Andrology: Epididymal sperm aspiration and use of donor spermatozoa when necessary for in-vitro fertilization, and vasoepididymostomy: an acceptable combination in the treatment of obstructive azoospermia

Infertility due to obstructive azoospermia in 24 men was treatedwith a combination of scrotal exploration, microsurgical spermaspiration and vasoepididymostomy, at the same operation. In-vitrofertilization (IVF) and embryo transfer were performed usingepididymal spermatozoa. Donor spermatozoa were used if no motileepididymal spermatozoa were obtained. With this combination,emotionally and economically acceptable pregnancy rates wereachieved: 24% per aspiration, 43% per embryo transfer, and 25%per couple. One twin pregnancy resulting in the birth of twohealthy female infants and one ongoing twin pregnancy were achievedwith epididymal spermatozoa; four pregnancies (one twin, twosingletons, one abortion) were achieved with donor spermatozoa.     

ORIGINAL ARTICLE: Sperm Antibodies,Intra‐Acrosomal Sperm Proteins,and Cytokines in Semen in Men from Infertile Couples

Problem The aim of this study was to investigate seminal sperm‐agglutinating antibodies, intra‐acrosomal proteins, sperm head abnormalities, and cytokines (IL‐1β, IL‐2, IL‐4, IL‐5, IL‐6, IL‐8, IL‐10, IL‐12p70 TNF‐α, and IFN‐γ) in men from infertile couples. Method of study The direct mixed anti‐immunoglobulin reaction test for IgG, IgA, and IgE in semen, and immunocytochemical method using monoclonal antibodies and indirect immunofluorescence for the examination of intra‐acrosomal proteins in the spermatozoa were used. Cytokines in seminal plasma were determined by multiplex immunoanalytic xMAP (LUMINEX) technology. Results Sperm‐agglutinating antibodies, IgG and IgA, in seminal plasma were found to be more in asthenospermatic and oligoasthenospermatic men than in normospermatic men. Sperm head pathology and very low amounts of acrosomal proteins were frequently detected in pathologic semen samples. Cytokine levels defined as ‘high’ (based on the 75 percentile for each cytokine in all groups) were obtained especially for IL‐8, IL‐5, IL‐6, and IL‐10. The high cellularity in semen was correlated with higher IL‐5. Conclusion Immunologic cause of male infertility is a very important risk factor in the pathogenesis of sperm cells. Sperm autoantibodies and the presence of intra‐acrosomal factors must be studied together, cytokines according to accessory cellularity in the semen.     

Human Sperm Anatomy: Ultrastructural Localization of the Cannabinoid1 Receptor and a Potential Role of Anandamide in Sperm Survival and Acrosome Reaction

Recently, the endocannabinoid (EC) system and the presence of CB1 receptor (CB1‐R), have been identified in human sperm. However, the effects of EC receptor ligands such as anandamide (N‐arachidonoylethanolamine) and the role of EC system in male fertility is still largely unexplored. In the present study, we investigated the ultrastructural compartmentalization of CB1‐R and analyzed the effects of its stimulation by using a stable analog of anandamide, 2‐methylarachidonyl‐2′‐fluoro‐ethylamide (MET‐F‐AEA). We focused particularly on sperm survival and acrosin activity. The study of human sperm anatomy by transmission electron microscopy with immunogold analysis revealed the location of the CB1‐R prevalently in the sperm membranes of the head and interestingly on the mitochondria. The effect of different concentrations of MET‐F‐AEA from 100 nM to 1 μM evidenced a significant decrease of sperm survival. Interestingly, we analyzed this negative effect at molecular level, testing the EC action on different known sperm survival targets. MET‐F‐AEA‐treatment decreased both pBCL2 and pAkt, two prosurvival proteins, and increased pPTEN expression which is the main regulator of the PI3K/Akt pathway. Moreover, a biphasic effect was observed with increasing MET‐F‐AEA concentrations on the acrosin activity. The blockage of the CB1‐R by using its selective antagonist SR141716 (rimonabant) induced an opposite action on sperm survival supporting a role for this receptor in the biology of the male gamete. Anat Rec, 2010. © 2009 Wiley‐Liss, Inc.     

