BlastAssist: a deep learning pipeline to measure interpretable features of human embryos.

STUDY QUESTION: Can the BlastAssist deep learning pipeline perform comparably to or outperform human experts and embryologists at measuring interpretable, clinically relevant features of human embryos in IVF? SUMMARY ANSWER: The BlastAssist pipeline can measure a comprehensive set of interpretable features of human embryos and either outperform or perform comparably to embryologists and human experts in measuring these features. WHAT IS KNOWN ALREADY: Some studies have applied deep learning and developed 'black-box' algorithms to predict embryo viability directly from microscope images and videos but these lack interpretability and generalizability. Other studies have developed deep learning networks to measure individual features of embryos but fail to conduct careful comparisons to embryologists' performance, which are fundamental to demonstrate the network's effectiveness. STUDY DESIGN, SIZE, DURATION: We applied the BlastAssist pipeline to 67 043 973 images (32 939 embryos) recorded in the IVF lab from 2012 to 2017 in Tel Aviv Sourasky Medical Center. We first compared the pipeline measurements of individual images/embryos to manual measurements by human experts for sets of features, including: (i) fertilization status (n = 207 embryos), (ii) cell symmetry (n = 109 embryos), (iii) degree of fragmentation (n = 6664 images), and (iv) developmental timing (n = 21 036 images). We then conducted detailed comparisons between pipeline outputs and annotations made by embryologists during routine treatments for features, including: (i) fertilization status (n = 18 922 embryos), (ii) pronuclei (PN) fade time (n = 13 781 embryos), (iii) degree of fragmentation on Day 2 (n = 11 582 embryos), and (iv) time of blastulation (n = 3266 embryos). In addition, we compared the pipeline outputs to the implantation results of 723 single embryo transfer (SET) cycles, and to the live birth results of 3421 embryos transferred in 1801 cycles. PARTICIPANTS/MATERIALS, SETTING, METHODS: In addition to EmbryoScope™ image data, manual embryo grading and annotations, and electronic health record (EHR) data on treatment outcomes were also included. We integrated the deep learning networks we developed for individual features to construct the BlastAssist pipeline. Pearson's χ2 test was used to evaluate the statistical independence of individual features and implantation success. Bayesian statistics was used to evaluate the association of the probability of an embryo resulting in live birth to BlastAssist inputs. MAIN RESULTS AND THE ROLE OF CHANCE: The BlastAssist pipeline integrates five deep learning networks and measures comprehensive, interpretable, and quantitative features in clinical IVF. The pipeline performs similarly or better than manual measurements. For fertilization status, the network performs with very good parameters of specificity and sensitivity (area under the receiver operating characteristics (AUROC) 0.84-0.94). For symmetry score, the pipeline performs comparably to the human expert at both 2-cell (r = 0.71 ± 0.06) and 4-cell stages (r = 0.77 ± 0.07). For degree of fragmentation, the pipeline (acc = 69.4%) slightly under-performs compared to human experts (acc = 73.8%). For developmental timing, the pipeline (acc = 90.0%) performs similarly to human experts (acc = 91.4%). There is also strong agreement between pipeline outputs and annotations made by embryologists during routine treatments. For fertilization status, the pipeline and embryologists strongly agree (acc = 79.6%), and there is strong correlation between the two measurements (r = 0.683). For degree of fragmentation, the pipeline and embryologists mostly agree (acc = 55.4%), and there is also strong correlation between the two measurements (r = 0.648). For both PN fade time (r = 0.787) and time of blastulation (r = 0.887), there's strong correlation between the pipeline and embryologists. For SET cycles, 2-cell time (P < 0.01) and 2-cell symmetry (P < 0.03) are significantly correlated with implantation success rate, while other features showed correlations with implantation success without statistical significance. In addition, 2-cell time (P < 5 × 10-11), PN fade time (P < 5 × 10-10), degree of fragmentation on Day 3 (P < 5 × 10-4), and 2-cell symmetry (P < 5 × 10-3) showed statistically significant correlation with the probability of the transferred embryo resulting in live birth. LIMITATIONS, REASONS FOR CAUTION: We have not tested the BlastAssist pipeline on data from other clinics or other time-lapse microscopy (TLM) systems. The association study we conducted with live birth results do not take into account confounding variables, which will be necessary to construct an embryo selection algorithm. Randomized controlled trials (RCT) will be necessary to determine whether the pipeline can improve success rates in clinical IVF. WIDER IMPLICATIONS OF THE FINDINGS: BlastAssist provides a comprehensive and holistic means of evaluating human embryos. Instead of using a black-box algorithm, BlastAssist outputs meaningful measurements of embryos that can be interpreted and corroborated by embryologists, which is crucial in clinical decision making. Furthermore, the unprecedentedly large dataset generated by BlastAssist measurements can be used as a powerful resource for further research in human embryology and IVF. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by Harvard Quantitative Biology Initiative, the NSF-Simons Center for Mathematical and Statistical Analysis of Biology at Harvard (award number 1764269), the National Institute of Heath (award number R01HD104969), the Perelson Fund, and the Sagol fund for embryos and stem cells as part of the Sagol Network. The authors declare no competing interests. TRIAL REGISTRATION NUMBER: Not applicable.

