Single-step Media Research Articles

P-234 The internal temperature of the catheter during the transfer: potential effects on the embryonic microenvironment

Abstract Study question Do the external environment and the different loading procedures affect temperature in the transfer catheter and embryo development during embryo transfer? Summary answer The inner temperature of transfer catheter dropped during the embryo transfer. Mouse 2-cell-embryos exposed to the transfer showed lower blastocyst and hatching rates. What is known already Embryo transfer is a crucial process composed of complex phases, which start from embryo loading into the catheter, and culminate in the transfer room in an open environment at room temperature. Although the transfer technique has been refined over time and several adjustments have been proposed towards its standardization (ASRM, 2017, D’Angelo et al., 2022), there is still great variability (Cirillo et al 2020). Few literature data are available regarding possible influences of the external environment on inner catheter conditions. Only recently, temperature drop during transfer has been described (Macklon et al 2021). Study design, size, duration We addressed possible temperature variations to which the embryo is exposed during transfer, by using a thin temperature probe placed inside a catheter. We simulated a total of 120 transfers (60 transfers with “fluid only” methodology and 60 with “air-fluid”). Additionally, we performed Mouse Embryo Assay (MEA) during transfer simulations and then cultured up to blastocyst to assess any impact of temperature drop exposure during transfer Participants/materials, setting, methods 120 mock transfers were performed, recording the temperature in the inner catheter were the embryos are loaded. In our setting, the procedure lasts 60-100 seconds. The internal temperature of the catheter was recorded after 15 min of preheating, at the loading and in the following 60 seconds. Environmental temperature and humidity values were recorded. A total of 73 mouse embryo were used for MEA (39 exposed to catheter, 34 controls) and cultured in time-lapse Main results and the role of chance The 120 simulations took place at an average environmental temperature of 22.43 °C± 0.85, and a relative humidity of 57.4%± 5.1. After 15 min of preheating the empty catheter reached an average temperature of 33.04 °C ±2.08. Upon loading, the average temperature inside the catheter was 31.22 °C± 1.67 and after 60 seconds, it dropped to 29.15 °C ±1.88. The comparison between the “fluid only” and “air-fluid” shows the following results: internal temperature at “fluid only” loading 31.97 °C ± 1.87, “air-fluid” 30.11 °C ± 1.48 (p < 0.001); Average temperature after 60 seconds “fluid only” 29.98 °C ± 1.71, “air-fluid” 27.91 °C ± 1.38 (p < 0.001). Seventy-three 2-cells mouse embryos were thawed and used to perform MEA tests. 39 of them were exposed to 5 transfer simulations of 200 seconds, and then put in culture in time-lapse incubator (single step medium, 6% CO2, 5%O2). The blastocyst rate was 82% in the control group and 74% in the exposed group (X2 (1, N = 73) = 0.6783, p >0.05). Hatching rate significantly differed (61% in controls vs. 34% in the exposed (X2 (1, N = 57) = 3.9, p = 0.04). Morphokinetics were not different among groups Limitations, reasons for caution The monocentric nature of this pilot study can represent the limitation of the single environmental conditions at which the catheters are exposed during the embryo transfer Wider implications of the findings The transfer procedure exposes embryos to a drastic decrease in temperature, directly proportional to the execution time. Mouse embryos showed an impaired development after being exposed to transfer simulations. Novel strategies to stabilize temperature during embryo transfer are needed. Trial registration number non-clinical trial

P-136 Non-invasive PGT (niPGT) using spent culture medium: Relationship between time lapse embryo dynamics and accuracy of niPGT analysis

Abstract Study question Do differences in the morphological characteristics of embryos or the dynamics of the developmental process affect the results of niPGT analysis? Summary answer Embryos with blastomeres that were excluded at compaction showed reduced accuracy of niPGT analysis, but other measured morphological characteristics had no impact. What is known already The PGT-A test, is invasive and requires training to be competent at the technique, aswell as the need for specialized equipment and consumables. In recent years, non-invasive PGT-A (niPGT-A), which analyzes Cell-Free DNA released from cultured embryos, has been attracting attention. Regarding the analysis accuracy of niPGT-A, there are reports that it is equivalent to or even superior to conventional PGT-A. However, at present, it is not clear how methodological differences or embryo dynamics impact results. Study design, size, duration From August 2021 to February 2022, 284 pronuclear-stage frozen embryos (with consent for research use), were thawed and cultured. They were individually cultured for at least 40 hours after Day 4. For the 150 embryos that reached the blastocyst stage, culture medium and the blastocysts were collected and subjected to chromosome aneuploidy analysis by NGS. For the 138 blastocyst with a result, the concordance rate was calculated with that obtained from the culture medium samples. Participants/materials, setting, methods The embryos were individually cultured in a single step medium using a 10 μl droplet from Day 4. The entire culture period was recorded in a time-lapse incubator. The concordance rate for all chromosomes between culture medium sample and the blastocyst was compared to recorded embryo dynamics, e.g. presence or absence of cumulus cell attachment, sperm attachment, fragmentation, blastomeres that excluded at compaction, shrinkage, and hatching. Chi-square test was used to compare the concordance rates. Main results and the role of chance The overall concordance rate for all chromosomes between the culture medium and the blastocysts samples was 46.4%(64/138). In the blastocysts where there were cumulus cells attached or not it was 43.6% (17/39) vs 47.5% (47/99) respectively, cases where there was still a sperm attached or not it was 53.1% (17/32) vs 44.3% (47/106) resepctively and in blastocysts where there had been fragmentation or no fragmenattion it was 46.9%(38/81) vs 45.6％(26/57) respectively. In cases where shrinkage of the blastocyst had been observed vs no shrinkage the respective concordance rates were 44.9% (48/107) vs 51.6% (16/31), whilst in cases where the blastocyst had hatched or not it was 40.6% (13/32) vs 48.1% (51/106) respectively.For the blastocysts where the blastomeres had been excluded at compaction or not it was 35.6％(31/87) vs 64.7% (33/51) respectively.Significant differences were only identified in cases with blastomere exclusion or no exclusion at compaction (P < 0.01). Limitations, reasons for caution In this study, the culture was continued to day 6 regardless of morphology of the embryo on day 5, which differs from the usual clinical culture process. Wider implications of the findings These results are useful in that they may aid in recognising which morphological features of embryos may impact the results obtained from niPGT and hence its reliability for clinical application. The blastomeres that excluded at compaction are clearly important for improving the accuracy of the niPGT analysis in the future. Trial registration number None

