Round spermatids from infertile men exhibit decreased protamine-1 and -2 mRNA

During spermiogenesis, histone-to-protamine exchange causes chromatin condensation. Spermatozoa from infertile men are known to exhibit an increased protamine-1 (PRM1) to protamine-2 (PRM2) protein ratio. Since patients undergoing testicular sperm extraction (TESE) followed by intracytoplasmic sperm injection (ICSI) reveal low fertilization rates, whether the outcome of ICSI could be related to the percentage of round spermatids expressing PRM1-mRNA and PRM2-mRNA was investigated. Applying in-situ hybridization, 55 testicular biopsies from men undergoing TESE/ICSI were investigated. The percentage of PRM1-mRNA and PRM2-mRNA positive spermatids was significantly (P < 0.0001) decreased in men with at least qualitatively normal spermatogenesis (PRM1-mRNA: 58.4 +/- 13.8%; PRM2-mRNA: 56.4 +/- 11.3%) and impaired spermatogenesis (PRM1-mRNA: 32.6 +/- 10.8%; PRM2-mRNA: 31.7 +/- 11.1%) compared with men with obstructive azoospermia and quantitatively normal spermatogenesis (PRM1-mRNA: 79.9 +/- 4.6%; PRM2-mRNA: 78.1 +/- 5.7%). A positive correlation (r(PRM1) = 0.733; r(PRM2) = 0.784; P < 0.001) was demonstrated between the score and the percentage of PRM1-mRNA and PRM2-mRNA positive spermatids. While successful fertilization was neither related to the score, nor to the percentage of PRM1-mRNA and PRM2-mRNA positive spermatids, a significant (P < 0.05) relationship was demonstrated between successful fertilization and the PRM1-mRNA to PRM2-mRNA ratio. Therefore, the PRM1-mRNA to PRM2-mRNA ratio in round spermatids may serve as a possible predictive factor for the outcome of ICSI.     

Flow of cells from polar to mural trophectoderm is polarized in the mouse blastocyst

During growth of the blastocyst there is a net flow of cells from the polar to the mural trophectoderm which is presumed to be radially symmetrical. However, such a pattern of cell movement is inconsistent with findings from a recent clonal analysis. To visualize the overall flow of cells directly, the polar trophectoderm of expanding blastocysts was labelled globally with fluorescent microspheres. Following further growth, the great majority of blastocysts that remained labelled throughout the polar trophectoderm exhibited a polarized rather than radial spread of label into the mural region. This was the case regardless of the labelling technique, whether the blastocysts were grown in utero or in vitro, or had the zona pellucida removed or left on. Intriguingly, where there were two foci of spread of label into the mural trophectoderm rather than one, these were diametrically opposite each other. In further experiments, fluorescent lineage labels were used to distinguish junctional trophectoderm cells with and without an extension onto the blastocoelic surface of the inner cell mass. The location of clones formed following further blastocyst growth provided no evidence that egress of cells from the polar trophectoderm is restricted circumferentially by the presence of junctional cells having an extension.     

Activated protein C resistance shows an association with pregnancy-induced hypertension

A common mutation in the factor V gene, the Leiden mutation, is the most frequent genetic cause of resistance to activated protein C (APC). Recent studies have shown that the prevalence of APC resistance is associated with severe pregnancy-induced hypertension (PIH). Our objective was to determine whether the factor V Leiden mutation is more prevalent in patients who developed severe PIH than in normotensive pregnant women. In 70 women with a history of severe PIH, of whom 15 had pre-eclampsia, we investigated common coagulation factors as well as APC resistance (factor V related). We found that seven of these 70 women showed low values for APC. Out of these, five were heterozygous and none was homozygous for factor V Leiden mutation. In a control group of normotensive pregnant women we found a 3.0% rate of APC resistance and a 3.0% rate of carriers of the Leiden mutation. These results indicate a significantly higher prevalence of both APC resistance and factor V Leiden mutation in women with severe PIH. Placental infarctions and micro-embolisms are considered to be one of the principle pathophysiological changes in severe PIH. Our results suggest that APC resistance is a risk factor for severe PIH, in addition to its well-known role in macrothrombo-embolism.     