A clinical predictive model for live birth in women of advanced age undergoing PGT cycles.

PURPOSE: The trend of delaying childbirth has resulted in a growing number of advanced-aged women who are opting for preimplantation genetic testing (PGT) to screen for monogenic diseases or structural chromosomal rearrangements (PGT-M and PGT-SR). This increase in demand necessitates the development of a clinical predictive model for live birth outcomes in these women. Therefore, the objective of this study is to construct a comprehensive predictive model that assesses the likelihood of achieving a successful live birth in advanced-aged women undergoing PGT-M and PGT-SR treatments. METHODS: A retrospective cohort study of 37-45-year-old women undergoing preimplantation genetic testing for monogenic disease or structural chromosomal rearrangement cycles from 2010 to 2021 was conducted at a university hospital reproductive centre. The purpose was to develop a clinical predictive model for live birth in these women. The main outcome studied was the cumulative live birth rate in the first or subsequent cycles. Developing a decision tree enabled a comprehensive study of clinical parameters and expected outcomes. RESULTS: The analysis included 158 women undergoing 753 preimplantation genetic testing cycles. The cumulative live birth rate was 37.342% (59/158). Decision tree analysis revealed that women aged ≤ 40.1 or women > 40.1 with one or more top-quality transferable embryos in their first cycle had the best chance for a live baby (56% and 41%, respectively). Those older than 40.1 without top-quality embryos and seven or fewer dominant follicles had no live births. A Kaplan-Meier curve showed that for autosomal dominant diseases, there was a negligible increase in live birth rate after three cycles, compared to six cycles in autosomal recessive inheritance. CONCLUSION: In older women, the chance of delivering after repeated cycles is higher in those with at least one top-quality unaffected embryo in their first preimplantation genetic testing cycle. Additional preimplantation genetic testing cycles after three in carriers of an autosomal dominant disorder and six in those with an autosomal recessive disorder should be considered prudently.

Isoform-resolved transcriptome of the human preimplantation embryo.

Human preimplantation development involves extensive remodeling of RNA expression and splicing. However, its transcriptome has been compiled using short-read sequencing data, which fails to capture most full-length mRNAs. Here, we generate an isoform-resolved transcriptome of early human development by performing long- and short-read RNA sequencing on 73 embryos spanning the zygote to blastocyst stages. We identify 110,212 unannotated isoforms transcribed from known genes, including highly conserved protein-coding loci and key developmental regulators. We further identify 17,964 isoforms from 5,239 unannotated genes, which are largely non-coding, primate-specific, and highly associated with transposable elements. These isoforms are widely supported by the integration of published multi-omics datasets, including single-cell 8CLC and blastoid studies. Alternative splicing and gene co-expression network analyses further reveal that embryonic genome activation is associated with splicing disruption and transient upregulation of gene modules. Together, these findings show that the human embryo transcriptome is far more complex than currently known, and will act as a valuable resource to empower future studies exploring development.

