Blastocyst Medium Research Articles

Quantitative Proteomics and Metabolomics of Culture Medium from Single Human Embryo Reveal Embryo Quality-Related Multiomics Biomarkers.

An effective tool to assess embryo quality in the assisted reproduction clinical practice will enhance successful implantation rates and mitigate high risks of multiple pregnancies. Potential biomarkers secreted into culture medium (CM) during embryo development enable rapid and noninvasive methods of assessing embryo quality. However, small volumes, low biomolecule concentrations, and impurity interference collectively preclude the identification of quality-related biomarkers in single blastocyst CM. Here, we developed a noninvasive trace multiomics approach to screen for potential markers in individual human blastocyst CM. We collected 84 CM samples and divided them into high-quality (HQ) and low-quality (LQ) groups. We evaluated the differentially expressed proteins (DEPs) and metabolites (DEMs) in HQ and LQ CM. A total of 504 proteins and 189 metabolites were detected in individual blastocyst CM. Moreover, 9 DEPs and 32 DEMs were identified in different quality embryo CM. We also categorized HQ embryos into positive implantation (PI) and negative implantation (NI) groups based on ultrasound findings on day 28. We identified 41 DEPs and 4 DEMs associated with clinical implantation outcomes in morphologically HQ embryos using a multiomics analysis approach. This study provides a noninvasive multiomics analysis technique and identifies potential biomarkers for clinical embryo developmental quality assessment.

P-195 Euploid embryo transfer in a hyaluronic acid-enriched media results in a higher ongoing pregnancy rate than euploid embryo transfer in media not containing hyaluronic acid

Abstract Study question Will using a hyaluronan-rich transfer medium increase the ongoing pregnancy rate and live birth rate when transferring euploid embryos cultivated in a dry incubator? Summary answer Transferring euploid embryos in a media with high concentrations of hyaluronic acid increases the ongoing pregnancy rate compared with media with no hyaluronic acid. What is known already Cochrane Review found that solutions with high concentrations of hyaluronic acid increase the chance of live birth by adding probably 1 additional live birth for every 14 embryos transferred in a high-concentration hyaluronic acid solution for patients aged 27 to 35. A study involving 85 frozen embryo transfer cycles showed a positive effect of high concentrations of hyaluronic acid on the results of transfer of morphologically poor euploid blastocysts. Previous studies were conducted in humidified incubators where hyaluronic acid-rich media was equilibrated without oil overlay. Our clinic uses dry incubators, necessitating oil overlay with all media, including hyaluronic acid-rich media. Study design, size, duration A randomized prospective study involving 255 patients aged 19-44 undergoing a transfer of a single euploid embryo between June 2023 and November 2023. Patients were randomly assigned to the study and control groups. Blastocysts were incubated after thawing for 3 hours either in a hyaluronic acid-enriched medium or in a medium without hyaluronic acid. Participants/materials, setting, methods Embryos underwent PGT-A at the blastocyst stage on either day 5 or day 6, using the EmbryoMap method. Trophectoderm biopsy was performed using the Flicking technique without a laser. 5-7 cells were removed for analysis. Blastocysts were vitrified following biopsy. Hyaluronic acid-enriched media was equilibrated for 16-18 hours in a 5%O2 6%CO2 atmosphere in a well of 4-well culture dish under oil overlay (600 microliters of media and 500 microliters of oil). Main results and the role of chance In this ongoing study, 127 transfers of euploid blastocysts in a hyaluronic acid-rich media resulted in 70 ongoing pregnancies, with a 55.1% ongoing pregnancy rate. In comparison, 128 transfers of euploid blastocysts in a media with no hyaluronic acid resulted in 65 ongoing pregnancies, with a 50.8% ongoing pregnancy rate. The relative improvement in the ongoing pregnancy rate is 8.5%. Our data suggests that using a hyaluronic acid-rich medium for the transfer of euploid blastocysts of good morphology can improve clinical pregnancy rates, although the effect is less dramatic than was shown in a 2023 study by Nakagawa et al. for morphologically poor euploid blastocysts, where more than twofold increase in clinical pregnancy rates was detected. Limitations, reasons for caution Specific media equilibration protocol is required for the incubation and transfer of embryos in a hyaluronan-rich transfer medium for IVF clinics utilizing dry incubators. Further research is required to confirm results and to establish whether transferring embryos in hyaluronic acid-enriched media increases the live birth rate after euploid embryo transfer. Wider implications of the findings Our results show that it is feasible to use a hyaluronic acid-enriched medium in a dry incubator with an oil overlay and that using it according to described protocol could increase the ongoing pregnancy rate after a single euploid blastocyst transfer. Trial registration number not applicable

P-513 Non-invasive PGT (niPGT) of vitrified blastocysts cultured for 24 hours after thawing

