Quantifying the calcification of abdominal aorta and major side branches with deep learning.

AIM: To explore the possibility of a neural network-based method for quantifying calcifications of the abdominal aorta and its branches. MATERIALS AND METHODS: In total, 58 computed tomography (CT) angiography volumes were selected from a dataset of 609 to represent different stages of sclerosis. The ground truth segmentations of the abdominal aorta, coeliac trunk, superior mesenteric artery, renal arteries, common iliac arteries, and their calcifications were delineated manually. Two V-Net ensemble models were trained, one for segmenting arteries of interest and another for calcifications. The branches of interest were shortened algorithmically. The volumes of calcification were then evaluated from the arteries of interest. RESULTS: The results indicate that automatic detection is possible with a high correlation to the ground truth. The scores for the ensemble calcification model were dice score of 0.69 and volumetric similarity (VS) of 0.80 and for the arteries of interest segmentations: aorta: dice 0.96, VS 0.98; aortic branches: dice 0.74, VS 0.87; and common iliac arteries: dice 0.72, VS 0.91. CONCLUSIONS: The presented neural network model is the first to be capable of automatically segmenting, in addition to calcification, both the aorta and its branches from contrast-enhanced CT angiography. This technology shows promise in addressing limitations inherent in earlier methods that relied solely on plain CT.

Ethinylestradiol in combined hormonal contraceptive has a broader effect on serum proteome compared with estradiol valerate: a randomized controlled trial.

STUDY QUESTION: Does an estradiol-based combined oral contraceptive (COC) have a milder effect on the serum proteome than an ethinylestradiol (EE)-based COC or dienogest (DNG) only? SUMMARY ANSWER: The changes in serum proteome were multifold after the use of a synthetic EE-based COC compared to natural estrogen COC or progestin-only preparation. WHAT IS KNOWN ALREADY: EE-based COCs widely affect metabolism, inflammation, hepatic protein synthesis and blood coagulation. Studies comparing serum proteomes after the use of COCs containing EE and natural estrogens are lacking. STUDY DESIGN, SIZE, DURATION: This was a spin-off from a randomized, controlled, two-center clinical trial. Women (n = 59) were randomized to use either EE + DNG, estradiol valerate (EV) + DNG or DNG only continuously for 9 weeks. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants were healthy, young, white volunteer women. Serum samples were collected before and after 9 weeks of hormonal exposure. Samples from 44 women were available for analysis (EE + DNG n = 14, EV + DNG n = 16 and DNG only n = 14). Serum proteins were analyzed by quantitative, discovery-type label-free proteomics. MAIN RESULTS AND THE ROLE OF CHANCE: Altogether, 446 proteins/protein families with two or more unique peptides were detected and quantified. The number of proteins/families that altered over the 9-week period within the study groups was 121 for EE + DNG and 5 for EV + DNG, while no changes were detected for DNG only. When alterations were compared between the groups, significant differences were detected for 63 proteins/protein families, of which 58 were between the EE + DNG and EV + DNG groups. The most affected functions during the use of EE + DNG were the complement system, acute phase response signaling, metabolism and the coagulation system. The results were validated by fetuin-B and cortisol-binding globulin ELISA and sex hormone-binding globulin immunoassay. LARGE SCALE DATA: Data are available via ProteomeXchange with identifiers PXD033617 (low abundance fraction) and PXD033618 (high abundance fraction). LIMITATIONS, REASONS FOR CAUTION: The power analysis of the trial was not based on the proteomic analysis of this spin-off study. In the future, targeted proteomic analysis with samples from another trial should be carried out in order to confirm the results. WIDER IMPLICATIONS OF THE FINDINGS: The EE-based COC exerted a broader effect on the serum proteome than the EV-based COC or the DNG-only preparation. These results demonstrate that the effects of EE in COCs go far beyond the established endpoint markers of estrogen action, while the EV combination is closer to the progestin-only preparation. The study indicates that EV could provide a preferable option to EE in COCs in the future and signals a need for further studies comparing the clinical health outcomes of COCs containing EE and natural estrogens. STUDY FUNDING/COMPETING INTEREST(S): Funding for this researcher-initiated study was obtained from the Helsinki University Hospital research funds, the Hospital District of Helsinki and Uusimaa, the Sigrid Juselius Foundation, the Academy of Finland, the Finnish Medical Association, the University of Oulu Graduate School, the Emil Aaltonen Foundation, the Swedish Cultural Foundation in Finland, the Novo Nordisk Foundation, Orion Research Foundation and the Northern Ostrobothnia Regional Fund. The funders had no role in study design, data collection and analysis, publishing decisions or manuscript preparation. T.P. has received honoraria for lectures, consultations and research grants from Exeltis, Gedeon Richter, MSD, Merck, Pfizer, Roche, Stragen and Mithra Pharmaceuticals. O.H. occasionally serves on advisory boards for Bayer AG and Gedeon Richter and has designed and lectured at educational events for these companies. The other authors have nothing to disclose. O.H. occasionally serves on advisory boards for Bayer AG and Gedeon Richter and has designed and lectured at educational events for these companies. The other authors have nothing to disclose. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov NCT02352090. TRIAL REGISTRATION DATE: 27 January 2015. DATE OF FIRST PATIENT'S ENROLMENT: 1 April 2015.

