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Purpose: To compare obstetrical and neonatal outcomes of embryo transfer cycles using fresh vs. frozen-thawed testicular sperm derived from microTESE in non-obstructive azoospermia (NOA) patients., Design: The retrospective cohort study included a total of 48 couples diagnosed with NOA who underwent 93 ET cycles, both fresh and frozen-thawed embryos, and resulted in pregnancy. ET cycles were divided into two groups according to sperm type, fresh (46 cycles, 49.5%) or frozen (47 cycles, 50.5%) testicular sperm. The primary outcome was the birth weight of newborns correlated with gestational week (birth weight percentile)., Results: A comparison of patients' basic characteristics and ET cycle parameters showed no significant clinical differences between the groups. A total of 172 embryos were transferred, 86 (50%) in each group. A higher rate of good-quality blastocysts was found in the fresh testicular group (83.3% vs. 50%, p = 0.046). A comparison of pregnancy outcomes showed no significant differences in clinical pregnancy, implantation, or live birth rates. A total of 53 cycles resulted in live birth, 26 (49%) and 27 (51%) in the fresh and frozen groups, respectively. No difference was found in pregnancy length, delivery mode, or obstetrical complications. A total of 61 newborns were included, 31 (51%) and 30 (49%) in fresh and frozen testicular groups, respectively. No significant differences were found in mean birth weight or birth weight percentile between the groups., Conclusion: No significant differences were found in obstetrical outcomes when comparing ET cycles using fresh or frozen-thawed testicular sperm retrieved from microTESE. Moreover, there is no association between the sperm source and the birth weight of newborns., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: To determine if an explainable artificial intelligence (XAI) model enhances the accuracy and transparency of predicting embryo ploidy status based on embryonic characteristics and clinical data., Methods: This retrospective study utilized a dataset of 1908 blastocyst embryos. The dataset includes ploidy status, morphokinetic features, morphology grades, and 11 clinical variables. Six machine learning (ML) models including Random Forest (RF), Linear Discriminant Analysis (LDA), Logistic Regression (LR), Support Vector Machine (SVM), AdaBoost (ADA), and Light Gradient-Boosting Machine (LGBM) were trained to predict ploidy status probabilities across three distinct datasets: high-grade embryos (HGE, n = 1107), low-grade embryos (LGE, n = 364), and all-grade embryos (AGE, n = 1471). The model's performance was interpreted using XAI, including SHapley Additive exPlanations (SHAP) and Local Interpretable Model-agnostic Explanations (LIME) techniques., Results: The mean maternal age was 38.5 ± 3.85 years. The Random Forest (RF) model exhibited superior performance compared to the other five ML models, achieving an accuracy of 0.749 and an AUC of 0.808 for AGE. In the external test set, the RF model achieved an accuracy of 0.714 and an AUC of 0.750 (95% CI, 0.702-0.796). SHAP's feature impact analysis highlighted that maternal age, paternal age, time to blastocyst (tB), and day 5 morphology grade significantly impacted the predictive model. In addition, LIME offered specific case-ploidy prediction probabilities, revealing the model's assigned values for each variable within a finite range., Conclusion: The model highlights the potential of using XAI algorithms to enhance ploidy prediction, optimize embryo selection as patient-centric consultation, and provides reliability and transparent insights into the decision-making process., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Muse cells, identified as cells positive for the pluripotent surface marker SSEA-3, are pluripotent-like endogenous stem cells located in the bone marrow (BM), peripheral blood, and organ connective tissues. The detailed characteristics of SSEA-3(+) cells in extraembryonic tissue, however, are unknown. Here, we demonstrated that similar to human-adult tissue-Muse cells collected from the BM, adipose tissue, and dermis as SSEA-3(+), human-umbilical cord (UC)-SSEA-3(+) cells express pluripotency markers, differentiate into triploblastic-lineage cells at a single cell level, migrate to damaged tissue, and exhibit low telomerase activity and non-tumorigenicity. Notably, ~ 20% of human-UC-SSEA-3(+) cells were negative for X-inactive specific transcript (XIST), a naïve pluripotent stem cell characteristic, whereas all human adult tissue-Muse cells are XIST-positive. Single-cell RNA sequencing revealed that the gene expression profile of human-UC-SSEA-3(+) cells was more similar to that of human post-implantation blastocysts than human-adult tissue-Muse cells. The DNA methylation level showed the same trend, and notably, the methylation levels in genes particularly related to differentiation were lower in human-UC-SSEA-3(+) cells than in human-adult tissue-Muse cells. Furthermore, human-UC-SSEA-3(+) cells newly express markers specific to extraembryonic-, germline-, and hematopoietic-lineages after differentiation induction in vitro whereas human-adult tissue-Muse cells respond only partially to the induction. Among various stem/progenitor cells in living bodies, those that exhibit properties similar to post-implantation blastocysts in a naïve state have not yet been found in humans. Easily accessible human-UC-SSEA-3(+) cells may be a valuable tool for studying early-stage human development and human reproductive medicine., (© 2024. The Author(s).)

Early trophoblast differentiation is crucial for embryo implantation, placentation and fetal development. Dynamic changes in DNA methylation occur during preimplantation development and are critical for cell fate determination. However, the underlying regulatory mechanism remains unclear. Recently, we derived morula-like expanded potential stem cells from human preimplantation embryos (hEPSC-em), providing a valuable tool for studying early trophoblast differentiation. Data analysis on published datasets showed differential expressions of DNA methylation enzymes during early trophoblast differentiation in human embryos and hEPSC-em derived trophoblastic spheroids. We demonstrated downregulation of DNA methyltransferase 3 members (DNMT3s) and upregulation of ten-eleven translocation methylcytosine dioxygenases (TETs) during trophoblast differentiation. While DNMT inhibitor promoted trophoblast differentiation, TET inhibitor hindered the process and reduced implantation potential of trophoblastic spheroids. Further integrative analysis identified that glutamyl aminopeptidase (ENPEP), a trophectoderm progenitor marker, was hypomethylated and highly expressed in trophoblast lineages. Concordantly, progressive loss of DNA methylation in ENPEP promoter and increased ENPEP expression were detected in trophoblast differentiation. Knockout of ENPEP in hEPSC-em compromised trophoblast differentiation potency, reduced adhesion and invasion of trophoblastic spheroids, and impeded trophoblastic stem cell (TSC) derivation. Importantly, TET2 was involved in the loss of DNA methylation and activation of ENPEP expression during trophoblast differentiation. TET2-null hEPSC-em failed to produce TSC properly. Collectively, our results illustrated the crucial roles of ENPEP and TET2 in trophoblast fate commitments and the unprecedented TET2-mediated loss of DNA methylation in ENPEP promoter., (© 2024. The Author(s).)

Purpose: Zygotes with 2.1 pronuclei (2.1PN) present with two normal-sized pronuclei, and an additional smaller pronucleus, that is approximately smaller than two thirds the size of a normal pronucleus. It remains unclear whether the additional pronucleus causes embryonic chromosome abnormalities. In the majority of cases, in vitro fertilization (IVF) clinics discarded 2.1PN zygotes. Thus, the present study aimed to evaluate the developmental potential and value of 2.1PN zygotes., Methods: 2.1PN-derived embryos from 164 patients who underwent IVF or intracytoplasmic sperm injection (ICSI) treatment between January 2021 and December 2022 were included in the present study. All embryos were monitored using a time-lapse system, and blastocyst formation was used to assess 2.1PN-derived embryo developmental potential. The blastocyst formation was quantified using generalized estimating equations, and chromosome euploidy was analyzed using next-generation sequencing (NGS). In addition, the potential association between age and occurrence of 2.1PN zygotes was determined., Results: The present study demonstrated that numerous 2.1PN zygotes developed into blastocysts. Early cleavage patterns and embryo quality on Day 3 were the independent predictors for the blastocyst formation of 2.1PN-derived embryos. The 2.1PN zygotes displayed a comparable developmental potential compared to 2PN zygotes in advanced age patients (≥ 38). Moreover, there was a tendency that 2.1PN-derived blastocysts showed a similar euploidy rate compared to 2PN-derived blastocysts., Conclusion: Clinicians should consider using 2.1PN-derived euploid embryos for transfer after preimplantation genetic testing in the absence of available 2PN embryo cycles. 2.1PN-derived embryos could be a candidate, particularly beneficial for patients at advanced age., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: Are human embryos arising from two plus one small pronucleated zygotes, called 2.1 pronuclei (PN), clinically useful?, Methods: In a retrospective embryo cohort study and prospective experimental study, a total of 287 cycles in which at least one 2.1PN was identified in the fertilization check were included. Embryonic development and clinical outcome were compared for the 1395 2PN zygotes and 304 2.1PN zygotes that were siblings. All embryos were individually cultured in time-lapse systems. Twenty-five 2.1PN-derived blastocysts, donated for research, were used in focused single-nucleotide variant ploidy analysis to identify the distribution pattern of heterozygosity., Results: The average diameter of PN was 24.9 ± 2.4 µm for large PN and 10.2 ± 2.4 µm for small PN; 79.9% of small PN was derived from female pronuclei. Blastocyst formation rate and good-quality blastocyst rate were significantly lower with 2.1PN embryos than with 2PN embryos (40.0% vs. 57.7%, 21.4% vs. 33.5%, respectively). A total of 13 embryos derived from 2.1PN were transferred, and three healthy babies were born. In ploidy constitutions of trophectoderm (TE), 2.1PN-derived blastocyst TE was shown to be mostly diploid (95.8%, 23/24), and only one blastocyst showed triploid., Conclusions: It was suggested that 2.1PN embryos have lower embryonic developmental potential than 2PN embryos, but most of the 2.1PN were diploid, indicating that they are likely to be clinically usable. It is recommended to perform embryo transfer following a combination of PGT-A and ploidy analysis., (© 2024. The Author(s).)

