Clinical Relevance of Secreted Small Noncoding RNAs in an Embryo Implantation Potential Prediction at Morula and Blastocyst Development Stages

Despite the improvements in biotechnological approaches and the selection of controlled ovarian hyperstimulation protocols, the resulting pregnancy rate from in vitro fertilization (IVF) protocols still does not exceed 30–40%. In this connection, there is an acute question of the development of a non-invasive, sensitive, and specific method for assessing the implantation potential of an embryo. A total of 110 subfertile couples were included in the study to undergo the IVF/ICSI program. Obtained embryos for transfer into the uterine cavity of patient cohort 1 (n = 60) and cohort 2 (n = 50) were excellent/good-quality blastocysts, and small noncoding RNA (sncRNA) content in the corresponding spent culture medium samples at the morula stage (n = 43) or at the blastocyst stage (n = 31) was analyzed by deep sequencing followed by qRT-PCR in real time. Two logistic regression models were developed to predict the implantation potential of the embryo with 100% sensitivity and 100% specificity: model 1 at the morula stage, using various combinations of hsa_piR_022258, hsa-let-7i-5p, hsa_piR_000765, hsa_piR_015249, hsa_piR_019122, and hsa_piR_008112, and model 2 at the blastocyst stage, using various combinations of hsa_piR_020497, hsa_piR_008113, hsa-miR-381-3p, hsa_piR_022258, and hsa-let-7a-5p. Protein products of sncRNA potential target genes participate in the selective turnover of proteins through the ubiquitination system and in the organization of the various cell cytoskeleton and nucleoskeleton structures, regulating the activity of the Hippo signaling pathway, which determines the fate specification of the blastomers.

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243 IN VITRO PRODUCTION OF LLAMA EMBRYOS BY IVF OR ICSI

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab243 ◽

2007 ◽

Vol 19 (1) ◽

pp. 237

Author(s):

P. A. Conde ◽

C. Herrera ◽

M. G. Chaves ◽

S. M. Giuliano ◽

A. Director ◽

...

Keyword(s):

Defined Medium ◽

Blastocyst Stage ◽

In Vitro Production ◽

Blastocyst Development ◽

Chi Square ◽

Vitro Fertilization ◽

South American Camelids ◽

Lama Glama ◽

Initial Description

Interest in South American camelids has increased in the last few years. Assisted reproduction techniques, such as in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) with epididymal spermatozoa, have shown poor results in these species (Tibary et al. 2005 Theriogenology 64, 618–638). The aim of the present study was to compare the efficacy of IVF vs. ICSI for in vitro embryo production in llama (Lama glama) using oocytes collected from ovarian follicles of different sizes. A total of 193 oocytes were collected from 223 follicles aspirated from 21 adult females by flank laparotomy after ovarian stimulation. Before aspiration, the diameter of each follicle was measured, and oocytes from each female were randomly assigned to either IVF or ICSI treatment. Semen samples were collected by electroejaculation and incubated in 25% (v/v) collagenase solution at 37°C to reduce viscosity. For IVF, spermatozoa were either non-treated or treated with heparin, penicillamine, and hypotaurine as capacitating agents. For ICSI, some oocytes were activated immediately after sperm injection with 5 µM of ionomycin for 10 min and 2 mM of 6-DMAP for 3 h, while others were subjected only to sperm injection. Spermatozoa used for ICSI were not treated with capacitating agents. All presumptive zygotes were cultured in SOFaas for 8 days. The percentage of total oocytes and mature (MII) oocytes recovered from follicles <7 mm and >7 mm in diameter were compared in each female. The proportion of oocytes inseminated via IVF and ICSI that cleaved and developed to the blastocyst stage was compared. The proportion of total oocytes, MII-oocytes, cleaved embryos, and blastocysts were compared between treatments by chi-square and Fisher's tests. The percentages of total (77/100; 77%) and MII (9/31; 29%) oocytes collected from <7 mm follicles were significantly lower than those of total (116/126; 92%) and MII (43/55; 78%) oocytes collected from >7 mm follicles (P < 0.01). The highest cleavage rates were observed in oocytes collected from follicles >7 mm in diameter and fertilized by IVF with (56%) or without (50%) capacitating agents; these rates were significantly different from those of the other treatments (P < 0.05, Table 1). Further studies will determine if the present results can be obtained with a higher number of oocytes. The results of the present study provide the first demonstration that Lama glama embryos can be produced in vitro using fresh semen. In addition, we have provided the initial description of blastocyst development after culture of ICSI-derived embryos in a defined medium. Table 1.Cleavage and blastocyst formation in different-sized llama oocytes inseminated via IVF or ICSI