A pathogenic variant in the uncharacterized RNF212B gene results in severe aneuploidy male infertility and repeated IVF failure.

Quantitative and qualitative spermatogenic impairments are major causes of men's infertility. Although in vitro fertilization (IVF) is effective, some couples persistently fail to conceive. To identify causal variants in patients with severe male infertility factor and repeated IVF failures, we sequenced the exome of two consanguineous family members who underwent several failed IVF cycles and were diagnosed with low sperm count and motility. We identified a rare homozygous nonsense mutation in a previously uncharacterized gene, RNF212B, as the causative variant. Recurrence was identified in another unrelated, infertile patient who also faced repeated failed IVF treatments. scRNA-seq demonstrated meiosis-specific expression of RNF212B. Sequence analysis located a protein domain known to be associated with aneuploidy, which can explain multiple IVF failures. Accordingly, FISH analysis revealed a high aneuploidy rate in the patients' sperm cells and their IVF embryos. Finally, inactivation of the Drosophila orthologs significantly reduced male fertility. Given that members of the evolutionary conserved RNF212 gene family are involved in meiotic recombination and crossover maturation, our findings indicate a critical role of RNF212B in meiosis, genome stability, and in human fertility. Since recombination is completely absent in Drosophila males, our findings may indicate an additional unrelated role for the RNF212-like paralogs in spermatogenesis.

The association between a carrier state of FMR1 premutation and numeric sex chromosome variations.

PURPOSE: Women carriers of FMR1 premutation are at increased risk of early ovarian dysfunction and even premature ovarian insufficiency. The aim of this study was to examine a possible association between FMR1 permutation and numeric sex chromosome variations. METHODS: A retrospective case-control study conducted in the reproductive center of a university-affiliated medical center. The primary outcome measure was the rate of sex chromosomal numerical aberrations, as demonstrated by haplotype analyses, in FMR1 premutation carriers compared to X-linked preimplantation genetic testing for monogenic/single gene defect (PGT-M) cycles for other indications that do not affect the ovarian follicles and oocytes. RESULTS: A total of 2790 embryos with a final genetic analysis from 577 IVF PGT-M cycles were included in the final analysis. Mean age was similar between the groups, however, FMR1 carriers required more gonadotropins, and more women were poor responders with three or less oocytes collected. The ratio of embryos carrying a numeric sex chromosome variation was similar: 8.3% (138/1668) of embryos in the FMR1 group compared to 7.1% (80/1122) in the controls. A subgroup analysis based on age and response to stimulation has not demonstrated a significant difference either. CONCLUSIONS: Although carriers of FMR1 premutation exhibit signs of reduced ovarian response, it does not seem to affect the rate of numeric sex chromosomal variation compared to women undergoing PGT-M for other indications. This suggests that the mechanism for chromosomal number aberrations in women at advanced maternal age are different to those FMR1 premutation carriers with poor ovarian reserve.

Cleavage stage at compaction-a good predictor for IVF outcome.

OBJECTIVE: To analyze whether cleavage stage at compaction, and not only kinetics, can serve as a reliable predictor for clinical outcome. METHODS: A retrospective cohort study including 1194 embryos, classified by compaction initiation stage (Group 1: compaction at fewer than eight cells, Group 2: compaction at eight cells, Group 3: compaction at more than eight cells). Of these, 815 embryos were evaluated for morphokinetic preimplantation parameters, and 379 embryos were analyzed for clinical implantation following thawing and transfer of single blastocysts during the same period. RESULTS: In total, 1194 embryos were analyzed. Embryos that underwent compaction from more than eight cells (Group 3) exhibited more synchronous cleavage compared with Groups 1 and 2 (at both S2 and S3; P < 0.001), and displayed a significantly lower fragmentation rate. The likelihood of obtaining top-quality blastocysts decreased by 73% and 44% when comparing Group 3 embryos with those of Groups 1 and 2, respectively, (P < 0.03). Clinical validation of the results shows that while compaction from fewer than eight cells barely produced blastocysts for transfer, compaction at eight or more cells is crucial for implantation and birth (birth rates 11.1% and 18.5% for Groups 2 and 3, respectively). CONCLUSION: Cleavage stage at compaction has a direct effect on blastocyst quality and subsequent pregnancy, so can be included in newly developed deep learning models for embryo selection.

