Blastocyst Day Research Articles

Effect of cumulin and super-GDF9 in standard and biphasic mouse IVM.

In vitro maturation (IVM) is a technology that generates mature oocytes following culture of immature cumulus-oocyte complexes (COC) in vitro. IVM is characterized by minimal patient stimulation, making it attractive for certain patient groups. Recently, a biphasic IVM system, capacitation (CAPA)-IVM, has shown improved clinical outcomes relative to standard IVM; however, it remains less efficient than IVF. This study assessed whether supplementation of CAPA-IVM culture media with the novel TGFβ superfamily proteins cumulin and super-GDF9 improves subsequent mouse embryo development. Immature mouse COCs were cultured by standard IVM or biphasic IVM ± cumulin or super-GDF9. Both cumulin and super-GDF9 in standard IVM significantly improved day-6 blastocyst rate (53.9% control, 73.6% cumulin, 70.4% super-GDF9; p = 0.006; n = 382-406 oocytes). Cumulin or super-GDF9 in CAPA-IVM did not alter embryo yield or blastocyst cell allocation in an unstimulated model. Moreover, cumulin did not alter these outcomes in a mild PMSG stimulation model. Cumulin in CAPA-IVM significantly increased cumulus cell expression of cumulus expansion genes (Ptgs2, Ptx3, Adamts1, Gfat2) and decreased Lhr expression relative to control. However, cumulin-induced mRNA expression of cumulus cell (Ptgs2, Ptx3) and oocyte genes (Gdf9, Bmp15, Oct4, Stella) in CAPA-IVM remained significantly lower than that of in vivo matured cells. Cumulin did not provide an additional beneficial effect in biphasic IVM in terms of blastocyst yield and cell allocation; however in standard IVM, cumulin and super-GDF9 significantly improve oocyte developmental competence.

The novel incorporation of aromatase inhibitor in hormonal replacement therapy cycles: a randomized controlled trial

Research questionDoes the incorporation of the aromatase inhibitor, letrozole, in hormone replacement therapy (HRT) improve the pregnancy outcome in vitrified–warmed blastocyst transfer cycles? DesignA randomized controlled trial; HRT was used in all cycles. Exogenous oestradiol, 6 mg daily started on day 2 or day 3 of the cycle. Tri-laminar endometrium 9 mm or thicker was the targeted cut-off. Thereafter, participants were randomized into two groups. Group A (HRT plus letrozole): 2.5 mg oral letrozole was given twice daily for 5 days only with continuation of daily oestradiol. Then, daily intramuscular progesterone was started with continuation of oestradiol. Group B (HRT only): daily intramuscular progesterone was administered in addition to daily oestradiol. In both groups, good-quality day-5 blastocyst transfer was planned on the sixth progesterone day with continuation of oestradiol and progesterone. Ongoing pregnancy rate was the primary outcome. ResultsA total of 112 patients were randomized, 56 in each group. Three participants did not have good-quality blastocyst after warming (one in group A and two in group B) and were excluded from the study. Group A and B included 55 and 54 participants, respectively. Ongoing pregnancy rate was significantly higher in group A than group B (RR 1.39, 95% CI 1.04 to 1.86, P = 0.023). Additionally, clinical pregnancy rate was significantly higher in group A (RR 1.31, 95% CI 1.02 to 1.68, P = 0.030). ConclusionsA new protocol of incorporating letrozole in HRT cycles seems to significantly increase probability of pregnancy, compared with HRT alone.

Do clinical outcomes differ for day-5 versus day-6 single embryo transfers controlled for endometrial factor?

Research questionIs there is a difference in clinical outcomes between day-5 versus day-6 blastocysts when transferred in a personalized embryo transfer (PET) cycle guided by Endometrial Receptivity Analysis (ERA)? DesignMulticentre, retrospective study; 260 patients who underwent a single embryo transfer with either a day-5 or day-6 blastocyst in a PET cycle guided by ERA between January 2017 and December 2019. ResultsA total of 260 blastocysts were transferred in a single embryo PET cycle guided by ERA. Of those, 183 (70.4%) were day-5 blastocysts and 77 (29.6%) were day-6 blastocysts. Clinical outcomes were similar when transferring day-5 blastocysts versus day-6 blastocysts: pregnancy rate was 75.4% (138/183) and 70.1% (54/77) (P = 0.465); implantation rate was 67.8% (124/183) and 63.6% (49/77) (P = 0.476); and ongoing pregnancy rate was 57.9% (106/183) and 58.4% (45/77) (P = 0.728), respectively. ConclusionsThe data suggest that the clinical potential of day-5 and day-6 blastocysts are similar, as no difference in clinical outcomes are observed when transferring at the time of optimal endometrial receptivity as determined by ERA.

Mitochondrial DNA Content May Not Be a Reliable Screening Biomarker for Live Birth After Single Euploid Blastocyst Transfer.

