177. ANTIBODIES AGAINST BMPR-IB UNCOVERS A PARACRINE FUNCTION FOR THE RECEPTOR IN MALE MOUSE LEYDIG CELL TESTOSTERONE PRODUCTION IN VITRO

2010 ◽  
Vol 22 (9) ◽  
pp. 95
Author(s):  
I. M. Ciller ◽  
U. A. Ciller ◽  
J. R. McFarlane
Keyword(s):  
Cell Culture ◽  
Leydig Cell ◽  
Leydig Cells ◽  
Chorionic Gonadotrophin ◽  
Slice Culture ◽  
Type I ◽  
Paracrine Signalling ◽  
Testosterone Production ◽  
Bmp Receptors

The male reproductive system is regulated by pituitary gonadotrophins and local testicular factors including the transforming growth factor-β superfamily members which up-regulate and modulate testosterone production respectively. Type I and type II bone morphogenetic protein (BMP) receptors have been identified in both steroidogenic and non-steroidogenic cells in the testis and have been reported to impact steroid production via their effect on steroidogenic enzymes. In this study we investigated if BMPR-IB had an autocrine or paracine role in testosterone production using BMPR-IB antibodies in testis tissue and Leydig cell culture in vitro. Immature (21d) and mature (60d) mice were sacrificed by CO2 asphyxiation and the testis removed, decapsulated and cultured in basal and equine chorionic gonadotrophin (eCG)-conditioned media for three hours at 32 °C in 5 % CO2, in the presence and absence of anti-BMPR-IB. Additionally, Leydig cells were Percoll purified from adult mouse testicular interstitial cells and cultured for three hours with and without anti-BMPR-IB under human chorionic gonadotrophin (hCG) stimulated or unstimulated conditions. After three hours of incubation the culture media was aspirated into labeled vials and assayed for testosterone using a radioimmunoassay. In adult testicular slice culture, treatment with anti-BMPR-IB resulted in a significant decrease in basal and eCG-stimulated testosterone production by 37 % and 41 % respectively, while having no significant effect on basal or eCG-stimulated testosterone production by the immature testis. In purified Leydig cell culture from adult male mice BMPR-IB immunization had no effect on testosterone production in basal or hCG-stimulated conditions. In conclusion, anti-BMPR-IB significantly reduced testosterone production in adult testicular slice culture but not in cell culture, demonstrating that BMP paracrine signalling from the seminiferous tubules is likely to be important in modulating testosterone production by Leydig cells. Additionally, the paracrine signalling appears to be developmentally regulated only occurring in the adult testis.

scholarly journals Mumps Virus Decreases Testosterone Production and Gamma Interferon-Induced Protein 10 Secretion by Human Leydig Cells

2003 ◽  
Vol 77 (5) ◽  
pp. 3297-3300 ◽  
Author(s):  
Ronan Le Goffic ◽  
Thomas Mouchel ◽  
Annick Ruffault ◽  
Jean-Jacques Patard ◽  
Bernard Jégou ◽  
...  
Keyword(s):  
Cell Cultures ◽  
Leydig Cell ◽  
Leydig Cells ◽  
Mumps Virus ◽  
Gamma Interferon ◽  
Testosterone Secretion ◽  
Testosterone Production

ABSTRACT Mumps virus is responsible for sterility. Here, we show that the mumps virus infects Leydig cells in vitro and totally inhibits testosterone secretion and that ribavirin in mumps virus-infected Leydig cell cultures completely restores testosterone production. Moreover, we show that gamma interferon-induced protein 10 (IP-10) is highly expressed by mumps virus-infected Leydig cells and that ribavirin does not block IP-10 production.