The impact of fragile X premutation carrier status on embryo morphokinetic development.

RESEARCH QUESTION: Does inheritance of the fragile X mental retardation 1 (FMR1) premutation allele affect embryo morphokinetic development? DESIGN: A retrospective cohort analysis of 529 embryos from 126 IVF cycles of 39 FMR1 premutation female carriers undergoing preimplantation genetic testing for monogenic/single gene defects (PGT-M). Morphological and morphokinetic parameters obtained using a time-lapse monitoring system were compared between embryos that inherited the FMR1 premutation allele (FMR1 group, n = 271) and those who received the normal allele (normal group, n = 258). The following embryo outcome measures were compared: morphokinetic parameters up to day 3, start of blastulation time (tSB) for day 5 embryos and the rate of top-quality embryos on days 3 and 5. RESULTS: No differences were found in morphokinetic parameters between the groups from the time of intracytoplasmic sperm injection (ICSI) until a biopsy on day 3. The blastulation rate in the two groups was comparable. However, the start of blastulation was delayed in FMR1 embryos compared to that in the genetically normal embryos (median tSB: 104.2 h [99.3-110.3] versus 101.6 h [94.5-106.7], P = 0.01). In addition, the rate of top-quality FMR1 embryos was lower than that of genetically normal embryos (25.6% versus 38.8%, P = 0.04). CONCLUSION: Embryos that inherit the FMR1 premutation allele are of lower quality at the blastocyst stage compared with those that do not inherit the mutated allele.

Similar fertilization rates and preimplantation embryo development among testosterone-treated transgender men and cisgender women.

RESEARCH QUESTION: What are the effects of testosterone treatment on oocyte fertilization and preimplantation embryo development among transgender men who have undergone fertility preservation? DESIGN: A retrospective study was undertaken in a university-affiliated tertiary hospital between April 2016 and November 2021. Embryos were divided into three groups by source: 210 embryos from 7 testosterone-exposed transgender men, 135 from 10 cisgender women who cryopreserved embryos, and 276 from 24 cisgender women who underwent fertility treatment. Statistical analyses compared assisted reproductive technology outcomes between the group of transgender men and both groups of cisgender women. Morphokinetic and morphological parameters were compared between the embryos derived from these three groups. RESULTS: The transgender men (30.2 ± 3.5 years of age) were significantly younger than the cisgender women who cryopreserved embryos (35.1 ± 1.8 years; P = 0.005) and the cisgender women who underwent fertility treatment (33.8 ± 3.2 years; P = 0.017). After adjusting for participant age, the fertilization rate was comparable between the transgender men and both groups of cisgender women (P = 0.391 and 0.659). There were no significant differences between the transgender men and the cisgender women who preserved fertility in terms of number of cryopreserved embryos (7.2 ± 5.1 and 3.5 ± 2.6; P = 0.473) or the distribution of embryo age at cryopreservation (P = 0.576). All morphokinetic parameters evaluated by time-lapse imaging, as well as the morphological characteristics, were comparable for the embryos in all three groups. CONCLUSIONS: Testosterone exposure among transgender men has no adverse impact upon fertilization rates or preimplantation embryo development and quality.

Maternal underweight does not adversely affect the outcomes of IVF/ICSI and frozen embryo transfer cycles or early embryo development.

