Embryo Day Research Articles

Embryo Density and Its Neutrality in Day-3 Embryo Development: A Retrospective Cohort Study.

Introduction Many studies have shown that embryo density has an impact on day-3 embryo-developmental outcomes; however, embryo density remains controversial in clinical practice. We aimed to evaluate the association between embryo density and day-3 embryo-developmental outcomes in real world with the largest sample size. Methods In 2018, we identified 10941 day-3 embryos from all female patients (n = 1568) in the study. The embryos were allocated to three embryonic densities: 30 μl/embryo (individual culture), 15 μl/embryo, and 10 μl/embryo (group culture). The primary outcomes were cleaving speed, quality, and proportion of successful implantations. The generalized estimate equation (GEE) model was used both in the univariate analysis and multivariable logistic regression analyses to investigate the relationship between embryo density and embryo-developmental outcomes. Results There were 3064, 5695, and 2182 embryos in the 30 μl/embryo group, 15 μl/embryo group, and 10 μl/embryo group, respectively. The proportions of 7–10 cell embryos were 57.2%, 56.1%, and 58.3% in three densities with no statistical significance (P=0.37), respectively. The proportions of morphologically good embryos were 20%, 20.3%, and 20% in three densities with no statistical significance (P=0.85), respectively. Proportions of implanted embryos were 37.7%, 37.1%, and 27.8% with no statistical significance (P=0.36), respectively. After adjustment for confounders, which were significant in the univariate analysis, the embryo density was still not associated with day-3 embryo-cleaving speed, day-3 embryo quality, and day-3 embryo-implanting potential (all P > 0.05). Conclusion In a 30 μl microdrop, the culturing embryos with embryo densities of 15, 10, and 30 μl/embryo (from zygotes to day 3) had similar developmental outcomes. The embryo density had no impact on day-3 embryo development.

Whole-genome risk prediction of common diseases in human preimplantation embryos

Preimplantation genetic testing (PGT) of in-vitro-fertilized embryos has been proposed as a method to reduce transmission of common disease; however, more comprehensive embryo genetic assessment, combining the effects of common variants and rare variants, remains unavailable. Here, we used a combination of molecular and statistical techniques to reliably infer inherited genome sequence in 110 embryos and model susceptibility across 12 common conditions. We observed a genotype accuracy of 99.0–99.4% at sites relevant to polygenic risk scoring in cases from day-5 embryo biopsies and 97.2–99.1% in cases from day-3 embryo biopsies. Combining rare variants with polygenic risk score (PRS) magnifies predicted differences across sibling embryos. For example, in a couple with a pathogenic BRCA1 variant, we predicted a 15-fold difference in odds ratio (OR) across siblings when combining versus a 4.5-fold or 3-fold difference with BRCA1 or PRS alone. Our findings may inform the discussion of utility and implementation of genome-based PGT in clinical practice.

Value of Meiotic Spindle Imaging on Fertilization and Embryo Development Following Human Oocyte Piezo-ICSI

Background: The objective of this study was to investigate accurately the effect of the meiotic spindle imaging of human oocytes on their fertilization and embryo development by using the Piezo-ICSI.Methods: We retrospectively assessed 529 oocytes with the first polar body retrieved from 124 infertile couples (147 cycles) who attended our clinic between May 2016 and December 2018. Of these, 489 oocytes (92.4%) with visible meiotic spindle comprised the Spindle (+) group and 40 oocytes (7.6%) not observed meiotic spindle comprised the Spindle (−) group. The meiotic spindle was imaged using polarized light microscopy. The rates of fertilization, high-quality day-3 embryo, day-5 blastocyst, and high-quality day-5 blastocyst were evaluated in both groups.Results: The fertilization rates of Spindle (+) and Spindle (−) were 92.0% (450/489) and 70.0% (28/40), respectively; high-quality day-3 embryo rates were 62.9% (283/450) and 35.7% (10/28); day-5 blastocyst rates were 53.7% (205/382) and 32.1% (9/28); high-quality day-5 blastocyst rates were 29.8% (114/382) and 3.6% (1/28). Fertilization, high-quality day-3 embryo, day-5 blastocyst, and high-quality day-5 blastocyst rates were significantly higher in the Spindle (+) group than in the Spindle (−) group.Conclusion: Spindle imaging (i.e., oocytes with a visible meiotic spindle or with not observed meiotic spindle) influences the outcome of Piezo-ICSI in human oocytes, including fertilization and embryo development. To our knowledge, this study is the first to evaluate the effect of meiotic spindle imaging on fertilization or embryo development among oocytes after Piezo-ICSI. The meiotic spindle imaging could be the indicator for the quality management of medical doctors and embryologists.

Day-3 vs. Day-5 fresh embryo transfer.