Sperm pathology: a step beyond descriptive morphology. Origin, characterization and fertility potential of abnormal sperm phenotypes in infertile men

Sperm pathology is presented as the discipline of characterizing structural and functional deficiencies in abnormal spermatozoa. This concept complements that of sperm morphology mainly concerned with the appearance of spermatozoa. These two notions collaborate in providing correlations of prognostic value with sperm fertilizing capacity, explaining the mechanisms of sperm inefficiency, suggesting strategies to improve fertilization and opening a door to molecular genetic studies. Phenotypes of genetic origin involving sperm heads, flagella and the neck region are presented describing their clinical manifestations, sperm structure, cytochemistry and genetic background. When available, animal models are used to highlight possible genetic mechanisms. Sperm pathologies secondary to andrological conditions or environmental factors are described, stressing the non-specific nature of the sperm response to noxious agents. The available literature on the prognostic value of sperm pathologies in ICSI is also reviewed. Flagellar anomalies bear a good prognosis, but those affecting the acrosome, sperm chromatin and the neck region entail an increasing chance of failure, which highlights the differential roles played by specific sperm components in fertilization, implantation and early embryonic development. A final discussion is devoted to genetic counselling and the risks involved in using immotile or abnormal spermatozoa in assisted reproduction.     

Comparison of methods for detecting mitomycin C- and ethyl nitrosourea-induced germ cell damage in mice: Sperm enzyme activities,sperm motility,testis weight

Testes weights, sperm motility and enzyme activities in single sperm were compared with respect to their ability to detect either developmental or mutational damage to germ cells. Male mice were injected i.p. with 2.5 mg/kg mitomycin C (MC) or 50 or 100 mg/kg ethylnitrosourea (ENU) or saline and were then killed at times such that sperm derived from treated vas sperm (SZ), spermatids (ST), preleptotene-late-spermatogonial cells (PLSG), spermatogonial cells (SG), or spermatogonial stem cells (SGS) could be evaluated. Testis weights decreased significantly as early as 1 wk after treatment, with the greatest decrease reached 3–4 wk after treatment, followed by recovery to normal levels 10–15 wk after treatment. We conclude that testis weight, which is easily obtained, is a sensitive indicator of germ cell damage by these agents. Sperm from each animal were evaluated for sperm motility, acrosin activity, succinic dehydrogenase (SDH) activity with or without the competitive inhibitor malonate or after exposure to 60°C for 10 min. The latter two assays were to detect sperm enzymes resistant to the inhibitor or heat. The presence of the acrosin protein was also detected immunologically. Sperm motility decreased most from treatment of PLSG and SG. After MC or ENU treatment, the greatest loss of acrosin activity and of the acrosin protein was also noted in sperm derived from treated PLSG and SG. MC and ENU failed to induce SDH activity in single sperm resistant to 60°C heat inhibition or to inhibition by malonate. Of the sperm assays, acrosin activity proved to be the most sensitive indicator of germ cell damage and was the simplest to measure.     

Ultrastructure of Anterior Uterus of the Oviduct and the Stored Sperm in Female Soft‐Shelled Turtle,Trionyx sinensis

