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Purpose: The purpose of this study was to determine if there is an increased incidence of hypospadias in male offspring conceived by in vitro fertilization (IVF).Materials and Methods: A retrospective institutional chart review from 1988 to 1992 and data from the Maryland Birth Defects Registry were statistically analyzed to assess the risk of hypospadias with IVF.Results: The data for the 5-year period indicated a 5-fold increased risk of hypospadias after IVF, with an incidence of approximately 1.5% in the IVF group and 0.3% in the control group. The only recognized difference between the groups was maternal progesterone administration in the IVF group but the cause of the increased risk of hypospadias was unknown. The distribution of hypospadias severity was similar in both groups.Conclusions: Male newborns conceived by IVF have a 5-fold increased risk of hypospadias, which may be related to maternal progesterone administration, or other maternal or fetal endocrine abnormalities that may or may...

Objective To determine if nitric oxide synthase activity within the male reproductive tract is regulated by androgen. Design Nitric oxide synthase activity was measured in the reproductive organs of three groups of mature rats: unoperated controls, 1-week castrates, and 1-week castrates given T capsules at the time of surgery. The presence of nitric oxide synthase activity was confirmed by using the nitric oxide synthase-specific inhibitor N -nitro-l-arginine methyl ester (l-NAME). Results After castration, nitric oxide synthase activity was significantly reduced by 88%, 73%, and 54% in the caput, corpus, and cauda epididymidis, respectively. In the penis, nitric oxide synthase activity decreased 45% and nitric oxide synthase protein decreased 57% after castration. In the seminal vesicle and lateral prostate, nitric oxide synthase activity increased significantly after castration from nondetectable levels in controls. Nitric oxide synthase activity in the coagulating gland and ventral and dorsal prostate did not change after castration. The changes in nitric oxide synthase activity in all organs after castration were prevented by T replacement. Additionally, the activity measured in every organ in all three treatment groups was >90% inhibited by l-NAME. Conclusion These data demonstrate that androgen differentially affects nitric oxide synthase activity in the male reproductive tract. To the best of our knowledge this is the first time that nitric oxide synthase activity has been shown to be influenced by androgen in any tissue.

We employed retrograde axonal tracing techniques and nitric oxide synthase (NOS) and choline acetyltransferase (ChAT) immunohistochemistry to identify NOS-containing neuronal populations within the lumbosacral spinal cord and determine whether these project to the major pelvic ganglion in the adult male Sprague-Dawley rat. Immunohistochemical localizations of NOS included neurons situated at the L5 to S2 segments of the spinal cord, which corresponded to the sacral parasympathetic nucleus. Another prominent locus for NOS was a group of neurons identified in the L1 segment corresponding to the dorsal commissural nucleus. These regions correlated directly with preganglionic parasympathetic and sympathetic neuronal origins, respectively, which were established with ChAT colocalizations. Retrograde tracing verified the projection of these neurons to the pelvis. Additional neuronal localizations of NOS were observed throughout the intermediolateral cell column, involving the superficial laminae of the dorsal horn, in the region surrounding the central canal and occasionally in the medial area of the ventral horn. These results indicate that the regulation of pelvic visceral activity may involve NO-based neuronal mechanisms operating at the level of the lumbosarcal spinal cord.

Nitric oxide synthase (NOS), which catalyzes the production of nitric oxide (NO), was characterized within the reproductive tract of adult male Sprague-Dawley rats by means of biochemical and immunohistochemical techniques. Tissues examined included the testis, epididymis (caput, corpus, and cauda regions), vas deferens, ejaculatory duct, seminal vesicle, and coagulating gland. NOS activity was measured by use of an assay based on the stoichiometric conversion of [3H]-L-arginine to [3H]-L-citrulline and NO, catalyzed by NOS. Low levels of NOS activity were detected in the testis and seminal vesicle (< 0.5 fmol [3H]-L-citrulline formed/min/mg protein in each tissue). The highest levels of NOS activity were present in the cauda segment of the epididymis and in the vas deferens, each having a sevenfold greater amount of NOS activity than the testis (p < 0.05). Intermediate levels of NOS activity were detected in the coagulating gland (0.863 +/- 0.248 fmol [3H]-L-citrulline formed/min/mg protein), caput epididymidis (0.457 +/- 0.180 fmol [3H]-L-citrulline formed/min/mg protein), and corpus epididymidis (0.631 +/- 0.215 fmol [3H]-L-citrulline formed/min/mg protein). NADPH diaphorase histochemistry and NOS immunohistochemistry localized NOS to neuronal fibers coursing throughout the smooth musculature and subepithelial regions of the epididymis, vas deferens, and ejaculatory duct. Endothelial cells and nerve plexuses within the adventitia of blood vessels supplying reproductive tissues were also positive for NOS. Additional localizations of NOS were within epithelial cells of the epididymis and coagulating gland.(ABSTRACT TRUNCATED AT 250 WORDS)