Embryo transfer on day-5 has been associated with higher success rates, therefore our IVF clinics started to extend embryo culture until blastocyst stage. This study aimed to compare the success rates of day-3 vs. day-5 embryo transfers. We had 266 patients included, all having undergone ICSI, with 221 patients having undergone day-3 embryo transfers, and 45 patients having undergone day-5 embryo transfers. Patients with more than five good quality embryos on day-3 were chosen to prolong the culture of embryos into day-5. There were no significant differences in patient characteristics, including baseline LH, FSH, Prolactin and Estradiol hormone levels. In addition, there were also no significant differences in rFSH total dosage and duration of stimulation day. Final estradiol levels, number of follicles, retrieved oocytes, matured oocytes, fertilized oocytes and number of embryos were significantly higher in day-5 compared to day-3 embryo transfer groups. Number of embryos transferred on day-3, were significantly higher compared to day-5. Neither group showed any significant differences in clinical pregnancy, implantation, multiple pregnancy or living birth rates. There were no differences in birth weights and lengths, head circumstances and Apgar Scores between both groups either in singleton or twin group. Transferring embryos at day-3 may provide the same benefits as day-5 embryo transfers to patients. However, more embryos were required to be transferred to achieve these comparable results.

Effect of post-warming culture time on the live birth rate after frozen embryo transfer.

PurposeThis study evaluated the influence of post‐warming culture time on the live birth rate in day‐3 and day‐5 frozen embryo transfer (FET) cycles.MethodsThis multicenter, retrospective cohort study was performed at IVFMD, My Duc Hospital and IVFMD Phu Nhuan, My Duc Phu Nhuan Hospital in Vietnam between October 2019 and October 2020. Women who underwent FET cycles with the transfer of ≤2 day‐3 or day‐5 embryos were included in the study. FET cycles were divided into four groups based on the quartiles for the time between embryo warming and embryo transfer. The primary outcome was live birth after FET.ResultsOf 2548 FET cycles, 885 and 1663 cycles, respectively, had transfer of day‐3 or day‐5 embryos. Post‐warming culture time ranged from 0.07 to 6.1 h. There were no significant differences between the post‐warming culture time quartiles with respect to the number of embryos thawed, the number of embryos transferred, and the number of top‐quality embryos transferred. Post‐warming culture time was not significantly associated with the live birth rate in FET cycles using either day‐3 or day‐5 embryos.ConclusionsPost‐warming culture time did not affect live birth rate in FET cycles. Therefore, IVF centers should consider scheduling workflows to best suit the patient.

Non-invasive Metabolomic Profiling of Embryo Culture Medium Using Raman Spectroscopy With Deep Learning Model Predicts the Blastocyst Development Potential of Embryos.

Purpose: This study aimed to establish a non-invasive predicting model via Raman spectroscopy for evaluating the blastocyst development potential of day 3 high-quality cleavage stage embryos.Methods: Raman spectroscopy was used to detect the metabolic spectrum of spent day 3 (D3) embryo culture medium, and a classification model based on deep learning was established to differentiate between embryos that could develop into blastocysts (blastula) and that could not (non-blastula). The full-spectrum data for 80 blastula and 48 non-blastula samples with known blastocyst development potential from 34 patients were collected for this study.Results: The accuracy of the predicting method was 73.53% and the main different Raman shifts between blastula and non-blastula groups were 863.5, 959.5, 1,008, 1,104, 1,200, 1,360, 1,408, and 1,632 cm–1 from 80 blastula and 48 non-blastula samples by the linear discriminant method.Conclusion: This study demonstrated that the developing potential of D3 cleavage stage embryos to the blastocyst stage could be predicted with spent D3 embryo culture medium using Raman spectroscopy with deep learning classification models, and the overall accuracy reached at 73.53%. In the Raman spectroscopy, ribose vibration specific to RNA were found, indicating that the difference between the blastula and non-blastula samples could be due to materials that have similar structure with RNA. This result could be used as a guide for biomarker development of embryo quality assessment in the future.

Do clinical outcomes differ for day-5 versus day-6 single embryo transfers controlled for endometrial factor?

Research questionIs there is a difference in clinical outcomes between day-5 versus day-6 blastocysts when transferred in a personalized embryo transfer (PET) cycle guided by Endometrial Receptivity Analysis (ERA)? DesignMulticentre, retrospective study; 260 patients who underwent a single embryo transfer with either a day-5 or day-6 blastocyst in a PET cycle guided by ERA between January 2017 and December 2019. ResultsA total of 260 blastocysts were transferred in a single embryo PET cycle guided by ERA. Of those, 183 (70.4%) were day-5 blastocysts and 77 (29.6%) were day-6 blastocysts. Clinical outcomes were similar when transferring day-5 blastocysts versus day-6 blastocysts: pregnancy rate was 75.4% (138/183) and 70.1% (54/77) (P = 0.465); implantation rate was 67.8% (124/183) and 63.6% (49/77) (P = 0.476); and ongoing pregnancy rate was 57.9% (106/183) and 58.4% (45/77) (P = 0.728), respectively. ConclusionsThe data suggest that the clinical potential of day-5 and day-6 blastocysts are similar, as no difference in clinical outcomes are observed when transferring at the time of optimal endometrial receptivity as determined by ERA.