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98 Assessing Endangered Felid Puma concolor Sperm Fertility by In Vitro Fertilization with Domestic Cat Oocytes

Reproduction Fertility and Development ◽

10.1071/rdv30n1ab98 ◽

2018 ◽

Vol 30 (1) ◽

pp. 188

Author(s):

M. Duque ◽

A. Sestelo ◽

D. F. Salamone

Keyword(s):

Puma Concolor ◽

Blastocyst Stage ◽

Domestic Cat ◽

Oocyte Activation ◽

Cleavage Rate ◽

Vitro Fertilization ◽

Sperm Suspension ◽

Morula Stage ◽

Cryopreserved Sperm

The Puma concolor population has been decreasing during the last 30 years. Semen cryopreservation of this species has been accomplished successfully and offers the possibility of preserving endangered species. We previously showed that fertilizing capability of wild felid spermatozoa can be evaluated using intracytoplasmic sperm injection (ICSI) with in vitro-matured domestic cat oocytes (Moro et al. 2014 Reprod. Domest. Anim. 49, 693-700). Due to the lack of homologous oocytes, we evaluated the capability of the Puma concolor sperm to induce domestic cat oocyte fertilization and subsequent pre-implantation embryo development. In the present study, cryopreserved sperm obtained by electroejaculation from five different males were used for IVF of in vitro-matured (IVM) domestic cat oocytes. Straws were thawed by exposing them to air for 10 s and then immersing in a 37°C water bath for 30 s. The contents of the straws were poured into a sterile 1.5-mL microtube pre-warmed to 37°C. The sperm suspension was diluted (1:3 v/v) by the slow (drop-by-drop) addition of a modified Tyrode’s solution. For IVF, IVM oocytes (n = 370) were co-incubated with 0.5 × 105 motile spermatozoa mL−1 in an atmosphere of 21% O2 in air at 38.5°C for 18 to 20 h. Presumptive zygotes were cultured in vitro in 50-μL drops of modified Tyrode’s medium on 6.5% CO2 in air at 38.5°C. Cleavage was determined at 48 h post-fertilization, and 5% FBS was added at Day 5 of in vitro culture. Blastocyst stage was evaluated at Day 8. Results (mean ± SEM) showed a high cleavage rate (179/370, 49.0 ± 4.0%), and a high development to morula stage (137/370, 34.4 ± 7.2%), and to blastocyst stage (94/370, 23.4 ± 4.7%) for all males. These results indicated that Puma concolor spermatozoa can induce domestic cat oocyte activation and development to blastocyst stage in similar rates to domestic cat homologous IVF: IVM oocytes (n = 291), cleavage rate (199/291, 67.1 ± 6.1%), development to morula stage (144/291, 47.8 ± 4.9%), and to blastocyst stage (86/291, 30.1 ± 1.6%). In conclusion, we demonstrated that domestic cat oocyte can be used to evaluated cryopreserve sperm samples from another felid species.

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Bayesian Network Modeling of IVF Blastocyst Score Prediction

AJIT-e Online Academic Journal of Information Technology ◽

10.5824/ajite.2021.02.001.x ◽

2021 ◽

Vol 12 (45) ◽

pp. 12-28

Author(s):

Aslı Uyar ◽

Yasemin Atılgan Şengül

Keyword(s):

Embryo Transfer ◽

Nearest Neighbor ◽

Morphological Evolution ◽

Blastocyst Stage ◽

Single Embryo Transfer ◽

Blastocyst Development ◽

True Negative ◽

Vitro Fertilization ◽

Estimate Method

Embryo transfer may be performed at cleavage stage (on day 2-3) or at blastocyst stage (on day 5) in In-Vitro Fertilization (IVF) treatment. Elective single embryo transfer at blastocyst stage increases the pregnancy probability and reduces the number of multiple pregnancies. However, the extended culture of embryos in the laboratory may result in transfer cancelation if no high quality blastocyst develops by day 5. Predicting the blastocyst score of individual embryos may help physicians to decide whether or not to further culture the embryos in the laboratory. In this paper, we use Bayesian networks for predicting the blastocyst score by modeling the morphological evolution of IVF embryos. We propose a weighted nearest neighbor approach to adjust the frequency estimates in the conditional probability table. Experimental results show that the proposed method significantly increases the accuracy and reduces false positive rates in IVF data in comparison to the frequency estimate method. Our proposed model can also predict low quality blastocyst development with a 77.3% True Negative rate. Using this model can help preventing developmental failures of embryos during IVF treatment.

