This paper presents four innovative utilizations of calcium sulfoaluminate (CSA) cement:–development of concrete with high early strength: 40 MPa, 6 h after its preparation, and higher than 55 MPa ...after 24 h,–design of self-leveling screed with limited curling, when unbonded to its support,–design of self-leveling topping mortar presenting the following properties: time of workability higher than 30 min, set within 75 min, and low drying shrinkage (<250μm/m).–glass-fiber-reinforced cement (GFRC) composites that can be demolded 4 h after casting and present high ductility and durability after aging in different weathering conditions.
Three kaolins were heated between 500 and 850 °C. The samples were tested by X-ray diffraction (XRD), differential thermal analysis (DTA), and Infra-Red (IR) spectroscopy in order to determine their ...mineralogical composition and degrees of crystallinity and dehydroxylation. The reactivity of the heated samples was evaluated by the determination of the residual quantity of Ca(OH)
2 by differential thermal analysis (DTA) performed on hydrated mixtures of 50% metakaolin and 50% Ca(OH)
2. There was no direct relationship between the pozzolanic activity of metakaolin and the degree of dehydroxylation. Highest activity was obtained when the degree of dehydroxylation was >
95%.
STUDY QUESTION
Is an organotypic culture system able to provide the appropriate testicular microenvironment for in-vitro maturation of human immature testicular tissue (ITT)?
SUMMARY ANSWER
Our ...organotypic culture system provided a microenvironment capable of preserving seminiferous tubule (ST) integrity and Leydig cell (LC) functionality and inducing Sertoli cell (SC) maturation.
WHAT IS KNOWN ALREADY
Cryopreservation of human ITT is a well-established strategy to preserve fertility in prepubertal boys affected by cancer, with a view for obtaining sperm. While spermatogenesis in mice has been replicated in organotypic culture, yielding reproductively efficient spermatozoa, this process has not yet been achieved in humans.
STUDY DESIGN, SIZE, DURATION
The aim of this study was to in vitro mature frozen-thawed ITT. To this end, 1 mm3 tissue fragments from three prepubertal patients aged 2 (P1), 11 (P2) and 12 (P3) years were placed in organotypic culture for 139 days. Culture media, supplemented with either testosterone or hCG, were compared.
PARTICIPANTS/MATERIALS, SETTING, METHODS
ST integrity and tissue viability were assessed by histological score and lactate dehydrogenase (LDH) levels in supernatants. Spermatogonia (SG), proliferating cells and proliferating SG were identified by the use of MAGE-A4 and Ki67 immunohistochemical markers. Glial cell line-derived neurotrophic factor (GDNF) was used as a marker of SC functionality, while SC maturation was evaluated by androgen receptor (AR), anti-Müllerian hormone (AMH) immunohistochemistry (IHC) and AMH immunoenzymatic assay. LC functionality was determined by testosterone levels in supernatants and by 3β-hydroxysteroid dehydrogenase (3β-HSD) IHC. Apoptosis was studied by IHC with active caspases 3 and 8 and by TUNEL (terminal deoxynubocleotidyl transferase-mediated dUTP nick end labeling) analysis.
MAIN RESULTS AND THE ROLE OF CHANCE
Tissue viability was preserved, as demonstrated by the decrease in and stabilization of LDH release, and evolution of ST scoring, with the percentage of well-preserved STs showing no statistical differences during culture in either medium. GDNF was expressed until Day 139, demonstrating SC functionality. Moreover, a significant reduction in AMH expression and release indicated SC maturation. Testosterone concentrations in supernatants increased in both culture media, demonstrating LC functionality with paracrine interactions. SG were present up to Day 139, although the ratio between MAGE-A4-positive cells and well-preserved tubules was significantly reduced over the course of culture (P ≤ 0.001). SCs exhibited a decreased proliferation rate over time (P ≤ 0.05). The proliferation rate of SG remained stable until Day 64, but over the total culture period (139 days), it was found to have decreased (P ≤ 0.05). The number of apoptotic cells did not vary during culture, nor was any statistical difference observed between the two culture media for any of the studied parameters.
LARGE SCALE DATA
N/A
LIMITATIONS, REASONS FOR CAUTION
Loss of SG constitutes a limitation for evaluating full functionality of spermatogonial stem cells and warrants further investigation. The scarcity of human immature material is the reason for the limited amount of tissue available for experiments, precluding more comprehensive analysis.
