Blastocyst Development Research Articles

Clinical utilization and outcomes over eight consecutive years following oocyte cryopreservation.

To compare embryologic and clinical outcomes following oocyte cryopreservation (OC) and immediate fertilization for embryo cryopreservation (EC) in non-infertile patients. Retrospective cohort study SUBJECTS: Patients treated at a single fertility center from 2016 to 2023. OC MAIN OUTCOME MEASURES: Blastocyst development was compared between women undergoing OC and non-infertile women undergoing immediate fertilization for EC between 18 and 42years of age. ICSI, PGT-A, and eSET were used in all cycles. Outcomes from 3697 thawed MII oocytes obtained from 239 OC patients were compared to 28,959 fresh MII oocytes obtained from 2031 patients undergoing immediate fertilization for EC. Average thaw MII survival rate was 92% and high across age groups. Post-thaw oocytes demonstrate lower fertilization rate compared to fresh oocytes (85% v 90%, p < 0.05). As oocyte age increased, the mean number of MII used per blastocyst increased to a greater extent among thawed compared to fresh oocytes (2.9 v 2.2 MII age 18-34, 3.3 v 2.5 MII age 34-37, 3.9 v 2.9 MII age 38-40, 7.5 v 3.9 MII age 41-42, p < 0.05). Among patients achieving transfer of a euploid blastocyst, clinical pregnancy rate and live birth rate per transfer were similar using thawed and fresh oocytes (p = 0.82 and p = 0.79, respectively). Blastocyst conversion is lower from thawed oocytes compared to fresh oocytes. Differences in blastocyst development between thawed and frozen oocytes increase with advancing oocyte age, suggesting that older oocytes may be less efficient in their conversion to blastocysts after vitrification and warming.

Embryology outcomes of a device-based sperm separation technique compared to density gradient centrifugation using thawed spermatozoa-a sibling donor oocyte study.

To evaluate whether the ZyMōt™ Multi 850μl sperm separation device (SSD) effectively recovers motile spermatozoa from cryopreserved ejaculates and compare its effect on key embryology outcomes including fertilization, cleavage stage, and total and top-quality blastocyst formation rates to the traditional Density Gradient Centrifugation (DGC) method. In this prospective, single-center, controlled study, we used fresh sibling donor oocytes and non-donor cryopreserved ejaculates. In total, 150 couples participated in this study. At least eight MII donor oocytes were allocated to each couple split into two arms. One arm underwent ICSI with the control DGC-processed sample, and the other arm processed with SSD. No significant difference on fertilization and cleavage stage embryo rates was observed between the two techniques. We observed a significant increase in the percentage of total (SSD: 74.03 ± 23.47% vs. DGC: 67.86 ± 23.92%; p = 0.016) and top-quality (SSD: 66.38 ± 24.94% vs. DGC: 60.98 ± 24.40%; p = 0.035) blastocysts formed post-SSD processing. Sub-analysis showed that this increase remained significant for the WHO-normal group (n = 118), but not for the WHO-abnormal group (n = 32). The SSD was successfully applied in all 150 cases, providing adequate numbers of spermatozoa to undergo ICSI. Additionally, SSD significantly improved blastocyst development rates; however, this was of limited clinical impact considering the minor improvement on the average number of top-quality blastocysts. It can be hypothesized that this positive contribution may be stronger and clinically significant when a larger number of oocytes is used or in homologous oocyte ICSI cycles, where the repair mechanisms of the oocytes may insufficient for promoting healthy embryo development.

The Success Rate of Cryopreservation and Thawing of Embryos at Day 3 and Day 5 Following Intracytoplasmic Sperm Injection (ICSI)

Cryopreservation, the technique of freezing and thawing embryos, is essential in in vitro fertilization (IVF) treatments to ensure the long-term preservation of embryos for future use. This innovative study evaluates the effectiveness of these procedures conducted at a private hospital in Tangerang, Indonesia, over a period of seven years (2016-2023), involving 18,000 couples. The design research was conducted retrospectively. The study examined embryos that underwent cryopreservation and subsequent thawing, with data collected on the rates of cell division observed on Day 3 and the rates of blastocyst formation on Day 5 after thawing. The study investigated the rates of cellular division on Day 3 and the development of blastocysts on Day 5 following the thawing process. The findings demonstrated a remarkable cleavage rate of 95.24% and a blastocyst rate of 97.22%. The remarkable results underscore the efficacy of the advanced freezing and thawing techniques employed. This discovery is highly significant since there is an urgent requirement to improve the success rates of in vitro fertilization (IVF) for couples facing infertility. It offers a fresh sense of hope and increased opportunities for establishing successful pregnancies with the improvement of cryopreservation techniques.