Blastocyst Prediction of Day-3 Cleavage-Stage Embryos Using Machine Learning

Background: Embryo selection plays an important role in the success of in vitro fertilization (IVF). However, morphological embryo assessment has a number of limitations, including the time required, lack of accuracy, and inconsistency. This study determined whether a machine learning-based model could predict blastocyst formation using day-3 embryo images. Methods: Day-3 embryo images from IVF/intracytoplasmic sperm injection (ICSI) cycles performed at My Duc Phu Nhuan Hospital between August 2018 and June 2019 were retrospectively analyzed to inform model development. Day-3 embryo images derived from two-pronuclear (2PN) zygotes with known blastocyst formation data were extracted from the CCM-iBIS time-lapse incubator (Astec, Japan) at 67 hours post ICSI, and labeled as blastocyst/non-blastocyst based on results at 116 hours post ICSI. Images were used as the input dataset to train (85%) and validate (15%) the convolutional neural network (CNN) model, then model accuracy was determined using the training and validation dataset. The performance of 13 experienced embryologists for predicting blastocyst formation based on 100 day-3 embryo images was also evaluated. Results: A total of 1,135 images were allocated into training ([Formula: see text] 967) and validation ([Formula: see text] 168) sets, with an even distribution for blastocyst formation outcome. The accuracy of the final model for blastocyst formation was 97.72% in the training dataset and 76.19% in the validation dataset. The final model predicted blastocyst formation from day-3 embryo images in the validation dataset with an area under the curve of 0.75 (95% confidence interval [CI] 0.69–0.81). Embryologists predicted blastocyst formation with the accuracy of 70.07% (95% CI 68.12%–72.03%), sensitivity of 87.04% (95% CI 82.56%–91.52%), and specificity of 30.93% (95% CI 29.35%–32.51%). Conclusions: The CNN-based machine learning model using day-3 embryo images predicted blastocyst formation more accurately than experienced embryologists. The CNN-based model is a potential tool to predict additional IVF outcomes.

Number of Blastomeres in Day-2 Embryos Affect the Rates of Blastocyst Formation and Clinical Pregnancy During In Vitro Fertilization Cycles.

In the current in vitro fertilization and embryo transfer protocol, the 8 blastomeres in the day-3 embryo are selected for transfer because these embryos can produce high rates of blastocyst formation and clinical pregnancy. However, the relationship between the blastomere number in day-2 embryos and the rate of blastocyst formation or clinical pregnancy remains unclear. The purpose of this retrospective study is to explore the relationship between the blastomere number in day-2 embryos and the rate of blastocyst formation or clinical pregnancy. From January 2015 to April 2020, we collected 8126day-3 embryos (8 blastomeres) from 2282 patients. These embryos were classified into 8 groups (1 blastomere, 2 blastomeres, 3 blastomeres, 4 blastomeres, 5 blastomeres, 6 blastomeres, 7 blastomeres, and 8 blastomeres) based on their blastomeres number on day 2 after insemination. Of these groups, the 4 blastomeres group accounted for the largest proportion (74.44%). The 1 blastomere group accounted for the smallest proportion (0.22%). A total of 3554day-3 embryos (8 blastomeres) from 1648 patients developed into blastocysts. The rate of blastocyst formation from the 4 blastomeres group was the highest (94.06%). Finally, 800 patients received single day-3 embryos (8 blastomeres) transfer. The rate of clinical pregnancy from 4 blastomeres group was the highest (51.98%). In conclusion, our data provide evidence that the number of blastomeres in day-2 embryos affects the rate of blastocyst formation and clinical pregnancy.

Live birth after in-vitro maturation of oocytes in a patient with specific ovarian insufficiency caused by long-term mitotane treatment for adrenocortical carcinoma

Research questionHow should the fertility of a woman with persistent specific ovarian dysfunction after long-term mitotane exposure be managed? DesignCase report. A 33-year-old woman who underwent surgery for adrenocortical carcinoma and treated with mitotane was referred for infertility. She rapidly became amenorrhoeic while taking mitotane, a condition that persisted for 5 years after cessation. Repeated serum hormone evaluation showed collapsed androgen levels, low oestradiol, high gonadotrophins (LH 69 and 63; FSH 23 and 43 IU/l), relatively high inhibin B level and slightly decreased anti-Müllerian hormone levels (1.4 and 0.7 ng/ml). An ultrasound scan revealed an antral follicle count of 13, contrasting with high serum gonadotrophin levels. After failure to obtain follicular growth after ovarian stimulation, in-vitro maturation (IVM) of immature oocytes aspirated from the antral follicles was carried out for microinjection with the spermatozoa of the patient's partner. ResultsTwo cycles of unstimulated egg retrieval were carried out, producing seven IVM oocytes, which were microinjected. A total of three cleavage-stage embryos were vitrified and unsuccessfully transferred after endometrial preparation using hormone replacement therapy (HRT). After a 20-month break, two new attempts were carried out under HRT with the aim of achieving a fresh embryo transfer. The last attempt succeeded after transfer of a single day-2 embryo, and the patient delivered a healthy baby. ConclusionPersistent specific impaired ovarian function 5 years after withdrawal of mitotane, and the first live birth after IVM in this situation, are reported. The question of fertility preservation before long-term mitotane treatment is raised.