Purpose: The purpose of this study was to provide a detailed analysis of clinical and laboratory factors associated with skewed secondary sex ratio (SSR) after ART., Method: Retrospective cohort study of embryos resulting in live births, from frozen and fresh single blastocyst transfers. Embryos were cultured in either G-TL (n = 686) or Sage media (n = 685). Data was analyzed using a multivariate logistic regression model and a mixed model analysis., Results: Significantly more male singletons were born after culture in Sage media compared to G-TL media (odds ratio (OR) 1.34, 95% CI (1.05, 1.70), P = 0.02). Inner cell mass grade B vs A (OR 1.36 95% CI (1.05, 1.76), P = 0.02) and one previous embryo transfer (OR 1.49, 95% CI (1.03, 2.16), P = 0.03) were associated with a significantly higher probability of male child at birth. Factors associated with a reduced probability of male child were expansion grade 3 vs 5 (OR 0.66, 95% CI (10.45, 0.96), P = 0.03) and trophectoderm grade B vs A (OR 0.57, 95% CI (0.44, 0.74), P = 0.00). Male embryos developed significantly faster in Sage media compared to G-TL media for the stages of blastocyst (- 1.12 h, 95% CI (- 2.12, - 0.12)), expanded blastocyst (- 1.35 h, 95% CI (- 2.34, - 0.35)), and hatched blastocyst (- 1.75 h, 95% CI (- 2.99, - 0.52))., Conclusion: More male children were born after culture in Sage media compared to G-TL media. Male embryo development was affected by culture media. Our observations suggest that culture media impact male embryo quality selectively, thus potentially favoring the selection of male embryos., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: To analyze the fertilization, developmental, and pregnancy potentials in oocytes with narrow perivitelline space., Methods: Perivitelline space (PVS) of oocytes was evaluated at the time of ICSI, and those without sufficient PVS were judged as oocytes with narrow PVS (NPVS oocytes), and those with sufficient PVS formation were judged as oocytes with non-narrow PVS (non-NPVS oocytes). The analysis included 634 NPVS oocytes from 278 cycles and 12,121 non-NPVS oocytes from 1698 cycles. The fertilization and developmental potentials of NPVS and non-NPVS oocytes were compared by calculating odds ratios using a mixed-effects logistic regression model. We also compared the embryo transfer outcomes of those used for single vitrified-warmed blastocyst transfer after developing into the blastocyst stage., Results: NPVS oocytes had higher odds ratios for degeneration (adjusted odds ratio [aOR], 1.555; 95% confidence interval [CI], 1.096-2.206; p = 0.0133) and 0PN (aOR, 1.387; 95% CI, 1.083-1.775; p = 0.0095), resulting in a lower 2PN rate (aOR, 0.761; 95% CI, 0.623-0.929; p = 0.0072). Even embryos with confirmed 2PN had lower odds ratios for cleavage (aOR, 0.501; 95% CI, 0.294-0.853; p = 0.0109) and blastocyst development (Gardner criteria; CC-AA) rates (aOR, 0.612; 95% CI, 0.476-0.788; p = 0.0001). Blastocysts developed from NPVS oocytes had significantly lower odds ratios for clinical pregnancy (aOR, 0.435; 95% CI, 0.222-0.854; p = 0.0156) than those developed from non-NPVS oocytes., Conclusions: Oocytes with NPVS have low fertilization and developmental potential, as well as low likelihood of pregnancy., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: To study the effectiveness of whole-scenario embryo identification using a self-supervised learning encoder (WISE) in in vitro fertilization (IVF) on time-lapse, cross-device, and cryo-thawed scenarios., Methods: WISE was based on the vision transformer (ViT) architecture and masked autoencoders (MAE), a self-supervised learning (SSL) method. To train WISE, we prepared three datasets including the SSL pre-training dataset, the time-lapse identification dataset, and the cross-device identification dataset. To identify whether pairs of images were from the same embryos in different scenarios in the downstream identification tasks, embryo images including time-lapse and microscope images were first pre-processed through object detection, cropping, padding, and resizing, and then fed into WISE to get predictions., Results: WISE could accurately identify embryos in the three scenarios. The accuracy was 99.89% on the time-lapse identification dataset, and 83.55% on the cross-device identification dataset. Besides, we subdivided a cryo-thawed evaluation set from the cross-device test set to have a better estimation of how WISE performs in the real-world, and it reached an accuracy of 82.22%. There were approximately 10% improvements in cross-device and cryo-thawed identification tasks after the SSL method was applied. Besides, WISE demonstrated improvements in the accuracy of 9.5%, 12%, and 18% over embryologists in the three scenarios., Conclusion: SSL methods can improve embryo identification accuracy even when dealing with cross-device and cryo-thawed paired images. The study is the first to apply SSL in embryo identification, and the results show the promise of WISE for future application in embryo witnessing., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: To assess the relation between number of inseminated oocytes and cumulative live birth rate (CLBR) in order to provide guidance for limiting the number of surplus blastocysts., Methods: The study was a retrospective, single-center cohort analysis of 1223 ART complete cycles. Cycles were stratified according to female age (≤ 34, 35-38, and 39-42 years) and number of inseminated oocytes (1-5, 6-10, and > 10). Inclusion criteria were indication for IVF/ICSI with own spermatozoa and blastocyst culture up to day 6 of all embryos., Results: In patients younger than 35 years, insemination of more than ten oocytes produced an increase in overall blastocyst number, CLBR (40.3%, 54.3%, and 75.8%, respectively, for each oocyte group) and surplus embryo rate (12.9%, 27.8%, and 49.7% of cases for each group). Instead, in the middle age group, the use of more than ten oocytes was solely associated with an increase in the rate of surplus embryos (1.25%, 21.33%, and 28.68% of cases after stratification for oocyte number). In older patients, neither CLBR (9.1%, 23.9%, and 24.7%, respectively) nor rate of surplus embryos (2.0%, 7.1%, and 13.4% of cases for each group) were higher in cycles with more than ten inseminated oocytes., Conclusion: In women up to 38 years, sustainable CLBR are achieved while limiting the number of inseminated oocytes and the resulting blastocysts remaining unused. Based on this notion, novel treatment strategies could pursue high outcome rates, while alleviating the problems derived from surplus stored embryos., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

We studied the influence of extracellular vesicles from the follicular fluid of a young donor on gene expression (MKI67, MYBL2, CCNB1, CCND1, CCNE1, CALM2, BAX, NDRG1, TP53I3, VEGF, VCAN, HAS2, CTSL2, PIBF1, RPL37, PFKP, GPX3, and AQP3) in embryos of women of different ages. According to nanoparticle tracking analysis data, the concentration of extracellular vesicles was 3.75±0.47×10 11 particles/ml and the mean particle size was 138.78±9.90 nm. During co-culturing of the follicular fluid extracellular vesicles with blastocysts of young women, we observed significantly increased expression of mRNA for genes CTSL2, CCND1, CCNE1, VEGF and reduced expression of BAX gene mRNA in comparison with embryos in women of late reproductive age. We hypothesized that addition of extracellular vesicles of the oocyte follicular fluid from a young donor to the culture medium of embryos could slow down apoptosis process typical of blastocyst cells in women above 36 years., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

The purpose of this study was to determine if morphometric parameters that can be measured quantitatively using a time-lapse embryo incubator are associated with aneuploidy. Embryos cultured in a time-lapse incubator and assessed with preimplantation genetic testing for aneuploidy (PGT-A) were analyzed retrospectively. Morphokinetic analysis included timing of cell divisions. Quantitative morphometric measurements included the distance between the second and first polar body, zona pellucida thickness at the pronuclear stage and at the 2-cell stage, and blastomere area at the 2- and 4-cell stages. Symmetry at the 2-cell stage was determined by percent difference between blastomeres; symmetry at the 4-cell stage was the percent difference between the smallest and largest blastomeres. Maternal age, blastocyst grade and day of biopsy were recorded. Euploid embryo characteristics were compared to aneuploid embryos. A receiver operating characteristic (ROC) curve was used to evaluate cell symmetry as a predictor of aneuploidy. Embryos (n = 182) from 21 patients (age 22-43; median = 34) were analyzed. Of the 182 embryos, 45% were euploid. Euploid and aneuploid embryos had similar morphokinetics and morphometry across many measures. As expected, age and blastocyst grade were associated with embryo ploidy. It was notable that, additionally, symmetry at the 4-cell stage (27% vs 31%, p = 0.01) was also associated with embryo ploidy. The optimized cutoff from the ROC curve to predict aneuploidy was determined to be 21%. Embryos with > 21% asymmetry at the 4-cell stage had high rates of aneuploidy while morphokinetic parameters were similar. In conclusion, this suggests that embryo selection models using time-lapse parameters would improve if they incorporate cleavage-stage morphometrics., (© 2021. Society for Reproductive Investigation.)

