Peter F. Levay

The detection of blood contamination in human follicular fluid

Purpose:The aim of this study was to compare the reliability of the methods conventionally used to identify low levels of blood contamination in human follicular fluid (hFF) as applicable in the clinical environment.Methods:Follicular fluid (n=339) and plasma samples (n=20) were collected from patients (n=138) attending the Centre for Fertility Studies, HF Verwoerd Hospital, University of Pretoria, South Africa. hFF blood contamination was assessed by means of (a) visual inspection, (b) hematocrit (Hct), (c) spectrophotometric analysis, (d) spectrophotometric hemoglobin kit, and (e) Combur-9-test urine sticks.Results:(1) Neither hematocrit nor spectrophotometry provided reliable detection at low levels of blood contamination. (2) Visual inspection presented with a better discriminatory ability than either Hct or spectrophotometry. (3) Combur-9-test sticks identified up to 50% of blood-contaminated fluids. (4) Spectrophotometrically determined hemoglobin levels presented with weak discriminatory abilities for detecting blood-contaminated fluids.Conclusions:Visual inspection as performed in this study provides a fast and relatively reliable method for the determination of blood-contaminated hFFs. In a laboratory environment, however, it would be recommended that a combination of visual inspection, Hct, and spectrophotometric evaluation be employed for the selection of blood-free fluids.

Interleukin-1β, interleukin-6, and growth hormone levels in human follicular fluid

PurposeTo investigate possible relationships of interleukin-1Β (IL-1Β, interleukin-6 (IL-6), and growth hormone (GH) with biochemical variables in human follicular fluid (FF) and selected in vitro fertilization (IVF) parameters.MethodsA total of 67 FF samples (n=67 patients undergoing oocyte retrieval for IVF) was evaluated. IL-1Β, IL-6, GH, hLH, FSH, PRL, hCG, testosterone, total protein, fibrinogen, sialic acid, α1-antitrypsin, plasminogen levels, and spectrophotometric absorbance at 458 nm were analyzed for selected FF. IL-6 and GH levels of serum and FF samples were also compared (n=23).ResultsImmunoreactive levels of IL-1Β, IL-6, and GH were detected in all FF samples. A positive correlation existed for IL-6 (r=0.5069, P=0.0161 when serum-to-FF levels were compared (concentration ratio, 1∶1.857). Smaller-volume follicles (<4 ml) were associated with high IL-1Β levels (P=0.0229, and an additional tendency of IL-1Β to decrease with increasing embryo cleavage and scoring was observed. With the exception of a weak positive correlation between follicular IL-1Β and testosterone levels (r=0.3128, P=0.025, no other relationship with biochemical variables or IVF parameters (etiology, e.g., endometriosis) could be implicated.ConclusionsSubstantially higher IL-6 levels occurred in FF compared to serum, thus supporting intrafollicular production. Interleukin- 1Β,IL-6, and GH levels in FF are, however, unsuitable markers for in vitro fertilization outcome.

Pro- and Anti-Inflammatory Cytokines during Immune Stimulation: Modulation of Iron Status and Red Blood Cell Profile

Forty-eight patients were subdivided according to C-reactive protein (CRP) levels, resulting in 19 patients with normal (2.8 ± 2.8 mg/L) and 29 with elevated (82.2 ± 76.2 mg/L) CRP levels. The elevated CRP group had iron and red blood cell (RBC) profiles characteristic of chronic immune stimulation (CIS), and the normal CRP group, profiles of true iron deficiency. Normal relationships between storage iron, bioavailable iron, and RBC indices were absent in the elevated CRP group—implying the role of iron as major determinant of the RBC profile to be diminished during CIS. The elevated CRP group had significant increases in proinflammatory cytokines (INF-γ, TNF-α, Il-1β, Il-6, and Il-8). Anti-inflammatory cytokine levels were normal, except for Il-10, supporting previous indications that Il-10 contributes to reducing bioavailable iron. Regression analysis suggested decreases in transferrin to be related to increases in Il-8 and an increase in ferritin to be related to a decrease in Il-12 levels. TGF-β levels were positively related to transferrin and negatively to ferritin.

A non-specific biomarker of disease activity in HIV/AIDS patients from resource-limited environments.

BACKGROUND A general non-specific marker of disease activity that could alert the clinician and prompt further investigation would be of value in patients with HIV/AIDS, especially in resource limited environments. OBJECTIVE To investigate the potential of neopterin as non-specific biomarker in patients with advanced HIV/AIDS. METHODS Cross-sectional study in 105 HIV positive patients (75 on highly active antiretroviral treatment (HAART). Neopterin was assessed by enzyme linked immune-absorbent assay and cytokines by flow cytometry. RESULTS Neopterin levels were significantly higher (p<0.001) for the total patient than for the control group. Significant correlations between neopterin and plasma indicators of inflammation showed neopterin to be a good indicator of active inflammatory status and of the effect of HAART on the immune system. Neopterin was superior to C-reactive protein and to individual cytokines as indicator of immune deficiency. Increased neopterin levels were associated with a decline in albumin, haemoglobin and the albumin/globulin ratio, and with increases in red cell distribution width. CONCLUSIONS Plasma neopterin is a good non-specific biomarker of disease activity in HIV/AIDS patients. It is a good indicator of inflammatory activity, perpetuation of inflammation-associated co-morbidities, degree of immune deficiency and has predictive value for underlying disease, and for monitoring the HAART response.