OBJECTIVE: To compare assisted reproductive technology (ART) outcomes and preimplantation embryo development between underweight and normal-weight women. METHODS: This retrospective cohort study included 26 underweight women (body mass index [BMI] < 18.50 kg/m2) and 104 normal-weight women (BMI >20 and <24.9 kg/m2) who underwent a total of 204 in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycles and 358 fresh/frozen embryo transfers (ET) in our institution between January 2016 and December 2018. Statistical analyses compared selected ART outcomes (ovarian stimulation, fertilization, and pregnancy) between both weight groups. Morphokinetic and morphological parameters were also compared between 346 and 1467 embryos of underweight and normal-weight women, respectively. RESULTS: The mean ± standard deviation age of the underweight and normal-weight women was similar (31.6 ± 4.17 vs 32.4 ± 3.59 years; p = .323). There were no differences in the peak estradiol levels, the number of retrieved oocytes, the number of metaphase II oocytes, and the oocyte maturity rates between the two groups. The IVF/ICSI fertilization rates and the number of embryos suitable for transfer or cryopreservation were similar for both groups. All morphokinetic parameters that were evaluated by means of time-lapse imaging as well as the morphological characteristics were comparable between low and normal BMI categories. There were no significant differences in pregnancy achievement, clinical pregnancy, live births, and miscarriage rates between the suboptimal and optimal weight women. CONCLUSION: Underweight status has no adverse impacts on the outcomes of IVF/ICSI with either fresh or frozen ET or on preimplantation embryo development and quality.

A decision tree analysis applied to women aged 43-45: who should be referred for ovum donation?

RESEARCH QUESTION: In women at the advanced age of 43-45 years undergoing repeated IVF cycles with autologous oocytes, who has the highest chance for birth and who should be referred early to receive donor oocytes? DESIGN: A retrospective cohort study was conducted at a university hospital reproductive centre. The computerized database of 394 women aged 43-45 years undergoing 1528 non-donor IVF or intracytoplasmic sperm injection cycles between 2010 and 2019 was analysed. A decision tree was developed, enabling a comprehensive study of a set of clinical parameters and the expected outcomes. RESULTS: The cumulative clinical pregnancy rate was 15.0% (59/394) and the cumulative live birth rate was 8.4% (33/394). The decision tree developed to predict women who should be offered egg donation included age, poor ovarian response to stimulation, the number of top-quality embryos, dominant follicles, previous pregnancy or live birth, fertilized oocytes and body mass index. The model showed that a good ovarian response in the first cycle was the best predictor for live birth (13.3% gave birth). However, among women with poor responses, 7.1% of those who were younger than 43.5 years gave birth, and none of the women who were older than 43.5 years did. CONCLUSIONS: Women over 43.5 years old with fewer than four oocytes collected in their first IVF cycle should be offered ovum donation, since their live birth rate in subsequent cycles is negligible.

A decision tree model for predicting live birth in FMR1 premutation carriers undergoing preimplantation genetic testing for monogenic/single gene defects.

RESEARCH QUESTION: Can patient selection for successful preimplantation genetic testing for women who are fragile X (FMR1) premutation carriers be optimized using a decision tree analysis? This decision support tool enables a comprehensive study of a set of clinical parameters and the expected outcomes. DESIGN: A retrospective case-control study analysing the results of 264 fresh and 21 frozen preimplantation genetic testing for monogenic disorders/single gene defects (PGT-M) cycles in 64 FMR1 premutation carriers. Primary outcome was live birth per cycle start. Live birth rate was calculated for the start of the ovarian stimulation cycle. Fresh and frozen embryo transfers from the same cycle were included. RESULTS: The decision tree model showed that the number of cytosine guanine (CGG) repeats was only a moderate predictor for live birth, whereas an age younger than 36 years was the best predictor for live birth, followed by a collection of 14 or more oocytes. These findings were supported by the results of the logistic regression, which found that only age and oocyte number were significantly associated with live birth (P = 0.005 and 0.017, respectively). CONCLUSIONS: The number of CGG repeats is a relatively poor predictor for live birth in PGT-M cycles. FMR1 premutation carriers are no different from non-carriers. Age is the best identifier of live birth, followed by the number of retrieved oocytes.

Gonadotropin-Releasing Hormone Agonist Versus Recombinant Human Chorionic Gonadotropin Triggering in Fertility Preservation Cycles.

