P–268 Assessing the effect of media, oil and culture dishes on media osmolality and its dynamics in the culture system

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
D González-Abreu ◽  
E Mestre ◽  
M Escribá-Suárez ◽  
C Miret-Lucio ◽  
A García-Esteve ◽  
...  
Keyword(s):  
Embryo Development ◽  
Embryo Culture ◽  
Culture System ◽  
Freezing Point ◽  
Culture Media ◽  
Mineral Oil ◽  
Single Step ◽  
Oil Viscosity ◽  
Culture Dish ◽  
Low Viscosity

Abstract Study question Can lab-related variables (media type, oil viscosity, microdroplet volume and culture dish design) modulate media evaporation and improve its stability during culture? Summary answer Using dishes with pre-defined wells, big volume microdroplets and high-viscosity mineral oil can help to reduce media evaporation and improve osmolality stability during embryo culture. What is known already Osmolality measures the number of solute particles present in a solution and is an important variable of a human embryo culture system. High ambient temperature and low humidity may induce evaporation in culture media, increasing its osmolality. In addition, recent tendencies in IVF laboratories, such as extending the embryo culture uninterruptedly until day 6/7 or the use of dry benchtop incubators, may intensify evaporation. Surpassing a 300mOsm/kg threshold can result deleterious for embryo development and impair clinical results. Different strategies (e.g. oil type/volume, dish type, micro-drop volume) have been proposed to reduce evaporation and stabilize osmolality during culture. Study design, size, duration Four variables were analyzed in their capacity to reduce media evaporation: type of culture medium, micro-droplet volume, oil viscosity and type of culture dish. Dishes were prepared with 5ml of oil and 50µl microdroplets (25µl were used for the comparison of micro-droplet volumes). Dishes were cultured in parallel in a dry benchtop incubator (AD–3100, Astec), and osmolality measured daily for seven days with a freezing point depression osmometer (Osmo1®, Advanced Instruments, accuracy ≤2mOsm/kg). Participants/materials, setting, methods The following comparison groups were analyzed: 1) Seven commercial single-step media with three differing initial osmolalities (approximately 260, 280 and >290mOsm/kg); 2) oil with high, medium or low viscosity; 3) 50 vs. 25µl microdroplets; 4) 35mm flat Petri dish vs. 35mm dish with defined wells. Temperature in the incubator was monitored continuously (T+Button, BrightSentinel), as well as room temperature and humidity (Octax Log&Guard, Vitrolife). All were stable at 37.3±0.05oC, 22.1±0.6 oC and 67.4±7.4%, respectively. Main results and the role of chance Evaporation occurred in all the studied groups, but its rate was modulated by various parameters. Culture dishes designed with pre-defined wells reduced evaporation when compared to regular Petri dishes (Increase 11.3mOsm/kg and increase 12.5mOsm/kg, respectively from day 0 to 7 (P = 0.007)). Similarly, oil viscosity had an impact in osmolality stability during culture, with an increase of 14.7mOsm/kg, 16.3mOsm/kg and 19.2mOsm/kg observed when using mineral oil with high, medium and low viscosity, respectively (P = 0.009). Finally, reducing the volume of the medium microdroplets from 50 to 25µl derived in higher evaporation rates, but without significant differences (Increase 14.7mOsm/kg and increase 15.8mOsm/kg, respectively (P = 0.325)). Different evaporation rates were observed between the seven studied culture media attending their three-differing initial osmolalities. Significant differences were observed for a media respect another three media with differing initial osmolality (P = 0.001, P = 0.01 and P = 0.015). Their initial osmolality had a direct correlation with the maximum osmolality reached at the end of culture. Thus, media with a high initial osmolality (>290mOsm/kg) resulted in hyperosmotic media above the recommended 300mOsm/kg threshold by the end of culture and, by contrast, the studied media with lower initial values were able to maintain osmolality below 300mOsm/kg for the whole duration of the culture. Limitations, reasons for caution While a clear effect was observed by the studied variables, other parameters, such as oil volume or dish preparation techniques, could also play a role in osmolality maintenance and could be studied in the future. Additionally, these findings could vary between different centers and should be validated in each laboratory. Wider implications of the findings: Osmolality has been shown to have a direct impact on embryo development, embryo quality and clinical outcomes. Carefully defining the consumables and methodologies used in the IVF laboratory will improve the stability of the culture system and, consequently, reduce the stress imparted to the embryos and gametes under culture. Trial registration number Not applicable

P–197 Two different strategies for embryo culture and selection: time-lapse with single-step medium and conventional incubator with sequential media. Are there differences in clinical results?

