Production Of High-quality Embryos Research Articles

Expression analysis of genes and MicroRNAs involved in recurrent implantation failure: New noninvasive biomarkers of implantation

Background: Recurrent implantation failure (RIF) is defined as three or more failed in vitro fertilization attempts and is due to several factors such as oocyte and embryo quality. Methods: Fifty-one RIF patients and 19 controls were selected based on the inclusion criteria. EFNB2, CAMK1D, AREG, and PTGS2 as well as miR-34, miR-145, miR-204-5p, and miR-26-5p were selected since the microRNAs (miRNAs) targeted the genes based on bioinformatic predictions and literature review. Total RNA was extracted from cumulus cells (CCs) and follicular fluid (FF) of the oocytes. We performed real-time polymerase chain reaction to evaluate the expression of the genes and the miRNAs in CC and FF of pregnant and nonpregnant RIF patients. The expression of CAMK1D, AREG, miR-34-5p, and miR-26-5p was higher in CC than FF. Results: The expression of CAMK1D, PTGS2, and miR-26-5p in CC of the pregnant group was higher than FF. The expression of EFNB2, PTGS2, miR-145, and miR-204-5p was lower in the CC, and the expression of EFNB2, AREG, miR-34-5p, mR-145, and miR-204-5p was lower in the FF of the pregnant group. The expression of CAMK1D, AREG, PTGS2, miR-34-5p, and miR-26-5p was higher in the CC and FF of the high quality (HQ) embryos than non-HQ (NHQ) embryos. The expression of EFNB2, miR-145, and miR-204-5p was higher in the CC and FF of the NHQ embryos. The difference was statistically significant for EFNB2 in CC and FF as well as miR-145 in CC. The level of progesterone and prostaglandin E2 in the FF of the pregnant group was higher than their level in the nonpregnant group. Conclusion: CAMK1D expression and overexpression of miR-34-5p and miR-26-5p could be considered as markers of successful pregnancy. In addition, the results show that normal FF treatment of RIF patients may result in the production of high-quality embryos.

136 GameteGuard in maturation medium improves bovine embryo quality and quantity

The number of invitro-produced (IVP) bovine embryos generated in the United States increased 11% from 2017 to 2018, demonstrating a demand for new technology to rapidly and efficiently improve herd genetics. Unfortunately, embryo production on a per oocyte basis remains low, limiting the number of high-quality commercial embryos available for transfer or freezing. Reactive oxygen species (ROS) are a major contributor to low embryo production rates of invitro gametes and are especially problematic in maturation media. This is due to high numbers of mitochondria in the bovine oocyte producing ROS exacerbated by the invitro environment. GameteGuard® is an organic plant-based additive that provides protection against reactive oxygen species. The aims of this research were to evaluate the effects of GameteGuard addition to invitro bovine embryo production media to (1) mitigate the impact of ROS on oocyte maturation and competence, and (2) increase the production of high-quality embryos in our IVP system. Four rounds of invitro embryo production (Colorado State University protocol and media) with or without maturation medium supplemented with GameteGuard were performed (n=1175 total oocytes). Oocytes were aspirated from abattoir ovaries; those with homogeneous ooplasm and multiple layers of even cumulus cells were matured using control (n=585) or GameteGuard-supplemented (n=590) maturation medium. Oocytes were fertilized with frozen/thawed semen from proven IVF bulls (n=4) and then cultured in a two-step media system to Day 7. Blastocysts were evaluated on Day 7 according to IETS embryo grading standards. A subset of 80 blastocysts were fixed and stained after grading using an In Situ Cell Death Detection Kit (Fluorescein; Millipore Sigma) and Hoechst 33342 (Invitrogen) as apoptotic and counter-stains, respectively. Stained blastocysts were assessed by immunofluorescence microscopy to obtain the percentage of apoptotic cells and total cell count. Because multiple bulls were used, a linear mixed model was required for data analysis. Treatment was considered the fixed effect and bulls as a random effect to determine the impact of the treatment on stage and quality of blastocysts. Cleavage rates were not different (P>0.5) but GameteGuard supplementation of maturation medium resulted in a 22% increase in grade-1 embryos per total blastocysts produced (P<0.01; n=79/106 control vs. n=120/137 GameteGuard grade 1 blastocysts). Additionally, the percent of grade 2 and 3 embryos per total blastocysts decreased, (P<0.01; 27%, n=27/106 control and 11%, n=14/137 GameteGuard grade 2 and 3 blastocysts), suggesting that supplementation with GameteGuard promoted development of better-quality embryos overall. The percentage of apoptotic cells was also significantly higher in control blastocysts compared with treated blastocysts (P<0.02; 11.4%, n=38 control and 12.8%, n=42 GameteGuard). GameteGuard supplementation of the maturation medium significantly increased the production of high-quality IVP bovine embryos, thereby providing potential marketable gain for embryo producers.