#341 : Does Only the Stage Matters? Influence of a Blastocyst Scoring System on Clinical Pregnancy and Live Birth Outcomes with Fresh Set: A Concluding Study

Background: Blastocyst culture with single embryo transfer (SET) is now routinely offered to our IVF patients at KK IVF. We have been using a simplified scoring system since 2016 in our routine assessment of blastocysts. Aims: This retrospective study aims to conclude our centre’s scoring system for fresh blastocyst for Day 5(D5) ET. Method: Data of 694 patients with fresh Intracytoplasmic Sperm Injection (ICSI) cycle and SET on D5 done between Jan 2018 and Dec 2020 were analysed. Only patients with 1[Formula: see text] cycle stimulation attempt and embryos cultured in single-step media under pre-mixed incubator conditions were included in this study. Rates of clinical pregnancy (CPR), multiple pregnancy (MPR), miscarriage (MISCR) and livebirth (LBR) were investigated. Data were classified into stage and grade of blastocyst transferred respectively as: Fully Expanded (FE), Early Expanded (EE), Early Blastocyst (EBL); A, B, C and Pseudo (P-; with at least 20% blastomeres excluded from the formation of blastocyst) and subjected to Chi-square test for significance, p<0.05. Blastocysts are scored in our centre first according to the stage of blastocyst development, followed by a grading assessment on the cohesion and cell number of the inner cellular mass (ICM) and trophectoderm cells (TE). The blastocyst grade equivalent by Gardner’s classification system of the ICM and TE is in parentheses - A=(AA), B=(AB, BA,BB) and C=(AC,CA,BC,CB). Blastocysts void of ICM are deemed non-transferrable. Results: CPR significantly different across stages of BL transferred (p = 0.00001). Transferring EBL is associated with a significantly higher MISCR (p=0.004). Multiple pregnancy was observed in groups with expanded blastocyst transferred only and MPR appears higher when top grade BL transferred (p=0.45). LBR is incremental with better quality BL transferred (p=0.000049). Conclusion: Stage of blastocyst development and a graded ICM at ET improve pregnancy and livebirth outcomes. Miscarriage risk is incremental with early blastocyst transferred. Suboptimal embryos remain transferrable as can still result in viable pregnancies and livebirths when there’s no better selection.

Does the Human Embryo Culture Media Has Any Impact on Implantation and Live Birth Rate in ART Cycles? A Comparative Study

ABSTRACT Introduction: Culture media plays a pivotal role in the embryo culture system apart from the other crucial components as air quality, temperature, humidity etc., so that the selection of embryo culture media is a crucial step for the embryology team for the optimal blastocyst development and for achieving a viable pregnancy. There are two opposing views for the selection of embryo culture medium. One is embryo free choice’-“Let the embryo choose” (single-step media) and the other one is “back to nature” (sequential media) approach. Present study analyses the efficacy of these media in terms of embryo developments in vitro and pregnancy rates. Materials and Methods: Patients were randomly recruited for single step or sequential media culture post intracytoplasmic sperm injection. Inclusion criteria were patients with no severe male or female factors. To minimize the confounding variables, patients with surgically retrieved sperm samples were excluded from the study. Biochemical pregnancy, clinical pregnancy, implantation rate, live birth rate and miscarriage rate were analyzed. Results: Biochemical pregnancy, clinical pregnancy and live birth rate were favorable for group 2 (sequential media) but not statistically significant. None of the analyzed parameters differed significantly among the two media. Conclusion: There is a lack of solid scientific data to support the sequential media culture. However, moving embryos from one medium to another in a sequential media system probably adds significant stress to the preimplantation developing embryos in-culture apart from the unintentional humane errors. Moreover, this approach is quite labor-intensive and expensive.