Ultrastructure of sperm storage in female soft‐shelled turtle, Trionyx sinensis was examined under light and electron microscopes. Sperm storage tubules are restricted to the anterior of the uterus. These tubules developed either by folding or fusion of the oviductal mucosal folds and are lined by both ciliated and secretory cells. Ciliated cells are characterized by a few microvilli and prominent cilia in the apical membranes. A prominent feature of the secretory cell is the presence of secretory granules in the supranuclear region. The size, shape, and electron density of these granules vary markedly. The secretory product is released mainly by exocytosis into the oviductal lumen, where it appears as flocculent material. The unique structure in the base of the epithelium, the basal border of the cell—the basal lamina—and a blood vessel layer, is presumed to be a important barrier, by which the nourishment exchange and the microenvironment maintenance are ensured. The gland cell is presented with numerous, round, membrane‐bound secretory granules of moderate to high electron densities. We divide these granules into three types according to their appearance: (1) membrane bounded granules with high‐homogeneous electron density, (2) membrane bounded granules with moderate‐homogeneous electron density, (3) membrane bounded, electron dense granules with concentric structures. These granules are presented as different stages of the secretions in the gland cell. The junction complexes are markedly distributed between cells, which are important in keeping stability and the microenvironment maintenance of the sperm storage tubules. Sperm stored in the tubules are heterogeneous in cytology. In addition to the mature sperm in the lumen, sperm with large chromatic granules are found, which are presumed to be immature sperm and are being in the process of nuclear condensation. Several spermatozoa in the tubules are exhibited with definitive indications of degeneration of the nuclei. The nuclear volume increases. The electron density of the central cores in mitochondria declines, combined with the deterioration of concentric membrane structure. Those changes are possibly due to the long time storage of the sperm in sperm storage tubules, and the leakage of reactive oxygen species is suggested to be a major cause. We conclude that the ultrastructure character of sperm storage in the oviduct of Trionyx sinensis is unique, in addition to having a basal function in secretion and the cilia swing, the tubules also provide an available microenvironment for the sperm to long time stored. The degenerative sperm in the tubules might be related to paternity‐specific reproductive adaptations, and the sperm competition might occur during long time storage. Anat Rec, 291:335–351, 2008. © 2008 Wiley‐Liss, Inc.     

REVIEW ARTICLE: Uterine NK Cells,Spiral Artery Modification and the Regulation of Blood Pressure During Mouse Pregnancy

Citation Burke SD, Barrette VF, Gravel J, Carter ALI, Hatta K, Zhang J, Chen Z, Leno‐Durán E, Bianco J, Leonard S, Murrant C, Adams MA, Anne Croy B. Uterine NK cells, spiral artery modification and the regulation of blood pressure during mouse pregnancy. Am J Reprod Immunol 2010 Reproductive success in mammals involves coordinated changes in the immune and cardiovascular as well as in the neuroendocrine and reproductive systems. This review addresses studies that identify potential links for NK cells and T cells with the local and systemic cardiovascular adaptations of pregnancy. The studies reviewed have utilized immunohistochemisty and in vivo analyses of vascular parameters by ultrasound, chronic monitoring of hemodynamics via radiotelemetric recording and intravital microscopy. At the uterine level, functional subsets of uterine natural killer cells were identified. These included subsets expressing molecules important for vasoregulation, in addition to those previously identified for angiogenesis. Spiral arteries showed conducted responses that could account for conceptus control of vasoactivity and mouse gestational blood pressure 5‐phase pattern. Vascular immunology is an emerging transdisciplinary field, critical for both reproductive immunology and cardiovascular disease.     

Sperm chromosome complements in a man heterozygous for a reciprocal translocation 46,XY,t(9;13)(q21.1;q21.2) and a review of the literature

Sperm chromosome complements were studied in a man heterozygous for a reciprocal translocation t(9;13)(q21.1;q21.2). A total of 89 spermatozoa were karyotyped after in vitro penetration of hamster eggs. The frequencies of alternate, adjacent 1 and adjacent 2 segregations were 46.9%, 35.8% and 17.3% respectively. For alternate segregation, the number of normal spermatozoa (21) was not significantly different from the number of spermatozoa carrying a balanced form of the translocation (17), as theoretically expected. The proportion of spermatozoa with an unbalanced form of the translocation was 53.1%. There was no evidence for an interchromosomal effect since the frequency of numerical abnormalities (unrelated to the translocation) was within the normal range of control donors. Data from a total of 31 reciprocal translocations studied by sperm chromosomal analysis were reviewed.     

