Prospective-randomized study comparing clinical outcomes of IVF treatments where embryos were cultured individually or in a microwell group culture dish.

Culturing embryos together in a microdrop of media may improve embryo quality, based on the results of animal studies, however individual identification of the embryos in such a system is not possible. The microwell group culture dish contains 9 or 16 microwells with a minimal well-to-well distance and a specific well morphology that facilitates paracrine and autocrine effects. The microwell group culture dish enables individual identification of the embryos while providing the environment that comes with similar benefits as group culture. Our aim was to investigate whether embryo culture in the microwell group culture dish (Primo Vision Dish, Vitrolife) improves IVF outcomes compared to individual culture in human IVF treatment. Five hundred thirty-two IVF-ET cycles were enrolled in this prospective randomized study in a university hospital. IVF cycles were randomized into microwell group culture and individual culture groups. Primary outcome measure was clinical pregnancy rate and secondary outcome measures were embryo quality, fertilization, implantation, delivery and embryo utilization rates. Fertilization rate in ICSI cycles was significantly higher in the microwell group culture group (70.6% vs. 64.9%, P = 0.001). Clinical pregnancy rate was 50.8% in the group culture and 40.6% in the individual culture (P = 0.022). Live birth rate was 41.5% in microwell and 32.9% in individual culture (P = 0.0496). Embryo utilization rate was higher in microwell group culture than in individual culture (80.6% vs. 75.0%; P < 0.001). Microwell group culture has a beneficial effect on IVF outcome and it also allows following up individual embryo development.ClinicalTrials.gov: NCT01774006.

Non-invasive embryo evaluation and selection using time-lapse monitoring: Results of a randomized controlled study.

OBJECTIVE: Continuous monitoring of embryos via time-lapse (TL) provides more information on embryo kinetics and morphology compared to standard daily evaluation. Embryo selection by TL could support single embryo transfer (SET). With SET multiple gestations are avoided and perinatal outcome is improved. Our primary goal was to determine whether selection of a single blastocyst based on an algorithm comprising kinetic and morphologic scores assessed through continuous TL monitoring results in superior clinical outcome compared to embryo selection based on morphology alone. A secondary goal was to assess whether a time-lapse score based on kinetic and morphologic parameters was predictive of implantation. STUDY DESIGN: Randomized controlled trial performed in two private IVF centers in Hungary. Infertile couples scheduled to undergo 1st or 2nd IVF cycles were enrolled. Female age had to be under 36 years. The intervention was embryo evaluation/selection based on TL algorithm. Patients were randomized to SET with TL monitoring (TL-eSET) vs. SET with standard evaluation (control-eSET). Assuming an increase in pregnancy from 44% to 58%, a sample size of 202 per group was calculated based on the interim analysis at 10% information fraction. The primary outcome of the study was pregnancy rate. Secondary outcomes were miscarriage rates, live birth, perinatal outcome and the ability of a time-lapse score constructed based on kinetic and morphologic parameters to predict implantation. Chi-square tests, likelihood-ratio tests and exact tests were used for the analysis of categorical variables. Continuous variables were compared using independent group t-test and analysis of variance. RESULTS: The study was closed after three years. Eventually 161 patients were randomized and analyzed (N = 80 TL-eSET and N = 81 control-eSET). Pregnancy rate did not significantly differ between the groups though there was a trend favoring TL selection (TL-eSET: 46.3% vs control-eSET: 34.6%, p = 0.150; OR: 1.628 (95% CI: 0.857-3.092)). The time-lapse score based on morphologic and kinetic parameters was significantly higher for blastocysts that implanted vs. those that did not (14.5 ± 1.8 vs. 12.1 ± 2.9, p = 0.0001). There were no adverse effects of the intervention. CONCLUSIONS: Selection of a single blastocyst based on information derived from time-lapse monitoring can aid embryo selection for SET.

Investigation of hydrostatic pressure-induced stress preconditioning of boar semen using modified cryopreservation.