Increase in Leydig cell responsiveness in the unilaterally cryptorchid rat testis and its relationship to the intratesticular levels of testosterone

1984 ◽  
Vol 102 (3) ◽  
pp. 319-327 ◽  
Author(s):  
R. M. Sharpe ◽  
I. Cooper ◽  
D. G. Doogan
Keyword(s):  
Leydig Cell ◽  
Leydig Cells ◽  
Venous Blood ◽  
Cell Interaction ◽  
Adult Rats ◽  
Similar Reduction ◽  
Testosterone Production ◽  
Lh Releasing Hormone ◽  
Unilateral Cryptorchidism

ABSTRACT Adult rats were made unilaterally cryptorchid (UCD) and 6–7 weeks later Leydig cells were isolated from the scrotal and abdominal testes and their capacity to secrete testosterone in vitro was compared. Basal testosterone production by Leydig cells from the abdominal testes of UCD rats was lowered, compared with cells from the contralateral scrotal testes, whilst their responsiveness to both human chorionic gonadotrophin and an LH releasing hormone agonist was enhanced two- to threefold (P< 0·001) compared both with cells from the contralateral scrotal testes and with cells isolated from untreated rats of the same age. In the UCD rats, concentrations of testosterone in testicular interstitial fluid (IF) were reduced (P< 0·001) by 70–90% in abdominal, compared with scrotal, testes. A similar reduction was evident in the levels of testosterone in spermatic venous blood, and both this decrease and that in IF levels of testosterone varied according to the degree of testicular involution. The ontogeny of the above changes was investigated. After induction of unilateral cryptorchidism, the weight of the abdominal compared with the scrotal testis declined slowly, such that by day 5 there was only a 25% reduction in weight compared with a 70% reduction by day 40. In contrast, the levels of testosterone in IF from abdominal testes declined rapidly, such that by day 5 an 80% reduction was attained, compared with scrotal testes, with little further change by day 40. Hormone-stimulated testosterone production by Leydig cells isolated from the abdominal testes was unchanged or marginally reduced over the first 3 days compared with cells from the scrotal testes, but by day 5 there was a significant increase in responsiveness; this increase was of smaller magnitude than that evident at day 40. These results suggest a possible association between the fall in intratesticular levels of testosterone induced by unilateral cryptorchidism and the Leydig cell hypertrophy and hyper-responsiveness that occurs in the same testes. The implications with respect to altered Sertoli–Leydig cell interaction are discussed. J. Endocr. (1984) 102, 319–327

Effects of clodronate-containing liposomes on testicular macrophages and Leydig cells in vitro

1997 ◽  
Vol 155 (1) ◽  
pp. 87-92 ◽  
Author(s):  
DE Brigham ◽  
G Little ◽  
YO Lukyanenko ◽  
JC Hutson
Keyword(s):  
Direct Effect ◽  
Dose Response ◽  
Leydig Cell ◽  
Leydig Cells ◽  
Direct Effects ◽  
Testosterone Production ◽  
Identical Response ◽  
Testicular Macrophages

We undertook the present studies to determine if clodronate-containing liposomes have direct effects on Leydig cells. Macrophages and Leydig cells were isolated and maintained separately in culture. Following treatment with clodronate-containing liposomes, macrophages were killed in a dose-response fashion over a range of 5-200 microliters liposomes. By comparison, a 500 microliters dose was required to kill Leydig cells, but this was not dependent upon clodronate since liposomes containing buffer elicited an identical response. The concentration of testosterone in medium from Leydig cells treated with clodronate-containing liposomes was significantly reduced compared with untreated cells. However, we subsequently found that liposomes can adsorb testosterone. Therefore, testosterone production was determined at various times following removal of liposomes from Leydig cells, thereby circumventing this complication. It was found that testosterone production was not altered by liposomes under these conditions. Finally, free clodronate had no effect on testosterone production, even at doses representing the amount present within the 500 microliters dose of liposomes. In summary, clodronate-containing liposomes killed testicular macrophages at a far smaller dose than required to kill Leydig cells. Most importantly, neither liposomes no free clodronate had a direct effect on testosterone production. Thus, clodronate-containing liposomes represent a valuable tool to study Leydig cell-macrophage interactions.

scholarly journals Adrenocorticotropic Hormone Directly Stimulates Testosterone Production by the Fetal and Neonatal Mouse Testis

Endocrinology ◽  
2003 ◽  
Vol 144 (8) ◽  
pp. 3279-3284 ◽  
Author(s):  
P. J. O’Shaughnessy ◽  
L. M. Fleming ◽  
G. Jackson ◽  
U. Hochgeschwender ◽  
P. Reed ◽  
...  
Keyword(s):  
Leydig Cell ◽  
Leydig Cells ◽  
Cell Function ◽  
Expressed Sequence Tag ◽  
Acth Stimulation ◽  
Testosterone Production ◽  
Testicular Cells ◽  
Testicular Steroidogenesis ◽  
Fetal Leydig Cells