Clinical,hematologic, and immunologic effects of interleukin-10 in humans

We conducted a double-blind, placebo-controlled study to investigate the safety, pharmacokinetics, and immunological properties of interleukin-10 (IL-10) administration in healthy humans. Volunteers received a single intravenous bolus injection of recombinant human IL-10 (1, 10, or 25g/kg) or placebo. Cytokine production in whole blood and peripheral blood mononuclear cells (PBMC) was assessed before and 3, 6, 24, and 48 hr after the injection. Peak serum concentrations of IL-10 (15±1.1, 208±20.1, and 505±22.3 ng/ml) occurred after 2–5 min for 1, 10, and 25g/kg IL-10, respectively. The terminal-phase half-life was 3.18 hr. A transient leukocytosis (24–63% above baseline) was observed 6 hr after injection, which coincided with a dose-dependent decrease (12–24%) in neutrophil superoxide generation. There was a marked inhibition (60–95%) of endotoxin-induced IL-6 production from whole blood in each group receiving IL-10. Production of IL-8 in endotoxin-stimulated blood was reduced in the 10g/kg group. In PBMC stimulated with phytohemagglutinin and phorbol ester, there was a decrease (72–87%) in interferon- (IFN) production 6 hr after IL-10 with a return to pre-IL-10 levels after 24 hr. This reduction was only partially associated with a decrease in the number of CD2-bearing cells. We conclude that IL-10 administration into humans is without significant side effects, and a single injection reducesex vivo production of IL-6, IL-8, and IFN.     

Reducing the time of sperm-oocyte interaction in human in-vitro fertilization improves the implantation rate

Human oocyte development was evaluated after a reduced timeexposure to spermatozoa in vitro. A total of 119 patients wereassigned to two study groups in a randomized prospective studyin which each patient‘s oocytes were exposed to spermatozoafor either 1 h (group 1 – 58 patients) or the standard16 h incubation period (group 2 – 61 patients). The fertilizationrate obtained in group 1 was higher than in group 2 (285/393,73%, and 272/410, 66% respectively), suggesting that the spermatozoa-oocyteinteraction occurs within 1 h. This was confirmed in a studyin vitro using fluorescently labelled spermatozoa and normaloocyte-cumulus complexes. Spermatozoa enter the cumulus complexwithin 15 min, traverse the cumulus layer within 3 h, and firstappear in the oocyte cortex at 4 h post-insemination. The incidenceof polyspermy was higher in oocytes exposed to spermatozoa for16 h (3%) than for 1 h (1%). There was no difference in thecleavage rate or morphological characteristics of embryos fromboth study groups. However, when evaluating the timing of embryodevelopment, group 1 generated a significantly higher percentageof four to five cell embryos when compared to group 2 (55 versus39%; P < 0.001), documented at 40 h post-insemination. Theimplantation and pregnancy rates for group 1 were 11 and 28%,while the corresponding rates for group 2 were 8 and 15%. Thissuggests that a reduced exposure of oocyte to spermatozoa favoursembryo viability, possibly due to a decrease in potential damagefrom sperm metabolic waste products.     

Molecular mechanisms underlying IFN-{gamma}-mediated tumor growth inhibition induced during tumor growth inhibition induced during tumor immunotherapy with rIL-12

The present studnt Investigates the molecular by which IFN-produced as a result of in vitroIL-12 addministration exertsits anty-tumor,rIL-12 was administered three or five times intomice bearing CDA1M fibrosarcoma, OV-HM ovarian carcinoma orMCH-1-A1 fibosarcoma. This regimen induced complete regressionof CSA1M and OV-HM tumors but only transient growth inhibitionof MCH-1-A1 tumors. The anty-tumor effects of Il-12 were associatatedwith enhanced induction of IFN-becouse these effects were abrogatedby pretreatment of hosts with anti-IFN- antibody.Exposure inin vitro of the three types of tumor cells to rIFN- resultedin moderate to potent inhibition of tumor cell growth.IFNstimulatedthe expression of mRNAs for an inducible type of NO synthasa(INOS)in CSA1M cells and indoleamine 2,3-dioxygenasa (IDO),an enzyme capable of degrading tryptophan, in OV-HM cells ,but induced only marginal levels of these mRNAs in MCH-I-ALcells. In association withiNOS gene expression, INF--stimulatedCSA1M cells produced a large amount of NO which functioned toinhibit their own growth in vitro. Although OV-HM and MCH-1-A1cells did not produce NO, they also exhibited NO susceptibility.Whereasthe tumor masses from IL-12-treated CSA1M-bearing mice inducedhigher levels of INOS (for CSA1M) or IDO and iNOS (for OV-HM)mRNAs,the MCH-1-A1 tumor mass expressed lower levels of iNOS mRNAalone.Moreover, massive infiltration of CD4⁺and CD8⁺ T cellsand Mac-1⁺ cells was seen only in the CSA1M and OV-HM tumors.Thus, these results indicate that IFN- produced after IL-12treatment induces the expression of various genes with potentialto modulate tumor cells and growth by acting directly on tumorecells or stimulating tumor-infiltrating lymphold cells and thatthe effectiveness of IL12 therapy is assoiated with the operation if these mechanisms.     