Variation in Timing,Duration, Intensity,and Direction of Adolescent Growth in the Mandible,Maxilla, and Cranial Base: The Fels Longitudinal Study

There is considerable individual variation in the timing, duration, and intensity of growth that occurs in the craniofacial complex during childhood and adolescence. The purpose of this article is to describe the extent of this variation between traits and between individuals within the Fels Longitudinal Study (FLS). Polynomial multilevel models were used to estimate the ages of onset, peak velocity, and cessation of adolescent growth, the time between these ages, the amount of growth between these ages, and peak velocity. This was done at both the group and individual levels for standard cephalometric measurements of the lengths of the mandible, maxilla, and cranial base, the gonial angle, and the saddle angle. Data are from 293 untreated boys and girls age 4–24 years in the FLS. The timing of the adolescent growth spurt was, in general, not significantly different between the mandible and the maxilla, with each having an earlier age of onset, later age of peak velocity, and later age of cessation of growth as compared to the cranial base length. Compared to lengths, angles had in general later ages of onset, peak velocity, and cessation of growth. Accurate characterization of the ontogenetic trajectories of the traits in the craniofacial complex is critical for both clinicians seeking to optimize treatment timing and anatomists interested in examining heterochrony. Anat Rec, 297:1195–1207, 2014. © 2014 Wiley Periodicals, Inc.     

Comparative ontogeny in humans and chimpanzees: Similarities, differences and paradoxes in postnatal growth and development of the skull

The hypothesis of retarded development is a classic and controversial issue in human evolution. It depends directly on the understanding of ontogenetic trajectories and their basic constituents: timing, rate and associated patterns of maturation. In the present study, we applied geometric morphometrics to investigate postnatal ontogeny in human and chimpanzee skulls (N = 302). We evaluated postnatal ontogenetic rates, based on comparisons of properties of size and shape in adults. At different dental ages the percentage of the adult mean size (growth) and adult mean shape (development) was used to quantify patterns of maturation. We found significantly higher levels of ontogenetic maturity in humans than chimpanzees during pre-M1 and M1 eruption. However, during this ontogenetic period the human increments were lower than those of chimpanzees suggesting lower rates. During and after M2-eruption species did not differ in their ontogenetic trajectories. The results indicate that higher prenatal and lower peri- and postnatal maturation rates characterize human ontogeny when compared with chimpanzees. If mandibular ontogeny is considered alone, a paradox was found. Whereas growth maturation proceeded in an expected trajectory continuously approximating 100% adult mean size, developmental maturity was different. After M1-eruption in both species the morphological distance, which had increased before, became reduced again, and reached adult mean shape in a second developmental peak. Such a tendency was found in humans and chimpanzees. This indicates that both size and shape maturation must be considered to understand the complexity of postnatal mandibular ontogeny.     

Primary pulmonary ganglioneuroblastoma in an adult: maturation, involution and the immune response

The features of a primary pulmonary ganglioneuroblastoma occurring in an adult are presented. The tumour showed evidence of both maturation and involution. Maturation appeared to be occurring in a centrifugal manner, a rim of mature ganglioneuromatous tissue enclosing the primitive neuroblastoma. Necrosis of the neuroblastomatous element was widespread and associated with deposition in the walls of numerous small vessels of an amorphous eosinophilic amyloid-like material. Accumulation of this material had led to occlusion of some vessels with resultant necrosis of related tumour. A collarette of lymphocytes surrounded the tumour, and lymphocytic aggregates were prominent at the interface between neuroblastoma and ganglioneuroma. Despite widespread vascular invasion, the patient remains well and apparently tumour-free, 25 years post-resection. The appearances may represent a combined cellular and humoral host response, and a possible relationship of this response to tumour maturation is suggested. The potential role of immunostimulation in the treatment of neuroblastoma is discussed.     

