Faculty Bibliography

Metaphase II oocytes (MII) from polycystic ovary syndrome (PCOS) frequently have impaired oocyte competence. Since telomere maintenance is important for folliculogenesis, oocyte maturation, and early embryonic development, we sought to verify the implications of PCOS on telomere length and telomerase activity in immature oocytes and cumulus cells. 43 PCOS and 67 control women were included, and anthropometric, biochemical, and hormonal characteristics were evaluated. The telomere length in germinal vesicle stage (GV) and in metaphase I (MI) oocytes, as well as in the cumulus cells of immature (CCI) and mature oocytes (CCM), and in leukocytes was measured by qPCR. The telomerase activity in reproductive cells was evaluated by the TRAPeze® XL Kit. The body mass index (p = 0.001), LH (p = 0.015), estradiol (p = 0.004), insulin (p = 0.002), testosterone (p < 0.0001), androstenedione (p = 0.001), free androgen index (p < 0.0001), and c-reactive protein (p = 0.003) were greater, while the FSH (p = 0.0002) was lower in the PCOS group. The telomere length in the CCI (p = 0.649) and CCM (p = 0.378) did not differ between the PCOS and the control groups. On the other hand, telomerase activity in the CCI (p = 0.003) and CCM (p = 0.022) was higher in the PCOS group. In the leukocyte's cells, the telomere length was reduced in the PCOS group (p = 0.025). In the GV and MI oocytes, no differences were observed in telomere length and telomerase activity between the groups. We showed that telomere length is not altered in reproductive cells from PCOS. However, higher telomerase activity in the CCI and CCM may be required for telomere length maintenance.

PURPOSE/OBJECTIVE:To determine the impact of accelerated telomere shortening on the fertility parameters and treatment outcomes of a woman with dyskeratosis congenita (DKC). METHODS:A case study of the clinical data, blood, discarded oocytes, and arrested embryos of a woman with DKC and donated cryopreserved embryos from unaffected patients. Mean telomere length in blood cells was analyzed by flow cytometry-fluorescence in situ hybridization (flow-FISH) and qPCR. The load of short telomeres in blood cells was measured by universal single telomere length analysis (Universal STELA). The mean telomere length in embryos was analyzed by single-cell amplification of telomere repeats (SCATR) PCR. RESULTS:Comparison of clinical parameters revealed that the DKC patient had reduced anti-Mullerian hormone (0.3 vs 4.1 ± 5.7 ng/ML), reduced oocytes retrieved (7 vs 18.5 ± 9.5), reduced fertilization rate, and reduced euploidy rate relative to unaffected patients. Additionally, mean telomere length in DKC embryos were shorter than unaffected embryos. However, hormone treatment led to increased leukocyte telomere length, while the load of short telomeres was also shown to decrease during the course of treatment. CONCLUSIONS:We demonstrate for the first time the direct detrimental impacts of short telomeres on female fertility. We further demonstrate positive effects of hormone treatments for people with telomere disorders.

Long interspersed element-1 (LINE-1, L1) sequences, which comprise about 17% of human genome, are the product of one of the most active types of mobile DNAs in modern humans. LINE-1 insertion alleles can cause inherited and de novo genetic diseases, and LINE-1-encoded proteins are highly expressed in some cancers. Genome-wide LINE-1 mapping in single cells could be useful for defining somatic and germline retrotransposition rates, and for enabling studies to characterize tumour heterogeneity, relate insertions to transcriptional and epigenetic effects at the cellular level, or describe cellular phylogenies in development. Our laboratories have reported a genome-wide LINE-1 insertion site mapping method for bulk DNA, named transposon insertion profiling by sequencing (TIPseq). There have been significant barriers applying LINE-1 mapping to single cells, owing to the chimeric artefacts and features of repetitive sequences. Here, we optimize a modified TIPseq protocol and show its utility for LINE-1 mapping in single lymphoblastoid cells. Results from single-cell TIPseq experiments compare well to known LINE-1 insertions found by whole-genome sequencing and TIPseq on bulk DNA. Among the several approaches we tested, whole-genome amplification by multiple displacement amplification followed by restriction enzyme digestion, vectorette ligation and LINE-1-targeted PCR had the best assay performance. This article is part of a discussion meeting issue 'Crossroads between transposons and gene regulation'.

