Use of electively cryopreserved microsurgically aspirated epididymal sperm with IVF and intracytoplasmic sperm injection for obstructive azoospermia.

OBJECTIVE: To investigate the efficacy of using intentionally cryopreserved epididymal sperm in selected cases of obstructive azoospermia. DESIGN: A retrospective, nonrandomized study. SETTING: Academic research environment. PATIENTS: One hundred forty-one couples undergoing first-time IVF/ICSI using either fresh or cryopreserved epididymal sperm. INTERVENTIONS: The epididymides were microsurgically aspirated. MAIN OUTCOME MEASURES: Clinical pregnancy rates. RESULTS: Motile sperm were obtained from all men. For the fresh group, the mean total sperm aspirated was 99 x 10(6) with 5.5 vials frozen per patient after ICSI and 82 x 10(6) with 4.7 vials frozen per patient in the cryopreserved group. No statistically significant difference in oocyte fertilization rate or number of embryos transferred was noted between groups. Of 108 patients using freshly aspirated sperm, 72 (66.7%) achieved clinical pregnancy. Of 33 patients in the group using cryopreserved sperm, 20 (60.6%) achieved clinical pregnancy (P=0.47). CONCLUSIONS: In selected ideal cases of unreconstructable azoospermia, elective open microsurgical epididymal sperm aspiration with cryopreservation yields pregnancy rates similar to that employing fresh sperm. The advantages of this method are: (1) Use of cryopreserved sperm obviates the logistics problems associated with the use of fresh sperm. (2) Abundant high-quality sperm can be cryopreserved in a single procedure for all future attempts at IVF/ICSI. Rarely, viable sperm will not be present after thawing, and fresh retrieval will be necessary.

ICSI and its outcome.

Since its introduction in 1992, intracytoplasmic sperm injection (ICSI) has become a popular assisted fertilization technique proved very efficient in treating male factor infertility. Many healthy children have been born worldwide from this procedure, and their physical and mental development appears to be within the normal limits. However, because of the peculiarity of the technique and the poor characteristics of the spermatozoa used, concern about the safety of ICSI still exist. In this article, we analyze the in vivo development of embryos conceived after ICSI as well as the obstetric outcome, occurrence of chromosomal abnormalities, and rate of congenital malformations in neonates born as a result of this treatment. A total of 2435 couples were studied in whom the male partners were presumed to be the cause of repeated failed attempts at in vitro fertilization (IVF) or had semen parameters that were unacceptable for conventional IVF treatment. Pregnancies resulting from 3573 ICSI cycles were analyzed; pregnancy outcome data were obtained from the records of obstetrician-gynecologists and/or pediatricians. The overall clinical pregnancy (fetal heartbeat) rate was 44.8% with a resultant delivery rate of 39.2% per ICSI cycle (n = 1388). In 37 of the 77 miscarriages for which cytogenetic data were available, an autosomal trisomy was found in each and 29 additional pregnancies were terminated because of a chromosomal abnormality revealed by prenatal diagnosis. There was an equal distribution of vaginal deliveries and cesarean sections (n = 682 and n = 658, respectively). Of the 2059 neonates resulting from ICSI treatment, 38 (1.8%) presented with congenital abnormalities (22 major and 16 minor). When the frequency of miscarriages and congenital malformations was analyzed in terms of semen origin, the outcome was no different between ICSI and IVF. The course of pregnancies and occurrence of congenital malformations following treatment by ICSI are within the ranges obtained following conventional IVF.

Incidence of sperm aneuploidy in relation to semen characteristics and assisted reproductive outcome.

OBJECTIVE: To evaluate the incidence of sperm aneuploidy in men screened for infertility and identify any eventual relation with assisted reproductive outcome. DESIGN: Controlled prospective study. SETTING: University hospital-based IVF program. PATIENT(S): Infertile couples who were screened for sperm aneuploidy and evaluated for IVF treatment. INTERVENTION(S): Fluorescence in situ hybridization was used to identify chromosomes 18, 21, X, and Y. The assisted reproductive techniques of IVF and intracytoplasmic sperm injection were used for infertility treatment. MAIN OUTCOME MEASURE(S): The incidence of sperm aneuploidy, semen parameters, fertilization rate, pregnancy characteristics, and rate of neonatal malformations were determined. RESULT(S): Oligozoospermic and teratozoospermic men had a significantly higher incidence of chromosomal abnormalities than men with normal semen parameters (2.7% vs. 1.8%). The increased frequency of sperm aneuploidy did not appear to affect pregnancy losses or the occurrence of neonatal malformations. CONCLUSION(S): Suboptimal semen samples had a higher incidence of aneuploidy. In this study, the increased frequency of chromosomal abnormalities did not have a direct effect on the fertilization rate, pregnancy characteristics, or neonatal outcome.

