Busulfan Group Research Articles

Secretions released from mesenchymal stem cells improve spermatogenesis restoration of cytotoxic treatment with busulfan in azoospermia mice.

This study aimed at the efficacy of sequential treatment of bone marrow-derived mesenchymal stem cell secretion for busulfan-treated azoospermia in mice. The conditioned media (CM) was obtained from bone marrow mesenchymal stem cells (MSCs) or 293 cells. Chemically induced azoospermia mice received 200μl MSC-CM or 293-CM twice a week intravenously for three consecutive weeks. The histological assessment of spermatogenic recovery quantifying the expression of meiosis-associated genes, and Sertoli cell barrier functional factors were assessed. The characteristics of TM4 cells (Sertoli cell line) after pre-incubation of MSC-CM in vitro were also obtained. The MSC-CM group had the most spermatogenic colonies among the three groups (p<.05), but no spermatids were seen. Expressions of the meiosis-associated genes Dazl, Vasa, Miwi, Stra8, CyclinA1, Pgk2 and Scp3 in MSC-CM testis were remarkably higher compared with 293-CM and busulfan groups respectively (p<.05). The levels of Sertoli cell barrier functional factors, for example ICAM-1 and N-cadherin, were significantly increased during MSC-CM treatment (p<.05). Moreover, pre-incubation of MSC-CM particularly accelerated the CD54 (ICAM-1) and CD44 expressions of TM4 cells and promoted cell inherent adhesion. MSC-CM treatment can significantly improve the short-term restoration of spermatogonial structures of chemically induced azoospermia related to facilitating Sertoli cell adhesion integrity.

Impaired spermatogenesis caused by busulfan is partially ameliorated by treatment with conditioned medium of adipose tissue derived mesenchymal stem cells

ABSTRACT Busulfan (BSU) is a chemotherapeutic drug that can cause subfertility or sterility in males. We investigated the effects of adipose tissue-derived mesenchymal stem cells (AT-MSC) conditioned medium (CM) (AT-MSC-CM) on histopathological and molecular characteristics of mouse testes exposed to BSU using stereology. We used adult male mice divided randomly into five groups: control, Dulbecco’s modified Eagle’s medium (DMEM), dimethyl sulfoxide (DMSO), BSU, and BSU + CM. Thirty-five days following BSU injection, sperm and testis tissues were harvested for stereological and molecular studies. The BSU group exhibited significantly reduced testis volume, interstitium and tubules compared to the other groups, although the volume of the testis remained unchanged for BSU and CM groups. The number of testis cells was reduced in the BSU group compared to the other groups. The CM group exhibited a significantly increased number of testis cells compared to the BSU group. Sperm count and motility, and length density of seminiferous tubules were increased in CM group compared to the BSU group. AT-MSC-CM exhibited ameliorative effects on histopathologic changes of mouse testes exposed to BSU.

Protective effect of curcumin on busulfan-induced renal toxicity in male rats

Aim The aim of this study was to evaluate the effects of curcumin in an experimental model of busulfan-induced renal toxicity with emphasis on importance of histological alterations. Methods In this study, we utilized 32 adult male Wistar rats (250 ± 10 g). All the animals were divided into four experimental groups randomly: (I) Control; (II) Busulfan (40 mg/kg); (III) Olive oil; and (IV) Curcumin (80 mg/kg/day). Finally, the rats were euthanized and kidney tissues were taken for histopathology experiments, serum BUN, and creatinine level, reactive oxygen species (ROS) production and glutathione disulfide (GSH) activity. Results Our result showed that the reduction in body weight and kidney weight in busulfan groups in comparison with the control and curcumin groups. The result in this study also showed that the reduction in BUN, creatinine, and ROS production in curcumin groups in comparison with the busulfan group together with an increasing of GSH activity compared to busulfan induced rats. Conclusion Our results of this study indicated that that the reduction in body weight, kidney weight, total volume of kidney, total length of nephron tubules, and numerical density of glomeruli and nephron tubules in busulfan groups in comparison with the control and curcumin groups However, curcumin can be an alternative choice for therapeutically and research purposes in the disturbing kidney after treatment with busulfan.

