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.

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.

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.

Mice with a deletion in the first intron of the Col1a1 gene develop dissection and rupture of aorta in the absence of aneurysms: high-resolution magnetic resonance imaging, at 4.7 T, of the aorta and cerebral arteries.

Deletion of the majority of the first intron of the Col1a1 gene in mice leads to decreased type I collagen synthesis and content in the aortic wall. In 54% of cases, mice homozygous for the Col1a1 mutation die of thoracic hemorrhage by the age of 18 months. It is unknown whether the fatal bleeding results from an acute dissection of the aortic wall or a gradually developing dilatation of the medial layer prior to rupture. We optimized high-resolution MRI methods using a 4.7 T MR scanner to obtain in vivo images of the entire mouse aorta. The MR images were acquired in three imaging planes using gradient echo, spin echo, and spin echo with inversion recovery pulse sequences with a maximum in-plane resolution of 68 x 68 microm and acquisition times less than 10 min. In five Col1a1 mutated mice aged 16 months, the MR images showed no signs of aneurysmal dilatation, wall defects, or former dissection, suggesting that the mechanism for aortic rupture is an acute dissection of the aortic medial layer. Cerebral arteries were imaged using a three-dimensional time of fight pulse sequence. The resolution of 73 x 73 x 94 microm showed normal cerebral arteries. Histology showed a 22% thinner cerebral artery wall in Col1a1 mutated mice.

Magnetic resonance imaging of experimental testicular torsion.

We investigated the feasibility of contrast enhanced (CE)-dynamic magnetic resonance imaging (MRI) for the detection of testicular torsion induced hypoperfusion in an experimental rat model. Adult Sprague-Dawley rats were subjected to unilateral testicular torsion of 360 or 720 degrees. After 1 h, the tail veins of the anaesthetized rats were cannulated and T2 -, diffusion-weighted and T1-weighted CE-dynamic MRI were subsequently performed by a 1.5 T MRI scanner. On apparent diffusion coefficient (ADC) images, the region of interest values of the ischaemic and control testes was compared. From CE-dynamic MR images, the maximal slopes of contrast enhancement were calculated and compared. In testicular torsion of 360 degrees, the maximal slope of contrast enhancement was 0.072%/s vs. 0.47%/s in the contralateral control testis (p < 0.001). A torsion of 720 degrees diminished the slope of contrast enhancement to 0.046%/s vs. 0.37%/s in the contralateral testis (p < 0.001). Diminished blood flow during torsion also followed in decreased ADC values in both 360 degrees (12.4% decrease; p < 0.05) and 720 degrees (10.8% decrease; p < 0.001) of torsion. Torsion of the testis causes ipsilateral hypoperfusion and decreased gadolinium uptake in a rat model that can be easily detected and quantified by CE-dynamic MRI. In diffusion-weighted MRI images, acute hypoperfusion results in a slight decrease of ADC values. Our results suggest that CE-dynamic MRI in combination with diffusion-weighted MRI can be used to detect compromised blood flow due to acute testicular torsion.

Technical developments for cerebral thermal treatment: water-cooled diffusing laser fibre tips and temperature-sensitive MRI using intersecting image planes.

The aim was to determine if water-cooled diffusing tips could produce larger and safer (better controlled) thermal lesions than non-cooled diffusing tips at 980 nm. Thermal lesions were induced in beef myocardium in vitro with and without water cooling using a 980 nm diode laser at various power levels. Seven intracerebral treatments were performed in six canines using water-cooled diffusing tips with four animals having intracerebral transmissible venereal tumours grown from inoculate. Magnetic resonance thermal imaging (MRTI)-based feedback software using a fast, radio frequency-spoiled gradient echo acquisition with two intersecting image planes was used for on-line monitoring and control of treatment and for the evaluation of in vivo laser lesion production. In cases where two-plane MRTI was employed, the maximum calculated temperature was compared in each plane. Using water-cooled tips and 400 micro m core diameter laser diffusing fibres in in vitro beef myocardium, power of up to 9.5 W was applied for 8 min without tip failure. Without cooling, tip failure occurred in under 4 min at 6 W, in under 2 min at 7 W and instantaneously at 8 W. Additionally, char accompanied lesions made with uncooled tips while cooled application resulted in only minimal char at only the highest thermal dose. Achieved lesion cross-sectional diameters in in vitro samples were up to 26.5 x 23.3 mm when water cooling was used. In canine brain and transmissible venereal tumours, up to 18.1 x 21.4 mm lesions were achieved. It is concluded that water cooling allows safe application of higher power to small core diameter diffusing tip fibres, which results in larger thermal lesions than can be achieved without cooling. Two-plane MRTI enhances on-line monitoring and feedback of thermal treatment.