Antibodies to Sperm in Benign and Malignant Diseases of the Prostate in Man: Incidence,Disease-Associated Specificity,and Implications

ABSTRACT: Initial investigation demonstrated antibodies to sperm (ASA) in patients with benign prostatic hypertrophy (BPH) and prostate cancer (PCa). The occurrence of ASA under a variety of normal and pathological circumstances indicated the need for confirmation and extension, including delineation of their possible disease-associated specificity and implications. As countercurrent Immunoelectrophoresis (CIEP) employing sonicated allogeneic sperm (Sp) extracts appeared most efficient from initial studies of ASA, CIEP was employed for the present further study of 200 serum specimens from patients with and without prostatic disease. While ubiquitous, the continuing presence of ASA in BPH and PCa, with a combined incidence in this study of 57(52%) of 109 vs. 9 (10%) of 91 in the absence of prostatic disease remains provocative in view of the hypothesized role of Sp in the development of BPH and PCa. The presence, however, of ASA in patients with genitourinary neoplasms other, than prostate, raises doubt as to their disease specificity. Implications of ASA, other than in their more commonly related role in infertility, including their cross-reactivity with foetal antigens and lymphocytes and higher incidence in association with tumours and the presence of tumour-associated immunity are considered. However, pending further investigation, the present data may most appropriately be viewed as being reflective of a host response (marker?) to aberrant genitourinary cellular alterations.     

MPTP Susceptibility in the Mouse: Behavioral, Neurochemical, and Histological Analysis of Gender and Strain Differences

To investigate the impact of strain and sex in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) animal model of Parkinson's disease, C57BL/6 and BALB/c mice were treated with either systemic MPTP-HCl (4 × 15 mg/kg) or saline and were examined in a number of behavioral tests. Furthermore, neostriatal and ventral striatal monoamine contents were determined, and the numbers of tyrosine hydroxylase-immunostained cells were counted in the substantia nigra and ventral tegmental area. Open-field testing showed that locomotor activity was drastically reduced as an acute effect of MPTP in both strains; however, subsequent recovery to control levels was faster in BALB/c mice than in C57BL/6. Nest building also indicated strain-dependent effects, since it was delayed only in C57BL/6 mice treated with MPTP. The other tests (grip test, pole test, rotarod, elevated plus-maze), although partly sensitive for overall strain or gender differences, turned out not to be useful to compare MPTP effects in these two strains. Neurochemically, MPTP led to more severe neostriatal dopamine depletions in C57BL/6 (–85%) than in BALB/c mice (–58%). Histologically, a loss of tyrosine hydroxylase immunoreactivity (–25%) was observed only in the substantia nigra of C57BL/6 animals. Thus, our analysis consistently showed that the C57BL/6 mouse strain is more susceptible to MPTP than the BALB/c strain. Sex differences in MPTP sensitivity were not observed in our mice. The implications of these findings for the search for genes related to susceptibility to neurodegeneration are discussed.     

A new, simple and rapid method for enumerating human T lymphocytes in full blood: the E. coli (ATCC 11303) rosette test

A new bacterial rosette technique for enumerating T lymphocytes is described. E. coli (strain B; ATCC 11303), fixed in formaldehyde after overnight growth in thioglycolate medium, are mixed with washed whole blood cells (100 μl) and after incubation at 4°C, slides are made, stained and counted. The nature of the lymphocytes forming E. coli rosettes was demonstrated by comparing their cytochemical staining characteristics with those of E rosetted lymphocytes, and by mixed E. coli and E, mouse E rosette and Fc receptor tests, and by mixed E. coli rosette tests and anti-Ig staining. E. coli and E rosette tests in controls and pediatric patients were also compared. The results show that T_μ and T_γ cells rosette with E. coli.     

