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Preview: Molecular Human Reproduction - current issue

MHR: Basic science of reproductive medicine Current Issue





Published: Fri, 05 Jan 2018 00:00:00 GMT

Last Build Date: Tue, 09 Jan 2018 08:49:55 GMT

 






Preclinical validation of a targeted next generation sequencing-based comprehensive chromosome screening methodology in human blastocysts

Sat, 25 Nov 2017 00:00:00 GMT

Abstract
STUDY QUESTION
Can a novel targeted next generation sequencing (tNGS) platform accurately detect whole chromosome aneuploidy in a trophectoderm biopsy and provide additional information to improve testing?
SUMMARY ANSWER
Karyotypes obtained by tNGS were concordant with other validated platforms and single nucleotide polymorphism genotyping information obtained can be used for improved detection and quality control.
WHAT IS KNOWN ALREADY
qPCR-based whole chromosome aneuploidy screening is highly accurate in comparison to other common methods and has been shown to improve IVF success in two randomized clinical trials. With aneuploidy screening becoming standard of care in many IVF centres, there is a need to develop platforms with high throughput, low cost capabilities.
STUDY DESIGN SIZE, DURATION
Twelve well-characterized cell lines were obtained from a commercial cell line repository and 31 discarded human blastocysts were obtained from 17 IVF patients who underwent comprehensive chromosome screening (CCS).
PARTICIPANTS/MATERIAL, SETTING, METHODS
All samples were processed using a unique amplification strategy which directly incorporated sequencing library adapters and barcodes. Sequencing was performed on an Ion Torrent Proton. A custom bioinformatics pipeline was used to determine the karyotype for each sample. The consistency of tNGS diagnoses with either conventional karyotyping of cell lines or quantitative real-time PCR based CCS of blastocyst biopsies was evaluated.
MAIN RESULTS AND THE ROLE OF CHANCE
Overall consistency per sample of tNGS based CCS in 5-cell samples from a variety of cell lines was 99.2%. In the blinded analysis of rebiopsies of aneuploid blastocysts, an overall targeted tNGS CCS consistency of 98.7% was observed per sample. These data demonstrate the ability of tNGS based CCS to provide an accurate and high throughput evaluation of aneuploidy in the human blastocyst.
LARGE SCALE DATA
Not applicable.
LIMITATIONS REASONS FOR CAUTION
This study is limited to whole chromosome aneuploidy, as mosaicism and segmental aneuploidy have not been investigated.
WIDER IMPLICATIONS OF THE FINDINGS
These data show an accurate, high throughput method, and with the greater depth of each amplicon sequenced in comparison to commercial kits, there is greater application available for single nucleotide polymorphism based analysis for quality control.
STUDY FUNDING/COMPETING INTERESTS
This study was funded through intramural research funds provided by the Foundation for Embryonic Competence. There are no competing interests.



Increased 27-hydroxycholesterol production during luteolysis may mediate the progressive decline in progesterone secretion