The aim of this study was to investigate the effect of varying hydrostatic pressure treatments (HP) on the boar semen quality during the modified cryopreservation. In Experiment I, combinations of pressure level (20/40/80 MPa) and duration of application (40/80/120 min) were used. Before freezing, only the magnitude but not the duration influenced the total (TM%) and progressive motilities (PM%). The 20/40 MPa levels yielded a significant (p < 0.05) improvement compared to control samples (atmospheric), but the 80 MPa was detrimental. The post-freezing-thawing (FT) motilities were influenced significantly by both the HP level and its duration. For TM%, the 40 MPa:120 min gave the highest post-FT result (54.8% ± 3.3%); however, the 40 MPa:80 min (41.0% ± 3.1%) application showed the largest and significant improvement (18.4% ± 3.1%) compared to its control (22.6% ± 3.1%) and compared to the improvement (12.9% ± 3.6%) achieved by 40 MPa:120 min. For PM%, the improvement with the 40 MPa:120 min application was slightly larger than with the 40 MPa:80 min one (15.2% ± 4.2% vs. 13.8% ± 3.3%); furthermore, the difference was not significant. In Experiment II, the 40 MPa:80 min combination was tested at four different stages of the semen handling. By pressurization after dilution with the freezing extender without glycerol, significantly higher post-FT values (TM%, intact acrosome% and head membrane%) were obtained. The two experiments demonstrated possible improvement in post-FT semen quality achievable through the appropriate application of HP to boar semen during cryopreservation.

Time-lapse culture with morphokinetic embryo selection improves pregnancy and live birth chances and reduces early pregnancy loss: a meta-analysis.

Embryo evaluation and selection is fundamental in clinical IVF. Time-lapse follow-up of embryo development comprises undisturbed culture and the application of the visual information to support embryo evaluation. A meta-analysis of randomized controlled trials was carried out to study whether time-lapse monitoring with the prospective use of a morphokinetic algorithm for selection of embryos improves overall clinical outcome (pregnancy, early pregnancy loss, stillbirth and live birth rate) compared with embryo selection based on single time-point morphology in IVF cycles. The meta-analysis of five randomized controlled trials (n = 1637) showed that the application of time-lapse monitoring was associated with a significantly higher ongoing clinical pregnancy rate (51.0% versus 39.9%), with a pooled odds ratio of 1.542 (P < 0.001), significantly lower early pregnancy loss (15.3% versus 21.3%; OR: 0.662; P = 0.019) and a significantly increased live birth rate (44.2% versus 31.3%; OR 1.668; P = 0.009). Difference in stillbirth was not significant between groups (4.7% versus 2.4%). Quality of the evidence was moderate to low owing to inconsistencies across the studies. Selective application and variability were also limitations. Although time-lapse is shown to significantly improve overall clinical outcome, further high-quality evidence is needed before universal conclusions can be drawn.

Stimulus-triggered enhancement of chilling tolerance in zebrafish embryos.

BACKGROUND: Cryopreservation of zebrafish embryos is still an unsolved problem despite market demand and massive efforts to preserve genetic variation among numerous existing lines. Chilled storage of embryos might be a step towards developing successful cryopreservation, but no methods to date have worked. METHODS: In the present study, we applied a novel strategy to improve the chilling tolerance of zebrafish embryos by introducing a preconditioning hydrostatic pressure treatment to the embryos. In our experiments, 26-somites and Prim-5 stage zebrafish embryos were chilled at 0°C for 24 hours after preconditioning. Embryo survival rate, ability to reach maturation and fertilizing capacity were tested. RESULTS: Our results indicate that applied preconditioning technology made it possible for the chilled embryos to develop normally until maturity, and to produce healthy offspring as normal, thus passing on their genetic material successfully. Treated embryos had a significantly higher survival and better developmental rate, moreover the treated group had a higher ratio of normal morphology during continued development. While all controls from chilled embryos died by 30 day-post-fertilization, the treated group reached maturity (~90-120 days) and were able to reproduce, resulting in offspring in expected quantity and quality. CONCLUSIONS: Based on our results, we conclude that the preconditioning technology represents a significant improvement in zebrafish embryo chilling tolerance, thus enabling a long-time survival. Furthermore, as embryonic development is arrested during chilled storage this technology also provides a solution to synchronize or delay the development.