The regulation and possible function of the preproenkephalin gene in testis were studied in vivo in transgenic mice containing: (1) bases -193 to +210 of the human proenkephalin gene and an additional one kilobase of 3' proenkephalin flanking sequence driving expression of bacterial chloramphenicol acetyltransferase (CAT), and (2) the same promoter and flanking sequences driving expression of a rat proenkephalin cDNA. Five lines of mice, designated HEC1-5, expressed the first construct and 10, HER1-10, the second. Each HEC male and many HER males showed dramatic expression of the transgene in the testis, although much lower expression was observed in the brain and other enkephalin-producing tissues. High levels of expression in testis can thus be achieved with a very short promoter region and do not require intron A sequences previously considered necessary. Altered enkephalin expression may affect testicular function. One founder, HER8, displayed grossly abnormal testicular morphology and was completely infertile. A second founder, HER6, had low sperm motility. Two offspring from other lines also displayed subnormal fertility. These studies support a role for specific promoter sequences in testis expression and may further support a significant role for proenkephalin in testicular function.

Objectives To determine the long-term effects of ranibizumab (RBZ) in patients with diabetic macular edema (DME). Design Prospective, randomized, interventional, multicenter clinical trial. Participants One hundred twenty-six patients with DME. Methods Subjects were randomized 1:1:1 to receive 0.5 mg RBZ at baseline and months 1, 3, and 5 (group 1), focal or grid laser photocoagulation at baseline and month 3 if needed (group 2), or a combination of 0.5 mg RBZ and focal or grid laser at baseline and month 3 (group 3). Starting at month 6, if retreatment criteria were met, all subjects could be treated with RBZ. Main Outcome Measures The mean change from baseline in best-corrected visual acuity (BCVA) at month 24. Results After the primary end point at month 6, most patients in all groups were treated only with RBZ, and the mean number of injections was 5.3, 4.4, and 2.9 during the 18-month follow-up period in groups 1, 2, and 3, respectively. For the 33 patients in group 1, 34 patients in group 2, and 34 patients in group 3 who remained in the study through 24 months, the mean improvement in BCVA was 7.4, 0.5, and 3.8 letters at the 6-month primary end point, compared with 7.7, 5.1, and 6.8 letters at month 24, and the percentage of patients who gained 3 lines or more of BCVA was 21, 0, and 6 at month 6, compared with 24, 18, and 26 at month 24. The percentage of patients with 20/40 or better Snellen equivalent at month 24 was 45% in group 1, 44% in group 2, and 35% in group 3. Mean foveal thickness (FTH), defined as center subfield thickness, at month 24 was 340 μm, 286 μm, and 258 μm for groups 1, 2, and 3, respectively, and the percentage of patients with center subfield thickness of 250 μm or less was 36%, 47%, and 68%, respectively. Conclusions Intraocular injections of RBZ provided benefit for patients with DME for at least 2 years, and when combined with focal or grid laser treatments, the amount of residual edema was reduced, as were the frequency of injections needed to control edema. Financial Disclosure(s) Proprietary or commercial disclosure may be found after the references.