PURPOSE/OBJECTIVE:To assess the accuracy and reliability of comprehensive chromosome screening by next-generation sequencing (NGS) of human trophectoderm (TE) biopsy specimens. METHODS:The reliability and accuracy of diagnoses made by preimplantation genetic testing for aneuploidy (PGT-A) from TE biopsy were tested. Repeat biopsies of TE and inner cell mass (ICM) samples were obtained from thawed blastocysts previously tested by NGS. To test for the reliability of the NGS assay, biopsy samples were compared with the original PGT-A results. Prior NGS testing classified the TE samples as euploid, aneuploid, or aneuploid-mosaic. The resulting re-biopsied samples underwent SurePlex whole genome amplification followed by NGS via the MiSeq platform, with copy number value (CNV) determined using BlueFuse Multi Software. The primary outcome measure was reliability, defined as concordance between initial TE result and the repeat biopsies. Accuracy was determined by concordance between the TE and ICM samples, and compared between three chromosome types (disomic, aneuploid, and mosaic). RESULTS:Re-biopsies were performed on 32 embryos with prior PGT-A showing euploidy (10 embryos), aneuploidy of one or two chromosomes (4 embryos), or aneuploid-mosaic with one aneuploid chromosome and one mosaic chromosome (18 embryos). One hundred twenty-nine biopsy samples completed NGS (90 TE and 39 ICM biopsies) and 105 biopsy results were included in the analysis. TE biopsies provide a highly accurate test of the future fetus, with the ICM disomic concordance rate of 97.6%. Clinical concordance rates indicate that TE biopsies provide a reliable test when the result is euploid (99.5%) or aneuploid (97.3%), but less reliable when the result is mosaic (35.2%). CONCLUSION/CONCLUSIONS:TE biopsies predict euploidy or aneuploidy in the ICM with a high degree of accuracy. PGT-A with NGS of TE biopsies is shown to be highly reliable, with clinically relevant concordance rates for aneuploidy and euploidy over 95%. TE biopsies indicating mosaicism were less reliable (35.2%), presumably because mitotic non-disjunction events are not uniformly distributed throughout the blastocyst. However, classification of TE biopsy of PGT-A with NGS results as either aneuploid or euploid provides a highly reliable test.

The testis expresses the largest number of genes of any mammalian organ, a finding that has long puzzled molecular biologists. Our single-cell transcriptomic data of human and mouse spermatogenesis provide evidence that this widespread transcription maintains DNA sequence integrity in the male germline by correcting DNA damage through a mechanism we term transcriptional scanning. We find that genes expressed during spermatogenesis display lower mutation rates on the transcribed strand and have low diversity in the population. Moreover, this effect is fine-tuned by the level of gene expression during spermatogenesis. The unexpressed genes, which in our model do not benefit from transcriptional scanning, diverge faster over evolutionary timescales and are enriched for sensory and immune-defense functions. Collectively, we propose that transcriptional scanning shapes germline mutation signatures and modulates mutation rates in a gene-specific manner, maintaining DNA sequence integrity for the bulk of genes but allowing for faster evolution in a specific subset.

During mammalian development, gonadotropin-releasing-hormone-1 neurons (GnRH-1ns) migrate from the developing vomeronasal organ (VNO) into the brain asserting control of pubertal onset and fertility. Recent data suggest that correct development of the olfactory ensheathing cells (OEC) is imperative for normal GnRH-1 neuronal migration. However, the full ensemble of molecular pathways that regulate OEC development remains to be fully deciphered. Loss-of-function of the transcription factor Gli3 is known to disrupt olfactory development, however, if Gli3 plays a role in GnRH-1 neuronal development is unclear. By analyzing Gli3 extra-toe mutants (Gli3^Xt/Xt), we found that Gli3 loss-of-function compromises the onset of achaete-scute family bHLH transcription factor 1 (Ascl-1) positive vomeronasal progenitors and the formation of OEC in the nasal mucosa. Surprisingly, GnRH-1 neurogenesis was intact in Gli3^Xt/Xt mice but they displayed significant defects in GnRH-1 neuronal migration. In contrast, Ascl-1^null mutants showed reduced neurogenesis for both vomeronasal and GnRH-1ns but less severe defects in OEC development. These observations suggest that Gli3 is critical for OEC development in the nasal mucosa and subsequent GnRH-1 neuronal migration. However, the non-overlapping phenotypes between Ascl-1 and Gli3 mutants indicate that Ascl-1, while crucial for GnRH-1 neurogenesis, is not required for normal OEC development. Since Kallmann syndrome (KS) is characterized by abnormal GnRH-1ns migration, we examined whole exome sequencing data from KS subjects. We identified and validated a GLI3 loss-of-function variant in a KS individual. These findings provide new insights into GnRH-1 and OECs development and demonstrate that human GLI3 mutations contribute to KS etiology.Significance statement. The transcription factor Gli3 is necessary for correct development of the olfactory system. However, if Gli3 plays a role in controlling GnRH-1 neuronal development has not been addressed. We found that Gli3 loss-of-function compromises the onset of Ascl-1+ vomeronasal progenitors, formation of olfactory ensheathing cells in the nasal mucosa, and impairs GnRH-1 neuronal migration to the brain. By analyzing Ascl-1^null mutants we dissociated the neurogenic defects observed in Gli3 mutants from lack of olfactory ensheathing cells in the nasal mucosa, moreover, we discovered that Ascl-1 is necessary for GnRH-1 ontogeny. Analyzing human whole exome sequencing data, we identified a GLI3 loss-of-function variant in a KS individual. Our data suggest that GLI3 is a candidate gene contributing to KS etiology.