Endometrial extracellular vesicles regulate processes related to embryo development and implantation in human blastocysts.

What is the transcriptomic response of human blastocysts following internalization of extracellular vesicles (EVs) secreted by the human endometrium? EVs secreted by the maternal endometrium induce a transcriptomic response in human embryos that modulates molecular mechanisms related to embryo development and implantation. EVs mediate intercellular communication by transporting various molecules, and endometrial EVs have been postulated to be involved in the molecular regulation of embryo implantation. Our previous studies showed that endometrial EVs carry miRNAs and proteins associated with implantation events that can be taken up by human blastocysts; however, no studies have yet investigated the transcriptomic response of human embryos to this EV uptake, which is crucial to demonstrate the functional significance of this communication system. A prospective descriptive study was performed. Primary human endometrial epithelial cells (pHEECs), derived from endometrial biopsies collected from fertile oocyte donors (n = 20), were cultured in vitro to isolate secreted EVs. Following EV characterization, Day 5 human blastocysts (n = 24) were cultured in the presence or absence of the EVs for 24 h and evaluated by RNA-sequencing. EVs were isolated from the conditioned culture media using ultracentrifugation, and characterization was performed using western blot, nanoparticle tracking analysis, and transmission electron microscopy. Human blastocysts were devitrified, divided into two groups (n = 12/group), and cultured in vitro for 24 h with or without previously isolated EVs. RNA-sequencing analysis was performed, and DESeq2 was used to identify differentially expressed genes (DEGs) (FDR < 0.05). QIAGEN Ingenuity Pathway Analysis was used to perform the functional enrichment analysis and integration with our recently published data from the pHEECs' EV-miRNA cargo. Characterization confirmed the isolation of EVs from pHEECs' conditioned culture media. Among the DEGs in blastocysts co-cultured with EVs, we found 519 were significantly upregulated and 395 were significantly downregulated. These DEGs were significantly enriched in upregulated functions related to embryonic development, cellular invasion and migration, cell cycle, cellular organization and assembly, gene expression, and cell viability; and downregulated functions related to cell death and DNA fragmentation. Further, the intracellular signaling pathways regulated by the internalization of endometrial EVs were previously related to early embryo development and implantation potential, for their role in pluripotency, cellular homeostasis, early embryogenesis, and implantation-related processes. Finally, integrating data from miRNA cargo of EVs, we found that the miRNAs carried by endometrial EVs targeted nearly 80% of the DEGs in human blastocysts. This is an in vitro study in which conditions of endometrial cell culture could not mimic the intrauterine environment. This study provides novel insights into the functional relevance of EVs secreted by the human endometrium, and particularly the role of EV-miRNA regulation on global transcriptome behavior of human blastocysts during early embryogenesis and embryo implantation. It provides potential biomarkers that could become useful diagnostic targets for predicting implantation success. This study was supported by the Spanish Ministry of Education through FPU awarded to M.S.-B. (FPU18/03735), Generalitat Valenciana through VALi+d Programme awarded to M.C.C.-G. (ACIF/2019/139), and Instituto de Salud Carlos III and cofounded by the European Social Fund (ESF) "Investing in your future" through the Miguel Servet Program (CP20/00120 [H.F.]; CP19/00149 [I.C.]). The authors have no conflicts of interest to disclose. N/A.

Fertility and early embryonic development in a CD46-edited Gir heifer with reduced susceptibility to BVDV.

Bovine viral diarrhea virus (BVDV) infection during pregnancy is a significant contributor to reproductive failures in cattle. The bovine receptor for BVDV (CD46) was previously edited with a six amino acid substitution (G82QVLAL to A82LPTFS) and shown to have significantly reduced BVDV susceptibility in a Gir heifer calf. Since a role for CD46 has been proposed in mammalian fertilization, our objective was to assess the edited heifer's fertilization rates, early embryonic development, and germline transmission conformation of the edit. Cumulus oocyte complexes were collected from the edited heifer and unedited females, fertilized with semen from an unedited bull and cultured until the blastocyst stage. Ultrasound examinations and serum progesterone concentration were also monitored to confirm estrous cyclicity in the CD46-edited heifer. Estrous cyclicity was normal with visualization of a corpus luteum and elevated progesterone concentrations. Fertilization rates and blastocyst development were not different in oocytes from edited and unedited controls. Genome sequence analysis of blastocysts confirmed germline transmission of either edited allele from the heifer. Subsequently, the CD46-edited heifer was artificially inseminated with semen from an unedited Gir bull and fertility status was confirmed with a diagnosed conception at day 35 of gestation. Thus, a six amino acid substitution in CD46 did not negatively affect fertilization of edited oocytes or early embryonic development when fertilized with semen from an unedited bull. An edited bull is still needed to similarly evaluate reproductive function of sperm cells carrying this CD46 edit.