Late Effects in Survivors of Infant Acute Lymphoblastic Leukemia from the 3 Consecutive Japanese Nationwide Clinical Trials

Background Very few studies have reported the incidence of and risk factor for late effects of infant leukemia. In addition, the long-term impact of a preparative regimen for very young children undergoing allogeneic stem cell transplantation (SCT) is not evident. Method To examine the late effects in survivors of infant acute lymphoblastic leukemia (ALL) treated on the Japanese Infant Leukemia Study Group (JILSG) trials MLL96, MLL98 and the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) trial MLL03, we performed a cross-sectional survey using questionnaires by the attending pediatricians from 2015 to 2017. In all 3 studies, infants with KMT2A gene rearranged (MLL-R) ALL were allocated to receive SCT in their first remission. Choice of preparative regimen was left to the physician's choice in the MLL96/98 studies, while use of non-total body irradiation (TBI) regimen with busulfan (BU) was mandatory in the MLL03 study. Results Seventy out of 102 (69%) eligible subjects were evaluated: 33 were males and 37 were females. 62 patients had an MLL-R ALL. The median age of diagnosis was 5.5 months (range, 0-12m) and 35 (50%) were &lt;6 months of age. At the time of analysis, the median age of the 70 patients was 13.5 years (range, 7-21y) and the median follow-up duration from diagnosis was 12.5 years (range, 7-20y). Fifty-seven (81%) of the 70 patients underwent SCT. Eight patients received the TBI-based regimen, while 35 received BU regimen and one received non-TBI, non-BU regimen. Thirteen patients had SCT twice and twelve patients received both TBI and BU regimen. Relapses occurred in seventeen patients (24%). Only two patients received cranial radiation therapy in 13 patients with central nervous system involved. At least one late effect was observed in 62 of the 68 patients (91%). Identified abnormalities included: underweight (body mass index [BMI]&lt;18.5) (69%), hypogonadism (56%), teeth mal-development (52%), short stature (52%), bone abnormalities (28%), thyroid dysfunctions (27%), skin abnormalities (24%), ophthalmic dysfunctions (24%), pulmonary dysfunctions(22%), neurocognitive dysfunctions (22%), and cardiovascular dysfunctions (14%), gastrointestinal dysfunctions (5%), renal dysfunctions (4%), second malignancy (3%), and overweight (3%). The mean standard deviation (SD) heights at last follow up with TBI regimen were -4.2 and those with multiple SCT (BU+TBI) were -3.8. Both were significantly lower than those with single SCT by BU regimen (-1.4) (TBI vs BU, P=0.014; BU+TBI vs BU, P&lt;0.001). The mean BMI at last follow up was lower in the TBI group (16.8) compared with that in the BU group (17.8) without statistical significance (TBI vs BU, P=0.342), whereas the mean BMI in the BU+TBI group (15.1) was significantly lower than that in the BU group (BU+TBI vs BU, P=0.020). Univariable analysis revealed that the risk of both thyroid dysfunctions (odds ratio [OR]=14.0, P=0.005) and the need of growth hormone (GH) treatment (OR=10.7, P=0.011) were significantly lower in the BU group than those in the TBI group. Hypothyroidism (OR=5.8, P=0.012), teeth mal-development (OR=2.8, P=0.066) and the need of GH treatment (OR=3.5, P=0.084) were more frequently observed in children who were diagnosed at less than 6 months of age. In addition, short stature (OR=23.3, P=0.003), thyroid dysfunctions (OR=4.1, P=0.023), cataract (OR=6.9, P=0.038) and alopecia (OR=6.8, P=0.038) were more frequently observed in relapsed patients. Overall, SCT significantly increased the incidence of short stature (OR=15.5, P=0.012) and underweight (OR=5.3, P=0.018). Conclusion In this study, survivors of infant leukemia had prolonged and various types of late effects. We observed serious growth failure in height and weight in patients treated with SCT and it was further pronounced in the TBI group and in the multiple SCT (BU+TBI) group. More than half of the patients had dental late effects and a third of all the patients had thyroid dysfunctions, which were more frequent in children diagnosed earlier. The number of patients over 20 years was limited in our study, more complications may increase in additional follow-up. Continuous long-term follow up and optimal intervention are crucial for survivors of infant leukemia who are transplanted. Disclosures Kada: Bayer Yakuhin: Other: personal fees for a member of independent data monitoring committee of clinical trials, outside of the submitted work..

Treosulfan or busulfan plus fludarabine as conditioning treatment before allogeneic haemopoietic stem cell transplantation for older patients with acute myeloid leukaemia or myelodysplastic syndrome (MC-FludT.14/L): a randomised, non-inferiority, phase 3 trial