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
C Alber. Rodriguez ◽  
M Valera ◽  
L Bori ◽  
F Meseguer ◽  
L Alegre ◽  
...  
Keyword(s):  
Logistic Regression ◽  
Embryo Development ◽  
Embryo Culture ◽  
Culture Media ◽  
Time Lapse ◽  
Clinical Results ◽  
Single Step ◽  
Ongoing Pregnancy ◽  
Media Change ◽  
Culture Strategy

Abstract Study question Is there a significant difference in the clinical results of embryos cultured in time-lapse systems with single-step medium and conventional benchtop incubators with sequential media? Summary answer Embryos cultured in time-lapse systems and single-step media are more likely to achieve an ongoing pregnancy and have higher implantation rates than those cultured otherwise. What is known already One of the strategies for embryo culture in IVF consisted in conventional benchtop incubators combined with sequential culture media (CI-Seq). New generation time-lapse systems provide useful information on the morphokinetics of embryo development, but also a stable culture environment where embryos can develop undisturbed until blastocyst stage when paired with single-step culture media (TLS-SS). These features have the potential to improve embryo development and selection. Nonetheless, there is inconclusive evidence of whether this new culture strategy has a significant effect on clinical results of ICSI treatments. Studies on the matter are heterogeneous and reduced in both number and sample size. Study design, size, duration Unicentric retrospective cohort study. We compared the results of 11471 blastocyst transferences from 10276 ICSI treatments performed during 4 consecutive years, where embryos were cultured either on CI with sequential media (N = 5255) or a TLS with single-step medium (N = 5021). 3922 of the totals were fresh embryo transfers (ET) and 7549 frozen-thawed ET. We compared the implantation rate (IR) and ongoing pregnancy rate (OGPR) in both study groups, stratifying by ovum origin. Participants/materials, setting, methods Three models of TLS were used for embryo culture: EmbryoScope, EmbryoScope Plus (Vitrolife) and GERI (Genea Biomedx), as well as one CI (ASTEC). Sequential media: Cook, Origio, Vitrolife; Single-step media: Gems, Irvine, Life Global. Embryo scoring and selection was performed by ASEBIR criteria in the CI group, and by morphological and morphokinetic assessment for embryos cultured in TLS. Embryos were extracted from the CI only for media change. Statistical analysis: ANOVA tests and Logistic regressions. Main results and the role of chance A general Logistic Regression was performed, including egg origin, PGT-A and culture strategy to explain their impact in OGPR. Egg origin (OR = 1,094 (95%CI: 1,015–1,179); P = 0,019) and culture strategy (OR = 1,141 (95%CI: 1,060–1,229); P < 0,001) were statistically significant, which confirms the need for stratification due to the heterogeneity of the groups. The total IR in the TLS-SS group was 54,68±48,84%, significantly higher than that of CI-Seq (49,18±47,91%; P < 0,001). In ovum-donation treatments, a complete Logistic Regression for OGPR, with all typical confounding variables (age, BMI, nº oocytes, fresh/frozen transfer, number and day of ET) resulted in an OR = 1,187 (95%CI: 1,074–1,313; P = 0,001) favoring culture in TL-SS. IR in these treatments were 61,98±47,68% in TL-SS vs 55,08±46,58% in CI-Seq (P < 0,001) in fresh transfers and 51,48±48,91% in TL-SS vs 44,39±47,67% in CI-Seq (P < 0,001) in frozen-thawed ET. In autologous treatments with PGT a similar regression yielded an OR = 1,055 (95%CI: 0,889–1,252; P = 0,542) for culture strategy. The IR of genetically tested ET was not significantly different: 53,08±49,49% for TL-SS, 50,90±49,07% for CI-Seq, P = 0,246. In autologous procedures without PGT, culture strategy was not significant for OGPR (OR = 0,998 (95%CI: 0,835–1,191), P = 0,979) nor IR of fresh (49,75±48,91% TL-SS vs 44,23±47,36% CI-Seq; P = 0,081) nor frozen-thawed transferences (50,77±48,33% TL-SS vs 50,67±47,33% CI-Seq; P = 0,970). Limitations, reasons for caution After fertilization check, embryos were evaluated exclusively on D5/6. On D3, embryos cultured in CI were taken out only for a quick media change, but not for evaluation, and all handling was done in isolette cabins with controlled environmental conditions. Being a retrospective study, there is high variability in population. Wider implications of the findings: A more homogenous prospective study, including comparison in life-birth rates, is necessary to extract final conclusions. However, our results suggest that the introduction of TLS and SS media in IVF laboratories might be a valid strategy to increase clinical results, especially in fresh embryo, thanks to an improved embryo selection. Trial registration number Not applicable

