Synthesis and antimicrobial activity of some new sugar-based monocyclic beta-lactams.

The syntheses of some new sugar-based monocyclic beta-lactams possessing several other functionalities in addition to the carbohydrate moiety are described. The key step was the Staudinger [2+2] cycloaddition of chiral carbohydrate Schiff base 5 with phthalimidoacetyl chloride to yield the sugar-based monocyclic beta-lactam 6 as a single isomer. Treatment of protected beta-lactams 6 and 8 with methylhydrazine afforded the free amino beta-lactams 9 and 10. Acylation of these free amino beta-lactams with benzoyl, phenoxyacetyl, cinnamoyl and phenylacetyl chloride in the presence of pyridine afforded beta-lactams 11a-d and 12a-d. Some of these novel beta-lactams were found to be active against Staphilococcus citrus, Klebsiella pneumoniae, Escherichia coli and Bacillus subtilis.

Asymmetric synthesis and antimicrobial activity of some new mono and bicyclic beta-lactams.

Reaction of the amino acid D-phenylalanine ethyl ester (4) with cinnamaldehyde gave chiral Schiff base 5, which underwent an asymmetric Staudinger [2+2] cycloaddition reaction with phthalimidoacetyl chloride to give the monocyclic beta-lactam 6 as a single stereoisomer. Ozonolysis of 6 followed by reduction with lithium aluminum tri(tert-butoxy) hydride afforded the hydroxymethyl beta-lactam 8. Treatment of 8 with methansulfonyl chloride gave the mesylated monocyclic beta-lactam 9, which was converted to the bicyclic beta-lactam 10 upon treatment with 1,8-diazabicyclo[5,4.0] undec-7-ene (DBU). Deprotection of the phthalimido group in beta-lactams 6 and 10 by methylhydrazine and subsequent acylation of the free amino beta-lactams with different acyl chlorides in the presence of pyridine afforded mono and bicyclic beta-lactams 14a-d and 15a-d respectively. The compounds prepared were tested against Escherichia coli, Staphilococcus citrus, Klebsiella pneumanie and Bacillus subtillis. Some of these compounds showed potential antimicrobial activities.

Value of clinical diagnosis in predicting the quality of cryopreserved sperm from cancer patients.

PURPOSE: Cryopreservation of semen from patients with malignant diseases (primarily testicular cancer, Hodgkin's disease and lymphoma) before specific chemotherapy, radiation or surgical therapy has become a realistic option to preserve fertility. Nevertheless, the post-thaw sperm quality in these patients is poor. Whether the clinical diagnosis of malignancy affects the cryopreservation of human sperm is unknown. Such an association could allow post-thaw sperm quality to be predicted. MATERIALS AND METHODS: We compared the effect of cryopreservation on sperm motility, velocity, linearity, amplitude of lateral head movement, motility index and motile sperm count in specimens from 30 normal men, 34 with testicular cancer, 39 with Hodgkin's disease, and 19 with leukemia and lymphoma who were referred for sperm banking during a 5-year period. RESULTS: The pre-freeze and post-thaw motility, motility index and motile sperm count were significantly lower in patients than in normal men (p < 0.01). No significant differences were noted among the patient groups. Similarly, no significant differences were noted after cryopreservation. Our results indicate that the poor pre-freeze sperm quality in some patients with malignancies explains the poor post-thaw results. However, the deterioration in sperm function after cryopreservation of semen among patients with different malignancies and normal donors appears to be similar. CONCLUSIONS: Clinical diagnosis of malignancy is not an adequate predictor of the effect of cryopreservation on human semen.

Excessive testosterone production in a patient with Nelson syndrome and bilateral testicular tumors.

Bilateral primary testicular tumors are rare and usually consist of either interstitial cells or hypertrophic testicular adrenal remnant tissue. Their differentiation on clinical presentation and histologic examination remains difficult but is essential because of the different therapeutic approaches. We report a rare case of excessive testosterone production by bilateral testicular tumors in a patient with Nelson syndrome (ACTH-secreting pituitary adenoma after bilateral adrenalectomy in patients with Cushing's disease). Increased ACTH stimulation in this patient supports the thesis of pluripotent cells within the testis which can undergo differentiation to cells which are not only morphologically similar to Leydig cells but also have the functional property of these cells. Our clinical findings support the diagnosis of hyperplasia of adrenal remnant or pluripotent cells rather than a true Leydig cell tumor. We emphasize the need for hormonal evaluations which should be assessed in the context of the size of these nodular tumors prior to therapeutic decisions. In cases with elevated serum ACTH and small nodular hyperplasia, we would favor a 'wait-and-see' strategy with appropriate hormonal therapy. In large tumors with clinical signs of hormonal activity, patient noncompliance with steroid replacement regimens or with local symptoms, scrotal exploration and tumor enucleation are indicated.