Glycoprotein B of bovine herpesvirus type 4: Its phylogenetic relationship to gB equivalents of the herpesviruses

In order to estimate the phylogenetic relationship of BHV-4 among the herpesviruses, we have cloned and sequenced its glycoprotein B (gB). The 2.6 kb open reading frame codes for a 874 amino acid long protein. The comparison of its deduced amino acid sequence with those of its counterparts in 19 distinct herpesviruses groups BHV-4 into the -herpesvirinae. The calculation of an evolutionary tree emphasized that BHV-4 is more closely related to herpesvirus saimiri (HVS) than to Epstein-Barr virus (EBV). However, in contrast to EBV and HVS, the gB of BHV-4 contains a putative protease cleavage site and 20 potential N-glycosylation sites. The alignment of the amino acid sequences revealed that 10 cysteine and 7 proline residues, as well as the motifs SPF and GQLG, were completely conserved among the 20 investigated gBs.     

Miscarriage rates following in-vitro fertilization are increased in women with polycystic ovaries and reduced by pituitary desensitization with buserelin

To assess the risk of miscarriage after in-vitro fertilization(IVF) with respect to age, cause of infertility, ovarian morphologyand treatment regimen, a retrospective analysis was performedof the first 1060 pregnancies conceived between June 1984 andJuly 1990 as a result of 7623 IVF cycles. Superovulation inductionwas achieved with human menopausal gonadotrophin (HMG) and/orpurified follicle stimulating hormone (FSH) together with eitherclomiphene citrate or the gonadotrophin hormone-releasing hormone(GnRH) agonist buserelin, the latter either as a short ‘flare’regimen or as a ‘long’ regimen to induce pituitarydesensitization. There were 282 spontaneous abortions (26.6%)and 54 ectopic pregnancies (5.1%). The mean age of women withongoing pregnancies was 32.2 (SD 3.9) years compared with 33.2(SD 4.1) years in those who miscarried, which were significantlydifferent (P = 0.008). There was no relation between the miscarriagerate and the indication for IVF. The miscarriage rate was 23.6%in women with normal ovaries compared with 35.8% in those withpolycystic ovaries [P = 0.0038, 95% confidence interval (CI)4.68–23.10%]. There was no difference in the miscarriagerate between treatment with HMG or FSH. Women whose ovarieswere normal on ultrasound were just as likely to miscarry ifthey were treated with clomiphene or with the long buserelinprotocol. Those with polycystic ovaries, however, had a significantreduction in the rate of miscarriage when treated with the longbuserelin protocol, 20.3% (15/74), compared with clomiphenecitrate, 47.2% (51/108) (P = 0.0003, 95% CI 13.82–40.09%).     

The regulatory role of FSH in steroid formation by luteinized human granulosa cells in culture

Granulosa cells were aspirated from human pre-ovulatory folliclesfollowing a combined clomiphene-gonadotrophin stimulation inan in-vitro fertilization (IVF) programme. The cells were culturedfor 8 days in medium M199 containing 10% bovine fetal calf serumunder 5% CO₂ in air. Pure human FSH and human LH were addedalone or in combination to the culture in various concentrationsand the progesterone (P) and oestradiol-17 (E₂) levels in themedium were measured every second day by a conventional RIAtechnique. In the presence of FSH or LH the formation of P increased2- to 3-fold with the pronounced effect after 4 to 6 days inculture. Addition of testosterone (T) (3 ? 10^–7 M) tothe culture medium affected neither basal nor gonadotrophinstimulated P formation. In this system, only minute amountsof E₂ were formed and neither FSH nor LH stimulated its formation.When the medium was fortified with T, basal E₂ formation increased50- to 100-fold. FSH further stimulated this conversion significantlyafter 6 and 8 days of culture, while LH had no significant influence.