Purpose: To determine if the change in endometrial thickness following exogenous progesterone (P) initiation correlates with outcome following autologous transfer of a single thawed blastocyst., Methods: The study is a retrospective observational cohort study conducted at a private fertility center. Patients scheduled for thawed blastocyst transfer received artificial endometrial preparation (artificial cycle FET) and underwent serial ultrasonography. The main outcomes were the rate of ongoing pregnancy (fetal heart motion at 12 weeks of gestation) and early pregnancy loss. Logistic regression was used to test for correlations between these outcomes and the change in endometrial thickness while adjusting for potential confounders (patient age, embryo quality, and the use of genetic testing)., Results: There were 232 qualifying autologous single-blastocyst transfers in the 20-month study period ending 31 December 2019. Mean endometrial thicknesses were 3.8 mm, 10.0 mm, and 11.2 mm at baseline, P initiation, and at transfer, respectively. The change in endometrial thickness after exogenous P exposure ranged from - 5 to + 9 mm and negatively correlated with ongoing pregnancy in logistic regression analyses. Specifically, ongoing pregnancy rates per transfer were 63.2% in 19 cases where endometria compacted by 10% or more, 64.2% in 95 cases where there was unchanged endometrial thickness, and 52.5% in 118 cases where endometria expanded., Conclusions: The change in endometrial thickness after P initiation was associated with the probability of ongoing pregnancy but not with early pregnancy loss. Ongoing pregnancy rates were greater in endometria with negative growth (compaction) when compared to endometria that grew (expanded) after P exposure., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: Does thawing cleavage embryos and culturing them for transfer as blastocysts improve pregnancy and perinatal outcomes compared to transferring thawed blastocysts?, Methods: Retrospective, observational cohort study performed at two assisted reproductive technology centers, 2014 to 2020. A total of 450 patients with 463 thawed embryo transfer cycles were divided into 2 groups according to the embryonic developmental stage at cryopreservation and transfer: 231 thawed blastocysts (day 5 group) and 232 thawed cleavage embryos that were cultured for 2 days and transferred as blastocysts (day 3-5 group). The two groups were compared for demographics, routine parameters of IVF treatment, pregnancy rates, and perinatal outcomes., Results: Multivariable logistic regression analysis for ongoing pregnancy and delivery demonstrated that the day 3-5 group had a greater likelihood of achieving ongoing pregnancy and delivery compared to the day 5 group (OR 1.58, 95%CI 1.062-2.361, p = 0.024). Perinatal outcomes were comparable between the three groups., Conclusion: Our results support culturing post-thaw cleavage embryos for 2 days and transferring them as blastocysts to increase chances of ongoing pregnancy and delivery., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objective: To determine whether euploidy rates and blastocyst development differ in a continuous culture medium under different CO 2 concentrations., Design and Method: A single-center retrospective study was performed from July 2018 to October 2019 including 44 fresh cycles with at least four fresh mature oocytes (MII) without severe male factor infertility. Sibling MII were injected and cultured in Global®Total®LP under 6.0% (pHe = 7.374 ± 0.014) or 7.0% (pHe = 7.300 ± 0.013) CO 2 , 5.0% O 2 , and 89.0% or 88.0% N 2 . Analyzed variables were normally fertilized oocytes (2PN), cleavage rate, blastulation rate on day 5/2PN, usable blastocyst (blastocysts biopsied/2PN), and euploidy rates. Blastocyst's trophectoderm biopsy was performed on day 5, 6, or 7 for genetic testing and mitochondrial DNA (mtDNA) quantification by next-generation sequencing., Results: Women's mean age was 33.0 ± 6.6 years old. From a total of 604 MII, no differences were found in normal fertilization and cleavage rates on day 3 between 6.0 and 7.0% CO 2 (72.3% vs 67.1%, p = 0.169 and 96.6% vs 96.3%, p = 0.897, respectively). Blastulation rate on day 5/2PN was comparable between 6.0 and 7.0% CO 2 (68.1% vs 64.2%, p = 0.409). Although usable blastocyst rate was not different (54.3% vs 55.3%, p = 0.922), total euploidy rates differed significantly (58.7% vs 42.8%, p = 0.016) between 6.0% and 7.0% CO 2 , respectively. The mean blastocyst mtDNA content was significantly lower in 6.0% CO 2 (30.4 ± 9.1 vs 32.9 ± 10.3, p = 0.037)., Conclusion: Blastocyst development is not affected when embryos are cultured in vitro at 6.0% or 7.0% CO 2 , while euploidy rates are significantly decreased at a higher CO 2 concentration, therefore at a lower pHe., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: A retrospective study examining the effects of embryo re-expansion before transfer on pregnancy outcomes for frozen embryo transfers (FET)., Methods: A total of 486 FET cycles from November 2017 through December 2019 were studied. These cycles included patients using autologous, donor oocytes, and donor embryo with patients ranging from ages 23 to 48 years with infertility diagnoses. Programmed FET priming was performed with exogenous estrogen and progesterone. All blastocysts were cultured in trigas incubators for 20 min to 4 h and 42 min. Pictures of each blastocyst after thaw and before transfer were taken utilizing the Hamilton Thorne Zilos laser software (Beverly, MA). The longest portion of the embryo was measured in µm. Pregnancy was defined by a positive hCG, and ongoing clinical pregnancy was defined by the presence of fetal cardiac activity. Wilcoxon rank sum tests were used to access differences in change parameters., Results: There is no significant difference in the amount of embryo expansion or contraction to achieve an ongoing pregnancy. The difference remained non-significant when stratified by embryo expansion or contraction. The amount of change over time and percent change from the first measurement were also not associated with achieving an ongoing pregnancy. This remained true after adjustment for patient age and whether or not a biopsy was performed., Conclusions: Embryos that do not re-expand after warming appear to have a similar chance of achieving a successful pregnancy as those that do re-expand., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Although in vitro culture of human embryos is a crucial step in assisted reproduction, the lack of focused research hampers worldwide standardisation and consistent outcomes. Only 1.2% of research papers published in five leading journals in human reproduction in 2019 focused on in vitro culture conditions, creating the impression that the optimisation process has approached its limits. On the other hand, in vitro culture of mammalian embryos is based on old principles, while there is no consensus on basic issues as density, time, medium change, gas atmosphere and small technical details including the way of drop preparation. This opinion paper aims to highlight and analyse the slow advancement in this field and stimulate research for simple and affordable solutions to meet the current requirements. A possible way for advancement is discussed in detail. Selection of embryos with the highest developmental competence requires individual culture and modification of the widely used "drop under oil" approach. Current use of three-dimensional surfaces instead of large flat bottoms is restricted to time-lapse systems, but these wells are designed for optical clarity, not for the needs of embryos. The size and shape of the original microwells (Well of the Well; WOW) offer a practical and straightforward solution to combine the benefits of communal and individual incubation and improve the overall quality of cultured embryos., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: To understand the clinical factors associated with embryo survival after vitrification in a cohort of human blastocysts screened by preimplantation genetic testing for aneuploidy (PGT-A)., Methods: Patient demographic, embryo, and cycle characteristics associated with failed euploid blastocyst survival were compared in a cohort of women (n = 6167) who underwent IVF-PGT-A., Results: Compared to those that survived warming, vitrified euploid embryos that failed to survive after warming came from IVF cycles with significantly higher estradiol levels at time of surge (2754.8 ± 1390.2 vs. 2523.1 ± 1190.6 pg/mL, p = 0.03), number of oocytes retrieved (19.6 ± 10.7 vs. 17.5 ± 9.8, p = 0.005), and basal antral follicle count (BAFC) (15.3 ± 8.5 vs. 13.9 ± 7.2, p = 0.05). Euploid embryos were less likely to survive warming if they came from cycles before 2015 (24.6% vs. 13.2%, p < 0.001), were cryopreserved on day 7 versus day 5 or 6 (9.1% vs. 3.0%, p < 0.001), underwent two trophectoderm biopsies (6.9% vs. 2.3%, p < 0.001), had a grade C inner cell mass (15.4% vs. 7.7%, p < 0.001), or were fully hatched (41.1% vs. 12.2%, p < 0.001). In the multivariate model, which controlled for relevant confounders, the association between decreased survival and increased BAFC, year of IVF cycle, double trophectoderm biopsy, and fully hatched blastocysts remained statistically significant., Conclusion: Euploid embryos that are fully hatched at time of vitrification, come from patients with high ovarian reserve, or require repeat trophectoderm biopsy are less likely to survive vitrification-warming. Our results provide a framework for reproductive counseling and offer realistic expectations to patients about the number of embryos needed to achieve family building goals., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: A deep learning artificial intelligence (AI) algorithm has been demonstrated to outperform embryologists in identifying euploid embryos destined to implant with an accuracy of 75.3% (1). Our aim was to evaluate the performance of highly trained embryologists in selecting top quality day 5 euploid blastocysts with and without the aid of a deep learning algorithm., Materials and Methods: A non-overlapping series of 200 sets of day 5 euploid embryo images with known implantation outcomes was distributed to 17 highly trained embryologists. One embryo in each set was known to have implanted and one failed implantation. They were asked to select which embryo to transfer from each set. The same 200 sets of embryos, with indication of which embryo in each set had been identified by the algorithm as more likely to implant was then distributed. Chi-squared, t-test, and receiver operating curves were performed to compare the embryologist performeance with and without AI., Results: Fourteen embryologists completed both assessments. Embryologists provided with AI results selected successfully implanted embryos in 73.6% of cases compared to 65.5% for those selected using visual assessments alone (p < 0.001). All embryologists improved in their ability to select embryos with the aid of the AI algorithm with a mean percent improvement of 11.1% (range 1.4% to 15.5%). There were no differences in degree of improvement by embryologist level of experience (junior, intermediate, senior)., Conclusions: The incorporation of an AI framework for blastocyst selection enhanced the performance of trained embryologists in identifying PGT-A euploid embryos destined to implant., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: To describe the pregnancy and neonatal outcomes using fresh and vitrified/warmed blastocysts obtained from ovarian stimulation with follitropin delta in controlled trials versus follitropin alfa., Methods: This investigation evaluated the outcome from 2719 fresh and frozen cycles performed in 1326 IVF/ICSI patients who could start up to three ovarian stimulations in the ESTHER-1 (NCT01956110) and ESTHER-2 (NCT01956123) trials, covering 1012 fresh cycles and 341 frozen cycles with follitropin delta and 1015 fresh cycles and 351 frozen cycles with follitropin alfa. Of the 1326 first cycle patients, 513 continued to cycle 2 and 188 to cycle 3, and 441 patients started frozen cycles after the fresh cycles. Pregnancy follow-up was continued until 4 weeks after birth., Results: The overall cumulative take-home baby rate after up to three stimulation cycles was 60.3% with follitropin delta and 60.7% with follitropin alfa (-0.2% [95% CI: -5.4%; 5.0%]), of which the relative contribution was 72.8% from fresh cycles and 27.2% from frozen cycles in each treatment group. Across the fresh cycles, the ongoing implantation rate was 32.1% for follitropin delta and 32.1% for follitropin alfa, while it was 27.6% and 27.8%, respectively, for the frozen cycles. Major congenital anomalies among the live-born neonates up until 4 weeks were reported at an incidence of 1.6% with follitropin delta and 1.8% with follitropin alfa (-0.2% [95% CI: -1.9%; 1.5%])., Conclusions: Based on comparative trials, the pregnancy and neonatal outcomes from fresh and frozen cycles provide reassuring data on the efficacy and safety of follitropin delta., Trial Registration: ClinicalTrials.gov Identifier: NCT01956110 registered on 8 October 2013; NCT01956123 registered on 8 October 2013., (© 2021. The Author(s).)

