Leptin and prolactin reduce cryodamage in normozoospermic human semen samples during cryopreservation / La leptina y la prolactina reducen el criodaño en muestras de semen humano normozoospérmico durante la criopreservación

Objectives: Cryopreservation has destructive effects on the function and structure of spermatozoa. It is known that leptin and prolactin play an active role in decreasing the rates of reactive oxygen species and DNA fragmentation, as well as enhancing sperm motility. Hence, this experiment aimed to investigate the effects of leptin and prolactin as pro-survival factors on the normozoospermic human semen samples during cryopreservation. Material and methods: Semen samples were collected from 15 healthy, fertile men ranging from 25 to 40 years. Cryopreservation of the samples was performed in liquid nitrogen over a period of two weeks, using five varying concentrations of leptin/prolactin, 0, 10, 100, 500, and 1000ng/ml respectively. Sperm motility, total caspase activity, and mitochondrial and cytosolic ROS were measured by flowcytometry, TUNEL, and other appropriate tests after thawing of the samples. Results: Both hormones were observed to have positive effects on the motility of the samples post-cryopreservation, the highest improvement being in the 100ng/ml concentration leptin and prolactin in comparison to the control group (P=0.01 and P=0.041, respectively). A significant reduction of mitochondrial ROS was also observed in 100 and 1000ng/ml of leptin (P=0.042), and there was a considerable decrease in the cytosolic ROS in the 100ng/ml of prolactin in comparison to the control group (P=0.048). Total caspase activity was also highly reduced in the 100, 500, and 1000ng/ml of leptin compared to the control group (P=0.039). Interestingly, both hormones also significantly decreased DNA fragmentation in 1000ng/ml compared to the control group (P=0.042). (AU)

Objetivos: La criopreservación tiene efectos destructivos sobre la función y estructura de los espermatozoides. Se sabe que la leptina y la prolactina desempeñan un papel activo en la disminución de las tasas de especies reactivas de oxígeno (ROS) y la fragmentación del ADN, así como en la mejora de la motilidad de los espermatozoides. Por lo tanto, este experimento tuvo como objetivo investigar los efectos de la leptina y la prolactina como factores de supervivencia en las muestras de semen humano normozoospérmico durante la criopreservación. Material y métodos: Se recolectaron muestras de semen de 15 hombres sanos y fértiles de entre 25 y 40 años. La crioconservación de las muestras se realizó en nitrógeno líquido durante un período de 2 semanas, utilizando 5 concentraciones variables de leptina/prolactina: 0, 10, 100, 500 y 1000ng/ml respectivamente. La motilidad de los espermatozoides, la actividad de caspasa total y las ROS mitocondriales y citosólicas se midieron mediante citometría de flujo, TUNEL y otras pruebas apropiadas después de descongelar las muestras. Resultados: Se observó que ambas hormonas tienen efectos positivos sobre la motilidad de las muestras después de la crioconservación, la mayor mejora se encuentra en la concentración de leptina y prolactina de 100ng/ml en comparación con el grupo de control (p=0,01 y p=0,041, respectivamente). También se observó una reducción significativa de las ROS mitocondriales en 100 y 1000ng/ml de leptina (p=0,042), y hubo una disminución considerable en las ROS citosólicas en los 100ng/ml de prolactina en comparación con el grupo de control (p=0,048). La actividad de la caspasa total también se redujo considerablemente en los 100, 500 y 1000ng/ml de leptina en comparación con el grupo de control (p=0,039). Curiosamente, ambas hormonas también redujeron significativamente la fragmentación del ADN en 1000ng/ml en comparación con el grupo de control (p=0,042). (AU)

Leptin and prolactin reduce cryodamage in normozoospermic human semen samples during cryopreservation.

