Embryo development is impaired by sperm mitochondrial-derived ROS

BACKGROUND: Basal energetic metabolism in sperm, particularly oxidative phosphorylation, is known to condition not only their oocyte fertilising ability, but also the subsequent embryo development. While the molecular pathways underlying these events still need to be elucidated, reactive oxygen species (ROS) could have a relevant role. We, therefore, aimed to describe the mechanisms through which mitochondrial activity can influence the first stages of embryo development. RESULTS: We first show that embryo development is tightly influenced by both intracellular ROS and mitochondrial activity. In addition, we depict that the inhibition of mitochondrial activity dramatically decreases intracellular ROS levels. Finally, we also demonstrate that the inhibition of mitochondrial respiration positively influences sperm DNA integrity, most likely because of the depletion of intracellular ROS formation. CONCLUSION: Collectively, the data presented in this work reveals that impairment of early embryo development may result from the accumulation of sperm DNA damage caused by mitochondrial-derived ROS.

Effect of advanced paternal age on reproductive outcomes in IVF cycles of non-male-factor infertility: a retrospective cohort study / 亚洲男科学杂志(英文版)

Advanced paternal age has been overlooked, and its effect on fertility remains controversial. Previous studies have focused mainly on intracytoplasmic sperm injection (ICSI) cycles in men with oligozoospermia. However, few studies have reported on men with semen parameters within reference ranges. Therefore, we conducted a retrospective cohort study analyzing the reproductive outcomes of couples with non-male-factor infertility undergoing in vitro fertilization (IVF) cycles. In total, 381 cycles included were subgrouped according to paternal age (<35-year-old, 35-39-year-old, or ≥40-year-old), and maternal age was limited to under 35 years. Data on embryo quality and clinical outcomes were analyzed. The results showed that fertilization and high-quality embryo rates were not significantly different (all P > 0.05). The pregnancy rate was not significantly different in the 35-39-year-old group (42.0%; P > 0.05), but was significantly lower in the ≥40-year-old group (26.1%; P < 0.05) than that in the <35-year-old group (40.3%). Similarly, the implantation rate significantly decreased in the ≥40-year-old group (18.8%) compared with that in the <35-year-old group (31.1%) and 35-39-year-old group (30.0%) (both P < 0.05). The live birth rate (30.6%, 21.7%, and 19.6%) was not significantly different across the paternal age subgroups (<35-year-old, 35-39-year-old, and ≥40-year-old, respectively; all P > 0.05), but showed a declining trend. The miscarriage rate significantly increased in the 35-39-year-old group (44.8%) compared with that in the <35-year-old group (21.0%; P < 0.05). No abnormality in newborn birth weight was found. The results indicated that paternal age over 40 years is a key risk factor that influences the assisted reproductive technology success rate even with good semen parameters, although it has no impact on embryo development.

Lipid Peroxidation Status in Embryo Culture Media: The Impact on Fertilization and Embryo Quality during IVF Cycles

Background: The process of preimplantation embryo development in vitro represents a key phase during in vitro fertilization (IVF) cycles. It involves several regulatory signaling pathways as well as an optimized in vitro culture system. The resulting embryo quality helps to determine embryo competence before implantation and pregnancy outcomes. Reactive oxygen species (ROS) are known to play a major role in influencing the process of embryo development. Their role can be reflected in the regulation of signaling pathways as second messengers or in the irreversible cell alterations due to oxidative stress following an excess of ROS levels. Methods: In this study, we investigated the association between morphological embryo quality (fertilization, cleavage quality, and fragmentation levels) and lipid peroxidation levels (Malondialdehyde) in embryo culture media. After intracytoplasmic sperm injection (ICSI), a total of 103 oocytes were evaluated on day 1 and day 3 of their development, and their corresponding culture media were analyzed by estimating MDA levels using thiobarbituric acid. Results: The results showed no significant association between MDA levels in culture media and fertilization rate (p=0.3), sperm quality (p=0.99; p= 0.17; p=0.46; p=0.30; p=0.65; p=0.44; p=0.09; p=0.15; p=0.56), embryo fragmentation levels (p=0.79; p=0.40), AMH levels (p=0.31; p=0.36) and female age (p=0.60; p=0.34). However, we revealed a significant association between cleavage quality and MDA levels in the embryo environment (p=0.03). Conclusion: We conclude that oxidative stress in IVF culture media might be mainly associated with delayed embryonic development.