P–168 RCT comparing the effect of Continuous ( Single Step ) embryo culture system versus a Sequential embryo culture system on the outcome of IVF/ICSI cycles

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
R Singh ◽  
M Singh
Keyword(s):  
Embryo Culture ◽  
Culture System ◽  
Culture Media ◽  
Poor Quality ◽  
Blastocyst Stage ◽  
Single Step ◽  
Blastocyst Formation ◽  
Cleavage Stage ◽  
Media Systems ◽  
Single Medium

Abstract Study question Is the outcome of IVF/ICSI cycles done with continuous (single step ) embryo culture system different from that with sequential embryo culture system ? Summary answer Yes the outcome of IVF / ICSI cycles done with continuous (single step ) embryo-culture system is better than that with sequential embryo-culture system . What is known already Embryo culture media are important factors in IVF, which can significantly influence the clinical outcome of IVF/ICSI cycles. However it is not clear which formulation is most optimal and whether sequential or continuous media (single step) should be favored. Sequential media complies with embryo demands based on developmental stage , taking into account metabolic changes embryos undergo in-vivo, while moving from the oviduct to the uterus. The embryos in the early cleavage stage prefer to use pyruvate to produce energy, whereas once development nears the blastocyst stage , the embryos start using glucose in the process of glycolysis . Study design, size, duration A prospective RCT was carried out at our centre between 2018–2019 and IVF-ICSI patients meeting inclusion criteria (at least six normal MII - Oocytes) were included in this study. The aim of study was to compare blastocyst formation rates after embryo-culture in two different culture media systems. 436 metaphase II Oocytes from 62 women were randomly and equally divided to be fertilized and cultured to the blastocyst stage in either sequential media or single-step media. Participants/materials, setting, methods In this prospective trial with sibling oocytes, 436 metaphase II oocytes from 62 women were randomly and equally divided to be fertilized and cultured to the blastocyst stage in either sequential media ( n = 218 MII oocytes) or a single medium ( n = 218 MII oocytes). In both groups, embryos were cultured in an interrupted fashion with media changes on day 3. Embryo transfer was performed on day 5. Main results and the role of chance Blastocyst formation rates on day 5 were significantly higher following culture in single step media 60.55% (132 / 218 ) as compared to sequential media 34.86% ( 76 / 218) . The percentage of good quality blastocysts was also significantly higher in single step media. In conclusion, culture in single step media was associated with higher blastocyst formation rates compared to sequential media , suggesting that the single medium may provide better support to the developing embryo. The proportion of poor quality embryos was significantly higher in the sequential media group. Results indicate that embryo culture in continuous media could be as efficient as embryo culture in sequential media. A significant difference observed was the proportion of poor quality embryos on day 5 , which was significantly higher when the embryos were cultured in sequential media. Our results suggest that the type of embryo culture media can influence the quality of embryos both at the cleavage stage and blastocyst stage. The use of continuous embryo culture media does not seem to cause an adverse effect; in fact, their use can lower the workload in busy IVF labs and lower the stress that embryos are exposed to during handling. Limitations, reasons for caution Although single-step-medium for extended culture has practical advantages and blastocyst formation rates appear to be higher, there is insufficient evidence to recommend either sequential or single-step media as being superior for the embryo-culture to days 5/6. Further studies comparing these two media systems in well-designed trials should be performed. Wider implications of the findings: When employing sequential media for embryo culture , it is necessary to transfer the embryos from one medium to another ( cleavage stage medium to blastocyst stage medium) which increases stress related embryo damage . Therefore, single-step media is beneficial as the embryos can develop undisturbed till blastocyst stage. Trial registration number Not applicable

scholarly journals Peroxidized mineral oil increases the oxidant status of culture media and inhibits in vitro porcine embryo development