Automatic detection of Crohn's disease using quantified motility in magnetic resonance enterography: initial experiences.

AIM: To report initial experiences of automatic detection of Crohn's disease (CD) using quantified motility in magnetic resonance enterography (MRE). MATERIALS AND METHODS: From 302 patients, three datasets with roughly equal proportions of CD and non-CD cases with various illnesses were drawn for testing and neural network training and validation. All datasets had unique MRE parameter configurations and were performed in free breathing. Nine neural networks were devised for automatic generation of three different regions of interests (ROI): small bowel, all bowel, and non-bowel. Additionally, a full-image ROI was tested. The motility in an MRE series was quantified via a registration procedure, which, accompanied with given ROIs, resulted in three motility indices (MI). A subset of the indices was used as an input for a binary logistic regression classifier, which predicted whether the MRE series represented CD. RESULTS: The highest mean area under the curve (AUC) score, 0.78, was reached using the full-image ROI and with the dataset with the highest cine series length. The best AUC scores for the other two datasets were only 0.54 and 0.49. CONCLUSION: The automatic system was able to detect CD in the group of MRE studies with lower temporal resolution and longer cine series showing potential in primary bowel disorder diagnostics. Larger ROI selections and utilising all available cine series for motility registration yielded slight performance improvements.

Endometrial stromal fibroblasts from women with polycystic ovary syndrome have impaired progesterone-mediated decidualization, aberrant cytokine profiles and promote enhanced immune cell migration in vitro.