P-202 Using artificial intelligence platform coupled to an existing time system; external validation of an automatic embryo score to assist in selection

Abstract Study question How does a novel artificial intelligence-based embryo evaluation system work in Geri® time-lapse systems? Summary answer Automatic score platform provided externally for embryos cultured in Geri® was associated with conventional morphology, euploidy, implantation and live birth. What is known already Artificial intelligence has been making headway in assisted reproduction in recent years. Many companies have developed different models for automated embryo evaluation and selection, such as CHLOETM software (Fairtility, Israel). According to the developers, it is an orchestration of cutting-edge morphological and morphokinetic AI algorithms, trained over 100,000 embryo videos and tens of millions of images. Different laboratories validated its use in specific time-lapse systems (Embryoscope, Vitrolife). However, this is the first time that an objective and independent review of CHLOETM has been performed on Geri® (Genea Biomedx, Australia) time-lapse system. Study design, size, duration This retrospective analysis included 3568 embryos from 417 patients that underwent IVF treatments in a single center. Embryos were cultured in Geri® (Genea Biomedx, Australia) time-lapse systems by single step media (Geri Medium, Genea Biomedx, Australia) and routinely evaluated by senior embryologists by using Connect and Assess software according to the ASEBIR criteria (from A-high quality to D-low quality and excluded embryos). Then, embryos were automatically scored by CHLOETM from 0 to 1. Participants/materials, setting, methods Automatic embryo score was compared with conventional morphology (n = 3568 embryos), ploidy (n = 467 embryos), and clinical outcomes for single blastocyst transfers (n = 461). Main results and the role of chance The comparison between the embryo score provided by CHLOETM and the category assigned by embryologists showed a direct association*. The means were 0.97 ± 0.10 for A (n = 123); 0.89 ± 0.21 for B (n = 842); 0.74 ± 0.30 for C (n = 607), 0.24 ± 0.31 for D (n = 997) and 0.15 ± 0.25 for excluded embryos (n = 403). The automatic embryo score for oocytes that failed to fertilize was 0.06 ± 0.19 (n = 596). Regarding the chromosomal status, embryos with normal content had significantly higher score than abnormal ones. Following results are presented per quartiles of similar sample size: the euploidy rates were 35.9% for score < 0.81 (n = 117), 40.8% for score 0.81-0.96 (n = 120), 48% for score 0.96-0.99 (n = 125) and 58.1% for score >0.99 (n = 105)*. Implanted embryos achieved significantly higher marks than non-implanted embryos: 0.93 ± 0.15 (n = 251) vs. 0.85 ± 0.25 (n = 210)*. Also, automatic embryo score was higher for embryos that led to a live birth than those that did not: 0.94 ± 0.15 (n = 188) vs. 0.86 ± 0.24 (n = 243)*. Focusing on top quality embryos (A+B), the score means were 0.94 ± 0.16 for implanted good quality embryos 0.88 ± 0.22 for non-implanted ones* (*p<0.05). Limitations, reasons for caution This project is limited by its retrospective and single-center nature. Multicenter validation would be necessary to ensure it is a safe and effective method of embryo assessment. Wider implications of the findings In addition to verifying that automatic scoring agrees with embryologists, this study demonstrated its ability to be applied in Geri Time-lapse incubator to distinguish between potential embryos with similar morphological characteristics helping embryologist to make decisions. Trial registration number NOT APPLICABLE

P-151 In women with recurrent implantation failures, can hyaluronan enriched media optimize reproductive outcomes?

Abstract Study question To evaluate the efficacy of Hyaluronan Enriched Media (HEM) to optimize Reproductive Outcomes in women with Recurrent Implantation Failures (RIF)? Summary answer In women with RIF, HEM seems to offer beneficial and safer reproductive outcomes. What is known already HEM has shown to enhance the implantation process of an embryo in-utero. Efficacy of HEM to optimize reproductive outcomes is still not proven. Study design, size, duration This retrospective study comprises data of 430 women with two failed IVF cycles or had RIF undergoing infertility treatment between 2016 to 2021. Study group included Young women (<37yrs) with RIF who opted elective-single blastocyst transfer (eSET) with HEM (n = 228). Control group had young women with RIF and eSET without use HEM (n = 202). Only self-gamete cycles with No PGT-A screening were included in this study. Women with known uterine pathologies were excluded from the study. Participants/materials, setting, methods All women had ICSI - freeze all culture strategy. In a FET, blastocyst showing 100% survival post warming was considered transfer. In study group, embryos were incubated in pre-equilibrated HEM for 10minutes before the intended embryo transfer. In control group embryo was transferred in single step media. ET procedure done as per the clinic’s SOP. Outcomes measured: Clinical Pregnancy Rates (CPR), Implantation rates (IR), Miscarriage Rates(MR), Live Birth rates(LBR) and Neonatal outcomes. Main results and the role of chance Following were the outcomes measured in HEM group Vs Control group: CPR- 66.22% Vs 61.38% (p = 0.2938) IR – 49.56% Vs 31.68% (p = 0.0002; Significant) MR – 11.4% Vs 16.33% (p = 0.1359) LBR – 54.82% Vs 45.05% (p = 0.0418; Significant) There was no significant adverse events with neonatal outcomes between the groups and data looked comparable. Use of HEM at embryo transfer for women with RIF seemed to be a beneficial intervention. It showed significant improvement with embryo implantation and live birth. Data in this study has also looked at Neonatal outcomes. Since HEM group had no reported adverse neonatal outcomes, it not only aids in improving reproductive outcomes but also is a safe intervention and can be considered for routine clinical use for indicated cases. Limitations, reasons for caution Retrospective data with small and unequal sample size. Wider implications of the findings Efficacy of various therapies is still unknown for women who had RIF. PGT-A and ERA (invasive therapies) have been tried for such cases, but its effectiveness is still up in question. Development for newer & safer therapies to improve embryo implantation and live birth for these patients is still awaited. Trial registration number Not applicable