Purpose: Different commercial human embryo culture mediums can alter embryo quality and change birthweight. One component that could be contributing to variations but is not widely investigated is human serum albumin (HSA). HSA plays a multitude of roles during embryo culture and is a carrier for molecules including lipids. It remains unclear if lipid composition of HSA varies among commercial products and its effects on embryo quality, implantation, and fetal outcomes are relatively unknown., Methods: Utilizing a mouse model of embryo culture, we cultured zygotes until the blastocyst stage (72-h culture) in G1/G2 containing either Vitrolife HSA, Sage HSA, or Recombinant HSA at 10%. Blastocyst quality (development, total cell number, superoxide generation), blastocyst lipid content (neutral lipids, non-esterified fatty acids, phospholipids, and triglycerides), implantation, and fetal lengths and weights were assessed. Fatty acid quantification of HSA source was assessed by standard thin-layer chromatography., Results: Sage HSA had the greatest fatty acid composition, with an eightfold increase in saturated fatty acids. This coincided with reduced blastocyst development, increased superoxide generation, neutral lipids and triglycerides levels of blastocysts, and decreased implantation rates (p < 0.05). Unexpectedly, while Recombinant HSA had the lowest overall lipids it had 70-fold increase in palmitoleic acid and the lowest fetal weights (p < 0.05)., Conclusion: Indicates the importance of a balance between different types/amount of lipids, and an "optimal ratio" required for embryo and fetal development. Therefore, the lipid content of HSA should be considered when choosing a suitable HSA source for use in clinical IVF., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: The goal is to determine if variations exist between male and female blastocysts in preimplantation measurements of quality and ploidy and in vitro fertilization elective single-embryo transfer (eSET) outcomes., Methods: A retrospective chart review was conducted from a private fertility center's database of blastocysts undergoing preimplantation genetic testing for aneuploidy, along with details of eSET from this screened cohort. Main outcomes included preimplantation embryo quality and sex-specific eSET outcomes., Results: A total of 3708 embryos from 578 women were evaluated, with 45.9% male and 54.1% female. The majority were High grade. No difference existed between embryo sex and overall morphological grade, inner cell mass or trophectoderm grade, or blastocyst transformation day. Female blastocysts had a higher aneuploidy rate than male blastocysts (P < 0.001). Five hundred thirty-nine eSETs from 392 women were evaluated, with High grade embryos more likely to have implantation (P < 0.001), clinical pregnancy (P < 0.001), and ongoing pregnancy (P = 0.018) than Mid or Low grade embryos. Day 5 blastocysts were more likely to have implantation (P = 0.018), clinical pregnancy (P = 0.005), and ongoing pregnancy (P = 0.018) than day 6 blastocysts. Male and female embryos had similar transfer outcomes, although female day 5 blastocysts were more likely to result in clinical pregnancy (P = 0.012), but not ongoing pregnancy, than female day 6 blastocysts. Male eSET outcomes did not differ by blastocyst transformation day., Conclusion: Male and female embryos have comparable grade and quality; however, female embryos were more likely to be aneuploid. Ongoing pregnancy rates did not differ by embryo sex. Day 5 embryos had more favorable transfer outcomes than day 6 embryos., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: This study aims to know what proportion of culture day 5 pre-blastocyst-stage embryos develop into blastocysts by culture day 6 and what patient and cycle characteristics are associated with delayed blastocyst formation., Methods: A retrospective observational cohort analysis was performed including a total of 9886 embryos from 1008 IVF cycles in 835 patients, who underwent treatment between January 1, 2016, and December 31, 2018. Autologous fresh in vitro fertilization (IVF) cycles at a single academic center were included in the analysis. Embryos were group-cultured using single-step culture media. Blastulation was defined as the presence of a new blastocyst. Usable blastulation was defined as the presence of a new good or excellent quality, expanded, hatching, or hatched blastocysts., Results: The mean blastulation rate between days 5 and 6 of extended embryo culture was 30.9%. The mean percentage of embryos developing into usable blastocyst-stage embryos was 19.8%. The factors associated with blastulation on day 6 included the total number of embryos and the number of pre-blastocysts on day 5, as well as the use of ICSI. Age, the number of total embryos, those remained in culture and pre-blastocysts, as well as the blastulation rate on day 5 were associated with usable blastulation., Conclusion: It is important to know the usable blastocyst development rate between culture days 5 and 6 in order to adequately counsel patients debating whether to proceed with fresh ET on day 5 or forego ET with the expectation that embryos will be biopsied for PGT and/or cryopreserved on culture day 6. Our findings provide evidence to help guide patients in this difficult decision., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: Oocytes and embryos can be vitrified with and without dimethyl sulfoxide (DMSO). Objectives were to compare no vitrification (No-Vitr), vitrification with DMSO (Vitr + DMSO), and vitrification without DMSO (Vitr - DMSO) on fresh/warmed oocyte survival, induced parthenogenetic activation, parthenogenetic embryo development, and embryonic maternal imprinted gene expression., Methods: In this prospective controlled laboratory study, mature B6C3F1 female mouse metaphase II oocytes were treated as: i) No-Vitr, ii) Vitr + DMSO/warmed, and iii) Vitr - DMSO/warmed with subsequent parthenogenetic activation and culture to the blastocyst stage. Oocyte cryo-survival, parthenogenetic activation and embryo development, parthenogenetic embryo maternal imprinted gene expression were outcome measures., Results: Oocyte cryo-survival was significantly improved in Vitr + DMSO versus Vitr - DMSO at initial warming and 2 h after warming. Induced parthenogenetic activation was similar between all three intervention groups. While early preimplantation parthenogenetic embryo development was similar between control, Vitr + DMSO, Vitr - DMSO oocytes, the development to blastocysts was significantly inferior in the Vitr - DMSO oocytes group compared to the control and Vitr + DMSO oocyte groups. Finally, maternal imprinted gene expression was similar between intervention groups at both the 2-cell and blastocyst parthenogenetic embryo stage., Conclusion(s): Inclusion of DMSO in oocyte vitrification solutions improved cryo-survival and developmental potential of parthenogenetic embryos to the blastocyst stage without significantly altering maternal imprinted gene expression., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: To determine whether blastocyst morphology has an impact on sex proportion at pre-implantation and birth in PGT-A and non-PGT-A cycles., Methods: A total of 1254 biopsied blastocysts from 466 PGT-A cycles were analyzed for sex proportion, day of biopsy, degree of expansion, inner cell mass (ICM), and trophectoderm (TE) morphology. From these, 197 frozen single embryo transfers (SET) were assessed for clinical outcomes and sex proportion of ongoing pregnancies and deliveries. In addition, we evaluated the day of vitrification/embryo transfer, degree of expansion, and TE morphology in a group of 229 births (217 cycles) from frozen or fresh transfers of non-biopsied blastocysts., Results: Sex proportion was impacted by day of biopsy and TE morphology, but not by ICM morphology, in PGT-A cycles. Therefore, biopsy on day 5 and TE "A" shifted the sex proportion towards males. Interestingly, we noted that our morphology-based embryo selection for SET of euploid blastocysts has favored the choice for XY embryos, generating a 54.3% XY proportion at transfer and 56.1% XY proportion at ongoing pregnancy/delivery. Our models indicate a weaker association between blastocyst morphology parameters and sex proportion of babies in non-PGT-A cycles., Conclusion: Blastocyst features associated with a skewed sex proportion towards XY embryos, such as biopsy on day 5 and top quality TE, are also parameters used for selecting euploid embryos for SET. Therefore, our data suggest that morphology-based embryo selection represents a strong factor responsible for a skewed male sex proportion at birth in PGT-A cycles., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: The aim of this study was to analyze the metabolic profiles of blastocoel fluid (BF) obtained from bovine embryos produced in vivo and in vitro., Methods: Expanded blastocysts (20/group) that were in vitro and in vivo derived at day 7 were used. BF was collected and analyzed under direct infusion conditions using a microTOF-Q ® mass spectrometer with electrospray ionization and a mass range of 50-650 m/z., Results: The spectrometry showed an evident difference in the metabolic profiles of BF from in vivo and in vitro produced embryos. These differences were very consistent between the samples of each group suggesting that embryo fluids can be used to identify the origin of the embryo. Ions 453.15 m/z, 437.18 m/z, and 398.06 m/z were identified as biomarkers for the embryo's origin with 100% sensitivity and specificity. Although it was not possible to unveil the molecular identity of the differential ions, the resulting spectrometric profiles provide a phenotype capable of differentiating embryos and hence constitute a potential parameter for embryo selection., Conclusion: To the best of our knowledge, our results showed, for the first time, an evident difference between the spectrometric profiles of the BF from bovine embryos produced in vivo and in vitro., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: To determine if euploidy rates and embryo development differ when blastocysts are cultured in CCM or SCM., Method: A single-center retrospective observational study was performed from September 2018 to March 2019. Patients [23-46 years] with at least four fresh mature oocytes (MII) without severe male factor infertility were included. Sibling MII were injected and cultured in Global®Total®LP (CCM) or Sage Quinn's Advantage® Cleavage and Blastocyst media (SCM) under 6% CO 2 , 5% O 2 , and 89% N 2 . Fertilization, cleavage, day (D) 5 blastulation, usable blastocyst (blastocysts biopsied/normally fertilized oocytes), and euploidy rates were recorded. Blastocysts were graded prior to trophectoderm (TE) biopsy on D5, 6, or 7 for genetic testing and mitochondrial DNA (mtDNA) quantification., Results: According to clinical practice, 1452 MII were randomly distributed: 751 in CCM and 701 in SCM. No differences were observed in fertilization and cleavages rates for CCM and SCM (77.4% vs 75.5%, p = 0.429 and 97.6% vs 99.1%, p = 0.094, respectively). Blastulation rate on D5 was higher in CCM (70.6% vs 62.2, p = 0.009); however, usable blastocyst rates were comparable (CCM: 58.3% vs SCM: 56.7%, p = 0.625). From a Poisson regression model adjusted for confounding factors, euploidy rates were not different between media (aOR = 1.18, [0.94-1.48], p = 0.157). Euploid blastocyst's mtDNA values were similar (CCM: 32.2, [30.5, 34.1] and SCM: 33.5, [31.8, 35.2], p = 0.345) and top-quality blastocysts (AA/BA) were increased in SCM (OR=1.04, [1.00-1.09], p = 0.037)., Conclusion: Under controlled in vitro conditions, euploidy rates and embryo development are comparable when embryos are cultured in CCM or SCM., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: Oocyte donor in vitro fertilization (IVF) represents an ideal model to study the effects of embryo stage on reproductive success, as embryos come from young women with high-quality oocytes. Our study aimed to determine if embryo transfer stage affected outcomes in oocyte donor IVF, including the common scenario where only a limited number of quality embryos are available after culture., Methods: This retrospective cohort analyzed anonymous vitrified donor oocyte cycles at a single clinic between 2008 and 2015. Overall, 983 recipients underwent 1178 warming cycles resulting in fresh transfer of one-to-two embryos. Our primary outcome was live birth; secondary outcomes included multiple birth, birthweight, and gestational age. Log binomial regression with cluster-weighted generalized estimating equations were used to calculate adjusted risk ratios (aRR) accounting for recipient age, race, and transfer year., Results: Among 132 cleavage and 1046 blastocyst transfer cycles, cleavage transfers were associated with lower probability of live birth (aRR 0.72, 95% CI 0.59-0.88). Subgroup analysis focused on cycles with a limited number of quality embryos 3 days post-fertilization (≤2), as clinically these women were most likely to be considered for cleavage transfers. Among these cycles (120 cleavage, 371 blastocyst), cleavage transfers were still associated with lower live birth rates compared to blastocyst (aRR 0.66, 95% CI 0.51-0.87) CONCLUSIONS: Even in a donor oocyte model with high-quality oocytes, there was a benefit to extended culture and blastocyst transfer, including when only one-to-two quality embryos were available after early culture. This is possibly owed to improved uterine synchronicity or decreased contractility., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