Analysis of the embryotoxic effects of blastocyst culture medium in vitro fertilization technology

Abstract. The culture medium for blastocysts contains components that may exhibit embryotoxicity, potentially affecting the developmental process of blastocysts and leading to abnormalities in the growth and development of offspring. This paper reviews and discusses the impact of blastocyst culture medium on the development of blastocysts and later-stage embryos. The results presented in the literature indicate that blastocyst media with different compositions not only affect development during the incubation period but may also have long-term effects on post-implantation development and maturation of embryos. The paper primarily summarizes the effects of the composition and concentration of blastocyst culture medium on embryonic development, aiming to further improve the quality of in vitro culture systems and optimize the culture conditions.

Oocyte maturity rate is an age-independent predictor of blastocyst development and euploidy rates: a multicenter retrospective study

Research Question Can oocyte maturity rate predict blastocyst development and euploidy rates independently of patient age? Design A multicenter retrospective study including 1547 consecutive women undergoing first in-vitro fertilization cycle of oocyte retrieval-intracytoplasmic sperm injection with preimplantation genetic testing for aneuploidy (IVF/ICSI/PGT-A). Both centers used the same PGT lab for PGT-A testing. Oocyte maturity rate as a predictor for cycle outcomes of blastocyst development and euploidy rates was analyzed by Poisson regression model, after controlling for other known confounders. Results Oocyte maturity rate is positively correlated with blastocyst development rate and euploidy rate after controlling for patient age at cycle start, BMI, AMH levels, total FSH dosage, number of days of ovarian stimulation, and peak estradiol level (p&lt;0.01). Conclusion This study demonstrated that oocyte maturity rate can predict blastocyst development and euploidy rates independently of other confounding factors including patient age. This finding suggests that oocyte maturity rate may serve as an important biomarker for oocyte quality. This can be used by physicians to evaluate and improve the efficacy of ovarian stimulation regimens for IVF. This may further enhance artificial intelligence (AI) capabilities in optimizing ovarian stimulation protocols and outcomes of Assisted Reproductive Technologies (ART).

Progesterone supplemented uterine epithelial cell co-culture improves in vitro quality embryo production in buffalo

Objectives: The present study was conducted to study the effect of progesterone and uterine luminal epithelial cells monolayer on blastocyst development and hatching rate. Material and Methods: The isolation, culture, and characterization of slaughterhouse-derived uterine epithelial cells were done using standard protocol. The steroid hormones estrogen and progesterone were supplemented in embryo developmental media (EDM), and day 04 embryos were cultured in different groups as progesterone supplementation (T1), co-cultured with epithelial cell monolayer (T2), co-cultured with progesterone supplemented epithelial cell monolayer (T3), or without any treatment (control). Finally, the effect of different treatments was analyzed in terms of blastocyst and hatching rate. Results: The isolated epithelial cells depicted compact cuboidal or columnar morphology at the confluence. Immunocytochemical localization and polymerase chain reaction study revealed positive expression of cytokeratin and absence of vimentin. Significantly higher blastocyst and hatching rates were noted in the T3 group, followed by T2, T1, and control groups. Conclusion: The present study revealed improved in vitro embryo production after co-culturing embryos with progesterone-supplemented uterine epithelial cells in buffalo.

Effect of biphasic in vitro maturation (CAPA-IVM) on EGF receptor and embryo development of prepubertal goat oocytes according to follicle size

Oocytes spontaneously resume meiosis following their liberation from follicles, preventing full competence acquisition. Biphasic IVM (CAPA-IVM) maintains oocytes in meiotic arrest to improve developmental competence, and it specially affects poorly developed oocytes. We assessed the effect of CAPA-IVM on oocytes from small (<3mm) and large (>3mm) follicles of prepubertal goats. Oocytes were cultured for 6h in pre-IVM with C-type natriuretic peptide (CNP) and estradiol as meiotic inhibitors, and germinal vesicle (GV) rate and chromatin configuration were assessed. Then, oocytes were cultured in conventional IVM (c-IVM) or CAPA-IVM (pre-IVM + c-IVM) and EGF receptor (EGFR) protein expression, intra-oocyte ROS and blastocyst development were assessed. GV rate was higher in CNP groups than control (69% vs 28%, and 67% vs 31%, small and large follicles, respectively), but GV chromatin configuration was similar. In large follicles, EGFR expression was higher in oocytes and cumulus cells after CAPA-IVM, and ROS levels were lower. In small follicles these differences were not observed. c-IVM and CAPA-IVM produced similar blastocyst rates in small (3.7% vs 2.6%, respectively) and large follicles (8.3% vs 2.5%). Overall, CAPA-IVM enhanced EGFR expression for EGF peptide signalling and antioxidant capacity in oocytes from large follicles but oocytes from small follicles were too immature to benefit from it.