P-186 Combination of proteomics and automatic scoring using artificial neural networks to detect aneuploid embryos

Abstract Study question How powerful is the combination of artificial neural networks that combine embryo's protein profile with its automatic score provided by time-lapse videos to predict ploidy? Summary answer An artificial neural network (ANN) that considers proteomics and automatic score assigned by deep learning achieves 71% accuracy in distinguishing between euploid and aneuploid embryos. What is known already Currently the most widely used technique for detecting chromosomal abnormalities involves biopsy of the developing embryo. However, it has several disadvantages related to invasiveness, technical difficulty, high economic costs, etc. Therefore, different non-invasive techniques are being studied for the detection of aneuploid embryos. The discovery of cell-free DNA (cfDNA) released by the embryo to the culture media during its development marked the beginning of a new era of noninvasive PGT (niPGT) but some factors require adaptation for the analysis. Also, artificial Intelligence (AI) represents a valuable alternative to developing new models for predicting PGT outcomes without disturbing the embryo. Study design, size, duration This study included 294 samples of culture medium from 81 treatments of the PGT-A program. Out of the total, 23 were control samples (medium in which no embryos had been cultured) and 271 were samples where there was a developing embryo. Embryos were cultured until the blastocyst stage in EmbryoScope systems (Vitrolife, Sweden) with single-step medium (Gems, Genea) and automatically scored by iDAScore v2 algorithm from 1 to 9.9. Participants/materials, setting, methods The spent culture medium was collected on day 5/6 of embryo development and chromosome analysis was performed using next-generation sequence technology (Juno Genetics, Valencia). The relative concentrations of 92 proteins were analysed using Proseek Multiplex Assays (Olink Bioscience) using 1 μl of each sample. The final assay readout was presented as Normalized Protein eXpression (NPX) values. Finally, we developed our own ANN algorithms considering protein profile and automatic embryo score. Main results and the role of chance For euploid embryos (n = 101), 35 protein samples analysed had different NPX values between conditioned and control media*. The relative concentration was reduced for 11 proteins (consumption by the embryo) and 24 proteins increased their levels (secretion). For aneuploid embryos (n = 170), 33 protein samples analysed had different NPX values between conditioned and control media*. The relative concentration was reduced for 4 proteins and 29 proteins increased their levels. Out of the total, only 6 proteins had on average different concentrations between normal and abnormal embryos*: MCP_1, IL_17A, CXCL1, IL18, IL_22RA1 and CSF_1. Additionally, the automatic embryo score provided by iDAScore v2 algorithm was higher for euploid embryos than for aneuploid embryos (5.9 ± 2.7 vs. 5.1 ± 2.6)*. For ploidy prediction, three architectures of ANN were developed with different input data ANN1 (six discriminatory proteins), ANN2 (automatic embryo score) and ANN3 (six discriminatory proteins and automatic embryo score). Our dataset was divided into 68% training, 16% validation and 16% test. The accuracy, sensibility and specificity for the test phase were as follows: 63.6%, 76.9% and 44% for ANN1; 61%, 11.8% and 95.8% for ANN2; and 71.1%, 62.5% and 77.3% for ANN3. Limitations, reasons for caution Only one laboratory by using single step culture medium from one brand was involved in this study. Wider implications of the findings Our study showed a new approach to avoid transferring aneuploid embryos in cases where embryo biopsy is not performed. In addition, further studies on this field may result in a new non-invasive methodology for detecting aneuploidy. Trial registration number PI21/00283

Mammalian embryo culture media: now and into the future

For over 70 years, since the culture of the first mammalian embryo in vitro, scientists have undertaken studies to devise and optimise media to support the manipulation and culture of gametes and embryos. This area of research became especially active in the late 1970s onwards following the successful birth of the first human in vitro fertilised embryo. This review summarises some of the key advances in mammalian embryo culture media over time based on a greater understanding of the biochemical milieu of the reproductive tract. It highlights how learnings from studies in mice and agricultural species have informed human culture media compositions, in particular the inclusion of albumin, growth factors, cytokines, and antioxidants into contemporary culture media formulations, and how these advances may then in turn help to inform and guide development of in vitro culture systems used in other arenas, in particular agriculture. Additionally, it will highlight how the introduction of new technologies, such as timelapse, can influence current trends in media composition and usage that may see a return to a single step medium.

A propensity score-based, comparative study assessing humid and dry time-lapse incubation, with single-step medium, on embryo development and clinical outcomes.

