8±7.5). Patients in LAH and controls had similar pregnancy rates (hCG >100) (46% versus 52%, respectively), CPR (37% versus 36%, respectively) (NS) and miscarriage rates. No difference was observed in CPR in relation to patients age. A significant increase in implantation was observed when blastocyst expansion took place 3 hours after warming, independently whether allocated in LAH (47% versus 17%, p < 0.01) or no-LAH group (51% versus 33%, p < 0.01). LAH did not influence cell expansion (83% in LAH versus 85% in no-LAH), however more blastocysts underwent hatching in LAH group (27% versus 12% in no-LAH, p < 0.06). Significantly more embryos that had hatched in LAH group led to pregnancy compared to no-LHA (83% versus 67%, respectively) (p = 0.05). Extra-cellular (EC) fragmentation not did not impact implantation in neither of the groups. LAH group had 46% of embryos implanted when absence of fragmentation, 40% when EC was present at ≤ 25%, 58% when EC was present at > 25% (NS). In no-LAH group, 52% of embryos implanted when absence of fragmentation, 49% when EC was present at ≤ 25%, 65% when EC was present at > 25% (NS). Limitations, reasons for caution A sample size of 700 blastocysts was first chosen calculating a 10% difference in clinical pregnancy rate (CPR) between LAH-group and no-LAH group. The study was interrupted following this interim analysis. Live birth outcomes should be considered in a further analysis to conclude on the null impact of LHA post-warming. Wider implications of the findings This study adds to the evidence of the existence of a limited potential of the application of LAH on vitrified-warmed blastocysts and its impact in terms of clinical pregnancy rates. Trial registration number not applicable
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Zhang, Ling, Yi-er Zhou, Yue-jin Wu, Li-mei Wu, Shi-shi Li, Lin Zhang, Zhen Jin, Chong-yi Shu, Wei-hai Xu, and Jing Shu. "Thinning or Opening: A Randomized Sibling-Embryo Pilot Trial on the Efficacy of Two Laser-Assisted Hatching Modes During the Extended Culture of Highly Fragmented Cleavage Embryos." Frontiers in Endocrinology 13 (June 27, 2022). http://dx.doi.org/10.3389/fendo.2022.927834.
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A randomized sibling-embryo pilot trial investigated whether two ways of laser-assisted hatching result in different blastulation and clinical outcomes after extended in vitro culture process of highly fragmented day-3 cleavage embryos. From 92 couples, a total of 315 highly fragmented day-3 embryos (the fragmentation >25%) were recruited and randomized into laser-assisted zona thinning (LAT, n=157) and opening (LAO, n=158) groups, and then underwent a blastocyst culture in vitro. The main endpoint measurements including blastocyst formation and grading as well as the clinical pregnancy after blastocyst transfer were obtained during the treatment procedure of in vitro fertilization and embryo transfer, and then analyzed with generalized estimating equation (GEE) and/or time-to blastocyst analysis models. A total of 166 day-3 embryos developed into blastocyst stage (52.70%), of which 97 were viable blastocysts (30.79%), and 42 top-quality ones (13.33%). LAT did not have any inferior or superior to LAO in the endpoints of either total, viable, top-quality or hatched blastocyst formation, with the ORs (95%CI) from GEE model as 0.89 (0.55-1.45), 0.71 (0.42-1.21), 1.12 (0.56-2.25) and 0.68 (0.42-1.12) respectively for LAT treatment. And the time-to-blastocyst analysis showed a similar result. Additionally, no difference in clinical outcomes after blastocyst transfer was found between the two groups. The author concluded that when applying the LAHs during the extended culture of highly fragmented embryos, both LAT and LAO can generate a promising clinical outcome, and the LAT operation be equivalent to the LAO. Future well-designed, multiple-center, larger-sample investigations are required to ascertain above conclusion.
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Wang, Xiaoxia, Jing Zhao, Zhongyuan Yao, Qiuping Xia, Tianli Chang, Jun Zeng, Jiaqi Liu, Yanping Li, and Huimin Zhu. "Arrested Cells/Cellular Debris Expelled from Blastocysts Is Self-Correction Phenomenon During Early Embryonic Development." Reproductive Sciences, January 10, 2023. http://dx.doi.org/10.1007/s43032-022-01159-8.
