Search

STUDY QUESTION As Syncytin 1 (human endogenous retrovirus (HERV-W)) is crucial for human embryo placentation is it expressed during preimplantation embryo development? SUMMARY ANSWER Syncytin 1 was expressed mainly in trophoblast cells of the blastocyst particularly in cells underlying the inner cell mass (ICM). WHAT IS KNOWN ALREADY Syncytin 1 (along with HERV-FRD or Syncytin 2) is expressed in first-trimester placenta and required for cell-cell fusion to enable formation of syncytiotrophoblast and effective placentation. STUDY DESIGN, SIZE AND DURATION Preimplantation human embryos donated for research were cultured in vitro and protein expression of Syncytin 1 at the blastocyst stage of development investigated. Comparisons were made with protein (Syncytin 1) and mRNA (Syncytin 1 and 2) expression in human embryonic stem cells (hESCs) undergoing differentiation to trophoblast-like cells in vitro. In total, 10 blastocysts (×3 or 4 replicates) were analysed and 4 hESC lines. The study was terminated after consistent observations of embryos were made. PARTICIPANTS/MATERIALS, SETTING, METHODS Donated embryos were thawed and cultured to blastocyst, fixed with 4% w/v paraformaldehyde. Syncytin 1 protein expression was determined by immunofluorescent localisation and confocal microscopy. Additionally, hESCs were differentiated to trophoblast-like cells in standard and conditioned culture medium with growth factors (bone morphogenetic protein 4 (BMP4) or fibroblast growth factor 4 (FGF4) and assessed for mRNA (Syncytin 1 and 2) by quantitative polymerase chain reaction (qPCR) and protein expression by immunolocalization and western blot. MAIN RESULTS AND ROLE OF CHANCE Syncytin 1 was expressed in cytoplasm and on the cell surface of some trophoblast cells, and consistently the trophectoderm underlying the ICM of the blastocyst. There was weak but consistent expression of Syncytin 1 in cells on the periphery of the ICM also displaying pluripotency antibody marker (Tra-1-60). Three-dimensional reconstruction of confocal slice data provided good visualization of expression. The time course of expression of Syncytin 1 was replicated in hESCs differentiated in vitro confirming the embryo observations and providing statistically significant differences in protein and mRNA level (P= 0.002) and (P< 0.05), respectively. LIMITATION, REASONS FOR CAUTION Culture of a limited number of embryos to blastocyst in vitro may not replicate the range and quality of development in situ. Probes (antibodies, PCR) were tested for specificity, but might have non-specific reactions. WIDER IMPLICATIONS OF FINDINGS Syncytin expression is a prerequisite for embryo implantation and placentation. Understanding when expression first occurs during embryo development may be informative for understanding conditions of abnormal gestations such as pre-clampsia. STUDY FUNDING/COMPETING INTERESTS The study was supported partly by an ERASMUS training grant and grant G0801059 from the Medical Research Council, U.K. There were no competing interests.

background: Sperm motility is regulated by mitochondrial enzymes that are partially encoded by mitochondrial DNA (mtDNA). MtDNA has therefore been suggested as a putative genetic marker of male fertility. However, recent studies in different populations have identified both significant and non-significant associations between mtDNA variation and sperm motility. Here, we tested whether mtDNA variation was associated with sperm motility in a large cohort of men from the UK, to test the robustness of previous studies and the reliability of mtDNA as a marker of poor sperm motility. methods: A total of 463 men attending for semen analysis as part of infertility investigations were recruited from a UK laboratory. Sperm motility was measured using both computer-assisted sperm analysis and traditional manual measurements. MtDNA haplogroup and haplotype were determined in 357 and 298 men, respectively, using single nucleotide polymorphism (SNP) markers throughout the mtDNA genome, and compared with sperm motility data. The linkage between the SNP markers, and possible associations between individual SNPs and motility, were also investigated. results: We found no statistical association between haplogroup or haplotype and sperm motility, regardless of how it was measured (P . 0.05 in all cases). Moreover, individual SNPs which were in linkage disequilibrium and dispersed across the mitochondrial genome, and therefore sensitive to mtDNA variation, were not predictive of sperm motility. conclusions: Mitochondrial haplotype is unlikely to be a reliable genetic marker of male factor infertility.