Does culture in a high relative humidity atmosphere improve clinical outcomes when using a time-lapse integrated incubator and single-step culture medium? Using an integrated time-lapse system and single-step culture medium, culture in a high relative humidity atmosphere increases the likelihood of embryos, especially those subjected to preimplantation genetic testing for aneuploidies, to achieve a pregnancy compared to those cultured in dry conditions. The use of a humid atmosphere inside incubators can reduce changes in culture media osmolality, which has been reported to have a significant effect on embryo quality and morphokinetics. Studies assessing the effect of humid culture (HC) in clinical outcomes are, however, scarce and inconclusive, mostly due to a high variability in culture conditions and reduced sample size. Retrospective cohort study performed over 1627 ICSI cycles performed during 3 consecutive years in which embryo cohorts were cultured in a time-lapse incubator with three dry and three humidified chambers, and using single-step culture medium. Clinical outcomes were compared between treatments in which embryo cohorts were cultured in either humid (n = 833) or dry (n = 794) conditions. The study includes autologous treatments, with (N = 492) and without (N = 372) preimplantation genetic testing for aneuploidies (PGT-A) and ovum donation treatments (N = 763), performed in three university-affiliated private IVF centres. Stimulation, oocyte pickup and fertilization were performed according to the standard procedures of the clinic. All embryo cohorts were cultured in the same model of time-lapse incubator, distributed to either a dry or humidified chamber, while the rest of the culture variables remained equal. The population was weighted by the inverse probability of treatment to control for all measured confounders. The association between HC and the main outcome was assessed by logistic regression over the weighted population. The E-value was reported as a way of considering for unmeasured confounders. Differences in embryo development and other secondary outcomes between the study groups were assessed by Pearson Chi-squared test, ANOVA test and Kaplan-Meier survival analysis. An univariable logistic regression analysis, weighted by the inverse probability of treatment, determined that embryos cultured in humid conditions are more likely to achieve a clinical pregnancy than those cultured in dry conditions (odds ratio (OR) = 1.236 (95% CI 1.009-1.515), P = 0.041, E = 1.460). Through stratification, it was determined that said effect is dependent on the type of treatment: no improvement in clinical pregnancy was present in ovum donation or autologous treatments, but a statistically significant positive effect was present in treatments with preimplantation genetic testing (OR = 1.699 (95% CI 1.084-2.663), P = 0.021, E = 1.930). Said increase does not relate with an improvement in later outcomes. Differences were also found in variables related to embryo developmental morphokinetics. The retrospective nature of the study makes it susceptible to some bias linked to the characteristics of the treatments. To lessen the effect of possible biases, cases were weighted by the inverse probability of treatment prior to the evaluation of the outcome, as means to assess for measured confounders. In addition, the E-value of the weighted OR was calculated as a sensitivity analysis for unmeasured confounders. A randomized prospective study could be performed for further assessing the effect of humid conditions in clinical outcome. These results support that embryo culture under conditions of high relative humidity contributes to optimize clinical results in undisturbed culture in a time-lapse incubator with single-step medium. To our knowledge, this is the largest study on the matter and the first performing a propensity score-based analysis. This work was supported by the ''Centro para el Desarrollo Tecnologico Industrial'' from the Spanish Ministry of Science, Innovation, and Universities (CDTI-20170310) and Generalitat Valenciana and European Social Fund (ACIF/2019/264). None of the authors have any competing interest to declare. N/A.

P-168 Does the concentration of Interleukin-6 (IL-6) in spent embryo culture medium tell us anything? Association with implantation potential after euploid embryo transfer

Abstract Study question Could Interleukin-6 (IL-6) secretion by euploid embryos provide extra information in the prediction of clinical outcomes after human blastocyst transfer? Summary answer The concentration of IL-6 in the spent embryo culture medium provides additional and non-redundant information associated with the success of a euploid embryo transfer. What is known already Previous studies revealed that the concentration of IL-6 in the culture medium could be useful in selecting the embryo for implantation (Dominguez et al., 2015) and higher concentrations of IL-6 was reported in embryos that reach blastocyst stage than in arrested ones (Lindgren et al., 2018). Recently, proteomic information gained from the analysis of the embryonic secretome have been used to predict the potential of a euploid embryo to lead to a live birth (Bori et al., 2021). Out of 92 proteins measured, IL-6 was one of the most relevant for the prediction of live birth along with blastocyst morphology. Study design, size, duration This prospective study included 157 euploid embryos from the preimplantation genetic testing for aneuploidies (PGT-A) program. Embryos were cultured until the blastocyst stage in EmbryoScope systems with single-step medium (Gems, Genea). All of them were routinely assessed by senior embryologists. Morphological quality was based on ASEBIR criteria and morphokinetic parameters were automatically annotated by guided annotations tool. The spent culture medium was collected on day 5/6 of embryo development (day of trophectoderm biopsy). Participants/materials, setting, methods Chromosome analysis was performed using next-generation sequence technology. In parallel, 20 µL of spent culture medium from each blastocyst was analyzed with Human IL-6 ELISA Kit. Only euploid embryos were used in the following analysis. We examined the association between IL-6 levels (pg/ul) and embryo morphology and morphokinetics. Out of the total, 77 embryos were selected and transferred according to ASEBIR morphological quality. Pregnancy, implantation and live birth rate were correlated to IL-6 concentration. Main results and the role of chance The average concentration of IL-6 in spent embryo culture medium was 1.76±0.93 pg/ul. The protein value was not related to embryo morphology (p = 0.332): 2.35±1.60 pg/ul for embryos graded as A; 1.72±0.95 pg/ul for embryos graded as B and 1.79±0.76 pg/ul for embryos graded as C. Likewise, IL-6 level was not related to morphokinetic parameters. Division time to 2 cells, 3 cells, 4 cells, 5 cells and time to blastulation were not statistically different among the following quartiles of IL-6: ≤ 1.06 pg/ul (n = 41), from 1.07 to 1.66 pg/ul (n = 39), from 1.67 to 2.21 pg/ul (n = 40) and >2.21 pg/ul (n = 37). However, the concentration of IL-6 was higher in those euploid embryos that achieved a clinical pregnancy than in those embryos that failed after transfer. For spent culture media with less than 1.66 pg/ul of IL-6, pregnancy rate was 50%*, implantation rate was 50%* and live birth rate 43.80%. For spent culture media with more than 1.66 pg/ul of IL-6, pregnancy rate was 77.80% *, implantation rate was 75,60%* and live birth rate was 64.40%. *p<0.05 Limitations, reasons for caution The detection limit in protein quantification and the specific culture medium used that may affect IL-6 concentrations are the main limitations of our study. Wider implications of the findings Our findings demonstrated that the concentration of IL-6 in spent culture medium is independent of embryo morphology and morphokinetics, but it is related to the success of a PGT-A treatment. This may provide a promising approach to predict implantation after euploid embryo transfer by measuring the IL-6 protein. Trial registration number CDTI (IDI-20191102)