The purpose of this research is to study the efficacy of GnRH-a versus r-hCG triggering in patients who go through fertility preservation cycles. This retrospective cohort study was performed in a tertiary university-affiliated medical center. It includes 191 patients undergoing fertility preservation cycles between May 2013 and September 2018, in which ovulation was induced by either GnRH-a or r-hCG. Main outcome measures were number and rate of mature oocyte. Among treatment cycles with medical indication, GnRH agonist significantly increases the odds for high mature rate by 3.55 (1.30-9.66), while in treatment cycles with social indication, there is no significant effect of the triggering agent. An advantage for GnRH-a triggering was observed in medically indicated preservation cycles.

Principles of signaling pathway modulation for enhancing human naive pluripotency induction.

Isolating human MEK/ERK signaling-independent pluripotent stem cells (PSCs) with naive pluripotency characteristics while maintaining differentiation competence and (epi)genetic integrity remains challenging. Here, we engineer reporter systems that allow the screening for defined conditions that induce molecular and functional features of human naive pluripotency. Synergistic inhibition of WNT/ß-CATENIN, protein kinase C (PKC), and SRC signaling consolidates the induction of teratoma-competent naive human PSCs, with the capacity to differentiate into trophoblast stem cells (TSCs) and extraembryonic naive endodermal (nEND) cells in vitro. Divergent signaling and transcriptional requirements for boosting naive pluripotency were found between mouse and human. P53 depletion in naive hPSCs increased their contribution to mouse-human cross-species chimeric embryos upon priming and differentiation. Finally, MEK/ERK inhibition can be substituted with the inhibition of NOTCH/RBPj, which induces alternative naive-like hPSCs with a diminished risk for deleterious global DNA hypomethylation. Our findings set a framework for defining the signaling foundations of human naive pluripotency.

Transfer of Day 6 Frozen-Thawed Blastocysts on Day 5 Compared with Day 6: Catching Up with the Window of Implantation-a Retrospective Study.

To compare clinical pregnancy rate (CPR) and live birth rate (LBR) after frozen-thawed embryo transfer (FET) of day (D-) 6 blastocysts on D-5 versus D-6. A retrospective cohort study. A university-affiliated single-center tertiary hospital. Women undergoing FET of D-6 blastocysts between August 2015 and March 2019 were included in the study. Exclusion criteria were endometriosis and maternal age ≥ 42. Cycles involving embryo transfer (ET) at D-6 were compared to cycles involving ET on D-5. Primary outcomes assessed were CPR and LBR, and the secondary outcomes were spontaneous abortion and chemical pregnancy rates. Forty-two cycles were assessed, 21 in which ET occurred on D-6 and 21 in which ET occurred on D-5. There were no significant differences between groups regarding age, body mass index (BMI), etiology of infertility, number of oocytes aspirated and blastocysts cryopreserved in the fresh cycle, reason for freezing on D-6, endometrial thickness before ET, and blastocyst grade. A comparison of outcomes of ET on D-5 with those involving ET on D-6 revealed that D-5 transfer produced significantly higher CPR (8, 38% vs. 2, 8.5%; P = 0.030) and LBR (6, 28.6% vs. 1, 4.8%; P = 0.038), respectively. FET of D-6 embryos on D-5 compared with D-6 is associated with increased CPR and LBR values. These findings might be related to the limited time window for optimal rates of implantation and indicate that transferring embryos on D-6 of a FET cycle is likely too late.

Effects of letrozole or tamoxifen coadministered with a standard stimulation protocol on fertility preservation among breast cancer patients.