Alterations of miR-16, miR-let-7a and their target genes expression in human blastocysts following vitrification and re-vitrification

Embryo cryopreservation is a widely used technique in infertility management and today is an essential part of assisted reproductive technology (ART). In some cases, re-vitrification can be applied to good quality supernumerary warmed embryos that have not been transferred in the present cycle. However, there is no study about re-vitrification impact on microRNA and gene expression in human embryos. The purpose of this study is to evaluate miR-16, miR-let7a and target genes expression in in vitro produced human blastocysts following re-vitrification.Day3 embryos obtained from ICSI cycles of fertile couples referring for family balancing program were biopsied and cultured individually. On the fourth day (post-ICSI) male ones (choices of their parents) were transferred and the females (good quality embryos) were donated for research. Donated embryos were cultured to blastocyst stage and assigned to three groups: fresh, vitrified and re-vitrification. Embryos were vitrified on Cryotech carriers. Then blastocysts of three groups were individually assessed for expression of miR-16, miR-let7a and target genes.The results showed that re-vitrification of human blastocysts did not affect the ability to re-expand in culture. In addition, significant decrease was observed in miR-16 and miR-let7a expression in re-vitrified group compared to fresh (p < 0.05). A significant upregulation of the target genes ITGβ3 and BCL-2 in re-vitrified and vitrified embryos was observed compared to the fresh group (p < 0.05). The expression of BAX as a pro-apoptotic gene showed a significant decrease in re-vitrification group comparing with the fresh one (P < 0.05).The results of this research indicated that re-vitrification of embryos changes the expression of miR-16, miR-let-7a and their target genes. These alterations include increased expression of BCl-2 and ITGβ3 genes which play important roles in embryo survival and implantation, respectively. Clinical proof of these effects requires further research.

BLASTOCYST CULTURE IN IN-VITRO FERTILIZATION IN WOMEN WITH POLYCYSTIC OVARY SYNDROME

Objective: This study aimed to evaluation of formation and development of blastocyst in in-vitro fertilization in women with polycystic ovary syndrome. Methods: Retrospective study on 53 women with PCOS and 52 women with non-PCOS, who underwent in-vitro fertilization with blastocyst culture at Center for Reproductive Endocrinology and Infertility, Hue University of Medicine and Pharmacy Hospital since January 2019 until December 2020. Results: In women with PCOS, ovarian stimulation resulted in a greater number of retrieved oocyte and blastocyst embryos. Study group with PCOS had a statistically significant lower fertilization rate and day-5 embryo rate of 74.8% and 54% compared with those in the control group (84% and 61.2%, respectively). No statistically significant differences were observed in the mature oocyte rate, the good cleavage embryo rate and the pregnancy rate in the group with PCOS compared with the control group. Conclusion: Women with PCOS have higher number of retrieved oocytes after stimulation. The ability to fertilization and quality of blastocyst was lower in the group with PCOS but the potential for implantation and pregnancy rate were similar to the control group after vitrified-warmed embryo transfer. Key words: Polycystic Ovary Syndrome (PCOS), blastocyst, in vitro fertilization, infertility

Use of Intravaginal Progesterone-Releasing Device Results in Similar Pregnancy Rates and Losses to Long-Acting Progesterone to Synchronize Acyclic Embryo Recipient Mares.

The objectives of this study were: (1) to assess uterine features and serum progesterone concentrations of acyclic mares synchronized and resynchronized with intravaginal progesterone release device (IPRD), and (2) to compare pregnancy rates and losses of cyclic and acyclic embryo recipient mares treated with different synchronization protocols. In Experiment 1, mares (n = 12) received estradiol for 3 days (E2-3d), and then 24 h after the last injection, an IPRD was inserted and kept in place for 9 days. Three days after IPRD removal, mares were treated with E2-3d, and then a new IPRD was inserted and maintained for three days. Serum progesterone concentrations were assessed 2, 6, and 12 h after insertion and removal of IPRD, and then daily from the insertion of the first IPRD to one day after removal of the second IPRD. Experiment 2 was conducted with embryo recipient mares randomly assigned to four groups: (1) Cyclic: mares (n = 75) had ovulation confirmed after receiving a single dose of histrelin when a periovulatory follicle was first detected, (2) LAP4: acyclic mares (n = 92) were treated with E2-3d and then administered a single dose of LAP4 24 h after the last estradiol injection, (3) IPRD: acyclic mares (n = 130) were treated with E2-3d and an IPRD for 4–8 days, and (4) RE-IPRD: acyclic mares (n = 32) were synchronized as in the IPRD group but not used for embryo transfer (ET), then 8 to 15 days later, the mares were resynchronized with E2-3d and an IPRD for 4–8 days. In vivo-produced Day-8 embryos were collected and transferred 4–8 days after ovulation or progesterone treatments. Mares in IPRD and RE-IPRD groups had the intravaginal device removed immediately before ET, and then a new IPRD was inserted right after ET. Pregnancy diagnosis was performed at 5, 30, and 60 days after ET. Once pregnancy was confirmed, mares in the three acyclic groups received weekly injections of LAP4 (1.5 g) until 120 days of pregnancy. Mares in IPRD and RE-IPRD groups had the device removed three days after the first pregnancy diagnosis. In Experiment 1, progesterone concentrations increased rapidly starting 2 h after insertion of IPRD (p < 0.05); then, concentrations plateaued well above pregnancy maintenance until removal on days 9 and 3, respectively. Progesterone concentrations were reduced to baseline 24 h after IPRD removal (p < 0.05). For experiment 2, there was no difference in pregnancy rates across groups (65–74%) or pregnancy losses by 60 days of gestation (7–12%) (p > 0.05). In conclusion, the IPRD used herein resulted in a rapid increase and a sharp decline in progesterone concentrations upon its insertion and removal, respectively. Finally, our results demonstrated that IPRD could be a compatible alternative to LAP4 to synchronize and resynchronize acyclic embryo recipient mares.