WIDER IMPLICATIONS OF THE FINDINGS
Our culture system, mimicking the peripubertal testicular microenvironment with SC maturation, LC functionality and preserved paracrine interactions, and the first to use human ITT, opens the door to a deeper understanding of niche and culture conditions to obtain sperm from cryostored ITT, with the ultimate goal of restoring fertility after gonadotoxic treatments.
STUDY FUNDING/COMPETING INTEREST(S)
This project was supported by a grant from the Fond National de la Recherche Scientifique de Belgique (grant Télevie N° 7.4554.14F and N° 7.4512.15F) and the Fondation Salus Sanguinis. No conflict of interest is declared.
Can human theca cells (TCs) be differentiated in vitro?
It is possible to differentiate human TCs in vitro using a medium supplemented with growth factors and hormones.
There are very few studies on ...the origin of TCs in mammalian ovaries. Precursor TCs have been described in neonatal mice ovaries, which can differentiate into TCs under the influence of factors from oocytes and granulosa cells (GCs). On the other hand, studies in large animal models have reported that stromal cells (SCs) isolated from the cortical ovarian layer can also differentiate into TCs.
After obtaining informed consent, ovarian biopsies were taken from eight menopausal women (53-74 years of age) undergoing laparoscopic surgery for gynecologic disease not related to the ovaries. SCs were isolated from the ovarian cortex and in vitro cultured for 8 days in basic medium (BM) (G1), enriched with growth factors, FSH and LH in plastic (G2) or collagen substrate without (G3) or with (G4) a GC line.
To confirm TC differentiation, relative mRNA levels for LH receptor (Lhr), steroidogenic acute regulatory protein (Star), cholesterol side-chain cleavage enzyme (Cyp11a1), cytochrome P450 17A1 (Cyp17a1), hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (Hsd3b1) and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 2 (Hsd3b2) were assessed. Immunohistochemistry was also performed for their protein detection and a specific marker was identified for TCs (aminopeptidase-N, CD13), as were markers for theca and small luteal cells (dipeptidyl peptidase IV (CD26) and Notch homolog 1, translocation-associated (NOTCH1)). Finally, we analyzed cell ultrastructure before (Day 0) and after in vitro culture (Day 8), and dehydroepiandrosterone (DHEA) and progesterone levels in the medium using transmission electron microscopy (TEM) and ELISA, respectively.
Results obtained from qPCR showed a significant increase (P < 0.05) in mRNA levels of Lhr in F2 (floating cells in G2) and G4, Cyp17a1 in G1 and F1 (floating cells in G1) and Hsd3b2 in G1, G2, G3 and G4. Immunohistochemistry confirmed expression of each enzyme involved in the steroidogenic pathway at the protein stage. However, apart from G1, all other groups exhibited a significant (P < 0.05) rise in the number of CD13-positive cells. There was also a significant increase (P < 0.05) in NOTCH1-positive cells in G3 and G4. Ultrastructure analyses by TEM showed a distinct difference between groups and also versus Day 0. A linear trend with time revealed a significant gain (q < 0.001) in DHEA concentrations in the medium during the culture period in G1, G2, G3 and G4. It also demonstrated a statistical increase (q < 0.001) in G2, G3 and G4 groups, but G1 remained the same throughout culture in terms of progesterone levels.
N/A.
Shorter periods of in vitro culture (e.g. 2, 4 and 6 days) could have led to increased concentrations of differentiated TCs in G2, G3 and G4. In addition, a group of cells cultured in BM and accompanied by COV434 cells would be necessary to understand their role in the differentiation process. Finally, while our results demonstrate that TCs can be differentiated in vitro from cells isolated from the cortical layer of postmenopausal ovaries, we do not know if these cells are differentiated from a subpopulation of precursor TCs present in ovarian cortex or ovarian SCs in general. It is therefore necessary to identify specific markers for precursor TCs in human ovaries to understand the origin of these cells.
This is a promising step toward understanding TC ontogenesis in the human ovary. Moreover, in vitro-generated human TCs can be used for studies on drug screening, as well as to understand TC-associated pathologies, such as androgen-secreting tumors and polycystic ovary syndrome.
This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS) (C.A.A. is an FRS-FNRS Research Associate; grant MIS #F4535 16 awarded to C.A.A.; grant 5/4/150/5 awarded to M.M.D.; grant ASP-RE314 awarded to P.A.) and Foundation Against Cancer (grant 2018-042 awarded to A.C.). The authors declare no competing interests.