PURPOSE: To assess the effects of letrozole or tamoxifen coadministration on fertility preservation treatment outcomes. METHODS: Retrospective cohort study of 118 breast cancer patients undergoing fertility preservation treatment between 2008 and 2018. Patients who received letrozole (n = 36) or tamoxifen (n = 30) were compared to controls (n = 52) who underwent standard ovarian stimulation protocols. The primary outcome measures included the number of retrieved oocytes, mature oocytes (MII), fertilization, and top-quality embryo rates. The secondary outcome measures included duration of stimulation, gonadotropin dose and peak estradiol level. RESULTS: The number of oocytes retrieved, MII oocytes, fertilization rate, duration of stimulation, or gonadotropin dose were similar in the letrozole and tamoxifen groups, compared to controls. Top-quality embryo rate was lower in the tamoxifen group compared to controls (25% vs 39.4%, respectively, P = 0.034). The abnormal fertilization rate was higher in the letrozole group compared to controls (7.8% vs 3.60%, respectively, P = 0.015). A stepwise logistic regression analysis revealed that letrozole and peak estradiol were significantly associated with abnormal fertilization (OR 11.94; 95% CI 2.35-60.4, P = 0.003 for letrozole and OR 1.075; 95% CI 1.024-1.12, P = 0.004 per 100 unit change in estradiol). CONCLUSIONS: There may be a negative effect of letrozole or tamoxifen on fertilization and embryo quality, in fertility preservation cycles. Further studies are needed to confirm these findings.

In-vitro maturation of oocytes recovered during cryopreservation of pre-pubertal girls undergoing fertility preservation.

RESEARCH QUESTION: In-vitro maturation (IVM) of oocytes recovered during ovarian tissue cryopreservation (OTC) is often practised, although it is still considered experimental. To date, only a few studies have examined the success of this maturation process in pre-menarche girls. The aim of this study was to examine the outcomes of IVM of oocytes recovered during OTC in pre-menarche patients scheduled for onco-therapy. DESIGN: A retrospective cohort study in a tertiary university-affiliated hospital. A total of 93 patients aged 0-25 years who underwent OTC as part of onco-fertility preservation between 2007 and 2019 were included in the study. Oocytes were recovered from the medium after OTC and matured over 48 h. Oocyte development and maturation rate were recorded and compared between different age groups. RESULTS: Patient's age was not correlated linearly with the total number of mature oocytes R = 0.2. The absolute maturation rate in post-menarche and pre-menarche patients differed significantly (38.0% versus 25.3%, respectively; P > 0.001), whereas the degeneration rate of the oocytes did not (39.8% versus 33.5%; P = 0.167). The pre-menarche group had significantly lower mean number of metaphase II oocytes compared with the post-menarche group (2.8 [±2.3] versus 5.6 [±4.6]; P = 0.01; 95% CI -4.62 to -0.46). Oocytes recovered from patients aged 1-5 years demonstrated low maturation rate. CONCLUSIONS: Oocytes recovered from pre-menarche girls, and especially those younger than the age of 5 years who undergo fertility preservation, have a lower chance of reaching maturity in IVM compared with older women. This may indicate a need for alternative methods for preserving fertility in these young patients.

The Effect of Advanced Maternal Age on Embryo Morphokinetics.

Purpose: To compare the morphokinetic parameters of pre-implantation development between embryos of women of advanced maternal age (AMA) and young women. Methods: Time-lapse microscopy was used to compare morphokinetic variables between 495 embryos of AMA women ≥ age 42 years and 653 embryos of young patients ( 0.05). Conclusions: While early morphokinetic parameters do not reflect dynamics unique to embryos of older women, a tendency toward developmental arrest was observed, which would likely be even more pronounced at later stages of development.

Morphokinetic characteristics of embryos originating from extremely small follicles: A prospective study.