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Abstract Arrested cells/ cellular debris is component left in the zona pellucida after blastocyst hatching. To identify whether expelling arrested cells/cellular debris from blastocysts is a process of human embryo self-correction by eliminating abnormal cells, 21 pairs of trophectoderm (TE) biopsies and the corresponding arrested cells/cellular debris expelled from the blastocysts from July to December 2020 were collected and analyzed using next-generation sequencing (NGS). Then, the NGS results of TE biopsies and the corresponding arrested cells/cellular debris were compared. We identified that 47.6% of blastocysts (10/21) were aneuploidies and mosaicism. A total of 18 groups of arrested cells/cellular debris (85.7%) expelled from blastocysts were abnormal, including nine aneuploid embryos and nine euploid embryos. In the arrested cells/cellular debris, all the chromosomes were affected. In conclusion, mosaicism and aneuploidies are common features of early embryonic development, and the arrested cells/cellular debris expelled from blastocysts provides evidence of early embryonic self-correction.
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Jiang, V., C. Bormann, I. Souter, I. Dimitriadis, M. K. Kanakasabapathy, P. Thirumalaraju, and H. Shafiee. "P-294 Use of Artificial Intelligence to Assess the Effects of Assisted Hatching on Embryo Development and Implantation Potential." Human Reproduction 37, Supplement_1 (June 29, 2022). http://dx.doi.org/10.1093/humrep/deac107.282.
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Abstract Study question Does the use of laser-assisted hatching (AH) on cleavage stage embryos affect in vitro preimplantation embryo development or implantation potential? Summary answer There is no difference in blastocyst conversion rate or implantation potential of embryos following AH at the cleavage stage for patients under age 35 years. What is known already Laser-AH is the process of creating an opening within the zona pellucida on cleavage stage embryos to facilitate biopsy of trophectoderm cells for preimplantation genetic testing (PGT). Studies have shown that PGT for aneuploidy (PGT-A) in patients under 35 years have reduced pregnancy rates compared to those not undergoing biopsy. This is attributed to the additional micromanipulation events involved with PGT-A may decrease the viability of embryos and compromise their implantation potential. We aimed to objectively compare the impact of AH on embryo development using an artificial intelligence (AI)-algorithm trained to assess embryo quality and predict developmental fate. Study design, size, duration A retrospective dataset from patients under 35 years was generated from two timepoints: cleavage stage embryos immediately before AH between 60-64 hours post insemination (hpi); and blastocyst stage embryos between 110-115 hpi prior to transfer or vitrification. Time-lapse imaging was obtained using the EmbryoScope (Vitrolife). Cleavage stage embryo images were used to train a convolutional neural networks (CNN) to predict and classify the development and implantation potential of cleavage and blastocyst stage embryos. Participants/materials, setting, methods Time-lapse images were collected for 1444 cleavage stage embryos spanning 189 in vitro fertilization (IVF) cycles between January 2014 – December 2021 at a single academic fertility center in Boston. Embryos were categorized into two groups: Day 3 embryos with AH (D3+AH) and without AH (D3-No AH). Each patient had a single blastocyst embryo transfer with a known outcome. Two-tailed t-tests were used to compare differences, with p-value less than 0.05 set for statistical significance. Main results and the role of chance The dataset included 1035 embryos with AH (D3+AH) and 409 embryos without AH (D3-No AH). There were no differences in AI-predicted blastocyst development between Day 3 embryos with AH and without AH (64.1% vs 64.1%) or AI-predicted high quality blastocyst development rate between these two groups (43.8% vs 40.8%), respectively. On Day 5 there were no differences in the AI-categorization of embryos at the blastocyst stage between embryos with or without AH (62.3% vs 62.5%) or AI-categorization of high-quality blastocyst development (45.2% vs 41.8%), respectively. AI predicted a similar implantation potential between embryos with and without AH at the cleavage stage (61.1% vs 69.9%). When stratifying to only the embryos transferred, there were no differences in the AI-predicted blastocyst development between Day 3 embryos with AH and without AH (96.0% vs 97.1%) or in the AI-predicted high quality blastocyst development rate between these two groups (72.0% vs 82.7%). AI predicted a similar implantation potential between embryos with and without AH at the cleavage stage (72.0% vs 69.0%). These results correspond with the true clinical pregnancy rate between the AH and Non-AH groups (68.0% vs 61.9%, p = 0.44). Limitations, reasons for caution These retrospective findings were of patients who had time-lapse imaging of cleavage stage and blastocysts available. Additionally, we focused on high prognosis patients that were eligible for single blastocyst stage embryo transfer. Clinical pregnancy rate was examined, not spontaneous abortion or live birth rates. Wider implications of the findings Utilization of AI technology allows for more objective and standardized methods for examining the impact of laboratory procedures on the developmental fate of embryos. This study demonstrated the safety of utilizing laser-assisted hatching on embryo development within this study population. Trial registration number None
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Watson, K., K. Ong, I. Korman, R. Turner, B. Vollenhoven, D. Zander-Fox, and Y. Liu. "O-213 Slow day 5 development affects implantation potential of fresh transferred embryos but not birthweight once pregnancy occurs: A multi-center retrospective cohort study." Human Reproduction 36, Supplement_1 (July 1, 2021). http://dx.doi.org/10.1093/humrep/deab128.024.