Effects of High Hydrostatic Pressure on Expression Profiles of In Vitro Produced Vitrified Bovine Blastocysts.

High hydrostatic pressure (HHP) has been used to pre-condition embryos before essential, yet potentially detrimental procedures such as cryopreservation. However, the mechanisms for HHP are poorly understood. We treated bovine blastocysts with three different HHP (40, 60 and 80 MPa) in combination with three recovery periods (0, 1 h, 2 h post HHP). Re-expansion rates were significantly higher at 40 and 60 but lower at 80 MPa after vitrification-warming in the treated groups than controls. Microarray analysis revealed 399 differentially expressed transcripts, representing 254 unique genes, among different groups. Gene ontology analysis indicated that HHP at 40 and 60 MPa promoted embryo competence through down-regulation of genes in cell death and apoptosis, and up-regulation of genes in RNA processing, cellular growth and proliferation. In contrast, 80 MPa up-regulated genes in apoptosis, and down-regulated protein folding and cell cycle-related genes. Moreover, gene expression was also influenced by the length of the recovery time after HHP. The significantly over-represented categories were apoptosis and cell death in the 1 h group, and protein folding, response to unfolded protein and cell cycle in the 2 h group compared to 0 h. Taken together, HHP promotes competence of vitrified bovine blastocysts through modest transcriptional changes.

Effects of high hydrostatic pressure on genomic expression profiling of porcine parthenogenetic activated and cloned embryos.

Handmade cloning (HMC) has been used to generate transgenic pigs for biomedical research. Recently, we found that parthenogenetic activation (PA) of porcine oocytes and improved HMC efficiency could be achieved by treatment with sublethal high hydrostatic pressure (HHP). However, the molecular mechanism underlying the effects of HHP treatment on embryonic development is poorly understood and so was investigated in the present study. Thus, in the present study, we undertook genome-wide gene expression analysis in HHP-treated and untreated oocytes, as well as in 4-cell and blastocyst stage embryos derived by PA or HMC. Hierarchical clustering depicted stage-specific genomic expression profiling. At the 4-cell and blastocyst stages, 103 and 163 transcripts were differentially expressed between the HMC and PA embryos, respectively (P<0.05). These transcripts are predominantly involved in regulating cellular differentiation, gene expression and cell-to-cell signalling. We found that 44 transcripts were altered by HHP treatment, with most exhibiting lower expression in HHP-treated oocytes. Genes involved in embryonic development were prominent among the transcripts affected by HHP. Two of these genes (INHBB and ME3) were further validated by quantitative reverse transcription-polymerase chain reaction. We also observed that HHP treatment activated expression of the imprinting gene DLX5 in 4-cell PA embryos. In conclusion, our genomic expression profiling data suggest that HHP alters the RNA constitution in porcine oocytes and affects the expression of imprinting genes during embryonic development.

Cells under pressure: how sublethal hydrostatic pressure stress treatment increases gametes' and embryos' performance.

The principal approach in in vitro embryo culture and manipulation has been a defensive one: procedures aim to satisfy passively the supposed or real physiological needs of gametes and embryos. Similarly, during cryopreservation the aim is to cause minimal damage to cells whilst attempting to obtain the highest achievable cell survival. However, carefully chosen and precisely controlled sublethal stress treatment of cells has been described to improve embryos' and gametes' performance, and, as a consequence, subsequent morphological survival, fertilisation, in vitro development, pregnancy and farrowing rates improved considerably compared with untreated controls. This review summarises studies that open up a new approach: instead of - and besides - trying to passively reduce the harm to cells during in vitro manipulations and culture, procedures may also prepare the cells themselves to ward off or reduce the damage by turning up the cells' own, inner capacities.