An increasing number of studies have related the mitochondrial DNA (mtDNA) content to embryo viability and transfer outcomes. However, previous studies have focused more on the relationship between mtDNA and embryo implantation, few studies have studied the effect of the mtDNA content on live birth. In the study, we investigated whether mtDNA content is a reliable screening biomarker for live birth after single blastocyst transfer. A total of 233 couples with 316 blastocyst stage embryos undergoing in vitro fertilization treatment and pre-implantation genetic testing analysis were included in the study. All embryos were chromosomally normal and had undergone single-embryo transfers. There was no significant difference observed in the blastocyst mtDNA content among the live birth, miscarriage and non-implanted groups (p=0.999), and the mtDNA content in blastocysts from the miscarriage and live birth groups was similar [median (interquartile range), 1.00*108(7.59*107- 1.39*108) vs 1.01*108 (7.37*107- 1.32*108)]. Similarly, no significant association was observed between mtDNA content and embryo implantation potential (p=0.965). After adjusting for multiple confounders in a logistic regression analysis with generalized estimating equations, no associations between mtDNA content and live birth were observed in all blastocysts, Day-5 and Day-6 blastocysts (p=0.567, p=0.673, p=0.165, respectively). The live birth rate was not significantly different between blastocysts with an elevated mtDNA content and blastocysts with a normal mtDNA content (26.7% vs 33.6% p=0.780). Additionally, there was no linear correlation between the mtDNA content and maternal age (p=0.570). In conclusion, the mtDNA content does not seem to be a potential biomarker for embryo transfer outcomes (i.e., implantation and live birth) based on the existing testing tools. Embryos with an elevated mtDNA content also have development potential for successful live birth.

First Baby Born in Brazil after Simultaneous Diagnosis through Non-Invasive and Conventional PGT-A.

Non-invasive preimplantation genetic testing for aneuploidies (niPGT-A) aiming to assess cell-free embryonic DNA in spent culture media is promising, especially because it might overcome the diminished rates of implantation caused by the inadequate performance of trophectoderm (TE) biopsy. Our center is part of the largest study to date assessing the concordance between conventional PGT-A and niPGT-A, and we report here the delivery of the first baby born in Brazil using niPGT-A. The parents of the baby were admitted to our center in 2018. They did not present history of infertility, and they were interested in using in vitro fertilization (IVF) and PGT-A in order to avoid congenital anomalies in the offspring. A total of 11 (3 day-5 and 8 day-6) expanded blastocysts were biopsied, and the spent culture media (culture from day-4 to day-6) from 8 day-6 blastocysts were collected for niPGT-A. Overall, 7 embryos yielded informative results for trophectoderm (TE) and media samples. Among the embryos with informative results, 5 presented concordant diagnosis between conventional PGT-A and niPGT-A, and 2 presented discordant diagnosis (1 false-positive and one false-negative). The Blastocyst 4, diagnosed as 46, XY by both niPGT-A and conventional PGT-A, was warmed up and transferred, resulting in the birth of a healthy 3.8 kg boy in February 2020. Based on our results and the recent literature, we believe that the safest current application of niPGT-A would be as a method of embryo selection for patients without an indication for conventional PGT-A. The approximate 80% of reliability of niPGT-A in the diagnosis of ploidy is superior to predictions provided by other non-invasive approaches like morphology and morphokinetics selection.

Early detection of pregnancy after IVF and embryo transfer with hyperglycosylated HCG versus Elecsys HCG+β assay

Research questionIs measurement of hyperglycosylated HCG (hHCG) superior to beta-HCG (HCG+β) for early pregnancy detection after IVF and embryo transfer? DesignBlood samples were collected on day 4 (+1), 7 (+1) and 11 (+2) after embryo transfer from women aged 18–45 years undergoing first or second fresh or frozen IVF embryo transfer cycles. Biochemical pregnancy was assessed on-site by HCG determination on day 11; clinical pregnancy was assessed by ultrasound on day 21 (+4/–3). Serum hHCG (immunochemiluminometric assay) and HCG+β (Elecsys® HCG+β assay) concentrations were measured. Performance of hHCG and HCG+β for predicting pregnancy was evaluated and cut-offs selected. ResultsIn total, 155 women were enrolled and underwent IVF and embryo transfer. Area under the curve (AUC) (95% CI) on day 4 was not significantly different for hHCG (AUC 0.88; 95% CI 0.83 to 0.94) and HCG+β (AUC 0.90; 95% CI 0.84 to 0.95), as was predictive performance on day 7 and 11, with higher AUC estimates compared with day 4. Applying cut-offs derived according to Youden's index on day 4 (hHCG, 100 pg/ml; HCG+β, 1.30 mIU/ml), both biomarkers demonstrated high negative predictive values for ruling out pregnancy (hHCG, 83.8%; HCG+β, 82.8%) and high positive predictive values for ruling in pregnancy (hHCG, 89.0%; HCG+β, 84.9%) on day 21. Diagnostic performance improved from day 4 to day 11. ConclusionsPredictive performance for early pregnancy post-IVF embryo transfer of day-5 blastocysts was not significantly different for hHCG and HCG+β; hHCG superiority over HCG+β was not shown.