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208 TIME LAPSE CINEMATOGRAPHIC ANALYSIS OF CLEAVAGE AND BLASTULATION IN BOVINE EMBRYOS OBTAINED BY OVUM PICKUP AND IN VITRO FERTILIZATION

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab208 ◽

2009 ◽

Vol 21 (1) ◽

pp. 202

Author(s):

K. Imai ◽

T. Somfai ◽

Y. Inaba ◽

Y. Aikawa ◽

M. Ohtake ◽

...

Keyword(s):

In Vitro Fertilization ◽

Calf Serum ◽

Development Program ◽

Time Lapse ◽

Blastocyst Stage ◽

Bovine Embryos ◽

Blastocyst Development ◽

Vitro Fertilization ◽

Cell Divisions

Since the 1980s, several different bovine in vitro embryo production systems have been developed, and more than 291 000 embryos have been transferred throughout the world (Thibier M 2007 IETS Newsletter 25(4), 15–20). However, we have limited knowledge about the cleavage pattern of the first, second, and third cell divisions and the developmental activities of embryos during in vitro culture (IVC). The present study was conducted to determine the developmental activities of bovine embryos obtained by ovum pickup (OPU), in vitro maturation (IVM), and in vitro fertilization (IVF). We analyzed embryonic development by time-lapse cinematography (TLC). A total of 92 cumulus–oocyte complexes were collected by OPU from Japanese Black cows and were subjected to IVM and IVF as reported previously (Imai et al. 2006 J. Reprod. Dev. 52(Suppl.), S19–S29). Inseminated oocytes were cultured in microdrops of CR1aa medium supplemented with 5% calf serum covered by mineral oil in 5% CO2 in air at 38.5°C. Kinetics of embryo development were measured by TLC for 168 h after IVF by using a Cultured Cell Monitoring System (CCM–M1.4ZS, Astec, Fukuoka, Japan). A total of 672 photographs of the embryos were taken (1 photograph every 15 min) during IVC. Image stacks were analyzed by the CCM–M1.4 software. Timing of the first, second, and third cell divisions, blastulation, and embryonic contractions were recorded. The results are reported as time (h) passed after insemination. In total, 75 (81.5%) embryos cleaved and 61 (66.3%) embryos developed to the blastocyst stage. The first, second, and third cell divisions in these viable embryos occurred at 24.0 ± 0.5, 32.1 ± 0.2, and 39.4 ± 0.4 h (mean ± SE) after IVF, respectively. On the other hand, in nonviable embryos (those that failed to develop to the blastocyst stage; n = 14), these cell divisions occurred at 29.5 ± 2.2, 41.3 ± 3.3, and 57.2 ± 7.6 h after IVF, respectively. There tended to be a difference (P = 0.06; paired t-test) in the timing of the first cell division between viable and nonviable embryos. Blastulation of embryos began at 114.4 ± 1.1 h, embryos developed to the blastocyst stage at 127.3 ± 1.4 h, and blastocysts began to expand at 138.4 ± 1.7 h after IVF, respectively. During blastocyst development, embryonic contractions (shrinkage attributable to the rupture of the blastocoele) and tight-shrinkage (shrinking of the embryo to less than 70% of its surface area) were observed in all embryos. The mean numbers of contractions and tight-shrinkages in blastocysts were 5.3 ± 2.7 and 2.1 ± 1.0 times, respectively. The frequency of contractions from the beginning of blastulation to the blastocyst stage was significantly lower (P < 0.01) than after the blastocyst stage. It took 6.9 ± 4.6 h for the embryos to re-expand after the tight-shrinkages. These results indicate that viable in vitro-produced embryos can be selected at early stages by TLC. Further studies are necessary to clarify the importance of the pulsating activity in OPU–IVF embryos. This work was supported by the Research and Development Program for New Bio-industry Initiatives.

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Lapatinib Decreases the Preimplantation Aneuploidy Rate of in vitro Fertilized Mouse Embryos without Affecting Completion of Preimplantation Development

Cytogenetic and Genome Research ◽

10.1159/000511371 ◽

2020 ◽

pp. 1-8

Author(s):

Parvaneh Maleki ◽

Hamid Gourabi ◽

Mohammad Tahmaseb ◽

Afsaneh Golkar-Narenji ◽

Masood Bazrgar

Keyword(s):

