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Can an understanding of the past influence research funding of the future?
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Editorial
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Why the Medical Research Council refused Robert Edwards and Patrick Steptoe support for research on human conception in 1971
BACKGROUNDIn 1971, Cambridge physiologist Robert Edwards and Oldham gynaecologist Patrick Steptoe applied to the UK Medical Research Council (MRC) for long-term support for a programme of scientific and clinical ‘Studies on Human Reproduction’. The MRC, then the major British funder of medical research, declined support on ethical grounds and maintained this policy throughout the 1970s. The work continued with private money, leading to the birth of Louise Brown in 1978 and transforming research in obstetrics, gynaecology and human embryology. METHODSThe MRC decision has been criticized, but the processes by which it was reached have yet to be explored. Here, we present an archive-based analysis of the MRC decision. RESULTSWe find evidence of initial support for Edwards and Steptoe, including from within the MRC, which invited the applicants to join its new directly funded Clinical Research Centre at Northwick Park Hospital. They declined the offer, preferring long-term grant support at the University of Cambridge, and so exposed the project to competitive funding mode. Referees and the Clinical Research Board saw the institutional set-up in Cambridge as problematic with respect to clinical facilities and patient management; gave infertility a low priority compared with population control; assessed interventions as purely experimental rather than potential treatments, and so set the bar for safety high; feared fatal abnormalities and so wanted primate experiments first; and were antagonized by the applicants’ high media profile. The rejection set MRC policy on IVF for 8 years, until, after the birth of just two healthy babies, the Council rapidly converted to enthusiastic support. CONCLUSIONSThis analysis enriches our view of a crucial decision, highlights institutional opportunities and constraints and provides insight into the then dominant attitudes of reproductive scientists and clinicians towards human conception research.
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Complicity in stem cell research: the case of induced pluripotent stem cells
Many who object to human embryonic stem cell (hESC) research because they believe it involves complicity in embryo destruction have welcomed induced pluripotent stem cell (iPSC) research as an ethical alternative. This opinion article aims to show that complicity arguments against hESC research are prima facie inconsistent with accepting iPSC research as it is currently done. Those who oppose hESC research on grounds of complicity should either (i) oppose iPSC research as well, (ii) advocate a radical change in the way iPSC research is done, (iii) demonstrate that complicity arguments against iPSC research are weaker than those against hESC research or (iv) reject complicity arguments against both hESC and iPSC research, either by adopting a more limited conception of complicity that allows acceptance of some hESC research, or by accepting that destroying embryos for important scientific research is not wrong.
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Glial cell line-derived neurotrophic factor is constitutively produced by human testicular peritubular cells and may contribute to the spermatogonial stem cell niche in man
BACKGROUNDTesticular peritubular cells form an ill-characterized cellular compartment of the human testis, which forms a border with Sertoli cells and spermatogonial stem cells (SSCs). A recently developed culture method has identified parts of the secretory repertoire of human testicular peritubular cells (HTPCs), which includes nerve growth factor. Whether peritubular cells produce glial cell line-derived neurotrophic factor (GDNF) and may thus contribute to the stem cell niche is not known. METHODSWe studied GDNF production in isolated peritubular cells from men with normal spermatogenesis (HTPCs) and impaired spermatogenesis and testicular fibrosis (HTPC-Fs). Human testicular biopsies and peritubular cells in culture were evaluated using immunohistochemistry, laser microdissection (LMD), RT–PCR and measurement of GDNF and cAMP by enzyme-linked immunosorbent assay. We also tested whether GDNF production is regulated by tumour necrosis factor- (TNF-) or tryptase, the products of mast cells or macrophages. RESULTSPeritubular wall cells are in close proximity to cells expressing the GDNF family co-receptor-1. GDNF mRNA was detected in LMD samples of the peritubular and tubular but not interstitial compartments. HTPCs and HTPC-Fs lack FSH- and LH-receptors but express receptors for TNF- and tryptase. Importantly, peritubular cells express GDNF and constitutively released GDNF into the medium in comparably high amounts. TNF- and tryptase had no effect on the secretion of GDNF by HTPCs or HTPC-Fs. CONCLUSIONSPeritubular cells in testes of normal and sub-/infertile men produce GDNF and are likely constitutive contributors of the SSC niche in the human testis.