TROPHECTODERM GRADE AND THE DAY OF TE BIOPSY SIGNIFICANTLY CORRELATE WITH PREGNANCY RATE IN EUPLOID BLASTOCYST TRANSFER

Currently, PGT-A becomes popular, and euploid blastocysts are usually selected for blastocyst transfer (BT). It is obvious that the morphological evaluation using trophectoderm (TE) grade and inner cell mass (ICM) grade are important for selecting a blastocyst, but it has been now controversial which grade can more contribute to achieve pregnancy even though using euploid blastocysts. The aim of this study is to evaluate which grade is more important, TE or ICM, to predict pregnancy among the euploid blastocysts. This study was a retrospective cohort study. From 1/2020 to 3/2021, 720 BT cycles in 520 patients were recruited. All blastocysts underwent TE biopsy on Day 5 (D5) or Day 6 (D6) for PGT-A, and those were cryopreserved. The extracted TE cells were analyzed by NGS. Clinical pregnancy rate (CPR) and miscarriage rates(MR) were evaluated according to the morphological evaluation and the day of TE biopsy. Fisher's exact test and multiple logistic regression analysis were utilized as appropriate. A p-value of < 0.01 was considered statistically significant. All statical analysis were performed with EZR. The maternal age was 36.6 ± 4.7 (mean ±S.D.). The CPR using D5 blastocyst showed 65.7%, and this was significantly higher than that of D6 blastocyst (43.8%, p<0.01). According to the TE grade, the CPRs of TE-A, B, and C were 71.6%, 52.8%, and 26.2%, respectively, with significant difference. In contrast, the CPRs of ICM-A, B and C were 67.8%, 56.8%, and 32.3%, respectively, without significant difference. The CPRs of the expansion stage were also no significant difference (Table1). The MR using D5 and D6 blastocysts were 10.6% and 17.0%, without significant difference, MR using TE-A (10.5%), B (12.2%) and C (36.4%), and using ICM-A (8.6%), B (15.0%) and C (50%) showed no significant difference neither. After adjusting for cofounders, not ICM grade but TE grade well correlated with CPR, and the day of TE biopsy also could be a good predictor to achieve pregnancy. The any morphological parameters and the day of TE biopsy had no relationship with miscarriage rates. Clinicians should focus on both the TE grade and the day of TE biopsy to select a good blastocyst for BT.

Blastocyst Transfer: A Risk Factor for Gestational Diabetes Mellitus in Women Undergoing In Vitro Fertilization.

BackgroundThe risk of developing gestational diabetes mellitus (GDM) is higher in women undergoing assisted reproductive treatment than in women conceiving spontaneously.ObjectivesTo determine whether the GDM risk after day-3 embryo transfer differs from the GDM risk after day-5 blastocyst transfer.MethodsProspective observational study in women becoming pregnant after first fresh embryo or blastocyst transfer.ResultsA total of 1579 women got pregnant and had live birth; 1300 women got day-3 embryo transfer only, whereas 279 women received at least 1 blastocyst. Of 1579 women, 252 developed GDM. Age, body mass index, baseline estradiol, baseline high-density lipoprotein, and progesterone on the day of human chorionic gonadotropin injection were not different in women receiving day-3 embryos only vs women receiving at least 1 blastocyst. The number and quality of retrieved oocytes were not different in women receiving day-3 embryo transfer from those receiving blastocysts. Our study confirmed already established GDM risk factors such as age and body mass index, baseline estradiol, and high-density lipoprotein, as well as progesterone after ovarian stimulation. We furthermore demonstrate that the GDM incidence in women receiving day-5 blastocyst transfer was significantly higher than those who received day-3 embryo transfer (21.15% vs 14.85%; P = 0.009). Considering confounding factors, we likewise saw that blastocyst transfer was an independent procedure-related GDM risk factor [P = 0.009, Exp (B): 1.56, 95% CI: 1.12-2.18].ConclusionBlastocyst transfer after in vitro fertilization/intracytoplasmic sperm injection increases the risk of developing GDM.

P–627 Optimal timing of day 6 blastocyst transfer in artificially prepared frozen-thawed embryo transfer cycles