EFFICACY OF USING CULTURE MEDIA IN ASSISTED REPRODUCTIVE TECHNOLOGY PROGRAMS

One of the steps to successfully solve the problem of human infertility is the development and improvement of culture media used for the cultivation of embryos in ART programs.  In the past, significant progress has been made in this direction - single-step and two-step culture media have been developed. Both media are widely used in practice to improve the efficiency of individual ART results. The aim of this work was to determine the effectiveness in usage of commercial culture media (Continuous Single Culture Irvine and Origio Sequential Cleav/Blast) in IVF and ICSI programs. A comparative analysis of their impact on embryo quality, fertilization and pregnancy outcomes in IVF and ICSI programs at the Embryology Laboratory of LLP “Centre ECO” in 2017-2021 was conducted. 2650 programs took place, of which 760 programs accounted for IVF and 1890 programs for ICSI. The results of the analysis revealed no significant differences in the use of the above-mentioned media to obtain good quality blastocysts in the IVF program. The usage of the studied culture media showed a higher level of obtaining blastocysts of optimal quality in the ICSI programs when cultured in single-step medium compared to sequential medium. The difference between the two relative values was statistically significant (t=8.08; p0.001). Comparative analysis of the results of embryo cultivation in different nutrient media allow us to conclude that both single-step and sequential culture media, equally contribute to the quality development of blastocysts and demonstrate positive clinical results. Program efficacy based on the factor of ART program ending in pregnancy using CSC Irvine nutrient media was 46.44% for IVF, and 41.42% for ICSI.  Culturing embryos in Origio Sequential Cleav/Blast medium in IVF programs showed pregnancy outcomes at 49.51%, in ICSI programs - 42.85%.

Duration of dry and humidified incubation of single-step embryo culture medium and oxygen tension during sham culture do not alter medium composition.

Background: The extended embryo culture using single-step medium gained popularity in clinical in vitro fertilisation (IVF). However, there are concerns about the degradation of unstable medium components and their negative effects on the developing embryos. Further, dry-incubation can increase osmolality, which can in-turn enhance the concentration of constituents of the media and their stability. Hence, this study was conducted to understand the immediate changes in the culture media constituents in relation to clinically comparable situations such as single-step extended embryo culture and use of dry and humidified-incubation in two-different gaseous conditions. Methods: Commercially available single-step medium was sham-cultured in droplets under oil in two different conditions viz. dry (37°C; 6%CO 2; 5%O 2) and humidified (37°C; 6% CO 2; atmospheric O 2) for 0h, 72h, and 120h intervals. Droplets were subjected to the sensitivity-enhanced nuclear magnetic resonance (NMR)-based profiling using 800 MHz NMR equipped with a cryogenically cooled micro-coil (1.7mm) probe. NMR profile of the embryo culture medium between the two groups were comprehensively assessed. Results: A total of ten amino acids and four energy substrates were identified from the culture medium. The medium constituents identified showed a non-significant increase in the dry-incubation group at 72h and then declined at 120h. Humidified incubation had no effects on the level of the identified medium constituents until 120h. No significant differences in the levels of medium constituents identified were observed between the dry and humidified-groups at various time-points tested. Conclusions: A non-significant variation in the levels of medium constituents observed in the dry-incubation of single-step medium most unlikely to influence a clinical outcome. However, the impact of these subtle changes on the (epi)genetic integrity of the embryos in a clinical set-up to be addressed.

Duration of dry and humidified incubation of single-step embryo culture medium and oxygen tension during sham culture do not alter metabolomics signature

Background: The extended embryo culture using single-step medium gained popularity in clinical in vitro fertilisation (IVF). However, there are concerns about the degradation of unstable medium components and their negative effects on the developing embryos. Further, dry-incubation can increase osmolality, which can in-turn enhance the concentration of constituents of the media and their stability. Hence, this study was conducted to understand the immediate changes in the culture media metabolites in relation to clinically comparable situations such as single-step extended embryo culture and use of dry and humidified-incubation in two-different gaseous conditions. Methods: Commercially available single-step medium was sham-cultured in droplets under oil in two different conditions viz. dry (37°C; 6%CO 2; 5%O 2) and humidified (37°C; 6% CO 2; atmospheric O 2) for 0h, 72h, and 120h intervals. Droplets were subjected to the sensitivity-enhanced nuclear magnetic resonance (NMR)-based profiling using 800 MHz NMR equipped with a cryogenically cooled micro-coil (1.7mm) probe. Metabolomic signatures between the two groups were comprehensively assessed. Results: A total of ten amino acids and four energy substrates were identified from the culture medium. Metabolite levels showed a non-significant increase in the dry-incubation group at 72h and then declined at 120h. Humidified incubation had no effects on the level of the metabolite until 120h. No significant differences in the levels of metabolites were observed between the dry and humidified-groups at various time-points tested. Conclusions: A non-significant variation in the levels of metabolites observed in the dry-incubation of single-step medium most unlikely to influence a clinical outcome. However, the impact of these subtle changes on the (epi)genetic integrity of the embryos in a clinical set-up to be addressed.