Influence of breed on the quality of in vivo produced embryos from Boran and Holstein Friesian cross dairy breed in Ethiopia

The variation of the dairy breed can determine the success of bovine embryo transfer by influencing the quantity and quality of in vivo embryo production. In this experiment, output and quality of in vivo produced embryos using semen of progeny tested Holstein Friesian (HF) sire in Boran and HF*Boran F1 cross cows, and semen from purebred Boran sire in HF*Boran F1 cross and Boran cows were evaluated. Boran (n=18) and HF*Boran cross (n=18) breed donor dams were superovulated using a previously optimized follicular stimulating hormone (FSH) (Pluset®) dose regimen: 650 IU for HF*Boran cross and 250 IU for Boran breeds. Each cow was flushed on Day-7 post insemination and embryos were evaluated for their developmental stages and quality. Superovulatory response rates were 88.9% and 83.3%, respectively, for Boran and HF*Boran with no significant (P&gt;0.05) breed differences. Total recovery rates were relatively lower (56.5%) in Boran compared to in HF*Boran (67.4%). The mean (±SE) embryo flush outputs were 6.5±0.8 for Boran and 6.9±0.7 forHF*Boran with no significant breed difference. Recovery of a transferrable embryo was significantly higher (68.0%; P&lt;0.05) in HF*Boran dam inseminated with HF sire semen. Boran cows yielded a significantly higher (P&lt;0.05) proportion of unfertilized ovum (57.6 %) irrespective of the sire breeds. Comparatively, a higher number of degenerated embryos were produced by HF*Boran cows. This study demonstrated that the presence of breed-related differences in both the quality and quantity of in vivo produced Bovine embryos.

USE OF INVOCELL INTRAVAGINAL EMBRYO CULTURE SYSTEM FOR FIVE DAYS OF INCUBATION: A PROSPECTIVE STUDY OF SAFETY AND TOLERABILITY

The purpose of this single-center study was to prospectively evaluate the INVOcell culture device with its retention device in planned day-5 embryo transfer cycles, as to its retention, comfort, and possible vaginal mucosal irritation. INVOcell is presently approved in the US for up to 3 days. The study was prospectively approved by WCG IRB (#1295533). Women scheduled for oocyte retrieval planning blastocyst transfer at a single center (N=31) were invited to participate in the study; all gave written informed consent. Two failed screening; 29 were enrolled in the study aged 26-41 (median 36) years with body mass index of 20.1-39.5 (median 30.5) kg/m2. A vaginal speculum exam was done at the completion of each retrieval, and after INVOcell removal at 5 days; before examination of INVOcell contents, all women completed the study questionnaire. The primary endpoint of the study was device retention, determined by patient report. Secondary endpoints of patient comfort and vaginal tissue reaction to the INVOcell system were assessed after its removal by patient questionnaire and speculum exam. The INVOcell system was retained by 100% of participants. One woman felt that the system was dislodged; she was able to readjust the system to maintain it in position. A conservative analytic approach thus gives a 96% rate of successful retention. All women completed the questionnaire, which assessed overall subject comfort and satisfaction with wearing the INVOcell system. Only two subjects reported any discomfort, and that was at a tolerable level not requiring medical intervention. The overall Likert rating for discomfort of 0.17 (pain scale of 0-10 with 0 being no pain/discomfort and 10 being severe pain/discomfort) demonstrates the favorable comfort level for the INVOcell and retention device during 5-day incubation. Whereas bleeding was noted immediately after oocyte retrieval in the majority of women and considered to be a normal sequela of this procedure, at Day-5 vaginal speculum exam no lesions, ulcerations, erythema, or bleeding were noted. In conclusion, our study demonstrates that the INVOcell system can be safely used for a 5-day embryo incubation without causing vaginal irritation or mucosal injury. Our results also verify that the system is well tolerated.