Stress tolerance and transcriptional response in mouse embryos treated with high hydrostatic pressure to enhance cryotolerance.

Sublethal high hydrostatic pressure (HHP) treatment of cells was reported to enhance stress tolerance and to increase post-thawing survival after cryopreservation in mouse, swine and cattle. The goal of this study was to define if HHP stress tolerance depends on the embryos' stage of development and culture conditions, to describe long term in vivo effects and transcriptional alterations of selected stress related genes. Studies showed that impacts greater than 60 MPa caused blastomere and membrane injuries to the two-cell stage embryos, while even 80 MPa was well tolerated by blastocysts. HHP treatment caused significant upregulation of Azin1, Sod2 and Gadd45g genes, detected by RT-qPCR. The transfer of HHP treated blastocysts revealed normal in vivo development and reproductive function in a two generation study. The cell type and the embryos' development stage shall be taken into account when optimizing sublethal HHP stress treatment protocol of different cells.

High hydrostatic pressure treatment of porcine oocytes induces parthenogenetic activation.

An innovative technique called high hydrostatic pressure (HHP) treatment has recently been reported to improve the cryosurvival of gametes and embryos in certain mammalian species, including the mouse, pig, and cattle. In the present study the parthenogenetic activation (PA) of pig oocytes caused by HHP treatment was investigated in different holding media with or without Ca(2+). The efficiency of activation was tested at different pressure levels and media including T2 (HEPES-buffered TCM-199 containing 2% cattle serum), and mannitol-PVA fusion medium with (MPVA + Ca(2+)) or without Ca(2+) and Mg(2+)(MPVA). The results showed that HHP cannot induce PA in T2, but only in MPVA + Ca(2+) with low Ca(2+) concentration and MPVA without Ca(2+). The highest activation efficiency was achieved with 10 min HHP treatment using 100 or 200 bars for oocytes in MPVA + Ca(2+) or MPVA, respectively. In the light of these results, the possible source of Ca(2+) during activation was investigated. It was found that even after a total of 30-min wash with TL-HEPES-PVA buffer without Ca(2+) before HHP treatment in MPVA, the oocytes could still be activated, indicating the possibility of an intracellular Ca(2+) source caused cytoplasmic free Ca(2+) elevation. In conclusion, parthenogenetic activation could be induced by HHP in certain holding media with low or zero Ca(2+) content. Further experiments are needed to identify the exact mechanisms of activation.

Pregnancy achieved by transfer of a single blastocyst selected by time-lapse monitoring.

Appropriate selection of a single blastocyst for transfer decreases the risk of multiple gestations. By using a compact time-lapse microscope system placed inside a regular incubator, combined with a microwell embryo culture dish, the development of all the embryos from a patient was continuously monitored by obtaining images at 10 min intervals. The embryos were not moved during the time-lapse observation. The system was switched off completely between image acquisitions in order to avoid exposure to electromagnetic radiation. The analysis of time-lapse records was used to choose a single blastocyst for transfer, which resulted in a singleton pregnancy and birth of a healthy boy on term.

Stress for stress tolerance? A fundamentally new approach in mammalian embryology.

In vitro culture, storage, and manipulation of gametes and embryos require meticulously adjusted conditions to avoid or minimize the harmful effects of uncontrolled stress. However, recent work indicates that a well-defined and properly applied stress may induce general adaptation and increase tolerance to various in vitro procedures. The aim of this review is to summarize reports on the effects of stress on gametes and embryos of several species. Treatment with sublethal doses of high hydrostatic pressure (HHP), or osmotic, heat, or oxidative stress resulted in increased morphological survival, fertilizing ability, or developmental potential after various in vitro or in vivo procedures. HHP treatment of spermatozoa, oocytes, embryos, and embryonic stem cells increased fertilizing ability, developmental competence, and differentiation and improved results after cryopreservation, parthenogenetic activation, intracytoplasmic sperm injection, and somatic cell nuclear transfer. Osmotic stress of oocytes resulted in higher developmental rates after cryopreservation, parthenogenetic activation, and somatic cell nuclear transfer. Heat shock was reported to increase developmental competence of parthenogenetically activated oocytes. Although cellular and subcellular mechanisms supposedly contributing to these processes require further research, the new principle, i.e., to improve the stress tolerance by a defined sublethal stress, may outline a completely new strategy in mammalian embryology, as well as cryopreservation of other cells and tissues with remarkable theoretical and practical consequences.

