Chromosomal and functional characterization of the early stages of human embryogenesis

The main objective of modern IVF is to maximize the effectiveness of the times to achieve a pregnancy and at the same time also manage the risks, looking for new predictive parameters of the embryonic developmental competence. The analysis of embryo morphodynamic growth is not associated with its euploidy or implantation competence. However, some static parameters of embryo quality might exist that could be associated with embryo competence beyond its chromosomal constitution. The aims of this project are: i) To study from a morphodynamic, genetic and clinical point of view, embryos that show an abnormal development during preimplantation growth, in particular, the exclusion of cells (ExC) from embryonic mass at the moment of morulation. ii) Trying to understand if the morphodynamic characterization of euploid blastocyst development allows a higher prediction of live-birth (LB) after single-embryo-transfers (SET). For both the aims set in this Ph.D. project, preimplantation development and morphodynamic growth of embryos were observed in a time-lapse culture system (Embryoscope, Vitrolife). For the first aim, our preliminary data show that the exclusion of cells from the body of the blastocyst could be not-intuitively associated to a higher competence resulting from the embryonic capacity to overcome an abnormal cleavage pattern occurred in the very first divisions before the activation of the embryonic genome (4 to 8cell stage in humans). It is exciting the future perspective of collecting the ExC aiming at analyzing them through the karyomapping technology as well as biochemical assays, to better describe both the chromosomal segregation and the cellular physiology. For the second aim, we have divided the study into two phases in collaboration with 3 IVF Centers. In phase1, 511 first euploid SETs from 1069 patients undergoing preimplantation-genetic-testing-for-aneuploidies (PGT-A) cycles at 2 IVF centers were investigated (training set). All embryos were cultured in a specific time-lapse incubator with continuous media. The data from the time of polar-body-extrusion to time starting-blastulation were collected. Trophectoderm (TE) and inner-cell-mass (ICM) static morphology were also assessed. Logistic regressions were conducted to outline a predictive model of LB, whose power was estimated through a ROC-curve. In phase2, this model was tested in an independent dataset of 319 consecutive SETs from 546 PGT-A cycles at 3 IVF centers (validation set). The average LB-rate in the training set was 40% (N=207/511). Only time-of-morulation (tM) and trophectoderm quality were outlined as putative predictors of LB at both centers. The model showed a significant AUC (area under the curve) of 0.65. In the validation set, the euploid blastocysts characterized by tM<80hr and high-quality trophectoderm resulted in an LB-rate of 55.2% (n=37/67), while those with tM≥80hr and a low-quality trophectoderm resulted in an LB-rate of 25.5% (N=13/51;p<0.01). The ROC-curve analysis pictured an AUC of 0.6. A model including tM and trophectoderm quality involves a better prediction of euploid blastocyst reproductive competence. This model was reproducible across different centers under specific culture conditions. These data support the crucial role of morulation for embryo development, a stage that involves massive morphological, cellular and molecular changes requiring more investigations. Moreover, important guidelines for IVF laboratories that do not conduct a time-lapse-based embryo culture may arise from these two studies

Human blastocyst biopsy and vitrification

Blastocyst biopsy is performed to obtain a reliable genetic diagnosis during IVF cycles with preimplantation genetic testing. Then, the ideal workflow entails a safe and efficient vitrification protocol, due to the turnaround time of the diagnostic techniques and to transfer the selected embryo(s) on a physiological endometrium in a following natural cycle. A biopsy approach encompassing the sequential opening of the zona pellucida and retrieval of 5-10 trophectoderm cells (ideally 7-8) limits both the number of manipulations required and the exposure of the embryo to sub-optimal environmental conditions. After proper training, the technique was reproducible across different operators in terms of timing of biopsy (~8 min, ranging 3-22 min based on the number of embryos to biopsy per dish), conclusive diagnoses obtained (~97.5%) and live birth rates after vitrified-warmed euploid blastocyst transfer (>40%). The survival rate after biopsy, vitrification and warming was as high as 99.8%. The re-expansion rate at 1.5 h from warming was as high as 97%, largely dependent on the timing between biopsy and vitrification (ideally ≤30 min), blastocyst morphological quality and day of biopsy. In general, it is better to vitrify a collapsed blastocyst; therefore, in non-PGT cycles, laser-assisted artificial shrinkage might be performed to induce embryo collapse prior to cryopreservation. The most promising future perspective is the non-invasive analysis of the IVF culture media after blastocyst culture as a putative source of embryonic DNA. However, this potential avant-garde is still under investigation and a reliable protocol yet needs to be defined and validated