Forty to fifty percent of patients with locally advanced squamous cell carcinoma of the head and neck (LA SCCHN) relapse despite multimodal treatment. Circulating tumor DNA (ctDNA) has the potential ...to detect minimal residual disease (MRD) after curative-intent therapy and to identify earlier which patients will progress. We developed a tumor-agnostic plasma ctDNA assay to detect MRD in unselected LA SCCHN with the aim of predicting progression-free survival (PFS) and overall survival without the need for tumor sequencing.
A 26-gene next-generation sequencing panel was constructed that included the most frequently mutated genes in SCCHN and two HPV-16 genes. MRD was assessed in each patient through an in-house informatic workflow informed by somatic mutations identified in the corresponding pre-treatment plasma sample. The presence of MRD was defined as the detection of ctDNA in one plasma sample collected within 1-12 weeks of the end of curative treatment. The primary endpoint was the PFS rate at 2 years. At least 32 patients were planned for inclusion with the hypothesis that PFS at 2 years was >80% in MRD-negative patients and <30% in MRD-positive patients (α = 0.05, β = 0.9).
We sequenced DNA from 116 plasma samples derived from 53 LA SCCHN patients who underwent curative-intent treatment. ctDNA was detected in 41/53 (77%) patients in the pre-treatment samples. Out of these 41 patients, 17 (41%) were MRD positive after treatment. The 2-year PFS rate was 23.53% (9.9% to 55.4%) and 86.6% (73.4% to 100%) in MRD-positive and MRD-negative patients, respectively (P < 0.05). Median survival was 28.37 months (14.30 months-not estimable) for MRD-positive patients and was not reached for the MRD-negative cohort (P = 0.011).
Our ctDNA assay detects MRD in LA SCCHN and predicts disease progression and survival without the need for tumor sequencing, making this approach easily applicable in daily practice.
•ctDNA may detect MRD after curative-intent therapy and allow the early identification of patients likely to progress.•A tumor-agnostic ctDNA assay was designed for LA SCCHN.•The assay detects MRD in LA SCCHN and predicts PFS and survival without the need for tumor sequencing.•This tumor-agnostic approach has the potential to be used broadly, and further studies are warranted.
How does the formation of the blood-testis barrier (BTB), as reflected by the expression of connexin 43 and claudin 11 proteins during the pubertal transition period, take place in vitro compared to ...samples from a large cohort of pre/peripubertal boys?
The BTB connexin 43 and claudin 11 expression patterns appeared to be partially achieved in organotypic culture when compared to that in samples from 71 pre/peripubertal patients.
Although alterations in the protein expression patterns of the BTB, whose main components are connexin 43 and claudin 11, are known to be associated with impaired spermatogenesis in mice and adult men, there is a lack of knowledge on its formation in pre-peripubertal human tissue both in vitro and in vivo. Moreover, despite Sertoli cell (SC) maturation during long-term organotypic culture of immature testicular tissue (ITT), initiation of spermatogenesis has not yet been achieved.
Histological sections from 71 pre-peripubertal patients were evaluated for the formation of the BTB acting as in vivo controls according to age, SC maturation, clinical signs of puberty and germ cell differentiation. Testicular tissue fragments retrieved from three prepubertal boys were cultured in a long-term organotypic system to analyze the BTB formation and expression pattern in correlation with SC maturation.
Testicular histological sections from 71 patients aged 0-16 years who underwent a biopsy between 2005 and 2014 to preserve their fertility before gonadotoxic treatment were examined. Immunohistochemistry (IHC) results for connexin 43 and claudin 11 as BTB markers, using a semi-quantitative score for their expression, and for Anti-Mullerian hormone (AMH), as SC maturation marker, were analyzed. Germ cell differentiation was evaluated on Hematoxylin-Eosin sections. Tanner stages at the time of biopsy were recorded from medical files. A longitudinal analysis of connexin 43, claudin 11 and AMH expressions on immunohistological sections of organotypic cultured testicular tissue from three prepubertal boys who underwent a biopsy for fertility preservation was performed. Immunostaining was evaluated at culture Days 0, 1, 3, 10, 16, 27, 32, 53, 64 and 139 for two different types of culture media.