Prediction of in-vitro developmental competence of early cleavage-stage mouse embryos with compact time-lapse equipment.

Single blastocyst transfer is regarded as an efficient way to achieve high pregnancy rates and to avoid multiple pregnancies. Risk of cancellation of transfer due to a lack of available embryos may be reduced by early prediction of blastocyst development. Time-lapse investigation of mouse embryos shows that the time of the first and second cleavage (to the 2- and 3-cell stages, respectively) has a strong predictive value for further development in vitro, while cleavage from the 3-cell to the 4-cell stage has no predictive value. In humans, embryo fragmentation during preimplantation development has been associated with lower pregnancy rates and a higher incidence of developmental abnormalities. Analysis of time-lapse records shows that most fragmentation is reversible in the mouse and is resorbed in an average of 9 h. Daily or bi-daily microscopic checks of embryo development, applied routinely in human IVF laboratories, would fail to detect 36 or 72% of these fragmentations, respectively. Fragmentation occurring in a defined time frame has a strong predictive value for in-vitro embryo development. The practical compact system used in the present trial, based on the 'one camera per patient' principle, has eliminated the usual disadvantages of time-lapse investigations and is applicable for the routine follow-up of in-vitro embryo development.

Hydrostatic pressure pre-treatment affects the protein profile of boar sperm before and after freezing-thawing.

A sublethal environmental stress, high-hydrostatic pressure (HHP) was reported to significantly improve the motility, viability and fertility parameters of frozen bull and boar semen. However, the mechanism of how HHP treatment improves survival rates at sperm cryopreservation remains unclear. The purpose of this study was to evaluate the effect of HHP treatment of fresh boar semen on the protein profile of boar sperm before and after freezing. Fresh, extended semen of eight boars was split, one part was treated with 200, 300 or 400bar for 90min using a custom made pressuring device before the start of the semen freezing procedure, and the other part was prepared without HHP treatment. After thawing, samples were checked for motility. The effect of HHP treatment on the post-thaw motility of frozen semen was significant (P=0.02). Post-thaw motility of each treatment groups increased compared to control (46% vs. 52%, 56% and 56%; control vs. 200bar, 300bar and 400bar treatments). Samples for protein analysis were collected from the 300bar treatment group before HHP treatment at room temperature (25+/-3 degrees C), at 5 degrees C of the cooling process and after thawing with or without HHP treatment. The sperm were lysed using a urea-pyranoside-dithiothreitol buffer to extract their proteins for protein analysis. Approximately 800microg total proteins were assayed by two-dimensional gel electrophoresis and stained with colloidal Coomassie blue. The levels of 125 protein spots were quantified. The results revealed that the levels of 7 protein spots differed significantly among treatments. The identities of various protein constituents were identified by mass spectrometry and database searching. Ubiquinol-cytochrome c reductase complex core protein 1, perilipin, and carbohydrate-binding protein AWN precursor were identified as HHP response proteins being significantly higher in HHP-treated samples. Testis-specific glyceraldehyde 3-phosphate dehydrogenase, outer dense fiber of sperm tails 2 isoform 10, cytosolic 5'-nucleotidase 1B, and quinone oxidoreductase represented the cooling and freezing related proteins. The differing levels of these identified proteins could be valuable for further exploring the protective mechanism of the HHP treatment in frozen-thawed porcine sperm.

High hydrostatic pressure treatment of porcine oocytes before handmade cloning improves developmental competence and cryosurvival.

