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Insemination of recipient sows improves the survival to term of vitrified and warmed porcine expanded blastocysts transferred non-surgically.

ABSTRACT: This study was performed to evaluate reproductive performance after non-surgical embryo transfer (Ns-ET) of 10-15 porcine expanded blastocysts (ExBs) that had been vitrified and warmed (V/W) using the micro volume air cooling (MVAC) method. The effect of asynchrony between the donor and recipient estrous cycle was investigated. Ns-ET was conducted in recipients whose estrous cycle was asynchronous to that of donors by a delay of 2, 1, or 0 days. In the 2-day and 1-day groups, the similar farrowing rates (27.3% and 25.0%) and survival rates to term (13.9% and 15.7%) were obtained after Ns-ET of V/W ExBs. None of the recipients in 0-day group farrowed. Artificial insemination (AI) prior to Ns-ET was then evaluated. Ten-15 V/W ExBs were transferred non-surgically to 12 recipients whose estrous cycles were asynchronous to that of donors by a 2-day delay. All of the recipients produced piglets, and all (100.0%) delivered piglets were derived from the transferred V/W ExBs. The survival rate of V/W ExBs to term was 25.2%. These results demonstrate that Ns-ET of V/W ExBs using MVAC can facilitate piglet production, even if 10-15 embryos are transferred. Moreover, piglets were obtained stably when AI was performed prior to Ns-ET.

SUBMITTER: Tajima S

PROVIDER: S-EPMC7539913 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Insemination of recipient sows improves the survival to term of vitrified and warmed porcine expanded blastocysts transferred non-surgically.

Tajima Shigeyuki S Uchikura Kenzo K Kurita Takayuki T Kikuchi Kazuhiro K

Animal science journal = Nihon chikusan Gakkaiho 20200101 1

This study was performed to evaluate reproductive performance after non-surgical embryo transfer (Ns-ET) of 10-15 porcine expanded blastocysts (ExBs) that had been vitrified and warmed (V/W) using the micro volume air cooling (MVAC) method. The effect of asynchrony between the donor and recipient estrous cycle was investigated. Ns-ET was conducted in recipients whose estrous cycle was asynchronous to that of donors by a delay of 2, 1, or 0 days. In the 2-day and 1-day groups, the similar farrowi ...[more]

PMID: 32926526

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Project description:Blastocyst vitrification has significantly improved embryo transfer methods, leading to higher implantation success rates and better pregnancy outcomes in subsequent frozen embryo transfer cycles. This study aimed to simulate the transcriptional changes caused by vitrifying human blastocysts using mouse blastocysts as a model and to further investigate these changes' effects. Utilizing a human vitrification protocol, we implanted both vitrified and fresh embryos into mice. We observed the implantation success rates and performed transcriptomic analysis on the blastocysts. To validate the results from messenger RNA sequencing, we conducted reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) to measure the expression levels of specific genes. Based on mRNA profiling, we predicted the microRNAs responsible for the regulation and used qPCR basic microRNA assays for validation. Our observations revealed a higher implantation success rate for vitrified embryos than fresh embryos. Transcriptomic analysis showed that vitrified-warmed blastocysts exhibited differentially expressed genes (DEGs) primarily associated with thermogenesis, chemical carcinogenesis-reactive oxygen species, oxidative phosphorylation, immune response, and MAPK-related signaling pathways. RT-qPCR confirmed increased expression of genes such as Cdk6 and Nfat2, and decreased expression of genes such as Dkk3 and Mapk10. Additionally, gene-microRNA interaction predictions and microRNA expression analysis identified twelve microRNAs with expression patterns consistent with the predicted results, suggesting potential roles in uterine epithelial cell adhesion, trophectoderm development, invasive capacity, and immune responses. Our findings suggest that vitrification induces transcriptomic changes in mouse blastocysts, and even small changes in gene expression can enhance implantation success. These results highlight the importance of understanding the molecular mechanisms underlying vitrification to optimize embryo transfer techniques and improve pregnancy outcomes.