Abstract Study question When is the optimal timing of day 6 (D6) blastocyst transfer between the 6thday (P6)and the 7th(P7) day of progesterone administration in artificially prepared frozen-thawed embryo transfer(FET) cycle Summary answer When transferring D6 blastocysts in artificially prepared FET cycles, live birth rate tended to be higher in P6 group than in P7 group. What is known already Blastocyst transfer in FET cycles has increased due to several reasons including convenience for optimization of endometrial synchronization, improvement of laboratory techniques and preimplantation genetic testing. Meanwhile, D6 blastocyst which cryopreserved on day 6 after being developed to the full blastocyst stage, presented lower pregnancy outcomes in FET cycle than D5 blastocysts. However, there have been few studies on the optimal duration of progesterone administration when transferring D6 blastocysts. Study design, size, duration This was a retrospective cohort study including patients who underwent frozen-thawed blastocyst transfer in artificially prepared cycles from January 2000 to May 2020. Patients with D6 blastocyst transfer on the 6th day of progesterone administration were included in D6-P6 group, and patients with D6 blastocyst transfer on the 7th day of progesterone administration were included in D6-P7 group. Participants/materials, setting, methods Increasing dose of estradiol valerate was administered from the 3rd day of menstruation: 4 mg/day for the first four days, 6 mg/day for next four days, and then 8 mg/day until the confirmation of pregnancy. Progesterone was administered from the 14th day of menstruation if the endometrial thickness reached ≥7 mm. The independent t-test or Mann-Whitney test, chi-square test, and logistic regression analysis were performed. Main results and the role of chance A total of 50 patients were included, and 13 patients underwent FET on P6 and 37 patients underwent FET on P7. Live birth rate was comparable between the P6 group and the P7 group (18.9% vs. 15.4%, p = 0.775). Live birth rate was higher in the D6-P6 group than in the D6-P7 group after adjusting for age, AMH, endometrial thickness on the starting day of progesterone administration and good embryo rate transferred with statistical significance (OR: 6.716, p = 0.005). Limitations, reasons for caution Limitations of the present study is the retrospective design and the small sample size. Caution is needed in extrapolating results of this study because only intramural and vaginal progesterone supplementations were included in this study. Wider implications of the findings: Even if the duration of blastocyst formation was delayed, frozen-thawed D6 blastocyst may need to be considered for on P6 rather than P7. The difference of live birth rate is not statistically significant. This study should be acknowledged for the underestimation of the difference because of the small sample size. Trial registration number Not applicable

P–159 Slow-growing embryos should be frozen on day 5

Abstract Study question What is the live birth rate after frozen embryo transfer (FET) of slow-growing embryos frozen on day 5 (D5) or on day 6 (D6)? Summary answer The live birth rate after single FET is significantly higher for slow-growing embryos frozen on D5 compared to those frozen on D6. What is known already Most data on the outcomes of blastocyst transfer stem from studies that evaluate fresh transfer from normal growing D5 blastocyst ET. However not all embryos will begin blastulation nor reach the fully expanded stage by D5; those are the slow-growing embryos. Studies that compare D5 to D6 embryos in FET cycles show contradictory results. Some have reported higher clinical pregnancy rates after D5 FET, while others have reported similar outcomes for D5 and D6 cryopreserved blastocyst transfers. There is a lack of evidence regarding the best approach for vitrifying embryos that exhibit a slow developmental kinetic. Study design, size, duration This retrospective cohort study included 821 single FET of slow-growing embryos frozen on D5 or D6, belonging to patients undergoing in vitro fertilization with donor oocytes between January 2011 and October 2019, in a single fertility center. The origin of blastocysts was either supernumerary embryos after fresh embryo transfer or blastocysts from freeze-all cycles. All embryos were transferred 2- 4h after thawing. Participants/materials, setting, methods We compared reproductive outcomes of slow-growing embryos frozen on D5 versus (n = 442) slow-growing embryos frozen on D6 (n = 379). D5 group consisted in embryos graded 0, 1, 2 of Gardner scale and frozen on D5. Similarly, D6 group consisted in embryos graded 3, 4, 5 of Gardner scale (blastocyst stage) and frozen on D6. Differences in pregnancy rates between study groups were compared using a Chi2 test. A p-value &amp;lt;0.05 was considered statistically significant. Main results and the role of chance Baseline characteristics were comparable between study groups. Overall, mean age of the woman was 42.3±5.4 years old; donor sperm was used in 25% of cycles, and it was frozen in 73.2% of cycles. Pregnancy rates were significantly higher when transferring slow D5 embryos compared to D6 for all the pregnancy outcomes analyzed: biochemical pregnancy rate was 27.7% vs 20.2%, p &amp;lt; 0.016; clinical pregnancy rate was 17.5% vs 10.2%, p &amp;lt; 0.004); ongoing pregnancy rate was: 15.7% vs 7.8% (p &amp;lt; 0.001); live birth rate was: 15.4% vs 7.5%, (p &amp;lt; 0.001). These results suggest that when embryos exhibit a slow development behavior (not reaching full blastocysts at D5), waiting until D6 for blastulation and expansion does not improve clinical outcomes. Vitrification at D5 will should the preferred option in cases where the oocyte is assumed of high quality Limitations, reasons for caution The retrospective design of the study is its main limitation. Also, morphology as sole selection criterion for transfer. However, blastocyst morphology is a very good predictor of implantation and pregnancy, and a good indicator of the embryo’s chromosomal status (higher euploidy rate in higher morphological quality blastocysts). Wider implications of the findings: These results can help to the standardization of laboratory protocols. As the decision of vitrifying slow developing embryos on D5 or D6 is made by the laboratory team or by the gynaecologist in agreement with the patient, having an evidence based strategy simplifies patient counselling and decision making. Trial registration number Not applicable

The Metabolic Signature of In Vitro Produced Bovine Embryos Helps Predict Pregnancy and Birth after Embryo Transfer.