Further improvement of preparative regimens before allogeneic haemopoietic stem cell transplantation (HSCT) is an unmet medical need for the growing number of older or comorbid patients with acute myeloid leukaemia or myelodysplastic syndrome. We aimed to evaluate the efficacy and safety of conditioning with treosulfan plus fludarabine compared with reduced-intensity busulfan plus fludarabine in this population. We did an open-label, randomised, non-inferiority, phase 3 trial in 31 transplantation centres in France, Germany, Hungary, Italy, and Poland. Eligible patients were 18-70 years, had acute myeloid leukaemia in first or consecutive complete haematological remission (blast counts <5% in bone marrow) or myelodysplastic syndrome (blast counts <20% in bone marrow), Karnofsky index of 60% or higher, and were indicated for allogeneic HSCT but considered at an increased risk for standard myeloablative preparative regimens based on age (≥50 years), an HSCT-specific comorbidity index of more than 2, or both. Patients were randomly assigned (1:1) to receive either intravenous 10 g/m2 treosulfan daily applied as a 2-h infusion for 3 days (days -4 to -2) or 0·8 mg/kg busulfan applied as a 2-h infusion at 6-h intervals on days -4 and -3. Both groups received 30 mg/m2 intravenous fludarabine daily for 5 days (days -6 to -2). The primary outcome was event-free survival 2 years after HSCT. The non-inferiority margin was a hazard ratio (HR) of 1·3. Efficacy was assessed in all patients who received treatment and completed transplantation, and safety in all patients who received treatment. The study is registered with EudraCT (2008-002356-18) and ClinicalTrials.gov (NCT00822393). Between June 13, 2013, and May 3, 2016, 476 patients were enrolled (240 in the busulfan group received treatment and transplantation, and in the treosulfan group 221 received treatment and 220 transplanation). At the second preplanned interim analysis (Nov 9, 2016), the primary endpoint was met and trial was stopped. Here we present the final confirmatory analysis (data cutoff May 31, 2017). Median follow-up was 15·4 months (IQR 8·8-23·6) for patients treated with treosulfan and 17·4 months (6·3-23·4) for those treated with busulfan. 2-year event-free survival was 64·0% (95% CI 56·0-70·9) in the treosulfan group and 50·4% (42·8-57·5) in the busulfan group (HR 0·65 [95% CI 0·47-0·90]; p<0·0001 for non-inferiority, p=0·0051 for superiority). The most frequently reported grade 3 or higher adverse events were abnormal blood chemistry results (33 [15%] of 221 patients in the treosulfan group vs 35 [15%] of 240 patients in the busulfan group) and gastrointestinal disorders (24 [11%] patients vs 39 [16%] patients). Serious adverse events were reported for 18 (8%) patients in the treosulfan group and 17 (7%) patients in the busulfan group. Causes of deaths were generally transplantation-related. Treosulfan was non-inferior to busulfan when used in combination with fludarabine as a conditioning regimen for allogeneic HSCT for older or comorbid patients with acute myeloid leukaemia or myelodysplastic syndrome. The improved outcomes in patients treated with the treosulfan-fludarabine regimen suggest its potential to become a standard preparative regimen in this population. medac GmbH.

Sertoli cell-conditioned medium restores spermatogenesis in azoospermic mouse testis.

The current study evaluates potential applications of Sertoli cell (SC)-conditioned medium (CM) and explores the effects of the conditioned medium on the spermatogenesis process in azoospermic mice. For this study, 40 adult mice (28-30g) were divided into 4 experimental groups: (1) control, (2) DMSO 2% (10μl), (3) busulfan (40mg/kg single dose) and (4) busulfan/CM (10μl). SCs were isolated from 4-week-old mouse testes. After using anesthetics, 10μl of CM was injected over 3-5min into each testis and subsequently, sperm samples were collected from the tail of the epididymis. Afterward, the animals were euthanized and testis samples were taken for histopathology experiments and RNA extraction in order to examine the expression of c-kit, STRA8 and PCNA genes. The data showed that CM notably increased the total sperm count and the number of testicular cells, such as spermatogonia, primary spermatocytes, round spermatids, SCs and Leydig cells compared with the control, DMSO and busulfan groups. Furthermore, the results showed that expression of c-kit and STRA8 was significantly decreased in the busulfan and busulfan/SC groups at 8weeks after the last injection (p < 0.001) but no significant difference was found for PCNA compared with the control and DMSO groups (p < 0.05). These findings suggest that the Sertoli cell-conditioned medium may be beneficial as a practical approach for therapeutic strategies in reproductive and regenerative medicine.

Melatonin synergistically enhances protective effect of atorvastatin against busulfan-induced spermatogenesis injuries in a rat model

