Time-lapse imaging of human embryos fertilized with testicular sperm reveals an impact on the first embryonic cell cycle

Abstract Testicular sperm is increasingly used during in vitro fertilization treatment. Testicular sperm has the ability to fertilize the oocyte after intracytoplasmic sperm injection (ICSI), but they have not undergone maturation during epididymal transport. Testicular sperm differs from ejaculated sperm in terms of chromatin maturity, incidence of DNA damage, and RNA content. It is not fully understood what the biological impact is of using testicular sperm, on fertilization, preimplantation embryo development, and postimplantation development. Our goal was to investigate differences in human preimplantation embryo development after ICSI using testicular sperm (TESE-ICSI) and ejaculated sperm. We used time-lapse embryo culture to study these possible differences. Embryos (n = 639) originating from 208 couples undergoing TESE-ICSI treatment were studied and compared to embryos (n = 866) originating from 243 couples undergoing ICSI treatment with ejaculated sperm. Using statistical analysis with linear mixed models, we observed that pronuclei appeared 0.55 h earlier in TESE-ICSI embryos, after which the pronuclear stage lasted 0.55 h longer. Also, significantly more TESE-ICSI embryos showed direct unequal cleavage from the 1-cell stage to the 3-cell stage. TESE-ICSI embryos proceeded faster through the cleavage divisions to the 5- and the 6-cell stage, but this effect disappeared when we adjusted our model for maternal factors. In conclusion, sperm origin affects embryo development during the first embryonic cell cycle, but not developmental kinetics to the 8-cell stage. Our results provide insight into the biological differences between testicular and ejaculated sperm and their impact during human fertilization.

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60 TELOMERE LENGTH DYNAMICS DURING BOVINE PREIMPLANTATION EMBRYO DEVELOPMENT

Reproduction Fertility and Development ◽

10.1071/rdv24n1ab60 ◽

2012 ◽

Vol 24 (1) ◽

pp. 142

Author(s):

C. de Frutos ◽

P. Bermejo-Alvarez ◽

D. Rizos ◽

A. Gutierrez-Adan

Keyword(s):

Developmental Stage ◽

Telomere Length ◽

Embryo Development ◽

Relative Abundance ◽

Preimplantation Embryo ◽

Telomerase Activity ◽

Alternative Mechanism ◽

Cell Stage ◽

Preimplantation Embryo Development ◽

Early Embryos

The establishment of telomere length (TL) during embryogenesis determines telomere reserves in newborn mammals. However, limited information is available on TL dynamics during preimplantation in contrast to the extensive existing data on telomerase activity in germ cells and embryogenesis. Telomerase activity is high in the male germ line, low or absent in oocytes and early stage embryos and high in blastocysts (Bl). Mechanisms allowing early embryos to reset TL remain poorly understood. The documented telomere lengthening at the morula/Bl transition in mice and bovines is dependent on telomerase activity. A recombinant-based mechanism termed alternative lengthening of telomeres (ALT) has been postulated to be responsible for the lengthening of telomeres in early embryos. The aims of the present study were to analyse the telomere dynamics during preimplantation embryo development in 2 species of known different TL: mice and bovines and the relative expression of 2 components of telomerase [telomerase reverse transcriptase (Tert; the key factor that controls the activity of the telomerase) and telomerase RNA component (Terc)]. Twenty samples for each developmental stage with an equivalent number of cells (matured oocytes/zygotes: 8 and 32; 2-cell embryos: 4 and 16; 4-cell embryos: 2 and 8; 8-cell embryos: 1 and 4; 16-cell embryos: 2 only for bovine; morulae: 1 and 1; and Bl: 1 and 1 for mice and bovines, respectively) were analysed for relative TL measurement using the real-time quantitative PCR method described previously (Bermejo-Alvarez et al. 2008 Physiol. Genomics 32, 264272). For measuring the mRNA, 3 groups of 10 oocytes/embryos for each developmental stage were used. Data were analysed by 1-way ANOVA. In mice, matured oocytes had the shortest telomeres of all stages examined (P < 0.01); a slight increase up to the 4-cell stage and a decrease at the 8-cell and morula stages was noted (P < 0.05), while a marked increase was observed in Bl, as expected (P < 0.01). In contrast, bovine matured oocytes had longer telomeres than zygotes and this length gradually decreased up to the 4-cell stage and increased again at the 16-cell stage (P < 0.05). Then, telomeres shortened at the morula stage (P < 0.05) and a significant increase was observed at the Bl stage like in mice (P < 0.01). The relative abundance of mTerc increased throughout development with a marked up-regulation at the morula and Bl stages (P < 0.01). On the other hand, the relative abundance of mTert was significantly higher in the mature oocytes and zygotes compared to later stages (P < 0.01); however, it should be noted that there was a gradual increase from the 2-cell stage up to Bl. In conclusion, in contrast to mice, bovine oocytes have longer telomeres than zygotes. Knowing that the telomerase activity is low or absent until the Bl stage (indicated by the low expression of Tert), the TL increase detected in 16-cell bovine embryos indicates an alternative mechanism for telomere elongation during early development, like that observed in mice. Understanding how telomeres reset during early embryo development has implications for the study of stem cells and regenerative biology.