OBJECTIVE: To investigate the developmental potential of oocytes and embryos derived from extremely small follicles (<10 mm) in comparison to those originated in larger follicles. STUDY DESIGN: A prospective study, undertaken in a university affiliated single center tertiary hospital. The study included 98 patients undergoing infertility treatments. On the day of ovum pickup (OPU) follicles were counted and measured. Aspiration of follicles larger and smaller than 10 mm was undertaken separately and the development of embryos originating from oocytes from these follicles was followed up using different wells for each embryo. There was no low limit of size for aspiration. Each oocyte retrieved was marked for its origin and numbered for further follow up. We recorded: Oocytes retrieved, maturation stage, fertilization rate, cleavage rate, morphokinetic parameters, embryo transfers, embryo freezing, oocyte freezing and biopsy rate for preimplantation genetic diagnosis (PGD). Quality was evaluated by the morphokinetic parameters of the embryos developed using time-lapse imaging technology. Day 3 KIDScore was calculated to all embryos. RESULTS: Small follicles compared to large follicles displayed lower recovery rate (45% vs. 74%, P < 0.0001), fewer matured oocytes (37.5% vs. 61.7%, P < 0.0001), higher rates of GV oocytes (20.7% vs., 3.7%, P < 0.0001), and lower fertilization rate (43.7% vs. 63.3%, P < 0.0001. However, morphokinetic variables were similar between embryos that originated from either small or large follicles. Median KIDscores were identical for embryos from small or large follicle origin. CONCLUSIONS: Embryos originated from small follicles were not different than embryos from larger follicles, as assessed by morphokinetic parameters in time lapse system. In view of our findings, physicians should bear in mind that small follicle aspiration might yield good quality embryos.

Systematic interrogation of human promoters.

Despite much research, our understanding of the architecture and cis-regulatory elements of human promoters is still lacking. Here, we devised a high-throughput assay to quantify the activity of approximately 15,000 fully designed sequences that we integrated and expressed from a fixed location within the human genome. We used this method to investigate thousands of native promoters and preinitiation complex (PIC) binding regions followed by in-depth characterization of the sequence motifs underlying promoter activity, including core promoter elements and TF binding sites. We find that core promoters drive transcription mostly unidirectionally and that sequences originating from promoters exhibit stronger activity than those originating from enhancers. By testing multiple synthetic configurations of core promoter elements, we dissect the motifs that positively and negatively regulate transcription as well as the effect of their combinations and distances, including a 10-bp periodicity in the optimal distance between the TATA and the initiator. By comprehensively screening 133 TF binding sites, we find that in contrast to core promoters, TF binding sites maintain similar activity levels in both orientations, supporting a model by which divergent transcription is driven by two distinct unidirectional core promoters sharing bidirectional TF binding sites. Finally, we find a striking agreement between the effect of binding site multiplicity of individual TFs in our assay and their tendency to appear in homotypic clusters throughout the genome. Overall, our study systematically assays the elements that drive expression in core and proximal promoter regions and sheds light on organization principles of regulatory regions in the human genome.

Time-lapse imaging reveals delayed development of embryos carrying unbalanced chromosomal translocations.

PURPOSE: The purpose of the study was to compare the morphokinetic parameters of embryos carrying balanced chromosomal translocations with those carrying unbalanced chromosomal translocations using time-lapse microscopy. METHODS: The study group included 270 embryos that underwent biopsies on day 3 for preimplantation genetic diagnosis (PGD) for chromosomal translocations in our unit between 2013 and 2015. All embryos were incubated under time-lapse microscopy and evaluated for timing of developmental events up to day 5. The timing of these events was compared between balanced and unbalanced embryos, potentially viable and nonviable variants, and maternal versus paternal inheritance of the translocation. RESULTS: The PGD analysis found that 209 (77%) of the 270 biopsied embryos carried an unbalanced translocation. Embryos carrying unbalanced translocations, which are expected to lead to implantation failure or miscarriage, cleaved less synchronously and were delayed in time of cleavage to the 4-cell stage (t4) and in time of start of blastulation (tSB) compared with balanced embryos (P < 0.05). Furthermore, embryos carrying nonviable translocations demonstrated a significant delay at the time of pronuclei fading (tPNf) compared with those carrying potentially viable translocations (P < 0.05). Embryos whose unbalanced translocations were of maternal origin were significantly delayed in most of the morphokinetic parameters (including tPNf, t2, t3, t4, t6, t7, t8, cc2, s2, and tSB) compared with embryos carrying balanced translocations (P < 0.05). CONCLUSIONS: Embryos carrying unbalanced chromosomal translocations mainly of maternal origin undergo delayed development and asynchronous cleavage that may lead to implantation failure or miscarriage.