An innovative technique, called the high hydrostatic pressure (HHP) treatment, has been recently reported to improve the cryosurvival of gametes or embryos in certain mammalian species. The aim of the present study was to investigate the in vitro and in vivo developmental competence and cryotolerance of embryos produced by handmade cloning (HMC) after pressure treatment of recipient oocytes. In vitro-matured porcine oocytes were treated with a sublethal hydrostatic pressure of 20 MPa (200 times greater than atmospheric pressure) and recovered for either 1 or 2 h (HHP1 and HHP2 groups, respectively) before they were used for HMC. After 7 days of in vitro culture, blastocyst rates and mean cell numbers were determined. Randomly selected blastocysts were vitrified with the Cryotop method based on minimum volume cooling procedure. The blastocyst rate was higher in the HHP2 group than in the control group (68.2 +/- 4.1% vs. 46.4 +/- 4.2%; p < 0.01), while there was no difference between HHP1 and control group (52.1 +/- 1.2% vs. 49.0 +/- 2.7%; p > 0.05). Similar mean cell numbers of produced blastocysts were obtained in HHP2 and control groups (56 +/- 4 vs. 49 +/- 5; p > 0.05). Subsequent blastocyst vitrification with the Cryotop method resulted in significantly higher survival rate after thawing in the HHP2 group than in the control group (61.6 +/- 4.0% vs. 30.2 +/- 30.9%; p < 0.01). Fifty-six and 57 day 5 to day 7 fresh blastocysts in HHP1 group were transferred into two recipient sows on day 5 of the estrous cycle. One recipient was diagnosed pregnant and gave birth to two healthy piglets by naturally delivery on day 122 of gestation. This pilot study proved that the sublethal HHP treatment of porcine oocytes before HMC results in improved in vitro developmental competence and cryotolerance, and supports embryonic and fetal development as well as pregnancy establishment and maintenance up to the birth of healthy piglets.

Improving post-thaw survival of cryopreserved mouse blastocysts by hydrostatic pressure challenge.

The purpose of the experiments was to study the effect of high hydrostatic pressure treatment prior to vitrification to the survival of expanded mouse blastocysts. High hydrostatic pressure has been reported to induce the production of "shock proteins" in bacteria, which can provide a possibility of cross-protection to other environmental stresses. The possible beneficial effects of this alleged principle was examined on embryo vitrification. First, the behaviour of blastocysts was studied at altered pressure conditions. In the second part of the study, pressure treatment was combined with a cryopreservation protocol. Our results indicate that the survival of pressurized mouse embryos depends on the magnitude and the duration of pressure applied. We demonstrated that a preceding pressure treatment strikingly increases the survival of the frozen blastocysts as well as the speed of resumption of the development, and hatching rate.

Survival of rapidly frozen hatched mouse blastocysts.

The objective of the present study was to examine the effect of rapid freezing on the in vitro and in vivo survival of zona-pellucida-free hatched mouse blastocysts. Hatched blastocysts were rapidly frozen in a freezing medium containing either ethylene glycol (EG) or glycerol (G) in 1.5 M or 3 M concentration. Prior to freezing, embryos were equilibrated in the freezing medium for 2 min, 10 min, 20 min or 30 min at room temperature. To freeze them, embryos were held in liquid nitrogen vapour [approximately 1 cm above the surface of the liquid nitrogen (LN2)] for 2 minutes and then immersed into LN2. After thawing, embryos were transferred either to rehydration medium (DPBS + 10% foetal calf serum +0.5 M sucrose) for 10 minutes or rehydrated directly in DPBS supplemented with foetal calf serum. In vitro survival of embryos frozen with EG was higher than those frozen with G. The highest survival was obtained with 3 M EG and 2 min or 10 min equilibration prior to freezing, combined with direct rehydration after thawing. Frozen blastocysts developed into normal foetuses as well as unfrozen control ones did, with averages of 30% (control), 26% (EG) and 15% (G). The results show that hatching and hatched mouse blastocysts can be cryopreserved by a simple rapid freezing protocol in EG without significant loss of viability. Our data indicate that the mechanical protection of the zona pellucida is not needed during freezing in these stages.