Diffusion weighted magnetic resonance imaging of rat testes: a method for early detection of ischemia.

PURPOSE: We investigated the feasibility of diffusion weighted magnetic resonance imaging (MRI) for the early detection of ischemia in the testis. MATERIALS AND METHODS: Circulation to the right testis in Wistar rats was occluded by surgical ligation of the right funicle. The left side was sham operated and served as a control. The diffusion and T2-weighted MRI images of the 2 testes was performed postoperatively by a 1.5 Tesla MRI unit using a knee coil. On apparent diffusion coefficient images and T2-weighted images the region of interest values in the 2 testes were measured and statistically compared. RESULTS: At 1 hour after testicular funicle ligation the apparent diffusion coefficient was 18% lower in the ischemic than in the sham operated testis (p <0.0098). At 2 hours the difference was 20% (p <0.0017). In the signal-to-noise ratio on T2-weighted images there was no difference in the left and right testes. CONCLUSIONS: Altered diffusion occurs in an ischemic testis, which can be measured on MRI at 1.5 Tesla. Thus, diffusion-weighted MRI may be a helpful method for the differential diagnosis of acute testicular torsion.

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.

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.

Failure of spermatogenesis to recover despite the presence of a spermatogonia in the irradiated LBNF1 rat.

The dose and time response of LBNF1, rat testis to gamma irradiation was studied with use of single doses from 2.5 Gy to 6.0 Gy. Germ cells were initially depleted as a result of killing the radiosensitive differentiating spermatogonia. Some recovery of spermatogenesis was observed at 4 and 6 wk after irradiation as indicated by the repopulation of tubules with germ cells derived from surviving stem spermatogonia. Although spermatogenesis showed additional recovery and was maintained throughout the 60-wk follow-up period after 2.5 Gy, at doses from 3.5 Gy to 6.0 Gy, repopulation indices declined after 6 wk to less than 2%. The numbers of Sertoli cells per nonrepopulating tubule were constant, independent of radiation dose or time. In addition, the nonrepopulating tubules contained an average of one A spermatogonium per 100 Sertoli cells. The size and shape of these cells corresponded to undifferentiated A spermatogonia in nonirradiated control tests. Despite high labeling (40%) and mitotic (20%) indices, the numbers of A spermatogonia changed very little with time, and no differentiated cells were produced in these tubules. The failure of spermatogenesis to recover was not due to hormone deficiency: serum gonadotropin levels increased after irradiation, and serum testosterone remained at control levels. The irradiated LBNF1 rat model may be useful for studying the regulation of differentiation of A spermatogonia.

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.

Stage-specific distribution of the spermatid-specific histone 2B in the rat testis.

Male germ cells contain a number of histone variants, most of which are synthesized during either the mitotic or the meiotic stages of spermatogenesis. A spermatid-specific H2B (ssH2B) variant has been identified in mouse round spermatids that has an additional 12 amino acids at its carboxyl terminus as compared to somatic H2Bs. Until now, the presence of this protein in other mammals has not been known. Northern blot analysis using an oligonucleotide probe complementary to a unique coding region of the C-terminus of mouse ssH2B showed that mRNA encoding this protein was present in isolated rat round spermatids. Furthermore, immunoblot analysis of basic nuclear proteins from round spermatids probed with an anti-ssH2B antiserum that recognizes the novel peptide sequence indicated that the protein was present in round spermatids but not in pachytene spermatocytes. The ssH2B comigrated with H2A.X by acid-urea (2.5 M) PAGE and migrated slightly more slowly than TH2B by acid-urea-Triton PAGE. When seminiferous tubules were microdissected stage-specifically and basic nuclear proteins were immunoblotted, ssH2B was detected in tubule sections of stages II-VI. It reached a maximum level in stages VII-VIII and then decreased to minimal levels in stages XIII-I. Since ssH2B was not detected in pachytene spermatocytes, the stage-specific levels of ssH2B corresponded to the respective steps of spermatids present in the tubules. While ssH2B constituted a relatively small amount (approximately 2%) of total H2B protein in round spermatids, its presence in both the mouse and the rat suggests that its function may be conserved in mammalian spermatogenic cells.

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.

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.

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.