33 BUFFALO (BUBALUS BUBALIS) EMBRYOS PRODUCED BY HAND-MADE CLONING AND IN VITRO FERTILIZATION DIFFER IN THEIR GLOBAL TRANSCRIPTOME PROFILE

2017 ◽  
Vol 29 (1) ◽  
pp. 124
Author(s):  
T. J. Sood ◽  
S. Viviyan ◽  
S. K. Singla ◽  
M. Mukesh ◽  
M. S. Chauhan ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Vitro Fertilization ◽  
Birth Rate ◽  
Differentially Expressed ◽  
Gene Expression Level ◽  
Transcriptome Profile ◽  
Vitro Fertilization ◽  
Functional Classes ◽  
Global Transcriptome

Although the blastocyst rate obtained with nuclear transferred (NT) embryos is higher than that obtained following in vitro fertilization (IVF) in buffalo, the live birth rate of NT embryos is <2% across different farm animal species compared with a birth rate >40% obtained with IVF embryos. This is believed to be due primarily to incomplete or incorrect nuclear reprogramming of the donor somatic cell by the oocyte, which results in aberrant embryonic gene expression. We compared the global transcriptome profile of buffalo blastocysts produced by hand-made cloning (HMC) and IVF using next-generation sequencing (NGS) for discovering transcripts that are differentially expressed between the 2 types of embryos. NT blastocysts were produced using fibroblast donor cells obtained from ear skin of a buffalo bull. The semen of the same bull was used for producing genetically half-identical IVF blastocysts. Total RNA was isolated from 3 pools of Day 8 NT and IVF blastocysts, with each pool containing 40 blastocysts. Complementary DNA library was prepared and subjected to NGS on Illumina HiSEqn 2000 (Illumina Inc., San Diego, CA, USA). The reads generated were aligned to Bos taurus reference genome, UMD 3.1. Differential expression analysis between the 2 blastocysts types at a minimum of 2-fold change revealed that 5557 transcripts were differentially expressed, of which 584 were unique to NT blastocysts, 709 were unique to IVF blastocysts, and 4264 were expressed in both types of blastocysts. Among these transcripts, at a significance level of P < 0.05, 331 transcripts were differentially expressed between the 2 blastocyst types, of which 19 were unique, 188 were down-regulated, and 143 were up-regulated in NT blastocysts. One-way ANOVA with Benjamini and Hochberg false discovery rate (FDR) correction was applied to determine the statistically significant differentially expressed transcripts. Nine of the differentially expressed transcripts (at minimal 2-fold change, P < 0.05), from different functional classes (RELN, NDRG1, SULT1A1, MAP1LC3A, MTHFD1L, PCBD1, PPA2, MGST1 and PRPH) were subjected to quantitative real-time PCR analysis for validation of NGS data. Gene expression level of RELN, NDRG1, SULT1A1, MAP1LC3A, PPA2, MGST1, and PRPH was found to be up-regulated while that of MTHFD1L and PCBD1 was down-regulated (P < 0.05) in NT embryos compared with IVF embryos. This pattern and the magnitude of relative gene expression level were found to be similar to that observed in NGS. These results indicate that the gene expression profile of NT embryos is very different from that of their IVF counterparts. Further analysis of these differentially expressed transcripts can help in identification of gene functional classes and pathways that are affected by the inefficient reprogramming of donor nuclei in NT embryos. Normalizing the expression of some of the differentially expressed genes may help in improving the cloning efficiency.