Sperm DNA damage compromises embryo development, but not oocyte fertilisation in pigs

BACKGROUND: The assessment of sperm DNA integrity has been proposed as a complementary test to conventional mammalian semen analysis. In this sense, single-strand (SSB) and double-strand (DSB) DNA breaks, the two types of sperm DNA fragmentation (SDF), have been reported to have different aetiologies and to be associated to different fertility outcomes in bovine and humans. Considering that no studies in porcine have addressed how SDF may affect sperm quality and fertility outcomes, the present work aimed to determine the impact of global DNA damage, SSB and DSB on sperm quality and in vitro fertilising ability. To this end, 24 ejaculates (one per boar) were split into three aliquots: the first was used to assess sperm quality parameters through a computer-assisted sperm analysis (CASA) system and flow cytometry; the second was used to perform in vitro fertilisation, and the third, to evaluate sperm DNA integrity using alkaline and neutral Comet assays. RESULTS: The results showed that global DNA damage negatively correlates (P 0.05). CONCLUSION: Considering all these findings, this work sets a useful model to study how SDF negatively influences fertility.

Insights into epigenetic patterns in mammalian early embryos

Mammalian fertilization begins with the fusion of two specialized gametes, followed by major epigenetic remodeling leading to the formation of a totipotent embryo. During the development of the pre-implantation embryo, precise reprogramming progress is a prerequisite for avoiding developmental defects or embryonic lethality, but the underlying molecular mechanisms remain elusive. For the past few years, unprecedented breakthroughs have been made in mapping the regulatory network of dynamic epigenomes during mammalian early embryo development, taking advantage of multiple advances and innovations in low-input genome-wide chromatin analysis technologies. The aim of this review is to highlight the most recent progress in understanding the mechanisms of epigenetic remodeling during early embryogenesis in mammals, including DNA methylation, histone modifications, chromatin accessibility and 3D chromatin organization.

Effects of sequential and single-step culture media systems on human oocyte fertilization and early embryo development / 吉林大学学报(医学版)

Objective: To compare the effects of sequential and single-step culture media systems on the development of human early embryo in in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) cycles, and to provide a reference for the selection and evaluation of the human embryo culture system of assisted reproductive technology (ART). Methods, A total of 155 patients received IVF/ICSI treatment were selected and the ova from the same patient were divided into two groups. The ova were cultured in Vitrolife sequential media system G-IVF/G1/G2 culture solution (sequential culture group) and Irvine single-step media system CSCC culture solution (single-step culture group) for IVF and embryo culture. The fertilization and early embryo development in the different culture systems in two groups were observed. Results: Compared with single-step culture group, the fertilization rate, 2PN fertil ization rate and multi-nuclear fertilization rate of the patients in sequential culture group had no significant differences (P>0. 05). In the IVF patients, there were no significant differences in the cleavage rates, quality embryo rates, high quality embryo rates and blastocyst formation rates between sequential culture group and single-step culture group (P>0. 05). In the ICSI patients, there were also no significant differences in the cleavage rates, quality embryo rates and blastocyst formation rates between sequential culture group and singlestep culture group (P>0. 05). However, the high quality embryo rate of the patients in sequential culture group was significantly higher than that in single-step culture group (P=0. 015). Both in the IVF and ICSI patients, the percents of densification embryos in single-step culture group were significantly higher than those in sequential culture group (P = 0.001). Moreover, the embryos cultured in sequental culture group were smooth and homogeneous, but the embryos cultured in single-step culture group were rough and granular. Conclusion: There are no significant differences in fertilization and early embryo development between the two culture media systems.

Differences between NMRI and DBA/2J mice in the development of somites and susceptibility to methylnitrosourea-induced skeleton anomalies

ABSTRACT The development of DBA/2J mouse strain embryos is nearly 12 h - or 6 somite pairs - delayed as compared to the outbred NMRI mouse embryos of the same age on gestation days (GD) 8-12. To evaluate inter-strain differences in susceptibility to teratogens, dams were treated with methylnitrosourea (MNU, 5 mg/kg body weight i.p.) on defined gestation days (NMRI: GD 9, 91/2 or 10; DBA/2J: GD 10 or 101/2). Skeletal anomalies produced by MNU on both mouse strains varied with the GD of treatment. The pattern of anomalies produced by MNU on a given GD markedly differed between the two mouse strains, yet they were similar -with a few exceptions- when exposures at equivalent embryonic stages are compared. Findings from this study indicated that strain-dependent differences in the developmental stage of mouse embryos of the same gestational age occur, a possibility that has been often neglected when inter-strain differences in susceptibility to developmental toxicants are interpreted.