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257. Activation of a calcium-activated chloride channel by paf is required for normal preimplantation mouse embryo development in vitro

Reproduction Fertility and Development ◽

10.1071/srb08abs257 ◽

2008 ◽

Vol 20 (9) ◽

pp. 57

Author(s):

Y. Li ◽

M. L. Day ◽

C. O.'Neill

Keyword(s):

Embryo Development ◽

Mouse Embryo ◽

Preimplantation Embryo ◽

Platelet Activating Factor ◽

Outward Current ◽

Normal Embryo ◽

Cell Stage ◽

Preimplantation Embryo Development ◽

Cl Channel

Platelet activating factor (paf) is an autocrine survival factor for preimplantation embryo. Binding of paf to its receptor activates PI3kinase, causing an IP3-dependent release of Ca2+ from intracellular stores as well as activation of Ca2+ influx via a dihydropyridine-sensitive Ca2+ channel. These actions result in the generation of a defined intracellular calcium ([Ca2+]i) transient in the 2-cell embryo[1]. By using combined whole-cell patch-clamp and real-time [Ca2+]i analyses, we have shown that paf also induces a concomitant hyperpolarisation of the membrane potential in 2-cell embryos, accompanied by an increased net outward ion current. Both the membrane hyperpolarisation and outward current were dependent upon the occurrence of the paf-induced [Ca2+]i transient[2]. The aim of this study was to investigate the characteristics of the paf-induced outward current in 2-cell embryos and to assess whether it has a role in normal mouse preimplantation development. We show that: (1) removal of extracellular anions or treatment with niflumic acid (NFA, 100 μM, a Ca2+-activated Cl- channel blocker) prevented activation of the outward current by paf but had no effect on the paf-induced [Ca2+]i transient; and (2) The culture of embryos with NFA (100 μM) from the 1-cell to late 2-cell stage significantly reduced their development to the blastocyst stage (P < 0.001), but treatment with NFA from the late 2-cell stage had no effect on development. The results show that paf induces an increase in [Ca2+]i which in turn activates a Ca2+-activated Cl- channel. The activity of this NFA-sensitive channel during the zygote to 2-cell stage is required for normal embryo development. (1) C. O’Neill (2008) The potential roles of embryotrophic ligands in preimplantation embryo development. Hum Reprod Update 14:275–288. (2) Y. Li, M.L. Day & C. O’Neill (2007) Autocrine activation of ions currents in the two-cell mouse embryo. Exp Cell Res. 313:2785–2794.

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Spindle Assembly Checkpoint Regulates Mitotic Cell Cycle Progression during Preimplantation Embryo Development

PLoS ONE ◽

10.1371/journal.pone.0021557 ◽

2011 ◽

Vol 6 (6) ◽

pp. e21557 ◽

Cited By ~ 39

Author(s):

Yanchang Wei ◽

Saima Multi ◽

Cai-Rong Yang ◽

Junyu Ma ◽

Qing-Hua Zhang ◽

...