Effect of cryopreservation on semen quality in patients with testicular cancer.

OBJECTIVES: Current techniques in cryopreservation of human semen substantially decrease sperm quality. In addition, the pregnancy rate using cryopreserved sperm obtained from testicular cancer patients is lower than when sperm from normal fertile men is used. However, it is still unclear whether cryopreserved sperm from these patients is inherently defective or if the sperm loses its motility after thawing. This study was undertaken to assess the effect of cryopreservation on the quality and motion characteristics of semen from patients with testicular cancer before definitive therapy compared with semen from normal volunteers. METHODS: We compared the sperm quality before and after cryopreservation in samples from 34 patients with testicular cancer and 30 normal volunteers who were referred for sperm banking over a 7-year period. The effects of cancer stage and histologic type on various semen parameters were also examined. A computer-assisted semen analysis was performed before and after cryopreservation on each specimen. The nitrogen vapor technique using Test yolk buffer with glycerol as a cryoprotective agent was used for cryopreservation. The motile sperm count and motion characteristics (motility, velocity, linearity, amplitude of the lateral head movement, motility index) were analyzed before and after cryopreservation and compared between the groups. RESULTS: Semen quality did not significantly differ among patients with Stage I, II, or III cancer. However, semen quality tended to be poorer at higher cancer stages. In general, semen quality was better among patients with pure seminomas than with pure embryonal tumors; quality was worst among patients with mixed germ cell tumors. However, 71.4% of patients with mixed tumors presented with Stage III disease, whereas all patients with seminomas presented with Stage I disease. Significant differences were also seen in prefreeze motility (median, 42% [interquartile range, 24 to 51] versus 60.5% [range, 49 to 73]; P = 0.0004) and motile sperm count (6.7 x 10(6)/mL [range, 3.4 to 14.4] versus 50.0 [range, 24.6 to 72.0]; P = 0.0001) in patients compared with controls, respectively. The motile sperm count and percent motility significantly decreased in both patients and controls after cryopreservation (P = 0.0001). However, the percentage decline in motile sperm count and motion characteristics after cryopreservation did not differ significantly between patients and controls (P > 0.01). CONCLUSIONS: We conclude that the effect of cryopreservation on sperm quality in patients with testicular cancer is identical to its effect on sperm from normal fertile men. Differences in values after preservation are explained by poor semen characteristics before freezing; semen quality declines with more extensive disease. Stage I patients also had poorer quality than control subjects. Thus, we recommend that routine sperm banking be encouraged among all patients with testicular cancer before the initiation of specific medical treatment. We also recommend that future efforts be focused on improving the technique of sperm banking.

Optimum abstinence time for cryopreservation of semen in cancer patients.

Although an abstinence period of 48 to 72 hours is the most commonly prescribed interval for diagnostic semen analysis, to our knowledge the association between the abstinence period and sperm quality after cryopreservation has not been identified. Patients with a malignant disease who are consulting for semen banking most often require urgent therapy. Consequently, defining the shortest abstinence period that allows for frequent semen collection within a limited interval and the best post-thaw sperm quality is necessary. We investigated the relationship between abstinence period, and the pre-freeze and post-thaw motility variables in semen specimens obtained from cancer patients for sperm banking. Samples collected from 95 patients were divided according to abstinence period: group 1-15 patients at 24 to less than 48 hours, group 2-53 at 48 to less than 72 hours and group 3-27 at more than 72 hours. Pre-freeze and post-thaw motile sperm count and motion variables (motility, velocity, linearity, amplitude of lateral head movement and motility index), and percentage decrease in sperm variables after cryopreservation were analyzed. Semen volume, the pre-freeze and post-thaw motile sperm count, motion parameters and the percentage decrease in semen variables did not differ significantly among the groups. We conclude that semen collection for cryopreservation after 24 to less than 48 hours of abstinence results in post-thaw quality comparable to that after an abstinence of 48 to less than 72 hours or longer. Thus, an abstinence period of 24 to less than 48 hours can be recommended for sperm banking in cancer patients.

Cryopreservation and semen quality in patients with Hodgkin's disease.