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Abstract Study question Does slow development of fresh transferred day 5 embryos lead to decreased implantation potential and birthweight? Summary answer Slow day 5 development was associated with reduced implantation potential when transferred fresh but the subsequent birthweight of the resulting baby was not impacted. What is known already Slow development of in vitro cultured cleavage stage embryos is associated with reduced blastocyst development and implantation rates. There is no current consensus regarding whether to transfer fresh slow developing day 5 embryos or to extend culture for a subsequent day with potential for cryopreservation. It is therefore important to understand the true prognosis of fresh transferred day 5 embryos at less advanced developmental stages. This would provide evidence based guidelines for the decision making process in regard to embryo transfer. Study design, size, duration This is a retrospective multi-center cohort study, including 1213 consecutive patients undergoing autologous oocyte in vitro fertilization (IVF) treatment during 2016-2019,with fresh transfer of a single day 5 embryo (selection based on developmental stage and inner cell mass and trophectoderm morphology if blastocyst was at the ≥expanding stage). Cycle data were collected from 4 associated private clinics, with repeat cycles of same patients excluded to avoid clustering effect at statistical analysis. Participants/materials, setting, methods Live birth and birthweight were followed up in all 1213 fresh day 5 SETs. Multiple regression (logistic or linear) was performed to investigate association between slow day 5 development (defined as ≤ early blastocyst) and (a)live birth, (b) birthweight, and (c) gestation-adjusted birthweight (Z score) to account for gestational age, gender and compared to embryos at ≥ expanded stage. Results were expressed as adjusted odds ratio (aOR) with 95% confidence interval (CI)or coefficients (β). Main results and the role of chance No implantation was achieved following single fresh transfer of day 5 embryos that failed to reach early blastocyst stage (n = 76) and were transferred as ≤ morula stage. Live birth rate was significantly lower following single day 5 fresh transfer of an early blastocyst (n = 237, 16%), in comparison to expanding (n = 329, 27%, P = 0.001), expanded(n = 392, 41%, P = 0.000), and hatching/hatched blastocysts (n = 169, 44%, P = 0.000). After adjusting for potential confounding factors including; maternal age, hours post insemination at day 5 assessment, number of oocytes collected, number of 2PN embryos, and number of embryos frozen; multiple logistic regression showed significantly reduced likelihood of live birth resulting from early blastocysts in reference to those at the expanding (aOR=0.584, 0.371-0.917, P = 0.020), expanded (aOR=0.322, 0.208-0.501, P = 0.000), or hatching/hatched stages (aOR=0.255, 0.147-0.443, P = 0.000). However, multivariate linear regression indicated that early blastocysts resulting in a live birth (n = 39) did not lead to altered birthweight (β=-9.091, P = 0.904; β=-34.960, P = 0.343; β=-26.074, P = 0.414; respectively) or Z score (β = 0.045, P = 0.706; β=-0.051, P = 0.426; β=-0.028, P = 0.506; respectively) in reference to the expanding (n = 90), expanded (n = 160), or hatching/hatched stages (n = 75). Limitations, reasons for caution The retrospective nature of this study does not allow controlling of unknown confounders. The 4 participating clinics are associated within the same network with shared protocols, therefore, results may not be generalized to other clinics with different settings. Wider implications of the findings The findings suggest no clinical value of fresh day 5 transfer of embryos ≤morula stage. Although early blastocysts implant at reduced rate, assuring birthweight outcomes suggest clinical value. Future studies intend to investigate slow growing day 5 fresh transfers versus embryos that were slow growing but transferred after day 6. Trial registration number NA
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Belchin, P., Y. Cabello, M. Sanche. d. Burgos, J. Guerrero, M. D. Riva, A. Garcia-Enguidanos, E. Izquierdo, and D. Ordonez. "P–158 Assisted Hatching on D + 3 in order to facilitate trophectoderm biopsy in blastocyst for PGT-A is not advisable in all patients." Human Reproduction 36, Supplement_1 (July 1, 2021). http://dx.doi.org/10.1093/humrep/deab130.157.