2017 ◽  
Vol 103 ◽  
pp. 17-23 ◽  
Author(s):  
C.A. Martinez ◽  
A. Nohalez ◽  
J.J. Ceron ◽  
C.P. Rubio ◽  
J. Roca ◽  
...  
Keyword(s):  
Embryo Development ◽  
Culture Media ◽  
Mineral Oil ◽  
Porcine Embryo ◽  
Oxidant Status

P–221 Prospective randomized sibling study on gamete preparation, insemination and subsequent culture of human oocytes in a time-lapse system using media systems with and without antioxidants

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
S Mizumoto ◽  
H Watanabe ◽  
Y Nagao ◽  
K Tanaka ◽  
M Murakami ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Clinical Outcome ◽  
Elderly Patients ◽  
Embryo Development ◽  
Embryo Culture ◽  
Age Groups ◽  
Time Lapse ◽  
Single Step ◽  
Sperm Preparation ◽  
Media System

Abstract Study question Does the addition of antioxidants for gamete preparation, insemination and embryo culture lead to differences in embryo development and clinical outcome Summary answer Using an antioxidant-containing media system for sperm preparation, insemination and embryo culture imparts significantly higher good-quality blastocyst rates and improved clinical outcome in elderly patients. What is known already A previous study showed that adding combined antioxidants for sequential embryo culture in conventional incubators (interrupted culture) improves embryo viability and clinical outcome, especially for elderly patients. Here we investigated the combined effect of three antioxidants Acetyl-L-Carnitine (10 µM), N-Acetyl-L-Cysteine (10 µM), and α-Lipoic Acid (5 µM) during sperm preparation, insemination, and time-lapse culture in a single step medium on human embryo development and clinical outcome. Study design, size, duration Prospective randomized single center study including 143 couples for IVF/ICSI between August 2018 and December 2019. Inclusion required at least eight cumulus-oocyte-complexes (COCs) after retrieval. Cycles involving PGT, split IVF/ICSI, and surgically retrieved sperm were excluded. Immediately after retrieval oocytes were randomly distributed to a study or control media system with or without antioxidants (Vitrolife). Similarly, ejaculates were split and prepared with and without antioxidants. Participants/materials, setting, methods Sibling oocytes were inseminated in the respective group with accordingly prepared sperm. Single step embryo culture was conducted in medium with (Gx-TL) and without (G-TL) antioxidants in the EmbryoScope+. Embryo quality and clinical outcome were assessed in relation to maternal age (<35/>35 years). Good-quality embryos on day 3 were defined as 8- to 10-cells with even cells and low fragmentation; good-quality blastocysts as > 3BB. Clinical outcome was assessed after single vitrified blastocyst transfer (SVBT). Main results and the role of chance From 143 participants (female age, 34.7±3.2 years), a total of 2424 COCs were collected; 1180 COCs/916 metaphase-II (MII) oocytes were allocated to Gx-TL media and 1244 COCs/981 MII oocytes to G-TL media. Age-related analysis in Gx-TL compared with G-TL in relation to allocated MII oocytes revealed a trend for higher fertilization rates in Gx-TL for both age groups (<35: 72.1% vs. 66.9%; >35: 70.7% vs. 64.9%, P < 0.1). Good-quality day 3 embryo development/MII oocytes was higher, albeit not significant, in the elderly patients in Gx-TL (<35: 35.9% vs. 34.4%; >35: 31.1% vs. 27.9%). Overall day 5/6 blastocyst rate was similar for both media (<35: 48.2% vs. 49.9%; >35: 42.3% vs. 39.5%). Day 5/6 GQB rate was comparable for younger patients (<35: 23.8% for Gx-TL vs. 26.0% for G-TL) but significantly higher in Gx-TL in elderly patients (>35: 20.7% vs. 14.4%; P < 0.05). A total of 200 SVBT were performed; 99 in the Gx-TL- and 101 in the G-TL-arm. We noted almost similar implantation and ongoing pregnancy rates between Gx-TL vs G-TL in the younger (<35) age group (50.0% vs. 55.4%; 50.0% vs. 55.6%) but higher albeit not significant rates for Gx-TL in older (>35) patients (44.1% vs. 33.3%; 44.1% vs. 33.3%). Limitations, reasons for caution In almost 95% of the cycles, oocytes were inseminated by ICSI; thus results may not equally apply for cycles with IVF. The use of a closed time-lapse system may have prevented from some environmental oxidative stress. Therefore results may come out different with a similar study using standard incubation. Wider implications of the findings: Supplementation of antioxidants to media for gamete isolation and preparation, as well as subsequent single step time-lapse culture may improve GQE/B rates and clinical outcomes in certain age groups, plausibly through the reduction of oxidative stress. Further studies in selected sub-groups (severe OAT syndrome / testicular cases) may be indicated. Trial registration number UMIN000034482