Keyword(s):

Cell Cycle ◽

Embryo Development ◽

Cell Cycle Progression ◽

Spindle Assembly Checkpoint ◽

Preimplantation Embryo ◽

Spindle Assembly ◽

Mitotic Cell ◽

Mitotic Cell Cycle ◽

Cycle Progression ◽

Preimplantation Embryo Development

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Maternal Underweight Does Not Adversely Affect The Outcomes of IVF/ICSI and Frozen Embryo Transfer Cycles or Early Embryo Development

10.21203/rs.3.rs-640651/v1 ◽

2021 ◽

Author(s):

Dana Hoffman ◽

Yael Kalma ◽

Nivin Samara ◽

Einat Haikin Herzberger ◽

Sagi Levi ◽

...

Keyword(s):

Embryo Development ◽

Preimplantation Embryo ◽

Morphological Characteristics ◽

Time Lapse ◽

Early Embryo ◽

Normal Weight ◽

Oocyte Maturity ◽

Vitro Fertilization ◽

Preimplantation Embryo Development

Abstract Purpose To compare assisted reproductive technology (ART) outcomes and preimplantation embryo development between underweight and normal weight women. Methods This retrospective cohort study included 26 underweight women (body mass index [BMI] < 18.50 kg/m2) and 104 normal weight women (BMI > 20 and < 24.9 kg/m2) who underwent a total of 204 in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycles and 358 fresh/frozen embryo transfers (ET) in our institution between January 2016 and December 2018. Statistical analyses compared selected ART outcomes (ovarian stimulation, fertilization, and pregnancy) between both weight groups. Morphokinetic and morphological parameters were also compared between 346 and 1467 embryos of underweight and normal weight women, respectively. Results The mean ± standard deviation age of the underweight and normal weight women was similar (31.6 ± 4.17 vs 32.4 ± 3.59 years; p = 0.323). There were no differences in the peak estradiol levels, the number of retrieved oocytes, the number of metaphase II oocytes, and the oocyte maturity rates between the two groups. The IVF/ICSI fertilization rates and the number of embryos suitable for transfer or cryopreservation were similar for both groups. All morphokinetic parameters that were evaluated by means of time-lapse imaging as well as the morphological characteristics were comparable between low and normal BMI categories. There were no significant differences in pregnancy achievement, clinical pregnancy, live births, and miscarriage rates between the suboptimal and optimal weight women. Conclusion Underweight status has no adverse impacts on the outcomes of IVF/ICSI with either fresh or frozen ET or on preimplantation embryo development and quality.

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l-Carnitine affects preimplantation embryo development toward infertility in mice

Reproduction ◽

10.1530/rep-16-0290 ◽

2016 ◽

Vol 152 (4) ◽

pp. 283-291 ◽

Cited By ~ 5

Author(s):

Christiana Kyvelidou ◽

Dimitris Sotiriou ◽

Tania Antonopoulou ◽

Margarita Tsagkaraki ◽

George J Tserevelakis ◽

...

Keyword(s):

Embryo Development ◽

Preimplantation Embryo ◽

Day Treatment ◽

Intraperitoneal Administration ◽

Blastocyst Development ◽

Cell Stage ◽

Preimplantation Embryo Development ◽

Early Stages

l-Carnitine (l-Cn), despite the beneficial role as energy-generating substance delivering long-chain fatty acids to the β-oxidation pathway in mitochondria, has been accused to cause an endometriosis-like state to BALB/c mice manifested by increased inflammatory cytokines in serum and peritoneal fluid, accumulation of immune cells in the peritoneal cavity and uterine walls and most importantly, correlating to infertility. Exploring this type of infertility, the effect of l-Cn on preimplantation embryo development, ovarian integrity and systemic maternal immunity was studied. Using nonlinear microscopy analysis, which was shown to be a powerful tool for determining embryo quality by quantitatively estimating the lipid body (LB) content of the cells, it was shown that in vitro and in vivo administration of l-Cn significantly decreased LB mean area in zygotes. Daily intraperitoneal administration of 2.5mg l-Cn for 3, 4 and 7days to mice significantly decreased the percent of normal zygotes. However, only the 7-day treatment persisted by affecting 2- and 8-cell stage embryos, while almost abolishing blastocyst development. Such effects were accompanied by abnormal ovarian histology, showing increased numbers of corpora luteus and elevated progesterone concentration in the serum. In addition, it was shown that the 7-day l-Cn treatment pushed maternal systemic immunity toward inflammation and immunosuppression by increasing CD11b-, CD25- and CD11bGr1-positive cells in spleen, which opposed the necessity for immunostimulation at these early stages of pregnancy. In conclusion, the results presented here demonstrated that elevated doses of l-Cn affect early stages of embryo development, leading to infertility.