Fertilization and pregnancy outcome with intracytoplasmic sperm injection for azoospermic men.

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.

H-Y antigen expression patterns in human X- and Y-chromosome-bearing spermatozoa.

PROBLEM: Restricted expression of H-Y antigen on Y-chromosome-bearing sperm has been reported in some species, although such preferential expression for H-Y antigen in human sperm has yet to be described. In this study, an immunomagnetic approach was used to characterize antigen expression patterns as a function of sex-chromosome content. METHOD OF STUDY: Human sperm was treated with monoclonal immunoglobulin (Ig) M antibodies directed against H-Y antigen. This preparation then was incubated with sheep antimouse IgM antibody affixed to paramagnetic beads, which then were exposed to a magnetic field and sorted. X- and Y-chromosome frequencies in the two subgroups of sperm were assayed by multiprobe fluorescent in situ hybridization (FISH). RESULTS: Sperm were immunomagnetically separated into two populations: a reactive group (presumably, H-Y Ag+); and a nonreactive group (presumably, H-Y Ag-). Triple-color FISH analysis of 1,600 spermatozoa (800 in each group) showed the antigen's expression to be somewhat more prevalent among Y-chromosome-bearing sperm (54.1%), but a large proportion of Y-chromosome-bearing sperm (49.0%) did not express this antigen. The difference was not significant (P = 0.43). CONCLUSIONS: The expression of H-Y antigen has a slightly higher frequency in human sperm containing the Y-chromosome, but its expression among X-chromosome-bearing sperm also is considerable. Current immunologic techniques relying on this antigen are unlikely to effect the sex selection of human sperm.

Behavior of visceral Leishmania donovani in an experimentally induced T helper cell 2 (Th2)-associated response model.

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-gamma (IFN-gamma)-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-gamma) readily restored resistance. In primed mice with established visceral infection, treatment with either IL-12 or IFN-gamma 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-gamma) have therapeutic action in an established systemic infection despite the presence of a disease-exacerbating Th2-type response.

Pentavalent antimony-mannan conjugate therapy of experimental visceral leishmaniasis.

Pentavalent antimony-mannan (Sb[V]-mannan) was 10-fold more potent than sodium stibogluconate in a murine model of visceral leishmaniasis. Liver antimony concentrations were six-fold higher after Sb[V]-mannan therapy compared with a dose of sodium stibogluconate that was equipotent in reducing liver parasite burdens. Murine toxicity of Sb[V]-mannan was variable, with a 50% lethal dose (LD50) for one preparation that was well above the concentration that killed 90% of the parasites, and for another preparation was only modestly higher than the concentration that killed 90% of the parasites.

Induction of macrophage antiprotozoal activity by monocyte chemotactic and activating factor.

To determine if monocyte chemotactic and activating factor (MCAF) induces intracellular antimicrobial activity, human monocyte-derived macrophages were treated with MCAF and challenged with Toxoplasma gondii and Leishmania donovani. Pretreatment with MCAF induced macrophages to inhibit protozoal replication by approximately 50%. These findings suggest a potential host defense role for MCAF in the inflammatory response to intracellular pathogens.

Models of relapse of experimental visceral leishmaniasis.

To establish models for studying recurrence of visceral leishmaniasis, a growing problem in T cell-deficient patients, two approaches were investigated: treatment of euthymic BALB/c mice with quiescent Leishmania donovani infection with T cell-depleting or anti-cytokine antibodies and serial observation of acutely infected nude BALB/c mice after an initial antileishmanial response induced by amphotericin B treatment. In chronically infected euthymic mice, maintenance of acquired immunity and prevention of relapse required CD4 cells and a multicytokine-dependent mechanism involving endogenous interleukin-2, interferon-gamma, and tumor necrosis factor-alpha. Acutely infected nude mice responded to amphotericin B with a > or = 85% reduction in liver parasite burdens; however, after a brief lag, visceral infection readily recurred in the posttreatment period. Both models may be useful for testing experimental interventions designed to reduce relapse of previously controlled visceral leishmaniasis in T cell-deficient hosts.