P–227 Fatty acid regulation of Nrf2/Keap1 pathway during mouse preimplantation embryo development

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
G Dionne ◽  
A J Watson ◽  
D H Betts ◽  
B A Rafea
Keyword(s):  
Oleic Acid ◽  
Fatty Acid ◽  
Palmitic Acid ◽  
Embryo Development ◽  
Embryo Culture ◽  
Preimplantation Embryo ◽  
Culture Media ◽  
Control Group ◽  
Blastocyst Development ◽  
Preimplantation Embryo Development

Abstract Study question Our objective is determining whether supplementing embryo culture media with palmitic acid and/or oleic acid impacts Nrf2/Keap1 antioxidant response pathways during preimplantation mouse embryo development. Summary answer Supplementation of embryo culture media with palmitic acid increases cellular Nrf2 levels per embryo after 48-hour culture, while oleic acid reverses this effect. What is known already Obese women experience higher incidence of infertility than women with healthy BMIs. The obese reproductive tract environment supporting preimplantation embryo development is likely to include enhanced free fatty acid (FFA) levels and increased accumulation of reactive oxygen species. Exposure to palmitic acid (PA) in vitro significantly impairs mouse embryo development while increasing ER stress mRNAs. Oleic acid (OA) reverses these effects. To further define effects of FFA exposure, we are characterizing the influence of FFAs on the Nrf2–Keap1 pathway and its downstream antioxidant defense systems. We hypothesize that PA treatment induces Nrf2-Keap1 activity, while OA treatment alleviates pathway activity. Study design, size, duration Female CD–1 mice (4–6 weeks) were super-ovulated via intraperitoneal injections of PMSG, followed 48 hours later by hCG. Female mice were mated with male CD–1 mice (6–8 months) overnight. Females were euthanized using CO2 and two-cell embryos were collected by flushing oviducts. Two-cell embryos were placed into KSOMaa-based treatment groups: 1) BSA (control); 2) 100µM PA; 3) 100µM OA; 4) 100µM PA+OA, and cultured for 48 hours (37 °C; 5% O2, 5% CO2, 90% N2). Participants/materials, setting, methods After 48-hour embryo culture, developmental stages of all mouse embryos were recorded. Immunofluorescence analysis of Nrf2 and Keap1 localization was performed for embryo treatments (BSA, 100µM PA, 100µM OA & 100µM PA+OA) using rabbit polyclonal anti-Nrf2 antibody, with Rhodamine-Phalloidin and DAPI staining. Embryos were imaged using confocal microscopy and Nrf2-positive cells were counted using ImageJ. Nrf2 and Keap1 mRNA abundances were assessed after culture in each treatment condition using RT-qPCR and the delta-delta Ct method. Main results and the role of chance Inclusion of 100µM PA in embryo culture significantly decreased blastocyst development frequency from 70.06±16.38% in the BSA (control) group to 11.61±8.19% in the PA-treated group (p < 0.0001). Embryo culture with 100µM OA and 100µM PA+OA co-treatment did not significantly impair blastocyst development (OA: 61.59±8.07%, p = 0.4053; PA+OA: 63.53±7.63%, p = 0.6204). Embryo culture with PA treatment significantly increased the mean percentage of Nrf2-positive cells to 56.83±30.49% compared with 21.22±15.63% in the control group (p < 0.0001). Conversely, 100µM OA and 100µM PA+OA treatments did not significantly affect Nrf2-positive cell frequencies compared with the control group (OA: 33.28±21.83%, p = 0.1825; PA+OA: 34.84±12.66%, p = 0.0691). Immunofluorescence results show that treating embryos with 100µM PA for 48 hours results in increased levels of cellular Nrf2, while combining 100µM PA with 100µM OA reversed these effects. Preliminary qPCR analysis showed no significant differences in Nrf2 or Keap1 relative transcript abundance between any embryo treatment groups. Nrf2 and Keap1 mRNA levels were both higher after embryo culture with 100µM OA than all other culture groups (p = 0.6268; p = 0.3201). Notably, Keap1 relative transcript levels dropped to undetectable levels after culture with 100µM PA, which suggests an increase in Nrf2 activation.Limitations, reasons for caution: While immunofluorescence localization of Nrf2/Keap1 provides insight into how the proteins behave during preimplantation embryo development, confocal images cannot determine protein-protein interactions or activity levels. Similarly, transcript information from RT-qPCR analysis only provides information about Nrf2 and Keap1 at the transcript level. Nrf2 activity will be assessed via downstream targets. Wider implications of the findings: The Nrf2–Keap1 pathway coordinates numerous cellular defence mechanisms, and is implicated in various diseases, including cancer. Establishing an impact of free fatty acid exposure on Nrf2–Keap1 during preimplantation embryo development will provide valuable information regarding the effects of maternal obesity on outcomes for embryos produced from these patients. Trial registration number Not applicable