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Whole-Genome DNA Methylation Dynamics of Sheep Preimplantation Embryo Investigated by Single-Cell DNA Methylome Sequencing

Frontiers in Genetics ◽

10.3389/fgene.2021.753144 ◽

2021 ◽

Vol 12 ◽

Author(s):

Zijing Zhang ◽

Jiawei Xu ◽

Shijie Lyu ◽

Xiaoling Xin ◽

Qiaoting Shi ◽

...

Keyword(s):

Dna Methylation ◽

Single Cell ◽

Embryo Development ◽

Molecular Mechanisms ◽

Preimplantation Embryo ◽

Preimplantation Embryos ◽

Whole Genome ◽

Cell Stage ◽

Preimplantation Embryo Development ◽

Developmental Block

The early stages of mammalian embryonic development involve the participation and cooperation of numerous complex processes, including nutritional, genetic, and epigenetic mechanisms. However, in embryos cultured in vitro, a developmental block occurs that affects embryo development and the efficiency of culture. Although the block period is reported to involve the transcriptional repression of maternal genes and transcriptional activation of zygotic genes, how epigenetic factors regulate developmental block is still unclear. In this study, we systematically analyzed whole-genome methylation levels during five stages of sheep oocyte and preimplantation embryo development using single-cell level whole genome bisulphite sequencing (SC-WGBS) technology. Then, we examined several million CpG sites in individual cells at each evaluated developmental stage to identify the methylation changes that take place during the development of sheep preimplantation embryos. Our results showed that two strong waves of methylation changes occurred, namely, demethylation at the 8-cell to 16-cell stage and methylation at the 16-cell to 32-cell stage. Analysis of DNA methylation patterns in different functional regions revealed a stable hypermethylation status in 3′UTRs and gene bodies; however, significant differences were observed in intergenic and promoter regions at different developmental stages. Changes in methylation at different stages of preimplantation embryo development were also compared to investigate the molecular mechanisms involved in sheep embryo development at the methylation level. In conclusion, we report a detailed analysis of the DNA methylation dynamics during the development of sheep preimplantation embryos. Our results provide an explanation for the complex regulatory mechanisms underlying the embryo developmental block based on changes in DNA methylation levels.

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Parental diet-induced obesity leads to retarded early mouse embryo development and altered carbohydrate utilisation by the blastocyst

Reproduction Fertility and Development ◽

10.1071/rd11256 ◽

2012 ◽

Vol 24 (6) ◽

pp. 804 ◽

Cited By ~ 48

Author(s):

Natalie K. Binder ◽

Megan Mitchell ◽

David K. Gardner

Keyword(s):

Cell Cycle ◽

Embryo Development ◽

Maternal Obesity ◽

Preimplantation Embryo ◽

Male Mice ◽

Cell Stage ◽

Female Mice ◽

The Impact ◽

Paternal Obesity

Maternal obesity results in reproductive complications, whereas the impact of paternal obesity is unclear. In the present study, the effects of parental obesity on preimplantation embryo cell cycle length and carbohydrate utilisation were investigated. Maternal and paternal obesity were assessed independently by deriving zygotes from normal or obese C57BL/6 female mice mated with normal Swiss male mice (maternal obesity), or from normal Swiss female mice mated with normal or obese C57BL/6 male mice (paternal obesity). Zygotes were cultured in vitro and development was then assessed by time-lapse microscopy and metabolism determined using ultramicrofluorescence. Maternal obesity was associated with a significant delay in precompaction cell cycle kinetics from the 1-cell stage. A significant increase in glucose consumption by embryos from obese compared with normal females occurred after compaction, although glycolysis remained unchanged. Similarly, paternal obesity led to significant delays in cell cycle progression during preimplantation embryo development. However, this developmental delay was observed from the second cleavage stage onwards, following embryonic genome activation. Blastocysts from obese males showed disproportionate changes in carbohydrate metabolism, with significantly increased glycolysis. Overall, metabolic changes were not inhibitory to blastocyst formation; however, blastocyst cell numbers were significantly lower when either parent was obese. These data suggest that both maternal and paternal obesity significantly impacts preimplantation embryo physiology.

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