background: Investigating the mechanisms of human primordial germ cell (PGC) and gamete development are important for understanding the causes of infertility and effects of environmental chemicals on reproductive development. However, there are practical and ethical difficulties associated with obtaining human tissue in early development. The aim of this study was to investigate whether human embryonic stem cell-hESC-generated germ cells could provide an in vitro model of gamete development. method: Human ESCs were differentiated as embryoid bodies (EBs) in vitro. Gene and protein marker expression profiles of EBs in different periods of culture were analysed by quantitative polymerase chain reaction (Q-PCR) and immunolocalization to monitor germ cell development. Secretion of hormones involved in germ cell maturation was measured, to detect the existence of a germ cell niche within EBs. results: Q-PCR revealed gene expression profiles consistent with PGC formation and germ cell development. A small population of post-meiotic spermatid cells were identified using sperm-specific antibodies (Protamine 1 and 1.97). Although gene expression profiles characteristic of oocyte development and follicle-like structures were detected, a committed oocyte with extra-cellular zona pellucida was not recognized with zona pellucida-specific monoclonal antibody. conclusions: hESCs can form PGCs and post-meiotic spermatids in vitro, however, there remains doubt about oocyte development. Levels of steroid hormones produced by EBs were significant when compared with known values for a similar quantity of human testis, suggesting that hESC may intrinsically create a favourable hormonal niche for spermatogenesis.

BACKGROUND: An effective embryonic–maternal interaction is crucial for successful human pregnancy. Failure of this process is a major cause of infertility and can lead to placental dysfunction resulting in recurrent miscarriage, fetal retardation and pre-eclampsia. Research is severely constrained by ethical and practical considerations; therefore, we aimed to generate cytotrophoblast stem (CTBS) cell lines from human embryonic stem cells (HESCs). METHOD: beta-HCG was used as a marker of viable trophoblast cells. In defined culture, embryoid bodies were generated from HESCs and selected for trophoblast enrichment by rounds of cellular aggregation and disaggregation. Distinct CTBS cell lines were isolated and characterized. Spheroid cytotrophoblast bodies were generated and their interaction with luteal-phase endometrial stroma was analysed by real-time image analysis. RESULTS: Three CTBS cell lines were derived, which were maintained in the absence of residual HESCs, fibroblast feeder cells or extracellular matrix. CTBS cells displayed typical cytotrophoblast and syncytiotrophoblast characteristics and exhibited further differentiation to invasive endovascular cell phenotype. One cell line was generated with constitutive expression of enhanced green fluorescent protein (eGFP). Spheroid trophoblast bodies mimicked closely the early invasive stages of implantation when incubated with human endometrial stromal preparations in vitro. CONCLUSION: These human CTBS cell lines are a significant new model for investigating human placentation and may have considerable potential in cell therapy applications.

Although testicular biopsy for sperm extraction is a procedure with a potential for complications, sperm retrieval is successful in 30-70% of patients with non-obstructive azoospermia. In order to predict the probability of retrieving at least one testicular spermatozoon we conducted a prospective study of a set of variables in 40 patients with non-obstructive azoospermia. Using the receiver operating characteristic curves, we determined the probability estimates of testicular volume, plasma follicle stimulating hormone (FSH) concentration, Johnsen score and visualization of testicular spermatids in discriminating between patients with successful and failed testicular sperm extraction. Visualization of testicular spermatids provided the best estimate of success of testicular sperm extraction. Of the factors studied using logistic-regression analysis (age, maternal and paternal age at birth, body mass index, luteinizing hormone, testosterone, FSH, testicular volume, the presence of testicular spermatids and Johnsen score), only the presence of spermatids and Johnsen score were independent variables able to predict the success of testicular sperm extraction. The visualization of the presence of spermatids gave a correct prediction of 77% and Johnsen score of 71%. The diagnostic model derived from these independent predictors when validated in 40 patients using the Jackknife technique gave a correct overall prediction of 87%. The probability of successful testicular sperm extraction in patients with non-obstructive azoospermia could be objectively predicted on the basis of simple histopathological criteria represented by the visualization of testicular spermatids and Johnsen score.