Blastocyst Stage ◽

Mouse Embryos ◽

Preimplantation Embryos ◽

Control Group ◽

Aneuploid Cell ◽

Vitro Fertilization ◽

Aneuploidy Rate ◽

Morula Stage ◽

And Control

One of the major reasons for implantation failure and spontaneous abortion is a high incidence of preimplantation chromosomal aneuploidy. Lapatinib simultaneously inhibits EGFR and HER2, leading to apoptosis. We hypothesized a higher sensitivity for aneuploid cells in preimplantation embryos to lapatinib based on reports of aneuploid cell lines being sensitive to some anticancer drugs. Late 2-cell mouse embryos were treated with lapatinib after determining a nontoxic dose. Morphologies were recorded 24, 48, and 60 hours later. The effect of lapatinib on the aneuploidy rate was evaluated by studying blastocyst cells using FISH. Although the rate of development to 8-cell and morula stage was higher in the control group (p < 0.05), there was no difference in development to the blastocyst stage at the same studied intervals between lapatinib-treated and control groups (p = 0.924). The mean number of cells in morula and blastocyst stages were not different between the groups (p = 0.331 and p = 0.175, respectively). The frequency of aneuploid cells and diploid embryos was, respectively, significantly lower and higher in lapatinib-treated embryos, (p < 0.001). Since lapatinib treatment reduced the aneuploidy rate without impact on the development of mouse preimplantation embryos to the blastocyst stage and number of total cells, lapatinib seems useful for prevention of preimplantation aneuploidy in in vitro fertilization.

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Small Noncoding RNA Signatures for Determining the Developmental Potential of an Embryo at the Morula Stage

International Journal of Molecular Sciences ◽

10.3390/ijms21249399 ◽

2020 ◽

Vol 21 (24) ◽

pp. 9399

Author(s):

Angelika Timofeeva ◽

Yulia Drapkina ◽

Ivan Fedorov ◽

Vitaliy Chagovets ◽

Nataliya Makarova ◽

...

Keyword(s):

Noncoding Rna ◽

Culture Medium ◽

Early Embryo ◽

Gene Target ◽

Developmental Potential ◽

Development Potential ◽

Vitro Fertilization ◽

Small Noncoding Rna ◽

Morula Stage

As part of the optimization of assisted reproductive technology programs, the aim of the study was to identify key small noncoding RNA (sncRNA) molecules that participate in maternal-to-zygotic transition and determine development potential and competence to form a healthy fetus. Small RNA deep sequencing followed by quantitative real-time RT-PCR was used to profile sncRNAs in 50 samples of spent culture medium from morula with different development potentials (no potential (degradation/developmental arrest), low potential (poor-quality blastocyst), and high potential (good/excellent quality blastocyst capable of implanting and leading to live birth)) obtained from 27 subfertile couples who underwent in vitro fertilization. We have shown that the quality of embryos at the morula stage is determined by secretion/uptake rates of certain sets of piRNAs and miRNAs, namely hsa_piR_011291, hsa_piR_019122, hsa_piR_001311, hsa_piR_015026, hsa_piR_015462, hsa_piR_016735, hsa_piR_019675, hsa_piR_020381, hsa_piR_020485, hsa_piR_004880, hsa_piR_000807, hsa-let-7b-5p, and hsa-let-7i-5p. Predicted gene targets of these sncRNAs included those globally decreased at the 8-cell–morula–blastocyst stage and critical to early embryo development. We show new original data on sncRNA profiling in spent culture medium from morula with different development potential. Our findings provide a view of a more complex network that controls human embryogenesis at the pre-implantation stage. Further research is required using reporter analysis to experimentally confirm interactions between identified sncRNA/gene target pairs.

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144 EFFECT OF ANTRAL FOLLICLE COUNT IN BEEF HEIFERS ON IN VITRO FERTILIZATION/PRODUCTION

Reproduction Fertility and Development ◽

10.1071/rdv29n1ab144 ◽

2017 ◽

Vol 29 (1) ◽

pp. 180

Author(s):

C. C. Chase ◽

R. A. Cushman ◽

A. K. McNeel ◽

E. C. Wright-Johnson ◽

G. A. Perry ◽

...