Abstract Adult Leydig cell steroidogenesis is dependent on LH but fetal Leydig cells can function independently of gonadotropin stimulation. To identify factors that may be involved in regulation of fetal Leydig cells expressed sequence tag libraries from fetal and adult testes were compared, and fetal-specific genes identified. The ACTH receptor [melanocortin type 2 receptor (Mc2r)] was identified within this fetal-specific group. Subsequent real-time PCR studies confirmed that Mc2r was expressed in the fetal testis at 100-fold higher levels than in the adult testis. Incubation of fetal or neonatal testes with ACTH in vitro stimulated testosterone production more than 10-fold, although ACTH had no effect on testes from animals aged 20 d or older. The steroidogenic response of fetal and neonatal testes to a maximally stimulating dose of human chorionic gonadotropin was similar to the response shown to ACTH. The ED50 for ACTH, measured in isolated fetal and neonatal testicular cells, was 5 × 10−10m and the lowest dose of ACTH eliciting a response was 2 × 10−11m. Circulating ACTH levels in fetal mice were around 8 × 10−11m. Neither α-MSH nor γ-MSH had any effect on androgen production in vitro at any age. Fetal testosterone levels were normal in mice that lack circulating ACTH (proopiomelanocortin-null) indicating that ACTH is not essential for fetal Leydig cell function. Results show that both LH and ACTH can regulate testicular steroidogenesis during fetal development in the mouse and suggest that fetal Leydig cells, but not adult Leydig cells, are sensitive to ACTH stimulation.

Stimulatory and inhibitory factors of Leydig cell steroidogenesis are secreted simultaneously by the rat seminiferous tubules and do not affect Leydig cell inhibin production in vitro

1994 ◽  
Vol 6 (6) ◽  
pp. 693 ◽  
Author(s):  
JR McFarlane ◽  
Kretser DM de ◽  
GP Risbridger
Keyword(s):  
Conditioned Medium ◽  
Leydig Cell ◽  
Leydig Cells ◽  
Seminiferous Tubules ◽  
Low Doses ◽  
Adult Rat ◽  
Testosterone Production ◽  
Significant Reversal ◽  
The Mean

The effect of conditioned medium from rat seminiferous tubules (at Stages VII-VIII and Stages IX-VI) cultured with or without follicle-stimulating hormone (FSH) on the production of testosterone and immunoactive inhibin by Leydig cells was examined. Low doses of conditioned medium from unstimulated tubules at Stages VII-VIII significantly (P < 0.05) increased the mean testosterone production to greater than 31 +/- 11% over that achieved with luteinizing hormone (LH) alone. At the highest dose, the conditioned medium significantly inhibited (P < 0.05) LH-stimulated testosterone production by 13 +/- 7%. Low doses of conditioned medium from unstimulated tubules at Stages IX-VI increased the mean testosterone production to 22 +/- 10%, whereas at higher doses, a significant reversal in the stimulation occurred although not to the same extent as that found with medium from tubules at Stages VII-VIII. Conditioned medium from FSH-stimulated tubules at Stages VII-VIII and Stages IX-VI, significantly increased testosterone production to 39 +/- 7% and 31 +/- 13% respectively. Immunoactive inhibin production by the Leydig cells remained unaffected by exposure to conditioned medium from FSH stimulated and unstimulated tubules at Stages VII-VIII and Stages IX-VI. The data demonstrate that tubule culture medium contains FSH-modulated activities which can specifically stimulate and inhibit testosterone synthesis by adult rat Leydig cells in vitro and therefore explains the contradictory reports in the literature.

scholarly journals Cyclooxygenases in Rat Leydig Cells: Effects of Luteinizing Hormone and Aging

Endocrinology ◽  
2007 ◽  
Vol 148 (2) ◽  
pp. 735-742 ◽  
Author(s):  
Haolin Chen ◽  
Lindi Luo ◽  
June Liu ◽  
Barry R. Zirkin
Keyword(s):  
Leydig Cell ◽  
Leydig Cells ◽  
Close Correlation ◽  
Dibutyryl Camp ◽  
Protein Levels ◽  
Testosterone Production ◽  
Age Related ◽  
The Relationship