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Abstract Study question Is it useful or beneficial to perform Assisted Hatching (AH) on D + 3 previously to biopsy for PGT-A on blastocyst stage on D + 5? Summary answer The routine use of AH on D + 3 to facilitate the embryo biopsy on D + 5 could negatively influence the development of the embryos to blastocyst stage. What is known already The blastocyst stage is the optimal stage for performing biopsies for PGT-A, which has been reported as a key factor determining the growing clinical application of this strategy worldwide. For trophectoderm (TE) biopsy, laser-assisted drilling is used to create a zona opening on D + 3 or D + 5 of development. The method of zona opening on D + 3 allows some of the TE cells to herniate during blastocyst formation and expansion, which facilitates the biopsy process. However, this method may result in herniation of inner cell mass cells instead of TE or maybe could affect the development of the embryo to blastocyst stage. Study design, size, duration A total of 100 PGT-A cycles were performed in 2019 and 2020. In 78 of them laser-assisted drilling was used to create a zona opening on D + 5 only in those embryos which arrived to blastocyst stage for TE biopsy (Group No-AH). In 22 cycles the same drilling was achieved on D + 3 in all embryos, independently of their quality (Group AH). The average of embryos per cycle in each group was 5 and 4.3 respectively. Participants/materials, setting, methods A total of 100 PGT-A cycles coming from 65 patients were studied. The average of the age of the patients was 40.83 (SD 3.45) in the group No-AH vs 42.18 (SD 3.42) in the Group AH (p = 0.108), so the age was not a determining factor for the development of the embryos. We analyzed by χ 2 test differences between groups on fertilization rates, number of embryos, development to blastocyst stage, euploidy and pregnancy rates. Main results and the role of chance The fertilization rate was 74.79% (No-AH group) and 68.53% (AH group) with no significative statistical differences (p = 0.12). In the No-AH group, the TE biopsy was performed on D + 5 in 63 cycles (81%). In the AH group, 41% of cycles didn’t reach the blastocyst stage, obtaining statistical differences between groups (p = 0.035). We found also significant differences in the number of cycles with biopsied blastocyst when we had 1 to 6 embryos/cycle on D + 3 between groups (p = 0.002), without obtaining any blastocyst to be diagnosed in 53% of the cycles in AH group vs 27% in No-AH group. When the number of embryos on D + 3 per cycle was > 6, at least 1 embryo reached the blastocyst stage in both groups, although this number was higher in No-AH group. The rate of biopsied blastocysts was significantly higher in the No-AH group compared to the AH group (46.61 vs 34.69) with a p = 0.031. The rate of euploid embryos analyzed was 23.30% in the No-AH group compared to 29.41% in the AH group, although no significant differences were found (p = 0.44) between groups. In the No-AH group, a clinical pregnancy rate of 52.94% was obtained (n = 34) vs 50% in the AH group (n = 4) (p = 0.91). Limitations, reasons for caution We have recently started to perform AH on D + 3, so the number of cases is smaller than No-AH group. We use a time lapse incubator in all cases, so in the No-AH the culture dish is changed, disturbing the stable incubation environment, while in the other group it is not. Wider implications of the findings: The use of AH on D + 3 in order to facilitate the TE biopsy on D + 5 could affect negatively the development of the embryos to blastocyst stage. Its routine use should be avoided based on laboratory workload, mainly if the patient has less than 7 embryos at D + 3. Trial registration number Not applicable
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Cimadomo, D., A. Marconetto, F. Innocenti, S. Trio, V. Chiappetta, D. Soscia, L. Albricci, et al. "O-101 Elucidation of blastocyst collapse and its consequences: a comprehensive artificial intelligence-powered analysis of 1943 embryos from 643 couples." Human Reproduction 37, Supplement_1 (June 29, 2022). http://dx.doi.org/10.1093/humrep/deac105.124.
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Abstract Study question What are the causes and consequences of blastocyst collapse? Summary answer ∼50% of blastocysts collapsed, especially if they are aneuploid and/or morphologically-poor. Yet, no impact on the live-birth-rate (LBR) per vitrified-warmed euploid single-embryo-transfer (SET) was reported. What is known already Time-lapse-microscopy (TLM) is a powerful tool to describe the peculiar dynamics of preimplantation development. Lately, artificial intelligence (AI) has been also implemented to automatize and standardize such description. Here, we adopted AI to comprehensively portray blastocyst collapse, namely the phenomenon of embryo contraction with an efflux of blastocoel fluid and the detachment of the trophectoderm (TE) from the Zona Pellucida (ZP). Although, the causes of this event are still undetermined, small blastocyst contractions have been reported beneficial for the hatching process, while a full collapse has been associated with lower competence. Study design, size, duration Observational study including 1943 blastocysts from 643 couples cultured in the Embryoscope between January-2013 and December-2020. TE biopsy without day3 ZP drilling and comprehensive-chromosome-testing were performed. The