Fertility Preservation Through Oocyte Vitrification: Clinical and Laboratory Perspectives

: Preserving female fertility is crucial in a multifunctional healthcare system that takes care of patients' future quality of life. Oocyte cryopreservation is recognized by several international scientific societies as the gold standard for fertility preservation in postpubertal women, for both medical and non-medical indications. The main medical indications are oncologic diseases, gynecologic diseases such as severe endometriosis, systemic diseases compromising the ovarian reserve, and genetic conditions involving premature menopause. This paper describes the whole clinical and laboratory work-up of a fertility preservation treatment by outlining recommendations for objective and evidence-based counseling. Furthermore, it focuses on the effectiveness of the procedure and describes the most appropriate strategies to fully exploit the ovarian reserve and maximize the number of oocytes retrieved in the shortest possible time. The evaluation of the ovarian reserve, the definition of an ideal stimulation protocol, as well as oocyte retrieval, denudation, and vitrification procedures have been detailed along with approaches to maximize their efficacy, efficiency, and safety

Biochemical pregnancy loss after frozen embryo transfer seems independent of embryo developmental stage and chromosomal status

Biochemical pregnancy loss (BPL), defined as serum beta-human chorionic gonadotropin levels ≥50 IU/l in at least two pregnancy tests, not associated with any ultrasonographical evidence of pregnancy, is often attributed to chromosomal abnormalities; however, no hard evidence exists to support this hypothesis. Are any IVF cycle parameters associated with the occurrence of a BPL? Retrospective study aimed at evaluating the effect of embryo developmental stage at transfer and chromosomal assessment on the BPL rate in IVF after frozen embryo transfer (FET). Specifically, 641 FET of 1179 cleavage stage untested embryos (Group A), 1021 FET of 1259 untested blastocyst stage embryos (Group B), and 789 blastocyst stage FET of 803 euploid embryos (Group C) were performed in a 6-year period. Only FET were evaluated to avoid a potential effect of ovarian stimulation on endometrial receptivity. The BPL rates were similar (n = 30/217, 13.8% in Group A; n = 37/412, 9.0% in Group B; n = 42/433, 9.7% in Group C). Neither embryo developmental stage at FET nor chromosomal assessment showed a correlation with BPL. Furthermore, logistic regression analyses did not show any association between BPL and patient, cycle and/or transfer characteristics. BPL seems independent of the embryo's developmental stage, the use of trophectoderm biopsy and the chromosomal constitution at FET. Similar BPL rates after transferring euploid blastocysts compared with both untested cleavage and blastocyst stage embryos suggest investigating the role of endometrial and other embryonic factors putatively involved in the process of implantation

Definition and validation of a custom protocol to detect miRNAs in the spent media after blastocyst culture: searching for biomarkers of implantation