Embryotrophic effect of exogenous protein contained adipose-derived stem cell extracellular vesicles

BackgroundExtracellular vesicles (EVs) regulate cell metabolism and various biological processes by delivering specific proteins and nucleic acids to surrounding cells. We aimed to investigate the effects of the cargo contained in EVs derived from adipose-derived stem cells (ASCs) on the porcine embryonic development.MethodsASCs were isolated from porcine adipose tissue and characterized using ASC-specific markers via flow cytometry. EVs were subsequently extracted from the conditioned media of the established ASCs. These EVs were added to the in vitro culture environment of porcine embryos to observe qualitative improvements in embryonic development. Furthermore, the proteins within the EVs were analyzed to investigate the underlying mechanisms.ResultsWe observed a higher blastocyst development rate and increased mitochondrial activity in early stage embryos in the ASC-EVs-supplemented group than in the controls (24.8% ± 0.8% vs. 28.6% ± 1.1%, respectively). The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay of blastocysts also revealed significantly reduced apoptotic cells in the ASC-EVs-supplemented group. Furthermore, through proteomics, we detected the proteins in ASC-EVs and blastocysts from each treatment group. This analysis revealed a higher fraction of proteins in the ASC-EVs-supplemented group than in the controls (1,547 vs. 1,495, respectively). Gene analysis confirmed that ASC-EVs showed a high expression of tyrosine-protein kinase (SRC), whereas ASC-EVs supplemented blastocysts showed a higher expression of Cyclin-dependent kinase 1 (CDK1). SRC is postulated to activate protein kinase B (AKT), which inhibits the forkhead box O signaling pathway and activates CDK1. Subsequently, CDK1 activation influences the cell cycle, thereby affecting in vitro embryonic development.ConclusionASC-EVs promote mitochondrial activity, which is crucial for the early development of blastocysts and vital in the downregulation of apoptosis. Additionally, ASC-EVs supply SRC to porcine blastocysts, thereby elongating the cell cycle.

IVF with frozen-thawed sperm after prolonged capacitation yields comparable results to ICSI in horses: A morphokinetics study

Intracytoplasmic sperm injection (ICSI) is the current clinical practice for the in vitro production of equine embryos. The use of conventional fertilization methods such as in vitro fertilization (IVF), has historically been associated with poor success in horses. However, recent improvements have led to better outcomes with IVF, though only when using fresh semen, which limits its use in clinical practice. IVF remains in its infancy in equine reproduction, and several unknowns remain about the technique. One significant gap in knowledge concerns the morphokinetics of IVF embryos and how they differ from their ICSI counterparts. To address this, we performed IVF using frozen-thawed sperm from five different stallions following sperm selection and a prolonged capacitation period of 10 h, on a total of 109 oocytes. We then analyzed the cleavage rate (cleaved/initial oocytes), blastocyst rate (blastocyst/initial zygotes), and blastocyst development (blastocyst/cleaved zygotes) of the IVF cycles, and compared them with those of the clinical ICSI cycles during the same period. We also evaluated time-lapse images of the developed embryos to assess developmental time points such as time to morula compaction and blastocyst expansion, as well as morula and blastocyst sizes. Overall, developmental rates were not different between IVF and ICSI cycles (blastocyst rate 41.1 % IVF and 41.8 % ICSI, p > 0.05). However, development proceeded faster in IVF cycles (blastocyst expansion IVF 155.5 ± 18.5 h; ICSI 167.2 ± 19.6 h; p < 0.05) and IVF embryos were also larger (blastocyst area IVF 22608 ± 2857 μm2; ICSI 20806 ± 1505 μm2; p < 0.05). The faster development and larger size might suggest a more advanced developmental stage. The implications of these findings need to be further evaluated to assess their association with pregnancy potential. The successful developmental rates achieved in IVF cycles demonstrate the potential of this technique for clinical application, although the amount of frozen-thawed semen required is significantly higher in IVF than in ICSI, which is an important consideration for mare and stallion owners. Nonetheless, the use of frozen-thawed semen in equine IVF, coupled with comparable blastocyst rate, presents promising potential for broader clinical adoption of the IVF technique.