Previous studies suggested that increased Leydig cell cyclooxygenase (COX)2 expression may be involved in the reduced testosterone production that characterizes aged Leydig cells. Our objective herein was to further elucidate the relationships among LH stimulation, Leydig cell COX2 and COX1 expression, aging, and testosterone production. Incubation of Leydig cells from young or aged rats with LH or dibutyryl cAMP resulted in increases in both intracellular COX2 protein expression and testosterone production. COX1 expression did not respond to LH or dibutyryl cAMP. Incubation of adult cells with a protein kinase A inhibitor suppressed the stimulatory effects of LH on COX2 and testosterone production. Short-term incubation of Leydig cells with TGF-α or IL-1β also increased COX2 protein levels; IGF-I had no effect. In vivo, LH also was found to stimulate both COX2 and testosterone, but not COX1. As reported previously, COX2 expression was greater in old than in young cells, and old Leydig cells responded to inhibition of COX2 in vitro with increased testosterone production. However, the effects of the COX2 inhibitors were not restricted to old cells; young Leydig cells also responded to COX2 inhibition with increased testosterone production. This and the observation that the incubation of young or old cells with LH resulted in increased COX2 and testosterone production in both cases suggests that the relationship between COX2 and testosterone production is not unique to aged Leydig cells. Moreover, the close correlation between increases in COX2 and testosterone in LH-stimulated young and aged Leydig cells is difficult to reconcile with the contention that the increased expression of COX2 in aged cells is responsible for age-related suppression of Leydig cell testosterone production.

Leptin inhibits basal but not gonadotrophin-stimulated testosterone production in the immature mouse and sheep testis

2008 ◽  
Vol 20 (4) ◽  
pp. 519 ◽  
Author(s):  
Muren Herrid ◽  
Yin Xia ◽  
Tim O'Shea ◽  
James R. McFarlane
Keyword(s):  
Leydig Cell ◽  
Developmental Stages ◽  
Physiological State ◽  
Inhibitory Effect ◽  
Slice Culture ◽  
Testosterone Production ◽  
Adult Mice ◽  
High Concentrations

The mechanisms whereby leptin regulates testosterone secretion are complex and are likely to involve actions at different levels of the hypothalamus–pituitary–gonadal axis. In the present study, the effect of leptin on testicular steroidogenesis at different developmental stages in mice and sheep was investigated. Testosterone data from testicular slice and Leydig cells of immature and adult mice testes demonstrated that the action of leptin in the regulation of steroidogenesis appears to be dependent on the developmental stage of the testis. Leptin biphasically modulates basal testosterone production in immature testicular slice cultures: at relatively low concentrations (6.25–12.5 ng mL–1) leptin exerts a significant inhibitory effect, but has less of an effect at very low (1.25 ng mL–1) or high concentrations (25 ng mL–1). However, leptin failed to modulate basal testosterone levels in Leydig cell preparations. In contrast with immature testes, leptin was unable to regulate either basal or human chorionic gonadotrophin (10 IU mL–1)-stimulated testosterone production in adult testicular slices or Leydig cell cultures. The age- and concentration-dependent regulation pattern was confirmed using sheep testicular slice culture. Leptin (1.56–25 ng mL–1) significantly inhibited basal testosterone production in the testis from birth to Day 21, but had no effect on Day 27 or older testes. However, the plasma and testicular concentrations of leptin and testosterone data in the ram indicate that such a regulatory effect of leptin on testis steroidogenesis in vitro is unable to efficiently influence testosterone concentrations in vivo. This does not exclude the possibility of a non-competitive mechanism of interaction between leptin and luteinising hormone to regulate testosterone production. Thus, we hypothesise that leptin is not an important independent regulator of testosterone concentration in the normal physiological state. The physiological significance and mechanism of leptin regulation of basal testosterone production are not known; further studies are required to elucidate these important issues.

scholarly journals Primary human testicular PDGFRα+ cells are multipotent and can be differentiated into cells with Leydig cell characteristics in vitro

2019 ◽  
Vol 34 (9) ◽  
pp. 1621-1631 ◽  
Author(s):  
J Eliveld ◽  
E A van den Berg ◽  
J V Chikhovskaya ◽  
S K M van Daalen ◽  
C M de Winter-Korver ◽  
...  
Keyword(s):  
Gene Expression ◽  
Leydig Cell ◽  
Leydig Cells ◽  
Culture Medium ◽  
Testicular Tissue ◽  
Interstitial Cells ◽  
Testosterone Levels ◽  
Testicular Interstitial Cells