In vitro produced (IVP) embryos show large metabolic variability induced by breed, culture conditions, embryonic stage and sex and gamete donors. We hypothesized that the birth potential could be accurately predicted by UHPLC-MS/MS in culture medium (CM) with the discrimination of factors inducing metabolic variation. Day-6 embryos were developed in single CM (modified synthetic oviduct fluid) for 24 h and transferred to recipients as fresh (28 ETs) or frozen/thawed (58 ETs) Day-7 blastocysts. Variability was induced with seven bulls, slaughterhouse oocyte donors, culture conditions (serum + Bovine Serum Albumin [BSA] or BSA alone) prior to single culture embryonic stage records (Day-6: morula, early blastocyst, blastocyst; Day-7: expanding blastocyst; fully expanded blastocysts) and cryopreservation. Retained metabolite signals (6111) were analyzed as a function of pregnancy at Day-40, Day-62 and birth in a combinatorial block study with all fixed factors. We identified 34 accumulated metabolites through 511 blocks, 198 for birth, 166 for Day-62 and 147 for Day-40. The relative abundance of metabolites was higher within blocks from non-pregnant (460) than from pregnant (51) embryos. Taxonomy classified lipids (12 fatty acids and derivatives; 224 blocks), amino acids (12) and derivatives (3) (186 blocks), benzenoids (4; 58 blocks), tri-carboxylic acids (2; 41 blocks) and 5-Hydroxy-l-tryptophan (2 blocks). Some metabolites were effective as single biomarkers in 95 blocks (Receiver Operating Characteristic – Area Under the Curve [ROC-AUC]: 0.700–1.000). In contrast, more accurate predictions within the largest data sets were obtained with combinations of 2, 3 and 4 single metabolites in 206 blocks (ROC-AUC = 0.800–1.000). Pregnancy-prone embryos consumed more amino acids and citric acid, and depleted less lipids and cis-aconitic acid. Big metabolic differences between embryos support efficient pregnancy and birth prediction when analyzed in discriminant conditions.

Euploid Day-5 Blastocysts Versus Euploid Day-6 Blastocysts — Will the Reproductive Outcomes Differ? An Observational Study

Background and objective: Day-5 blastocyst embryos are usually chosen for assisted reproductive therapy. We compared the reproductive outcomes of the euploid blastocysts developed on Day 5 versus Day 6. Methods: This single-center, retrospective observational study analyzed patients aged 25–45 years, who underwent intracytoplasmic sperm injection from December 2014 to November 2018. Depending on the day of trophectoderm biopsy, patients were categorized into Day-5 and Day-6 groups. Percentages of euploid embryos were calculated for both groups, and elective single euploid blastocysts were transferred in a frozen embryo transfer (FET) cycles. The study endpoints were the comparisons of the reproductive outcomes including clinical pregnancy rate (CPR), implantation rate (IR), miscarriage rate (MR), and live birth rate (LBR) between Day-5 and Day-6 euploid FET groups. Results: A total of 801 embryos from 184 patients were evaluated [Day 5 ([Formula: see text]); Day 6 ([Formula: see text]); 42.45% were euploid] with the rate of euploidy in Day-5 and Day-6 groups at 42.52% and 40.62%, respectively. A total of 126 patients underwent FET with 126 elective single euploid embryos (Day 5: 117; Day 6: 9). For Day-5 versus Day-6 groups, a significantly higher IR (61.54% vs. 44.44%; [Formula: see text]), CPR (61.54% vs. 44.44%; [Formula: see text]), and LBR (61.54% vs. 33.33%; [Formula: see text]) were reported. Multivariate analysis on ANOVA suggested, comparable pregnancy rates at Day 5 and Day 6 ([Formula: see text]). Conclusions: Day-5 euploid blastocysts seem to offer better reproductive outcomes than Day-6 euploid blastocysts. Further research is recommended to evaluate the reproductive outcomes of Day-6 blastocysts.

Effects of Donor Cell Types on the Development of Bovine Embryos Using Cytoplasm Injection Cloning Technology

Cytoplasm injection cloning technology (CICT) is an efficient technique for evaluating the developmental potential of cloned embryos. In this study, we investigated the effects of donor cell type on the developmental potential and quality of cloned bovine embryos. Adult fibroblasts (AFs) and embryonic cells (ECs) were used as donor cells to clone bovine embryos using CICT. We initially used AF cells to develop cloned embryos and then cultured the cloned day-8 blastocysts for 10 days to obtain ECs as donor cells for second embryo cloning. We found that the bovine blastocysts cloned using AF cells had significantly reduced developmental rates, embryo quality, and ratios of inner cell mass (ICM) to the total number of cells compared to those using ECs as donor cells. Furthermore, there were significant differences in the DNA methyltransferase-, histone deacetylation-, apoptosis-, and development-related genes at the blastocyst stage in embryos cloned from AFs compared to those in embryos cloned from ECs. Our results suggest that using ECs as donor cells for nuclear transfer enhances the quantity and quality of cloned embryos. However, further investigation is required in terms of determining pregnancy rates and developing cloned embryos from different donor cell types.

Constitutive Expression of TERT Enhances β-Klotho Expression and Improves Age-Related Deterioration in Early Bovine Embryos.