STUDY QUESTION: Do endometrial stromal fibroblasts (eSF) in women with polycystic ovary syndrome (PCOS) (eSFpcos) exhibit altered estrogen and/or progesterone (P4) responses, which may explain some of the adverse reproductive outcomes and endometrial pathologies in these women? SUMMARY ANSWER: In vitro, eSF from women with PCOS exhibit an aberrant decidualization response and concomitant changes in pro-inflammatory cytokine, chemokine and matrix metalloproteinase (MMP) release and immune cell chemoattraction. In vivo these aberrations may result in suboptimal implantation and predisposition to endometrial cancer. WHAT IS KNOWN ALREADY: The endometrium in women with PCOS has several abnormalities including progesterone (P4) resistance at the gene expression level, likely contributing to subfertility, pregnancy complications and increased endometrial cancer risk in PCOS women. STUDY DESIGN, SIZE, DURATION: Prospective, university-based, case-control, in vitro study. PARTICIPANTS/MATERIALS, SETTING, METHODS: Cultures of eSFPCOS (n = 12, Rotterdam and NIH criteria) and eSFControl (Ctrl) (n = 6, regular cycle length, no signs of hyperandrogenism) were treated with vehicle, estradiol (E2, 10 nM) or E2P4 (10 nM/1 µM) for 14 days. Progesterone receptor (PGR) mRNA was assessed with quantitative real-time PCR (qRT-PCR) and eSF decidualization was confirmed by insulin-like growth factor-binding protein-1 (IGFBP-1) transcript and protein expression. Fractalkine (CX3CL1), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL) 6, 8 and 11, macrophage chemoattractant protein (MCP) 1 and 3, CCL5 (RANTES) and MMPs (MMP1, 2, 3, 7, 9, 10 and 12) were measured in conditioned media by Luminex multiplex assays, and chemotactic activity of the conditioned media was tested in a migration assay using CD14+ monocyte and CD4+ T-cell migration assay. Effects of IL-6 (0.02, 0.2, 2 or 20 ng/ml) or IL-8 (0.04, 0.4, 4, or 40 ng/ml) or combination (0.2 ng/ml IL-6 and 4.0 ng/ml IL-8) on 14-d decidualization were also tested. ANOVA with pre-planned contrasts was used for statistical analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Hormonal challenge with E2P4 to induce decidualization revealed two distinct subsets of eSFPCOS. Eight eSFPCOS (dPCOS) and all eSFCtrl (dCtrl) cultures showed a normal decidualization response to E2P4 as determined by morphology and IGFBP-1 secretion. However, 4 eSFPCOS cultures showed blunted decidualization (ndPCOS) in morphological assessment and low IGFBP-1 levels even though all three groups exhibited normal estrogen-mediated increase in PGR expression. Interestingly dPCOS had decreased IL-6 and GM-SCF secretion compared with dCtrl, whereas the ndPCOS cultures showed increased IL-6 and 8, MCP1, RANTES and GM-CSF secretion at base-line and/or in response to E2 or E2P4 compared with dCtrl and/or dPCOS. Furthermore, even though PGR expression was similar in all three groups, P4 inhibition of MMP secretion was attenuated in ndPCOS resulting in higher MMP2 and 3 levels. The conditioned media from ndPCOS had increased chemoattractic activity compared with dCtrl and dPCOS media. Exogenously added IL-6 and/or 8 did not inhibit decidualization in eSFCtrl indicating that high levels of these cytokines in ndPCOS samples were not likely a cause for the aberrant decidualization. LIMITATIONS, REASONS FOR CAUTION: This is an in vitro study with a small sample size, utilizing stromal cell cultures from proliferative and secretory phase endometrium. The effect of PCOS on endometrial epithelium, another major histoarchitectural cell compartment of the endometrium, was not evaluated and should be considered in future studies. Furthermore, results obtained should also be confirmed in a larger data set and with mid/late secretory phase in vivo samples and models. WIDER IMPLICATIONS OF THE FINDINGS: The alterations seen in ndPCOS may contribute to endometrial dysfunction, subfertility and pregnancy complications in PCOS women. The results emphasize the importance of understanding immune responses related to the implantation process and normal endometrial homeostasis in women with PCOS. STUDY FUNDING/COMPETING INTERESTS: Sigrid Juselius Foundation, Academy of Finland, Finnish Medical Foundation, Orion-Farmos Research Foundation (to T.T.P.), the NIH Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) U54HD 055764-07 Specialized Cooperative Centers Program in Reproduction and Infertility Research (to L.C.G.), the NICHD the Ruth L. Kirschstein National Research Service Awards grant 1F32HD074423-03 (to J.C.C.). The authors have no competing interests.

Imaging of blood flow in cerebral arteries with dynamic helical computed tomography angiography (DHCTA) using a 64-row CT scanner.

BACKGROUND: Cerebral computed tomography angiography (CTA) depicts a structural image of intracranial arteries without providing much time-resolved information on blood flow dynamics. Current CT technology allows obtaining of rapidly repeated helical scans during the arterial contrast filling phase after an intravenous contrast injection. PURPOSE: To report our experience on dynamic CT imaging in determining the direction of contrast filling within proximal intracranial arteries of operated cerebral artery aneurysm patients. Such dynamic information can help detect vascular occlusion or severe spasm. The method is here referred to as dynamic helical CT angiography (DHCTA). MATERIAL AND METHODS: We retrospectively collected image and related technical data for 23 patients who underwent DHCTA and CTA during their first postoperative day after cerebral artery aneurysm surgery. For DHCTA, we had helically scanned a 4-cm tissue volume three times in succession with a 64-row CT scanner at intervals of 2.6 s during arterial contrast filling after an intravenous contrast injection. We assessed how well DHCTA succeeded in demonstrating the direction of contrast filling in the proximal intracranial arteries, evaluated clinically relevant structural information provided by DHCTA and CTA, and compared radiation doses for the two methods. RESULTS: For 21 patients, DHCTA outlined the direction of contrast filling in proximal intracranial arteries. As to arterial spasm and residual filling of the operated aneurysm, CTA and DHCTA gave similar information. Radiation doses were higher (P<0.000001) for DHCTA than for CTA at 120 kV tube voltage. At 100 kV, the difference was smaller, but doses for DHCTA still exceeded (P<0.05) those for CTA. CONCLUSION: DHCTA gave dynamic information unobtainable with CTA and could prove useful in selected clinical settings.

Rapid protection of rat spermatogenic stem cells against procarbazine by treatment with a gonadotropin-releasing hormone antagonist (Nal-Glu) and an antiandrogen (flutamide).