Fairtility® software automatically registered: (i)time of starting-blastulation (tSB), (ii)starting and ending time of each collapse (tSC and tEC), (iii)blastocysts’ areas, (iv)shrinkage% [(area at SC – area at EC)/area at SC)], (v)embryo:ZP ratio at EC (area of the collapsed embryo/area of the ZP), and (vi)time of biopsy (t-biopsy). Participants/materials, setting, methods Blastocyst quality was defined according to Istanbul Consensus (11, excellent; 12-21, good; 22-13-31, average; 33-23-32, poor) and with the Fairtility implantation score (IS) as well, i.e., a continuous variable from 0 to 1 generated by the KID+ software based on the TLM videos of preimplantation development. The main outcome was the LBR per euploid SET adjusted for confounders through logistic regressions. All couple and embryo features were also investigated for their association with blastocyst collapse. Main results and the role of chance 47.3% of the blastocysts collapsed 1- to 9-times (interval between collapses: 4-8hr), and 73% of the couples had ≥1 collapsed blastocyst (1.8±1.1, range:1-8). No couple feature, though, was associated with blastocyst collapse. The longest collapses lasted 1.5±1.1 (0.13-5.1)hr, while the largest shrinkage% and embryo:ZP ratio at EC were 35±14% (10-78%) and 81±9% (33-90%), respectively. In ∼50-60% of collapses a 20-40% blastocyst volume reduction was registered, 40-60% or 20-40% in ∼15-30%, 60-80% in 0-4%. In case of multiple collapses, the first three involved smaller shrinkages. Blastocysts undergoing ≥1 collapse showed similar tSB as not-collapsing blastocysts, but progressively longer tEB and t-biopsy. The earlier the first event, the more the consecutive collapses. Notably, the poorer the morphology, the higher the risk (excellent, good, average, and poor not-collapsing blastocysts were 64%,50%,44% and 37%), number (e.g.,≥4 collapses were 0.4%,2%,4% and 8%) and duration (1.2±1.0,1.4±1.0,1.6±1.1 and 1.9±1.3hr) of blastocyst collapse. Collapsing blastocysts were significantly less euploid than non-collapsing (35% vs 47%; multivariate-OR:0.75,95%CI 0.6-0.92,p<0.01); conversely, their LBR per euploid SET (39% vs 46%) and miscarriage rate per clinical pregnancy (17% vs 11%), were not significantly different (adjusted-OR:1.0,95%CI 0.69-1.48,p=0.96 and adjusted-OR:1.65,95%CI 0.79-3.42,p=0.18, respectively). All data were confirmed also by defining blastocyst quality through the Fairtility IS. Limitations, reasons for caution Gestational and perinatal outcomes were not assessed. Other culture strategies and media shall be assessed for their association with blastocyst collapse. Perhaps, future studies from other groups and with a larger sample size might unveil a significant impact on the clinical outcomes. Wider implications of the findings Collapse is common and delays blastocyst full-expansion. Moreover, poor morphology and aneuploidies involve a higher risk of collapse(s); however, no impact was reported on the clinical outcomes after euploid SET. AI appears to increase the throughput of the analysis, but additional data are required to research the causes of collapse. Trial registration number Not applicable
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Trio, S., F. Innocenti, G. Saturno, M. Taggi, C. Hickman, B. Kantor, D. Taub, et al. "P-297 Comprehensive artificial intelligence-powered investigation of blastocyst expansion dynamics: associations with competence." Human Reproduction 38, Supplement_1 (June 1, 2023). http://dx.doi.org/10.1093/humrep/dead093.655.
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Abstract Study question Are blastocyst expansion dynamics from time of starting blastulation (tSB) to time of biopsy (t-biopsy) indicative of embryo competence? Summary answer Early expansion dynamics across 5hours after tB, embryo-proper area (emb-A), zona-pellucida-area (zp-A), ZP-thickness (zp-T) at t-biopsy and their t-biopsy/tB ratios are associated with competence. What is known already Blastocyst expansion is the very first morphogenetic event common to several species. Time-lapse-technology (TLT) implementation in IVF allowed deeper understanding of blastocyst expansion process. Some studies leveraged TLT and Artificial-Intelligence to investigate blastocyst expansion timings and dynamics for their association with embryo competence. Huang’s group, in particular, designed a quantitative standard expansion assay (qSEA) showing promising results. However, data about qSEA reproducibility are missing. Here we comprehensively investigated the expansion processes between tSB and t-biopsy through Artificial-Intelligence, and adapted the qSEA to our setting that encompasses PGT-A without day3-hatching and single-euploid-blastocyst-transfer. Study design, size, duration Retrospective study including 2184 blastocysts cultured in EmbryoScope during 786 PGT-A cycles conducted across 2013-2020. Videos were analyzed through an Artificial-Intelligence-powered tool (CHLOE™, Fairtility). The software automatically extracted timings in hours-post-insemination and measures as proportions of video frames occupied by each feature under investigation (single pixel = 300µm; wells’ area = 90,000µm2) recorded every 30min from tSB. These data were tested for their association with euploidy and live-birth after 548 euploid transfers via multivariate regressions. Participants/materials, setting, methods ICSI, trophectoderm biopsy on fully-expanded blastocysts without day3 zp drilling, and