In vitro fertilization has been regarded as a forefront solution in treating infertility for over four decades, yet its effectiveness has remained relatively low. This could be attributed to the lack of advancements for the method of observing and selecting the most viable embryos for implantation. The conventional morphological assessment of embryos exhibits inevitable drawbacks which include time- and effort-consuming, and imminent risks of bias associated with subjective assessments performed by individual embryologists. A combination of these disadvantages, undeterred by the introduction of the time-lapse incubator technology, has been considered as a prominent contributor to the less preferable success rate of IVF cycles. Nonetheless, a recent surge of AI-based solutions for tasks automation in IVF has been observed. An AI-powered assistant could improve the efficiency of performing certain tasks in addition to offering accurate algorithms that behave as baselines to minimize the subjectivity of the decision-making process. Through a comprehensive review, we have discovered multiple approaches of implementing deep learning technology, each with varying degrees of success, for constructing the automated systems in IVF which could evaluate and even annotate the developmental stages of an embryo., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Staff competency is a crucial component of the in vitro fertilization (IVF) laboratory quality management system because it impacts clinical outcomes and informs the key performance indicators (KPIs) used to continuously monitor and assess culture conditions. Contemporary quality control and assurance in the IVF lab can be automated (collect, store, retrieve, and analyze), to elevate quality control and assurance beyond the cursory monthly review. Here we demonstrate that statistical KPI monitoring systems for individual embryologist performance and culture conditions can be detected by artificial intelligence systems to provide systemic, early detection of adverse outcomes, and identify clinically relevant shifts in pregnancy rates, providing critical validation for two statistical process controls proposed in the Vienna Consensus Document; intracytoplasmic sperm injection (ICSI) fertilization rate and day 3 embryo quality., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Embryo selection within in vitro fertilization (IVF) is the process of evaluating qualities of fertilized oocytes (embryos) and selecting the best embryo(s) available within a patient cohort for subsequent transfer or cryopreservation. In recent years, artificial intelligence (AI) has been used extensively to improve and automate the embryo ranking and selection procedure by extracting relevant information from embryo microscopy images. The AI models are evaluated based on their ability to identify the embryo(s) with the highest chance(s) of achieving a successful pregnancy. Whether such evaluations should be based on ranking performance or pregnancy prediction, however, seems to divide studies. As such, a variety of performance metrics are reported, and comparisons between studies are often made on different outcomes and data foundations. Moreover, superiority of AI methods over manual human evaluation is often claimed based on retrospective data, without any mentions of potential bias. In this paper, we provide a technical view on some of the major topics that divide how current AI models are trained, evaluated and compared. We explain and discuss the most common evaluation metrics and relate them to the two separate evaluation objectives, ranking and prediction. We also discuss when and how to compare AI models across studies and explain in detail how a selection bias is inevitable when comparing AI models against current embryo selection practice in retrospective cohort studies., (© 2021. The Author(s).)