Assessment of loliolide extracted from Biserula pelecinus, present during in vitro oocyte maturation, on fertilisation and embryo development in sheep

Context As a ‘duty of care’, it is important to test whether new forage plants for ruminants contain secondary compounds (PSCs) that affect reproductive performance. We have previously observed, a posteriori, that the presence of a methanolic extract of Biserrula pelecinus during maturation of sheep oocytes increased fertilisation rate and blastocyst development. This result needed to be verified a priori and, if the outcome was repeated, we needed to identify the plant secondary metabolite responsible. Aims To test whether PSCs from B. pelecinus, when added to the oocyte maturation medium, improve fertilisation rate and blastocyst development; to test whether loliolide is the active molecule produced by B. pelecinus. Methods Methanol–chloroform extracts of B. pelecinus were fractionated using rapid silica filtration and solvents of increasing polarity. Fractions at final concentrations of 0, 100 or 200 μg mL−1 were added to the medium used to mature sheep cumulus–oocyte complexes (COCs) and effects were determined for maturation, subsequent cleavage rate, blastocyst rate, hatching rate, blastocyst efficiency and total blastocyst cell number (TCN). Results Fraction BP-6 at 100 μg mL−1 reduced blastocyst rate (P &lt; 0.05), but had no effect when the dose was doubled to 200 μg mL−1. Further fractionation using semi-preparative high-performance liquid chromatography showed loliolide as the most abundant compound in BP-6. Supplementation of the in vitro maturation medium with loliolide (0, 2.5, 5, 10 and 25 μg mL−1) did not affect any measure of embryo development. All COCs treated with B. pelecinus fractions reached the final stage of embryo development, blastocyst hatching. Total blastocyst cell number was not affected. Conclusion The presence of fractions of B. pelecinus extract during in vitro oocyte maturation can reduce embryo development. Implications In vitro techniques can detect potential effects of forages on reproduction. Some fractions from an extract of B. pelecinus when present during oocyte maturation can reduce embryo development. The abundant PSC, loliolide, was not responsible. There was no indication that a PSC in B. pelecinus improves outcomes.

Fertilization and developmental competence of in vitro fertilized embryos from C57BL/6J mice of different ages and the impact of vitrification.

Prepubertal animals are often preferred as sources of oocytes for assisted reproductive technologies (ARTs) in laboratory mice, but the normality and developmental competence of these oocytes remain controversial. This study systematically examined in vitro fertilization competence, embryo development, and fetal development after embryo transfer (ET) using oocytes from C57BL/6J mice aged 3 to 10 weeks. Oocytes were collected from superovulated females, fertilized, and cultured in vitro for 96 h or transferred at 2-cell stage to recipient females. Additionally, fetal development was compared between unfrozen and frozen-thawed in vitro-fertilized 2-cell embryos across different age groups. The number of ovulated oocytes per animal decreased with age, while the percentage of morphologically normal oocytes was highest in 3-week-old mice (99%) compared to older ages (70-86%, P < 0.05). Although fertilization percentages were consistently high (≥ 97%), blastocyst development in vitro, the nuclear counts of blastocysts and fetal development after ET were lowest for embryos from 3-week-old mice. Development of frozen-thawed embryos to fetuses was significantly reduced compared to unfrozen embryos in all age groups, except for those from 10-week-old mice. These findings suggest that oocytes from prepubertal mice, particularly from 3-week-old mice, are less developmentally competent than those from older mice. Therefore, the age of animals for oocyte source should be carefully considered based on the specific requirements of the research or ART applications.

Time will tell: time-lapse technology and artificial intelligence to set time cut-offs indicating embryo incompetence.