OBJECTIVES: Cryopreservation has destructive effects on the function and structure of spermatozoa. It is known that leptin and prolactin play an active role in decreasing the rates of reactive oxygen species and DNA fragmentation, as well as enhancing sperm motility. Hence, this experiment aimed to investigate the effects of leptin and prolactin as pro-survival factors on the normozoospermic human semen samples during cryopreservation. MATERIAL AND METHODS: Semen samples were collected from 15 healthy, fertile men ranging from 25 to 40 years. Cryopreservation of the samples was performed in liquid nitrogen over a period of two weeks, using five varying concentrations of leptin/prolactin, 0, 10, 100, 500, and 1000ng/ml respectively. Sperm motility, total caspase activity, and mitochondrial and cytosolic ROS were measured by flowcytometry, TUNEL, and other appropriate tests after thawing of the samples. RESULTS: Both hormones were observed to have positive effects on the motility of the samples post-cryopreservation, the highest improvement being in the 100ng/ml concentration leptin and prolactin in comparison to the control group (P=0.01 and P=0.041, respectively). A significant reduction of mitochondrial ROS was also observed in 100 and 1000ng/ml of leptin (P=0.042), and there was a considerable decrease in the cytosolic ROS in the 100ng/ml of prolactin in comparison to the control group (P=0.048). Total caspase activity was also highly reduced in the 100, 500, and 1000ng/ml of leptin compared to the control group (P=0.039). Interestingly, both hormones also significantly decreased DNA fragmentation in 1000ng/ml compared to the control group (P=0.042). CONCLUSION: It can be concluded that leptin and prolactin act as protective agents against cryodamage to spermatozoa during cryopreservation.

Cyclic adenosine monophosphate (cAMP) analog and phosphodiesterase inhibitor (IBMX) ameliorate human sperm capacitation and motility.

OBJECTIVES: Human sperm quality is decreasing progressively. One of the foremost reasons for infertility is the failure in sperm capacitation. We examined the influence of a cAMP (cyclic-adenosine mono phosphate analog)+IBMX (3-isobutyl-1-methylxanthine) on the motility and capacitation rate of human sperm over time. MATERIAL AND METHODS: Samples were gotten from 20 asthenozoospermic infertile patients referring to the Academic Center for Education, Culture and Research unit of the infertility research center, Qom, Iran. Samples were processed with a Density Gradient Centrifuging. Spermatozoa were divided into 4 groups: control, experimental 1, 2 and 3 (E1, E2, E3) based on the dose/time schedules (cAMP 5mmol+IBMX 0.2mmol/2, 4, and 6h, respectively). The computer-assisted sperm analysis and chlortetracycline assays were used to measure sperm motility and capacitation. RESULTS: After incubation with a cAMP analog and IBMX, the levels of progressive motile sperms considerably improved in all experimental groups compared to the control group (E1=18.89±7.1, E2=30±9.7, E3=26.3±9.6 vs Control=10.28±6.2, P<0.05) especially in E2 group (P<0.05), indicating a greater effect of db cAMP (5mmol) and IBMX (0.2mmol) for 4h compared to the same doses at 2 and 6h. Also, non-progressive motile sperms significantly decreased in E2 group compared to the other groups (P<0.05). Moreover, both patterns C and B were substantially improved in all experimental groups especially in E2 group (P<0.05). CONCLUSION: Our findings support that the supplementation of sperm with db cAMP+IBMX specially for 4h, could be useful for men with asthenozoospermia to improve the success of assisted reproductive technology.

Expression of RXFP2 receptor on human spermatozoa and the anti-apoptotic and antioxidant effects of insulin-like factor 3.

