Y.G. Kramer

Assessing morphokinetic parameters via time lapse microscopy (TLM) to predict euploidy: are aneuploidy risk classification models universal?

PurposeTo determine if Aneuploidy Risk Classification Models are predictive of euploidy/aneuploidy amongst IVF facilities.MethodsWe retrospectively applied key time lapse imaging events of embryos (Campbell et al.[5, 6]) to stratify embryos into 3 groups: low, medium and high risk of aneuploidy. The actual ploidy results (from array comparative genomic hybridization) were compared with expectations [5, 6]. Sources of variability in morphokinetic parameters were determined using Analysis of Variance (ANOVA).ResultsThe model failed to segregate euploid embryos from aneuploid embryos cultured at our facility. Further analysis indicated that the variability of embryos among patients was too great to allow selection of euploid embryos based on simple morphokinetic thresholds. Clinical selection of embryos based on morphokinetics alone is unlikely to identify euploid embryos accurately for transfer or yield higher rates of live delivery.ConclusionsThe use of non-invasive morphokinetics is unlikely to discriminate aneuploid from euploid embryos. Further, it does not approach the accuracy of preimplantation genetic screening with array comparative genomic hybridization.

Progesterone receptor membrane component-1 regulates hepcidin biosynthesis

Iron homeostasis is tightly regulated by the membrane iron exporter ferroportin and its regulatory peptide hormone hepcidin. The hepcidin/ferroportin axis is considered a promising therapeutic target for the treatment of diseases of iron overload or deficiency. Here, we conducted a chemical screen in zebrafish to identify small molecules that decrease ferroportin protein levels. The chemical screen led to the identification of 3 steroid molecules, epitiostanol, progesterone, and mifepristone, which decrease ferroportin levels by increasing the biosynthesis of hepcidin. These hepcidin-inducing steroids (HISs) did not activate known hepcidin-inducing pathways, including the BMP and JAK/STAT3 pathways. Progesterone receptor membrane component-1 (PGRMC1) was required for HIS-dependent increases in hepcidin biosynthesis, as PGRMC1 depletion in cultured hepatoma cells and zebrafish blocked the ability of HISs to increase hepcidin mRNA levels. Neutralizing antibodies directed against PGRMC1 attenuated the ability of HISs to induce hepcidin gene expression. Inhibiting the kinases of the SRC family, which are downstream of PGRMC1, blocked the ability of HISs to increase hepcidin mRNA levels. Furthermore, HIS treatment increased hepcidin biosynthesis in mice and humans. Together, these data indicate that PGRMC1 regulates hepcidin gene expression through an evolutionarily conserved mechanism. These studies have identified drug candidates and potential therapeutic targets for the treatment of diseases of abnormal iron metabolism.

A single-cell assay for telomere DNA content shows increasing telomere length heterogeneity, as well as increasing mean telomere length in human spermatozoa with advancing age

PurposeThe effect of age on telomere length heterogeneity in men has not been studied previously. Our aims were to determine the relationship between variation in sperm telomere length (STL), men’s age, and semen parameters in spermatozoa from men undergoing in vitro fertilization (IVF) treatment.MethodsTo perform this prospective cross-sectional pilot study, telomere length was estimated in 200 individual spermatozoa from men undergoing IVF treatment at the NYU Fertility Center. A novel single-cell telomere content assay (SCT-pqPCR) measured telomere length in individual spermatozoa.ResultsTelomere length among individual spermatozoa within an ejaculate varies markedly and increases with age. Older men not only have longer STL but also have more variable STL compared to younger men. STL from samples with normal semen parameters was significantly longer than that from samples with abnormal parameters, but STL did not differ between spermatozoa with normal versus abnormal morphology.ConclusionThe marked increase in STL heterogeneity as men age is consistent with a role for ALT during spermatogenesis. No data have yet reported the effect of age on STL heterogeneity. Based on these results, future studies should expand this modest sample size to search for molecular evidence of ALT in human testes during spermatogenesis.

Next generation sequencing for preimplantation genetic screening improves pregnancy outcomes compared with array comparative genomic hybridization in single thawed euploid embryo transfer cycles

OBJECTIVE To evaluate whether the use of next generation sequencing (NGS) for preimplantation genetic screening (PGS) in single thawed euploid embryo transfer (STEET) cycles improves pregnancy outcomes compared with array comparative genomic hybridization (aCGH). DESIGN Retrospective cohort study. SETTING Single university-based fertility center. PATIENT(S) A total of 916 STEET cycles from January 2014 to December 2016 were identified. Cases included 548 STEET cycles using NGS for PGS and controls included 368 STEET cycles using aCGH for PGS. INTERVENTION(S) Patients having a STEET after undergoing IVF and PGS with either NGS or aCGH. MAIN OUTCOME MEASURE(S) Primary outcomes were implantation rate, ongoing pregnancy/live birth rate (OP/LBR), biochemical pregnancy rate (PR), and spontaneous abortion (SAB) rate. RESULT(S) The implantation rate was significantly higher in the NGS group compared with the aCGH group (71.6% vs. 64.6%). The OP/LBR was also significantly higher in the NGS group (62% vs. 54.4%), and there were significantly more biochemical pregnancies in the aCGH group compared with the NGS group (15.1% vs. 8.7%). After adjustment for confounding variables with a multiple logistic regression analysis, OP/LBR remained significantly higher in the NGS group. The SAB rate was not significantly different in the NGS group compared with the aCGH group (12.4% vs. 12.7%). CONCLUSION(S) Preimplantation genetic screening using NGS significantly improves pregnancy outcomes versus PGS using aCGH in STEET cycles. Next-generation sequencing has the ability to identify and screen for embryos with reduced viability such as mosaic embryos and those with partial aneuploidies or triploidy. Pregnancy outcomes with NGS may be improved due to the exclusion of these abnormal embryos.

Human blastocysts of normal and abnormal karyotypes display distinct transcriptome profiles

Unveiling the transcriptome of human blastocysts can provide a wealth of important information regarding early embryonic ontology. Comparing the mRNA production of embryos with normal and abnormal karyotypes allows for a deeper understanding of the protein pathways leading to viability and aberrant fetal development. In addition, identifying transcripts specific for normal or abnormal chromosome copy number could aid in the search for secreted substances that could be used to non-invasively identify embryos best suited for IVF embryo transfer. Using RNA-seq, we characterized the transcriptome of 71 normally developing human blastocysts that were karyotypically normal vs. trisomic or monosomic. Every monosomy and trisomy of the autosomal and sex chromosomes were evaluated, mostly in duplicate. We first mapped the transcriptome of three normal embryos and found that a common core of more than 3,000 genes is expressed in all embryos. These genes represent pathways related to actively dividing cells, such as ribosome biogenesis and function, spliceosome, oxidative phosphorylation, cell cycle and metabolic pathways. We then compared transcriptome profiles of aneuploid embryos to those of normal embryos. We observed that non-viable embryos had a large number of dysregulated genes, some showing a hundred-fold difference in expression. On the contrary, sex chromosome abnormalities, XO and XXX displayed transcriptomes more closely mimicking those embryos with 23 normal chromosome pairs. Intriguingly, we identified a set of commonly deregulated genes in the majority of both trisomies and monosomies. This is the first paper demonstrating a comprehensive transcriptome delineation of karyotypic abnormalities found in the human pre-implantation embryo. We believe that this information will contribute to the development of new pre-implantation genetic screening methods as well as a better understanding of the underlying developmental abnormalities of abnormal embryos, fetuses and children.