Abstract STUDY QUESTION Is it possible to differentiate primary human testicular platelet-derived growth factor receptor alpha positive (PDGFRα+) cells into functional Leydig cells? SUMMARY ANSWER Although human testicular PDGFRα+ cells are multipotent and are capable of differentiating into steroidogenic cells with Leydig cell characteristics, they are not able to produce testosterone after differentiation. WHAT IS KNOWN ALREADY In rodents, stem Leydig cells (SLCs) that have been identified and isolated using the marker PDGFRα can give rise to adult testosterone-producing Leydig cells after appropriate differentiation in vitro. Although PDGFRα+ cells have also been identified in human testicular tissue, so far there is no evidence that these cells are true human SLCs that can differentiate into functional Leydig cells in vitro or in vivo. STUDY DESIGN, SIZE, DURATION We isolated testicular cells enriched for interstitial cells from frozen–thawed fragments of testicular tissue from four human donors. Depending on the obtained cell number, PDGFRα+-sorted cells of three to four donors were exposed to differentiation conditions in vitro to stimulate development into adipocytes, osteocytes, chondrocytes or into Leydig cells. We compared their cell characteristics with cells directly after sorting and cells in propagation conditions. To investigate their differentiation potential in vivo, PDGFRα+-sorted cells were transplanted in the testis of 12 luteinizing hormone receptor-knockout (LuRKO) mice of which 6 mice received immunosuppression treatment. An additional six mice did not receive cell transplantation and were used as a control. PARTICIPANTS/MATERIALS, SETTING, METHODS Human testicular interstitial cells were cultured to Passage 3 and FACS sorted for HLA-A,B,C+/CD34−/PDGFRα+. We examined their mesenchymal stromal cell (MSC) membrane protein expression by FACS analyses. Furthermore, we investigated lineage-specific staining and gene expression after MSC trilineage differentiation. For the differentiation into Leydig cells, PDGFRα+-sorted cells were cultured in either proliferation or differentiation medium for 28 days, after which they were stimulated either with or without hCG, forskolin or dbcAMP for 24 h to examine the increase in gene expression of steroidogenic enzymes using qPCR. In addition, testosterone, androstenedione and progesterone levels were measured in the culture medium. We also transplanted human PDGFRα+-sorted testicular interstitial cells into the testis of LuRKO mice. Serum was collected at several time points after transplantation, and testosterone was measured. Twenty weeks after transplantation testes were collected for histological examination. MAIN RESULTS AND THE ROLE OF CHANCE From primary cultured human testicular interstitial cells at Passage 3, we could obtain a population of HLA-A,B,C+/CD34−/PDGFRα+ cells by FACS. The sorted cells showed characteristics of MSC and were able to differentiate into adipocytes, chondrocytes and osteocytes. Upon directed differentiation into Leydig cells in vitro, we observed a significant increase in the expression of HSD3B2 and INSL3. After 24 h stimulation with forskolin or dbcAMP, a significantly increased expression of STAR and CYP11A1 was observed. The cells already expressed HSD17B3 and CYP17A1 before differentiation but the expression of these genes were not significantly increased after differentiation and stimulation. Testosterone levels could not be detected in the medium in any of the stimulation conditions, but after stimulation with forskolin or dbcAMP, androstenedione and progesterone were detected in culture medium. After transplantation of the human cells into the testes of LuRKO mice, no significant increase in serum testosterone levels was found compared to the controls. Also, no human cells were identified in the interstitium of mice testes 20 weeks after transplantation. LARGE SCALE DATA N/A LIMITATIONS, REASONS FOR CAUTION This study was performed using tissue from only four donors because of limitations in donor material. Because of the need of sufficient cell numbers, we first propagated cells to passage 3 before FACS of the desired cell population was performed. We cannot rule out this propagation of the cells resulted in loss of stem cell properties. WIDER IMPLICATIONS OF THE FINDINGS A lot of information on Leydig cell development is obtained from rodent studies, while the knowledge on human Leydig cell development is very limited. Our study shows that human testicular interstitial PDGFRα+ cells have different characteristics compared to rodent testicular PDGFRα+ cells in gene expression levels of steroidogenic enzymes and potential to differentiate in adult Leydig cells under comparable culture conditions. This emphasizes the need for confirming results from rodent studies in the human situation to be able to translate this knowledge to the human conditions, to eventually contribute to improvements of testosterone replacement therapies or establishing alternative cell therapies in the future, potentially based on SLCs. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by Amsterdam UMC, location AMC, Amsterdam, the Netherlands. All authors declare no competing interests.