Purpose: Some women undergoing stimulated cycles have elevated serum progesterone (P) on the day of ovulation trigger, but its effect on embryo quality is unclear. We analyze embryo quality among patients with high and low serum P undergoing preimplantation genetic testing for aneuploidy (PGT-A)., Methods: This retrospective study included 1597 patients divided into two groups by serum P values: < 1.5 ng/mL or ≥ 1.5 ng/mL. A gonadotrophin-releasing hormone (GnRH) antagonist protocol was established for each patient. Serum P levels were measured on the day of triggering. Propensity score matching and Poisson regression were done. Age, body mass index, and ovarian sensitivity index were also compared., Results: Elevated serum P was not significantly associated with euploid embryo rate or other embryo-quality variables evaluated in our study. Age was the only variable associated with euploidy rate (per MII oocyte, P < 0.001; per biopsied embryo, P = 0.008), embryo biopsy rate (P < 0.001), absolute number of euploid embryos (P = 0.008), and top-quality embryo rate (P = 0.008). Categorical variables decreased in value for every year of increased age in patients with high serum P., Conclusions: Elevated serum P did not affect the number of euploid and good-quality embryos for transfer in GnRH antagonist intracytoplasmic sperm injection (ICSI) cycles. Contrary to the clear influence of premature P elevation on endometrial receptivity based on literature, our results may help to tip the balance towards the absence of a negative effect of P elevation on embryo competence. More studies are needed to fully understand the effect of P elevation on reproductive outcomes., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: To study the relationship between the migration speed of nucleolus precursor bodies (NPBs) in male and female pronuclei (mPN; fPN) and human embryo development during assisted reproduction., Methods: The migration speed of 263 NPBs from 47 zygotes was quantitated, and embryonic development was observed until the blastocyst stage. The central coordinates of mPN, fPN, and NPBs were noted at multiple timepoints. Then, the distance traveled by the NPBs between two sequential images was measured, and migration speed was calculated. Additionally, we investigated the relationship between NPB migration speed and ploidy status (N = 33) or live birth/ongoing pregnancy (LB/OP) (N = 60) after assisted reproduction., Results: The NPB migration speed in both mPN and fPN was significantly faster in the zygotes that developed into blastocysts (N = 25) than that in the zygotes that arrested (N = 22). The timing of blastulation was negatively correlated with NPB migration speed in the mPN. Faster NPB migration was significantly correlated with LB/OP. In multivariate logistic analysis, NPB migration speed in the mPN was the only morphokinetic parameter associated with LB/OP. In a receiver-operating characteristic curve analysis of LB/OP by the NPB migration speed in the mPN, the cut-off value was 4.56 μm/h. When this cut-off value was applied to blastocysts with preimplantation genetic testing for aneuploidy, 100% of the blastocysts faster than or equal to the cut-off value were euploid., Conclusion: The NPBs migrated faster in zygotes having the potential to develop into a blastocyst, and eventually into a baby. This predictor could be an attractive marker for non-invasive embryo selection., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: To assess whether utilization of a mathematical ranking algorithm for assistance with embryo selection improves clinical outcomes compared with traditional embryo selection via morphologic grading in single vitrified warmed euploid embryo transfers (euploid SETs)., Methods: A retrospective cohort study in a single, academic center from September 2016 to February 2020 was performed. A total of 4320 euploid SETs met inclusion criteria and were included in the study. Controls included all euploid SETs in which embryo selection was performed by a senior embryologist based on modified Gardner grading (traditional approach). Cases included euploid SETs in which embryo selection was performed using an automated algorithm-based approach (algorithm-based approach). Our primary outcome was implantation rate. Secondary outcomes included ongoing pregnancy/live birth rate and clinical loss rate., Results: The implantation rate and ongoing pregnancy/live birth rate were significantly higher when using the algorithm-based approach compared with the traditional approach (65.3% vs 57.8%, p<0.0001 and 54.7% vs 48.1%, p=0.0001, respectively). After adjusting for potential confounding variables, utilization of the algorithm remained significantly associated with improved odds of implantation (aOR 1.51, 95% CI 1.04, 2.18, p=0.03) ongoing pregnancy/live birth (aOR 1.99, 95% CI 1.38, 2.86, p=0.0002), and decreased odds of clinical loss (aOR 0.42, 95% CI 0.21, 0.84, p=0.01)., Conclusions: Clinical implementation of an automated mathematical algorithm for embryo ranking and selection is significantly associated with improved implantation and ongoing pregnancy/live birth as compared with traditional embryo selection in euploid SETs., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: To study embryo morphokinetics in relation to release in spent media of molecules with possible roles in development and implantation (miR-20a, miR-30c, and sHLA-G)., Methods: Data were obtained from embryos generated in standard IVF and ICSI cycles. The Eeva system was used for embryo assessment, based on early morphokinetic parameters and producing a score (1-5, best-worst) corresponding to higher/medium/lower chances of development to blastocyst. miRNAs - mm miR-20a-5p and miR-30c-5p - and sHLA-G were quantified in 25 μl of spent blastocyst media (SBM) collected before vitrification or transfer. Statistical analyses were performed applying Kolmogorov-Smirnov, Shapiro-Wilk, and Spearman's correlation coefficient tests, where appropriate., Results: SBM were collected from a total of 172 viable blastocysts. Their analysis showed that concentration of miR-20a was progressively lower as Eeva score increased and probability of development to blastocyst decreased (P = 0.016). The opposite trend was observed in the case of miR-30c, i.e., concentration was higher as score increased and chances of development to blastocyst decreased (P = 0.004). Analysis of sHLA-G revealed a negative correlation with Eeva score, i.e., levels were progressively lower as Eeva score increased and probability of development to blastocyst decreased (R = - 0.388, N = 141, P = 0.001)., Conclusion: Our data suggest that morphokinetic algorithms that predict development to blastocyst stage, in fact, also identify embryos with molecular and cellular profiles more consistent with developmental functions., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: We aimed to assess whether home collection and increased time to semen processing are associated with altered sperm parameters, fertilization rates (FR), day 5 usable quality blastocyst development rates (D5-UQBR), or pregnancy rates (PR) in patients undergoing IVF/ICSI., Methods: This was a retrospective cohort study of patients undergoing IVF/ICSI before the coronavirus disease 2019 (COVID-19) pandemic ("clinic" collection, n = 119) and after COVID-19 ("home" collection, n = 125) at an academic fertility practice. Home collection occurred within 2 h of semen processing. Patient sperm parameters, FR (#2PN/MII), D5-UQBR (# transferable and freezable quality blastocysts/# 2PN), and PR in fresh transfer cycles were compared between clinic and home groups with t-tests. The association between time to processing on outcomes was assessed with regression modeling, controlling for potential confounders., Results: Mean male age was 37.9 years in the clinic group and 37.2 years in the home group (p = 0.380). On average, men were abstinent for 3.0 days (SD 1.7) in the clinic group and 4.1 days (SD 5.4) in the home group (p = 0.028). Mean time to semen processing was 35.7 min (SD 9.4) in the clinic group and 82.6 min (SD 33.8) in the home group (p < 0.001). There was no association between collection location and increased time to processing on sperm motility, total motile count, FR, D5-UQBR, or PR., Conclusions: Our data suggest that increased time to processing up to 2 h with home semen collection does not negatively impact sperm parameters or early IVF/ICSI outcomes.

In order to maintain pregnancy rates following single embryo transfer, optimisation of embryo culture and selection is vital. Time-lapse monitoring (TLM) has the potential to play a crucial role by providing sequential images of embryo development and minimal disturbance. Therefore, in this study morphometric assessment of blastocyst area and maximum width was performed in order to evaluate if these parameters are associated with pregnancy outcomes in IVF/ICSI cycles. This is a retrospective study of 664 patients who had elective single blastocyst transfer (eSBT). The EmbryoScope drawing tools were used to measure specific variables such as the maximum blastocyst width and blastocyst area. Our results show that women who were pregnant had significantly (P < 0.01) larger blastocyst width [median (range) μm] 184 (125-239) versus non-pregnant, 160 (120-230)] and area [median (range) μm 2 ] 26099 (12101-45,280) versus non-pregnant women, 22,251 (10992-37,931)]. A univariate logistic regression performed showed that blastocyst width [(OR = 1.026, 95% CI = (1.019, 1.033)] was significant (P < 0.01) and for every μm increase of blastocyst width, the odds of clinical pregnancy increase by 2.6%. A univariate logistic regression performed showed that blastocyst area [(OR = 1.00008, 95% CI = (1.00006, 1.00011)] was significant with P < 0.01. For every μm 2 increase of blastocyst area, our data showed the odds of clinical pregnancy increase by 0.008%. Hosmer-Lemeshow tests of calibrations were performed to verify calibration. Although our findings show a clear correlation between blastocyst dimensions and the clinical pregnancy rate, further studies are necessary to confirm these observations.