Insulin-like factor 3 (INSL3) has an important role in the human reproductive system; however, its detailed function is still mysterious. We aimed to investigate the possibility of expression of RXFP2 receptor on human spermatozoa and to determine the anti-apoptotic and antioxidant mechanism derived the binding of INSL3 and RXFP2. In this experimental study, the expression/location of the RXFP2 receptor was determined on the spermatozoa of fertile and infertile men. Twenty samples from 20 fertile men were collected and divided into 6 parts (control group, and five groups treated with INSL3 10, 100, 250, 500, 1,000 ng/ml). DNA damage, active caspase, reactive oxygen species (ROS) and sperm parameters were evaluated by TUNEL, flow cytometry, optical microscope and computer-assisted sperm analysis. The expression of RXFP2 was confirmed by Western blot. Immunocytochemistry illustrated that this receptor is expressed in the posterior half of the spermatozoa's head. The INSL3 at concentrations of 500 and 1,000 ng/ml reduced the active caspase and mitochondrial ROS, and also reduced DNA fragmentation at 1,000 ng/ml. Besides, INSL3 500 and 1,000 ng/ml significantly increased the sperm motility. This study confirmed the presence of RXFP2 receptor in fertile and infertile men's spermatozoa, indicating the highly dose-dependent efficacy of the INSL3, which may have promising impacts on the in-vitro fertilisation outcomes.

Effect of insulin on functional parameters of human cryopreserved sperms.

Cryopreservation of sperms is common therapy but with multiple damages to sperms. The aim of this study was to assess the effect of insulin as a prosurvival factor on the most important functional parameters of human spermatozoa during cryopreservation. Semen samples were obtained from 15 normozoospermic men at age 25-40 years of old through masturbation. Cryopreservation of sperms was conducted along with adding 10, 100, 500 and 1000 (ng/ml) insulin and a control group was also considered by adding distilled water. Samples were cryopreserved for 2 weeks in liquid nitrogen. Then, after thawing sperm motility; cytosolic/mitochondrial reactive oxygen species (ROS) levels; and DNA fragmentation were analyzed. Data were analyzed by SPSS software using one-way ANOVA. Results showed that insulin at all doses significantly decreased cytosolic ROS especially in 10 ng/ml group (P˂0.05). Mitochondrial ROS also decreased by adding insulin in comparison to the control group, although unmeaningfully (P˃0.05). Insulin at 1000 (ng/ml) decreased DNA fragmentation, significantly (P˂0.05). Also, the number of motile sperms increased in all insulin groups but it wasn't meaningful (P˃0.05). Based on our findings adding insulin to semen leads to protecting effects against cryopreservation damages and increases sperms motility. Therefore, using insulin for human semen seems to could be suggested for future clinical applications.

Development of a High-Resolution Melting Analysis Method for CYP2C19*17 Genotyping in Healthy Volunteers.

BACKGROUND: Genetic polymorphisms of drug metabolisms by cytochrome P450 (P450s) could affect drug response, attracting particular interest in the pharmacogenetics. Due to the importance of CYP2C19* 17 allele and its capability of super- fast metabolism and also lack of information about distribution of the alleles in Iranian population, this research aimed to use High Resolution Melting (HRM) method compared to PCR-RFLP for genotyping healthy Iranian population. METHODS: Blood samples were collected from 100 healthy Iranian volunteers. DNA was extracted by salting out method. Real-time PCR was used for amplification of the CYP2C19 gene and the alleles were identified by HRM. Sequencing was used to confirm the amplified DNA fragments and data were analyzed using SPSS software ver.18. RESULTS: The frequency of alleles CYP2C19*1/*1, CYP2C19*1/*17 and CYP2C19*17/*17 were estimated as 58.33, 29.1 and 11.1%, respectively. Specificity and sensitivity of HRM method were 90% and 100%, with respect to PCR-RFLP. Also, HRM analysis has been evaluated as a faster and more effective approach. CONCLUSION: Comparison of our results based on HRM analysis with PCR-RFLP showed that our developed method is rapid, accurate, fast and economic to study the CYP2C19*17 allele and it is appropriate for other similar population genetic studies.

Opium could be considered an independent risk factor for lung cancer: a case-control study.