Surgical ablation of the inferior mesenteric plexus in the rat results in an excessive accumulation of sperm within the cauda epididymidis. The present study examined the effect of ablation of the inferior mesenteric plexus on the motility of cauda epididymal spermatozoa. One to 8 weeks after ablation of the inferior mesenteric plexus, sperm curvilinear and straight line swimming velocities were reduced significantly compared with sperm curvilinear and straight line swimming velocities from sham-operated controls. Sperm swimming linearity also changed significantly versus controls after inferior mesenteric plexus ablation. Additional experiments using a vasectomy model suggested that the reductions in sperm curvilinear and straight swimming velocities after inferior mesenteric plexus ablation were not primarily the result of tubal obstruction. These data suggest that the sympathetic nervous system may influence epididymal sperm function.

Background: Subfoveal neovascular membranes cause significant visual loss in age-related macular degeneration (AMD) and the ocular histoplasmosis syndrome. The frequency of post-laser treatment persistence or recurrence of subfoveal membranes in AMD is as high as 51 %. The reason for the high incidence of failure after laser treatment is unknown. The authors performed a histopathologic study of subfoveal membranes to determine the distribution of blood vessels within the neovascular complex, and to see if the blood vessel pattern would provide insight into the reason for laser treatment failure. Methods: The authors used light microscopy to examine serial sections of subfoveal membranes from six patients (4 with AMD, 2 with the ocular histoplasmosis syndrome). The data from this examination were used to create detailed two-dimensional vascular maps of each membrane. Results: The authors found that subfoveal membranes from patients with AMD and the ocular histoplasmosis syndrome, whether occurring de novo or after laser treatment, have a nonuniform distribution of blood vessels, and that large areas which include the membrane margin may be avascular. Conclusions: Using current laser treatment protocols, it is likely that avascular or poorly perfused peripheral areas of the neovascular complex would be left untreated after laser photocoagulation. Partial treatment of the neovascular complex may contribute to the high rate of post-laser treatment persistence or recurrence of subfoveal membranes.

To determine whether or not there is an association between testicular histologic changes and antisperm antibodies in vasectomized men.Morphometry was performed on testicular biopsy specimens obtained from 19 vasectomized men and 21 fertile control subjects. Antisperm antibody status was determined on the serum of each patient and control subject using the indirect immunobead assay.Significant increases in seminiferous tubule wall thickness (p0.001), focal interstitial fibrosis (p0.001), and percent composition of interstitium (p0.01) were observed in vasectomized men as compared with control subjects. Serum antisperm activity was present in 74 percent of the vasectomized men but none in the control subjects (p0.001). There was no association between testicular histologic changes and immune status.Vasectomized men exhibit significant testicular histologic changes and increased autoimmune activity as compared with fertile control subjects. These histologic changes are not directly associated with antisperm antibody status, suggesting that some other pathophysiologic process must be responsible.

The clinicopathologic features of age-related macular degeneration (AMD) with occult choroidal neovascularization (CNV) detected by digital indocyanine green (ICG) videoangiography in an 82-year-old woman are discussed.Serial sections through the macula of both eyes were prepared, and two-dimensional reconstruction maps depicting the histopathologic features were drawn. A technique by which electron microscopic examination of sections removed from glass slides was performed is described.Histopathologic examination of the lesion disclosed a 3.5 mm x 0.02 mm thick fibrovascular subretinal pigment epithelial choroidal neovascular membrane in an eye with diffuse basal laminar deposit in the macula.This case represents the first clinicopathologic correlation involving ICG videoangiography of CNV. The findings support the growing clinical impression that ICG videoangiography is of value in identifying what has been previously described as ill-defined or occult CNV.

An improved understanding of the physiology of penile erection has resulted from recent evidence that implicates nitric oxide as the principal mediator of erectile function. Previously, the neuroanatomy of erection in man was established with descriptions of the autonomic innervation of the pelvic organs and external genitalia. The basis upon which novel physiological concepts of erection relate to earlier neuroanatomical principles remains to be determined. In the present study these relationships were explored with nitric oxide synthase immunohistochemistry and reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry of select pelvic tissue specimens obtained from 4 men (3 at radical prostatectomy and 1 at autopsy). Nitric oxide synthase, the enzyme that catalyzes nitric oxide production, was identified in discrete neuronal locations, including the pelvic plexus, cavernous nerves and their terminal endings within the corporeal erectile tissue, branches of the dorsal penile nerves and nerve plexuses in the adventitia of the deep cavernous arteries. This distribution of nitric oxide synthase-containing nerves suggests that nitric oxide neuronally modulates local vascular smooth musculature of the penis. On this basis, nitric oxide is identified as a neuronal mediator of penile erection in man.