Immunohistochemical control sections showed progressive maturation of SCs, as shown by the decrease in AMH expression, with increasing age (P ≤ 0.01) and the AMH expression was negatively correlated with the expression of connexin 43 and claudin 11 (P ≤ 0.01 for both proteins). Androgen receptor (AR) expression increased with age (P ≤ 0.01) and was significantly correlated with the expression of connexin 43 (P = 0.002) and claudin 11 (P = 0.03). A statistical correlation was also found between the reduction of AMH expression and both the advancement of Tanner stages (P ≤ 0.01) and the differentiation of germ cells (P ≤ 0.01). Furthermore, positive correlations between BTB formation (using connexin 43 and claudin 11 expression) and age (P ≤ 0.01 for both the proteins), higher Tanner stages (P ≤ 0.001 and P ≤ 0.01 for connexin 43 and claudin 11, respectively), and presence of more advanced germ cells (P ≤ 0.001 for both proteins) were observed. In the subanalysis on organotypic cultured ITT, where a significant decrease in AMH expression as a marker of SC maturation was already reported, we showed the onset of expression of connexin 43 at Day 16 (P ≤ 0.001) and a constant expression of claudin 11 from Days 0 to 139, for all three patients, without differences between the two types of culture media.
N/A.
Accessibility of prepubertal human testicular tissue is a major limiting factor to the analysis of cultured tissue samples from a wide number of patients, as would be needed to assess the in vitro development of the BTB according to the age. The impossibility of performing longitudinal studies on in vivo BTB formation in the same patient prevents a comparison of the time needed to achieve effective BTB formation and protein expression patterns in vivo and in vitro.
To the best of our knowledge, this is the first report describing the expression of two BTB proteins in samples from a cohort of prepubertal and peripubertal boys, for the in vivo pattern, and in cultured ITT from a few prepubertal boys, for the in vitro evaluation. Since the formation of this barrier is essential for spermatogenesis and because little is known about its protein expression patterns and development in humans, a deeper understanding of the testicular microenvironment is essential to improve ITT in vitro culture conditions. The final aim is to restore fertility by acheiving in vitro differentiation of spermatogonial stem cells, using cryopreserved ITT collected before gonadotoxic therapies.
Funding was received from Fonds National de la Recherche Scientifique de Belgique (Grant Télevie Nos. 7.4554.14F and 7.6511.16) and Fondation Salus Sanguinis. No conflict of interest has to be disclosed.
STUDY QUESTION
What is the best source of ovarian cells for the artificial ovary: medulla or cortex, cryopreserved or fresh?
SUMMARY ANSWER
Ovarian cells from fresh medullary tissue, which can be ...isolated in larger numbers, show higher viability and are able to improve graft vascularization.
WHAT IS KNOWN ALREADY
In a previous study, addition of endothelial cells along with ovarian cells was found to be crucial for formation of a well-vascularized ovary-like structure. This study is the first to evaluate both the effect of cryopreservation and the source of ovarian tissue on isolated ovarian cells.
STUDY DESIGN, SIZE, DURATION
Prospective experimental study in an academic research unit using ovarian tissue from seven patients undergoing surgery for benign gynecologic disease.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Ovarian tissue was retrieved from seven patients, with one half processed as fresh (fresh group) and the other half frozen and thawed before processing (frozen group). In each group, ovarian cells from the cortex and medulla were isolated separately, and their viability was tested using a calcein AM/ethidium homodimer viability assay. Fifty thousand cells were then encapsulated in fibrin and grafted to peritoneal pockets in nude mice (14 in all). Grafts recovered after 7 days were analyzed by immunohistochemistry for the presence of ovarian cells (vimentin), proliferation (Ki67) and graft vascularization (double CD34). Cell apoptosis was analyzed by TUNEL assay.
MAIN RESULTS AND THE ROLE OF CHANCE
Cryopreservation decreased ovarian cell yield (−2804 cells/mg, P = 0.015) and viability (−9.72%, P = 0.052) before grafting and had a considerable (5-fold, P = 0.2) but non-significant negative impact on ovarian cell presence in grafts. The medulla yielded many more cells (+3841 cells/mg, P < 0.001) with higher viability (+18.23%, P < 0.001) than did the cortex. Moreover, grafts with cells from the medulla exhibited a statistically significant 6.44- and 2.47-fold increase in human and total vascular surface area, respectively. P-values were adjusted for multiple testing using the Benjamini–Hochberg method to achieve a 10% false discovery rate and adjusted P-values < 0.1 were therefore considered significant.
LIMITATIONS, REASONS FOR CAUTION
Pilot study involving a limited number of experiments.
WIDER IMPLICATIONS OF THE FINDINGS
Knowing that fresh medullary tissue is the best source of stromal cells is important for construction of the artificial ovary, as isolated follicles require structural support and a rich vascular network for their survival and development.