BACKGROUND: Cryopreservation of semen from patients with Hodgkin's disease yields fewer motile sperm than from fertile men without Hodgkin's disease. However, although poor sperm quality and subfertility have been associated with Hodgkin's disease, whether the disease adversely affects sperm quality is not clear because many studies evaluated semen quality after chemotherapy or radiation therapy had begun. Furthermore, the effect of cryopreservation on semen quality in these patients is unknown. This study investigated pretreatment sperm quality and the effect of cryopreservation on semen quality in patients with Hodgkin's disease. METHODS: Specimens from 39 patients with Hodgkin's disease and 30 normal volunteers who underwent sperm banking over a 5-year period were analyzed. No patient had undergone chemotherapy or radiation therapy before sperm banking. The nitrogen vapor technique, using Test-Yolk buffer with glycerol as a cryoprotective agent, was used for cryopreservation. Prefreeze and postthaw motile sperm count (MSC) and motion characteristics, namely motility, curvilinear velocity (VCL), linearity, amplitude of lateral head movement (ALH), and motility index, were compared between the two groups. RESULTS: Prefreeze values for MSC (P = 0.0001), motility (P = 0.0001), motility index (P = 0.0001), and VCL (P = 0.0019) differed significantly between patients and donors. Except for linearity and ALH, postthaw sperm MSC, motility, VCL, and motility index decreased significantly (P = 0.0001) in both groups. However, the percentage decline in semen quality from prefreeze to postthaw values did not differ significantly between donors and patients. CONCLUSION: The pretreatment semen quality in patients with Hodgkin's disease is poor compared with that of normal fertile men. However, half the patients had a normal MSC, so a clinical diagnosis of Hodgkin's disease does not predict cryopreservation outcome adequately. Semen cryopreservation should be encouraged as a routine part of the therapeutic management of men of reproductive age who will undergo chemotherapy or radiation therapy for Hodgkin's disease.

Effects of time and sperm concentration on reactive oxygen species formation in human semen.

Although generally accepted standards exist for routine semen analysis, recent methods of assessing reactive oxygen species (ROS) in human semen lack a standardized protocol. The purpose of this study was to investigate (1) the relationship between ROS level and the time interval between semen collection and analysis, and (2) the effect of sperm concentration on the level of ROS formation. Semen specimens from men (n = 40) consulting for infertility treatment were divided in two groups: in 20, routine semen analysis was performed and ROS formation evaluated at 1, 3, 5, and 24 h after semen collection; in the other 20, ROS formation was evaluated at four sperm concentrations (60, 30, 15, and 7.5 x 10(6)/mL). White blood cell (WBC) concentration was assessed before ROS measurement using a myeloperoxidase staining technique (Endtz test). ROS level was measured by a chemiluminescence method. ROS formation decreased significantly over time. The mean ROS level 343.4 (1 h), 133.5 x 10(4) cpm (3 h, p = .004), 66.0 x 10(4) cpm (5 h, p < or = .001), and 22.2 x 10(4) cpm (24 h, p < or = .001), respectively. In the first group of 20 specimens, 14 were positive for ROS formation at 1 h after collection, and 4 of these were positive for the Endtz test (> 1 x 10(6) WBC/mL). The number of ROS-positive specimens after 3, 5, and 24 h was eight, six, and two, respectively. In the second group, eight patients were positive for ROS formation at 1 h after collection.(ABSTRACT TRUNCATED AT 250 WORDS)

Positive myeloperoxidase staining (Endtz test) as an indicator of excessive reactive oxygen species formation in semen.

PURPOSE: Although the significance of leukocytospermia in semen remains controversial, evidence exists that white blood cells (WBCs) may adversely affect sperm function and act as a potential cofactor in male infertility. Nevertheless, the mechanisms by which leukocytes may alter sperm function in vitro is unknown. Recent investigations suggest that reactive oxygen species (ROS) generated by the polymorphonuclear (PMN) granulocytes could adversely affect sperm function. The objective of this study was to investigate the relationship between the presence of leukocytospermia as determined by the Endtz test and the excessive formation of ROS. METHODS: The WBC concentration and ROS formation in human semen, obtained from men consulting for infertility, were assessed and compared to that of normal donors. ROS was measured by a chemiluminescence assay using luminol and a Berthold luminometer. The WBC concentration was determined with a myeloperoxidase staining technique (Endtz test). Specimens were obtained from 94 subjects (20 donors, 74 patients). RESULTS: Of the 20 donors, 2 were Endtz positive and ROS positive; 18 were Endtz negative with 2 (11%) ROS positive. In the patient group (n = 74), 26 (35%) were ROS positive, and 17 were Endtz positive and found to be ROS positive. Of the 57 Endtz-negative patients, 9 (15%) were ROS positive. The positive Endtz test results correlated strongly with positive ROS formation in patients and donors (P < 0.001). CONCLUSIONS: Our results indicate that the simple, cost-efficient Endtz test could be used as an indicator of excessive ROS formation in semen.