260 COMPREHENSIVE IDENTIFICATION OF RELATIVE GENES CAUSING ABNORMAL DEVELOPMENT OF BOVINE EMBRYOS PRODUCED BY SOMATIC CELL NUCLEAR TRANSFER

2006 ◽  
Vol 18 (2) ◽  
pp. 237
Author(s):  
J. Park ◽  
N. Minami ◽  
H. Imai
Keyword(s):  
Gene Expression ◽  
In Vitro Fertilization ◽  
Somatic Cell ◽  
Differentially Expressed Genes ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Blastocyst Stage ◽  
Differentially Expressed ◽  
Vitro Fertilization

Developmental failure of a cloned animal using somatic cell nuclear transfer (SCNT) procedures is considered to be the result of abnormal expression of developmentally important genes caused by incomplete reprogramming of the donor cell nuclei. However, there are few reports about stage-specific gene expression during cleavage progression of cloned embryos. The aim of this study was to identify using fluorescein differential display method, the differentially expressed genes in cloned embryos at early developmental stages compared with those produced by in vitro fertilization. Bovine cumulus-oocytes complexes (COCs) were aspirated from follicles (2-8 mm in diameter) of slaughterhouse ovaries and cultured in TCM-199 supplemented with 10% fetal calf serum (FCS) for 18 h for somatic cell nuclear transfer (NT) or 24 h for in vitro fertilization (IVF) at 39�C. Removal of oocyte nuclei for NT was performed by squeezing out a small amount of the cytoplasm laying beneath the first polar body by means of a glass needle. Donor cells for NT were obtained from skin cells of an adult cow and cultured in DMEM supplemented with 10% FCS. After the transfer of somatic cell into enucleated oocytes, DC electric pulses at 200 V/mm for 2 � 10 �s were used for fusion, and the reconstructed embryos were treated with 10 �g/mL cycloheximide for 6 h. The embryos were then cultured for 120 h (morula stage) or 168 h (blastocyst stage) in modified SOF medium under 5% CO2, 5% O2 and 90% N2 at 39�C. Total RNA obtained from NT and IVF embryos were analyzed by differential display RT-PCR (DDRT-PCR) as previously described (Minami et al. 2001 Biol. Reprod. 64, 30-35). We obtained several differences in gene expression patterns between NT and IVF embryos at the morula and blastocyst stage. A total of 52 cDNA fragments were isolated and analyzed. Semiquantitative analysis revealed that some genes (NADH dehydrogenase subunit 1, SR rich protein, KIAA0107, ribosomal protein L19) were highly expressed in IVF embryos compared with NT embryos, whereas other genes (CASK) were highly expressed in NT embryos compared with IVF embryos. These results indicate that the differentially expressed genes observed in NT embryos may be representative of marker genes for the production of normal NT offspring and DDRT-PCR procedure is quite useful for identification of several genes that are differentially expressed between NT and IVF embryos.Although the detailed function of the genes and their products remains to be determined, it is likely that the reprogramming mechanisms can be elucidated genetically by the analysis of differentially expressed genes in the future.

scholarly journals Expression of mRNAs for Pro-and Anti-Apoptotic Factors in Granulosa Cells and Follicular Fluid of Women Undergoing in Vitro Fertilization. A Pilot Study

2021 ◽  
Author(s):  
Jozsef Bodis ◽  
Endre Sulyok ◽  
Akos Varnagy ◽  
Viktória Prémusz ◽  
Krisztina Godony ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Vitro Fertilization ◽  
Mrna Expression ◽  
Granulosa Cells ◽  
Follicular Fluid ◽  
Vitro Fertilization ◽  
Expression Levels ◽  
The Impact ◽  
Apoptotic Factors

Abstract BackgroundThis observational clinical study evaluated the expression levels and predictive values of some apoptosis-related genes in granulosa cells (GCs) and follicular fluid (FF) of women undergoing in vitro fertilization (IVF).Methods GCs and FF were obtained at oocyte retrieval from 31 consecutive patients with heterogeneous infertility diagnosis (age: 34.3±5.8 years, body mass index: 24.02±3.12 kg/m2, duration of infertility: 4.2±2.1 years). mRNA expression of pro-apoptotic (BAX, CASP3, CASP8) and anti-apoptotic (BCL2, AMH, AMHR, FSHR, LHR, CYP19A1) factors was determined by quantitative RT-PCR using ROCHE LightCycler 480. Results No significant difference in GC or FF mRNA expression of pro- and anti-apoptotic factors could be demonstrated between IVF patients with (9 patients) or without (22 patients) clinical pregnancy. Each transcript investigated was detected in FF, but their levels were markedly reduced and independent of those in GCs. The number of retrieved oocytes was positively associated with GC AMHR (r=0.393, p=0.029), but the day of embryo transfer was negatively associated with GC LHR (r=-0.414, p=0.020) and GC FSHR transcripts (r=-0.535, p=0.002). When pregnancy positive group was analysed separately the impact of apoptosis- related gene expressions on some selected measures of IVF success could be observed. Strong positive relationship was found between gene expression levels of pro- and anti-apoptotic factors in GCs.ConclusionOur study provides only marginal evidences for the apoptosis dependence of IVF outcome and suggests that the apoptosis process induces adaptive increases of the anti-apoptotic gene expression to attenuate apoptosis and to protect cell survival.

