Differential regulation of H3S10 phosphorylation, mitosis progression and cell fate by Aurora Kinase B and C in mouse preimplantation embryos

Coordination of cell division and cell fate is crucial for the successful development of mammalian early embryos. Aurora kinases are evolutionarily conserved serine/threonine kinases and key regulators of mitosis. Aurora kinase B (AurkB) is ubiquitously expressed while Aurora kinase C (AurkC) is specifically expressed in gametes and preimplantation embryos. We found that increasing AurkC level in one blastomere of the 2-cell embryo accelerated cell division and decreasing AurkC level slowed down mitosis. Changing AurkB level had the opposite effect. The kinase domains of AurkB and AurkC were responsible for their different ability to phosphorylate Histone H3 Serine 10 (H3S10P) and regulate metaphase timing. Using an Oct4-photoactivatable GFP fusion protein (Oct4-paGFP) and fluorescence decay after photoactivation assay, we found that AurkB overexpression reduced Oct4 retention in the nucleus. Finally, we show that blastomeres with higher AurkC level elevated pluripotency gene expression, which were inclined to enter the inner cell mass lineage and subsequently contributed to the embryo proper. Collectively, our results are the first demonstration that the activity of mitotic kinases can influence cell fate decisions in mammalian preimplantation embryos and have important implications to assisted reproduction.

Pathogenic variant in NLRP7 (19q13.42) associated with recurrent gestational trophoblastic disease: Data from early embryo development observed during in vitro fertilization / 대한생식의학회지

OBJECTIVE: To describe in vitro development of human embryos derived from an individual with a homozygous pathogenic variant in NLRP7 (19q13.42) and recurrent hydatidiform mole (HM), an autosomal recessive condition thought to occur secondary to an oocyte defect. METHODS: A patient with five consecutive HM pregnancies was genomically evaluated via next generation sequencing followed by controlled ovarian hyperstimulation, in vitro fertilization (IVF) with intracytoplasmic sperm injection, embryo culture, and preimplantation genetic screening. Findings in NLRP7 were recorded and embryo culture and biopsy data were tabulated as a function of parental origin for any identified ploidy error. RESULTS: The patient was found to have a pathogenic variant in NLRP7 (c.2810+2T>G) in a homozygous state. Fifteen oocytes were retrieved and 10 embryos were available after fertilization via intracytoplasmic sperm injection. Developmental arrest was noted for all 10 embryos after 144 hours in culture, thus no transfer was possible. These non-viable embryos were evaluated by karyomapping and all were diploid biparental; two were euploid and eight had various aneuploidies all of maternal origin. CONCLUSION: This is the first report of early human embryo development from a patient with any NLRP7 mutation. The pathogenic variant identified here resulted in global developmental arrest at or before blastocyst stage. Standard IVF should therefore be discouraged for such patients, who instead need to consider oocyte (or embryo) donation with IVF as preferred clinical methods to treat infertility.

Effect of gonadotropins on chromosome aneuploidy, chromosome mosaicism & sex ratio in mouse preimplantation embryos.

Background & objectives: There are potential risks of major birth defect in IVF (in vitro fertilization) pregnancy as well as IVF-ICSI (intra cytoplasmic sperm injection) pregnancies in comparison with naturally conceived human pregnancies. This increase risk could be due to either gonadotropins used for ovarian stimulation or in vitro culture conditions or multiple pregnancy or combinations of all the factors. The effects of gonadotropins on chromosome aneuploidy, chromosome mosaicism and sex ratio on mouse preimplantation embryos were evaluated through the use of fl uorescence in situ hybridization (FISH). Methods: The study material consisted of 111 preimplantation mouse embryos (2-16 cell stage) in control group and 405 preimplantation mouse embryos in gonadotropin stimulated group from genetically identical Swiss Albino young (6-8 wk) mouse kept in a similar environmental conditions. The study was designed to investigate effect of gonadotropins on chromosome aneuploidy, chromosome mosaicism and sex ratio through the use of FISH technique using chromosome X, Y and 19 probes. All blastomeres of embryos in both groups were assessed. Results: Interpretable FISH results were obtained in 66 embryos in control group and 128 embryos in gonadotropin stimulated group. There was no excess of chromosome aneuploidy (only one case of sex chromosome trisomy in study group; 19, 19, X, Y, Y) or chromosome mosaicism or deviations in sex ratio between the two groups. However, deviation (1.36 M: 1 F in control group & 1.25 M : 1 F in study group) was seen from expected sex ratio (1 M : 1 F) i.e., skewed sex ratio in both the groups. Interpretation & conclusions: Our results showed that gonadotropins used for ovarian stimulation had no effects in causing increase in chromosome X, Y, 19 aneuploidy and mosaicism and skewing of sex ratio in mouse model. A large scale study with more FISH probes on a larger sample size need to be done to confi rm the findings.