Desarrollo embrionario preimplantacional, en ciclos de fecundación in vitro con ovodonación y estudio genético / Preimplantational embryo development in in vitro fertilization cycles with oocyte donation and genetic studies

Introducción. Los estudios genéticos preimplantacionales son cada vez más utilizados con la esperanza de conseguir mejores tasas de implantación y nacido vivo, así como una disminución en la tasa de abortos; por ello resulta necesario analizar estos procedimientos. Objetivo. Evaluar el desarrollo preimplantacional in vitro con ovodonación y estudio genético Diseño. Cohortes retrospectivas. Lugar. Laboratorio Pranor, 20082013. Material. Ciclos de fecundación in vitro con ovodonación (FIV-OD). Intervenciones. Se evaluaron 2 077 ciclos de FIV-OD, los cuales fueron clasificados en tres cohortes: 1) ciclos con biopsia embrionaria en día 3, mediante una incisión en la zona pelúcida (ZP) embrionaria, para exéresis de una blastómera (n=527); 2) ciclos con incisión láser en día 4 del desarrollo embrionario, como parte del procedimiento para la biopsia de trofoblasto (n=131); y, 3) ciclos sin intervención (n=1 419). Principales medidas de resultado. Tasa de fecundación, tasa de blastulación. Resultados. No existió diferencia significativa en la tasa de fecundación de las 3 cohortes (75,0%, 74,6% y 75,9%, respectivamente, p=0,31). La tasa de blastulación en la cohorte 1 fue 42,5%, mientras que en la cohorte 3 fue 47% (RR=1,085; IC=1,051 a 1,120; p<0,0001). Adicionalmente, la cohorte 2 tuvo 51,9%, con una diferencia estadísticamente significativa de prevención del riesgo de no blastular con respecto a la cohorte 3 (RR=0,906; IC=0,851 a -0,965; p=0,0017). Conclusiones. El desarrollo preimplantacional hasta blastocisto puede mejorar cuando se utiliza el láser embrionario en día cuatro. Es necesario realizar más estudios para confirmar nuestros resultados.

Introduction: Preimplantational genetic studies are used to achieve a better implantation rate and live birth, as well as to decrease the abortion rate; these techniques should be evaluated. Objective: To evaluate preimplantational embryo development in in vitro fertilization cycles with oocyte donation and genetic studies.. Design: Retrospective cohort study. Setting: Pranor Laboratory, 2008-2013. Material: In vitro fertilization with oocyte donation (IVF-OD) cycles. Interventions: 2 077 cycles of IVF-OD were evaluated, separated in 3 cohorts: Cohort 1, cycles with embryo biopsy on day 3, by means of an incision in the zona pellucida (ZP), for excision of a blastomer (n = 527); Cohort 2, cycles with a laser incision at day 4 of embryo development, for biopsy of the trophoblast in the blastocyst stage (n = 131) and; cohort 3, FIV-OD cycles without any intervention (n = 1 419). Main outcome measures: Fertilization rate, blastulation rate. Results: There was no difference in the fertilization rate among the three groups studied (75.0%, 74.6% and 75.9% respectively, p = 0.31). Blastulation rate in cohort 1, was 42.5%, whereas, in cohort 3, it was 47% (RR = 1.085, CI = 1.0511.120; p <0.0001). In addition, the rate for cohort 2 was 51.9%, with statistical significance, which prevents from non-blastulation risk compared with cohort 3 (RR = 0.906; IC = 0.851-0.965; p = 0.0017). Conclusions: Preimplantational embryo development would improve blastocyst formation when laser is performed on day 4. Further studies are needed to confirm our results.