GnRH antagonist (Nal-Glu) treatment combined with the antiandrogen flutamide was used to suppress rat spermatogenesis to achieve protection of spermatogonial stem cells against the anticancer drug procarbazine. Daily injections with Nal-Glu alone suppressed spermatogenesis in a dose-responsive manner. However, it was necessary to combine Nal-Glu (600 micrograms/kg.day) with flutamide at 20 mg/kg.day to decrease testicular weight in 2 weeks to less than 0.6 g, a level previously demonstrated sufficient to protect stem cells in our model system. The Nal-Glu-flutamide pretreatment suppressed serum gonadotropin levels and intratesticular testosterone levels (6% of control) and action, resulting in a reversible decrease in the number of late spermatids to 1% of control levels. When rats were given Nal-Glu-flutamide for 2 weeks before a 250 mg/kg dose of procarbazine, recovery of spermatogenesis, as measured by testis weight, testicular sperm head counts, and repopulation indexes, was significantly better than in control rats (no hormonal pretreatment). The protection achieved with Nal-Glu-flutamide was better than that achieved with 2 weeks of testosterone and estradiol treatment. The present results show that Nal-Glu-flutamide protects spermatogonial stem cells against procarbazine and suggest a method of hormonal pretreatment to achieve rapid and efficient protection of spermatogenesis in humans.

Protection against procarbazine-induced testicular damage by GnRH-agonist and antiandrogen treatment in the rat.

Suppression of spermatogenesis in LBNF1 rats by treatment with the GnRH agonist Zoladex combined with the antiandrogen flutamide was evaluated in order to rapidly achieve protection of spermatogenic stem cells against procarbazine with clinically used drugs. Zoladex-flutamide treatment required 3 weeks to suppress the completion of spermatogenesis; only a small degree of suppression was observed with Zoladex alone. The suppression of spermatogenesis was reversible. In rats pretreated for 3 weeks with Zoladex-flutamide, the recovery of spermatogenesis at 9 weeks after a single injection of procarbazine as measured by testis weight, testicular sperm head counts, or a histological end point was significantly better than without hormonal pretreatment. Thus Zoladex-flutamide treatment enhanced the recovery of spermatogenesis from stem spermatogonia after procarbazine treatment in the rat and might be applicable to protect spermatogenesis in patients undergoing chemotherapy for cancer.

In vitro, follicle-stimulating hormone prevents apoptosis and stimulates deoxyribonucleic acid synthesis in the rat seminiferous epithelium in a stage-specific fashion.

The effects of FSH on stage-specific apoptosis and DNA synthesis in the adult rat seminiferous epithelium were studied in vitro. Seminiferous tubular segments from stages I, V, VIIa, and VIII-IX were cultured for 24, 48, and 72 h in different concentrations of FSH. Apoptotic cells were detected by in situ end labeling of DNA strands and quantified from squash preparations. After 48 h of culture, a FSH concentration of 2 ng/ml prevented apoptosis of early (steps 1-3) spermatids. In stage VIII-IX tubules cultured for 72 h, FSH decreased the apoptosis of pachytene spermatocytes. An apoptotic type of cell death of germ cells was confirmed by DNA laddering, electron microscopy, supravital acridine orange staining, and phase contrast microscopy of unstained living cells. The effects of FSH on stage-specific DNA synthesis were studied using the same culture system. FSH increased [3H]thymidine incorporation specifically at stages I and VIII-IX, and autoradiography confirmed stimulation of mitotic and meiotic DNA synthesis in type B spermatogonia and preleptotene spermatocytes, respectively. Increased thymidine incorporation also suggested that FSH stimulated DNA synthesis of type A and intermediate spermatogonia. Most effects exerted by FSH were seen in stages containing high levels of FSH receptors and FSH-stimulated cAMP production. In conclusion, the results suggest that FSH, probably acting via Sertoli cells, has a regulatory function in spermatogenic apoptosis and DNA synthesis in stages previously demonstrated to be preferentially dependent on FSH stimulation.

Suppression of mouse spermatogenesis by a gonadotropin-releasing hormone antagonist and antiandrogen: failure to protect against radiation-induced gonadal damage.