Initial ovarian sensitivity index predicts embryo quality and pregnancy potential in the first days of controlled ovarian stimulation

BackgroundTo determine if a modified ovarian sensitivity index (MOSI), based on initial follicular measurements and the initial follicle-stimulating hormone (FSH) dose, can predict the production of high-quality embryos for successful implantation during in vitro fertilization (IVF).MethodsThis study consisted of two phases: 1) a retrospective study and 2) a prospective observational study. For the first phase, 363 patients charts were reviewed, of which 283 had embryos transferred. All women underwent a standardized antagonist-based IVF protocol. At the first follow-up (Day 3/4), the number and size of the follicles were determined. MOSI was calculated as ln (number follicles (≥6 mm) × 1000 / FSH initial dose). Afterward, the number and quality of the ova, embryo development, and the number and quality of the blastocysts were determined. Embryo implantation was confirmed by β-hCG. For the second phase, 337 IVF cycles were followed to determine MOSI’s accuracy.ResultsMOSI could predict the production of ≥4 high-quality embryos by Day 2 (AUC = 0.69, 95%CI:0.63–0.75), ≥2 blastocysts (AUC = 0.74, 95%CI:0.68–0.79), and ≥ 35% rate of blastocyst formation (AUC = 0.65, 95%CI:0.58–0.72). Using linear regression, MOSI was highly associated with the number of ova captured (β = 5.15), MII oocytes (β = 4.31), embryos produced (β = 2.90), high-quality embryos (β = 0.98), and the blastocyst formation rate (β = 0.06, p < 0.01). Using logistic regression, MOSI was highly associated with achieving ≥4 high-quality embryos (odds ratio = 2.80, 95%CI:1.90–4.13), ≥2 blastocysts (odds ratio = 3.40, 95%CI:2.33–4.95), and ≥ 35% blastocysts formation rate (odds ratio = 1.96, 95%CI:1.31–2.92). This effect was independent of age, BMI, and antral follicle count. For implantation, MOSI was significantly associated with successful implantation (odds ratio = 1.79, 95%CI:1.25–2.57). For the prospective study, MOSI was highly accurate at predicting ≥6 high-quality embryos on Day 2 (accuracy = 68.5%), ≥6 blastocysts (accuracy = 68.0%), and a blastocyst formation rate of ≥35% (accuracy = 61.4%).ConclusionMOSI was highly correlated with key IVF parameters that are associated with achieved pregnancy. Using this index with antagonist cycles, clinicians may opt to stop an IVF cycle, under the assumption that the cycle will fail to produce good blastocysts, preventing wasting the patient’s resources and time.

Interspecific variation in the physiological and reproductive parameters of porcelain crabs from the Southeastern Pacific coast: potential adaptation in contrasting marine environments

Porcelain crabs inhabit from upper intertidal to subtidal habitats. These environments are characterized by highly variable environmental conditions, which subject species found in these habitats to stress. In this study, we compared reproductive traits of mothers [i.e. fecundity, reproductive output (RO), dry weight, organic matter] and physiological parameters of their offspring (i.e. wet weight, water content, dry weight, organic matter, lactate content of embryos) of three species of porcelain crabs that inhabit the Southeastern Pacific: Petrolisthes laevigatus (upper intertidal); P. violaceus (low intertidal); Allopetrolisthes punctatus (subtidal). Overall, female P. laevigatus had lower fecundity (802 ± 115 vs. 4181 ± 1097 embryos) and amount of organic matter in their embryo masses (0.053 ± 0.006 vs. 0.27 ± 0.025 g) but higher RO values (1.34 ± 0.34 vs. 0.20 ± 0.07) than Allopetrolisthes punctatus. In addition, P. laevigatus embryos had higher organic matter content (81.09 ± 28.8 vs. 64.54 ± 6.1 μg), higher water content (188.6 ± 91.9 vs. 152.4 ± 30.8 μL) and higher lactate content (0.26 ± 0.04% vs. 0.07 ± 0.01% dry weight) than that found in A. punctatus embryos. Furthermore, females and embryos of P. violaceus showed low values and similar to those observed in P. laevigatus. As a potential strategy to increase survival of the offspring, P. laevigatus seems to invest a large portion of its energy in production of high quality embryos, despite costs to fecundity. This study reveals that porcelain crabs have physiological adaptations during their ontogeny that allow them to survive in fluctuating environments.