Duration of dry and humidified incubation of single-step embryo culture medium and oxygen tension during sham culture do not alter metabolomics signature.

Background: The extended embryo culture using single-step medium gained popularity in clinical in vitro fertilisation (IVF). However, there are concerns about the degradation of unstable medium components and their negative effects on the developing embryos. Further, dry-incubation can increase osmolality, which can in-turn enhance the concentration of constituents of the media and their stability. Hence, this study was conducted to understand the immediate changes in the culture media metabolites in relation to clinically comparable situations such as single-step extended embryo culture and use of dry and humidified-incubation in two-different gaseous conditions. Methods: Commercially available single-step medium was sham-cultured in droplets under oil in two different conditions viz. dry (37°C; 6%CO 2; 5%O 2) and humidified (37°C; 6% CO 2; atmospheric O 2) for 0h, 72h, and 120h intervals. Droplets were subjected to the sensitivity-enhanced nuclear magnetic resonance (NMR)-based profiling using 800 MHz NMR equipped with a cryogenically cooled micro-coil (1.7mm) probe. Metabolomic signatures between the two groups were comprehensively assessed. Results: A total of ten amino acids and four energy substrates were identified from the culture medium. Metabolite levels showed a non-significant increase in the dry-incubation group at 72h and then declined at 120h. Humidified incubation had no effects on the level of the metabolite until 120h. No significant differences in the levels of metabolites were observed between the dry and humidified-groups at various time-points tested. Conclusions: A non-significant variation in the levels of metabolites observed in the dry-incubation of single-step medium most unlikely to influence a clinical outcome. However, the impact of these subtle changes on the (epi)genetic integrity of the embryos in a clinical set-up to be addressed.

Duration of dry and humidified incubation of single-step embryo culture medium and oxygen tension during sham culture do not alter medium composition.

Background: The extended embryo culture using single-step medium gained popularity in clinical in vitro fertilisation (IVF). However, there are concerns about the degradation of unstable medium components and their negative effects on the developing embryos. Further, dry-incubation can increase osmolality, which can in-turn enhance the concentration of constituents of the media and their stability. Hence, this study was conducted to understand the immediate changes in the culture media constituents in relation to clinically comparable situations such as single-step extended embryo culture and use of dry and humidified-incubation in two-different gaseous conditions. Methods: Commercially available single-step medium was sham-cultured in droplets under oil in two different conditions viz. dry (37°C; 6%CO 2; 5%O 2) and humidified (37°C; 6% CO 2; atmospheric O 2) for 0h, 72h, and 120h intervals. Droplets were subjected to the sensitivity-enhanced nuclear magnetic resonance (NMR)-based profiling using 800 MHz NMR equipped with a cryogenically cooled micro-coil (1.7mm) probe. NMR profile of the embryo culture medium between the two groups were comprehensively assessed. Results: A total of ten amino acids and four energy substrates were identified from the culture medium. The medium constituents identified showed a non-significant increase in the dry-incubation group at 72h and then declined at 120h. Humidified incubation had no effects on the level of the identified medium constituents until 120h. No significant differences in the levels of medium constituents identified were observed between the dry and humidified-groups at various time-points tested. Conclusions: A non-significant variation in the levels of medium constituents observed in the dry-incubation of single-step medium most unlikely to influence a clinical outcome. However, the impact of these subtle changes on the (epi)genetic integrity of the embryos in a clinical set-up to be addressed.

Duration of dry and humidified incubation of single-step embryo culture medium and oxygen tension during sham culture do not alter metabolomics signature.

Background: The extended embryo culture using single-step medium gained popularity in clinical in vitro fertilisation (IVF). However, there are concerns about the degradation of unstable medium components and their negative effects on the developing embryos. Further, dry-incubation can increase osmolality, which can in-turn enhance the concentration of constituents of the media and their stability. Hence, this study was conducted to understand the immediate changes in the culture media metabolites in relation to clinically comparable situations such as single-step extended embryo culture and use of dry and humidified-incubation in two-different gaseous conditions. Methods: Commercially available single-step medium was sham-cultured in droplets under oil in two different conditions viz. dry (37°C; 6%CO 2; 5%O 2) and humidified (37°C; 6% CO 2; atmospheric O 2) for 0h, 72h, and 120h intervals. Droplets were subjected to the sensitivity-enhanced nuclear magnetic resonance (NMR)-based profiling using 800 MHz NMR equipped with a cryogenically cooled micro-coil (1.7mm) probe. Metabolomic signatures between the two groups were comprehensively assessed. Results: A total of ten amino acids and four energy substrates were identified from the culture medium. Metabolite levels showed a non-significant increase in the dry-incubation group at 72h and then declined at 120h. Humidified incubation had no effects on the level of the metabolite until 120h. No significant differences in the levels of metabolites were observed between the dry and humidified-groups at various time-points tested. Conclusions: A non-significant variation in the levels of metabolites observed in the dry-incubation of single-step medium most unlikely to influence a clinical outcome. However, the impact of these subtle changes on the (epi)genetic integrity of the embryos in a clinical set-up to be addressed.