Spatial Allocation and Specification of Cardiomyocytes during Zebrafish Embryogenesis

Incomplete development and severe malformation of the heart result in miscarriage of embryos because of its malfunction as a pump for circulation. During cardiogenesis, development of the heart is precisely coordinated by the genetically-primed program that is revealed by the sequential expression of transcription factors. It is important to investigate how spatial allocation of the heart containing cardiomyocytes and other mesoderm-derived cells is determined. In addition, the molecular mechanism underlying cardiomyocyte differentiation still remains elusive. The location of ectoderm-, mesoderm-, and endoderm-derived organs is determined by their initial allocation and subsequent mutual cell-cell interactions or paracrine-based regulation. In the present work, we provide an overview of cardiac development controlled by the germ layers and discuss the points that should be uncovered in future for understanding cardiogenesis.

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 the quality of embryos on biochemical pregnancy or arrest of embryo development in the freezing and thawing cycles / 上海交通大学学报（医学版）

Objective · To investigate whether the quality of embryos will result in biochemical pregnancy or arrest of embryo development in the freezing and thawing cycles of in-vitro fertiliazation-embryo transfer (IVF-ET). Methods · The clinical data of patients who accepted IVF-ET in Center of Reproductive Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine from January 2015 to June 2016 were retrospectively studied. The data includes 115 cycles of biochemical pregnancy, 64 cycles of arrest of early embryonic development and 871 cycles of ongoing pregnancy after frozen thawed embryo transfer. We compared the embryo score on the third day after embryo transfer (D3), the blastocyst development rate and the blastocyst grade in the three groups. Results · There were no significant differences in the period of infertility, the age of the patients and their spouses, the endometrial thickness, the estrogen and progestogen levels of the day of transplantation among the three groups (P > 0.05). The scores of most frozen thawed embryos on D3 were from 6 to 8, and the scores were not statistically significant among the three groups (P > 0.05). The proportion of transplanted blastocyst on D5 was higher than that on D6 in the three groups, but there was no significant difference among the three groups (P > 0.05). There was no significant difference in the proportion of inner cell mass of blastocysts which were scored as Grade A&B or Grade C among the three groups. Nevertheless, in the arrest of early embryonic development group, the proportion (52.2%) of the trophoblast of blastocysts which were cored as Grade C was significantly higher than the proportion (35%) in biochemical pregnancy group and the proportion (29.3%) in ongoing pregnancy group (P<0.05). Conclusion · The quality of embryos is not necessarily related to biochemical pregnancy, but the score of trophoblastic may be related to the arrest of early embryo growth.

Application of chromosomal microarray technique in genetic analysis of patients with embryo development arrest / 实用医学杂志

Objective To investigate the value of chromosomal microarray technique in genetic analysis of patients with embryo development arrest. Methods A total number of 273 patients with embryo development arrest were recruited for the chromosomal microarray testing.Results 41.4% of the 273 patients were chromosomal abnormalities. Among which 61(22.34%)were numerical chromosomal abnormalities,43 were structural anoma-lies,including which 15.75% were terminal deletion or duplication and microdeletion or microduplication. And 9 (3.3%)were mosaicisms.Conclusions Chromosomal microarray technique is highly accurate and specific,which can offer more genetic information than conventional karyotyping. And chromosomal microarray technique can also facilitate estimation of recurrence risk of future pregnancies for patients with embryo development arrest.

Comparative study of fertilization rates of C57BL/6NKorl and C57BL/6N mice obtained from two other sources / 한국실험동물학회지

C57BL/6N is the most widely used inbred mouse strain applied in a wide variety of research areas including cancer, cardiovascular biology, developmental biology, diabetes and obesity, genetics, immunology, neurobiology, and sensorineural research. To compare the fertilization rates of C57BL/6NKorl mice with two commercial C57BL/6N stocks, differences in reproductive organ structures, sperm and egg numbers, fertilization rates, and embryo development rates among C57BL/6NKorl (Korea FDA source), C57BL/6NA (USA source), and C57BL/6NB (Japan source) mice were determined. Among the stocks, no significant differences were detected in organ weight and histological structure of male and female reproductive organs, although body weight was higher in C57BL/6NKorl mice than that in the other groups. The concentration and morphology of sperm and eggs in C57BL/6NKorl mice were similar to those of C57BL/6NA and C57BL/6NB mice. Furthermore, the three stocks had similar in vitro fertilization and embryo development rates, although these rates tended to be higher in C57BL/6NB mice. Pup body weight was higher in C57BL/6NKorl and C57BL/6NB mice than that in C57BL/6NA mice. The results of the present study suggest that C57BL/6NKorl, C57BL/6NA, and C57BL/6NB mice obtained from three different sources have similar fertilization and embryo development rates, although there were slight differences in the magnitude of their responses rates.