Abstract Study question Is niPGT on culture medium exposed for 24 hours to vitrified-thawed blastocysts useful? Summary answer niPGT using a culture medium from vitrified blastocysts cultured for 24hours after thawing showed a high concordance rate with the PGTa results. What is known already Conventional PGTa, which is widely practiced around the world, requires specialized techniques and involves embryonic trophectoderm (TE) biopsy. Recently, niPGT using cell free DNA released from embryos into culture medium has been reported as an alternative to conventional PGT, and has been found to correlate well with TE-biopsy analysis results (Carmen. Rubio, 2020). Although there have been numerous reports of niPGT accuracy based on the timing of culture medium collection, there have been few comparisons of its accuracy based on different time points of medium collection from the same embryo. Study design, size, duration From March to April 2023, 128 vitrified pronuclear stage embryos from 33 patients with consent for research use, were thawed and cultured, and from Day 4, they were individually cultured for over 40 hours. 60 embryos that reached blastocyst by Day 6 were vitrified, and the spent culture medium (SCM1) was collected. The vitrified blastocysts were later thawed and cultured individually for 24 hours, and then the embryos and spent culture medium (SCM2) were collected. Participants/materials, setting, methods Embryos were cultured in a 30 μl droplet until Day 4 and a 10 μl droplet for individual culture. Culture medium was collected and embryos were subjected to chromosomal analysis by NGS. We compared the results of niPGT taken from the same embryos on days 4- 6 (SCM1) and after thawing of vitrified blastocysts (SCM2), with PGTa analysis of the embryos. Main results and the role of chance Among all media samples analyzed, results were obtained for 80.0% (48/60) SCM1 and 96.7% (58/60) SCM2 samples. The ploidy concordance rate was 81.3% (39/48) for SCM1 and 98.3% (57/58) for SCM2. The overall concordance rate, defined as the combined rate of the total concordance for all chromosomes and the partial concordance for some chromosomes was 72.9% (35/48) for SCM1 and 94.8% (55/58) for SCM2 respectively. Significant differences were identified in the informative sample rate, the ploidy concordance rate, and the overall concordance rate(p < 0.05) between SCM1 and SCM2. Fisher’s exact test was used for statistical analysis. Limitations, reasons for caution In this study, embryo culture was continued to day 6, regardless of morphology of the embryo at day 5, which differs from the usual clinical culture process. Additionally, the timing of sample collection differed between the day 4-6 culture medium sample and that for a blastocyst. Wider implications of the findings The results demonstrated that accuracy of ploidy status was higher in the SCM2 compared to the SCM1 sample, suggesting that analysis of thawed vitrified blastocyst medium maybe a more precise predictor and therefore a non-invasive alternative to conventional PGTa. Trial registration number not applicable

P-543 Multicenter concordance study of embryo cell-free DNA and trophectoderm biopsies: impact on clinical outcomes

Abstract Study question Are reproductive outcomes of transfers from euploid day-6 biopsied blastocysts different depending on results of the corresponding embryo cell-free DNA (cfDNA) in the culture medium? Summary answer Transfers with euploid trophectoderm biopsies but aneuploid cfDNA showed higher miscarriage rates than those with euploid trophectoderm biopsies and cfDNA, although without reaching statistical significance. What is known already During development, embryos release cfDNA. There is, therefore, genetic material in the droplet of culture medium in which they develop. When analyzing, for the same embryo, the spent blastocyst medium containing cfDNA, trophectoderm (TE) and inner cell mass biopsies or the whole blastocyst, high concordance rates have been observed. This high representativeness shown by cfDNA on the chromosomal content of the blastocysts opens a new era of possibilities for non-invasive preimplantation genetic testing for aneuploidy (niPGT-A) as its analysis avoids the need of an invasive trophectoderm biopsy for aneuploidy detection. Study design, size, duration Prospective, observational, multicenter study conducted in 10 assisted reproductive centers after approval by each local institutional review board. From April 2018 to December 2022, 2539 embryos from 716 patients included in PGT-A cycles were cultured following a niPGT-A culture protocol. Blastocysts underwent TE biopsy and media collection that were subsequently analyzed and compared. 441 frozen single embryo transfers (SET) from euploid embryos, based on the TE biopsy result, were performed and their clinical data registered. Participants/materials, setting, methods Embryos were cultured in routine conditions up to day 4, then washed and transferred to a new 10μl droplet. On day 6, media were collected and frozen at -20 °C; and blastocyst biopsy and vitrification were performed. Samples were analyzed by NGS (Ion ReproSeq PGS kit, ThermoFisher Scientific) and the results processed with customized algorithms for TE/cfDNA. SET were performed based on TE biopsy results, with blinded cfDNA results at the moment of transfer. Main results and the role of chance Clinical outcomes were compared according to whether the TE and cfDNA result was euploid (eu-eu) or with euploid TE and aneuploid cfDNA (eu-aneu). Transfers where cfDNA analysis had provided non-informative results were excluded. The eu-eu group included 288 transfers (mean female age = 35.3 ± 4.9 years), with an ongoing pregnancy rate (OPR) of 44.4% and a miscarriage rate (MR) of 14.7%. Whereas the eu-aneu group included 95 transfers (mean female age = 34.4 ± 5.2 years), with an OPR of 43.2% and a MR of 25.5%. The comparisons of OPR and MR between both groups did not reach statistical significance (Chi-square test), but MR showed a two-fold increase in transfers with aneuploid cfDNA. A sub-analysis was performed, while only considering transfers from patients without endometrial factor history. Eu-eu and eu-aneu groups included 231 and 73 transfers, respectively. The differences in OPR (49.4% vs 43.8%) and MR (14.3% vs 25.6%) were slightly higher between both groups but did not reach statistical significance (Chi-square test). Limitations, reasons for caution To better understand the trends observed, especially on the miscarriage rate, it would be necessary to collect clinical outcomes from more SET. This study could not determine the benefit of embryo selection based only in cfDNA, since SET was performed according to TE biopsy results. Wider implications of the findings The analysis of embryo cfDNA could provide useful information to help select the best embryo for transfer and, therefore, decrease miscarriage rates and increase the chances of having a healthy newborn at home. Trial registration number NCT03520933