An expert commentary on essential equipment, supplies and culture media in the assisted reproductive technology laboratory

The assisted reproductive technology (ART) laboratory is a complex system designed to sustain the fertilization, survival, and culture of the preimplantation embryo to the blastocyst stage. ART outcomes depend on numerous factors, among which are the equipment, supplies and culture media used. The number and type of incubators also may affect ART results. While large incubators may be more suitable for media equilibration, bench-top incubators may provide better embryo culture conditions in separate or smaller chambers and may be coupled with time-lapse systems that allow continuous embryo monitoring. Microscopes are essential for observation, assessment, and micromanipulation. Workstations provide a controlled environment for gamete and embryo handling and their quantity should be adjusted according to the number of ART cycles treated in order to provide a steady and efficient workflow. Continuous maintenance, quality control and monitoring of equipment are essential and quality control devices such as the thermometer, and pH-meter are necessary to maintain optimal culture conditions. Tracking, appropriate delivery and storage conditions, and quality control of all consumables are recommended so that adequate quantity and quality are available for use. Embryo culture media have evolved: preimplantation embryos are cultured either by sequential media or single-step media that can be used for interrupted or uninterrupted culture. There is currently no sufficient evidence that any individual commercially-available culture system is better than others in terms of embryo viability. In this review, we aim to analyze the various parameters that should be taken into account when choosing the essential equipment, consumables and culture media systems that will create optimal culture conditions and provide the most effective patient treatment.

Can Culture Media Impact Preimplantation Embryo Aneuploidy?

With continued improvements in blastocyst culture, cell sampling approaches, and genetic analysis platforms, the resulting improvements in embryo development and the resolution and accuracy of chromosome analysis have provided valuable insights into the preimplantation embryo. This includes the impact of in vitro culture conditions on chromosomal dynamics. Specifically, through analysis of embryo aneuploidy and mosaicism, a growing number of reports indicate that rates of chromosomal abnormalities can vary between IVF centers. Because differences in mosaicism reflect mitotic errors, this endpoint analysis suggests that IVF laboratory-controlled variables during embryo development may be influencing chromosome separation and segregation. A growing body of literature suggests that culture media may be one variable influencing preimplantation embryo aneuploidy and mosaicism. However, these data are far from definitive in demonstrating cause-and-effect. Whether reported differences may be due to media formulation, use of sequential media or single-step media, or uninterrupted culture approaches is unknown. Importantly, variables directly impacting media performance and embryo development, including pH, temperature, osmolality, and oxygen concentration, must also be considered and make it difficult to isolate the impact of culture media as the sole factor responsible. These IVF laboratory variables will be reviewed and literature suggesting a possible link to mitotic aneuploidy/mosaicism will be discussed.

Investigation of Stem Cell Applications on In Vitro Fertilization in Rats

We aimed to search the effects of bone marrow-derived mesenchymal stem cell-conditioned media on in vitro fertilization by investigation of lifetime of germ cells cleavage, degeneration rates and embryo quality. For this purpose, firstly MSCs were isolated from femurs and tibias of the rat, and cells were cultured until the fourth passage. Sperm and oocytes were collected from male and female rats. Oocytes were added in Human Tubal Fluid Media (HTFM), Single Step Media (SSM), Alpha-MEM Media (AMM) and Bone Marrow-Derived Mesenchymal Stem Cell-Conditioned Media (CM). Thousand sperm were added into the media which including oocytes. Embryos were allowed to produce by IVF. The development of the embryos was followed until the 11th day, and the arrest, degeneration rates and alive embryos were established. The embryos reached 2, 4, 8, 16 cells stages and morula stage in the CM. While AMM had a negative effect on fertilization and embryo development, the most favourable effect was shown to be caused by CM in comparison with the other medias. These results have shown that the beneficial effects of CM in IVF would be a significant increase in the rate of fertility and development of embryos.

Effect of sequential versus single-step culture medium on IVF treatments, including embryo and clinical outcomes: a prospective randomized study.

Sequential media G5 series (Vitrolife) and single-step medium Continuous Single Culture Complete (CSC-C) (Irvine Scientific) are two different culture media. We want to examine difference between culturing effects of the two media. To compare the fertilization and early embryo development, a prospective randomized controlled trial with sibling oocytes in infertile patients, aged ≤ 45years with ≥ 8 oocytes (226 cycles) was conducted. Each half of the retrieved oocytes from the same patient were randomly allocated to two culture media separately. The remaining fresh cycles were randomly assigned to two culture media during the same period (179 cycles). We compared the clinical outcomes based on the total fresh ET cycles in this periods, in which the transferred embryos were only from one culture medium. Embryo outcomes: 226 cycles, included 176 IVF and 50 ICSI cycles, were analyzed, which correspond to 3518 inseminated or micro-injected oocytes. 71 (CSC-C) and 71 (G5 series) fresh ET cycles were compared. There were no significant differences in clinical outcomes and general fertilization rate. However, the fertilization rate was superior in the CSC-C when compared with G5 in ICSI cycles (76.51% vs. 67.25%, P = 0.008). In addition, the compacted embryo development rate was significantly higher in CSC-C on day 3. The cycles that had compacted embryos on day 3 demonstrated better outcomes both in embryos as well as clinically. CSC-C had higher fertilization rates than G5 series in ICSI cycles. In addition, the compaction rates of day 3 embryos were significantly higher in CSC-C.