N. Zaninovic

Embryo development after heterotopic transplantation of cryopreserved ovarian tissue

BACKGROUND Cancer treatments, including chemotherapy, radiotherapy, and radical surgery, can induce premature menopause and infertility in hundreds of thousands of women of reproductive age every year. One of the ways to possibly preserve fertility before these treatments is to cryopreserve ovarian tissue for later transplantation. We aimed to restore fertility by cryopreservation and transplantation of ovarian tissue. METHODS Ovarian tissue was cryopreserved from a 30-year-old woman with breast cancer before chemotherapy-induced menopause, and this tissue was transplanted beneath the skin of her abdomen 6 years later. FINDINGS Ovarian function returned in the patient 3 months after transplantation, as shown by follicle development and oestrogen production. The patient underwent eight oocyte retrievals percutaneously and 20 oocytes were retrieved. Of the eight oocytes suitable for in-vitro fertilisation, one fertilised normally and developed into a four-cell embryo. INTERPRETATION Fertility and ovarian endocrine function can be preserved in women by long-term ovarian tissue banking.

Expansion and maintenance of human embryonic stem cell–derived endothelial cells by TGFβ inhibition is Id1 dependent

Previous efforts to differentiate human embryonic stem cells (hESCs) into endothelial cells have not achieved sustained expansion and stability of vascular cells. To define vasculogenic developmental pathways and enhance differentiation, we used an endothelial cell–specific VE-cadherin promoter driving green fluorescent protein (GFP) (hVPr-GFP) to screen for factors that promote vascular commitment. In phase 1 of our method, inhibition of transforming growth factor (TGF)β at day 7 of differentiation increases hVPr-GFP+ cells by tenfold. In phase 2, TGFβ inhibition maintains the proliferation and vascular identity of purified endothelial cells, resulting in a net 36-fold expansion of endothelial cells in homogenous monolayers, which exhibited a transcriptional profile of Id1highVEGFR2highVE-cadherin+ ephrinB2+. Using an Id1-YFP hESC reporter line, we showed that TGFβ inhibition sustains Id1 expression in hESC-derived endothelial cells and that Id1 is required for increased proliferation and preservation of endothelial cell commitment. Our approach provides a serum-free method for differentiation and long-term maintenance of hESC-derived endothelial cells at a scale relevant to clinical application.

Promoter-Bound Trinucleotide Repeat mRNA Drives Epigenetic Silencing in Fragile X Syndrome

Repeat Silencing Fragile X syndrome, a genetic cause of many cases of autism and mental retardation, involves expansion of a trinucleotide repeat in the fragile X mental retardation 1 (FMR1) gene. Working with human embryonic stem cells, Colak et al. (p. 1002) found that the expanded repeat region was transcribed into the untranslated region of FMR1 messenger RNA, which then bound to the DNA repeat region in the FMR1 gene, inactivating the gene. The findings explain how the trinucleotide repeat expansion causes RNA-directed gene silencing during development in fragile X syndrome. An abnormal activation of gene silencing underlies fragile X syndrome. Epigenetic gene silencing is seen in several repeat-expansion diseases. In fragile X syndrome, the most common genetic form of mental retardation, a CGG trinucleotide–repeat expansion adjacent to the fragile X mental retardation 1 (FMR1) gene promoter results in its epigenetic silencing. Here, we show that FMR1 silencing is mediated by the FMR1 mRNA. The FMR1 mRNA contains the transcribed CGG-repeat tract as part of the 5′ untranslated region, which hybridizes to the complementary CGG-repeat portion of the FMR1 gene to form an RNA·DNA duplex. Disrupting the interaction of the mRNA with the CGG-repeat portion of the FMR1 gene prevents promoter silencing. Thus, our data link trinucleotide-repeat expansion to a form of RNA-directed gene silencing mediated by direct interactions of the trinucleotide-repeat RNA and DNA.

The DNA Replication Program Is Altered at the FMR1 Locus in Fragile X Embryonic Stem Cells

Fragile X syndrome (FXS) is caused by a CGG repeat expansion in the FMR1 gene that appears to occur during oogenesis and during early embryogenesis. One model proposes that repeat instability depends on the replication fork direction through the repeats such that (CNG)n hairpin-like structures form, causing DNA polymerase to stall and slip. Examining DNA replication fork progression on single DNA molecules at the endogenous FMR1 locus revealed that replication forks stall at CGG repeats in human cells. Furthermore, replication profiles of FXS human embryonic stem cells (hESCs) compared to nonaffected hESCs showed that fork direction through the repeats is altered at the FMR1 locus in FXS hESCs, such that predominantly the CCG strand serves as the lagging-strand template. This is due to the absence of replication initiation that would typically occur upstream of FMR1, suggesting that altered replication origin usage combined with fork stalling promotes repeat instability during early embryonic development.