119 Genetic Highlights for Reproduction and Health Traits in Russian Black-and-White and Holstein Animals Selected for Production of High-Quality Embryos

The aim of genetic improvement in cattle breeding is to create animals, which are characterised by high economic efficiency while maintaining the high breeding values for health and reproduction traits. In Russia dairy cattle industry the integrated approaches based on application of genomic methods for cows’ fertility and health traits along with milk production are being developed. However, their efficiency depends largely on the ability for multiple replicating outstanding individuals by embryo transfer. Therefore, the target of our research was to develop a strategy for the integrated genetic improvements of fertility and udder health traits towards the production of embryos with high estimated breeding values (EBV). We analysed the culling factors of more than 23,400 Black-and-White and Holstein cows of the Moscow region cattle population. Using the genotyping procedure by Illumina Bovine SNP50 BeadChip (Illumina Inc., San Diego, CA, USA), the regional training group consisted of 401 proven bulls and 60 pre-selected cows was formed. After the quality check based on Plink 1.90, we selected 39,631 single nucleotide polymorphisms for the analysis. The GBLUP methodology was used to calculate genomic EBV (GEBV). The cows were additionally tested by PCR or PCR-RFLP (restricted fragment length polymorphism) for LoF mutations in the genes FANCI, APAF1, SMC2, GART, ITGB2, SLC35A3, and APOB, associated with fertility haplotypes HH0, HH1, HH34, HH4, HHB, HHC, and HCD, respectively. The evaluation of genetic variances and EBV for days open (DO), breeding per conception (BC), calving ease (CE), somatic cell score (SCS), milk yield (MY), fat yield (FY), and protein yield (PY) was carried out using the BLUPF90 family of programs. Cows’ culling rate for reproductive traits was 21% and for udder diseases was 8%. The genetic correlations, rg, between SCS and cows’ fertility traits were rg = 0.132 for BC, rg = 0.067 for DO, and rg = –0.056 for CE that can be used as an indicator for the monitoring reproductive disorders at the different stages of lactation. The frequencies of fertility haplotypes’ carriers among the potential donor dams were 6.6, 1.1, 8.8, 0.0, 1.1, 0.0, and 4.4% for HH0, HH1, HH3, HH4, HHB, HHC, and HCD, respectively. Based on EBV and carrier status for LoF mutations, we selected 46 dams and 28 sires to produce embryos with high GEBV for MY (+633, ..., +884 kg), FY (+26 kg), PY (+18 kg), and SCS (106 points) and higher as population average GEBV for fertility and udder health traits. Genome-wide association study analysis by the reliability of Bonferroni correction test (P &lt; 1.2 × 10−6) showed the presence of quantitative trait loci on BTA1, BTA5, BTA9, BTA11, BTA12, BTA27, and BTX. Our results will be used to construct Russian fitness index for dairy cattle and for implementing embryo production technology into the Russian breeding system. Research supported by the Russian Science Foundation within Project no. 15-16-00020.

ICSI with the Assistance of Meiotic Spindle Imaging for the Production of High Quality Embryos

Abstract To avoid multiple pregnancies, it is important to select a single high quality embryo for transfer. Observation of meiotic spindles in oocytes is one way of evaluating embryo developmental competence. The spindle is non-invasively visualized by polarized light microscopy. It is possible to perform ICSI more safely and with optimal timing during meiosis by visualizing the meiotic spindle just before ICSI. In some human studies, it has been reported that oocytes with a spindle show significantly higher fertilization, blastocyst formation and pregnancy rates. Recently, it has been reported that morphological spindle normality is related to the live birth rate. In mice, the areas of the spindles in aged oocytes (23 h after hCG injection) are larger than those in young oocytes (15 h after hCG injection), and the blastocyst formation rate of aged oocytes is lower than that of young oocytes. Addition of MG132, a ubiquitin-mediated proteasome proteolysis inhibitor, to the medium prevents the spindle from...

Abscisic Acid Effect on Improving Horse Chestnut Secondary Somatic Embryogenesis

The effect of abscisic acid on the development of primary androgenic embryo and secondary somatic embryogenesis was investigated with the aim of improving multiplication rates and secondary somatic embryo quality in horse chestnut microspore and anther culture. The early embryo stage (globular) had a better response than late stages (heart, torpedo, and cotyledonary) in both types of cultures. Also, microspore culture had a high potential for mass secondary embryo production. The number of secondary somatic embryos was three times higher on hormone-free medium than on medium enriched with 0.01 mg·L−1 abscisic acid. However, most of the embryos on hormone-free medium had abnormal morphology. For this reason, abscisic acid was added to the media to improve embryo quality. The morphology of abscisic acid treated embryos was better than abscisic acid non-treated embryos. The optimal abscisic acid concentration for secondary somatic embryo induction and production of high-quality embryos was 0.01 mg·L−1. Overall, the effect of abscisic acid on the induction of secondary somatic embryogenesis and plant regeneration of androgenic embryos of this species may be helpful for the further synthesis of secondary metabolites in vitro and their application in the pharmaceutical industry.