Purpose: In vitro developing embryos may apparently show no developmental progress during 24 h and resume their development up to the blastocyst stage. The present study was conducted to assess their ability to implant and to give rise to a live birth when replaced at day 5 (fresh or vitrified/warmed) as compared to continuously developing embryos., Methods: Embryo development follow-up and grade were prospectively recorded in a photo database. The studied period was from April 2011 to July 2017. The studied embryos included transient arrested embryos (TAE) that showed the same developmental stage at two subsequent observations, i.e. between day 2 and day 3 (d2 and d3), between day 3 and day 4 (d3 and d4) and between day 4 and day 5 (d4 and d5). TAE were compared to continuously developing embryos (CDE). Elective day 5 embryo transfers were performed., Results: Woman age was higher in TAE (34.3±3.9) than in CDE (32.9±4.8) (p<0.01). TAE were more frequently (63.1%) observed after ICSI than after conventional IVF (55.9%) (p<0.01). Implantation rate was reduced in TAE as compared to CDE, after both fresh (10.0% vs 23.8% [p<0.01]) and vitrified/warmed (12.9% vs 19.0% [p<0.01]) embryo transfers. Delivery rate was also lower after the transfer of fresh (8.3% vs 19.4% [p<0.01]) and vitrified/warmed (8.5% vs 14.1% [p<0.01]) TAE as compared to CDE. Implantation and delivery rates were not statistically different whether embryo arrested between day 2 and day 3 (d2 and d3), between day 3 and day 4 (d3 and d4) or between day 4 and day 5 (d4 and d5)., Conclusion: TAE may be considered for transfer at a lower priority than CDE and associated with inferior prognosis than CDE.

Purpose: To determine whether the blastocyst mitochondrial DNA (mtDNA) content is related to the miscarriage rate in patients undergoing single euploid frozen embryo transfer (SEFET)., Methods: A total of 355 single euploid frozen embryo transfer cycles were studied retrospectively between April 2017 and December 2018. A trophectoderm biopsy was performed on day 5/6 blastocysts. Post next-generation sequencing (NGS), the mtDNA content was calculated as the ratio of mitochondrial DNA over nuclear DNA, and the association between blastocyst mtDNA content and miscarriage rate was evaluated., Result(s): Three hundred fifty-five euploid blastocysts were selected for SEFET in 314 patients with an average age of 33.7 ± 5.6 years; 255 were biopsied on day 5 (71.8%) and 100 on day 6 (28.2%). Frozen embryo transfer (FET) was performed either in a hormone replacement therapy (HRT) cycle (71.8%; n = 255) or in a natural cycle (NC) (28.2%; n = 100). A pregnancy rate of 66.2% (235/355) was obtained with clinical pregnancy and miscarriage rates of 52.4% (n = 186) and 5.6% (n = 20), respectively. There was no significant difference neither between the blastocyst mtDNA content of pregnant and nonpregnant patients (27.7 ± 9.2 vs. 29.4 ± 8.6, P = 0.095) nor between patients with a clinical pregnancy and miscarriage (30.5 ± 9.3 vs. 27.3 ± 9.2, P = 0.136). Multivariate logistic regression analysis showed the same nonsignificant relationship, except for the miscarriage rate and BMI (OR 1.149, 95% CI 1.03-1.28; P = 0.012)., Conclusion(s): Mitochondrial DNA content is unable to predict the miscarriage of implanted human euploid blastocysts.

Purpose: To evaluate the effectiveness, efficiency, and safety of a transnational gamete donation (TGD) programme based on the shipping of vitrified donor oocytes., Methods: A retro-prospective observational study was conducted in the Assisted Reproductive Technology Center of the University Hospital of Florence, Italy. The study population included 622 consecutive donor oocyte cycles. A mean number of 6 vitrified oocytes per couple were shipped from two Spanish biobanks. In the receiving centre, gametes were warmed and inseminated and the subsequent embryo transfer (ET) was performed. The main outcome measurement was LBR. Secondary outcomes included oocyte survival rate, ICSI damage rate, normal fertilization, cleavage, and implantation rate (IR) in both 'fresh' and cryotransfer cycles., Results: A total number of 3536 mature oocytes were warmed with 81.4% surviving oocytes. 2PN oocytes were 1941 with an ICSI normal fertilization rate of 70.4% and a cleavage rate of 93.4%; 857 day-3 embryos were transferred in 498 women, 63 blastocysts in 44. Couples with at least one vitrified embryo were 181 (32.3%). IR was 25.1% and 33.1% for day-3 ET and blastocyst stage respectively. Crude pregnancy rate and LBR after the first ET were 35.5% and 27% correspondingly with a conservative cumulative LBR of 34% and an optimal LBR of 51.4%., Conclusion: Imported vitrified donor oocytes retain their competence and are capable of resulting in ongoing pregnancies and healthy babies in a proportion comparable to other existing systems as egg donation with vitrification/warming in the same laboratory and transnational fresh oocyte donation.

Purpose: This study aimed to determine the effects of drilling and thinning treatment of laser-assisted hatching on the expression and methylation of imprinted gene IGF2/H19 in embryos and offspring., Methods: The prehatching blastocysts with treatment of drilling or thinning, or control prehatching blastocysts, were transplanted in surrogate uteri. The DNA methylation of IGF2/H19 imprinting control region (ICR) and the expression of IGF2 and H19 were respectively evaluated using bisulfite conversion-mediated sequencing and real-time PCR., Results: The drilling group showed a significant increase in the development rate of hatched blastocysts in comparison with the control and thinning group. DNA methylation level of IGF2/H19 ICR of hatched blastocysts in the thinning group was 27.33% in comparison with the 38.67% and 36% observed in the control and drilling group. The thinning treatment increased the DNA methylation level of IGF2/H19 ICR in the placenta in comparison with the control and drilling group. The drilling and thinning treatment decreased the expression level of H19 mRNA in prehatching and hatched blastocysts as well as placenta, while a significant increase in the expression level of H19 mRNA of offspring was observed in the thinning group. The thinning treatment increased the expression level of IGF2 mRNA of prehatching blastocysts and offspring and a significant decrease in placenta, while the drilling treatment resulted in a significant increase in the expression level of IGF2 mRNA of hatched blastocysts and placenta., Conclusion: These observations suggested that drilling used for hatching of in vitro cultured mouse blastocysts may improve the production of offspring.

Spontaneous in vitro hatching of human blastocysts starts with the formation of a tunnel through the zona pellucida (ZP) by cellular projections of trophoblast cells. Our aim was to identify the proteins that are upregulated in these initially hatching cells as compared to trophectoderm (TE) cells from blastocysts that had not yet hatched. Forty seven women that underwent assisted reproduction treatment donated their ICSI-derived polyploid blastocysts for the study. In polyploid blastocysts that started spontaneous hatching, hatched clusters of cells were collected from the outer side of the ZP. Liquid chromatography mass spectrometry was applied to determine the proteins that were upregulated in these cells as compared to TE cells obtained from inside the ZP. Whole non-hatched polyploid blastocysts were used as controls. Overall 1245 proteins were identified in all samples. Forty nine proteins were significantly upregulated in hatching cells and 17 in the TE cells. There was minimal overlap between hatching and TE samples; only serine protease inhibitors (SERPINS) and lipocalin were detected in both samples. Myosin and actin were highly upregulated in the hatching cells as well as paraoxonase, N-acetylmuramoyl alanine amidase, and SERPINS clade A and galectin. In the TE cells, gamma butyrobetaine dioxygenase, lupus La protein, sialidase, lysosomal Pro-X carboxypeptidase, phospholipase b, and SERPINS clade B and A were among the most highly upregulated proteins. These findings may contribute to the basic knowledge of the molecular behavior of the specific cells that actively perforate the glycoprotein matrix of the ZP.

Trophectoderm biopsy is increasingly performed for pre-implantation genetic testing of aneuploidies and considered a safe procedure on short-term clinical outcome, without strong assessment of long-term consequences. Poor biological information on human trophectoderm is available due to ethical restrictions. Therefore, most studies have been conducted in vitro (choriocarcinoma cell lines, embryonic and pluripotent stem cells) and on murine models that nevertheless poorly reflect the human counterpart. Polarization, compaction, and blastomere differentiation (e.g., the basis to ascertain trophectoderm origin) are poorly known in humans. In addition, the trophectoderm function is poorly known from a biological point of view, although a panoply of questionable and controversial microarray studies suggest that important genes overexpressed in trophectoderm are involved in pluripotency, metabolism, cell cycle, endocrine function, and implantation. The intercellular communication system between the trophectoderm cells and the inner cell mass, modulated by cell junctions and filopodia in the murine model, is obscure in humans. For the purpose of this paper, data mainly on primary cells from human and murine embryos has been reviewed. This review suggests that the trophectoderm origin and functions have been insufficiently ascertained in humans so far. Therefore, trophectoderm biopsy should be considered an experimental procedure to be undertaken only under approved rigorous experimental protocols in academic contexts.