Artificial shrinkage before fresh blastocyst transfer and IVF outcomes: a pilot randomized controlled study

Research questionDoes artificial shrinkage before fresh blastocyst transfer improve clinical pregnancy rates in IVF? DesignIn this monocentric prospective, randomized, double-blind, controlled pilot study, 150 couples undergoing fresh single-blastocyst transfer were randomized between 20 May 2018 and 22 February 2022. In the artificial shrinkage group (AS group), a single laser pulse was directed to the cellular junction of the trophectoderm on the opposite side of the inner cell mass in each blastocyst. IVF outcomes were clinical pregnancy, multiple pregnancy and live birth rates. Cell-free DNA (cfDNA) concentration was also measured by quantitative real-time PCR in the blastocyst culture medium. ResultsIn total, 142 couples underwent fresh single-blastocyst transfer: control group, no artificial shrinkage, n = 47; and AS group, artificial shrinkage, n = 95; An intention-to-treat (ITT) analysis was employed. After a reassessment and the exclusion of patients with major protocol deviations, 139 couples underwent fresh single-blastocyst transfer under optimal conditions: control group, n = 47; and AS group, n = 92; a per-protocol analysis was used here. The clinical and laboratory characteristics were not significantly different between the groups. The clinical pregnancy rate was similar in the control and AS groups (ITT: 48.9% versus 49.5%, P = 0.97; per protocol: 48.94% versus 51.1%, P = 0.89). The multiple pregnancy rate and the live birth rate were also similar between the groups. No significant differences in gestational age, birthweight or proportion of male/female newborns were observed. The concentration of cfDNA in the blastocyst culture medium was not associated with IVF outcome. ConclusionsLarge-scale randomized controlled trials are required to confirm these preliminary results.

Development of porcine embryos cultured in media irradiated with ultraviolet-C.

Sterilization of the culture medium using ultraviolet (UV)-C reduces the potential adverse effects of microorganisms and allows for long-term use. In the present study, we investigated the effects of a medium directly irradiated with UV-C prior to invitro culture on the development and quality of porcine invitro-fertilized embryos and the free amino acid composition of the culture media. The culture media (porcine zygote medium [PZM-5] and porcine blastocyst medium [PBM]) were irradiated with UV-C at 228 and 260 nm for 1 and 3 days, respectively. Next, the culture media were irradiated with UV-C at 228 nm for 3, 7, or 14 days. After invitro fertilization, the embryos were cultured in the UV-C-irradiated media for 7 days. Free amino acid levels in culture media irradiated with 228 and 260 nm UV-C for 3 days were analysed. The blastocyst formation rate of embryos cultured in media irradiated with 260 nm UV-C for 3 days was significantly lower than that of embryos cultured in non-irradiated control media. However, 228 nm UV-C irradiation for up to 14 days did not affect blastocyst formation rates and quality in the resulting blastocysts. Moreover, 260 nm UV-C irradiation significantly increased the taurine concentration in both culture media and decreased methionine concentration in the PBM. In conclusion, UV-C irradiation at 228 nm before invitro culture had no detrimental effects on embryonic development. However, 260 nm UV-C irradiation decreased embryo development and altered the composition of free amino acids in the medium.

#237 : The Application of Noninvasive Implantation Capability Screening (NICS®) Resulted in Improved Clinical Outcomes in Patients with Recurrent Pregnancy Loss or Repeated Implantation Failure

Background and Aims: Chromosomal abnormalities are frequently observed in early human embryos, particularly in patients experiencing repeated pregnancy loss (RPL) or repeated implantation failure (RIF). Although preimplantation genetic testing for aneuploidy (PGT-A) is a common technique for screening embryo ploidy, it comes with the risks associated with embryo biopsy. To address this issue, we developed the NICS assay, which evaluates embryos using spent culture medium (SCM) and thus reduces the risks associated with embryo biopsy. However, further evaluation is required to determine the clinical applicability of NICS for patients with RPL or RIF. Method: This retrospective cohort study analyzed 303 patients with RPL or RIF between July 2018 and May 2021. These patients underwent aneuploidy screening using the NICS assay and were thus categorized into the NICS group. The non-NICS group consisted of patients who received conventional morphology embryo transfer during the same period. Routine intracytoplasmic sperm injection (ICSI) was performed, and the embryos were placed in droplets. Approximately 30 [Formula: see text] L of blastocyst medium from each embryo was transferred into cell lysis buffer, followed by DNA library preparation using the ChromInst kit from Yikon Genomics. Results: In patients with RPL, the NICS group demonstrated significantly lower rates of miscarriage per frozen embryo transfer (FET) than the non-NICS group, 17.9% vs 42.6%. Furthermore, the NICS group exhibited significantly higher rates of ongoing pregnancies (40.7% vs 25.0%) and live births (38.9% vs 20.6%) compared to the non-NICS group. In the case of patients with RIF, the NICS group showed higher clinical pregnancy rates per FET compared to the non-NICS group. Specifically, the clinical pregnancy rates, 46.9% vs 28.7%. Conclusion: The present study demonstrates that the use of NICS for selecting euploid embryos can effectively reduce the incidence of miscarriage in patients experiencing RPL, as well as improve the clinical pregnancy rate in those experiencing RIF.