Moral status of preimplantation embryos / 대한산부인과학회지

here have been continuing challenges to devaluate the moral status of early embryo. The preembryo-embryo distinction was mainly based on the lack of individuation. The term of preembryo was introduced by a frog embryologist and then was literally spread around the world because of policy reasons. The term "preembryo" has not yet been used in most medical textbooks including textbooks of human embryology. Preembryo is one period of continuum during human development and therefore should be regarded as valuable as an early form of human life. Preimplantation embryo is more accurate term for the early embryo.

Moral status of preimplantation embryos / 대한산부인과학회지

here have been continuing challenges to devaluate the moral status of early embryo. The preembryo-embryo distinction was mainly based on the lack of individuation. The term of preembryo was introduced by a frog embryologist and then was literally spread around the world because of policy reasons. The term "preembryo" has not yet been used in most medical textbooks including textbooks of human embryology. Preembryo is one period of continuum during human development and therefore should be regarded as valuable as an early form of human life. Preimplantation embryo is more accurate term for the early embryo.

Effects of Vero Cells Co-culture System on The In Vitro Development of Mouse Preimplantation Embryos in Media with Different Composition of Glucose and Pyruvate / 대한산부인과학회지

OBJECTIVE: The purpose of this study was to examine the effects of vero cells co-culture system on the in vitro development of mouse preimplantation embryos in culture media with different composition of glucose and pyruvate. METHODS: Two-cell embryos were collected from 4-5 weeks old ICR mice. Seven hundreds twenty two embryos were cultured with or without vero cells monolayer in four media with different compositions that was manufactured by two DMEM media with (DMEM-GGP) or without (DMEM-G) glucose and pyruvate. In control, DMEM-G medium which is currently using for human embryo culture in our infertility clinic was used. Group I (DMEM-G(1/4)GP) was cultured in medium which was mixed three volume of DMEM-G and one volume of DMEM-GGP, and group II (DMEM-G(1/2)GP) was cultured in medium which was mixed same volume of DMEM-G and DMEM-GGP, and group III was cultured in DMEM-GGP. All media were added to 20% hFF. Results between different groups were analyzed using a Chi-square test, and considered statistically significant when p value was less than 0.05. RESULTS: The developmental rate into 3-cell

Expression of Ids in Preimplantation Mouse Embryos / 대한불임학회지

OBJECTIVE: The Id family of helix-loop-helix proteins are thought to affect the balance between cell growth and differentiation by negatively regulating the function of basic-helix-loop-helix (bHLH) transcriptional factors. The aim of this study was to investigate the expression pattern of Ids (Id-1,-2,-3, and -4) in preimplantation mouse embryos at mRNA and protein levels. METHODS: Oocytes and preimplantation embryos were collected from reproductive organs of female ICR mice following superovulation. RT-PCR was performed to investigate the mRNA expression patterns of Id genes and their protein were localized by immunofluorescence analysis. RESULTS: Id-1 and Id-3 mRNAs were strongly expressed at the germinal vesicle (GV) oocyte and the blastocyst stages. Id-2 mRNA was expressed throughout preimplantation embryo development, but Id-4 was not expressed. Immunofluorescence showed that Id-1 and Id-2 were predominantly localized in cytoplasmic region, but the immunofluorescence signal of Id-3 was weak throughout preimplantation embryo development. CONCLUSION: These data show for the first time that Ids are expressed in preimplantation mouse embryos and suggest that Ids may play an important role in early preimplantation embryo development and uterine physiological changes.

Expression of Apoptotic Genes in Mouse Preimplantation Embryo Development / 대한불임학회지

OBJECTIVES: The aim of this study was to evaluate the influence of three different media on preimplatation embryo development and the expression of Bcl-2, Mcl-1, Bax, and Bok in mouse. MATERiALS AND METHODS: Two-cell embryos were retrieved from iCR female mice (4 weeks old) at 48 hr after hCG injection and cultured in Ham's F-10, HTF, and G1.2 media. The developmental rate of 2-cell embryos was evaluated from 24 hr to 72 hr after culture. RT-PCR was performed for the detection of Bcl-2, Mcl-1, Bax, and Bok gene expression. RESULTS: The rates of morula and blastocyst in HTF and G1.2 media (88%, 98.1%) were significantly higher than those in Ham's F-10 media (39.6%) at 48 hr. Likewise, the rates of hatching and hatched blastocyst in HTF and G1.2 media (21.9%, 52.9%) were higher than those in Ham's F-10 media (3.5%) at 72 hr. Bcl-2 and Bax mRNAs were highly detected in embryos cultured in Ham's F-10 when compared in embryos cultured in HTF and G1.2. in contrast, the expression of Mcl-1 and Bok was not significantly different. CONCLUSION: These results show that HTF and G1.2 culture media increase the rate of blastocyst formation and stimulate Bcl-2 and Bax gene expression in mouse preimplantation embryos.

