Surgical sperm retrieval and intracytoplasmic sperm injection as treatment of obstructive azoospermia

Male genital tract obstructions may result from infections, previous inguinal and scrotal surgery (vasectomy) and congenital bilateral absence of the vas deferens (CBAVD). Microsurgery can sometimes be successful in treating the obstruction. In other cases and in cases of failed surgical intervention, the patient can be treated by microsurgical or percutaneous epididymal sperm aspiration (MESA, PESA) or testicular sperm extraction (TESE) and intracytoplasmic sperm injection (ICSI). We present the results of 39 ICSI procedures for obstructive azoospermia in 24 couples. The aetiology of the obstruction was failed microsurgery in 11 patients, CBAVD in nine and genital infections in four. Sperm retrieval was accomplished via MESA in four cases, PESA in 18 cases and via TESE in 11 cases. TESE was only applied when PESA failed to produce enough spermatozoa for simultaneous ICSI. In six patients, the ICSI procedure was performed with cryopreserved spermatozoa after an initial PESA procedure. Fertilization occurred in 47% of the metaphase II oocytes; embryo transfer was performed in 92% of procedures and resulted in a clinical pregnancy in 13/39 procedures. Ongoing pregnancy was achieved in 10/39 procedures. One pregnancy was terminated early after prenatal investigation showed a cytogenetic abnormality (47,XX+18, Edwards syndrome). The other nine pregnancies resulted in the live birth of 10 children, without any congenital abnormalities. Epididymal and testicular retrieved spermatozoa were successfully used for ICSI to treat obstructive azoospermia, and resulted in an ongoing pregnancy in 10 of 24 couples (41.6%) after 39 ICSI procedures, a success rate of 25.6% per treatment cycle and of 27.7% per embryo transfer.      

Accuracy of supplementary serologic testing for human T-lymphotropic virus types I and II in US blood donors. Retrovirus Epidemiology Donor Study

Blood donations in the United States have been screened for antibody to human T-lymphotropic virus type I (HTLV-I) by HTLV-I enzyme immunoassay (EIA) since November 1988. Specimens repeatedly found to be reactive by EIA undergo confirmation by supplementary serologic tests. We assessed the accuracy of blood center testing of 994 HTLV-I EIA repeat-reactive specimens in five US blood centers between November 1988 and December 1991. Of 410 confirmed HTLV-I/II donations, 407 (99.3%) were infected with HTLV-I/II, as determined by polymerase chain reaction (PCR) (403 cases) and by repeat serologic testing (4 cases). The three false- positive results occurred in the first year of testing. Of 425 HTLV- indeterminate specimens, 6 (1.4%) were found to be infected by PCR (5 with HTLV-II and 1 with HTLV-I). None of 159 confirmatory test-negative donations was PCR positive. Of HTLV-I/II-seropositive specimens, 80.2% to 95.4% could be typed as HTLV-I or HTLV-II by type-specific serologic assays. These results support recommendations that HTLV-I/II- seropositive donors should be advised that they are infected with HTLV- I, HTLV-II, or HTLV-I/II (depending on results of type-specific assays). HTLV-indeterminate donors should be advised that their results only rarely indicate HTLV infection. HTLV confirmatory test-negative donors should be reassured that they are not infected with HTLV-I or HTLV-II.     

DLA-identical bone marrow grafts after low-dose total body irradiation: the effect of canine recombinant hematopoietic growth factors