Busulfan, a chemotherapy drug, causes a variety of adverse effects, such as damage to the reproductive system of male cancer patients. The aim of this study was to investigate the protective effect of intra-peritoneal (IP) injection of exogenous melatonin and atorvastatin alone and in combination on busulfan-induced spermatogenesis injuries and oxidative stress activity in a rat model. A total of 36 male Wistar rats (220–250 g) (n = 6) were randomly divided into six different groups, including atorvastatin, melatonine, sham (received DMSO or busulfan solvent), busulfan, atorvastatin + melatonine, and control groups. Busulfan was diluted in DMSO and intra-peritoneally (IP) administrated in two doses of 25 and 10 mg/kg with a 14-day interval in all groups except the control and sham groups. Administration of atorvastatin and melatonin started 1 day after injection of busulfan. After 28 days, the animals were euthanized and the semen was collected from epididymis and the testicular tissues were removed for sperm parameter assessment and biochemical analysis, including super oxide dismutase (SOD), SOD/total protein (TP), catalase, catalase/TP, and MDA. The highest and lowest values of sperm parameters belonged to the control and busulfan groups, respectively, followed by sham, atorvastatin + melatonine, melatonin, and atorvastatin groups as well as antioxidant enzyme activity. However, MDA activity in the busulfan group was significantly increased compared to that in the other groups. In conclusion, the results of the present study indicated that co-administration of melatonin and atorvastatin and monotherapy can attenuate busulfan-induced spermatogenesis injuries in rats via reducing oxidative stress.

Mesenchymal stem cell‐secreted factors delayed spermatogenesis injuries induced by busulfan involving intercellular adhesion molecule regulation

The present study was designed to investigate the therapeutic effect of bone marrow MSC-derived factors on gonadotropic toxicity induced by busulfan in vivo. The conditioned media (CM) was obtained from MSCs in serum-free incubation for 48hr and concentrated ~25-fold by ultrafiltration. The CM of HEK 293 cells was treated as control (293-CM). MSC-CM was injected into busulfan mice via caudal veins after 1day of busulfan treatment for 2weeks (200μl per dose/twice weekly）. Compared to the 293-CM group, testicular injury was delayed in MSC-CM group, including reduced vacuolations of cells in the basal compartment of the seminiferous epithelium and detachment of cells from basement membrane. Apoptotic spermatogenic cells were significantly decreased in MSC-CM group (p＜0.05). Interesting N-cadherin，ICAM-1 and P-cadherin expressions significantly increased in MSC-CM group, while occludin, ZO-1 and connexin 43 expressions showed no difference among MSC-CM, 293-CM and busulfan groups. Present results suggest MSC-secreted factors protect spermatogenesis impairment after busulfan treatment by reducing the apoptosis of spermatogenic cells and enhancing intercellular adhesion molecule expressions.

Effects of Bene (Pistacia atlantica) on Histopathology of Testis, Sperm Chromatin Quality and Stress Oxidative in Busulfan-Induced Infertile Mice

Background: Some plants stimulate spermatogenesis and increase fertility, while some cause spermatogenesis arrest. So far, the effects of bene (Pistacia atlantica) on male fertility have not been studied. The aim of this study was to investigate the effects of bene on sperm parameters, testicular histopathology, sperm quality, and oxidative stress in busulfan-induced infertile mice. Methods: Thirty-five male BALB/c mice were randomly assigned to control, sham, busulfan, bene, and bene + busulfan groups. The busulfan group received 10 mg/kg as a single dose and intraperitoneally. The bene group received pellets containing 10% of bene. Another group received 10 mg/kg busulfan and was fed with pellet containing 10% bene. Then, sperms, sperm chromatin quality, testicular histopathology, and oxidative stress levels were studied on the 35th day of the experiment. Results: Busulfan injection resulted in a significant reduction in sperm parameters compared to the control group (p&lt;0.001); it decreased after bene administration (p&lt;0.001). In addition, in the group treated with bene, the sperm count with damaged DNA was reduced and the level of malondialdehyde decreased compared to the busulfan group. A significant increase was observed in the mean level of superoxide dismutase and catalase enzymes in the bene + busulfan group compared to the busulfan group (p&lt;0.001). The histopathological improvement of the testis was observed in the bene + busulfan group. Conclusion: The administration of 10 mg/kg of bene powder for 35 days reduced the oxidative stress, improved testicular histopathology, sperm chromatin quality, and sperm parameters in the infertile mice model.

Effects of Sertoli Cell Transplantation on Spermatogenesis in Azoospermic Mice.

The aim of this study was to evaluate the potential and significant applications of Sertoli cells (SCs) transplantation, and to explore the effect of transplantation on spermatogenesis process, in azospermic mice. In this study, we utilized 18 adult mice (28‒30 g), divided into four experimental groups: (1) control, (2) vehicle (DMSO 2%) (10 µl) (3) busulfan and (4) busulfan+ SCs (1×104 cells/μL). SCs were isolated from the testis of 4-week-old mouse and after using anesthetics, 10 μl of SCs suspension (1×104 cells/μL) was injected over 3-5 min, into each testis and subsequently, sperm samples were collected from the tail of the epididymis. Afterward, the animals were euthanized and testis samples were taken for histopathology experiments, and RNA extraction, in order to examine the expression of c-kit, STRA8 and PCNA genes. Our data showed that SCs transplantation could notably increase the total sperm count and the number of testicular cells, such as spermatogonia, primary spermatocyte, round spermatid, SCs and Leydig cells, compared to the control, DMSO and busulfan groups. Furthermore, the result showed that the expression of c-kit and STRA8 were significantly decreased in busulfan and busulfan/SCs groups, at 8 weeks after the last injection (p<0.001), but no significant decrease was found for PCNA, compared to the control and DMSO groups (P<0.05). These findings suggest that SCs transplantation may be beneficial as a practical approach for therapeutic strategies in reproductive and regenerative medicine. We further highlighted the essential applications that might provide a mechanism for correcting fertility in males, suffering from cell deformity.