EMBRYONIC PREDICTOR FACTORS FOR A SUCCESSFUL BLASTOCYST BIOPSY PGT-A (PREIMPLANTATION GENETIC TESTING FOR ANEUPLOIDY) CYCLE

PGT-A has revealed to be a useful tool to select euploid embryos for transfer. Although some efforts have been made, more data are needed to determine the weight of embryonic parameters such as the embryo quality or the day of blastulation on the success of a PGT-A program. Our goal was to assess which are the embryonic factors that most affect the success of a blastocyst biopsy cycle. This is a retrospective study including 4072 biopsied blastocysts from 2108 PGT-A cycles performed in the same centre between February 2018 and March 2021. Biopsied embryos were divided according to the inner cell mass and trophectoderm (TE) quality Gardner’s scoring (Best: AA; Good: AB, BA, BB; Poor: AC, CA, BC, CB, CC) and the day of biopsy (day-5 or day-6). The independent relationship between each group and both euploidy and survival rate was assessed. Completely hatched blastocysts (HB) were analysed separately. Assisted hatching was performed on day 3 and biopsy was carried out when the herniation status of the embryos allowed retrieving 4 to 9 TE cells. TE biopsy was immediately followed by vitrification and Comprehensive Chromosome Screening was performed using high-resolution next generation sequencing. Only euploid embryos were thawed. Statistical report was performed under R version 3.6.1. (2019-07-05). A total of 3993 embryos resulted informative after genetic analysis (98.05%). Embryo ploidy showed a relationship with embryo quality as significant differences were found among the percentage of euploid embryos of the three quality groups (Best 63.7% n=351; Good 46.6% n=1391; Poor 31.4% n=379) (p<0.001). Euploidy did not correlate with the biopsy day independently of the embryo quality or with the hatching status of the embryo. Data show no differences between the euploidy rate of day-5 and day-6 (44.2% vs 39.84%) (odds ratio [OR]=0.61: p=0.2497) or between the ploidy of the HB and non-HB (47.31% vs 43.31%) (p=0.6074). Regarding survival rate, results showed no relationship with embryo quality (90.64% for Best, 90.44% for Good, 83.81% for Poor quality embryos) even if a trend was detected against Poor embryo quality group (Poor vs. Best: OR=0.48; p=0.2928 and Poor vs. Good: OR=0.57; p=0.3700) compared to differences between Best and Good quality groups (OR=0.85; p=0.8882). On the other hand, non-hatched day-6 biopsied embryos had lower survival rates (86.47%) than non-hatched day-5 embryos (92.43%) (OR=0.52; p=0.0308) independently of the embryo quality. Concerning the hatching status of the embryo, HB had fewer chances to survive (75.26%) than non-HB (91.01%) (OR= 3.5; p<0.001). Confirming findings of prior publications, good quality embryos are more prone to be euploid. Day-6 biopsied embryos and hatched blastocysts have fewer chances to survive after vitrification.

Blastocyst Transfer: A Risk Factor for Gestational Diabetes Mellitus in Women Undergoing In Vitro Fertilization.

BackgroundThe risk of developing gestational diabetes mellitus (GDM) is higher in women undergoing assisted reproductive treatment than in women conceiving spontaneously.ObjectivesTo determine whether the GDM risk after day-3 embryo transfer differs from the GDM risk after day-5 blastocyst transfer.MethodsProspective observational study in women becoming pregnant after first fresh embryo or blastocyst transfer.ResultsA total of 1579 women got pregnant and had live birth; 1300 women got day-3 embryo transfer only, whereas 279 women received at least 1 blastocyst. Of 1579 women, 252 developed GDM. Age, body mass index, baseline estradiol, baseline high-density lipoprotein, and progesterone on the day of human chorionic gonadotropin injection were not different in women receiving day-3 embryos only vs women receiving at least 1 blastocyst. The number and quality of retrieved oocytes were not different in women receiving day-3 embryo transfer from those receiving blastocysts. Our study confirmed already established GDM risk factors such as age and body mass index, baseline estradiol, and high-density lipoprotein, as well as progesterone after ovarian stimulation. We furthermore demonstrate that the GDM incidence in women receiving day-5 blastocyst transfer was significantly higher than those who received day-3 embryo transfer (21.15% vs 14.85%; P = 0.009). Considering confounding factors, we likewise saw that blastocyst transfer was an independent procedure-related GDM risk factor [P = 0.009, Exp (B): 1.56, 95% CI: 1.12-2.18].ConclusionBlastocyst transfer after in vitro fertilization/intracytoplasmic sperm injection increases the risk of developing GDM.