Effect of noninvasive embryo viability testing versus conventional IVF on the live birth rate in IVF/ICSI patients: a study protocol for a double-blind, multicenter, randomized controlled trial

BackgroundPreimplantation genetic testing for aneuploidy (PGT-A) was demonstrated to be superior to conventional IVF in reducing the incidence of miscarriage and abnormal offspring after the first embryo transfer (ET). PGT-A requires several embryo trophectoderm cells, but its negative impacts on embryo development and long-term influence on the health conditions of conceived children have always been a concern. As an alternative, noninvasive PGT-A (niPGT-A) approaches using spent blastocyst culture medium (SBCM) achieved comparable accuracy with PGT-A in several pilot studies. The main objective of this study is to determine whether noninvasive embryo viability testing (niEVT) results in better clinical outcomes than conventional IVF after the first embryo transfer. Furthermore, we further investigated whether niEVT results in higher the live birth rate between women with advanced maternal age (AMA, > 35 years old) and young women or among patients for whom different fertilization protocols are adopted.MethodsThis study will be a double-blind, multicenter, randomized controlled trial (RCT) studying patients of different ages (20–43 years) undergoing different fertilization protocols (in vitro fertilization [IVF] or intracytoplasmic sperm injection [ICSI]). We will enroll 1140 patients at eight reproductive medical centers over 24 months. Eligible patients should have at least two good-quality blastocysts (better than grade 4 CB). The primary outcome will be the live birth rate of the first embryo transfer (ET). Secondary outcomes will include the clinical pregnancy rate, ongoing pregnancy rate, miscarriage rate, cumulative live birth rate, ectopic pregnancy rate, and time to pregnancy.DiscussionIn this study, patients who undergo noninvasive embryo viability testing (niEVT) will be compared to women treated by conventional IVF. We will determine the effects on the pregnancy rate, miscarriage rate, and live birth rate and adverse events. We will also investigate whether there is any difference in clinical outcomes among patients with different ages and fertilization protocols (IVF/ICSI). This trial will provide clinical evidence of the effect of noninvasive embryo viability testing on the clinical outcomes of the first embryo transfer.Trial registrationChinese Clinical Trial Registry (ChiCTR) Identifier: ChiCTR2100051408. 9 September 2021.

P-231 The effect of warming blastocysts in Vitrolife media previously vitrified in Cook media on reproductive potential and clinical outcome

Abstract Study question Does the use of a different manufacturer’s media for vitrification and warming of blastocysts affect pregnancy, miscarriage and live birth rates. Summary answer Warming blastocysts in Vitrolife media, previously vitrified in Cook media, does not significantly affect the pregnancy or live birth rate. What is known already Current good practice is to vitrify and warm blastocysts in media manufactured by the same company. This is due to the optimisation of the protocols and media composition intending to maintain high survival rates and associated pregnancy rates. However, circumstances can arise, such as supply and production issues, whereby it is not possible to source the same media for warming vitrified blastocysts as the media they were vitrified in. Study design, size, duration This retrospective cohort study was conducted at TFP-Oxford Fertility. Frozen embryo transfer (FET) cycles included in the study were performed between 2018 and 2022. Embryos that had undergone genetic testing were excluded from the study. The mean women's age was 35 in the control group (Cook warming media) and 36 in the study group (Vitrolife warming media). The average number of embryos transferred was 1.06 vs 1.17 in control vs study group. Participants/materials, setting, methods For the present study 817 patients underwent FET between 2016 and 2018 whereby blastocysts were vitrified in cook media (Sydney IVF Blastocyst Vitrification Kit, K-SIBV-5000) and warmed in cook media (Sydney IVF Blastocyst Warming Kit, K-SIBW-5000). In comparison 704 patients underwent a FET between 2018 and 2022 whereby blastocysts were vitrified in cook media and warmed in vitrolife warming media (Rapidwarm™ Blast, 10120). The pregnancy, clinical pregnancy and live birth rates were compared. Main results and the role of chance Fisher’s exact test was used to calculate the p value and a level of 0.05 was used for significance. The results showed that there was no significant difference in the pregnancy rate, clinical pregnancy rate or live birth rate for blastocysts warmed in Vitrolife media vs Cook media (59.0% vs 57.0% p = 0.56, 47.2% vs 43.9% p = 0.44, 39.3% vs 37.0% p = 0.72) Limitations, reasons for caution This is a retrospective study at a single centre. The number of previous treatment cycles per couple was not included in the study. The study only looked at a comparison of Cook and Vitrolife warming media, no other commercially available media was tested. Wider implications of the findings Although good practice to vitrify and warm blastocysts in the same manufacturer media, our results show that the reproductive potential and clinical outcome may not be adversely affected by using a different manufacturer’s media. However, composition of the media and protocol should be considered and further studies should be conducted. Trial registration number not applicable

P-138 Protein concentration of 5 mg/mL in recovery medium for thawed blastocysts is equivalent to 10 mg/mL in post-thaw survival and pregnancy rates