The Effects of Olive Leaf Extract on The Testis, Sperm Quality and Testicular Germ Cell Apoptosis in Male Rats Exposed to Busulfan.

BackgroundBusulfan (BU) has a destructive effect on the male reproductive system. The goal of this study was to assess the effects of olive leaf extract (OLE) as a source of antioxidants and phenolic compounds, on BU-induced damages in rat testes.Materials and MethodsIn this experimental study, 40 male Wistar rats were randomly divided into 5 groups. The control group (CTL) received a single intraperitoneal (i.p.) injection of dimethyl sulfoxide (DMSO), followed by oral administration of distilled water for 5 weeks. In BU group, BU (10 mg/kg) was administrated i.p. once. In co- treatment groups, first, received BU (10 mg/kg, a single i.p. injection) then, OLE was administrated orally at different doses of 250 mg/kg (BU+OLE 250), 500 mg/kg (BU+OLE 500) and 750 mg/kg (BU+OLE 750), for 5 weeks. Next, blood and sperm samples were collected. The left testis was removed to investigate testicular parameters and apop- tosis by using H&E and TUNEL staining, respectively. All data were analyzed by SPSS software and a P<0.05 was considered significant.ResultsThere was a significant decline in sperm viability (P=0.017), number of primary spermatocyte (PS) (P=0.001) and Leydig cells (P=0.023) in the BU group versus the CTL group. OLE at three doses could repair these defects ver- sus BU group. Increases in apoptotic spermatogonia cells (SG) due to BU were significantly reduced by OLE 250 and 500 mg/kg (P<0.01). A reduction in germinal epithelium height and an increase in apoptotic SG were observed in BU+OLE 750 group vs. other groups (P<0.01) and alkaline phosphatase (ALP) was at the highest level, also Aspartate aminotransferase (AST) increased markedly vs. CTL (P=0.024).ConclusionOral administration of OLE at the doses of 250 and 500 mg/kg could be helpful in ameliorating BU- induced toxicity in rat testes, while OLE 750 mg/kg not only did not cause positive effects, but also could exacerbate the harmful effects.

The Safe Recipient of SSC Transplantation Prepared by Heat Shock With Busulfan Treatment in Mice

Safety is the chief consideration in recipient preparation of spermatogonial stem cell (SSC) transplantation in mammals, especially humans. In this study, we compared the safety of the SSC transplantation recipients that were prepared both by testes heat shock plus testes busulfan injection (heat shock+busulfan(t)) and by busulfan intraperitoneal injection (busulfan i.p.) only. Our results showed that heat shock+busulfan(t) treatment significantly (p < 0.05) reduced mortality in mice and did not produce bone marrow cell toxicity. Furthermore, heat shock+busulfan(t) treatment directly damaged SSCs and exhausted almost all of the germ cells in the testis; the exhaustion of these cells is considered a key factor in the successful preparation of the recipients. Therefore, we used heat shock+busulfan(t) treatment to prepare recipients of SSC transplantation. Two months after SSC transplantation, the number and length of donor SSC-derived colonies in the testis of recipient in heat shock+busulfan(t) group was closed to that in busulfan i.p. group. Therefore, compared with busulfan i.p. treatment, heat shock+busulfan(t) treatment improved the safety of recipient preparation without reducing the efficiency of SSC transplantation. Two GFP-positive offspring were produced from 1 of the 20 recipients that had mated with female mice 72 days after SSC transplantation. In conclusion, heat shock with busulfan treatment is a safe method to prepare the recipient of SSC transplantation in mice.