Can more reliable time cut-offs of embryo developmental incompetence be generated by combining time-lapse technology (TLT), artificial intelligence, and preimplantation genetics screening for aneuploidy (PGT-A)? Embryo developmental incompetence can be better predicted by time cut-offs at multiple developmental stages and for different ranges of maternal age. TLT is instrumental for the continual and undisturbed observation of embryo development. It has produced morphokinetic algorithms aimed at selecting embryos able to generate a viable pregnancy, however, such efforts have had limited success. Regardless, the potential of this technology for improving multiple aspects of the IVF process remains considerable. Specifically, TLT could be harnessed to discriminate developmentally incompetent embryos: i.e. those unable to develop to the blastocyst stage or affected by full-chromosome meiotic aneuploidies. If proven valuable, this application would prevent the non-productive use of such embryos, thereby improving laboratory and clinical efficiency and reducing patient stress and costs due to unnecessary embryo transfer and cryopreservation. The training dataset involved embryos of PGT-A cycles cultured in Embryoscope with a single media (836 euploid and 1179 aneuploid blastocysts and 1874 arrested embryos; 2013-2020). Selection criteria were ejaculated sperm, own (not donated) fresh oocytes, trophectoderm biopsy and comprehensive-chromosome-testing to diagnose uniform aneuploidies. Out-of-sample (30% of training), internal (299 euploid and 490 aneuploid blastocysts and 680 arrested embryos; 2021-2022) and external (97 euploid, 110 aneuploid and 603 untested blastocysts and 514 arrested embryos, 2018 to early 2022) validations were conducted. A training dataset (70%) was used to define thresholds. Several models were generated by fitting outcomes to each timing (tPNa-t8) and maternal age. ROC curves pinpointed in-sample classification values associated with 95%, 99% and 99.99% true-positive rate for predicting incompetence. These values were integrated with upper limits of maternal age ranges (<35, 35-37, 38-40, 41-42, and >42 years) in logit functions to identify time cut-offs, whose accuracy was tested on the validation datasets through confusion matrices. For developmental (in)competence, the best performing (i) tPNa cut-offs were 27.8 hpi (error-rate: 0/743), 32.6 hpi (error rate: 0/934), 26.8 hpi (error rate: 0/1178), 22.9 hpi (error-rate: 1/654, 0.1%) and 17.2 hpi (error rate: 4/423, 0.9%) in the <35, 35-37, 38-40, 41-42, and >42 years groups, respectively; (ii) tPNf cut-offs were 36.7 hpi (error rate: 0/738), 47.9 hpi (error rate: 0/921), 45.6 hpi (error rate: 1/1156, 0.1%), 44.1 hpi (error rate: 0/647) and 41.8 hpi (error rate: 0/417); (iii) t2 cut-offs were 50.9 hpi (error rate: 0/724), 49 hpi (error rate: 0/915), 47.1 hpi (error rate: 0/1146), 45.8 hpi (error rate: 0/636) and 43.9 hpi (error rate: 0/416); (iv) t4 cut-offs were 66.9 hpi (error rate: 0/683), 80.7 hpi (error rate: 0/836), 77.1 hpi (error rate: 0/1063), 74.7 hpi (error rate: 0/590) and 71.2 hpi (error rate: 0/389); and (v) t8 cut-offs were 118.1 hpi (error rate: 0/619), 110.6 hpi (error rate: 0/772), 140 hpi (error rate: 0/969), 135 hpi (error rate: 0/533) and 127.5 hpi (error rate: 0/355). tPNf and t2 showed a significant association with chromosomal (in)competence, also when adjusted for maternal age. Nevertheless, the relevant cut-offs were found to perform less well and were redundant compared with the blastocyst development cut-offs. Study limits are its retrospective design and the datasets being unbalanced towards advanced maternal age cases. The potential effects of abnormal cleavage patterns were not assessed. Larger sample sizes and external validations in other clinical settings are warranted. If confirmed by independent studies, this approach could significantly improve the efficiency of ART, by reducing the workload and patient impacts (extended culture and cleavage stage cryopreservation or transfer) associated with embryos that ultimately are developmentally incompetent and should not be considered for treatment. Pending validation, these data might be applied also in static embryo observation settings. This study was supported by the participating institutions. The authors have no conflicts of interest to declare. N/A.

Boric acid supplementation promotes the development of in vitro-produced mouse embryos by related pluripotent and antioxidant genes.

Boric acid (BA) is an important mineral for plants, animals and humans that assists metabolic function and has both positive and negative effects on biological systems. The present study aimed to investigate the effects of different concentrations of BA added to the culture media, the quality and in vitro development potential of mouse embryos. Superovulated C57Bl6/6j female mice were sacrificed ∼18 hours after human chorionic gonadotropin (hCG) injection. Single-cell-stage embryos were collected from the oviduct, divided into experiment groups and cultured in embryo medium with supplemented BA+ in 5% CO2 at 37 °C until 96 hours at the blastocyst stage. The blastocyst development rates of 0, 1.62 × 10-1, 1.62 × 10-2, 1.62 × 10-3 and 1.62 × 10-4 µM BA were 51.52%, 73.47%, 77.36% and 81.13%, respectively. The in vitro development rates were significantly higher in the 1.62 × 10-3 (p < 0.05) and 1.62 × 10-4 µM BA groups than in the control group (p < 0.001). These results indicated that low BA doses influenced embryo development by positively affecting in vitro development rates, embryo cell numbers, biochemical parameters and development at the molecular level by pluripotent and antioxidant genes. Therefore, BA seems to play an important role on in vitro embryo development.