A combined GnRH antagonist (Nal-Glu) and antiandrogen (flutamide) treatment was used to suppress mouse spermatogenesis in an attempt to enhance recovery from stem cells after irradiation, as observed previously in the rat. Two weeks of treatment suppressed the intratesticular testosterone concentration to 10% of the control value, decreased testicular weight to 16% of the control value, and sperm count 2600-fold. Suppression was at least as great as that produced in the rat by Nal-Glu-flutamide. Testicular weights, sperm counts, and histology were indistinguishable from those in normal controls 45 days after the end of the treatment. Despite the suppression of spermatogenesis, the treatment did not enhance recovery of spermatogenesis after damage produced by a 10-Gray dose of radiation; 45 days after irradiation, testicular weight, sperm head counts, and repopulation indexes were as low as in the mice that received no hormone treatment. In contrast to the situation in the rat, after irradiation of mice, almost no A spermatogonia were found in the 80% non-repopulating tubules, indicating that nearly all A spermatogonia remaining after irradiation were capable of differentiation. This absence of A spermatogonia that fail to differentiate in the mouse is proposed as the reason for failure to protect against radiation-induced gonadal damage by hormone treatment.

Effects of adenosine analog PIA (n-phenylisopropyladenosine) on FSH-stimulated cyclic AMP (cAMP) production in the rat seminiferous epithelium.

In rat seminiferous epithelium, FSH-stimulated cAMP production is cyclically modulated by spermatogenic cells and is highest in stages XIV-V and lowest in stages VII-VIII of the epithelial cycle. Adenosine has been proposed to be an inhibitory paracrine molecule in Sertoli cells. In this paper the effect of adenosine analog n-phenylisopropyladenosine (PIA) on FSH-stimulated cAMP production was studied in staged rat seminiferous tubules. In low responsive stages VII-VIII of the cycle, 100 nM and 10 microM PIA inhibited FSH-stimulated cAMP production by 24% and 28%, respectively. To study whether PIA effect is mediated through Gi-protein, pertussis toxin (PT) pretreatment was used to block the Gi-protein. PT pretreatments of 3 or 18 h caused 42% or 16% elevation in FSH-stimulated cAMP production, respectively. PIA blocked the stimulation caused by PT pretreatment. At 38 days post irradiation, when spermatocytes and round spermatids were decreased in number, in stages VII-VIII of the cycle the inhibitory effect of PIA was abolished. In high responsive stages XIV-V of the cycle, 100 nM PIA stimulated cAMP production by 27%, while 10 microM PIA had no effect. At 38 days post irradiation FSH response was decreased by 19% when compared to non-irradiated level, and PIA stimulated FSH-stimulated cAMP production by 22%. The results suggest that there are stage-specific mechanisms for adenosine-dependent regulation of FSH-stimulated cAMP production in the rat seminiferous epithelium. Advanced spermatogenic cells seem to maintain the mechanisms that include PIA-mediated inhibition of FSH response. Other mechanisms than PT-sensitive Gi-protein seem to be involved in the inhibition.

Stage- and cell-specific expression of the ornithine decarboxylase gene during rat and mouse spermatogenesis.

Ornithine decarboxylase (ODC) is an enzyme that has been shown to be induced in the growth, differentiation and proliferation of cells. We have used a cDNA probe to determine ODC mRNA levels in different stages of the cycle of rat and mouse seminiferous epithelium. For Northern and slot-blot hybridizations, RNA was isolated from microdissected staged seminiferous tubules. Cell-specific localization of ODC mRNA was studied by in situ hybridization. In the rat, in situ hybridization showed increasing mRNA levels during prophase of meiosis with the highest mRNA levels seen in late pachytene spermatocytes and step 3-5 spermatids. In the mouse, the mRNA levels increased in a similar fashion and the highest mRNA levels were found in step 1-8 spermatids. In the rat, Northern blot hybridizations revealed three molecular sizes of ODC mRNA: 2.2, 2.7 and 1.6 kb. The levels of all molecular sizes were highest in stages VII-VIII, and the lowest mRNA levels were seen in stage I of the seminiferous epithelial cycle. The level of the 2.2 kb transcript was low during stages XIII-I. In the mouse, the Northern blot hybridizations also showed three molecular sizes of ODC mRNA: 2.2 and 2.7 kb and very low levels of 1.6 kb transcript. The levels of the transcripts were steady throughout the cycle. In the mouse, the 2.2 kb transcript was more abundant than the 2.7 kb transcript indicating a species difference between rat and mouse in the usage of the two polyadenylation signals within the ODC gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Hormone pretreatment enhances recovery of spermatogenesis in rats after neutron irradiation.