Abstract Study question Are comparable survival and pregnancy rates obtained when recovery media are supplemented with 5mg/mL protein versus customary 10mg/mL protein in Frozen Embryo Transfer cycles? Summary answer Recovery medium containing 5mg/mL protein compared to 10mg/mL yielded similar outcomes in blastocyst survival and pregnancy rates. What is known already Accepted practice has been to culture thawed blastocysts in medium with the same protein concentration as the vitrification media (20% protein) to minimize osmotic stress and optimize survival and implantation rates. The potential to acquire similarly positive outcomes with supplementation of only 5 mg protein/ml has been explored and demonstrated, challenging the requirement for high protein levels in recovery media. Study design, size, duration From July through August 2022, a prospective study was conducted in a university laboratory to compare the survival and implantation rates in Frozen Embryo Transfer cycles using vitrified blastocysts recovered in Continuous Single Culture–NX Complete (CSCM-NXC) (5mg/mL Human Serum Albumin (HSA)) or global HP[LW1] (8.8mg/mL HSA and 1.2mg/mL α-and ß-globulins). 50 blastocysts were warmed per condition. Comparative analyses included survival and implantation rates, beta HCG levels, and clinical pregnancy rates by fetal cardiac activity. Participants/materials, setting, methods To evaluate the effect of protein level in recovery medium for vitrified blastocysts, CSCM-NXC and global HP, with 5mg/mL and 10mg/mL total protein, respectively, were compared. Blastocysts were vitrified in Irvine Scientific Vit Kit – Freeze and were warmed in Irvine Scientific Vit Kit – Warm. Minimum recovery time was the same for all blastocysts (30 minutes), and is defined as the time from completion of the warming procedure to the time of embryo transfer. Main results and the role of chance In both study arms, one blastocyst was removed from the data analysis due to embryo transfer cancellation that was not related to embryo survival or suitability for transfer, resulting in N = 49 for each group. Chi-square analysis was performed to assess statistical significance of all compared parameters, and a p value < 0.05 was considered significant. Average patient age (years) for CSCM-NXC was 36.1 and for global HP, 35.0. There was no difference between blastocysts warmed in CSCM-NXC and global HP in average recovery time, i.e., 3.8 and 3.9 hours, respectively, or survival rates, 98.4% and 98.1% respectively. Comparison of patients with blastocysts warmed in CSCM-NXC reflected a trend towards higher outcomes compared to those in global HP with initial positive beta HCG rates of 64.6% vs 56.3% and ongoing clinical pregnancy rates of 52.1% vs 45.8%, respectively. Implantation rates were 56.1% and 57.7% in CSCM-NXC and global HP, respectively. Incidence of monozygotic twinning of 0% in CSM-NXC and 3.3% in Global HP was observed. Limitations, reasons for caution As this is a challenge to accepted formulation of recovery media for vitrified blastocysts, specifically with respect to protein supplementation - a strategy for minimizing osmotic shock and potential damage to warming embryos - more expansive and robust studies must be undertaken to increase the statistical power of this study. Wider implications of the findings As frozen embryo transfers are utilized more routinely in assisted reproductive treatment, the clinical finding that a commercial recovery medium containing of 5mg/mL protein, the standard for routine embryo culture, can support the recovery of vitrified embryos and lead to successful pregnancy could increase the efficiency in the laboratory. Trial registration number na

O-008 The composition of commercially available human preimplantation embryo culture media

Abstract Study question What is the composition of currently available commercial human preimplantation embryo culture media from eleven different brands for each stage of preimplantation embryo development? Summary answer Besides similarities in embryo culture medium composition between brands, there were also differences in composition for which there does not seem to be a rationale. What is known already Although a lot remains uncertain, there seems to be some consensus in the scientific literature on the environmental and nutritional needs of preimplantation embryos in in vitro culture, at least for certain components. Since suppliers do not disclose the exact formulations of their media, it is unclear whether the embryo culture media currently used in IVF laboratories meet these suggested conditions. We previously published composition analyses of a limited number of culture media, but have now repeated this analysis and have included a larger number of media from eleven brands of human preimplantation embryo culture media. Study design, size, duration Forty-seven human embryo culture media and protein supplements were purchased between December 2019 and June 2020. Upon arrival, each complete medium (n = 23), unsupplemented medium (n = 14), and supplement (n = 10) was aliquotted in Eppendorf tubes, snap frozen in liquid nitrogen and stored in -80oC until composition analysis in November 2021. Additionally, unsupplemented media were supplemented with each available supplement from the same brand (n = 33 combinations) and then also aliquotted, snap frozen and stored in -80oC until analysis. Participants/materials, setting, methods The concentration of forty components was determined in all collected samples (n = 80). Seven ions, glucose, three immunoglobulins, uric acid, alanine aminotransferase (ASAT), aspartate aminotransferase (ALAT), albumin, and the total amount of proteins were determined in each sample using a Cobas 8000 Analyser (Roche Diagnostics). Analysis of pyruvate, lactate, carnitine and twenty-one amino acids was achieved with Ultra-high Performance Liquid Chromatography-Mass Spectometry (UPLC-MS/MS). Main results and the role of chance Our analysis of the concentration of 40 components in ready to use embryo culture media of most available brands on the market showed that in general the culture media follow generally accepted assumptions on the changing needs of an embryo during early development. However, there were clear differences between brands, and no two embryo culture media were the same. For example, pyruvate concentrations were higher in fertilization and cleavage stage media and lower in blastocyst stage media, whereas glucose was relatively high in fertilization media, lower in cleavage stage media, and higher again in blastocyst media. This follows previous findings that demonstrate that pyruvate is the main energy source until blastocyst stage and that glucose becomes the main energy source for blastocysts. Other components, like lactate for example, followed different patterns in the sequential media of different brands, and concentrations also differed between brands. The composition of continuous media resembled the composition of the other media of the same brand, but differed between brands. Interestingly, some of the culture media brands belong to the same parent company, but differ in their composition. The rationale of such differences is not clear. Research providing such rationale seems warranted. Limitations, reasons for caution The analysed embryo culture media may contain other components that were not investigated in this study. Wider implications of the findings Detailed information on human embryo culture medium composition, including the exact concentrations of each ingredient, is much needed to in the end be able to understand its effect on IVF outcomes, and to facilitate the scientific improvement of media for human preimplantation embryo culture. Trial registration number not applicable