Activity of oral atovaquone alone and in combination with antimony in experimental visceral leishmaniasis.

BALB/c mice with established visceral infection caused by the intracellular protozoan Leishmania donovani were treated with oral atovaquone for 7 days. Treatment with 100 mg/kg of body weight per day was optimal and halted parasite replication in the liver. In mice treated with atovaquone, the effect of a suboptimal dose of pentavalent antimony was converted from partially leishmanistatic to leishmanicidal. These results demonstrate the in vivo antileishmanial effect of atovaquone and suggest a potential role for this oral agent in visceral leishmaniasis as an adjunct to conventional antimony treatment.

Multiple host defense defects in failure of C57BL/6 ep/ep (pale ear) mice to resolve visceral Leishmania donovani infection.

Euthymic C57BL/L ep/ep (pale ear [PE]) mice halt the visceral replication of intracellular Leishmania donovani but fail to properly resolve infection. A previous study identified an isolated defect in tissue granuloma formation in these mice; CD4+ and CD8+ cell number, gamma interferon (IFN-gamma) production, and macrophage antimicrobial activity in vitro were all intact. New in vivo results reported here suggest a considerably more complex immune defect, with evidence indicating (i) enhanced control over L. donovani after transfer of normal C57BL/6 spleen cells, (ii) a partially suppressive Th2 cell-associated response mediated by interleukin-4 (IL-4) but not reversed by CD4+ cell depletion, (iii) absent responses to endogenous Th1 cell lymphokines (IFN-gamma and IL-2) but preserved responsiveness to endogenous tumor necrosis factor alpha, (iv) absent responses to exogenous treatment with recognized antileishmanial cytokines (IFN-gamma, IL-2, IL-12, and granulocyte-macrophage colony-stimulating factor [GM-CSF]) not corrected by transfer of C57BL/6 spleen cells, and (v) a deficient response to antimony chemotherapy. Defective hepatic granuloma formation was not corrected by transfer of C57BL/6 spleen cells or by anti-IL-4 administration. While treatment with IL-2 and GM-CSF modified the tissue reaction and induced selected effector cells to encase tissue macrophages, no antileishmanial activity resulted. Together, these observations suggest that the failure of PE mice to resolve visceral L. donovani infection likely represents expression of multiple suboptimal immune responses and/or partial defects, probably involving a combination of T-cell dysfunction, a Th2 cell response, and target cell (macrophage) hyporesponsiveness.

Antimicrobial response of a T cell-deficient host to cytokine therapy: effect of interferon-gamma in experimental visceral leishmaniasis in nude mice.

To determine if interferon (IFN)-gamma can enhance intracellular antimicrobial defense in a T cell-deficient host, nude BALB/c mice were infected with Leishmania donovani and treated with IFN-gamma. IFN-gamma induced killing of L. donovani in livers of euthymic mice but had no effect in nude mice despite activating peritoneal macrophages in vivo. Transfer of CD4+ or CD8+ T cells permitted nude mice to respond to IFN-gamma; treatment with T cell-regulated antileishmanial cytokines (interleukin-2, granulocyte-macrophage colony-stimulating factor, or tumor necrosis factor-alpha) could not substitute for T cells. NK cells played no apparent role. In reconstituted nude mice, the antileishmanial effect of IFN-gamma correlated with markedly enhanced mononuclear cell recruitment to infected liver foci. Thus, although IFN-gamma activates macrophages in the absence of host T cells, a T cell mechanism is required for antileishmanial activity in tissue. Provided one T cell subset is adequately preserved, IFN-gamma may prove useful in intracellular infections in the T cell-deficient host.

Effect of granulocyte-macrophage colony-stimulating factor in experimental visceral leishmaniasis.

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 micrograms/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-gamma. These effects were either largely or entirely T cell dependent. Treatment with human GM-CSF also induced anti-leishmanial 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.

Interleukin 12 is effective treatment for an established systemic intracellular infection: experimental visceral leishmaniasis.