Tue, 21 Nov 2017 00:00:00 GMT

AbstractSTUDY QUESTIONDoes 27-hydroxycholesterol (27OH) actively facilitate the progression of luteolysis?SUMMARY ANSWERThere is increased mRNA expression of the enzyme that produces 27OH during luteolysis in vivo in rhesus macaques and sheep, and 27OH reduces progesterone secretion from human luteinized granulosa cells.WHAT IS KNOWN ALREADYThere is an increase in mRNA expression of liver x receptor (LXR) and a decrease in sterol regulatory element binding protein 2 (SREBP2) target genes during spontaneous luteolysis in primates, which could result in reduced cholesterol availability for steroidogenesis. Concentrations of 27OH are also increased in primate corpora lutea (CL) during luteolysis, and 27OH is a dual LXR agonist and SREBP2 inhibitor.STUDY DESIGN SIZE, DURATIONThis was an in vitro study using primary human luteinized granulosa cells in a control versus treatment(s) design. Analyses of CL from sheep undergoing induced or spontaneous luteolysis were also performed, along with database mining of microarray data from rhesus macaque CL.PARTICIPANTS/MATERIALS, SETTING, METHODSPrimary luteinizing granulosa cells were obtained from 37 women aged 24–44 who were undergoing oocyte donation or IVF for male factor or idiopathic infertility, and cells were further luteinized in vitro using human chorionic gonadotropin. Three approaches to test the effect of 27OH produced via CYP27A1 (cytochrome p450, family 27, subfamily A, polypeptide 1) on luteinized granulosa cells were used: (i) direct 27OH supplementation, (ii) induction of endogenous CYP27A1 activity via pharmacologic inhibition of steroidogenesis, and (iii) siRNA-mediated knockdown to directly inhibit CYP27A1 as well as cholesterol transport into the mitochondria via the steroidogenic acute regulatory protein (STAR). Endpoints included: progesterone (P4) secretion into culture media determined by enzyme immunoassay, cholesterol efflux and uptake assays using fluorescent lipid analogs, and mRNA expression determined via semi-quantitative real-time PCR (QPCR). An additional experiment involved QPCR analysis of 40 CL collected from ewes undergoing induced or spontaneous luteolysis, as well as database mining of microarray data generated from 16 rhesus macaque CL collected during spontaneous luteolysis and 13 macaque CL collected during a luteinizing hormone ablation and replacement protocol.MAIN RESULTS AND THE ROLE OF CHANCEThe mRNA expression of CYP27A1 was significantly increased during luteolysis in rhesus macaques and sheep in vivo, and CYP27A1 transcription was suppressed by luteinizing hormone and hCG. There was a significant decrease in hCG-stimulated P4 secretion from human luteinized granulosa cells caused by 27OH treatment, and a significant increase in basal and hCG-stimulated P4 synthesis when endogenous 27OH production was inhibited via CYP27A1 knockdown, indicating that 27OH inhibits steroidogenesis. Pharmacologic inhibition of steroidogenesis by aminoglutethimide significantly induced LXR and inhibited SREBP2 target gene mRNA expression, indicating that increased oxysterol production occurs when steroidogenesis is suppressed. Inhibiting cholesterol delivery into the mitochondria via knockdown of STAR resulted in reduced SREBP2 target gene mRNA expression, indicating that STAR function is necessary to maintain SREBP2-mediated transcription. The effects of 27OH treatment on markers of LXR and SREBP2 activity were moderate, and knockdown of CYP27A1 did not prevent aminoglutethimide-induced changes in LXR and SREBP2 target gene mRNA expression. These observations indicate that 27OH inhibits P4 secretion partially via mechanisms separate from its role as an LXR agonist and SREBP2 inhibitor, and also demonstrate that other oxysterols are involved in modulating LXR and SREBP2-mediated transcription when steroidogenesis is suppressed.LARGE SCALE DATANone.LIMITATIONS REASONS FOR CAUTIONLuteinized granulosa cells may differ from luteal cells, and the effect on luteal function in vivo was not directly tested.[...]



Proteomic analysis of germinal vesicles in the domestic cat model reveals candidate nuclear proteins involved in oocyte competence acquisition

Wed, 08 Nov 2017 00:00:00 GMT

Abstract
STUDY QUESTION
Do nuclear proteins in the germinal vesicle (GV) contribute to oocyte competence acquisition during folliculogenesis?
SUMMARY ANSWER
Proteomic analysis of GVs identified candidate proteins for oocyte competence acquisition, including a key RNA processing protein–heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1).
WHAT IS KNOWN ALREADY
The domestic cat GV, which is physiologically similar to the human GV, gains the intrinsic ability to resume meiosis and support early embryo development during the pre-antral-to-antral follicle transition. However, little is known about nuclear proteins that contribute to this developmental process.
STUDY DESIGN SIZE, DURATION
GVs were enriched from pre-antral (incompetent) and antral (competent) follicles from 802 cat ovaries. Protein lysates were subjected to quantitative proteomic analysis to identify differentially expressed proteins in GVs from the two follicular categories.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Two biological replicates (from independent pools of ovaries) of pre-antral versus antral samples were labeled by tandem mass tags and then assessed by liquid chromatography–tandem mass spectrometry. Proteomic data were analyzed according to gene ontology and a protein–protein interaction network. Immunofluorescent staining and protein inhibition assays were used for validation.
MAIN RESULTS AND THE ROLE OF CHANCE
A total of 174 nuclear proteins was identified, with 54 being up-regulated and 22 down-regulated (≥1.5-fold) after antrum formation. Functional protein analysis through gene ontology over-representation tests revealed that changes in molecular network within the GVs during this transitional phase were related to chromatin reorganization, gene transcription, and maternal RNA processing and storage. Protein inhibition assays verified that hnRNPA2B1, a key nuclear protein identified, was required for oocyte meiotic maturation and subsequent blastocyst formation.
LARGE SCALE DATA
Data are available via ProteomeXchange with identifier PXD007211.
LIMITATIONS REASONS FOR CAUTION
Proteins identified by proteomic comparison may (i) be involved in processes other than competence acquisition during the pre-antral-to-antral transition or (ii) be co-expressed in other macrostructures besides the GV. Expressional and functional validations should be performed for candidate proteins before downstream application.
WIDER IMPLICATIONS OF THE FINDINGS
Collective results generated a blueprint to better understand the molecular mechanisms involved in GV competence acquisition and identified potential nuclear competence markers for human fertility preservation.
STUDY FUNDING AND COMPETING INTEREST(S)
Funded by the National Center for Research Resources (R01 RR026064), a component of the National Institutes of Health (NIH) and currently by the Office of Research Infrastructure Programs/Office of the Director (R01 OD010948). The authors declare that there is no conflict of interest.



