The evident ability of the intracytoplasmic sperm injection (ICSI) procedure to achieve high fertilization and pregnancy rates regardless of semen characteristics has induced its application with spermatozoa surgically retrieved from azoospermic men. Here, ICSI outcome was analysed in 308 cases according to the cause of azoospermia; four additional cycles were with cases of necrozoospermia. All couples were genetically counselled and appropriately screened. Spermatozoa were retrieved by microsurgical epididymal aspiration or from testicular biopsies. Epididymal obstructions were considered congenital (n = 138) or acquired (n = 103), based on the aetiology. Testicular sperm cases were assessed according to the presence (n = 14) or absence (n = 53) of reproductive tract obstruction. The fertilization rate using fresh or cryopreserved epididymal spermatozoa was 72.4% of 911 eggs for acquired obstructions, and 73.1% of 1524 eggs for congenital cases; with clinical pregnancy rates of 48.5% (50/103) and 61.6% (85/138) respectively. Spermatozoa from testicular biopsies fertilized 57.0% of 533 eggs in non-obstructive cases compared to 80.5% of 118 eggs (P = 0.0001) in obstructive azoospermia. The clinical pregnancy rate was 49.1% (26/53) for non-obstructive cases and 57.1% (8/14) for testicular spermatozoa obtained in obstructive azoospermia, including three established with frozen-thawed testicular spermatozoa. In cases of obstructive azoospermia, fertilization and pregnancy rates with epididymal spermatozoa were higher than those achieved using spermatozoa obtained from the testes of men with non-obstructive azoospermia.

SummaryWhen administered at or near the initiation of experimental intracellular infection caused by Leishmania major, Toxoplasma gondii, or Cryptococcus neoformans, treatment with the immunoregulatory cytokine interleukin 12 (IL-12), induces protective antimicrobial activity. In contrast, once infections are established, IL-12 exerts considerably less or no effect in the face of a suppressive Th2 cell-associated response (L. major) or rapidly progressive fatal infection (T. gondii). To test the efficacy of IL-12 in an established intracellular protozoal infection but under quite different immunologic conditions (Thl cell response, acquired resistance), L. donovani-infected BALB/c mice were treated starting 2 wk after challenge coincident with the onset of the Thl cell response. In this environment, 7 d of IL-12 treatment reduced liver parasite burdens by 47%, an effect comparable to that induced by exogenous interferon (IFN) % The in vivo mechanism responsive to IL-12 was complex, and required both CD4 + and CD8 + T cells as well as natural killer cells and the action of multiple endogenous antileishmanial cytokines (IFN-% IL-2, tumor necrosis factor oe). Early treatment with IL-12 before the expression of the Thl cell response was also effective and induced an accelerated, near-cure response via an IFN-'lr-dependent mechanism. These results extend the antimicrobial-inducing capacity of IL-12 beyond prophylaxis by indicating that IL-12 can exert clear-cut therapeutic activity in an established intracellular infection.N 'umerous recent reports have identified the pleiotropic cytokine, IL-12, as a critical component of the early events that initiate successful cell-mediated antimicrobial defense (1-3). The role and/or effect of IL-12 has thus far been tested in four models of intraceUular infection caused by Le/shmania major (4,5), Toxoplasma gondii (6-8), Listeria monocytogenes (9), and Cyptococcus neoformans (10). Results from these models suggest that the role of endogenous IL-12 and the treatment effect of exogenous IL-12 are both most prominent at or near the time infection is first introduced (4, 5, 7). Indeed, when tested after the initial stage of host-parasite interaction in two of these models, IL-12 in exogenous form exerted considerably less activity in L. major infection (4, 5) and no effect in acute toxoplasmosis (7). These latter results have prompted the impression that while IL-12 appears to be of clear-cut immunoregulatory importance (1-3) and effective early on or as prophylaxis (4-7, 10), there may nevertheless be a narrow therapeutic window for IL-12 in such infections.Specific features of the preceding models of uncontrolled progressive infection (4, 5, 7), however, likely influenced the efficacy of delayed IL-12 treatment: (a) the rapid establishment of an overwhelming and uniformly fatal infection in the acute T gondii model (7), and (b) the prompt emergence of a particularly suppressive Th2 cell-assodated response which characterizes and propagates L. major infection in BALB/c mice (4...

Although implicated in the clinical expression of human visceral leishmaniasis, a disease-exacerbating T helper cell 2 (Th2)-associated immune response involving interleukin-4 (IL-4) and/ or IL-10 is not readily detectable in experimental visceral infection. To overcome this obstacle to analyzing visceral Leishmania donovani in this relevant immunopathogenetic environment, we sought a model in which a Th2 response induces noncuring infection. Four initial approaches were tested primarily in BALB/c mice which control intracellular L. donovani via an IL-12– and interferon-γ (IFN-γ)–dependent Th1 mechanism: (a) modifying the cytokine milieu when the parasite is first encountered (treatment with exogenous IL-4 or anti–IL-12), (b) providing sustained endogenous exposure to a Th2 cytokine (infection of IL-4 transgenic mice), (c) increasing the parasite challenge inoculum, and (d) injecting heat-killed L. major promastigotes (HKLMP) to induce a cross-reactive Th2 response to live L. donovani. Only the last approach generated a functional Th2-type response that induced disease exacerbation accompanied by inhibition of tissue granuloma assembly. In HKLMP-primed BALB/c mice, prophylaxis with anti–IL-4, anti–IL-10, or exogenous IL-12 (but not IFN-γ) readily restored resistance. In primed mice with established visceral infection, treatment with either IL-12 or IFN-γ also successfully induced antileishmanial activity. The results in this model (a) suggest that rather than acting alone, IL-4 and IL-10 may act in concert to prevent acquisition of resistance to L. donovani, (b) reemphasize the capacity of IL-12 to reverse early Th2-related effects, and (c) demonstrate that Th1 cytokines (IL-12, IFN-γ) have therapeutic action in an established systemic infection despite the presence of a disease-exacerbating Th2-type response.

GM-CSF induces three effects potentially beneficial in visceral leishmaniasis: blood monocyte mobilization, macrophage activation, and amelioration of granulocytopenia. To determine the experimental role and effect of GM-CSF in this intracellular infection, livers from Leishmania donovani-infected BALB/c mice were tested for GM-CSF mRNA expression and mice were treated with anti-GM-CSF antiserum or GM-CSF. L donovani infection upregulated hepatic GM-CSF mRNA expression by 10-fold, and anti-GM-CSF treatment exacerbated visceral infection and tripled liver parasite burdens 4 wk after challenge. In euthymic mice with established infection, treatment with 1-5 jug/d murine GM-CSF induced three dose-related effects: peripheral blood leukocytosis, preferential accumulation of myelomonocytic cells at visceral foci of infection, and leishmanicidal activity comparable to that achieved by IFN-y. These effects were either largely or entirely T cell dependent. Treatment with human GM-CSF also induced antileishmanial activity but with little effect on peripheral leukocyte number or tissue myelomonocytic cell influx; human G-CSF stimulated marked peripheral granulocytosis and neutrophil tissue accumulation but induced little antileishmanial effect. These results identify a role for endogenous GM-CSF in the initial host defense response to L donovani, reemphasize the influxing monocyte as an effector cell, and indicate that GM-CSF can be used as an antileishmanial treatment. (J. Clin. Invest. 1995.95:1183-1192