Previous studies showed that a 6-week pretreatment of rats with testosterone plus estradiol enhanced the recovery of spermatogenesis 9 weeks after gamma irradiation, resulting in a dose-modifying factor (DMF) of about 2. To test whether the effect of the hormone treatment was mediated through changes in oxygen tension, thiol levels or DNA repair, we irradiated the testes of rats with neutrons, which depend less on these factors than does low-LET radiation for their cytotoxic action. Control rats and rats treated with testosterone plus estradiol were irradiated with 0.7-2.7 Gy of cyclotron-generated high-energy neutrons. The recovery of spermatogenesis was assessed 9 weeks after irradiation by testis weights, sperm counts and the tubule repopulation indices. Greater recovery of spermatogenesis was observed for all end points, with a DMF of about 2 for rats treated with testosterone plus estradiol compared to the irradiated, cholesterol-treated rats. The equal protection factors for neutrons and gamma rays indicate that oxygen, thiols and repair of DNA damage are unlikely to be involved in the protective effect of the hormone treatment.

Hormone treatment after irradiation stimulates recovery of rat spermatogenesis from surviving spermatogonia.

The possibility of stimulating the recovery of spermatogenesis after irradiation using hormone treatment was tested in LBNF, rats. At 10 weeks after irradiation with 3.5 Gy, the percentage of tubules showing recovery of spermatogenesis (repopulation index) was 37% in rats that received no hormone treatment. GnRH agonist (GnRH-Ag) treatment with Zoladex or continuous treatment with testosterone markedly stimulated the recovery of spermatogenesis. When GnRH-Ag treatment was started immediately after 3.5-Gy irradiation and maintained for 10 weeks, the repopulation index was 91%. When an additional 6.5 weeks without further treatment was allowed between the 10-week GnRH treatment and killing the rats, the repopulation index recovered to 100% and sperm counts to 83 x 10(6). These sperm counts were more than 100-fold higher than those in rats not given hormone treatment and 50% of normal nonirradiated control levels. GnRH-Ag for 10 weeks also stimulated spermatogenic recovery in rats irradiated with 6 Gy, even when the start of treatment was delayed until 18 weeks after irradiation. Without GnRH-Ag, the repopulation index was 0, but in GnRH-Ag-treated rats it was 14.5%. Since all of the hormone treatments suppress intratesticular testosterone, high levels of testosterone may be inhibiting differentiation and their suppression may stimulate recovery. Even though the exact mechanism is not yet known, this method may still be applicable for clinical use to activate spermatogenesis in patients rendered azoospermic by irradiation or possibly by other cytotoxic treatments.

Hormonal protection from cyclophosphamide-induced inactivation of rat stem spermatogonia.

Studies of protection of testicular function from cyclophosphamide with hormonal pretreatment have been limited by the lack of a convenient model for cyclophosphamide-induced inactivation of stem spermatogonia. In the rat, the mortality from cyclophosphamide had prevented the administration of sufficient dosages to produce detectible damage to stem spermatogonia. To overcome this problem, we used bone marrow transplantation and sodium 2-mercaptoethanesulfonate (Mesna) treatment to raise the lethal dose for 50% of the animals (LD50) for cyclophosphamide from 275 to > 400 mg/kg body weight. In addition we used irradiation, 2 weeks prior to injection of cyclophosphamide, to greatly enhance the measured toxicity of cyclophosphamide towards stem spermatogonia. Whereas sperm counts at 9 weeks after a 300 mg/kg cyclophosphamide dose were reduced by only a factor of 1.6 without prior irradiation, they were reduced by a factor of 60 when 2.5 Gy of irradiation had been given. Dramatic protection against this toxicity was produced by hormone treatment with a gonadotropin-releasing hormone (GnRH) antagonist (Nal-Glu) and an antiandrogen (flutamide) following the radiation but prior to cyclophosphamide. This hormone treatment did not modify the stem cell toxicity of the radiation and it therefore must be protecting stem cells against cyclophosphamide-induced damage. Because GnRH antagonist-antiandrogen treatment can protect stem spermatogonial survival and/or function in the rat from cyclophosphamide-induced damage, if the same principles are applicable in human, hormonal pretreatment should be useful for preventing the prolonged azoospermia caused by chemotherapy with cyclophosphamide-containing protocols.

Mechanism of protection of rat spermatogenesis by hormonal pretreatment: stimulation of spermatogonial differentiation after irradiation.