P–159 Slow-growing embryos should be frozen on day 5

Abstract Study question What is the live birth rate after frozen embryo transfer (FET) of slow-growing embryos frozen on day 5 (D5) or on day 6 (D6)? Summary answer The live birth rate after single FET is significantly higher for slow-growing embryos frozen on D5 compared to those frozen on D6. What is known already Most data on the outcomes of blastocyst transfer stem from studies that evaluate fresh transfer from normal growing D5 blastocyst ET. However not all embryos will begin blastulation nor reach the fully expanded stage by D5; those are the slow-growing embryos. Studies that compare D5 to D6 embryos in FET cycles show contradictory results. Some have reported higher clinical pregnancy rates after D5 FET, while others have reported similar outcomes for D5 and D6 cryopreserved blastocyst transfers. There is a lack of evidence regarding the best approach for vitrifying embryos that exhibit a slow developmental kinetic. Study design, size, duration This retrospective cohort study included 821 single FET of slow-growing embryos frozen on D5 or D6, belonging to patients undergoing in vitro fertilization with donor oocytes between January 2011 and October 2019, in a single fertility center. The origin of blastocysts was either supernumerary embryos after fresh embryo transfer or blastocysts from freeze-all cycles. All embryos were transferred 2- 4h after thawing. Participants/materials, setting, methods We compared reproductive outcomes of slow-growing embryos frozen on D5 versus (n = 442) slow-growing embryos frozen on D6 (n = 379). D5 group consisted in embryos graded 0, 1, 2 of Gardner scale and frozen on D5. Similarly, D6 group consisted in embryos graded 3, 4, 5 of Gardner scale (blastocyst stage) and frozen on D6. Differences in pregnancy rates between study groups were compared using a Chi2 test. A p-value &amp;lt;0.05 was considered statistically significant. Main results and the role of chance Baseline characteristics were comparable between study groups. Overall, mean age of the woman was 42.3±5.4 years old; donor sperm was used in 25% of cycles, and it was frozen in 73.2% of cycles. Pregnancy rates were significantly higher when transferring slow D5 embryos compared to D6 for all the pregnancy outcomes analyzed: biochemical pregnancy rate was 27.7% vs 20.2%, p &amp;lt; 0.016; clinical pregnancy rate was 17.5% vs 10.2%, p &amp;lt; 0.004); ongoing pregnancy rate was: 15.7% vs 7.8% (p &amp;lt; 0.001); live birth rate was: 15.4% vs 7.5%, (p &amp;lt; 0.001). These results suggest that when embryos exhibit a slow development behavior (not reaching full blastocysts at D5), waiting until D6 for blastulation and expansion does not improve clinical outcomes. Vitrification at D5 will should the preferred option in cases where the oocyte is assumed of high quality Limitations, reasons for caution The retrospective design of the study is its main limitation. Also, morphology as sole selection criterion for transfer. However, blastocyst morphology is a very good predictor of implantation and pregnancy, and a good indicator of the embryo’s chromosomal status (higher euploidy rate in higher morphological quality blastocysts). Wider implications of the findings: These results can help to the standardization of laboratory protocols. As the decision of vitrifying slow developing embryos on D5 or D6 is made by the laboratory team or by the gynaecologist in agreement with the patient, having an evidence based strategy simplifies patient counselling and decision making. Trial registration number Not applicable

P–261 The human embryo following biopsy on day5 vs day3: Implantation, cytoskeleton, ultrastructure and effects of endometrial damage/inflammation on receptivity as revealed by scanning electron microscopy

Abstract Study question Are there any differences in implantation, cytoskeleton and ultrastructure of embryos biopsied on day5 vs day3 and how endometrial damage/inflammation may affect receptivity and implantation? Summary answer No differences are observed in implantation rates but vitrification following day5 biopsy led to more cytoskeletal/ultrastructural anomalies. Infections and epithelial damage severely affected endometrial receptivity. What is known already Successful implantation is dependent on the correct synchronization of the window of implantantation with the transfer of chromosomally/genetically normal embryos, in a well prepared receptive endometrium. This is the first study to examine the effects of day5 vs day3 embryo biopsy by comparing implantation/pregnancy rates and by analysing cytoskeleton using Confocal Laser Scanning Microscopy (CLSM), and ultrastructure by Transmission Electron Microscopy (TEM). In addition, Scanning Electron Microscopy (SEM) was used on endometrial biopsies to assess possible uterine pathologies/inflammation that may be responsible for the failed implantation after PGT-A/M and if subsequent treatment can increase implantation/pregnancy rates in succeeding PGT-A/M cycles. Study design, size, duration 470 embryos were biopsied on day5 for PGT-A (n = 152–37 cycles) or on day3 for PGT-A (n = 162–29 cycles) and PGT-M (n = 156–22 cycles). Following transfer of normal embryos, spare embryos, rejected for transfer following day5 or day3 biopsy were processed for Cytoskeletal analysis (n = 30 fresh day3 biopsied, n = 30 day5 biopsied/vitrified) or TEM (n = 20 fresh day3 biopsied, n = 20 day5 biopsied/vitrified). Also, patients with a –ve hCG test, underwent endometrial biopsy to detect infection/inflammation and assess receptivity. Participants/materials, setting, methods Cytoskeletal analysis was performed by embryo immunostaining with α-tubulin, γ-tubulin, acetylated-tubulin antibodies and DAPI or/ PI to visualise DNA. TEM analysis was carried out following embryo fixation in gluteraldehyde, incubation in osmium, aqueous uranyl acetate, dehydration through ethanol series, and immersion in Epon. Endometrial biopsies were fixed in gluteraldehyde solution and processed for SEM using standard methods. The study was conducted in an academic hospital with an IVF/PGD laboratory and 3 private IVF Units. Main results and the role of chance 162 embryos were biopsied on day 3 for PGT-A (29 cycles), 10 cycles had no normal embryos for transfer, 30 normal embryos were transferred in 19 cycles leading to 12/19 (63.2%) +vehCG/ET and 11/19 (57.9%) Ongoing pregnancy rate/ET. 156 embryos were biopsied on day 3 for PGT-M (22 cycles), 2 cycles had no normal embryos for transfer, 34 normal or carrier embryos were transferred in 20 cycles leading to 15/20 (75.0%) +vehCG/ET and 13/20 Ongoing pregnancy rate/ET (65.0%). 152 embryos were biopsied on day 5 for PGT-A (37 cycles), 8 cycles had no normal embryos for transfer, 34 normal embryos were transferred in 29 cycles leading to 18/29 (62.1%) +vehCG/ET and 16/29 (55.2%) Ongoing pregnancy rate /ET. Analysis of endometrial biopsies with SEM revealed bacterial infections, inflammation and epithelial damage. So far, 33.3% of patients who received intracavitary infusions-antibiotic treatment per os achieved a +vehCG/ET in their next PGT-A/M cycle. Cytoskeletal analysis showed that the majority of spindles examined in both day3 and day5 biopsied embryos were normal (85/114(74.6%) and 87/137(63.5). However vitrification following day5 biopsy led to more cytoskeletal/ultrastructural anomalies which included multipolar/abnormally shaped spindles, chromosome-bridging, chromosome-lagging and more vacuoles, lipofuscins. and distension of mitochondria. Limitations, reasons for caution Patients undergoing PGT-A have various aetiologies (heterogeneous group). The embryos used for cytoskeletal and ultrastructural analysis in this study were all diagnosed with either chromosomal abnormalities or single gene defects following PGT-A or PGT-M. Wider implications of the findings: This is the first study to compare implantation/pregnancy rates, cytoskeleton and ultrastructure of day5 vs day3 biopsied embryos. The similarities observed in implantation/pregnancy rates, and the limited ultrastuctural and cytoskeletal anomalies identified confirm the procedures’ safety and indicate in certain cases endometrial factors/inflammation responsible for failed implantation following PGT-A/M. Trial registration number Not applicable