qPCR/NGS to assess full-chromosome non-mosaic aneuploidies were performed. The timings assessed were tSB, tB, tEB and t-biopsy ( = end of video). At these timings and every 30min across 5hours after tSB the software recorded the following measures emb-A, zp-A, zp-T, inner-cell-mass area (ICM-A), and ICM-to-trophectoderm ratio. Also increase/decrease ratios for all measures between timings were assessed. Putative confounders (e.g., maternal age, blastocyst quality) were considered. Main results and the role of chance Larger emb-A and zp-A at t-biopsy and zp-T at both tEB and t-biopsy were associated with euploidy. Similarly, the ratios zp-A at t-biopsy/tB and t-biopsy/tEB highlighted a larger expansion among euploid versus aneuploid blastocysts. All these differences were confirmed when adjusting for maternal age, morphological quality and tB (p < 0.01). zp-A t-biopsy/tB ratio (aneuploid:+68.8% versus euploid:+79.9%) showed a more relevant association than final zp-A at t-biopsy per se (24082 ± 5763 versus 25438 ± 5969µm2). The ratios zp-T at t-biopsy/tB and t-biopsy/tEB were also significantly associated with euploidy, even when adjusting for confounders (p < 0.01). In this case, zp-T at t-biopsy (8.1 ± 3.2 versus 7.1 ± 2.7µm) per se showed a stronger association than zp-T t-biopsy/tB ratio (-50% versus -55%). ICM-A and ICM-to-trophectoderm ratio showed no association with euploidy. All features showed no association with LBs (N = 233/548 euploid transfers). The qSEA every 30min across 5hours after tB outlined different early expansion dynamics between euploid and aneuploid blastocysts, with the former expanding more (larger areas and thinner zp) and sooner. The differences became significant already after 2.5-3 hours, due to rather constant expansion rates in both groups, but faster among euploid. The same significant trend was reported for euploid blastocysts resulting in a LB versus not. Limitations, reasons for caution Retrospective single-center study. Previous studies on qSEA were based on 10 measurements every hour from tB, instead of 10 measurements every 30min. To properly assess the association between expansion dynamics and timings with LB, more transfers are required. To outline a predictive power, instead, a prospective randomized design is warranted. Wider implications of the findings Blastocyst expansion dynamics, timings and ratios measured through Artificial-Intelligence, already during the 5hours following tB, provide objective quantitative data associated with embryo competence. qSEA is a promising clinical strategy, user-friendly and easily applicable, that deserves further appraisal. Basic research on the mechanisms that govern blastocyst expansion processes is warranted. Trial registration number None
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Hennes, Aurélie, Johanna Devroe, Katrien De Clercq, Martina Ciprietti, Katharina Held, Katrien Luyten, Nele Van Ranst, et al. "Protease secretions by the invading blastocyst induce calcium oscillations in endometrial epithelial cells via the protease-activated receptor 2." Reproductive Biology and Endocrinology 21, no. 1 (April 15, 2023). http://dx.doi.org/10.1186/s12958-023-01085-7.
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Abstract Background Early embryo implantation is a complex phenomenon characterized by the presence of an implantation-competent blastocyst and a receptive endometrium. Embryo development and endometrial receptivity must be synchronized and an adequate two-way dialogue between them is necessary for maternal recognition and implantation. Proteases have been described as blastocyst-secreted proteins involved in the hatching process and early implantation events. These enzymes stimulate intracellular calcium signaling pathways in endometrial epithelial cells (EEC). However, the exact molecular players underlying protease-induced calcium signaling, the subsequent downstream signaling pathways and the biological impact of its activation remain elusive. Methods To identify gene expression of the receptors and ion channels of interest in human and mouse endometrial epithelial cells, RNA sequencing, RT-qPCR and in situ hybridization experiments were conducted. Calcium microfluorimetric experiments were performed to study their functional expression. Results We showed that trypsin evoked intracellular calcium oscillations in EEC of mouse and human, and identified the protease-activated receptor 2 (PAR2) as the molecular entity initiating protease-induced calcium responses in EEC. In addition, this study unraveled the molecular players involved in the downstream signaling of PAR2 by showing that depletion and re-filling of intracellular calcium stores occurs via PLC, IP3R and the STIM1/Orai1 complex. Finally, in vitro experiments in the presence of a specific PAR2 agonist evoked an upregulation of the ‘Window of implantation’ markers in human endometrial epithelial cells. Conclusions These findings provide new insights into the blastocyst-derived protease signaling and allocate a key role for PAR2 as maternal sensor for signals released by the developing blastocyst. Graphical Abstract
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Pérez-Gómez, Alba, Leopoldo González-Brusi, Pablo Bermejo-Álvarez, and Priscila Ramos-Ibeas. "Lineage Differentiation Markers as a Proxy for Embryo Viability in Farm Ungulates." Frontiers in Veterinary Science 8 (June 15, 2021). http://dx.doi.org/10.3389/fvets.2021.680539.