Incidence and level of seminal reactive oxygen species in normal men.

OBJECTIVES: Excessive formation of reactive oxygen species (ROS) in human semen has been associated with impaired sperm function and fertility potential. The presence of ROS in semen specimens from normal fertile men emphasizes the importance of defining a normal range of ROS formation. The purpose of this study was to establish a normal range of ROS generation and to investigate the effect of sperm concentration on the ROS level. METHODS: ROS was determined in 15 healthy donors and 20 men with suspected infertility. After the sperm concentration in normal donors was adjusted to 20 x 10(6)/mL, ROS was measured by chemiluminescence using luminol in a Berthold luminometer. A specimen was regarded as positive (abnormal) when the value was at least 10 x 10(4) counts per minute (cpm). ROS was also evaluated at 4 sperm concentrations (60, 30, 15, and 7.5 x 10(6)/mL) from samples obtained from the patients with suspected infertility. In addition, ROS was measured in 7 ROS-positive specimens at a sperm concentration of 15 x 10(6)/mL and 60 x 10(6)/mL. RESULTS: Results showed that ROS formation was negative in all 15 healthy donors (median, 0.9 x 10(4) cpm; interquartile range, 0 to 1.48 x 10(4) cpm). The ROS formation value among all the donors was less than 5.5 x 10(4) cpm. ROS formation was positive in 8 (40%) of the suspected infertile patients. ROS levels were significantly lower at sperm concentrations of 15 x 10(6)/mL or 7.5 x 10(6)/mL compared with 30 x 10(6)/mL or 60 x 10(6)/mL (P = 0.05). The ROS level increased after centrifugation for 10 minutes at 500 g in all 7 specimens at both 15 and 60 x 10(6)/mL. However, the increase in ROS formation at 60 x 10(6)/mL was significantly greater than that at 15 x 10(6)/mL (P < 0.001). CONCLUSIONS: A range of ROS formation of 0 to 5.5 x 10(4) cpm at a sperm concentration of 20 x 10(6)/mL may be considered as normal for healthy donor semen. The positive relationship between ROS formation and sperm concentration at the time of measurement emphasizes the importance of concentration adjustment before analysis when comparing ROS levels between different specimens.

A method of human semen centrifugation to minimize the iatrogenic sperm injuries caused by reactive oxygen species.

Current techniques of sperm preparation for in vitro fertilization or intrauterine insemination require centrifugation of human semen to separate spermatozoa from the seminal plasma. Centrifugation increases reactive oxygen species (ROS) formation in semen. Moreover, high levels of ROS are associated with sperm membrane injury through spontaneous lipid peroxidation, which may alter sperm function. We investigated the relationship between centrifugation variables (time and g-force) and ROS production to establish an optimal centrifugation protocol for sperm preparation techniques. Semen from 38 men (24 patients and 14 normal volunteers) was evaluated for the formation of ROS before centrifugation and after centrifugation at 200 g for 2 or 10 min and after 500 g for 2 or 10 min. The absence of white blood cells in semen which can also produce ROS was determined with the myeloperoxidase technique (Endtz test). All specimens were negative (< 1 x 10(6)/ml) by the Endtz test. The formation of ROS was measured by chemiluminescence. ROS formation was regarded as high (positive) when the chemiluminescence response was at least 10 x 10(4) counted photons/min (cpm). The sperm concentration in each sample was adjusted to 15-20 x 10(6) cells/ml before analysis. Eight specimens (7 patients and 1 donor) exhibited high levels of ROS before centrifugation. All 8 showed further, significant increases in ROS formation regardless of g-force or time. The increase in ROS was significantly less when semen was centrifuged for 2 as compared to 10 min (p < 0.001). Six specimens previously ROS-negative became ROS-positive after centrifugation for 10 min at 200 and 500g. We conclude that the time of centrifugation is more important than g-force for inducing ROS formation in semen. Based on these results, we recommend a shorter centrifugation period in the preparation of sperm for assisted reproductive techniques.

Microsurgical vasoepididymostomy: a comparison between the end-to-side anastomosis and the invagination technique.

In this study we compared the invagination technique with the commonly used end-to-side anastomosis. A total of 30 invaginations and 30 end-to-side anastomoses was performed randomly in 30 Wistar rats. We checked the patency of the anastomoses 4 months after operation by macroscopic examination, spermiogram, methylene blue injection, and X-radiography studies as well as histology. The examination of the anastomoses showed patency in 19 (63.2%) compared with 24 patent invaginations (80%). The introduction of the simple time-saving technique of invagination could be useful for clinical purposes, since it is easier to learn and to practise and might also improve the clinical results concerning the patency of anastomoses.