Lysate of bovine adipose-derived stem cells accelerates in-vitro development and increases cryotolerance through reduced content of lipid in the in vitro fertilized embryos

Mesenchymal stem cells such as adipose-derived stem cells (ADSCs) are known to secrete factors that stimulate cell division and promote regeneration in neighboring cells. While conditioned medium from stem cells has been used in blastocyst production, no studies have examined the use of cell lysates. In this study we investigated the effects of adding ADSC lysate to in vitro culture (IVC) medium. ADSCs and fibroblasts were isolated from bovine adipose tissue and auricular tissue, respectively, and their lysates were prepared by freeze-thaw disruption. ADSC lysate was added to synthetic oviductal fluid medium. The effects on cleavage, blastocyst development rates, cell numbers, cryotolerance, gene expression (POU5F1, BAX, IGF1R, IGF2R, SOD2), lipid content, and membrane integrity were evaluated according to the experimental design. In Expt. 1, the comparison involved adding ADSC or fibroblast lysate alongside the control group. The total blastocyst rate increased when ADSC lysate was introduced (ADSCs: 40.1 %, fibroblasts: 33.1 %, control: 27.3 %). However, there were no significant differences in the number of trophoblast cells or in the inner cell mass. Experiment 2 confirmed that this increase in blastocyst development was not due to the solvent, PBS(−). In Expt. 3, addition of 10 % fetal calf serum (FCS) or ADSC lysate increased the total blastocyst rate compared to the control (control, 26.2 %; 10 % FCS, 43.4 %; 1 % ADSC lysate, 34.2 %; 10 % ADSC lysate, 48.1 %). After freezing and thawing, the survival and hatching rates of embryos with FCS were significantly lower than those of the control as well as those with added ADSC lysate. In Expt. 4, the addition of ADSC lysate or FCS had no significant effect on gene expression in blastocysts compared to control. However, the addition of FCS significantly increased the gray intensity, indicating higher lipid content compared to the control, with a significant increase in the number of dead cells in the blastocyst. These results indicate that the addition of ADSC lysate to the IVC medium accelerates bovine blastocyst development and that its 10 % addition, corresponding to 1 × 105 cells/mL, is as effective as 10 % FCS without a decrease in cryotolerance due to the increased lipid content.

Bta-miR-665 improves bovine blastocyst development through its influence on microtubule dynamics and apoptosis.

Extracellular vesicles (EVs) contain microRNAs (miRNAs), which are important regulators of embryonic development. Nevertheless, little is known about the precise molecular processes controlling blastocyst development and quality. In a previous study, we identified bta-miR-665 as one of the miRNAs more abundantly present in extracellular vesicles of embryo-conditioned culture media of blastocysts compared to degenerate ones. Here, we investigated the effect and regulatory roles of bta-miR-665 in blastocyst development by supplementation of bta-miR-665 mimics or inhibitors to the culture media. Supplementation of bta-miR-665 mimics improved cleavage and blastocyst rate (P < 0.01), and blastocyst quality as indicated by increased inner cell mass rates and reduced apoptotic cell ratios (P < 0.01). Furthermore, supplementation of bta-miR-665 inhibitors had the opposite effect on these phenotypes. Low input transcriptome analysis and RT-qPCR revealed that bta-miR-665 acts on genes linked to microtubule formation and apoptosis/cell proliferation. These insights not only elucidate the important role of bta-miR-665 in embryo development, but also underscore its potential in improving reproductive efficiency in bovine embryo culture.

Developmental and Molecular Effects of C-Type Natriuretic Peptide Supplementation in In Vitro Culture of Bovine Embryos.