Pretreatment of rats with hormones that suppress testosterone levels and sperm production enhances the recovery of spermatogenesis from stem cells after a cytotoxic insult. It is not known whether the enhanced recovery results from an increase in the numbers of surviving stem cells or whether their ability to differentiate is enhanced. In this study, untreated rats and rats pretreated with testosterone plus estradiol-17beta (T + E) were irradiated with 3.5 or 6 Gy, and the recovery of spermatogenesis from surviving stem cells was assessed at 6, 10, and 20 weeks after irradiation. T + E pretreatment did not significantly affect the numbers of A spermatogonia remaining in the tubules at 6 weeks after irradiation. In rats that were given irradiation alone, spermatogenesis steadily declined after 6 weeks because the stem cells lost their ability to differentiate. However, when rats were treated with T + E before irradiation, this decline was prevented, and in fact, at least at the lower dose of radiation, there was a progressive recovery of spermatogenesis. Given the similar spermatogonial counts at 6 weeks after irradiation in the irradiated-only and T + E-treated, irradiated rats, the hormone treatment appears not to protect stem cells from being killed by the cytotoxic agent. Rather, the later enhancement of spermatogenic recovery results from prevention of an injury-induced change in spermatogonia or in their environment, which would have otherwise resulted in failure of spermatogonial differentiation.

Basal and FSH-stimulated steady state levels of SGP-2, alpha 2-macroglobulin, and testibumin in culture media of rat seminiferous tubules at defined stages of the epithelial cycle.

Production of several proteins by rat Sertoli cells is dependent on the stage of the cycle of the seminiferous epithelium. The authors have determined steady state levels and follicle-stimulating hormone responsiveness of three Sertoli cell products in culture media of rat seminiferous tubule segments at different stages of the epithelial cycle: SGP-2 (sulfated glycoprotein-2), alpha 2-macroglobulin, and testibumin. Basal SGP-2 levels were twofold higher in stages VII through VIII compared with stages XIII to I to VI (P less than 0.05). Highest basal alpha 2-macroglobulin levels were found in stages II through VIII; this was about 35% greater than in stages XIII through I of the cycle (P less than 0.05). Basal testibumin levels were twofold higher in stages II through VI compared with stages IX through XII of the cycle. Follicle-stimulating hormone had no effect on SGP-2, but by contrast it (50 mg/L) increased the level of alpha 2-macroglobulin significantly (P less than 0.05) in stages XIII through I. Follicle-stimulating hormone treatment (10 mg/L) elevated testibumin levels at each stage-pool by about 40% (P less than 0.05). The current results using staged tubular segments in vitro demonstrate cyclic basal steady-state levels of the three proteins along the seminiferous tubules and follicle-stimulating hormone regulation of alpha 2-macroglobulin and testibumin.

DNA-flow cytometry of defined stages of rat seminiferous epithelium: effects of 3 Gy of high-energy X-irradiation.

Testes of adult Sprague-Dawley rats were irradiated locally by 3 Gy of 4 MeV X-rays produced by a linear accelerator. This type and dose of radiation gives an even distribution through the testis and selectively kills the proliferating spermatogonia. The seminiferous tubular cells were quantified by DNA flow cytometry at defined stages of the epithelial cycle at 7, 17, 22, 38, 52, and 80 days after irradiation. The flow cytometric technique was modified by using frozen instead of fresh samples. Freezing did not alter cell numbers when compared with fresh samples. At 7 days post-irradiation no significant changes were observed in any cell population by DNA flow cytometry, whereas histological analysis revealed a reduction in intermediate and type B spermatogonia. At 17 and 22 days post-irradiation, the number of cells at meiotic prophase (4C) was decreased, particularly in stages II-V of the cycle. In stages VII-VIII, cell numbers were 40 and 31%, and in stages IX-XIII, 24 and 43% of that in non-irradiated controls at 17 and 22 days, respectively. At 38 days after irradiation, both 4C and 1C (haploid) cells were decreased in number. The 4C cells were reduced to 24, 17, and 13% of that in non-irradiated controls in stages II-V, VII-VIII, and IX-XIII of the cycle, respectively. The corresponding numbers of 1C cells were 5, 17, and 4%. At 52 days after irradiation, 1C cells had declined to 38 and 19% of control values in stages II-V and IX-XIII, respectively. In stages II-V, 1C' cells (haploid cells with condensed nuclei) declined to 28% of controls at 52 days. The present data provide a quantitative basis for the use of X-ray-irradiated rat testes as a model system in experiments pursuing interactions between Sertoli cells and spermatogenic cells.