Interaction between endometrium and embryo under the condition of in vitro co-culture during the implantation window stage / 实用医学杂志

Objective To investigate the interaction between endometrium and embryo under the co-culture condition during the planting window stage. Methods Twenty patients who cancelled transplant for OHSS in the Reproductive Center of the second affiliated hospital of Zhengzhou university were enrolled.The discarded embryos were used to build the embryos co-culture system (Group A), with a single membrane (group B) and a single embryo(group C)as the control group. Levels of LIF and IGF expression and embryo growth were checked on 3, 4,5,6,7 day post-oocyte retrieval. Results Expressions of LIF mRNA and IGF mRNA in Group A were significantly higher than those in Group B. Embryo growth in the Group A was better than that in Group C. Conclusion Co-culture of endometrium and embryo supports the development of embryo and improves the receptivity of endometrium.

In vitro development and chimeric efficiency of mouse-porcine interspecies chimeric embryos in different culture systems / 生物工程学报

With the advancements of stem cells and regenerative medicine, interspecies chimera has become a hot topic and will pave a new way of providing donor sources in organ transplantation. However, the interspecies chimera is confronted with a number of scientific questions and technical obstacles, including selections of appropriate embryonic stage and appropriate culture medium; those factors will deeply influence the developmental balance between donor cells and receptor embryos. Due to its relatively rapid reproductive cycle and similar organ size to human's, porcine is a very potential donor candidate to study these questions. To compare the development and chimeric efficiency of interspecies embryos, we tested and evaluated three different culture systems, PZM-3 (Porcine zygotic medium), culture medium for iPSCs (N2B27) and 3.5 h of N2B27 before PZM-3 (N2B27(3.5 h)), and two different embryonic stages, 8-cell and blastocyst in mouse-porcine chimeric embryos using parthenogenetically activated porcine embryos and mouse induced pluripotent stem cells (miPS). The results showed that, PZM-3 was beneficial for both development of chimeric embryos and miPSCs proliferation in porcine embryos in the 8-cell injection group. After early blastocyst injection, the chimeric efficiency did not appear significantly different among the three culture systems but was lower than 8-cell injection. In summary, the results suggest that 8-cell injection and PZM-3 culture medium are more beneficial to the in vitro development and chimeric efficiency of mouse-porcine chimeric embryos.

Limitações e controvérsias na determinação do valor preditivo de critérios de morfologia oocitária e embrionária / Limitations and controversies in determining the predictive value of oocyte and embryo morphology criteria

Com o objetivo de aumentar as taxas de sucesso das pacientes que são submetidas a técnicas de reprodução humana assistida (RHA), numerosos estudos apresentam como foco a identificação do embrião com maior potencial de implantação. Apesar dos avanços tecnológicos significativos da Medicina Reprodutiva baseados no advento da era da genômica, proteômica e metabolômica (OMICS), técnicas rotineiramente aplicáveis ainda não estão disponíveis. Dessa forma, laboratórios de fertilização in vitro(FIV) de todo o mundo selecionam para transferência embriões humanos cultivados in vitrobaseados em parâmetros morfológicos avaliados em microscopia de luz. Diversos parâmetros morfológicos podem ser avaliados desde o estágio pronuclear até o estágio de blastocisto para embriões humanos cultivados in vitro. De modo geral, independentemente do dia da transferência, tais critérios parecem apresentar valor preditivo de viabilidade embrionária quando avaliados individualmente ou coletivamente. No entanto, a subjetividade da avaliação morfológica, bem como a ampla diversidade de sistemas de classificação embrionária aplicados por diferentes clínicas, implica em resultados contraditórios, tornando extremamente difícil a implementação de um consenso do valor preditivo dos diferentes parâmetros morfológicos avaliados. A otimização da seleção embrionária representa um grande potencial de aumento das taxas de sucesso do tratamento, além de possibilitar a realização da transferência de um número reduzido de embriões, minimizando os riscos derivados de estações múltiplas.