Human ESC-derived hemogenic endothelial cells undergo distinct waves of endothelial to hematopoietic transition

UNLABELLED Several studies have demonstrated that hematopoietic cells originate from endotheliumin early development; however, the phenotypic progression of progenitor cells during human embryonic hemogenesis is not well described. Here, we define the developmental hierarchy among intermediate populations of hematopoietic progenitor cells (HPCs) derived from human embryonic stem cells (hESCs). We genetically modified hESCs to specifically demarcate acquisition of vascular (VE-cadherin) and hematopoietic (CD41a) cell fate and used this dual-reporting transgenic hESC line to observe endothelial to hematopoietic transition by real-time confocal microscopy. Live imaging and clonal analyses revealed a temporal bias in commitment of HPCs that recapitulates discrete waves of lineage differentiation noted during mammalian hemogenesis. Specifically, HPCs isolated at later time points showed reduced capacity to form erythroid/ megakaryocytic cells and exhibited a tendency toward myeloid fate that was enabled by expression of the Notch ligand Dll4 on hESC-derived vascular feeder cells. These data provide a framework for defining HPC lineage potential, elucidate a molecular contribution from the vascular niche in promoting hematopoietic lineage progression, and distinguish unique subpopulations of hemogenic endothelium during hESC differentiation. KEY POINTS Live imaging of endothelial to hematopoietic conversion identifies distinct subpopulations of hESC-derived hemogenic endothelium. Expression of the Notch ligand DII4 on vascular ECs drives induction of myeloid fate from hESC-derived hematopoietic progenitors.

Fertility preservation using controlled ovarian hyperstimulation and oocyte cryopreservation in a premenarcheal female with myelodysplastic syndrome

OBJECTIVE To report the first case of fertility preservation in a premenarcheal female by use of controlled ovarian hyperstimulation and oocyte cryopreservation. DESIGN Case report. SETTING Reproductive endocrinology and infertility unit of a tertiary care university-based medical center. PATIENT(S) A 13-year-old premenarcheal female with Tanner stage 3 breast development and Tanner stage 1 pubic hair diagnosed with myelodysplastic syndrome, referred by her medical oncologist for fertility preservation before undergoing a potentially sterilizing antineoplastic therapy. INTERVENTION(S) Evaluation of ovarian reserve, ovarian stimulation, transvaginal oocyte aspiration, in vitro maturation of immature oocytes, and oocyte cryopreservation. MAIN OUTCOME MEASURE(S) Cryopreservation of mature oocytes. RESULT(S) Successful controlled ovarian hyperstimulation allowed for the cryopreservation of 18 mature oocytes before the patients gonadotoxic treatment. The oocyte retrieval and cryopreservation did not delay the patients planned chemotherapy. CONCLUSION(S) Ovarian stimulation and oocyte cryopreservation can be successfully performed in premenarcheal/peripubertal patients, thus providing a viable alternative to ovarian tissue freezing for fertility preservation in the pediatric population.

Cis-acting DNA sequence at a replication origin promotes repeat expansion to fragile X full mutation

An SNP upstream of the CGG repeats located at a replication initiation site may contribute to origin inactivation, to altered replication fork progression through the CGG repeats, and repeat expansion to fragile X full mutation.

Morphologic grading of euploid blastocysts influences implantation and ongoing pregnancy rates

OBJECTIVE To determine whether blastocyst grading can predict pregnancy outcomes in the frozen-thawed embryo transfer (FET) of euploid blastocysts. DESIGN Retrospective cohort study. SETTING Academic medical center. PATIENT(S) Women who underwent FET of euploid embryo(s) between January 2013 and December 2015, with blastocysts were divided into four groups based on their morphologic grading before cryopreservation: excellent (≥3AA), good (3-6AB, 3-6BA, 1-2AA), average (3-6BB, 3-6AC, 3-6CA, 1-2AB, 1-2BA), and poor (1-6BC, 1-6CB, 1-6CC, 1-2BB). INTERVENTION(S) FET. MAIN OUTCOMES MEASURE(S) Ongoing pregnancy rate (OPR). RESULT(S) A total of 417 FET cycles (477 embryos) were included. Excellent-quality embryos (n = 38) yielded a statistically significantly higher OPR than poor-quality embryos (n = 106) (84.2% vs. 35.8%; adjusted odds ratio 11.0; 95% confidence interval, 3.8-32.1) and average-quality embryos (n = 197) (84.2% vs. 55.8%; adjusted odds ratio 4.8; 95% confidence interval, 1.7-13.3). Good-quality embryos (n = 76) were associated with a statistically significantly higher OPR than poor-quality embryos (61.8% vs. 35.8%). These odds ratios were adjusted for patients age, body mass index, number of transferred embryos, type of frozen cycle, peak endometrial thickness, day of trophectoderm biopsy (5 or 6), and total number of euploid embryos for each patient. An inner cell mass grade of A yielded a statistically significantly higher OPR than ICM grade C (76.2% vs. 13.5%) or grade B (76.2% vs. 53.6%) after controlling for all confounders. CONCLUSION(S) Contrary to prior published studies, the current data suggest that blastocyst morphologic grading and particularly inner cell mass grade is a useful predictor of OPR per euploid embryo. Morphologic grading should be used to help in the selection among euploid blastocysts.