BACKGROUND: Lung cancer is the leading cause of cancer-related death worldwide, and half of all incident lung cancers are believed to occur in the developing countries, including Iran. OBJECTIVE: We investigated the association of opium with the risk of lung cancer in a case-control study. METHODS: We enrolled 242 cases and 484 matched controls in this study. A questionnaire was developed, containing questions on basic demographic characteristics, as well as lifelong history of smoking cigarettes, exposure to passive smoking, opium use and alcohol consumption. For smoking cigarettes and opium and also oral opium intake frequency, duration and cumulative use were categorized into three groups: no use, low use and high use. Conditional logistic regression was used to calculate the odds ratios (ORs) and 95% confidence intervals (CIs). RESULTS: Multivariate analysis in men showed that after adjusting for the effect of ethnicity, education and pack years of smoking cigarettes, smoking opium remained as a significant independent risk factor with an OR of 3.1 (95% CI 1.2-8.1). In addition, concomitant heavy smoking of cigarettes and opium dramatically increased the risk of lung cancer to an OR of 35.0 (95% CI 11.4-107.9). CONCLUSION: This study demonstrated that smoking opium is associated with a high risk of lung cancer as an independent risk factor.

Genetic Variation and Selection of Domain I of the Plasmodium vivax Apical Membrane Antigen-1(AMA-1) Gene in Clinical Isolates from Iran.

BACKGROUND: Apical Membrane antigen 1 (AMA-1) is positioned on the surface of merozoite and it may play a role in attack to red blood cells. The main aim of present study was to determine the genetic variation, as well as, to detect of selection at domain I of AMA-1 gene Plasmodium vivax isolates in Iran. METHODS: Blood samples were collected from 58 patients positive for P. vivax, mono infection and the domain I of AMA-1 gene was amplified by nested PCR and then sequenced. RESULTS: A total 33 different haplotypes were identified among 58 Iranian sequences. The 23 new haplotypes were determined in this study that was not reported previously in other regions of the world. There were totally observed 36 point mutations at the nucleotide level in the analyzed sequences. Sequences analyses indicated 25 amino acid changes at 20 positions in which 5 sites demonstrated thrimorphic polymorphism and the others were dimorphic in the domain I of the Iranian PvAMA-1 isolates. CONCLUSION: Our findings indicated relatively high level of allelic diversity at the domain I of PvAMA-1 among P.vivax isolates of Iran. Since, PvAMA-1 is considering as vaccine candidate antigen, these data provide valuable information for the development of a PvAMA-1 based malaria vaccine.

Role of endogenous vitamin E in renal ischemic preconditioning process: differences between male and female rats.

BACKGROUND: Antioxidants such as α-tocopherol (vitamin E) and ß-carotene (vitamin A) play an important role in protective effect of repeated brief periods of ischemia, namely ischemic preconditioning (IPC). Values of these antioxidants were investigated and compared after induction of ischemia reperfusion (IR) and kidney IPC in both male and female rats. METHODS: Forty eight Wistar rats were divided randomly into six groups of 8: groups A and B (male and female controls, respectively), group C (male IR or IR cases), group D (female IR cases) and groups E and F (male and female IPC cases, respectively). In groups C and D, ischemia was induced by clamping of left renal arteries for 45 min. In groups E and F, rats underwent four cycles of 4 min of arterial clamping and 11 min of de-clamping before final 45 min ischemia induction. Afterward, serum was collected to assess the blood urea nitrogen, creatinine and vitamins E and A values. Renal tissues were obtained for histological assessments. RESULTS: α-tocopherol levels in male and female rats showed a significant increase in IPC compared with IR group (P<0.01) and also in female IPC compared with male IPC group. ß-carotene levels had no significant variations. Histological evaluation showed that IR-induced renal injuries were less in female rats. Also, protective effects of IPC were more in female rats (P<0.01). CONCLUSIONS: Renal IPC reduced damages in both male and female rats, but tissue injuries in females were decreased much more along with the increase of endogenous vitamin E.

Ginger as a miracle against chemotherapy-induced vomiting.