Previous studies found that bone marrow (BM) allografts from DLA- identical littermates resulted in survival of two thirds of recipient dogs after otherwise lethal doses of 450 to 600 cGy of total body irradiation (TBI) because of successful allografts or autologous recovery after rejection of the allografts. The current study asked whether survival could be further improved by treating allograft recipients with recombinant canine granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF), or G-CSF/SCF. Of 21 dogs, 14 (67%) receiving allografts but no growth factors survived, 10 with successful allografts (including 5 mixed chimeras) and 4 with autologous recovery; whereas 7 animals died, 5 from infections during BM aplasia and 2 from acute graft-versus-host disease. By comparison, 30 of 34 dogs (88%) receiving hematopoietic growth factors in addition to the BM graft survived, 17 with successful allografts (including 10 mixed chimeras) and 13 with autologous recovery; whereas 4 died, all with infection related to BM aplasia after rejection of the allograft. Survival was similar for recipients of G-CSF, SCF, or the combination of G-CSF and SCF. Logistic regression analyses, which accounted for possible effects of TBI dose, showed a trend for improved survival in dogs receiving growth factors (P = .09), no change in allogeneic engraftment (P = .74), and a slight increase in autologous recovery (P = .22). In agreement with previous data, we found that grafts of BM from DLA-identical littermates improved survival of recipient dogs exposed to low but otherwise lethal doses of TBI. A further improvement in survival could be achieved by additional treatment with G-CSF, SCF, or G-CSF/SCF. Results suggest that treatment by hematopoietic growth factors along with BM grafts should be considered for victims of radiation accidents.     

Contrasting effects of recombinant human granulocyte-macrophage colony- stimulating factor (CSF) and granulocyte CSF treatment on the cycling of blood elements in childhood-onset cyclic neutropenia

Recombinant human granulocyte colony-stimulating factor (G-CSF) treatment has been shown to increase average neutrophil counts substantially in patients with childhood-onset cyclic neutropenia (or "cyclic hematopoiesis"), but not to eliminate the cyclic oscillations of neutrophil counts or those of other blood elements (monocytes, platelets, eosinophils, and reticulocytes) that are characteristic of this hematopoietic disorder. Indeed, oscillations of neutrophil counts are amplified during G-CSF treatment. We have compared the effects of recombinant granulocyte-macrophage-CSF (GM-CSF) with those of G-CSF in three patients with this disease (2 men and 1 woman, 17, 30, and 32 years of age). These patients were treated with GM-CSF (2.1 micrograms/kg/day, subcutaneously) for 6 weeks, preceded and followed by 6 to 13 weeks of detailed observation to document changes in the cyclic oscillations of blood neutrophils and other blood elements; two of the patients were subsequently treated with G-CSF (5.0 micrograms/kg/d, subcutaneously) and observed for comparable periods of time. Unlike G-CSF treatment, which increased average neutrophil counts more than 20-fold, GM-CSF increased neutrophil counts only modestly, from 1.6- to 3.9-fold, although eosinophilia of varying prominence was induced in each patient. However, at the same time, GM-CSF treatment dampened or eliminated the multilineage oscillations of circulating blood elements (neutrophils, monocytes, platelets, and/or reticulocytes) in each of the patients. In contrast, G-CSF treatment of the same patients markedly amplified the oscillations of neutrophil counts and caused the cycling of other blood elements (monocytes in particular) to become more distinct. These findings support the conclusion that the distinctive cycling of blood cell production in childhood-onset cyclic neutropenia results from abnormalities in the coordinate regulation of both GM-CSF-responsive, multipotential progenitor cells and G-CSF-responsive, lineage-restricted, neutrophil progenitors.     

Expression of specific granule markers on the cell surface of neutrophil cytoplasts

The widespread assumption that cytoplasts generated from human polymorphonuclear leukocytes (PMNs) are vesicles consisting solely of cytoplasm surrounded by plasma membrane and devoid of granule activity remains to be tested. PMN cytoplasts were prepared by centrifugation of intact cells on a Ficoll step gradient in the presence of cytochalasin B. Two granule membrane markers, Mol, a fluorometrically detectable antigen, and cytochrome b, both of which have been shown to translocate to the plasma membrane during granule release, were compared for their activity in cytoplasts and intact PMNs. We found that the amount of Mol detected on the plasma membrane of intact PMNs, as compared with other membrane markers (such as antigens LFA-1 and beta 2m), increased 1.6- fold upon exposure of PMNs to Ficoll plus cytochalasin B prior to centrifugation. Another twofold increase in Mol expression occurred upon cytoplast preparation. Release of the granule enzymes, vitamin B12- binding protein, and lysozyme were also followed and correlated well (r = .78 and .92) with the amount of Mol antigen present on the cell surface. Cytochrome b was also found to be higher (1.4-fold) on plasma membranes isolated from cytoplasts than on plasma membranes isolated from intact control cells. These results indicate that some fusion of granule membranes and plasma membranes occurred during treatment of PMNs with Ficoll plus cytochalasin b and during cytoplast preparation.     