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Embryonic losses constitute a major burden for reproductive efficiency of farm animals. Pregnancy losses in ungulate species, which include cattle, pigs, sheep and goats, majorly occur during the second week of gestation, when the embryo experiences a series of cell differentiation, proliferation, and migration processes encompassed under the term conceptus elongation. Conceptus elongation takes place following blastocyst hatching and involves a massive proliferation of the extraembryonic membranes trophoblast and hypoblast, and the formation of flat embryonic disc derived from the epiblast, which ultimately gastrulates generating the three germ layers. This process occurs prior to implantation and it is exclusive from ungulates, as embryos from other mammalian species such as rodents or humans implant right after hatching. The critical differences in embryo development between ungulates and mice, the most studied mammalian model, have precluded the identification of the genes governing lineage differentiation in livestock species. Furthermore, conceptus elongation has not been recapitulated in vitro, hindering the study of these cellular events. Luckily, recent advances on transcriptomics, genome modification and post-hatching in vitro culture are shedding light into this largely unknown developmental window, uncovering possible molecular markers to determine embryo quality. In this review, we summarize the events occurring during ungulate pre-implantation development, highlighting recent findings which reveal that several dogmas in Developmental Biology established by knock-out murine models do not hold true for other mammals, including humans and farm animals. The developmental failures associated to in vitro produced embryos in farm animals are also discussed together with Developmental Biology tools to assess embryo quality, including molecular markers to assess proper lineage commitment and a post-hatching in vitro culture system able to directly determine developmental potential circumventing the need of experimental animals.
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Karagianni, M., M. I. Papadopoulou, C. Oraiopoulou, N. Christoforidis, A. Papatheodorou, and A. Chatziparasidou. "P-160 The effect of laser assisted hatching (AHΑ) on vitrified-warmed blastocysts post warming on reproductive outcome." Human Reproduction 37, Supplement_1 (June 29, 2022). http://dx.doi.org/10.1093/humrep/deac107.155.
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Abstract Study question Does laser assisted hatching (ΑΗΑ) of vitrified-warmed blastocysts post warming improve the pregnancy rates? Summary answer This study suggests that although laser assisted hatching post warming shows a trend towards improving pregnancy rates, the difference is not significant. What is known already It is known that vitrification may alter the zona pellucida’s biochemical properties and possibly cause hatching failure and ultimately implantation failure. One proposed way in order to overcome this drawback, is the implementation of laser assisted hatching. During assisted hatching, multiple laser shots are performed in the perivitelline space creating an opening on the zona pellucida which facilitates the blastocyst’s herniation process. The efficiency of this method in regard to pregnancy outcomes though, remains controversial. Some studies report a significantly positive effect on pregnancy outcomes using laser assisted hatching whereas others report no significant difference. Study design, size, duration This prospective randomized study was performed at Embryolab Fertility Clinic, in Thessaloniki, Greece between January 2020 and October 2020 and included 2439 frozen embryo transfers. Patients with vitrified-warmed embryos were randomized and allocated to the study (ΑHA) group or control (NO AHA) group. Participants/materials, setting, methods Patients were divided in two groups: AΗΑ group (n = 1799) where laser assisted hatching was performed on the day of embryotransfer (Day 3 or Day 5) on the zona of the embryos post warming and control group (n = 640) where the embryos remained untreated after the warming procedure. The 2 groups were further divided in 3 subgroups depending on the women’s’ age (≤35, 36-40, ≥41) and in 2 more subgroups depending on the day of the transfer. Main results and the role of chance Mean pregnancy rate for all embryos’ stages in AHA group was 60.03% whereas in the NO AHA group was 58.28% (p = 0.4385). In the subgroup of ≤ 35-years, pregnancy rates were 70.23% and 66.49% respectively (p = 0.3363). In the 36-40years subgroup, rates were 58.24% and 60.09% respectively (p = 0.6418). And finally, in the ≥41-years subgroup, pregnancy rates were 54.26% and 49.79% respectively (p = 0.2331). In the group where the embryos were on cleavage stage, the overall pregnancy rate for the AHA group was 27.46 and for the NO AHA group was 35.42% (p = 0.2957). In the subgroup of ≤ 35-years, pregnancy rates were 44.44% and 58.33% respectively (p = 0.4231). In the 36-40years subgroup, rates were 33.33% and 42.86% respectively (p = 0.5236). And finally, in the ≥41-years subgroup, pregnancy rates were 18.42% and 18.18% respectively (p = 0.9796). In the group where the embryos were on blastocyst stage, the overall pregnancy rate for the AHA group was 62.82% and for the NO AHA group was 60.14% (p = 0.2486). In the subgroup of ≤ 35-years, pregnancy rates were 71.66% and 67.03% respectively (p = 0.2430). In the 36-40years subgroup, rates were 60.16% and 61.31% respectively (p = 0.7786). And finally, in the ≥41-years subgroup, pregnancy rates were 58.37% and 53.08% respectively (p = 0.1763). Limitations, reasons for caution Clinical pregnancy rates and live birth rates were not available so as to draw a safer conclusion regarding the effectiveness of the method. In contrast to all kind of cases (both homologous and heterologous cycles) having been pooled together, an appropriate subgrouping might would have been the optimal approach. Wider implications of the findings AHA before transfer of vitrified/warmed embryos does not improve pregnancy rates regardless of stage or age. Nevertheless of the trend towards improving pregnancy rates, the difference is not statistically significant. Large scale, well-designed and appropriately subgrouped studies are necessary in order to investigate if this trend can become statistically significant. Trial registration number N/A
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Campagnolo, L. "O-093 Endometrium-blastocyst cross talk: how far are we from understanding?" Human Reproduction 38, Supplement_1 (June 1, 2023). http://dx.doi.org/10.1093/humrep/dead093.112.