Project description:IntroductionRecent data suggest a higher clinical pregnancy rate performing assisted hatching (AH) on previously cryopreserved embryos but fail to demonstrate significant effects on live birth rate. However, current evidence is based on studies with a small sample size and may hide a type II error. Moreover, poor attention has been given to the specific effect of AH on frozen/thawed blastocysts. To shed light on this topic, we developed the present protocol for a randomised trial to investigate the benefits of the laser-mediated partial removal of the zona pellucida in vitrified/warmed blastocysts.Methods and analysisThe pArtiaL zonA pelluciDa removal by assisteD hatchINg of blastocysts (ALADDIN) study is a multicentric prospective comparative study with a parallel randomised controlled design aiming to investigate whether AH performed on warmed blastocysts before embryo transfer can improve live birth rate. Women allocated to the control group will undergo embryo transfer of blastocysts not previously subjected to AH. Two infertility units will be involved in the study. Enrolment of patients will last 18 months with quarterly monitoring and the entire study is foreseen to be closed in 36 months. Secondary outcomes include: proportion of transferred blastocysts/thawed blastocyst, morphological features of blastocysts before embryo transfer, implantation, biochemical pregnancy, clinical pregnancy (ultrasound visible gestational sac), miscarriage, multiple pregnancy, preterm birth (<37 weeks of gestation), obstetrical and neonatal complications and congenital anomaly rates.Ethics and disseminationThis protocol received a favourable ethical opinion from the Ethical Committee of IRCCS San Raffaele Scientific Institute and the Ethical Committee Area 2 Milan. Each participant will provide written consent to participate and remain encoded during the study. The trial results will be published in peer-reviewed journals and presented at conferences.Trial registration numberNCT03623659; Pre-results.

Project description:BackgroundCryopreservation of embryos plays a major role in the in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) treatment. However, the storage condition of the cryopreserved embryo can change temporarily due to repeated retrieval of the embryo from the liquid nitrogen (LN2) tank during the practical application during cryopreservation. Whether the implantation potential of a cryopreserved embryo will be damaged when the cane containing it is temporarily exposed to air due to the transfer between the LN2 tank and LN2 container is yet to be elucidated. Also, whether the exposed-to-air frequency (EAF) of cryopreserved embryos influences the clinical outcomes is unclear.ObjectiveTo investigate whether the EAF of cryopreserved embryo affects the clinical outcomes of vitrified-warmed embryo transfer.MethodsA total of 9200 vitrified-warmed embryo transfer cycles were included in this study. All cycles were divided into five groups according to different EAFs (2, 4, 6, 8, or ≥ 10). Post-warming survival rates and clinical outcomes, including implantation, clinical pregnancy and live birth rates were investigated. Kruskal-Wallis test and Pearson's chi-squared tests were used to compare the patient characteristics and clinical outcomes among the five groups. Furthermore, multivariate logistic regression analyses were conducted to investigate the association between EAF and clinical outcomes.ResultsNo significant differences were observed in the positive HCG rate, implantation rate and live birth rate (P > 0.05) among five EAF groups with respect to D3 embryo, D5 blastocyst and D6 blastocyst. Post-warmed survival rate of D3 embryos (P = 0.015) differed significantly among the five EAF groups, but it was not EAF-dependent. Although clinical pregnancy was different among the five groups with respect to D5 blastocyst (P = 0.042), multivariate logistic regression analysis adjusted for confounding variables suggested that EAF did not adversely affect clinical pregnancy or live birth.ConclusionThese findings indicated that human vitrified embryos in the open system could be repeatedly retrieved from the LN2 tank without affecting the implantation potential of the embryo.