O-205 Aneuploidy induces proteotoxic stress and autophagy-mediated apoptosis in human preimplantation embryos

Abstract Study question Are aneuploid cells in human preimplantation embryos eliminated by apoptosis due to proteotoxic stress and autophagy-mediated apoptosis? Summary answer Proteotoxic stress, autophagy and apoptosis are differentially activated in aneuploid embryos, showing that aneuploid cells are eliminated by these mechanisms during early human embryogenesis. What is known already Aneuploidies are a common feature of human preimplantation embryos which could explain low success rates after in vitro fertilization (IVF). While most aneuploidies of meiotic origin are detrimental, transfer of euploid-aneuploid mosaic embryos can lead to healthy live-births. Moreover, the proportion of aneuploid cells are lower in blastocysts when compared to cleavage stage embryos. In the mouse, aneuploid cells are eliminated from the epiblast by autophagy-mediated apoptosis in a p53-dependent manner. We propose that in human embryos, aneuploidy causes chronic protein misfolding which leads to autophagy-induced apoptosis. Study design, size, duration Eighty-one blastocysts that were diagnosed by PGT as euploid (n = 49) or uniformly combined abnormal (CA, n = 32), i.e. 2 or more chromosomes were abnormal in every cell, were warmed. Sixty-seven were suitable for trophectoderm (TE) biopsy, 54 biopsies were successfully tubed and sent for RNA-sequencing while the remainder of the embryos was fixed for immunostaining. Thirty-three day-3 embryos were overnight incubated in 0.5µM reversine allowed to develop into blastocysts and treated as the PGT embryos. Participants/materials, setting, methods After TE biopsy, we live-stained the embryos with either Caspase-3/7 or 8 and subsequently fixed them. The biopsies underwent RNA-sequencing using the SMART-seqv4 and the fixed embryos were immunostained for LC3B, p62 (autophagy) and HSP70 (proteotoxic stress). Confocal imaging was performed using a Zeiss LSM800 confocal microscope and the presence of signal was quantified using the Zen Blue 2.0 and Arivis software. Main results and the role of chance Forty-two percent of the embryos in which we induced aneuploidies using reversine developed into blastocysts, which is comparable to untreated embryos. After immunostaining, we observed that CA and reversine-treated (RT) embryos contained less cells than euploid embryos (median number of nuclei: 43.5, 47, 90, respectively). This correlates with a higher expression of apoptotic markers Caspase-3/7 in CA embryos (p = 0.0199) and Caspase-8 in both aneuploid groups (CA: p = 0.0085 and RT: p = 0.0394). Aneuploid embryos showed significantly increased HSP70 levels (median intensity per cell: euploid=165, CA = 313, RT = 400), LC3B (median puncta per cell: euploid=3.07, CA = 10.10, RT = 19.62) and p62 (median puncta per cell: euploid=17.60, CA = 30.53), suggesting increased proteotoxic stress and autophagy. Preliminary analysis of the RNA-sequencing data reveals enrichment for pathways such as the p53-pathway, protein secretion, TNFA signaling via NFkB and apoptosis, supporting the hypothesis of a link between aneuploidy and apoptosis. Limitations, reasons for caution No functional tests e.g. with inhibitors of autophagy were carried out. RNA-sequencing was carried out on a small sample; we will expand this sample in the near future. Wider implications of the findings This study shows for the first time the mechanism by which aneuploid cells are eliminated from the human preimplantation embryo, explaining how mosaic embryos can still lead to a healthy and genetically normal live birth. Trial registration number not applicable