Mast cell number and maturation in the central nervous system: influence of tissue type, location and exposure to steroid hormones

While it is well established that brain mast cells are usually associated with the cerebral vasculature, in ring doves mast cells lie directly in the neuropil of the medial habenula. During normal development mast cells enter the habenula and complete their differentiation in situ. In the present study, we asked what characteristics of the medial habenula contribute to mast cell entry and differentiation. Grafts of embryonic habenula or control optic tectal grafts were placed in the lateral ventricle or anterior chamber of the eye. Transplantation alters the location of the habenula as well as its neural and vascular connections. Three groups of hosts were used for the ventricular grafts: four-month-old and killed three months after transplantation; four-month-old and killed seven months later, and two- to three-year-old gonadectomized males killed three months later. Hosts for the intraocular grafts were four months of age and killed three months later. Mast cells were present in the habenular grafts but not in the control tissue. Mast cells in three- and seven-month-old grafts were phenotypically immature when compared to those of hosts. They contained fewer metachromatic granules, fewer granules immunoreactive to an antiserum against gonadotropin-releasing hormone, and no highly-sulphated proteoglycans. As previously described, gonadectomized adults had fewer mast cells in their medial habenula than did intact animals, but there was no change in mast cell number in habenular grafts.

The current experiments indicate that the occurrence and survival of mast cells can occur within the microenvironment of the medial habenula, but that maturation of these cells requires the normal connections of this nucleus. Furthermore, gonadectomy appears to alter mast cell number in the medial habenula by generating a secondary signal which the transplanted tissue is incapable of receiving or processing.     

Participation of the interstitium in acute immune-complex nephritis: interstitial antigen accumulation,cellular infiltrate,and MHC class II expression

Bovine serum albumin (BSA) injected into the rabbits induces acute immune complex glomerulonephritis. Since albumin is filtered and reabsorbed in the tubules, we investigated whether tubulointerstitial cells participate in the pathogenesis of this experimental condition. For this purpose, we induced immune-complex nephritis in 45 rabbits with the injection of 125I-BSA and urinary BSA excretion, glomerular and tubulointerstitial BSA accumulation, lymphocyte infiltration, proliferative activity and MHC class II antigens were examined 2, 4-5 and 6-8 days after immunization. Proteinuria developed day 6-8. BSA was found in urine from day 2 (mean +/- SE; 1089 +/- 339 micro g/24 h) and peaked on day 4 after immunization (2249 +/- 1106). BSA content (cpm/g tissue) in tubulointerstitium (TI) and glomeruli were similar at day 2 (457 +/- 45 and 407 +/- 75, respectively), but afterward increased significantly in TI, reaching a peak level on day 5 (1026 +/- 406) while remained unchanged in glomeruli (388 +/- 95). At the same time, preceding the onset of proteinuria, maximal intensity of the lymphocyte infiltration, proliferative activity and MHC class II antigen expression in tubular cells, monocytes/macrophages and interstitial cells were observed. Our study shows that antigen is excreted in the urine and concentrated in TI in association with overexpression of MHC class II molecules and lymphocyte infiltration. These findings occur prior to the development of proteinuria and suggest that the tubulointerstitial cells play a critical role in the pathogenesis of this model.     

Formation of immature and mature genomic RNA dimers in wild-type and protease-inactive HIV-1: Differential roles of the Gag polyprotein, nucleocapsid proteins NCp15, NCp9, NCp7, and the dimerization initiation site

Formation of immature genomic RNA (gRNA) dimers is exquisitely nucleocapsid (NC)-dependent in protease-inactive (PR-in) HIV-1. This establishes that Pr55gag/Pr160gag-pol has NC-dependent chaperone activity within intact HIV-1. Mutations in the proximal zinc finger and the linker of the NC sequence of Pr55gag/Pr160gag-pol abolish gRNA dimerization in PR-in HIV-1. In wild type, where the NC of Pr55gag is processed into progressively smaller proteins termed NCp15 (NCp7-p1-p6), NCp9 (NCp7-p1) and NCp7, formation of immature dimers is much swifter than in PR-in HIV-1. NCp7 and NCp15 direct this rapid accumulation. NCp9 is sluggish in this process, but it stimulates the transition from immature to mature gRNA dimer as well as NCp7 and much better than NCp15. The amino-terminus, proximal zinc finger, linker, and distal zinc finger of NCp7 contribute to this maturation event in intact HIV-1. The DIS is a dimerization initiation site for all immature gRNA dimers, irrespective of their mechanism of formation.