The outcome and costs of testicular sperm extraction under outpatient local analgesia or general anaesthesia were compared in men with non-obstructive azoospermia. Nineteen consecutive patients were allocated to receive general anaesthesia, while the subsequent 21 consecutive patients received outpatient analgesia in the form of i.v. midazolam sedation, lignocaine spray, scrotal infiltration with local anaesthetic and spermatic cord block. Blood pressure, pulse rate and respiratory rate were determined. Sedation and testicular pain were assessed by subjective scoring. Both groups showed haemodynamic stability with little alteration in blood pressure, pulse rate and oxygen saturation. Toxic symptoms of local anaesthetic were not encountered in the outpatient group. No relationship was found between testicular size and the duration of the operation. The median postoperative pain intensity, sedation scores and analgesic requirements were significantly less in the outpatient group (P < 0.05). These advantages led to a shorter recovery time (P < 0.0001), 3-fold cheaper care and greater patient satisfaction (P < 0.0001) in the outpatient group.

Limiting testicular biopsy for intracytoplasmic sperm injection (ICSI) to those with a high chance of having testicular spermatozoa has not been possible because of the poor predictive value of current clinical and laboratory methods. In order to predict testicular pathology and sperm extraction, we characterised the semen of 28 men with azoospermia due to gonadal failure in terms of the presence of spermatids using an immunological method. The results were compared with the assessment of testicular biopsies by histology and the extraction of spermatozoa into culture medium. Washed cellular elements in the ejaculate were smeared on microscope slides and fixed in 100% methanol, before incubation with acrosome-specific monoclonal antibody (18.6), fluorescein isothiocyanate-labelled anti-mouse goat IgG, and examination by epifluorescent microscopy. Semen from men with oligozoospermia and obstructive azoospermia served as positive and negative controls, respectively. Twelve patients who had positive immunofluorescence (one or more spermatids present) had spermatozoa retrieved from their testes (five hypospermatogenesis, seven focal spermatogenesis), and 16 patients with negative immunofluorescence (spermatids absent) had apparent Sertoli cell-only syndrome (12) or maturation arrest histological pattern (four). However, four patients with apparent Sertoli cell-only syndrome had testicular spermatozoa present after extraction from the biopsy. Plasma follicle stimulating hormone concentration and testicular volume did not predict retrieval of seminal spermatids or testicular spermatozoa. We conclude that the immunofluorescent localization of one or more spermatids in the ejaculate can be used to predict the likelihood of obtaining testicular spermatozoa for ICSI. However, in some patients with Sertoli cell-only syndrome, spermatozoa could still be recovered in the absence of apparent seminal spermatids.

Use of a cryostage has enabled direct observation of human spermatozoa as they are cryopreserved and thawed. Crystallization and recrystallization events are readily observed. In combination with computer-aided semen analysis (CASA) equipment it was possible to determine the consequence of altering the cooling, freezing and thawing rates of a temperature-rate profile on sperm motility. Increasing the cooling rate to 50 degrees C/min resulted in significantly lower pre-freeze to post-thaw ratios for average path velocity (VAP, 13%), mean straight line velocity (VSL, 35%), mean linearity (LIN, 28%) and straightness (STR, 24%), while the ratio of the number of cells crossing the field of view (NCF) significantly increased (30%) compared to a standard freeze-thaw temperature rate profile. The NCF pre-freeze to post-thaw ratio was associated with the percentage of cell recovery after cryopreservation. Faster thaw rates resulted in better survival of the cells, perhaps due to the shorter time during which recrystallization occurred. The NCF ratios were significantly higher (33 and 30% for thaw rates of 50 and 100 degrees C/min respectively) than for the standard profile samples. Previous studies on cell survival have shown a link between the cooling and thaw rates. The cryostage should prove invaluable in future studies to identify the causes of cryodamage to spermatozoa. When used in combination with CASA, changes to sperm function during cryopreservation can be accurately measured.