Endothelin-1 promotes human germinal vesicle-stage oocyte maturation by downregulating connexin-26 expression in cumulus cells

Wed, 08 Nov 2017 00:00:00 GMT

AbstractSTUDY QUESTIONDoes endothelin-1 (ET-1) promote human oocyte maturation and by what mechanism?SUMMARY ANSWERAddition of ET-1 to the medium in which human germinal vesicle (GV)-stage immature oocytes are cultured enhances the GV breakdown (GVBD) rate; the resumption of meiosis may be initiated by ET-1 downregulating the expression of connexin-26 (Cx26) in cumulus cells via endothelin receptor type B (ETRB), leading to decreased cAMP levels in the oocyte.WHAT IS KNOWN ALREADYThe paracrine factor ET-1 is secreted by ovarian somatic cells in pre-ovulatory follicles and regulates oocyte maturation in mice. Connexins, or gap junction proteins, form intercellular membrane channels that play important roles in the resumption of meiosis.STUDY DESIGN, SIZE, DURATIONThis laboratory study was conducted over a 1-year period. The effects of ET-1 on meiotic resumption were evaluated in human GV-stage cumulus-oocyte complexes (COCs; 70 oocytes/group). The transcriptome profiles of ET-1-treated or untreated cumulus cells were compared to explore the possible mechanisms by which ET-1 may regulate oocyte maturation.PARTICIPANTS/MATERIALS, SETTING, METHODSThe ET-1, ETRA and ETRB expression levels in human cumulus cells from oocytes at different stages of maturation were evaluated using real-time quantitative PCR. Human GV-stage COCs collected from patients undergoing IVF at a university-affiliated infertility centre were cultured with or without ET-1, and cumulus cells were subsequently denuded using hyaluronidase and cultured in α-MEM. A GeneChip® Human Transcriptome Array was applied to explore differences in the whole-genome transcriptome profiles of cumulus cells treated with or without ET-1. Real-time quantitative PCR and Western blotting were used respectively to examine Cx26 mRNA and protein levels in cumulus cells. Changes in cAMP levels in both oocytes and cumulus cells after ET-1 treatment were measured using an enzyme-linked immunosorbent assay.MAIN RESULTS AND THE ROLE OF CHANCECumulus cells from MII-stage oocytes exhibited upregulated ET-1 expression, compared to those from GV-stage oocytes. The addition of ET-1 to the culture medium enhanced the GVBD rate of cumulus cell-enclosed human oocytes. Whole-genome transcriptome microarray analyses revealed significantly downregulated Cx26 expression in cumulus cells after ET-1 treatment, and this action was blocked by an ETRB antagonist. The involvement of Cx26 was further supported by the finding that ET-1 treatment led to decreased cAMP levels in oocytes but increased cAMP levels in cumulus cells.LARGE SCALE DATAMicroarray data are published in the GEO database (GSE97684).LIMITATIONS, REASONS FOR CAUTIONThe heterogeneity of human COCs collected from patients undergoing IVF might affect the maturation results in vitro. Although we focused on the effects of ET-1 on human oocyte maturation in the present study, mammalian oocyte maturation is a complicated process involving many endocrine and paracrine factors.WIDER IMPLICATIONS OF THE FINDINGSOur present study suggests that in vitro, human GV-stage oocyte maturation could be enhanced by adding ET-1 to the culture medium. In the present study, we explored the molecular mechanisms by which ET-1 initiates the resumption of meiosis and demonstrated that ET-1 promotes oocyte maturation by downregulating the expression of the gap junction protein Cx26 in cumulus cells. These results expand our understanding of the molecular mechanisms underlying mammalian oocyte maturation and provide a basis for better in-vitro maturation strategies.STUDY FUNDING AND COMPETING INTERESTSThis work was supported by grants from the China Natural Science Foundation (Grant Nos. 81170567 and 81370761). The authors declare that they have no conflicts of interest associated with this manuscript.[...]