一种新的层析等电点聚焦技术提纯葡萄球菌肠毒素D

我们成功地建立了一个新的层析等电点聚焦的三步程序.细菌培养液首先以CG-50吸附,洗脱出的粗毒素溶液在PBE_(94)柱上进行层析等电点聚焦.分别用5mMTris-Base pH9.4和Buffalyte_(8-4) pH5.4缓冲液洗脱,出现多个洗脱峰,即刻测定pH值.收集pH6.8～7.1的洗脱液,混合、浓缩,最后通过Sephacryl s-200过滤,以0.05M PBS pH6.8洗脱,出现3个峰,毒素在第1峰.毒素纯度为98%,回收率89%,p17.6,MW28500,SDS-PAGE上1条带.以~(125)I标记SED作探针的电泳转移和放射自显影均证明了提纯SED的免疫学特异性.免疫扩散试验,即使200μg/ml SED与不产毒素的FRI-184抗血清,也未有可见沉淀线.     

Effects of cigarette smoking and age on the maturation of human oocytes

We investigated whether cigarette smoking, measured by follicular fluid concentrations of cotinine (a major metabolite of nicotine), affects the maturity of oocytes from women undergoing in-vitro fertilization (IVF) and embryo transfer. In 234 women, follicular fluid samples were assessed for cotinine and their 2020 oocytes were assessed for maturity stage. Data on individual proportions of oocytes which were mature (OM) and were fertilized (OF) were analysed by regression in relation to age and follicular fluid cotinine. OF gave an independent assessment of oocyte maturity. Both age and follicular fluid cotinine entered the OM and OF regressions and were significant. The age-adjusted regression coefficients for log cotinine were positive; greater cotinine concentrations usually accompanied greater OM and OF. The cotinine effect on OM was positive in younger women, but it became negative (decreased OM with increasing cotinine concentrations) in older women (> or = 40 years). We further found in older women an average reduction of approximately 50% in the number of mature oocytes; this reduced number was lower than the number of embryos usually transferred. Smoking can reduce the number of mature oocytes even further, therefore risking a negative IVF-embryo transfer outcome. This may be the reason why the negative effects of smoking become clinically detectable in older women.      

Synthesis of a cyclic retro analogue of somatostatin suitable for photoaffinity labelling

Cyclic somatostatin analogues containing the modified retro sequence of the amino acids Phe⁷ to Phe¹¹ of the natural compound have been found to exhibit high activity for cytoprotection of rat hepatocytes against cell poisons such as phallotoxins and galactosamine. Cyclo(-Phe(p-NH(1-¹⁴C)Ac)-Thr-Lys(CO(p-N₃)C₆H₄)-Trp-Phe-d -Pro-), a photoreactive and radioactive analogue of one of the most active cyclohexapeptides, was synthesized by a combination of solid phase technique and classical solution peptide synthesis. This peptide labels the same proteins in rat liver cell membrane that are modified by photolysable derivatives of bile acids, phalloidin and antamanide.     

Enhanced calcitonin secretion in the rat after parathyroidectomy and during chronic calcium deprivation

The thyroidal content of calcitonin (CT) and the serum calcitonin responses to acute hypercalcaemia were studied in female rats during chronic hypocalcaemia induced by parathyroidectomy (PTX), a low calcium (Ca) diet, or both. The thyroidal CT content of the PTX animals 50 days after surgery was twice that of intact rats. An acute intraperitoneal (i.p.) calcium load on day 50 after PTX resulted in an increase in serum CT twice as large as that observed in control animals. Reversal of the chronic hypocalcaemia with 1,25(OH)2D3 resulted in a reduction in thyroidal CT as well as a depression of the calcium-induced CT response. In each case the values were similar to those observed in normocalcaemic controls. A low calcium diet increased the thyroidal CT content in intact rats and induced a further increase in PTX rats. The CT response to an acute Ca load was exaggerated by a low calcium diet in intact as well as in PTX rats. These results suggest that in the rat chronic hypocalcaemia enhances CT storage and secretion.