Histomorphometric Evaluation of Allogeneic Transplantation of Bone Marrow Mesenchymal Stem Cells in Azoospermic Mice Model

BACKGROUND: Stem cell-based therapy is one of the newest and evolving techniques in reproductive medicine. The aim of this study was to investigate the effect of allogeneic bone marrow mesenchymal stem cells (BM-MSCs) transplantation on the testis of busulfan induced azoospermia in Balb/C mice.METHODS: Eighteen adult Balb/C mice were divided into three equal groups including control, busulfan and busulfan+cell therapy (busul+CT). For induction of azoospermia, busulfan and busul+CT groups received two injections of 10 mg/Kg of busulfan intraperitoneally with 21 days interval. In the cell therapy group 35 days after the last injection of busulfan, cluster of differentiation (CD)90+/CD34-/CD45- BM-MSCs were injected into the efferent duct of testis. Eight weeks after the BM-MSCs therapy, mice were sacrificed and tissues were taken for histological and histomorphometric evaluations.RESULTS: In busul+CT group, cellular and total diameters and cellular and cross-sectional areas significantly increased in comparison to busulfan group (p˂0.001), but there were no significant differences between busul+CT and control group (p˃0.05). Numerical density and tubular count per area unit in busul+CT and control groups were significantly less than busulfan group (p˂0.001), but there were no significant difference between busul+CT and control group (p˃0.05). The luminal diameter and area showed no significant change in all groups (p˃0.05). In busul+CT group, spermatogenesis index significantly increased when compared to busulfan and control groups (p˂0.001 and p˂0.05, respectively).CONCLOSION: Histomorphometric findings showed CD90+/CD34-/CD45- BM-MSCs transplantation on the testis of busulfan-induced azoospermic in Balb/C mice recovered spermatogenesis.KEYWORDS: mesenchymal stem cell, cell therapy, azoospermia, busulfan, mouse

The Effect of CoQ10 on Testicular Tissue in Rats Treating with Busulfan: Sperm Quality and Histological Changes.

Objective: Busulfan(Bus) is a chemotherapy drug that is widely used for cancer treatment. The protective effect of CoQ10 evaluated on testis and sperm parameters after busulfan treatment. Design: Experimental StudyAnimals: Thirty tow adult male Wistar rats Procedures: In this experimental study 32 adult male Wistar rats have randomly divided into four groups: Control group received normal saline (0.1 mL, daily, intraperitoneally). Sham group received a single dose of busulfan 10 mg/kg, IP. Positive control group received 0.1 mL CoQ10 (10 mg/kg, IP). The treatment group received busulfan along with CoQ10 (10 mg/kg, IP). All procedures were continued for 35 days. For histomorphometric analyses, the thickness of testicular capsule, the germinal epithelium height and the diameter of the seminiferous tubules were measured. Semen analysis was used for the assessment of sperm parameters. Results: Histomorphometric analyses showed the thickness of testicular capsule was increased in busulfan groups (P<0.05). Compared to the control group, germinal epithelium height was decreased significantly in Sham and treatment groups (P<0.05). There was a significant decrease in seminiferous tubules diameter in busulfan and treatment groups compared to the control group. In Sham group, the sperm count, motility, viability, nuclear maturation and sperm morphology were significantly decreased compared to control and CoQ10 treated groups (P<0.05). The percentage of sperm with DNA damage in the CoQ10 treated group was significantly increased compared to Sham groups (P<0.05).Conclusion and Clinical Relevance: Administration of CoQ10 in busulfan-treated animals improved histological and sperm quality.

Effects of Carob (Ceratonia siliqua) on Sperm Quality, Testicular Structure, Testosterone Level and Oxidative Stress in Busulfan-Induced Infertile Mice

Background: Ceratonia silique has antioxidant activities that may inactivate toxic factors and influence sperm quality. To the best of the authors’ knowledge, there is no available data on the effects of carob on male fertility. Hence, the purpose of this study was to investigate the effects of carob on sperm quality, testicular structure, and level of testosterone hormone in busulfan-induced infertile mice. Methods: Sixty-four adult male mice were randomly divided into 8 groups (control, sham, busulfan and carobs 1 to 5). The busulfan group was injected a single dose of 10 mg/kg busulfan intraperitoneally. Carobs 1 to 5 groups received intraperitoneal doses of 800, 400, 200, 100 and 50 mg/kg of carob extract plus a single dose of 10 mg/kg busulfan for 35 days. The sperm analysis, morphometric study, testosterone levels and oxidative stress determination were done on the 35th day of the experiment. Results: The lowest percentage of sperm parameters was related to the busulfan group and the highest was related to the carobs 1 and 2 groups. The seminal vesicles index of the carob 1 group showed a significant increase as compared to the busulfan group (p < 0.001). A significant increase was observed in the mean value of germinal epithelium thickness, as well as thiol and catalase levels in carobs 1 and 2 groups as compared to the busulfan group (p < 0.001). There was a significant increase in the mean level of testosterone in the carob groups as compared to the busulfan group (p < 0.001). Also, there was a significant decrease in the mean value of malondialdehyde level in the carobs 1 and 2 groups p < 0.001) and a significant increase in the mean value of superoxide dismutase enzyme in the carob groups as compared to the busulfan group (p < 0.001). Conclusion: Administration of 800 mg/kg of carob extract for 35 days improved sperm quality, biochemical parameters, thickness of germinal epithelium and testosterone levels in infertile mice induced by busulfan.