P–250 Mineral oil with high viscosity improves the stability of pH and osmolality in the human in vitro culture system

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
E Mestres ◽  
Q Matia-Algué ◽  
A Villamar ◽  
M García-Jiménez ◽  
A Casals ◽  
...  
Keyword(s):  
Embryo Culture ◽  
Culture System ◽  
Chemical Properties ◽  
High Viscosity ◽  
Mineral Oil ◽  
Culture Conditions ◽  
Physico Chemical ◽  
Temperature Loss ◽  
The Stability

Abstract Study question Do commercial mineral oil brands differ in their capacity to stabilize the human embryo culture system, and is this related to the oil’s viscosity? Summary answer While the oils’ viscosity only had minor effects on temperature maintenance, it showed a direct correlation with the stability of pH and osmolality during culture. What is known already Mineral oil is a key component of the in vitro embryo culture system, which stabilizes temperature, pH and osmolality of the media during culture. Its use has been implemented worldwide for several decades and many manufacturers currently produce and commercialize oil intended for human embryo culture. Unfortunately, oil remains as one of the less characterized products in the IVF laboratory due to a lack of standardized nomenclature, production and testing. With differing physico-chemical properties, such as viscosity, oils produced by various manufacturers could behave differently to the same culture conditions and, thus, its use may need to be adjusted accordingly. Study design, size, duration Viscosity was quantified in three high-viscosity (H-V) and three low-viscosity (L-V) oils with a viscosity-meter. The required time for media’s pH to equilibrate using each oil was studied, as well as its subsequent stability outside the incubator for 30min. In-drop temperature was assessed during 15min when taking a dish outside the incubator, and again when putting it back. Additionally, each oil’s capacity to avoid media evaporation was studied with daily osmolality measurements during 7 days. Participants/materials, setting, methods pH equilibration was measured with a continuous pHmeter (Log&Guard, Vitrolife) in 4-well dishes prepared with 600µl of medium and 500µl of oil. For the other experiments, 35mm dishes with 4ml of oil and 20µl media droplets were used. pH stability was assessed after 0, 15 and 30min outside the incubator with a blood-gas-analyzer (epoc,SiemensHelthineers). A fine-gauge thermocouple was used to measure in-drop temperature loss/recovery. Daily osmolality readings were taken with a vapor pressure osmometer (Vapro5600,Wescor). Main results and the role of chance The selected oil samples had a viscosity of 115, 111, 52, 22, 18, and 12cP. The medium’s pH took approximately 12h to completely equilibrate under H-V oils, while it took less than 4h in L-V. Similarly, the rise in pH after 30min on a heated stage outside of the incubator with room atmosphere was 0.03, 0.04, 0.06, 0.13, 0.17, and 0.26, respectively. Dishes were taken out of the incubator and placed on a heated surface. In the first five minutes, the in-drop temperature loss ranged between –0.22 and –0.13oC/min, with no significant differences observed between oil types. However, temperature plateaued at a significantly higher value in L-V oils (36.5oC), compared to H-V brands (36.25–36.1oC; p = 0.0005). By contrast, all samples followed a similar pattern when the dishes were returned to the benchtop incubator, with temperature taking around 7 minutes to completely recover. Some media evaporated in all oil groups during the 7-day culture in a dry benchtop incubator. The linear regression performed to compare the evaporation rate between groups showed a statistically significant correlation between oil viscosity and the rate of evaporation (p < 0.0001), with an osmolality rise ranging between +2.55mmol/kg/day in the most viscous oil and +6.29mmol/kg/day in the least viscous. Limitations, reasons for caution While the selected oils for this study represent a wide range of options in the market, future projects could widen this selection and include additional tests, such as optimized bioassays. Results may vary between centers, and thus each laboratory should test and optimize their culture system with their own settings. Wider implications of the findings: Different oil brands have shown differing physico-chemical properties that have a direct effect on the culture system and the stability of several culture conditions. These results may be of major importance to adapt the settings and methodologies followed in each IVF laboratory according to the type of oil being used. Trial registration number Not applicable