Age-related changes in human Leydig cell status

2020 ◽  
Vol 35 (12) ◽  
pp. 2663-2676
Author(s):  
Valentina Mularoni ◽  
Valentina Esposito ◽  
Sara Di Persio ◽  
Elena Vicini ◽  
Gustavo Spadetta ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Sertoli Cell ◽  
Leydig Cell ◽  
Leydig Cells ◽  
Cell Number ◽  
Organ Donors ◽  
Steroidogenic Enzymes ◽  
Age Related ◽  
Age Range

Abstract STUDY QUESTION What are the consequences of ageing on human Leydig cell number and hormonal function? SUMMARY ANSWER Leydig cell number significantly decreases in parallel with INSL3 expression and Sertoli cell number in aged men, yet the in vitro Leydig cell androgenic potential does not appear to be compromised by advancing age. WHAT IS KNOWN ALREADY There is extensive evidence that ageing is accompanied by decline in serum testosterone levels, a general involution of testis morphology and reduced spermatogenic function. A few studies have previously addressed single features of the human aged testis phenotype one at a time, but mostly in tissue from patients with prostate cancer. STUDY DESIGN, SIZE, DURATION This comprehensive study examined testis morphology, Leydig cell and Sertoli cell number, steroidogenic enzyme expression, INSL3 expression and androgen secretion by testicular fragments in vitro. The majority of these endpoints were concomitantly evaluated in the same individuals that all displayed complete spermatogenesis. PARTICIPANTS/MATERIALS, SETTING, METHODS Testis biopsies were obtained from 15 heart beating organ donors (age range: 19–85 years) and 24 patients (age range: 19–45 years) with complete spermatogenesis. Leydig cells and Sertoli cells were counted following identification by immunohistochemical staining of specific cell markers. Gene expression analysis of INSL3 and steroidogenic enzymes was carried out by qRT-PCR. Secretion of 17-OH-progesterone, dehydroepiandrosterone, androstenedione and testosterone by in vitro cultured testis fragments was measured by LC-MS/MS. All endpoints were analysed in relation to age. MAIN RESULTS AND THE ROLE OF CHANCE Increasing age was negatively associated with Leydig cell number (R = −0.49; P &lt; 0.01) and concomitantly with the Sertoli cell population size (R= −0.55; P &lt; 0.001). A positive correlation (R = 0.57; P &lt; 0.001) between Sertoli cell and Leydig cell numbers was detected at all ages, indicating that somatic cell attrition is a relevant cellular manifestation of human testis status during ageing. INSL3 mRNA expression (R= −0.52; P &lt; 0.05) changed in parallel with Leydig cell number and age. Importantly, steroidogenic capacity of Leydig cells in cultured testis tissue fragments from young and old donors did not differ. Consistently, age did not influence the mRNA expression of steroidogenic enzymes. The described changes in Leydig cell phenotype with ageing are strengthened by the fact that the different age-related effects were mostly evaluated in tissue from the same men. LIMITATIONS, REASONS FOR CAUTION In vitro androgen production analysis could not be correlated with in vivo hormone values of the organ donors. In addition, the number of samples was relatively small and there was scarce information about the concomitant presence of potential confounding variables. WIDER IMPLICATIONS OF THE FINDINGS This study provides a novel insight into the effects of ageing on human Leydig cell status. The correlation between Leydig cell number and Sertoli cell number at any age implies a connection between these two cell types, which may be of particular relevance in understanding male reproductive disorders in the elderly. However aged Leydig cells do not lose their in vitro ability to produce androgens. Our data have implications in the understanding of the physiological role and regulation of intratesticular sex steroid levels during the complex process of ageing in humans. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by grants from Prin 2010 and 2017. The authors have no conflicts of interest. TRIAL REGISTRATION NUMBER N/A.

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