The use of C-type natriuretic peptide (CNP) in the interaction with the oocyte and in the temporary postponement of spontaneous meiosis resumption has already been well described. However, its action in pre-implantation developmental-stage embryos is yet to be understood. Thus, our study aimed to detect the presence of the canonical CNP receptor (natriuretic peptide receptor, NPR2) in germinal vesicle (GV)-, metaphase II (MII)-, presumptive zygote (PZ)-, morula (MO)-, and blastocyst (BL)-stage embryos and, later, to observe possible modulations on the embryos when co-cultured with CNP. In Experiment I, we detected and quantified NPR2 on the abovementioned embryo stages. Further, in Experiment II, we intended to test different concentrations (100, 200, or 400 nM of CNP) at different times of inclusion in the in vitro culture (IVC; inclusion from the beginning, i.e., day 1, or from day 5). In Experiment III, 400 nM of CNP was used on day 1 (D1) in the IVC, which was not demonstrated to be embryotoxic, and it showed potentially promising results in the blastocyst production rate when compared to the control. Thus, we analyzed the embryonic development rates of bovine embryos (D7) and hatching kinetics (D7, D8, and D9). Subsequently, morula and blastocyst were collected and evaluated for transcript abundance of their competence and quality (apoptosis, oxidative stress, proliferation, and differentiation) and lipid metabolism. Differences with probabilities less than p < 0.05, and/or fold change (FC) > 1.5, were considered significant. We demonstrate the presence of NPR2 until the blastocyst development stage, when there was a significant decrease in membrane receptors. There was no statistical difference in the production rate after co-culture with 400 nM CNP. However, when we evaluated the abundance of morula transcripts, there was an upregulated transcription in ADCY6 (p = 0.057) and downregulated transcripts in BMP15 (p = 0.013), ACAT1 (p = 0.040), and CASP3 (p = 0.082). In addition, there was a total of 12 transcriptions in morula that presented variation FC > 1.5. In blastocysts, the treatment with CNP induced upregulation in BID, CASP3, SOX2, and HSPA5 transcripts and downregulation in BDNF, NLRP5, ELOVL1, ELOVL4, IGFBP4, and FDX1 transcripts (FC > 1.5). Thus, our study identified and quantified the presence of NPR2 in bovine pre-implantation embryos. Furthermore, 400 nM of CNP in IVC, a concentration not previously described in the literature, modulated some transcripts related to embryonic metabolism, and this was not embryotoxic morphologically.

Effect of refreezing extender on stallion sperm quality and embryo production after intracytoplasmic sperm injection

Intracytoplasmic sperm injection (ICSI) is a valuable assisted reproduction technology in clinical practice, especially when semen availability is limited. Since the number of sperm required per ICSI cycle is much less than the number of sperm available in a standard straw of frozen semen, refreezing semen at lower sperm concentrations could yield multiple straws for ICSI use. However, there is little data on the effect of sperm refreezing on ICSI outcomes, especially on the effect of extender used for refreezing. The objective of the present study was to evaluate the effect of refreezing extender on stallion sperm quality and embryo production after ICSI. Semen was frozen in a low egg yolk/glycerol/amide extender (Extender 1), then thawed, re-extended, and refrozen in each of three extenders. When sperm were refrozen in Extender 1, the cleavage rate was lower (P < 0.05) and the blastocyst development rate tended to be lower (P = 0.06) than for the once-frozen sperm. In contrast, when sperm were refrozen in high egg yolk/glycerol (Extender 2) or low egg yolk/milk/glycerol (Extender 3) extenders, cleavage and blastocyst development rates did not differ significantly from those for the once-frozen semen. Notably, sperm refrozen in Extender 1, which yielded the lowest ICSI outcomes, showed the highest sperm motility and viability, demonstrating that traditional measures of sperm quality were inadequate to assess the suitability of refrozen sperm for ICSI. In a follow-up experiment conducted to evaluate the effects of Extenders 1 and 3 when used for once-frozen semen, cleavage and blastocyst rates did not differ between extenders. In conclusion, the extender used to initially freeze stallion sperm may not significantly affect ICSI outcomes; however, the extender used for refreezing can significantly affect embryo production. Refrozen stallion semen can be effectively used for ICSI when low egg yolk/milk/glycerol extender is used for refreezing. Until further research is available, use of extenders without amides is recommended when refreezing stallion semen for ICSI.

In vitro maturation of bovine oocytes: the role of ROS, oxidative stress, and the impact of natural antioxidant supplementation

According to available data in the literature, Brazil has one of the largest cattle herds in the world and is one of the leading embryo producers, with a clear predominance of embryos produced in vitro. These data influence the ongoing pursuit of optimizing the results obtained in IVEP (in vitro embryo production). In this technique, in vitro maturation (IVM) is such a crucial stage that it justifies studies involving components that, when added to the media, can lead to improved cleavage rates and blastocyst development. It is well known that oocyte quality is influenced by oxidative stress, and in this context, the inclusion of antioxidants in the IVM medium may enhance these outcomes. Thus, the present study aimed to review oocyte maturation, the impact of oxidative stress, and the addition of antioxidants, such as Moringa oleifera, to the IVM media.