Age-associated decline in oocyte quality is one of the dominant factors of low fertility. Aging alters several key processes, such as telomere lengthening, cell senescence, and cellular longevity of granulosa cells surrounding oocyte. To investigate the age-dependent molecular changes, we examined the expression, localization, and correlation of telomerase reverse transcriptase (TERT) and β-Klotho (KLB) in bovine granulosa cells, oocytes, and early embryos during the aging process. Herein, cumulus-oocyte complexes (COCs) obtained from aged cows (>120 months) via ovum pick-up (OPU) showed reduced expression of β-Klotho and its co-receptor fibroblast growth factor receptor 1 (FGFR1). TERT plasmid injection into pronuclear zygotes not only markedly enhanced day-8 blastocysts’ development competence (39.1 ± 0.8%) compared to the control (31.1 ± 0.5%) and D-galactose (17.9 ± 1.0%) treatment groups but also enhanced KLB and FGFR1 expression. In addition, plasmid-injected zygotes displayed a considerable enhancement in blastocyst quality and implantation potential. Cycloastragenol (CAG), an extract of saponins, stimulates telomerase enzymes and enhances KLB expression and alleviates age-related deterioration in cultured primary bovine granulosa cells. In conclusion, telomerase activation or constitutive expression will increase KLB expression and activate the FGFR1/β-Klotho pathway in bovine granulosa cells and early embryos, inhibiting age-related malfunctioning.

Metabolites Secreted by Bovine Embryos In Vitro Predict Pregnancies That the Recipient Plasma Metabolome Cannot, and Vice Versa.

This work describes the use of mass spectrometry-based metabolomics as a non-invasive approach to accurately predict birth prior to embryo transfer (ET) starting from embryo culture media and plasma recipient. Metabolomics was used here as a predictive platform. Day-6 in vitro produced embryos developed singly in modified synthetic oviduct fluid culture medium (CM) drops for 24 h were vitrified as Day-7 blastocysts and transferred to recipients. Day-0 and Day-7 recipient plasma (N = 36 × 2) and CM (N = 36) were analyzed by gas chromatography coupled to the quadrupole time of flight mass spectrometry (GC-qTOF). Metabolites quantified in CM and plasma were analyzed as a function to predict pregnancy at Day-40, Day-62, and birth (univariate and multivariate statistics). Subsequently, a Boolean matrix (F1 score) was constructed with metabolite pairs (one from the embryo, and one from the recipient) to combine the predictive power of embryos and recipients. Validation was performed in independent cohorts of ETs analyzed. Embryos that did not reach birth released more stearic acid, capric acid, palmitic acid, and glyceryl monostearate in CM (i.e., (p < 0.05, FDR < 0.05, Receiver Operator Characteristic—area under curve (ROC-AUC) > 0.669)). Within Holstein recipients, hydrocinnamic acid, alanine, and lysine predicted birth (ROC-AUC > 0.778). Asturiana de los Valles recipients that reached birth showed lower concentrations of 6-methyl-5-hepten-2-one, stearic acid, palmitic acid, and hippuric acid (ROC-AUC > 0.832). Embryonal capric acid and glyceryl-monostearate formed F1 scores generally >0.900, with metabolites found both to differ (e.g., hippuric acid, hydrocinnamic acid) or not (e.g., heptadecanoic acid, citric acid) with pregnancy in plasmas, as hypothesized. Efficient lipid metabolism in the embryo and the recipient can allow pregnancy to proceed. Changes in phenolics from plasma suggest that microbiota and liver metabolism influence the pregnancy establishment in cattle.

Lycopene Improves In Vitro Development of Porcine Embryos by Reducing Oxidative Stress and Apoptosis

In vitro culture (IVC) for porcine embryo development is inferior compared to in vivo development because oxidative stress can be induced by the production of excessive reactive oxygen species (ROS) under high oxygen tension in the in vitro environment. To overcome this problem, we investigated the effect of lycopene, an antioxidant carotenoid, on developmental competence and the mechanisms involved in mitochondria-dependent apoptosis pathways in porcine embryos. In vitro fertilized (IVF) embryos were cultured in IVC medium supplemented with 0, 0.02, 0.05, 0.1, or 0.2 μM lycopene. The results indicate that 0.1 μM lycopene significantly increased the rate of blastocyst formation and the total cell numbers, including trophectoderm cell numbers, on Day In terms of mitochondria-dependent apoptosis, IVF embryos treated with 0.1 μM lycopene exhibited significantly decreased levels of ROS, increased mitochondrial membrane potential, and decreased expression of cytochrome c on Days 2 and Furthermore, 0.1 μM lycopene significantly decreased the number and percentage of caspase 3-positive and apoptotic cells in Day-6 blastocysts. In addition, Day-2 embryos and Day-6 blastocysts treated with 0.1 μM lycopene showed significantly reduced mRNA expression related to antioxidant enzymes (SOD1, SOD2, CATALASE) and apoptosis (BAX/BCL2L1 ratio). These results indicate that lycopene supplementation during the entire period of IVC enhanced embryonic development in pigs by regulating oxidative stress and mitochondria-dependent apoptosis.