Individual agents within each of the major classes of antibiotics have been shown to have significant adverse effects on spermatogenesis or spermatozoal function in mammals. For humans, infertility or significant alterations in semen parameters have been well documented for the nitrofurans and for patients on sulfasalazine. Other commonly used antibiotics, such as minocycline, have been shown to be toxic to sperm at any concentration. Until further information is available, clinicians must keep in mind that treatment with antibiotics may adversely affect the fertility potential of men. It is possible that some classes of antibiotic agents, such as the penicillins or the quinolones, may have minimal effects on male fertility and maintain the clinical efficacy for patients requiring long-term antibiotic suppressive therapy. Further investigation is needed into the relative toxicity of antibiotics and the mechanisms by which antibiotics affect spermatogenesis and spermatozoal function. A background of the current state of knowledge regarding the adverse effects of antibiotics on male fertility is presented in this review.

Progressive motility was induced in hamster caput epididymal spermatozoa incubated in Tyrodes medium containing 50 mM theophylline, 1.0% Fraction V bovine serum albumin, and 15% (v/v) heat-treated human seminal plasma. Under these induction conditions, however, the maximum percent of caput spermatozoa exhibiting progressive motility (21%) and the time during which motility was sustained (120 min) were significantly less (p less than 0.05) than that of controls from the cauda epididymidis. Moreover, in contrast to caudal spermatozoa, the majority of the induced caput spermatozoa exhibited some degree of flagellar bending at the neck or midpiece. In subsequent experiments the procedure for motility induction was modified to achieve levels of motility in caput spermatozoa equivalent to those observed for caudal spermatozoa. The addition of 5 microM diamide, a sulfhydryl oxidant, to the induction medium prevented the flagellar angularity observed in induced caput sperm preparations. The percentage of caput spermatozoa induced to progressive motility was increased to levels characteristic of caudal spermatozoa (48%) by the addition of hamster caudal epididymal fluid (CEF) to the induction medium. Finally, the viability of the induced caput spermatozoa was significantly enhanced (p less than 0.05) by the removal of Fraction V albumin from the induction medium. In the presence of CEF and in the absence of albumin, 50% of the caput spermatozoa acquired progressive motility and sustained this motility for 4 h. Moreover, when fatty acid-free, charcoal-extracted albumin instead of Fraction V albumin was utilized in the induction procedure, a maximum of 43% of the caput spermatozoa acquired progressive motility and maintained this motility for 4 h, suggesting that the decreased sperm viability observed in the presence of Fraction V albumin was due to a contaminant of albumin, possibly fatty acids. The studies described herein demonstrate for the first time that immature quiescent caput epididymal spermatozoa can be induced to acquire progressive and sustained motility equivalent to that observed in mature caudal epididymal spermatozoa.

The present studies were designed to identify mouse spermatid proteins into which intratesticularly injected [3 HI arginine and �3 HI lysine were initially incorporated and to determine the fates of those proteins during subsequent spermatid differentiation. At intervals between 2 h and 7 days after injections, elongated spermatid nuclei were isolated from the testes by virtue of their resistance to sonication, and mature sperm nuclei were isolated from the epididyrnides. Basic proteins were extracted from isolated spermatid and sperm nuclei and subjected to electrophoresis on acid-urea polyacrylamide gels. Two hours after injection, [3 HI arginine was seen in a number of spennatid basic proteins, including both the “testis’specific” protein (TP) and the protainines. As expected from previous studies, only one class of these labeled proteins, protamine, was retained through the completion of spermiogenesis and sperm maturation 7 days later. In striking contrast, [3 Hi lysine was initially incorporated only into the spermatid TP protein, was retained for only 3 days, and was then lost. Our previous autoradiographic study (Mayer and Zirkin, 1979) demonstrated that intratesticularly injected [3 HI lysine was initially incorporated into elongating sperinatid nuclei at the initiation of chromatin condensation (late step 12 and step 13), was retained for 3 days through the completion of chromatin condensation (step 14), and was then lost. The present results, taken together with the results of our previous autoradiographic study, demonstrate striking temporal relationships between the first appearance of newly synthesized TP protein and the initiation of chromatin condensation in spermatid nuclei of late step 12, and between the loss of TP protein and the completion of chromatin condensation in spermatid nudei of step 14.