O-291 Clinical Application of Noninvasive PGT for Patients with Recurrent Pregnancy Loss or Repeated Implantation Failure

Abstract Study question Has noninvasive PGT been allied with the patients experiencing recurrent pregnancy loss (RIF) and recurrent implantation failure (RPL)? What are the clinical beneficiaries? Summary answer Embryo selection through noninvasive PGT can reduce the miscarriage rate in patients experiencing RPL and improve the clinical pregnancy rate in patients experiencing RIF. What is known already Chromosomal abnormalities exist in early human embryos, especially in patients with RPL and RIF. Therefore, embryos are usually evaluated through pre-implantation genetic testing for aneuploidy (PGT-A). However, a biopsy is an important concern for undetermined health risks. Meanwhile, the researchers observed genomic DNA contents in the embryo culture medium. Since then, multiple studies have been published using culture medium for analyzing chromosomal ploidy. In 2016, Xu et al. first reported a noninvasive chromosome screening (NICS) assay using a spent blastocyst culture medium. Nevertheless, the clinical application of NICS has not been evaluated in patients experiencing RPL or RIF. Study design, size, duration We designed a retrospective cohort study including 303 subjects from July 2018 to May 2021, according to the records of the Reproductive Centre at the Second Affiliated Hospital of Wenzhou Medical University. Patients experiencing RPL or RIF who received the NICS for aneuploidy were included in the NICS group, while those who underwent conventional morphology embryo transfer during the same period were included in the non-NICS group. Participants/materials, setting, methods We included women with a history of RPL (≥2 pregnancies) or RIF (≥3 implantations), exclusion criteria were antiphospholipid syndrome (APS), diabetes, hypothyroidism, or other severe complications. Routine IVF/ICSI was performed based on sperm quality. The embryos were placed in droplets. Approximately 30 mL of blastocyst medium from each embryo was transferred into cell lysis buffer. Whole-genome amplification was performed using culture media, followed by library preparation using ChromInst (Yikon Genomics; EK100100724 NICS Inst Library Preparation Kit). Main results and the role of chance For the patients experiencing RPL, the miscarriage rate per FET was significantly lower in the NICS group than in the non-NICS group (17.9% vs. 42.6%), whereas the ongoing pregnancy rate (40.7% vs. 25.0%) and live birth rate (38.9% vs. 20.6%) were significantly higher in the NICS group compared to the non-NICS group. Nevertheless, no differences were identified in pregnancy rates per patient between the NICS and non-NICS groups (49.6% vs. 44.9%). For the patients experiencing RIF, the pregnancy rates per FET were significantly higher in the NICS group than in the non- NICS group (46.9% vs. 28.7%), whereas the live birth rate and ongoing pregnancy rate per FET and per patient were no significant difference in the NICS group than in the non-NICS group. Nevertheless, no differences were identified in the miscarriage rate per clinical pregnancy between the NICS and non-NICS groups (23.3% vs. 25.9%). Limitations, reasons for caution As a retrospective study, patients in the NICS groups had different clinical prognoses than those in the non-NICS groups, introducing bias in the study results. Wider implications of the findings Non-invasive PGT can be used not only for aneuploidy detection but also for a comprehensive evaluation of morphology and DNA concentration, mosaic ratio, resolution, etc. The accumulation of clinical outcomes can also be combined with the clinical data of patients as an index to predict the clinical outcomes of embryos. Trial registration number NA

Liquid storage of porcine in vitro-produced blastocysts; a practical approach for short storage.

Commercial application of embryo transfer in pig breeding is dependent on the storage of embryos. The aim of this study was to assess the embryo quality of in vitro-produced blastocysts after 3 h liquid storage at 37°C in CO2-free medium by evaluating morphology, in vitro developmental capacity and apoptosis. Blastocysts at days 5 and 6 post-fertilization were randomly allocated to the storage group (HEPES-buffered NCSU-23 medium including bovine serum albumin in a portable embryo transport incubator at 37°C) or a control group (porcine blastocyst medium in a conventional culture incubator). Thereafter, blastocysts were evaluated for morphology and stained to assess apoptosis straight after the 3 h storage period or after a further 24 h conventional incubation. There was no significant difference between the storage and control group after 3 h storage and the further 24 h conventional incubation for any of the parameters, nor for apoptosis straight after the 3 h storage. Embryos that reached the blastocyst stage at day 5 showed less apoptosis (6.6% vs 10.9%, P = 0.01) and a trend for a higher rate of developmental capacity (70.6% vs 51.5%, P = 0.089) than embryos reaching the blastocyst stage on day 6. In conclusion, in vitro-produced porcine blastocysts can be stored for 3 h at physiological temperature in transportable incubators using a CO2-independent medium without compromising quality.