Role of the Oviduct in Early Embryo Development

This review highlights the role of the oviduct in early embryo development, which has to fulfil many aligned and well-tuned tasks during early embryogenesis. The oviductal lining is subjected to dynamic changes to timely accomplish gamete transport, fertilization and embryo development and to deliver a competent and healthy conceptus to the endometrium which can implant and develop to term. Although knowledge about the role of the oviduct is limited, we know that embryos are very sensitive to the environment in which they develop. The success of in vitro embryo production techniques demonstrates that it is possible to bypass the oviduct during early development and, to a certain extent, replicate the conditions in vitro. However, comparative studies show that embryos developed in vivo are superior to their in vitro produced counterparts, underlining our relatively poor knowledge of the biology of the oviduct. Oviduct activity is orchestrated by various factors, depending on cyclic dynamics, which crucially affect the success of tubal transfer and/or (re-)collection of embryos in embryo transfer studies. This paper reviews data which demonstrate that in vivo culture of embryos in the bovine oviduct is a useful tool for the assessment of embryos developed under various conditions (e.g. superovulation vs single ovulation, lactating dairy cows vs non-lactating cows). It is concluded that more work in the field of early embryo development within the oviduct would contribute to improved ART protocols leading to healthy pregnancies and offspring.

Replacing liquid fyrite with digital to improve &lt;i&gt;in vitro&lt;/i&gt; fertilization laboratory quality control procedures

In clinical in vitro fertilization (IVF), optimal culture conditions are required for production of high quality embryos and for achieving high pregnancy rates. Cell culture systems require vigilant attention to quality control and quality assurance, and upgrades to equipment and procedures require strenuous deliberation. During a 2-week maintenance period, we undertook an extensive analysis of incubator carbon dioxide (CO2) monitoring and the effect on culture media pH by comparing our traditional liquid Fyrite instruments to a certified and calibrated digital CO2 analyzer. The digital analyzer produced consistently lower CO2 readings and significantly greater precision than the liquid Fyrite. Media pH measurements showed significant variation depending on CO2 calibration device; however pH remained within manufacturers’ specifications. After superior performance by the digital analyzer, we incorporated this device into the incubator calibration and daily quality control procedures. A retrospective comparison of overall lab performance before and after this equipment switch demonstrated improved clinical pregnancy and implantation rates. This report illustrates the necessary caution when altering established laboratory procedures and equipment while highlighting the benefits of judiciously updating techniques and equipment in a laboratory setting that is often stubborn to change pre-existing, ingrained methodology.

Effects of species and cellular activity of oviductal epithelial cells on their dialogue with co-cultured mouse embryos

An efficient co-culture system, especially with oviductal or uterine epithelial cells, is important not only for the production of high quality embryos, but also for the study of the molecular dialogue between embryos and their maternal environment. Although mouse embryos have been co-cultured successfully with oviductal epithelial cells (OECs) from several species, studies on the effects of species and functionality of OECs are few. Reports concerning the necessity of direct contact between the embryo and OECs and about the culture of mouse embryos in medium conditioned with heterologous OECs have been controversial. In this study, pronuclear embryos from Kunming mice, characterized by an obvious two-cell block in vitro, were co-cultured with mouse, goat, and chick OECs. The functionality of OECs was determined by analyzing the cell cycle, apoptosis, the numbers of mitochondria and cilia, and the ability both to support embryonic development and to remove hypoxanthine from the culture medium. The necessity of direct contact between OECs and embryos was studied by repeated renewal of culture medium with fresh conditioned medium, the culture of embryos in plastic wells connected by tunnels to wells with OEC monolayers, and the co-culture of embryos separated from OECs by a filter. Both goat and chick OECs supported mouse embryonic development, but their embryotrophic lifespan was shorter than that of the mouse OECs. Whereas media conditioned with mouse OECs supported mouse embryonic development satisfactorily, medium conditioned with goat OECs supported little development. Immediate dialogue between heterologous OECs and embryos was essential for efficient co-culture, whereas direct contact between the two cell types was not; neither dialogue nor contact was needed between isologous OECs and embryos. Embryotrophic activity and the ability to remove hypoxanthine from conditioned medium declined with time after confluence and number of passages of OECs, mainly because of apoptosis and dedifferentiation. Thus, the species and functionality of OECs have profound effects on their molecular dialogue with co-cultured embryos, and efficient co-culture depends upon both positive and negative conditioning.