The Effects of 5% Oxygen Condition and Superoxide Dismutase ( SOD ) on the in - vitro Development of Preimplantation Mouse Embryos / 대한산부인과학회잡지

OBJECTIVE: In the human body the embryo initially gmws in the fallopian tube which is maintained in an 3-8% O2 concentration environment, and various substances such as growth factors and antioxidants present in tbe tubal fluid assists in maintaining a healthy environment for embryo development. But in IVF programs embryo cultures are conducted in incubators with 21.9% O2 and 5% CO2 condition, and such high oxygen concentrations have been reported to increase the production of oxygen free radicals within the embryo and is detrimental to the growth and development of the embryo. The objective of this study, therefore, is to determine the culture conditions which will decrease oxygen free radical production and thereby minimize the injury to the embryo. METHODS: Six to eight week old ICR strain mice embryos were cultured in 5% or 21.9% O2 conditions and in culture media to which inaement concentrations of superoxide dismutase (SOD) had been and the H2O2 concentration within the embryo, embryo developmental rate, and degree of fragmentation of the embryos was investigated. RESULTS: The control gmup embryos which were cultured in 21.9% O2 condition without addition of SOD showed developmental arrest at the 2-cell stage or fragmentation, while those cultured in 21.9% O2 condition with addition of SOD showed development to the blastocyst stage with deaeased fragmentation. In particular, the blastulation and fragmentation rates were the lowest in the group to which 500 IU/ml of SOD was added, but in the 5% O2 enviranment group many embryos reached the blastocyst stage and with no difference in frapnentation with or without addition of SOD. The HO relative intensity (120.5+/-20.2) within the embryos cultured in 21.9% O2 environment without SOD was significantly higher than that (56.8+/-10.8) of group with SOD (p<0.05). As showing that in the 5% O2 environment group without SOD it was 43.8+/-7.8 and in the group with SOD it was 37.3+/-5.4, the H2O2 concentration within embryos cultured in 5% 02 condition was significantly lower those that of 21,9% 02 environment regardless of SOD addition (p<0.05). CONCLUSION: The optimal oxygen concentration in incubator for mice embryo cultures is that which is similar to the 5% 0 concentration in vivo. When 20% 02 incubators are routinely used, the addition of SOD to the culture media will decrease the H2O2 concentration within the embryos with subsequent improvement in development. The optimal concentration which should be used is thought to be 500 IU/ml. It is suggested that the use of the above method in human IVF-ET programs will lead to improved embryo quality and enhanced pregnancy rates.

Effects of Exposure to Nitrous Oxide After Fertilization on Embryo in Vitro and in Vivo Development in Mice / 대한마취과학회지

BACKGROUND: Nitrous oxide exposure for long periods during gestation causes the increased fetal wastage, growth retardation, morphological abnormalities in rodents. Most studies have explained deleterious effects of nitrous oxide on postimplantation embryo development. The aim of this study was to evaluate the effects of nitrous oxide on embryo after the fertilization in superovulated BALB/c mice. METHODS: Pregnant mice were exposed to 70% nitrous oxide in oxygen for 6, 12 and 24 hours on the day of gestation, and 2-cell stage embryos were cultured to blastocyst. Reproductive data were determined at cesarean section on 16th day of gestation based on embryonic developmental failure in vitro by 24 hours nitrous oxide. The protective effects of folinic acid or methionine against inhibition of 2-cell embryo development were also evaluated. RESULTS: Blastocyst development was significantly lower in 12 and 24 hours nitrous oxide group than in the control and 6 hours nitrous oxide group. The pregnancy rate and the mean number of implantations were significantly lower in 24 hours nitrous oxide group than in the control. No significant differences in percentage of the living fetus, the dead fetus, the resorption per implantation, the mean fetal weight and the crown-rump length were observed between nitrous oxide group and control group. There was no significant difference between the nitrous oxide group and the nitrous exposed group receiving methionine and folinic acid. CONCLUSIONS: The exposure to high concentration of nitrous oxide for a long time after the fertilization in mice may be possibility of the early abortion of embryos, whereas there is not any influence on fetus after the implantation.