Transfer of embryos following culture in continuous single-step or sequential-step embryo culture media

2013 ◽  
Vol 100 (3) ◽  
pp. S246
Author(s):  
M.A. Stout ◽  
V.A. Cholewczynski ◽  
S.G. Shukry ◽  
G.S. Karman ◽  
J.R. Graham ◽  
...  
Keyword(s):  
Embryo Culture ◽  
Culture Media ◽  
Single Step

scholarly journals Systematic Development, Validation and Optimization of a Human Embryo Culture System

2020 ◽  
Vol 1 (1) ◽  
pp. 1-14
Author(s):  
Mitchel C. Schiewe ◽  
Shane Zozula ◽  
Nancy L. Nugent ◽  
John B. Whitney ◽  
Ilene Hatch ◽  
...  
Keyword(s):  
Embryo Culture ◽  
Culture System ◽  
Age Groups ◽  
Clinical Effectiveness ◽  
Single Step ◽  
Human Embryos ◽  
Blastocyst Development ◽  
Paraffin Oil ◽  
Study Results

Objective: To develop and validate a reliable in vitro culture system for human embryos. Design: Retrospective analyses of a series of four studies were conducted between 2006 and 2010 to assess the effect of incubator type (CO2 box versus Tri-gas minibox), media type, oil type, and hyaluronate supplementation. Optimization of in vitro blastocyst development was verified by assessing our National CDC/ART Surveillance reports between 2010 and 2016. Material and Methods: All patients experienced controlled ovarian hyperstimulation, followed by egg retrieval 35 h post-hCG. Cumulus-oocyte complexes were temporarily cultured in P1 or LG Fert medium plus HSA. Eggs were moved to a more complex media (G-medium or Global®-LG medium) containing a synthetic protein and embryo adhesion supplement (SPS and EAS, respectively; mLG) post-ICSI insemination. Zygotes were assigned to group culture in 25 µl droplets under oil (light mineral oil or paraffin oil; 37 °C) and embryo development was evaluated on Days 3, 5, and 6 and transferred on Day 3 to 5 depending on the number/quality of embryos available and the IVF history of the patient. Transfers were performed under ultrasound guidance, primarily using a Sureview-Wallace catheter, and enriched ET medium containing 500 µg/mL EAS. Results: Pilot study results (Expt. 1) showed that a mLG single-step medium could be effectively used in combination with Sanyo MCO-5 tri-gas (TG) incubators. Once adapted to SCIRS Lab in 2007 (Expt. 2), the latter culture system yielded improved blastocyst production and pregnancy outcomes compared to CO2 in air sequential incubation in P1/Multi-blast medium. In Expt. 3, the mLG/TG system yielded high levels of ≥2BB quality blastocysts (51 to 66%) across all age groups, and greater (p < 0.05) pregnancy success/live birth rates using fewer embryos transferred on Day 5 versus Day 3. After validating its clinical effectiveness, mLG was then prospectively compared to a new generation G-media (1.5 & 2.5; Expt. 4) and determined that the crossover treatment using paraffin oil (Ovoil™) allowed the mLG system to be optimized. Subsequently, a compilation of our Annual CDC/ART reported data over six years verified the overall viability of in vitro cultured and vitrified blastocysts produced in the mLG/TG system. Conclusion: By systematically evaluating and implementing various components of an embryo culture system we were able to optimize blastocyst development over the last decade. Our mLG/TG culture system modified an exceptionally well designed KSOMAA LG medium using endotoxin-free EAS and SPS additives to support cellular membrane wellness under stressful in vitro conditions (e.g., culture, cell biopsy, vitrification). Our use of the mLG/TG culture system has proven to be effective, creating reliably high blastocyst production, implantation, and healthy live births.

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