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Expression of a truncated form of KIT tyrosine kinase in human spermatozoa correlates with sperm DNA integrity
BACKGROUNDTR-KIT, a truncated form of KIT (the KITL receptor), corresponding to the c-terminal half of the intracellular split tyrosine kinase domain, is expressed during the haploid stages of mouse spermatogenesis, and is one of the candidate sperm factors possibly involved in egg activation at fertilization. METHODSImmunocytochemistry of adult human testis, and studies of human semen samples from volunteer donors through immunofluorescence, confocal microscopy, flow cytometry, western blot and RT–PCR analyses were performed. RESULTSWe show that the TR-KIT is expressed during spermiogenesis in the human testis, and that it is maintained in human ejaculated spermatozoa. TR-KIT is localized both in the equatorial segment and in the sub-acrosomal region of the human sperm head. The equatorial localization of the TR-KIT persists after the spontaneous acrosome reaction. Cytometric analysis of several sperm samples from volunteer donors, showed variable degrees of the TR-KIT-specific immunolabeling, and a significant inverse correlation (Pearson's coefficient, r = –0.76, P < 0.0001, n = 23) of the TR-KIT positivity with markers of sperm damage, i.e. DNA fragmentation, as revealed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labeling (TUNEL) analysis and the intense clusterin positivity. We also found less significant inverse correlation with altered head morphology (r = –0.47, P < 0.05, n = 23) and direct correlation with sperm forward motility parameters (r = 0.59, P < 0.01, n = 23). CONCLUSIONSThe TR-KIT is present in the equatorial region of human spermatozoa, which is the first sperm component entering into the oocyte cytoplasm after fusion with the egg. This localization is consistent with the function previously proposed for this protein in mice. In addition, the TR-KIT represents a potential predictive parameter of human sperm quality.
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Intravenous immunoglobulin and idiopathic secondary recurrent miscarriage: a multicentered randomized placebo-controlled trial
BACKGROUNDIdiopathic secondary recurrent miscarriage may be associated with an abnormal maternal immune response to subsequent pregnancies. Intravenous immunoglobulin (IVIG) has been studied in randomized controlled trials (RCTs) with conflicting results. Therefore, a definitive trial was proposed. METHODSWe conducted an investigator-initiated, multicentered, randomized, double-blinded, placebo-controlled trial comparing IVIG with saline in women with idiopathic secondary recurrent miscarriage, defined as a history of at least one prior ongoing pregnancy followed by three or more consecutive unexplained miscarriages. Subjects received either IVIG 500 mg/kg or the equivalent volume of normal saline. Preconception infusions were administered 14–21 days from the projected next menstrual period. With documentation of pregnancy, the subject received the same infusion every 4 weeks until 18–20 weeks of gestation. The primary outcome was an ongoing pregnancy of at least 20 weeks of gestation. RESULTSA total of 82 patients enrolled, of whom 47 had an index pregnancy. All ongoing pregnancies resulted in live births. Therefore, the live birth rates were 70% (16/23) in the IVIG group and 63% (15/24) in the control group (P = 0.760); odds ratio (OR) 1.37 [95% confidence interval (CI) 0.41–4.61]. Including only clinical pregnancies (embryo with cardiac activity at 6 weeks of gestation), the live birth rates were equivalent, 94% (16/17) and (15/16), respectively (P > 0.999); OR 1.07 (95% CI 0.06–18.62). Meta-analysis of randomized controlled trials (RCTs) evaluating IVIG for idiopathic secondary recurrent miscarriage revealed live birth rates of 70% (31/44) in the IVIG group and 62% (28/45) in the control group (P = 0.503); common OR 1.44 (95% CI 0.59–3.48). CONCLUSIONSThis is the largest RCT to date in which IVIG was evaluated in women with idiopathic secondary recurrent miscarriage; no treatment benefit was found. The meta-analysis, which combined our study results with two prior RCTs, also showed no significant effect of treatment with IVIG. ClinicalTrials.gov NCT00606905.