Cellular regulation of follicle-stimulating hormone (FSH) binding in rat seminiferous tubules.

Stage-specific binding of follicle-stimulating hormone (FSH) was measured in rat seminiferous tubules. The binding in single-point assays was over 3-fold higher (P less than 0.05) in stages XIII to I than in stages VI to VII of the epithelial cycle. No difference was found between the equilibrium association constants (Ka) of FSH binding in stages XIV to IV (10 +/- 1.9 X 10(9) 1/mol) and VII to VIII (9.2 +/- 0.6 X 10(9) 1/mol, mean +/- SEM, n = 5). In another experiment, the testes were dosed locally with 3 Gy of 4 MV x-irradiation to selectively lower the number of spermatogonia. After irradiation, FSH binding in staged seminiferous tubule segments was measured when the desired types of spermatogenic cells were reduced in number. Seven days after irradiation when differentiating spermatogonia and preleptotene spermatocytes were reduced in number, FSH binding was decreased in all stages of the cycle, but the cyclic variation remained. Seventeen days after irradiation when intermediate and type B spermatogonia and spermatocytes up to diplotene of stage XIII showed low numbers, FSH binding was decreased in all stages of the cycle and the stage-dependent variation disappeared. At 38 days when pachytene spermatocytes and early spermatids were reduced in number, similar results were found. But at 52 days postirradiation when all spermatids were low in number, FSH binding was slightly elevated compared with days 17 and 38. There were no significant differences in serum FSH or LH levels between irradiated and non-irradiated animals. These findings suggest that all spermatogenic cell types may stimulate FSH binding in the Sertoli cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Stage-specific expression of nucleoprotein mRNAs during rat and mouse spermiogenesis.

The expression of mRNAs for a transition protein (TP1) and two variants of protamines (P1 and P2) during rat and mouse spermiogenesis was investigated using cDNA hybridization techniques. Slot-blot analyses from 1-mm segments of seminiferous tubules and in situ hybridization from testis sections showed that the levels of mRNA for TP1 increased in step-7 round spermatids at substage VIIb of the seminiferous epithelial cycle, earlier than that of P1 and P2 at substage VIIc. The mRNA levels of all transcripts remained high during steps 8-13 in both species. In the rat, the mRNA of TP1 disappeared during step 14 between substages XIVa and XIVb. The P1 mRNA levels decreased during steps 15-16 (stages I-III) and the P2 mRNA during step 15 (stage I). In the mouse, TP1 mRNA disappeared during step 13 (stage I). The P1 mRNA level decreased before P2 in step 14 (stage II), whereas P2 was detected up to step 15 (stage V). Northern-blot analyses with all three cDNA probes revealed two sizes of mRNA and their stage-specific expression. The shorter transcripts appeared later than the longer ones, at the steps of spermiogenesis where translation is known to begin. The results suggest that transcription of TP1, P1, and P2 mRNAs starts at specifically defined times during spermiogenesis and that the temporal translational regulation of these mRNAs is different.

Magnetic resonance imaging of acute scrotum.

BACKGROUND AND AIMS: The aim of the pilot study was to evaluate the feasibility of dynamic contrast enhanced (CE)-magnetic resonance imaging (MRI) in the detection of testicular ischemia and its ability to differentiate testicle torsion from other causes of acute scrotum. MATERIAL AND METHODS: Seventeen boys or young men with an acute scrotum were included in the prospective study during the time period from October 2001 to December 2005. The median age of the patients was 16,4 (7-44) years. The duration of the symptoms preceding the MRI study varied from six hours to 30 days. The study protocol included physical examination by a surgeon, laboratory tests and Doppler ultrasound (DUS) and finally testicles were imaged by using a 1,5 T MRI scanner; T1-weighted and diffusion weighted images were produced. The gadolinium uptake, reported as the region of interest (ROI) perfusion values and presented as curves, was compared between the affected and contralateral testicle. In testicles with normal blood circulation the ROI values increased during the imaging time. Nine patients were operated on, because the spermatic cord torsion could not be excluded by clinical or DUS findings. RESULTS AND CONCLUSIONS: All the normal testicles gave increasing ROI values meanwhile all three testicles with torsion gave constantly low values referring to no perfusion. Other causes of acute scrotum, such as epididymitis and torsion of testicular appendage seemed to be related with normal perfusion. Dynamic CE-MRI seems to show reliably ischemia of testicle and thus it may be helpful in selecting patients with acute scrotum for urgent operation.