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Abstract The early events characterising implantation of the human embryo represent a critical step in which proper activation of orchestrated molecular pathways regulate the success of pregnancy. Understanding this complex network is considered by many the last frontier of human reproduction. Although outstanding research advancements have provided clinicians tools to select the best quality embryo, identification of the optimal timing in which the endometrium is ready to accommodate it remains a challenge. Over the last decade several studies have identified many key players involved in the preparation of the endometrium to implantation and several diffusible signals guiding the embryo in its journey toward the maternal endometrial tissue have been uncovered (Massimiani et al. 2020). However, pieces of the puzzle are still missing. For convenience, the process of implantation is subdivided into several sequential processes, the completion of one being propaedeutic to the following. Once the blastocyst has arrived in close proximity to the uterine epithelium, the first event occurring is its escape from the zona pellucida. The molecular basis of blastocyst hatching remains poorly understood, and species-specific differences have been reported. Using the mouse model, we recently demonstrated that thyroid hormone up-regulates the expression of specific lytic enzymes (Isp1 and Isp2) known mediators of mouse embryo zona lysis. Interestingly, we observed that TH-mediated upregulation of Isp1 and Isp2 is strictly dependent on the presence of endometrial stromal cell feeder layer, clearly indicating that blastocyst–endometrium interaction is indispensable for TH-mediated increased expression of these proteases. So far, no molecular signals regulating hatching in humans have been identified, and only mechanical forces have been proposed to induce zona opening; however very recently the possibility that proteases may be involved also in humans has been suggested (Almagor et al., 2020), although further studies are needed to support this hypothesis. Once free from the zona, the embryo contacts the endometrial epithelium and starts its journey to further proceed in the consolidation of pregnancy. Hormones, pro- and anti-inflammatory mediators, growth factors and their receptors, adhesion molecules, and proteases and protease inhibitors have been reported in the regulation of the process of embryo apposition, adhesion, and invasion (Massimiani et al., 2020). Among the many molecular signals, the Notch pathway has been recognized key in embryo-endometrium cross talk (Cuman et al., 2014). Transient activation of the Notch1 receptor has been demonstrated in the process of decidualization (Afshar et al., 2012), a limiting step to implantation, and blastocyst-conditioned medium has been shown to induce the expression of Notch family members in decidual cells (Hess et al., 2007). Expression of notch receptors and ligands has been reported in the blastocyst trophectoderm as well (Cuman et al., 2014). We previously demonstrated that the secreted factor epidermal growth factor-like domain 7 (EGFL7) is a novel modulator of the Notch pathway, it is expressed by trophoblast cells of the mouse blastocyst (Fitch et al., 2004; Lacko et al., 2014) and it regulates human trophoblast migration and invasion ability by activating the Notch pathway (Massimiani et al., 2015). Data will be presented demonstrating that EGFL7 is expressed in the human endometrium and its expression is mainly localized in the glandular epithelium throughout the menstrual cycle. EGFL7 is significantly upregulated in whole endometrial tissue during the secretory phase, particularly in the stromal compartment; this observation is further supported by in vitro studies showing its upregulation in decidualized endometrial stromal cells, suggesting a role for EGFL7 in implantation. Further support is provided by the evidence that its levels are strongly downregulated in the endometrium of women with impaired fertility. We propose EGFL7 as a novel player involved in the embryo-endometrium cross talk.