Keyword(s):

Antral Follicle ◽

Antral Follicle Count ◽

Blastocyst Stage ◽

In Vitro Production ◽

Blastocyst Development ◽

University Of Florida ◽

Vitro Fertilization ◽

Frozen Semen ◽

Vitro Production

Our objective has been to compare the IVF and in vitro production (IVP) of embryos from low and high antral follicle count (AFC) heifers. This is the fourth year of the study with years 1 to 3 reported individually. For this report, we add data for the fourth year and present a combined analysis (years 1 to 4) for the first time. Each year, AFC was determined on ~120 Angus heifers using transrectal ultrasonography. Ten heifers with the lowest AFC and 10 heifers with the highest AFC and all with evidence of oestrous cyclicity were synchronized with two 5-mL injections of PGF2α 11 days apart. Half were harvested on Day 5 to 6 and half on Day 15 to 16 of the oestrous cycle. The IVF procedure was slightly modified each year. For year 4, the IVF procedure included protocols for semi-defined media and was as described (IVP Protocol, P. J. Hansen’s Laboratory, University of Florida). Cumulus-oocyte complexes (COC) from follicles less than 8 mm in diameter were cultured in maturation medium (5% CO2; 38.5°C) for 24 h. Matured COC were fertilized using thawed frozen semen from a bull that was purified using isolate. Motile spermatozoa were added to COC in fertilization medium at a final concentration of 1 × 106 spermatozoa per mL. About 24 h later, presumptive zygotes were placed in micro drops of development medium under oil, and cultured (5% CO2; 5% O2; balance N2; 38.5°C). On Day 3 and 8 after fertilization, cleavage and blastocyst development rates, respectively, were assessed. Data were analysed using the MIXED procedure of SAS (SAS Institute Inc., Cary, NC, USA) and the model included the effects of year (1 to 4), group (high or low AFC), and their interaction. The year × group interaction was not significant (P > 0.10). Low AFC heifers, compared with high AFC heifers, had fewer numbers of COC (P < 0.0001; 12.8 ± 1.83 v. 31.9 ± 1.86), fewer numbers of COC that cleaved (P < 0.0001; 8.0 ± 1.38 v. 21.6 ± 1.40), and fewer numbers of COC that developed to the blastocyst stage (P < 0.0001; 1.7 ± 0.58 v. 5.7 ± 0.58). Year affected the numbers of COC that cleaved (P < 0.003) and the numbers of COC that developed to the blastocyst stage (P < 0.0001). Year also influenced the percentage of COC that cleaved (P < 0.0002) and the percentage of COC that developed to blastocysts (P < 0.0001). Group (AFC) did not influence (P > 0.19) the percentage of COC that cleaved (61.2 ± 2.83 v. 66.4 ± 2.83%, for low v. high AFC, respectively). Low AFC heifers had a lower (P < 0.002) percentage of COC that developed to blastocysts (10.3 ± 1.52%) than high AFC heifers (17.6 ± 1.52%). These results indicate that high AFC heifers, compared to low AFC heifers, have more COC recovered, more COC cleaved, and more COC developed to the blastocyst stage. The percentage of COC cleaved did not differ between AFC groups; however, the percentage of COC that developed to the blastocyst stage was greater for high than low AFC heifers. This suggests a potential advantage in maternal to embryonic transition for high compared with low AFC heifers.

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Thickness of endometrium: predictor of the effectiveness of IVF/ICSI programs (literature review)

GYNECOLOGY ◽

10.26442/2079-5696_20.1.113-116 ◽

2018 ◽

Vol 20 (1) ◽

pp. 113-116

Author(s):

L A Bagdasaryan ◽

I E Korneyeva

Keyword(s):

Assisted Reproductive Technologies ◽

Endometrial Thickness ◽

Molecular Genetic ◽

Uterine Cavity ◽

Reproductive Technologies ◽

Genetic Characteristics ◽

Vitro Fertilization ◽

Need To Evaluate ◽

The Relationship

The aim of the study is to systematically analyze the data available in the modern literature on the relationship between endometrial thickness and the frequency of pregnancy in the program of assisted reproductive technologies (ART). Materials and methods. The review includes data from foreign and domestic articles found in PubMed on this topic. Results. The article presents data on the relationship between the thickness of the endometrium and the frequency of pregnancy in ART programs. The greatest number of studies is devoted to the evaluation of the relationship between the thickness of the endometrium and the frequency of pregnancy on the day of the ovulation trigger. Data are presented on the existence of a correlation between the thickness of the endometrium measured on the day of the ovulation trigger and the frequency of clinical pregnancy, as well as data on the need to evaluate the structure of the endometrium and the state of subendometric blood flow. The importance of multilayered (three-layered) endometrium as a prognostic marker of success in in vitro fertilization/intracytoplasmic sperm injection programs in the ovum is emphasized. The conclusion. The thickness of the endometrium can not be used as an argument for canceling the cycle or abolishing embryo transfer to the uterine cavity. Further studies in this direction are needed with a study of the morphological and molecular genetic characteristics of the endometrium, which in the future will allow us to evaluate the relationship between the thickness of the endometrium and the probability of pregnancy.

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