Application of stereological methods for unbiased estimation of sperm morphology in the mice induced by busulfan.

Busulfan is an anticancer drug, which causes the apoptosis germ cells and azoospermia in humans and animals. Abnormal morphology of spermatozoa related to the male infertility. The sperm morphology is evaluation of sperm size, shape and appearance characteristics should be assessed by carefully observing a stained sperm sample under the microscope. Evaluation of sperm morphology has been considered as one of the most important factors for a successful fertilization and determining sperm quality. The mice were assigned to tow experimental groups: control and busulfan. Each group included six mice that were housed under standard conditions. The volume was estimated using the nucleator method. The sperm's flegellum and mid-piece length was estimated by counting the number of intersections between the tails and Merz grid test line in an unbiased counting frame, superimposed on live images of sperms. Our results demonstrated a significant different in the volume and surface of the sperm's head and the length of the sperm's flagellum in the control and busulfan groups. Busulfan can effect on the volume of the sperm's head and the length of the sperm's flagellum in rat.

Comparison of Total Body Irradiation-Based with Intravenous Busulfan-Based Chemotherapy-Only Conditioning Regimens for Myeloablative Hematopoietic Cell Transplantation (HCT) in Adults with Acute Lymphoblastic Leukemia

Total body irradiation (TBI)-based conditioning regimens are considered the standard of care for patients with acute lymphoblastic leukemia (ALL) undergoing allogeneic hematopoietic cell transplantation (HCT). However, due to concerns regarding acute and long-term toxicities, non-TBI regimens, commonly including busulfan (BU), have been increasingly explored. We performed a retrospective cohort analysis with the hypothesis that there would be equivalence of these two myeloablative approaches by reviewing outcomes for adult patients (pts), aged 18-60 years, undergoing a first, well-matched sibling, related or unrelated donor HCT in CR1 or CR2 reported to the Center for International Blood and Marrow Transplant Research (CIBMTR) from 2005-2014. Eight hundred nineteen pts received TBI (63% 9-12 Gy, 37% ≥13 Gy) combined with etoposide (25%) or cyclophosphamide (Cy) (75%) and 299 pts received intravenous BU combined with a second alkylator Cy (15%) or melphalan (Mel) (13%), or a nucleoside analogue fludarabine (Flu) (41%) or clofarabine (Clo)(30%). The majority of the BU-based pts were treated at the Moffitt or MD Anderson Cancer Center. The BU-based regimens were grouped together for analyses since no significant differences in basic outcomes among the different chemotherapy-only regimens were noted. Patients in the BU-containing group were older but had better performance status, took longer to achieve CR1 and longer to receive HCT; more received peripheral blood than marrow grafts, peri-HCT ATG, pre- and post HCT tyrosine kinase inhibitors ( TKI), and were treated more recently than pts in the TBI-based group (Table 1). With median follow-up of 3.6 years for the BU-based group and 5.3 years for the TBI-based group, adjusted 3 year outcomes showed treatment-related mortality (TRM) BU 19% vs. TBI 25% (p=.04); relapse BU 37% vs. TBI 28% (p=.007); disease-free survival (DFS) Bu 45% vs. TBI 48% (p=.35); and overall survival (OS) BU 57% vs. TBI 53% (p=.35) (Figures A-B). Patients in the BU group had significantly more grade II-IV acute GVHD (47% vs. 40%, p=.025), but marginally less chronic GVHD (49% vs. 55% at 3 years, p=.073). In multivariate analysis, the BU group had a significantly higher rate of acute GVHD after day 50 (RR 1.75, 95% CI 1.19-2.58, p=.004), but marginally less chronic GVHD (RR 0.83, 95% CI 0.68-1.01 p=.059) and higher risk of relapse (RR 1.46, 95% CI 1.15-1.85 p=.002) compared with TBI-based regimens. Despite the observed higher risks of acute GVHD and relapse, BU-based conditioning led to similar TRM, OS, and DFS following HCT for ALL. [Display omitted] [Display omitted] [Display omitted] DisclosuresCiurea:Spectrum Pharmaceuticals: Other: Advisory Board; Cyto-Sen Therapeutics: Equity Ownership. Seftel:Otsuka: Research Funding. Pulsipher:Medac: Other: Housing support for conference; Chimerix: Consultancy; Jazz Pharmaceutical: Consultancy; Novartis: Consultancy, Other: Study Steering Committee. Champlin:Ziopharm Oncology: Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership, Patents & Royalties.

Effect of human recombinant granulocyte colony-stimulating factor on rat busulfan-induced testis injury.