Purpose: To study whether members of the miR-290-295 cluster in spent culture medium (SCM) of embryos are correlated with morphokinetics and apoptosis., Methods: Cryopreserved 1-cell stage mouse embryos were cultured to the blastocyst stage, development was monitored by time-lapse, 59 SCM were collected, and miR-291a and miR-294 were detected with polymerase chain reaction (PCR). Blastocysts were immuno-stained for sexing (H2AK119ub) and for apoptosis (TUNEL). Each embryo and SCM were individually processed. Correlations were run between the miRNAs and developmental events (t2, t3, t4, t5, t8, tSB, tB, ECC2, ECC3, s2, s3, dB) and apoptosis (apoptotic cells/total cell number %). MiR-294 SCM and cell levels were compared in 40 blastocysts. Apoptosis was induced in 15 blastocysts with UV radiation and SCM samples were analyzed for miR-294., Results: MiR-291a and miR-294 are released in variable levels by mouse blastocysts. Their release is similar between male and female embryos. No significant correlations were found between these miRNAs and development. MiR-294 was significantly positively correlated with apoptosis (r = 0.560, p < 0.001). Cellular expression was lower in blastocysts that released miR-294 in high levels compared with null, low, and medium release embryos (p < 0.01). UV radiation caused apoptosis which triggered higher secretion of miR-294 in 15 blastocysts versus 13 control embryos (p < 0.01)., Conclusion(s): MicroRNAs are important regulators of preimplantation development. Apoptosis triggers the release of miR-294 by blastocysts which possibly serves a secretory role for embryo-maternal communication. SCM miRNA analysis is possible for individually cultured embryos and future studies can investigate miRNAs as noninvasive markers of embryo quality.

Purpose: The purpose was to investigate the association between embryonic development or implantation and the content of interleukin-6 and 10 (IL-6, IL-10) and tumor necrosis factor-α (TNF-α) in single-blastocyst conditioned medium (SBCM)., Methods: Thirty-eight SBCM samples (SBCMs) were collected from blastocysts with different morphological scores. IL-6, IL-10, and TNF-α concentration in 38 SBCMs was detected by Single Molecule Array and compared according to the blastocyst quality: top-quality (TQ) and non-top quality (NTQ), or blastulation time: day 5 (D5) and day 6 (D6). In another experiment, 61 SBCMs were collected from TQ blastocyst transplanted on D5, and IL-6 concentration in SBCM was compared based on whether embryos are implanted or not (implanted and non-implanted)., Results: In the first experiment, IL-6, IL-10, and TNF-α concentration was not significantly different between the TQ-SBCM and NTQ-SBCM. The D6-SBCM had a higher IL-6 concentration compared with the D5-SBCM, while IL-10 and TNF-α concentration was not significantly different between the D5-SBCM and D6-SBCM. The IL-6 concentration in D5-NTQ or D6-TQ SBCM was higher than that in D5-TQ or D6-NTQ SBCM (P < 0.05), respectively. Furthermore, the spearman analysis demonstrated that IL-6 concentration in SBCM was negatively correlated with the blastocyst quality on D5 and positively correlated with the blastocyst quality on D6. In the second experiment, no significant difference in IL-6 concentration was found between SBCM from implanted and non-implanted blastocyst., Conclusion: IL-6 concentration in SBCM was associated with embryo quality depending on the blastulation time, although it might not be associated with the blastocyst implantation.

Purpose: Binovular follicles including a pair of conjoined oocytes within a common zona pellucida or their fusion in the zonal region gained some attentions due to its possible role in dizygotic twins. Although some cases in the literature been reported in which two conjoined oocytes arising from binovular follicles were mature, and injected with two separated sperm, no available evidence reported for dizygotic twin pregnancies., Methods: A case report of a 37-year-old female patient underwent embryo transfer cycle whereby a pair of conjoined blastocysts after ICSI of a pair of conjoined oocytes was transferred., Results: The β-hCG level was positive 15 days after embryo transfer. The subsequent pregnancy scan revealed a dizygotic pregnancy. The woman gave birth to two healthy boys in the mid of 38 weeks of gestation by cesarean section., Conclusions: Given the insufficient evidence on how to handle conjoined oocytes, this report acknowledges the first occurrence of dizygotic twin delivery resulted from transfer of a pair of conjoined blastocysts after ICSI of a pair of conjoined oocytes. This also confirms that we should be extremely conservative in discarding any mature oocyte without sufficient data about its useless future to result in a healthy baby.

Purpose: To evaluate whether mtDNA content at the blastocyst stage differs between embryos derived from fresh or vitrified sibling oocytes., Material and Methods: A retrospective analysis was performed between March 2017 and September 2018, including 504 blastocysts from 94 couples undergoing preimplantation genetic testing for aneuploidies (PGT-A), using fresh oocytes together with previously vitrified oocytes. Trophectoderm biopsies were performed and subjected to next generation sequencing., Results: On average, 1.8 ± 1.0 oocyte vitrification cycles were performed per patient. Between fresh and vitrified cycles, no difference was observed between the number of fertilized oocytes (5.3 ± 4.2 versus 5.5 ± 3.0). Blastulation rate on day 5 per fertilized oocyte was significantly higher in the fresh group (62% ± 29% versus 44% ± 31%; p < 0.001). For the 504 biopsied blastocysts, 294 fresh versus 210 vitrified, no significant differences were found in the euploid rate, 40.5% versus 38.6% (p = 0.667), and mtDNA content, 30.1 (± 10.6) versus 30.0 (± 12.5) (p = 0.871), respectively. Regardless of the origin of the oocytes, aneuploid blastocysts contained significantly higher mtDNA values compared with the euploid ones (31.4 versus 28.0; p = 0.001). Furthermore, top-quality blastocysts had a significantly lower mtDNA content compared with moderate and poor-quality blastocysts (p < 0.001) and blastocysts biopsied on day 5 showed significantly lower mtDNA content compared with day 6 or day 7 blastocysts (p < 0.001). However, when analyzing the blastocyst mtDNA content according to the ploidy state, no differences were found for blastocyst quality or day of biopsy between blastocysts originating from fresh or vitrified oocytes., Conclusion: Oocyte vitrification does not affect the mtDNA content of trophectoderm biopsies.

Purpose: The aim of this study was to establish a new method of decreasing cytoplasmic fragmentation in early-stage human embryos., Methods: The zona pellucida (ZP) of abnormally-fertilized oocytes (zygotes with three pronuclei (3PN)), which were donated by patients, was removed at the pronuclear stage. ZP-free embryos were observed in a time-lapse imaging and culturing system in order to examine developmental morphology and embryonic quality., Results: Based on a modification of Veeck's criteria, 47 of 69 ZP-free 3PN embryos (68.1%) showed fragmentation of less than 20% of the total volume of cytoplasm at the first cleavage (grades 1 and 2), 17 (24.6%) showed 20-40% cytoplasmic fragments (grade 3), and only 5 (7.2%) showed more than 40% fragments (grade 4). These results suggest that the rate of fragmentation is decreased by ZP removal before the first cleavage, compared with normal (ZP-intact) 3PN and 2-pronuclear/2-polar body embryos., Conclusions: This study revealed that the ZP is not always necessary for normal development after the pronuclear stage because the ZP-free embryos studied herein developed normally, maintained their cell adhesion well, and showed a decreased rate of fragmentation. This innovative culture system might provide the major breakthrough needed for patients who have difficulty obtaining good-quality embryos.

Purpose: To establish parameters during mouse extended embryo culture that accurately predict fetal developmental potential of a blastocyst without performing embryo transfer., Methods: Embryos of three varying qualities were produced: poor quality embryos produced from in vitro matured oocytes (IVM), intermediate quality embryos produced from in vivo matured oocytes followed by in vitro fertilization and embryo culture (IVF); high quality embryos developed in vivo (VIVO). Embryonic day (E) 3.5 embryos from each group with similar morphologies were used for surgical embryo transfer to assess implantation and fetal developmental potential, in addition to placing these embryos into extended culture until E 8.5 to examine outgrowth area, egg cylinder volume, epiblast cell number, and outgrowth morphologies by immunofluorescence and 3D confocal microscopy., Results: The proportional differences in epiblast cell number are strikingly similar to fetal development following embryo transfer, suggesting that this parameter may be indicative of the potential of an embryo to successfully develop into a fetus., Conclusion: Extended embryo culture provides more accurate information regarding developmental potential than blastocyst morphological assessment. Specifically, epiblast cell number is an accurate and valuable predictor of fetal developmental potential. This work sets the stage for routine evaluation of embryo quality past the time embryos would normally be transferred. The ability to determine post implantation potential without embryo transfer may greatly improve efforts to culture higher quality embryos in vitro for human IVF, as well as reducing animal use and eliminating confounding maternal factors associated with embryo transfer experiments in research.