In order to increase the success rate of in vitrofertilization cycles, several studies have focused on the identification of the embryo with higher implantation potential. Despite recent advances in the reproductive medicine, based on the OMICs technology, routinely applicable methodologies are still needed. Thus, in most fertilization centers embryo selection for transfer is still based on morphological parameters evaluated under light microscopy. Several morphological parameters may be evaluated, ranging from the pronuclear to blastocyst stage. In general, despite the day of transfer, some criteria are suggested to present a predictive value for embryo viability when analyzed independently or combined. However, the subjectivity of morphological evaluation, as well as the wide diversity of embryo classification systems used by different fertilization centers shows contrasting results, making the implementation of a consensus regarding different morphological criteria and their predictive value a difficult task. The optimization of embryo selection represents a large potential to increase treatment success rates, allowing the transfer of a reduced number of embryos and inimizing the risks of multiple pregnancy.

Challenges and perspectives to enhance cattle production via in vitro techniques: focus on epigenetics and cell-secreted vesicles / Desafios e perspectivas na produção in vitro de embriões bovinos: enfoque epigenético e em vesículas secretadas por células

This review aim to present some clinical problems found in IVP-derived animals focusing on NT procedures and to discuss the possible role of epigenetics in such process. Also, as cell-secreted vesicles have been reported as possible regulators of important physiological reproductive processes such as folliculogenesis and fertilization, it is also presented herein a new perspective of manipulating the pre-implantation period trough effector molecules contained in such vesicles.

Nesta revisão, apresentamos alguns problemas clínicos encontrados nos animais derivados de PIV, principalmente derivados de transferência de núcleo, e discutimos o possível papel da epigenética em tais processos. Além disso, uma vez que vesículas secretadas por células têm sido descritas como possíveis reguladores de processos reprodutivos fisiológicos importantes, tais como a foliculogênese e a fertilização, estas são aqui apresentadas como uma possível nova ferramenta para a manipulação do período embrionário pré-implantacional através de moléculas efetoras, contidas em tais vesículas.

Supplementation of fetal bovine serum alters histone modification H3R26me2 during preimplantation development of in vitro produced bovine embryos / Suplementação com soro fetal bovino altera a modificação de histona H3R26me2 durante o período pré-implantacional em embriões bovinos produzidos in vitro

In vitro production (IVP) of bovine embryos is not only of great economic importance to the cattle industry, but is also an important model for studying embryo development. The aim of this study was to evaluate the histone modification, H3R26me2 during pre-implantation development of IVP bovine embryos cultured with or without serum supplementation and how these in vitro treatments compared to in vivo embryos at the morula stage. After in vitro maturation and fertilization, bovine embryos were cultured with either 0 or 2.5% fetal bovine serum (FBS). Development was evaluated and embryos were collected and fixed at different stages during development (2-, 4-, 8-, 16-cell, morula and blastocyst). Fixed embryos were then used for immunofluorescence utilizing an antibody for H3R26me2. Images of stained embryos were analyzed as a percentage of total DNA. Embryos cultured with 2.5% FBS developed to blastocysts at a greater rate than 0%FBS groups (34.85±5.43% vs. 23.38±2.93%; P<0.05). Levels of H3R26me2 changed for both groups over development. In the 0%FBS group, the greatest amount of H3R26me2 staining was at the 4-cell (P<0.05), 16-cell (P<0.05) and morula (P<0.05) stages. In the 2.5%FBS group, only 4-cell stage embryos were significantly higher than all other stages (P<0.01). Morula stage in vivo embryos had similar levels as the 0%FBS group, and both were significantly higher than the 2.5%FBS group. These results suggest that the histone modification H3R26me2 is regulated during development of pre-implantation bovine embryos, and that culture conditions greatly alter this regulation...