Granulocyte colony-stimulating factor (G-CSF) is known for proliferation and anti-apoptotic activities. We aimed to use this growth factor in busulfan-injured testis. 32 male Wistar rats were injected with a double dosage of 15 ml/kg busulfan with 14 days interval. Administration of human recombinant G-CSF (100 µg/kg) subcutaneously was performed in two different time periods: 3 days before and 2 days after receiving busulfan, G-CSF1; and at days 14-18 of busulfan injection, G-CSF2. Animals were sacrificed at the end of week five. Histological analysis, testis weight and sperm parameters (sperm count and viability) has been checked. Expressions of DEAD (Asp-Glu-Ala-Asp) box polypeptide 4 (DDX4), deleted in azoospermia like (DAZL), transition protein 2 (TP2), proliferating cell nuclear antigen (PCNA) and 5-Bromo-20-deoxyuridine (BrdU) were assessed. Empty seminiferous tubules were apparent in the busulfan- and G-CSF2-injected rats, but not in the G-CSF1 group. The G-CSF1-treated animals showed an increase in testis weight and sperm count and viability along with high expressions of DDX4, DAZL, TP2, PCNA and BrdU; even so, the changes were reversed in the busulfan and G-CSF2 groups (for all p < 0.05). Our results revealed that G-CSF application prior to busulfan insult is a promising approach in fertility maintenance.

Incidence and risk factors for cataract after haematopoietic stem cell transplantation for childhood leukaemia: an LEA study.

Cataract was prospectively assessed by serial slip lamp tests in 271 patients included in the Leucémie Enfants Adolescents (LEA) programme, the French cohort of childhood leukaemia survivors. All had received haematopoietic stem cell transplantation (HSCT) after total body irradiation (TBI, n = 201) or busulfan-based (n = 70) myeloablative conditioning regimen. TBI was fractionated in all but six patients. The mean duration of follow-up from HSCT was 10·3 years. Cataract was observed in 113/271 patients (41·7%); 9/113 (8·1%) needed surgery. Cumulative incidence after TBI increased over time from 30% at 5 years to 70·8% and 78% at 15 and 20 years, respectively, without any plateau thereafter. The 15-year cumulative incidence was 12·5% in the Busulfan group. A higher cumulative steroid dose appeared to be a cofactor of TBI for cataract risk, in both univariate and multivariate Cox analysis. In the multivariate analysis, cataract had an impact in two quality of life domains: 'the role limitation due to physical problems' and 'the role limitation due to emotional problems'. These data suggest that with increasing follow-up, nearly all patients who receive TBI, even when fractionated, will suffer from cataract that can impact on their quality of life and that high cumulative steroid dose is a cofactor.

Melatonin and Testicular Damage in Busulfan Treated Mice

Background:Advancement in the treatment of various types of cancer has led to greater patient survival. These treatments essentially have toxic effects on different kinds of cells, such as germ cells. Infertility as one of the side effects of cancer treatment has changed the quality of life of young cancer survivors dramatically. Melatonin is an antioxidant with receptors in the reproductive systems.Objectives:We supposed that melatonin, as an antioxidant, may protect testis against the toxic effects of the drugs.Materials and Methods:In this experimental study, three groups with seven mice each, were allocated. The control group received normal saline for two months, and the busulfan group received a single dose of 40 mg/kg busulfan intra-peritoneally, and the melatonin group received 20 mg/kg melatonin daily for two months, 45 days after a single dose of busulfan. Next, after decapitation and removal of the testis, tissues were fixed in Bouin's solution and stained by H&E and TUNEL. The sections were evaluated, assessing morphology and spermatogenesis.Results:In this research, a significant reduction in Johnson’s criteria in the busulfan group (Mean rank = 15.50) was found versus the control group (Mean rank = 45.50), P < 0.001 and in the melatonin group (Mean rank = 45.50) compared to the busulfan group (Mean rank = 15.50), P < 0.001. There was a significant difference between the melatonin and control groups, P < 0.05. In addition, a significant decrease in seminiferous tubule diameter was observed in the busulfan group (763.2 ± 104.41) versus the control group (855.4 ± 52.35), P < 0.01 and melatonin group (834.2 ± 87.26), P < 0.05. Testicular epithelium height was significantly decreased in the busulfan group (Mean rank = 14.60) compared to the control group (Mean rank = 26.40), P < 0.01 and in the busulfan group (Mean rank = 14.95) in comparison with the melatonin group (Mean rank = 26.05), P < 0.01. Also melatonin group (Mean rank = 25.42) showed a significant reduction in epithelium height compared to the control group (Mean rank = 35.58), P < 0.05. Spermatogenesis was impaired in the busulfan group. Although melatonin reduced the rate of apoptosis in the busulfan group, yet it could not remove all apoptotic cells.Conclusions:This study indicated that melatonin ameliorates the cytotoxic effects of busulfan on germ cells.