When administered at or near the initiation of experimental intracellular infection caused by Leishmania major, Toxoplasma gondii, or Cryptococcus neoformans, treatment with the immuno-regulatory 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 (Th1 cell response, acquired resistance), L. donovani-infected BALB/c mice were treated starting 2 wk after challenge coincident with the onset of the Th1 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) gamma. 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-gamma, IL-2, tumor necrosis factor alpha). Early treatment with IL-12 before the expression of the Th1 cell response was also effective and induced an accelerated, near-cure response via an IFN-gamma-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.

Role and effect of TNF-alpha in experimental visceral leishmaniasis.

TNF-alpha has been implicated in cytokine-induced macrophage activation and tissue granuloma formation, two activities linked to control of intracellular visceral infection caused by Leishmania donovani. To determine the role of TNF-alpha in L. donovani-infected BALB/c mice, we measured TNF-alpha levels and treated mice with either anti-TNF-alpha antiserum or TNF-alpha. TNF-alpha activity in infected livers was increased by 2.7-fold 2 wk after challenge and by 5.5-fold at wk 8. In parallel, although control mice acquired resistance by wk 4 and resolved infection by wk 8, liver parasite burdens steadily increased in anti-TNF-alpha-treated animals. Hepatic granuloma formation, however, was not impaired by anti-TNF-alpha. Endogenous TNF-alpha levels provoked by L. donovani appeared sufficient and optimal because exogenous TNF-alpha administration had no beneficial effect on established infection and continuous high-dose treatment impaired antileishmanial activity. Thus, although not required for granuloma formation, endogenous TNF-alpha appears to be critical to both initial acquisition of resistance to L. donovani and resolution of experimental visceral infection.

Circulating Borrelia burgdorferi in patients with acute Lyme disease: results of blood cultures and serum DNA analysis.

To determine the usefulness of blood culture and polymerase chain reaction (PCR) analysis in detecting circulating Borrelia burgdorferi or its DNA, blood and serum from untreated patients with acute Lyme disease were examined. None of the cultures of blood or serum from the 7 patients tested demonstrated spirochetes. Similarly, all patient serum samples, assayed in two laboratories, were negative for B. burgdorferi DNA using PCR amplification. These results suggest that in patients with acute Lyme disease, spirochetes, spirochete DNA, or both circulate early, only intermittently, or at low levels and that neither culture nor PCR testing of blood or serum, as currently done, appears likely to prove generally useful in the diagnosis of Lyme disease.

Treatment of experimental visceral leishmaniasis in a T-cell-deficient host: response to amphotericin B and pentamidine.

In experimental visceral leishmaniasis, euthymic but not athymic (nude) BALB/c mice respond to conventional treatment with pentavalent antimony, indicating that the in vivo efficacy of antimony is T cell dependent. This finding correlates with frequent antimony treatment failures for T-cell-deficient patients with visceral leishmaniasis. To determine whether the in vivo efficacies of alternative antileishmanial agents also require T cells, Leishmania donovani-infected euthymic and nude BALB/c mice were treated with pentamidine or amphotericin B. Pentamidine induced leishmanistatic activity in euthymic mice but had little effect in nude mice. In contrast, amphotericin B exerted potent leishmanicidal activities in both euthymic and nude animals. These results suggest that amphotericin B may be of particular use for T-cell-deficient patients with visceral leishmaniasis.

Response to treatment for an intracellular infection in a T cell-deficient host: toxoplasmosis in nude mice.

To examine the experimental basis of treatment failures in T cell-deficient patients with intracellular infections, euthymic and athymic (nude) BALB/c mice were infected with Toxoplasma gondii and treated with sulfadiazine. All euthymic and nude mice survived during 2 weeks of sulfadiazine therapy. Once treatment was discontinued, 100% of euthymic mice survived while all nude mice died. Post-sulfadiazine treatment survival was enhanced in nude mice by reconstitution with either L3T4+ or Lyt-2+ cells and was reduced in euthymic mice by monoclonal antibody treatment directed at depleting either L3T4+ or Lyt-2+ cells or interleukin-2 (IL-2) or interferon-gamma (IFN-gamma). These results suggest that although T cells and their products are not required for an initial response (survival) to treatment in acute experimental toxoplasmosis, survival off drug is strictly T cell-dependent. Optimal posttreatment survival appears to involve both L3T4+ helper and Lyt-2+ cytotoxic cells, probably acting in concert, as well as the endogenous secretion of at least two T cell-derived lymphokines, IL-2 and IFN-gamma.