Project description:BackgroundHuman embryos express the prolactin (PRL) receptor at the morula and blastocyst stages. Treatment with PRL from cleavage to the blastocyst stage improves blastocyst outgrowth on fibronectin-coated dishes. However, whether post-warming PRL treatment of blastocysts cultured without PRL could improve outgrowth competence remains unknown. Furthermore, the optimal time for post-warming PRL treatment remains to be ascertained. This study investigated the effects of PRL treatment during recovery culture on human blastocyst outgrowth and its related genes.MethodsIn total, 374 discarded vitrified blastocysts were randomly allocated to two groups, to be cultured with (n = 208) or without PRL (control; n = 166) for 120 min for recovery, and then plated on fibronectin-coated dishes. The expression level of PRL-interacting genes, blastocyst adhesion rate, outgrowth area, distance of trophoblast migration, and outgrowth degeneration were examined.ResultsThe mRNA expression of ezrin, radixin, and moesin, which regulate cell adhesion and invasion by controlling actin reorganization during epithelial-to-mesenchymal transition (EMT), was stimulated by PRL treatment for 120 min. The expression of EMT-related genes, transforming growth factor β1, snail1, and twist1 was also promoted following treatment with PRL for 120 min. PRL-treated blastocysts also exhibited augmented expression of cadherin 2 and transcriptional repression of cadherin 1. Higher mRNA expression of integrin-based focal adhesion-related genes, ITGA5 and ITGB1, was observed after treatment with PRL for 120 min than in the non- and shorter-treatment groups. PRL treatment for 120 min did not alter the rate of blastocyst adhesion to fibronectin-coated dishes 96 h after the outgrowth culture assay. However, multiple linear regression analysis revealed that the outgrowth area was significantly increased in PRL-treated blastocysts. The migration distance of trophoblast cells was significantly increased and degeneration rate was significantly decreased after PRL treatment. Furthermore, a more beneficial effect of PRL treatment on blastocyst outgrowth was observed when the blastocysts were vitrified on day 5 than when they were vitrified on day 6.ConclusionsPost-warming culture of human vitrified blastocysts with PRL for 120 min promoted trophoblast outgrowth in vitrified human blastocysts. Furthermore, PRL treatment may reduce outgrowth degeneration by increasing resistance to apoptosis during trophoblast migration.

Project description:BackgroundWe had previously demonstrated that vitrification reduces the levels of certain phospholipid classes, and that oocytes from aged mice show a similar lipidome alteration, even without vitrification. In the current investigation, we examined if vitrification-warming of mouse oocytes from young and aged mice causes any changes in molecular aspects of lipid-associated features.MethodsMetaphase II (MII) stage oocytes were harvested from young (10-14-week-old) and aged (45-54-week-old) mice by a superovulation regime with PMSG followed by hCG. We examined the status of the intracellular lipid pool and the integrity of the plasma membrane by staining oocytes with BODIPY 500/510 and CellMask live dyes. Expression of lipid uptake- and necroptosis-associated genes was assessed by quantitative PCR analyses, in oocytes from young and old mice, before and after vitrification. Localization patterns of two crucial necroptosis proteins, phosphorylated MLKL (pMLKL) and phosphorylated RIPK1 (pRIPK1) were examined in mouse oocytes by immunofluorescence staining. Necrostain-1 (Nec1), an inhibitor of RIPK1, was used to examine if RIPK1 activity is required to maintain oocyte quality during vitrification.ResultsWe confirmed that vitrified-warmed oocytes from aged mice showed noticeable decrease in both CellMask and BODIPY 500/510 dyes. Among the lipid uptake-associated genes, Cd36 expression was higher in oocytes from aged mice. Necroptosis is a type of programmed cell death that involves damage to the plasma membrane, eventually resulting in cell rupture. The expression of necroptosis-associated genes did not significantly differ among groups. We observed that localization patterns of pMLKL and pRIPK1 were unique in mouse oocytes, showing association with microtubule organizing centers (MTOCs) and spindle poles. pMLKL was also localized on kinetochores of MII chromosomes. Oocytes treated with Nec1 during vitrification showed a decreased survival rate, indicating the importance of RIPK1 activity in oocyte vitrification.ConclusionsWe report that oocytes from aged mice show differential expression of CD36, which suggests that CD36-mediated lipid uptake may be influenced by age. We also show for the first time that pMLKL and pRIPK1 exhibit unique localization pattern in mouse oocytes and this may suggest role(s) for these factors in non-necroptosis-associated cellular processes.

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Insemination of recipient sows improves the survival to term of vitrified and warmed porcine expanded blastocysts transferred non-surgically.

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Insemination of recipient sows improves the survival to term of vitrified and warmed porcine expanded blastocysts transferred non-surgically.

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