Glycans in spent embryo culture medium are related to the implantation ability of blastocysts

Research questionDoes glycan profile in spent blastocyst culture medium have the potential to be used as a biomarker to predict implantation outcome. DesignA nested case-control study was conducted in Northwest women’s and children’s Hospital, Xi’an, China. The patients underwent fresh IVF/ICSI cycles with single blastocyst transfer were included. Total 78 cases were included and separated into groups according to success (n = 39) and failure (n = 39) implantation outcomes. The glycosylation patterns in spent blastocyst culture medium were detected by lectin microarray containing 37 lectins using pooled samples and confirmed by reversed lectin microarray using individual sample. ResultsBinding signals of 10 lectins were found to be different between samples from successful and failed implantation. And 8 of them were confirmed that glycans binding to lectin NPA, UEA-I, MAL-I, LCA and GNA were significantly increased while DBA and BPL were decreased in the successful implantation compared to failed implantation. The glycan binding to lectin PHA-E + L had no difference between two groups. No significant differences in the glycan profile were found in spent culture medium of embryos with different morphological grades except the glycan binding to UEA-I between blastocysts of Poor and blastocysts of Medium. ConclusionDetection of glycan profile in spent culture medium may lead to a novel non-invasive assessment assay of embryo viability. In addition, these results may be helpful to further understanding molecular mechanisms in embryo implantation.

Implementation of Noninvasive Preimplantation Genetic Testing for Aneuploidy with Spent Blastocyst Medium into Clinical and Laboratory Practice: The First Successful Experience in Taiwan

Noninvasive preimplantation genetic testing for aneuploidy (niPGT-A) using cell-free DNA (cfDNA) present in the spent blastocyst medium (SBM) of human blastocysts has emerged as an alternative for classical preimplantation genetic testing for aneuploidy (PGT-A). The major benefit is to avoid possible damage to the trophectoderm and subsequently increase the implantation potential of the embryo. niPGT-A provides more information including the ploidy state of both trophectoderm and inner cell mass compared to invasive PGT-A in which only a few trophectoderm cells are assessed. This study was to report our first successful experience solely using niPGT-A to select embryos to transfer resulting in a successful delivery. This practical application is believed to be the first case report in Taiwan and can serve as an innovative model for other clinics to apply niPGT-A.

A comprehensive characterization of cell-free RNA in spent blastocyst medium and quality prediction for blastocyst.

The rate of pregnancy can be affected by many factors in assisted reproductive technology (ART), and one of which is the quality of embryos. Therefore, selecting the embryos with high potential is crucial for the outcome. Fifteen spent blastocyst medium (SBM) samples were collected from 14 patients who received in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), seven from high-grade embryos and eight from low-grade embryos. Cell-free RNA (cf-RNA) profile of SBM samples were analyzed by RNA sequencing in the present study. It was found that a large amount of cf-RNA were released into SBM, including protein-coding genes (68.9%) and long noncoding RNAs (lncRNAs) (17.26%). Furthermore, a high correlation was observed between blastocyst genes and SBM genes. And the cf-mRNAs of SBM were highly fragmented, and coding sequence (CDS) and untranslated (UTR) regions were released equally. Two hundred and thirty-two differentially expressed genes were identified in high-grade SBM (hSBM) and low-grade SBM (lSBM), which could be potential biomarker in distinguishing the embryos with different quality as an alternative or supplementary approach for subjective morphology criteria. Hence, cf-RNAs sequencing revealed the characterization of circulating transcriptomes of embryos with different quality. Based on the results, the genes related to blastocyst quality were screened, including the genes closely related to translation, immune-signaling pathway, and amino acid metabolism. Overall, the present study showed the types of SBM cf-RNAs, and the integrated analysis of cf-RNAs profiling with morphology grading displayed its potential in predicting blastocyst quality. The present study provided valuable scientific basis for noninvasive embryo selection in ART by RNA-profiling analysis.

MiRNAs secreted by human blastocysts could be potential gene expression regulators during implantation.

Micro RNAs (miRNAs) are small non-coding RNAs known as essential regulators of cell-cell communication. Recent studies have revealed that miRNAs are secreted by a blastocyst in culture media. We hypothesized that endometrial epithelial cells take up embryo-derived miRNAs as well as other soluble factors and regulate their receptivity-related gene expression. Blastocyst culture media (BCM) were collected from the individually cultured embryos, while human endometrial epithelial cells (HEECs) were collected from healthy fertile volunteers. To evaluate the effect of BCM on the endometrial receptivity gene expression, HEECs were co-cultured with implanted BCM, non-implanted BCM, and a control culture medium. After determining altered gene expression in the HEECs, the miRNAs-related genes through bioinformatics databases were identified and evaluated in the BCM. Co-culture of primary HEECs with BCM significantly stimulated the expression levels of VEGFA, HBEGF, HOXA10, and LIF in the implanted group compared with non-implanted and control groups. The fold changes of miR-195 significantly diminished in the implanted BCM group compared with the non-implanted BCM group. Reduced fold changes of miR-29b, 145 and increased miR-223 were also observed in the implanted BCM group compared with the non-implanted ones. miRNAs could function as potential gene expression regulators during implantation. These molecules are secreted by human blastocyst, taken up by endometrial epithelial cells, and cause a change in the endometrial function. We found that BCMs can be effective in implantation process by stimulating related receptivity gene expression.