O-107 Searching for the optimal number of oocytes to reach a life birth following in vitro fertilization: a systematic review with meta-analysis

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
N Sermondade ◽  
C Sonigo ◽  
M Pasquier ◽  
N Yata-Ahdad ◽  
E Fraison ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Live Birth ◽  
Birth Rate ◽  
Meta Analysis ◽  
Live Birth Rate ◽  
Cumulative Live Birth Rate ◽  
Continuous Increase ◽  
Vitro Fertilization ◽  
The Relationship

Abstract Study question To investigate the relationship between the number of oocytes and both the live birth rate after fresh embryo transfer and the cumulative live birth rate. Summary answer Above a 15-oocyte threshold, live birth rate (LBR) following fresh transfer plateaus, whereas a continuous increase in cumulative live birth rate (CLBR) is observed. What is known already Several lines of evidence indicate that number of oocytes represents a key point for in vitro fertilization (IVF) success. However, consensus is lacking regarding the optimal number of oocytes for expecting a live birth. This is a key question because it might impact the way practitioners initiate and adjust COS regimens. Study design, size, duration A systematic review and meta-analysis was performed. MEDLINE, EMBASE, and Cochrane Library were searched for studies published between January 01, 2004, and August 31, 2019 using the search terms: “(intracytoplasmic sperm injection or icsi or ivf or in vitro fertilization or fertility preservation)” and “(oocyte and number)” and “(live birth)”. Participants/materials, setting, methods Two independent reviewers carried out study selection, quality assessment using the adapted Newcastle-Ottawa Quality Assessment Scales, bias assessment using ROBIN-1 tools, and data extraction according to Cochrane methods. Independent analyses were performed according to the outcome (LBR and CLBR). The mean-weighted threshold of optimal oocyte number was estimated from documented thresholds, followed by a one-stage meta-analysis on articles with documented or estimable relative risks. Main results and the role of chance After reviewing 843 records, 64 full-text articles were assessed for eligibility. A total of 36 studies were available for quantitative syntheses. Twenty-one and 18 studies were included in the meta-analyses evaluating the relationship between the number of retrieved oocytes and LBR or CLBR, respectively. Given the limited number of investigations considering mature oocytes, association between the number of metaphase II oocytes and IVF outcomes could not be investigated. Concerning LBR, 7 (35.0%) studies reported a plateau effect, corresponding to a weighted mean of 14.4 oocytes. The pooled dose-response association between the number of oocytes and LBR showed a non-linear relationship, with a plateau beyond 15 oocytes. For CLBR, 4 (19.0%) studies showed a plateau effect, corresponding to a weighted mean of 19.3 oocytes. The meta-analysis of the relationship between the number of oocytes and CLBR found a non-linear relationship, with a continuous increase in CLBR, including for high oocyte yields. Limitations, reasons for caution Statistical models show a high degree of deviance, especially for high numbers of oocytes. Further investigations are needed to assess the generalization of those results to frozen mature oocytes, especially in a fertility preservation context, and to evaluate the impact of female age. Wider implications of the findings Above a 15-oocyte threshold, LBR following fresh transfer plateaus, suggesting that the freeze-all strategy should probably be performed. In contrast, the continuous increase in CLBR suggests that high numbers of oocytes could be offered to improve the chances of cumulative live births, after evaluating the benefit–risk balance. Trial registration number Not applicable

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