A produção in vitro (PIV) de embriões de bovinos não é apenas de grande importância econômica para a pecuária, mas é também um importante modelo para estudar o desenvolvimento embrionário. O objetivo deste estudo foi avaliar a modificação de histona, H3R26me2 durante o desenvolvimento pré-implantacional em embriões bovinos produzidos in vitro, cultivados com ou sem suplementação de soro fetal bovino (SFB), bem como comparar essa modificação específica entre mórulas produzidas in vitro e in vivo. Após a maturação in vitro e fertilização, embriões foram cultivados com suplementação de 0 ou 2,5% SFB. O desenvolvimento embrionário foi avaliado e embriões foram coletados e fixados em diferentes fases durante o desenvolvimento (2, 4, 8 e 16 células, mórula e blastocisto). Os embriões fixados foram avaliados por imunofluorescência utilizando um anticorpo para H3R26me2. Imagens de embriões corados foram analisadas baseadas na porcentagem do DNA total. Embriões cultivados com 2,5% SFB tiveram uma taxa de desenvolvimento ao estágio de blastocisto maior que o grupo que não recebeu suplementação com SFB (34.85±5,43% vs 23.38±,93%; P<0,05). Níveis de H3R26me2 variaram para ambos os grupos ao longo do desenvolvimento. No grupo 0% SFB, a marcação para H3R26me2 foi mais intensa nos estágios de 4 células (P<0,05), 16 células (P<0,05) e mórula (P<0.05). No grupo 2.5% SFB, apenas os embriões de 4 células tiveram marcação significativamente maior que todas as outras fases (P<0,01). Mórulas produzidas in vivo apresentaram níveis de H3R26me2 semelhantes ao grupo 0% SFB, e ambos foram significativamente maiores que o grupo 2.5% SFB. Estes resultados sugerem que a modificação de histona H3R26me2 é regulada durante o desenvolvimento pré-implantacional de embriões bovinos, e que as condições de cultura alteram de maneira importante esta regulação...

The role of ripply1 in zebrafish dorsal-ventral development / 中国实验动物学报

Objective To explore the role of ripply1 in zebrafish dorsal-ventral development .Methods Using ze-brafish whole-mount in situ hybridization to examine the ripply1 expression pattern in early embryo development .To analyse the expression pattern changes of dorsal-ventral marker genes at shield stage and the morphological changes at 24 hpf (hours post-fertilization) after overexpression of ripply1 by injecting synthetic mRNA at 1-cell stage.Using Tol2 transposon technology to obtain a ripply1 promoter driven GFP transgenic fish and to identify promoter region that recapitulates endoge -nous ripply1 expression pattern .Results The in situ hybridization results revealed that ripply1 specifically expresses in the future dorsal region at shield stage .Overexpression of ripply1 caused an enhanced expression of dorsal marker genes and a reduction of ventral marker genes .Embryos overexpressing ripply1 also showed severely dorsalized phenotype , with enlarged head, reduced ventral yolk extension , and shortened posterior trunk and tail regions , and the formation of a secondary trunk axis.Transgenic fish revealed the maternal expression of ripply1 and suggested that a 1.2 kb promoter-driven GFP is able to recapitulate the endogenous gene expression pattern .Conclusion ripply1 may participate in the early development of dor-sal-ventral axis in zebrafish embryo .

Culture media influenced laboratory outcomes but not neonatal birth weight in assisted reproductive technology / 华中科技大学学报（医学）（英德文版）

Whether the type of culture media utilized in assisted reproductive technology has impacts on laboratory outcomes and birth weight of newborns in in-vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) was investigated. A total of 673 patients undergoing IVF/ICSI and giving birth to live singletons after fresh embryo transfer on day 3 from Jan. 1, 2010 to Dec. 31, 2012 were included. Three types of culture media were used during this period: Quinn's Advantage (QA), Single Step Medium (SSM), and Continuous Single Culture medium (CSC). Fertilization rate (FR), normal fertilization rate (NFR), cleavage rate (CR), normal cleavage rate (NCR), good-quality embryo rate (GQER) and neonatal birth weight were compared using one-way ANOVA and χ (2) tests. Multiple linear regression analysis was performed to determine the impact of culture media on laboratory outcomes and birth weight. In IVF cycles, GQER was significantly decreased in SSM medium group as compared with QA or CSC media groups (63.6% vs. 69.0% in QA; vs. 71.3% in CSC, P=0.011). In ICSI cycles, FR, NFR and CR were significantly lower in CSC medium group than in other two media groups. No significant difference was observed in neonatal birthweight among the three groups (P=0.759). Multiple linear regression analyses confirmed that the type of culture medium was correlated with FR, NFR, CR and GQER, but not with neonatal birth weight. The type of culture media had potential influences on laboratory outcomes but did not exhibit an impact on the birth weight of singletons in ART.