STUDY QUESTION: Can miRNAs be reliably detected in the spent blastocyst media (SBM) after IVF as putative biomarkers of the implantation potential of euploid embryos? SUMMARY ANSWER: Adjustment of the data for blastocyst quality and the day of full-expansion hinders the predictive power of a fast, inexpensive, reproducible and user-friendly protocol based on the detection of 10 selected miRNAs from SBM. WHAT IS KNOWN ALREADY: Euploidy represents so far the strongest predictor of blastocyst competence. Nevertheless, ∼50% of the euploid blastocysts fail to implant. Several studies across the years have suggested that a dialogue exists between the embryo and the endometrium aimed at the establishment of a pregnancy. MicroRNAs have been proposed as mediators of such a dialogue and investigated in this respect. Several expensive, time-consuming and complex protocols have been adopted and promising results have been produced, but conclusive evidence from large clinical studies is missing. STUDY DESIGN, SIZE, DURATION: This study was conducted in two phases from September 2015 to December 2017. In Phase 1, the human blastocyst miRNome profile was defined from the inner cell mass (ICM) and the corresponding whole-trophectoderm (TE) of six donated blastocysts. Two different protocols were adopted to this end. In parallel, 6 pools of 10 SBM each were run (3 from only implanted euploid blastocysts, IEBs; and 3 from only not-implanted euploid blastocysts, not-IEBs). A fast, inexpensive and user-friendly custom protocol for miRNA SBM profiling was designed. In Phase 2, 239 SBM from IEB and not-IEB were collected at three IVF centres. After 18 SBM from poor-quality blastocysts were excluded from the analysis, data from 107 SBM from not-IEB and 114 from IEB were produced through the previously developed custom protocol and compared. The data were corrected through logistic regressions. PARTICIPANT/MATERIALS, SETTINGS, METHODS: Donated blastocysts underwent ICM and whole-TE isolation. SBM were collected during IVF cycles characterized by ICSI, blastocyst culture in a continuous media, TE biopsy without zona pellucida opening in Day 3, quantitative PCR (qPCR)-based aneuploidy testing and vitrified-warmed single euploid embryo transfer. Not-IEB and IEB were clustered following a negative pregnancy test and a live birth, respectively. The Taqman Low Density Array (TLDA) cards and the Exiqon microRNA human panel I+II qPCR analysis protocols were adopted to analyse the ICM and whole-TE. The latter was used also for SBM pools. A custom Downloaded from https://academic.oup.com/humrep/article-abstract/34/9/1746/5550837 by Sapienza Università di Roma user on 28 October 2019 Customized miRNA analysis in spent IVF media 1747 protocol and plate was then designed based on the Exiqon workflow, validated and finally adopted for SBM analysis in study Phase 2. This custom protocol allows the analysis of 10 miRNAs from 10 SBM in 3 hours from sample collection to data inspection. MAIN RESULTS AND ROLE OF THE CHANCE: The TLDA cards protocol involved a higher rate of false positive results (5.6% versus 2.8% with Exiqon). There were 44 miRNAs detected in the ICM and TE from both the protocols. One and 24 miRNAs were instead detected solely in the ICM and the TE, respectively. Overall, 29 miRNAs were detected in the pooled SBM: 8 only from not-IEB, 8 only from IEB and 13 from both. Most of them (N = 24/29, 82.7%) were also detected previously in both the ICM and TE with the Exiqon protocol; two miRNAs (N = 2/29, 6.9%) were previously detected only in the TE, and three (N = 3/29, 10.3%) were never detected previously. In study Phase 2, significant differences were shown between not-IEB and IEB in terms of both miRNA detection and relative quantitation. However, when the data were corrected for embryo morphology and day of full development (i.e. SBM collection), no significant association was confirmed. LIMITATIONS, REASONS FOR CAUTION: This study did not evaluate specifically exosomal miRNAs, thereby reducing the chance of identifying the functional miRNAs. Ex-vivo experiments are required to confirm the role of miRNAs in mediating the dialogue with endometrial cells, and higher throughput technologies need to be further evaluated for miRNA profiling from clinical SBM samples. WIDER IMPLICATIONS OF THE FINDINGS: Although no clinical predictive power was reported in this study, the absence of invasiveness related with SBM analysis and the evidence that embryonic genetic material can be reliably detected and analysed from SBM make this waste product of IVF an important source for further investigations aimed at improving embryo selection. STUDY FUNDING/COMPETING INTEREST(S): This project has been financially supported by Merck KgaA (Darmstadt, Germany) with a Grant for Fertility Innovation (GFI) 2015. The authors have no conflict of interest to declare related with this stud

Time of morulation and trophectoderm quality are predictors of a live birth after euploid blastocyst transfer: a multicenter study

Objective: To investigate whether the morphodynamic characterization of a euploid blastocyst's development allows a higher prediction of a live birth after single-embryo-transfer (SET). Design: Observational cohort study conducted in two phases: training and validation. Setting: Private in vitro fertilization centers. Patient(s): Euploid blastocysts: 511 and 319 first vitrified-warmed SETs from 868 and 546 patients undergoing preimplantation genetic testing for aneuploidies (PGT-A) in the training and validation phase, respectively. Intervention(s): Data collected from time of polar body extrusion to time of starting blastulation, and trophectoderm and inner-cell-mass static morphology in all embryos cultured in a specific time-lapse incubator with a continuous medium. Logistic regressions conducted to outline the variables showing a statistically significant association with live birth. In the validation phase, these variables were tested in an independent data set. Main outcome measure(s): Live births per SET. Result(s): The average live birth rate (LBR) in the training set was 40% (N = 207/511). Only time of morulation (tM) and trophectoderm quality were outlined as putative predictors of live birth at two IVF centers. In the validation set, the euploid blastocysts characterized by tM <80 hours and high-quality trophectoderm resulted in a LBR of 55.2% (n = 37/67), while those with tM ≥ 80 hours and a low-quality trophectoderm resulted in a LBR of 25.5% (N = 13/51). Conclusion(s): Time of morulation and trophectoderm quality are better predictors of a euploid blastocyst's reproductive competence. Our evidence was reproducible across different centers under specific culture conditions. These data support the crucial role of morulation for embryo development, a stage that involves massive morphologic, cellular, and molecular changes and deserves more investigation