123 Efficiency of embryo production using ovum pickup oocytes recovered from stimulated and nonstimulated cows

Recovery of oocytes from live animals through ultrasound-guided transvaginal follicular aspiration (ovum pickup, OPU) is an essential element of modern livestock development. To increase the number of OPU oocytes retrieved, hormonal stimulation is broadly applied; however, the results are ambiguous. The aim of the present study was to compare recovery rate, quality of oocytes, and efficiency of invitro production of cattle embryos using OPU oocytes derived from hormonal treated and untreated cows. The study was performed in Simmental heifers at the age of 17 to 23 months. The heifers in the first group (n=7) were previously synchronized using prostaglandin F2α (Estrumate, MSD Animal Health) and gonadotrophin-releasing hormone (GnRH; Receptal, MSD Animal Health) and then underwent superstimulation with Stimofol (Reprobiol SPRL, Belgium) in a total amount of 0.25mg of FSH and 0.05mg of LH in a treatment of 6 injections on 3 consecutive days starting on Day −4 before OPU. The heifers of the second group (n=2) underwent OPU once a week for 5 weeks (10 OPU sessions). Groups of recovered cumulus–oocyte complexes (COCs) were matured in TCM-199 containing 10% fetal calf serum, 1mM Na-pyruvate, 50μg mL−1 gentamycin, 10μg mL−1 FSH, and 10μg mL−1 LH for 24h, fertilized in Fert-TALP (Tyrode’s-albumin-lactate-pyruvate) for 18 to 20h, and cultured in CR1aa medium for 7 days. All steps of IVP were performed at 38.5°C with 5% CO2 and 90% humidity. The cleavage and blastocysts rates were assessed on Day 2 and 7, respectively. In addition, Day-7 blastocysts were fixed, and the total cell number was determined using 4′,6-diamidino-2-phenylindole (DAPI) staining. The data were analysed by ANOVA using SigmaStat software package. As expected, the number of aspirated follicles per session in the first group of cows was higher than that in the second group: 14.6±1.2 vs. 6.8±0.5 (P&amp;lt;0.05). In total, 54 and 42 COCs were collected, which corresponds to recovery rates of 54.5±7.7 and 61.3±4.9% for stimulated and nonstimulated cows, respectively. After the quality evaluation, 36 COCs of SS heifers (67.2±1.9%) and 31 COCs of non-SS heifers (75.3±5.0%) were selected for IVP. We did not observe a significant difference in cleavage rate between two groups: 77.8% in the SS group vs. 71.0% in the non-SS group, whereas the rate of blastocyst production was higher (P&amp;lt;0.05) for SS heifers (25.0%) compared with non-SS animals (16.1%). The total number of cells in Day 7 blastocysts was significantly (P&amp;lt;0.01) higher in the first group (89.8±1.3) compared with the second group (70.6±3.2). Thus, superstimulation of heifers before OPU increases the efficiency of oocyte retrieval by ultrasound-guided transvaginal follicular aspiration as well as the developmental competence of OPU-derived bovine oocytes invitro. These studies were performed under financial support of the Russian Science Foundation (project No. 19-16-00115) and the Ministry of Science and Higher Education of Russia.

63 Effect of sire conception rate on bovine early embryo development

Currently, the only measure of sire fertility in the bovine is sire conception rate (SCR), which is determined by Day 70 pregnancy diagnosis and not reflective of early embryo development. Therefore, this study aimed to establish the relationship between SCR and early embryo development. In the first experiment, 65 sires of negative (&amp;lt;−1, n=25), average (−1 to 1, n=19), and high (&amp;gt; +1, n=21) SCR were characterised for their ability to produce embryos using an invitro embryo production (IVP) system. For each sire, 100 cumulus–oocyte complexes (COCs) were used. COCs were matured for 22h, fertilized by co-incubation with sperm selected from density gradient centrifugation for 18h, and then placed in culture medium. A sire of known IVP performance was used as a control in each run. Cleavage and blastocyst rates (BL) were measured on Days 3 and 8 post-insemination, respectively. Photographs were taken on Days 3, 5, and 8 to identify arrest stages of non-blastocyst embryos. Sires were ranked based on their blastocyst rate and grouped into quartiles for statistical analysis. Differences in BL were determined by ANOVA using sire, IVP run, and a sire×IVP run interaction. In addition, the correlation between SCR and BL was determined. All data were analysed using SAS software version 9.4 (SAS Institute Inc.). Mean BL between each quartile was significant (P&amp;lt;0.05), with rates ranging from 8 to 22% and 32 to 62% for the lowest and highest quartile, respectively. There was no correlation (P=0.90) between SCR and BL. Arrest stage was measured by subtracting the number of Day-8 blastocysts from, first, embryos that were morulas on Day 5, and then embryos that were 8- to 16-cell stage embryos on Day 5. This method is based on the assumption that embryos closer to the blastocyst stage on Day 5 are more likely to contribute to the Day 8 blastocyst population. The most frequent arrest stage was the 4- to 6-cell stage (39/52 sires). It has been shown that decreased rates of autophagy are associated with embryonic arrest at the 4- to 8-cell stage in humans, leading us to investigate this mechanism in the second experiment. Select high (n=3) and low (n=4) performing sires identified in experiment 1 were used to generate 4- to 6-cell embryos, and autophagy rates were measured using live immunofluorescence with CYTO-ID autophagy dye (n=20 embryos/sire). The mean fluorescent intensity of each embryo was divided by the number of cells within the embryo. Differences in autophagy between high and low sires were determined by ANOVA using SAS. Interestingly, low-performing sires had a significantly higher autophagy rates than high-performing sires (77.8±3.1 vs. 50.0±3.5). This could indicate that embryos produced with low-performing sires had higher levels of stress than their counterparts. In summary, the effect of sire on embryonic development seems to be independent of the SCR classification. The most common arrest stage observed is the 4- to 6-cell stage, right before embryonic genome activation. Further research is required to elucidate the mechanisms by which sires influence pre-implantation development. This research was supported by USDA-NIFA AFRI Competitive Grant No. 2019-67015-28998.