Direct Unequal Cleavages: Embryo Developmental Competence, Genetic Constitution and Clinical Outcome

Objective To investigate the prevalence, developmental potential, chromosomal constitution and clinical outcome of embryos with direct unequal cleavages (DUC). Design A retrospective observational study. Setting Academic Institution. Participant 21,261 embryos from 3,155 cycles cultured in EmbryoScope®. Results The total incidence of DUCs per embryo occupying the first three cleavages were 26.1%. Depending of the cell stage, DUC rate was 9.8% at first cleavage (DUC-1), 9.1% at second cleavage (DUC-2), and 3.7% at third cleavage (DUC-3) with 3.6% of embryos exhibiting multiple DUCs (DUC-Plus). The occurrence of DUCs was not correlated with female gamete age or source. The incidence of DUC-1 was significantly higher in embryos fertilized by epididymal and testicular sperm (13.6% and 11.4%, respectively) compared to ejaculated sperm (9.1%, all p<0.05). The total incidences of DUCs were strongly correlated with the onset of blastomere multinucleation (MNB) during the first three divisions. In MNB embryos, DUCs incidence are two to three times more likely to develop when compared to non-MNB embryos (OR = 3.11, 95% CI [2.64, 3.67] at 1-cell stage, OR = 2.64, 95% CI [2.39, 2.91] at 2-cell stage and OR = 2.51, 95% CI [1.84, 3.43] at 4-cell stage). The blastocyst formation rates gradually decreased from 61.0% in non-DUC to 40.2% in DUC-3, 18.8% in DUC-2, 8.2% in DUC-1 and 5.6% in multiple DUC embryos (DUC-Plus). The known implantation rates (FH) for day 3 (D3) transfers were 12.42% (n = 3172) in Non-DUC embryos, 6.3% (n = 127) in DUC-3, and 2.7% (n = 260) in DUC-2 embryos. No live births resulted from either DUC-1 (n = 225) or DUC-Plus (n = 100) embryo transfers. For blastocyst transfers, lower implantation rates (33.3%) but similar live birth (LB) rates (40%) were observed if DUC blastocysts were transferred. Comparatively rates in Non-DUC blastocyst were 45.2% and 34.8%, respectively. The euploid rate gradually increased from DUC-1, -2, -3 to Non-DUC (13.3%, 19.5%, 33.3%, 45.6%, p<0.001) for D3 biopsied embryos. Interestingly, the trend of decreased euploidy disappeared in DUC D5/6 biopsied embryos and similar rates were exemplified in DUC (D5 56.3%, D6 35.6%) vs. non-DUC (D5 51.4%, D6 33.8%) embryos. Conclusion Blastocyst formation, implantation potential and euploid rate were significantly reduced in DUC embryos. DUC embryos should be deselected for D3 transfers, but should be culture to blastocyst stage for possible ET.

Follicular flushing and in vitro fertilization outcomes in the poorest responders: a randomized controlled trial

STUDY QUESTION Does follicular flushing during oocyte retrieval improve the number of oocytes retrieved in the poorest responders? SUMMARY ANSWER Follicular flushing in the poorest responders does not increase the number of oocytes retrieved and may result in lower implantation and clinical pregnancy rates. WHAT IS KNOWN ALREADY Although previous studies have shown no beneficial effect of follicular flushing in normal responders, no study has demonstrated a detrimental effect and many IVF centers continue to perform this technique in poor responders. Data on follicular flushing in this patient group are limited, with no randomized trial to date assessing its utility in the poorest responders. STUDY DESIGN, SIZE, DURATION This randomized controlled trial compared the effects of follicular flushing and direct aspiration on IVF outcomes in the poorest responders, defined as having four or fewer follicles ≥12 mm on the day of hCG administration. Fifty patients were randomized during the 12-month enrollment period. PARTICIPANTS/MATERIALS, SETTING, METHODS The patients were treated at an academic fertility center at Weill Cornell Medical College, New York. MAIN RESULTS AND THE ROLE OF CHANCE Fifty women were randomized to follicular flushing (n = 25) or direct aspiration (n = 25). One patient in the direct aspiration group was canceled prior to oocyte retrieval for premature ovulation and was included in the intent-to-treat analysis. There was no difference in the number of oocytes retrieved with a median (IQR) of 4 (2-6) in the aspiration group versus 3 (2-5) in the flushing group (95% CI: -0.78, 1.98; P = 0.41). Patients who underwent follicular flushing had significantly fewer embryos transferred {1.7 [standard deviation (SD) 0.6] versus 2.5 (SD 1.2), P = 0.03}, a lower implantation rate (5.3 versus 34.2%, P = 0.006) and a lower clinical pregnancy rate (4 versus 36%, P = 0.01). The difference in pregnancy rates remained significant after adjusting for embryos transferred. LIMITATIONS, REASONS FOR CAUTION Findings, including results for secondary outcome measures, may not be generalizable to natural IVF cycles as these were excluded from the study. WIDER IMPLICATIONS OF THE FINDINGS This is the first randomized trial to evaluate the utility of follicular flushing in the poorest responders, and the first to demonstrate a potentially detrimental effect of flushing on IVF outcomes. STUDY FUNDING/COMPETING INTEREST(S) None. TRIAL REGISTRATION NUMBER NCT 01558141.