BACKGROUND: Vomiting is one of the most prevalent side effects of chemotherapy in cancer patients. The aim of this study was to evaluate the effect of ginger plant on chemotherapy-induced vomiting, since the previous studies were somehow imperfect and have provided controversial results. MATERIALS AND METHODS: This randomized double-blind placebo-controlled clinical trial was conducted on 80 women with breast cancer undergoing chemotherapy and suffering from vomiting in Imam Khomeini Hospital, Tehran, Iran, between July and December 2009. During a convenience sampling the participants were randomly allocated into treatment and placebo groups after taking a written informed consent. Two groups were matched based on the age and emetic risk of chemotherapy drugs. The treatment group received 250 mg ginger powder capsules (Zintoma) and placebo group 250 mg starch capsules 4 times a day (1 g/day) for 6 days since 3 days before chemotherapy session. A two-part self-made questionnaire was used to assess the effect of ginger. Patients completed the instrument every day. Then by STATA software version 8, the gathered data were analyzed using Fisher's exact, Kruskal-Wallis, and Chi-square tests. RESULTS: The 2 groups had no significant age differences and were matched (ginger: 41.8±8.4 vs placebo: 45.1±10, P = 0.1). Vomiting cases were significantly lower in ginger group at anticipatory (P = 0.04), acute (P = 0.04), and delayed (P = 0.003) phases. Also, heartburn was the only and venial reported side effect (P > 0.05). CONCLUSIONS: Taking ginger capsules (for 6 days since 3 days before chemotherapy) accompanied by the routine antiemetic treatment could relieve chemotherapy-induced vomiting in all phases.

Consequences of Ischemic Preconditioning of Kidney: Comparing between Male and Female Rats.

OBJECTIVES: Ischemia-reperfusion injury (IRI) is a leading cause of kidney transplantation failure, and ischemic-preconditioning (IPC) is a protective method against the IRI. In the present study, the defensive effect of IPC on rats' kidney was investigated and more importantly the differences between two genders were appraised. MATERIALS AND METHODS: THIRTY TWO WISTAR RATS WERE RANDOMLY ALLOCATED TO FOUR GROUPS: group A (8 male IR), B (8 female IR), C (8 male IPC) and D (8 female IPC). Ischemia was induced by clamping of left renal arteries for 45 min in groups A and B. Rats in groups C and D experienced four cycles of 4 min arterial clamping followed by 11 min of de-clamping prior to the final 45 min of ischemia. 24 hr later, serum was provided to assess the blood urea nitrogen (BUN) and creatinine values. Also, renal tissues were obtained for histological measurements. RESULTS: Induction of IPC in both male and female rats led to significant decrease in creatinine levels in comparison with sham groups (P<0.01). The same results were seen in BUN levels (P<0.01). However, there were no significant difference between two genders. Besides, histological protective effects of IPC was proved especially in female rats (P<0.01). CONCLUSION: Findings of our study confirmed that renal IPC reduces the damages in both genders especially females. Thus, the IPC procedure seems to be a useful method mainly in females.

Self-care behavior and affecting factors among patients with heart failure in Iran.

OBJECTIVE: To diagnose self-care behavior of heart failure (HF) patients and their correlations with the affecting variables such as age, gender, marriage, educational status, disease duration, left ventricular ejection fraction, and contextual chronic diseases. METHODS: This descriptive-analytical study was conducted in 5 hospitals of Tehran and Iran Universities of Medical Sciences, Tehran, Iran between March to April 2006. After providing written consent, 250 eligible patients participated. We used the Persian version of the European Self-Care Behavior questionnaire to collect information. The gathered data were analyzed by descriptive statistics, one-way ANOVA, independent-t, and other appropriate tests using SPSS software version 11.5. RESULTS: We found out that only 26% of patients had good self-care and the behavior of `I take my medications as prescribed` was the most performed behavior. These behaviors had a significant reverse relationship with age and re-hospitalization rate, and a direct relationship with other variables (p=0.0001). CONCLUSION: It is necessary to understand self-care behaviors of HF patients, and then offer individual and special educational programs regarding these behaviors, and also to follow these behaviors. Administration of these programs by nurses will raise HF patients' capabilities and quality of life.