Successful implantation is associated with a transient increase in serum pro-inflammatory cytokine profile followed by a switch to anti-inflammatory cytokine profile prior to confirmation of pregnancy

To compare the changing peripheral levels of inflammation-related cytokine profile during a 9-day period after blastocyst transfer between women who did and did not conceive. Prospective, observational, and longitudinal study. University-affiliated hospital. Forty-seven women with infertility who were undergoing single day-5 blastocyst transfer were recruited. This prospective observational and longitudinal study on 47 women with infertility was performed in an invitro fertilization unit from December 2018 to August 2019. The amounts of a range of cytokines was measured on serial blood samples obtained during a 9-day period after blastocyst transfer. Serial blood samples were obtained on the day of embryo transfer, and 3, 6, and 9 days afterward for measurement of serum interferon gamma (IFN-γ), tumor necrosis factor alpha, interleukin (IL)-2, IL-4, IL-10, IL-12, IL-13, IL-17, IL-18, and IL-22 using cytometric bead arrays; transforming growth factor beta 1 (TGF-β1) was measured using commercial enzyme-linked immunosorbent assay kits. The cytokine profile was similar between the women who conceived and those who did not on the day of blastocyst transfer. In women who conceived, IFN-γ and IL-17 (pro-inflammatory cytokines) exhibited a transient and significant increase on day 3 after blastocyst transfer, which decreased to the baseline levels by day 6. Meanwhile, IL-10 (anti-inflammatory cytokine) was increased significantly on days 6 and 9, and TGF-β1 (anti-inflammatory cytokine) was increased significantly on day 9 after blastocyst transfer. In women who did not conceive, there was a more pronounced increase in IFN-γ and IL-17 (pro-inflammatory cytokines) on day 3, which was sustained on days 6 and 9 without a switch to an anti-inflammatory cytokine profile. Among women who conceived after blastocyst embryo transfer, there was a transient and modest increase in serum pro-inflammatory cytokine profile (IFN-γ and IL-17) 3 days after blastocyst transfer, which was followed by a switch to anti-inflammatory cytokine profile (increase IL-10 and TGF-β1) by 6 days after blastocyst transfer and the latter increase was sustained 9 days after blastocyst transfer, when pregnancy was confirmed.

Blastocoel fluid removal and melatonin supplementation in the culture medium improve the viability of vitrified bovine embryos

In this study, we investigated the effects of melatonin supplementation in the culture medium and blastocoel fluid removal (BFR) before vitrification on the quality and viability of in vitro-derived bovine embryos. After fertilization, presumptive zygotes were assigned to one of the following treatments: control, in vitro standard culture (IVC) medium; IVC + M10−9, IVC medium supplemented 10−9 M melatonin; or IVC + M10−9 BFR, IVC medium supplemented with 10−9 M melatonin plus BFR on day 7 (D7) of culture. D7 blastocysts were vitrified by the Cryotop method and, after 5 mo of storage, were warmed and incubated for an additional 72 h. The re-expansion rate was evaluated after 2 and 24 h, and the hatching rate was evaluated after 24, 48, and 72 h. At 72 h, the total number of cells (TNC); number of apoptotic cells (NAC); and expression of genes related to oxidative stress (HSPA5), cell metabolism (SLC2A3), cell repair (MSH6), placentation (KRT8 and PLAC8), and implantation (FOSL1) were assessed in the blastocysts. Less than 30% of the control blastocysts re-expanded until 2 h, whereas more than 85% of the IVC + M10−9 and IVC + M10−9 BFR blastocysts re-expanded (P < 0.05). The hatching rate of IVC + M10−9 BFR blastocysts increased at all time points (P < 0.05), reaching 66.8% at 72 h of incubation. The TNC was similar among treatments (P > 0.05), regardless of vitrification/warming and re-cultivation. The NAC:TNC was smaller for melatonin-treated blastocysts (P < 0.05). BFR increased HSPA5 (P = 0.0118) expression and did not affect SLC2A3, MSH6, KRT8, and FOSL1 expression (P > 0.05). In conclusion, melatonin (10−9 M) supplementation in the culture medium and BFR on D7 of culture increased the hatching rate 24, 48, and 72 h after warming of the vitrified embryos, indicating an improvement in cryotolerance.