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An innovative virtual reality technique for automated human embryonic volume measurements
BACKGROUNDThe recent introduction of virtual reality (VR) enables us to use all three dimensions in a three-dimensional (3D) image. The aim of this prospective study was to evaluate an innovative VR technique for automated 3D volume measurements of the human embryo and yolk sac in first trimester pregnancies. METHODSWe analysed 180 3D first trimester ultrasound scans of 42 pregnancies. Scans were transferred to an I-Space VR system and visualized as 3D ‘holograms’ with the V-Scope volume-rendering software. A semi-automatic segmentation algorithm was used to calculate the volumes. The logarithmically transformed outcomes were analysed using repeated measurements ANOVA. Interobserver and intraobserver agreement was established by calculating intraclass correlation coefficients (ICCs). RESULTSEighty-eight embryonic volumes (EVs) and 118 yolk sac volumes (YSVs) were selected and measured between 5+5 and 12+6 weeks of gestational age (GA). EV ranged from 14 to 29 877 mm3 and YSV ranged from 33 to 424 mm3. ANOVA calculations showed that when the crown-rump length (CRL) doubles, the mean EV increases 6.5-fold and when the GA doubles, the mean EV increases 500-fold (P < 0.001). Furthermore, it was found that a doubling in GA results in a 3.8-fold increase of the YSV and when the CRL doubles, the YSV increases 1.5-fold (P < 0.001). Interobserver and intraobserver agreement were both excellent with ICCs of 0.99. CONCLUSIONWe measured the human EV and YSV in early pregnancy using a VR system. This innovative technique allows us to obtain unique information about the size of the embryo using all dimensions, which may be used to differentiate between normal and abnormal human development.
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Activation of Toll-like receptor 5 decreases the attachment of human trophoblast cells to endometrial cells in vitro
BACKGROUNDEmbryo implantation in the uterus involves the trophoblast cells apposing and adhering to, then invading across the epithelium lining of the endometrium. However, ethical concerns regarding experimentation with primary human tissue during this period of life necessitates creation of in vitro models for understanding the basic mechanisms involved. Toll-like receptors (TLRs) play a crucial role in defence against pathogens invading the female reproductive tract. The objective of this study is to establish and optimize an in vitro model for studying human endometrial embryonic interactions and to understand the effect of TLR5 stimulation on the attachment of trophoblast cells to endometrial cells. METHODSBy using a human telomerase immortalized endometrial epithelial cell line (hTERT-EECs) and choriocarcinoma human trophoblast cells (JAr cells), an in vitro assay of human implantation was established. In order to investigate the impact of TLR5 stimulation on attachment in this assay, bacterial flagellin was applied to the endometrial and trophoblast cells. In order to block TLR5 in the endometrial and trophoblast cells, TLR5 function-blocking antibody was applied to the cells prior to flagellin treatment. RESULTSThe results demonstrated that JAr spheroids attached to hTERT-EECs in a time and concentration-dependent manner. Our results also demonstrated that treatment of endometrial cells with flagellin, suppressed the attachment of JAr spheres to the endometrial cells. Application of TLR5 function-blocking antibody significantly restored the attachment of JAr spheres to the endometrium. CONCLUSIONSThese data suggest a novel mechanism by which the presence of intrauterine infection through TLR5 activation may result in implantation failure. These data may provide a new opportunity in the management of infertility cases.
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Possible role of the exchange protein directly activated by cyclic AMP (Epac) in the cyclic AMP-dependent functional differentiation and syncytialization of human placental BeWo cells
BACKGROUNDThe mononuclear villous cytotrophoblast (CTB) differentiates and fuses to the multinucleated syncytiotrophoblast (STB), which produces hCG and progesterone. cAMP-mediated intracellular pathways are involved in the process of endocrine differentiation and fusion (syncytialization). The exchange protein directly activated by cAMP (Epac) is a mediator of cAMP signaling. We examined the differential roles of Epac and protein kinase A (PKA) signaling in the cell fusion and differentiation of trophoblast-derived BeWo cells. METHODSEpac1 and Epac2 were localized in human placental tissue (n = 9) by immunohistochemistry. The PKA-selective cAMP analog (N6-phenyl-cAMP, Phe) or Epac-selective cAMP analog (CPT) was tested for effects on hCG and progesterone production, and syncytialization in BeWo cells. The effect of knockdown of Epac or its downstream target molecule (Rap1) on syncytialization was evaluated. RESULTSEpac1 and Epac2 proteins were expressed in villous CTB, STB, stroma, blood vessels and extravillous CTB of the placenta. Phe increased the expression of hCG/β mRNA and secretion of hCG protein in BeWo cells (P < 0.01 versus control). CPT-stimulated production of hCG (P < 0.05), albeit to a lesser extent than Phe. Progesterone production was also enhanced by Phe or CPT (P < 0.01 and P < 0.05, respectively). CPT or a stable cAMP analog (dibutyryl-cAMP: Db) increased the number of syncytialized BeWo cells (P < 0.01), whereas Phe did not stimulate fusion. CPT- or Db-induced syncytialization was observed, even in the presence of a PKA inhibitor. Knockdown of Epac1 or Rap1 repressed the Db-, CPT- or forskolin-induced cell fusion. CONCLUSIONSThe Epac signaling pathway may be associated with the cAMP-mediated functional differentiation and syncytialization of human trophoblasts.
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