Immotile spermatozoa from the caput epididymidis become progressively motile when incubated in medium containing theophylline, seminal plasma, and albumin. We previously reported that under these incubation conditions the spermatozoa induced to acquire motility exhibited a marked flagellar angularity, with the sperm head or midpiece bent 90-180 degrees towards the tail. In addition, we demonstrated that sperm flagellar bending did not occur when the sulfhydryl oxidant diamide was added to the motility induction medium. In the present study, we examined further the effect of sulfhydryl oxidation on the morphology and sulfhydryl content of immature caput spermatozoa induced to acquire motility in vitro. We found that flagellar bending was prevented and sperm flagellar straightness was maintained in a dose-dependent manner by diamide. Moreover, flow cytometric analysis of caput sperm sulfhydryls using the sulfhydryl reagent monobromobimane (mBBr) revealed that 1) diamide oxidizes caput sperm sulfhydryls, and 2) less than 15% of the total reactive sperm sulfhydryls were oxidized at diamide concentrations capable of preventing sperm angulation. Sodium tetrathionate (NaTT), another sulfhydryl oxidant, and hamster cauda epididymal fluid (CEF) containing sulfhydryl oxidase enzyme activity also maintained flagellar straightness in induced caput spermatozoa and oxidized sperm sulfhydryls. The flagellar straightness in caput spermatozoa treated with sulfhydryl oxidants, however, was temporary; with extended incubation, diamide- or CEF-treated spermatozoa exhibited flagellar bending. Additional studies showed that the flagellar straightness observed in sulfhydryl-oxidized spermatozoa was sustained when nitrofurantoin, an inhibitor of glutathione reductase, was included in the induction medium. Flow cytometric analysis of nitrofurantoin-treated spermatozoa showed that nitrofurantoin maintained the sperm disulfides formed by diamide and prevented the reduction of sperm disulfides back to sulfhydryls. Taken together, these studies demonstrate the significance of sulfhydryl oxidation in maintaining the morphology of immature caput epididymal spermatozoa induced to acquire motility in vitro and suggest that sulfhydryl oxidation may be important in the development of motility during sperm epididymal maturation in vivo.

Rat and hamster testes, epididymides, vas deferentia, seminal vesicles, prostates and coagulating glands were assayed for sulfhydryl oxidase activity. The activity, present primarily in the fluids of these organs, was lowest in the testis, increased through the epididymis and was highest in the seminal vesicle. The localization and reactivity of sulfhydryl oxidase activity suggests that it may play a role in regulating the degree of disulfide bonding in spermatozoa during epididymal maturation and storage and after ejaculation.

A unilateral microsurgical vasoepididymostomy utilizing 35 mm of epididymas was performed in a patient with postinflammatory epididymal obstruction. Success was verified with multiple semen analyses, the hamster egg penetration test, and a pregnancy. These results demonstrate that surgical bypass of inflammatory tubal obstruction in the distal epididymas can result in the return of normal epididymal function and fertility.

Treatment of rabbit spermatozoa with Triton X-100 and dithiothreitol for increasing periods of time results in progressive increases in both the percentages of spermatozoa with swollen nuclei and in the ability of the swollen nuclei to serve as template for DNA synthesis. The inwilvement of endogenous proteolytic activity in this thiol-induced swelling and release of template restrictions is suggested by the fact that when spermatozoa are incubated in solutions containing various inhibitors of proteolysis (soybean trypsin inhibitor, lima bean trypsin inhibitor, or p-arninobenzamidine) in addition to Triton X-100 and dithiothreitol, inhibition of nuclear swelling and DNA template activity occur. The degree of inhibition is dependent upon the concentration of inhibitor employed. These results demonstrate a direct relationship between release of DNA template restrictions and nudear swelling, and suggest that both processes depend upon some degree of endogenous proteolytic activity as well as upon disulfide reduction.