STUDY FUNDING/COMPETING INTEREST(S)
This work was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (5/4/150/5 and 7.4518.12F), Fonds Spéciaux de Recherche, Fondation Saint Luc and Foundation Against Cancer, and donations from Mr Pietro Ferrero, Baron Frère and Viscount Philippe de Spoelberch. None of the authors have any conflicting interests to declare.
Purpose
Spasmodic dysphonia (SD) or laryngeal dystonia is as a rare vocal disorder characterized by involuntary action-induced endolaryngeal contraction. In the last decade, botulin toxin injection ...has become the standard treatment in adductor spasmodic dysphonia necessitating repetitive injections. The purpose of this study is to analyze retrospectively data from patients treated with the minimal-invasive transoral radiofrequency-induced thermotherapy (RFITT) of the terminal branches of the recurrent nerve.
Methods
Between 2009 and 2015, 11 patients (six females and five males aged from 32 to 91 years) with adductor SD were treated with RFITT. Pre-operative and post-operative vocal assessments (VHI-30, GRBASI, and acoustic-aerodynamics measurements), number of surgical revisions, delay between procedures, and post-operative complications were recorded. Statistical analyses were carried out on the first vocal assessment performed 2–8 weeks after the first procedure.
Results
Based on available data from ten patients, voice handicap index (VHI) showed improvement with a mean value of −17.7 points (
p
-value (
p
val) = 0.014, adjusted
p
-value (adj
p
val) = 0.21); instability has also revealed improvement in six patients (
p
val = 0.05, adj
p
val = 0.31). Four patients underwent only one procedure including one patient showing still long-term beneficial results after 5 years of follow-up. Other patients required one to three new procedures with an average time between procedures of 15.3 months. Over 24 surgeries performed on a total of 11 patients, one definitive treatment-related severe adverse event was reported.
Conclusion
Thanks to long-lasting effect, repetitive treatments are less frequent compared to botulin toxin therapy. In our opinion, RFITT is a promising alternative to botulin toxin as a second-step procedure in case of toxin resistance or patient’s lack of compliance.
Purpose
Housekeeping genes (HKGs), reference or endogenous control genes, are vital to normalize mRNA levels between different samples. Since using inappropriate HKGs can lead to unreliable results, ...selecting the proper ones is critical for gene expression studies. To this end, normal human ovaries, as well as those from patients diagnosed with ovarian endometrioid adenocarcinoma (OEA), ovarian mucinous adenocarcinoma (OMA), ovarian serous papillary carcinoma (OSPC), and polycystic ovary syndrome (PCOS), were used to identify the most suitable housekeeping genes.
Methods
RNA was isolated from 5 normal human ovaries (52–79 years of age), 9 cancerous ovaries (3 OEA, 3 OMA, 3 OSPC; 49–75 years of age), and 4 PCOS ovaries (18–35 years of age) in women undergoing hysterectomy. cDNA was synthesized using a whole transcriptome kit, and quantitative real-time PCR was performed using TaqMan array 96-well plates containing 32 human endogenous controls in triplicate.
Results
Among 32 HKGs studied, RPS17, RPL37A, PPIA, 18srRNA, B2M, RPLP0, RPLP30, HPRT1, POP4, CDKN1B, and ELF1 were selected as the best reference genes.
Conclusions
This study confirms recent investigations demonstrating that conventional HKGs, such as GAPDH and beta-actin, are not suitable reference genes for specific pathological conditions, emphasizing the importance of determining the best HKGs on a case-by-case basis and according to tissue type. Our results have identified reliable HKGs for studies of normal human ovaries and those affected by OEA, OMA, OSPC, or PCOS, as well as combined studies of control subjects vs. each cancer or PCOS group.
•Calcium Carbide method is a classical field technic for residual water evaluation.•Study focuses on residual water evaluation on model materials for SLU applications.•Comparison of residual water ...measurements techniques evidence biases.•Residual water evaluation is highly dependents of hydrates microstructure.•New evaluation techniques are proposed for material with weakly bound water.
A self-leveling underlayment is a thin layer used to smooth floors and has to contain a very low level of residual water prior to covering with the final coating. Therefore, the evaluation of residual water is a key point. The aim of this study is to compare two residual water evaluation techniques: the calcium carbide method (CCM) and oven drying at 40 °C. The following model materials are investigated: silica, gypsum, plaster, and ettringite. The CCM measurements provide an effective indicator of free water for silica and gypsum. Although not a field measurement, oven drying offers a reliable and truly representative measure of the residual water, regardless of the material used.
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