Concordance of PGT for aneuploidies between blastocyst biopsies and spent blastocyst culture medium

Research questionNon-invasive preimplantation genetic testing for aneuploidies (niPGT-A) avoids the possible detrimental impact of invasive PGT-A on embryo development and clinical outcomes. Does cell-free DNA (cfDNA) from spent blastocyst culture medium (BCM) reflect embryonic chromosome status better than trophectoderm (TE) biopsy? DesignIn this study, 35 donated embryos were used for research and the BCM, TE biopsy, inner cell mass (ICM) and residual blastocyst (RB) were individually picked up from these embryos. Whole genome amplification (WGA) was performed and amplified DNA was subject to next-generation sequencing. Chromosome status concordance was compared among the groups of samples. ResultsThe WGA success rates were 97.0% (TE biopsy), 100% (ICM), 97.0% (RB) and 88.6% (BCM). Using ICM as the gold standard, the chromosomal ploidy concordance rates for BCM, TE biopsy and RB were 58.33% (14/24), 68.75% (22/32) and 78.57% (22/28); the diagnostic concordance rates were 83.33% (20/24), 87.50% (28/32) and 92.86% (26/28); and the sex concordance rates were 92.31% (24/26), 100% (32/32) and 100% (28/28), respectively. Considering RB the gold standard, the chromosome ploidy concordance rates for BCM and TE biopsy were 61.90% (13/21) and 81.48% (22/27); the diagnostic concordance rates were 71.43% (15/21) and 88.89% (24/27); and the sex concordance rates were 91.30% (21/23) and 100% (27/27), respectively. ConclusionsThe results of niPGT-A of cfDNA of spent BCM are comparable to those of invasive PGT-A of TE biopsies. Modifications of embryo culture conditions and testing methods will help reduce maternal DNA contamination and improve the reliability of niPGT-A.

Non -invasive embryo selection strategy for clinical IVF. New prioritization tool in embryo transfer

Background and Aim: The biggest challenges in IVF is selecting viable embryos that are most likely to produce a healthy child at home. A non-invasive alternative is to analyze (the cfDNA) in blastocyst culture medium. Several studies have shown that cfDNA testing on blastocyst culture medium at day 6 of development allows detection of aneuploidies with high concordance rates compared to TE biopsy and inner cell mass. The aim is if the niPGTA could be new prioritization tool in embryo transfer compared to morphological embryo selection without aneuploidy testing.

Developmental potential of different embryos on day 3: a retrospective study

To investigate how different quality of day 3 (D3) embryos affect blastocyst formation and clinical outcomes. This retrospective study analysed 699 patients undergoing assisted reproductive technology (ART) between January 2017 and February 2021. A total of 2517 D3 embryos were transferred to blastocyst medium for extended culture. D3 embryos were divided into five groups. Grade A, 6–10 cells, symmetrical blastomeres and <20% fragmentation; grade B, 6–10 cells, uneven blastomeres and ≥20% fragmentation; grade C, >10 cells, symmetrical blastomeres and <20% fragmentation; grade D, >10 cells, uneven blastomeres and ≥20% fragmentation; grade E, <6 cells. Status of day 5 (D5) and day 6 (D6) blastocysts and the clinical outcomes (blastocyst transfer) of each D3 embryo were recorded. The grade C group showed a higher D5 blastocyst formation rate and a high-quality blastocyst rate than other groups (p<.05). However, the clinical pregnancy rates in the grade A group were higher than other groups (p<.05). Embryos with low speed of development (grade E group) showed considerable clinical outcomes that were still worth investigating. D3 embryos with less fragmentation and ≥6 symmetrical blastomeres revealed a higher developmental potential, while embryos with 6–10 blastomeres showed the ideal clinical outcomes. Impact Statement What is already known on this subject? Accurate embryo evaluation can effectively reflect the developmental potential of different embryos. The number of blastomeres, proportion of fragmentation, and blastomere symmetry are three important and popular morphologic parameters used for evaluating day 3 (D3) embryos. However, in existing reports, combining these three parameters for embryo evaluation often results in different results. This is because different researchers have chosen different criteria for these three parameters. What do the results of this study add? In this retrospective study, we summarised the medical records of our reproductive centre in the past three years, redefined the evaluation method of the D3 embryos, and analysed the corresponding developmental potential and clinical outcomes. We conclude that although the embryonic development potential of grade C embryos (>10 cells, symmetrical blastomeres and/or <20% fragmentation) is relatively good, the results of grade A embryos (6–10 cells, symmetrical blastomeres and/or <20% fragmentation) are better in terms of clinical outcomes. What are the implications of these findings for clinical practice and/or further research? We believe this is meaningful for embryologists to choose embryos for transfer and predict the clinical outcome of IVF cycles.