Expression of the H- and L-subunits of ferritin in bone marrow macrophages of patients with osteoarthritis

Osteoarthritis is a disease characterized by an increase in the production of reactive oxygen species (ROS) in afflicted joints. Excess iron, due to its role in the production of ROS and crystal deposition in the joints, is implicated in the disease progression of osteoarthritis. Ferritin is a major regulator of the bioavailability of iron, and its functions are determined largely by the combination of H- and L-subunits present in its outer protein shell. The purpose of the study was to investigate the expression of the H- and L-subunits of ferritin in bone marrow macrophages of osteoarthritis patients. The cytokine profiles were assessed as cytokines play an important role in the expression of the ferritin subunits. The H-subunit of ferritin in the bone marrow macrophages was significantly higher (P value = 0.035) in the osteoarthritis patients compared with the controls (107.84; 69.25–167.94 counts/μm2; n= 7 versus 71.07; 58.56–86.26 counts/μm2; n= 19). A marginally significant increase (P value = 0.059) was shown for the expression of the L-subunit in the osteoarthritis patients compared with the controls (133.03; 104.04–170.10 counts/μm2; n= 7 versus 104.23; 91.53–118.70 counts/μm2; n= 19). The osteoarthritis and control groups had comparable C-reactive protein, as well as proinflammatory and anti-inflammatory cytokine concentrations. The major exception was for transforming growth factor-β (TGF-β), which was higher (P value = 0.014) in the plasma of the osteoarthritis patients (16.69; 13.09–21.28 ng/mL; n= 7 versus 8.60; 6.34–11.67 ng/mL; n= 19). Up-regulation of the ferritin subunits decreases the levels of bioavailable iron and provides protection against the unwarranted production of ROS and crystal deposition. A role for TGF-β in the up-regulation of the expression of the H-subunit, and possibly the L-subunit, of ferritin is postulated in osteoarthritis.

Expression of the H-subunit and L-subunit of ferritin in bone marrow macrophages and cells of the erythron during cellular immune activation

BACKGROUND The expression of the two types of ferritin subunits, the H-subunit and L-subunit, has been shown to be differentially regulated by cytokines. The primary aim of the present study was to quantitatively measure the expression of the H-subunit and L-subunit of ferritin in bone marrow macrophages and cells of the erythron in patients with chronic T-helper cell type-1 immune stimulation. METHODS The expression of the H-subunit and L-subunit of ferritin in bone marrow macrophages and cells of the erythron was quantitatively evaluated by post-embedding immunolocalisation with immunogold transmission electron microscopy. RESULTS The present study showed up-regulation of the H-subunit of ferritin in the bone marrow macrophage in patients with pronounced cellular immune activation (94.7±37.3 counts/μm(2); n=31 vs 72.4±34.0 counts/μm(2); n=13, p-value=0.037). CONCLUSION This supports a possible role for H-subunit rich ferritins in the hypoferraemia of chronic disease.

Spectrophotometric analysis of human follicular fluid with regard to in vitro fertilization (IVF) parameters, follicular protein, and hormone content

PurposeOur purpose was to investigate possible relationships with spectrophotometric absorbance (458-nm region) and biochemical variables in follicular fluid (FF) as well as in vitro fertilization (IVF) outcome.MethodsThis study included 227 normal ovulatory women undergoing oocyte retrieval for IVF. Blooduncontaminated fluid samples, identified by spectrophotometry, were investigated. Spectrophotometric absorbance of FF at 458 nm (n = 426), as well as hLH, FSH, PRL, hCG, testosterone, sialic acid, α1-antitrypsin and plasminogen of selected fluids, was analyzed.ResultsSmall-volume follicles (≤2 ml) were associated with higher absorbance profiles (P <0.05), when compared to volumes greater than 2 ml. Our data suggest that the presence or absence of an oocyte, the potential of an oocyte to fertilize or cleave, failed to show any relationship with maximum FF absorbance at 458 nm. Maximum absorbances were significantly lower in FF from patients who subsequently became clinically pregnant (P =0.039). No correlation between FF absorbances and biochemical parameters (P >0.15) were established.ConclusionsAbsorbance of clear FF at 458 nm should not be viewed as the single parameter to predict oocyte development in vitro.

Electron microscopic study of human sperm membrane isolation

ObjectOur purpose was to isolated pure, homogeneous human sperm membranes, free of cellular contaminants.MethodsDonor semen samples collected after masturbation were stored at −70°C and eventually pooled. Each attempt at sperm membrane isolation required 800 × 106 spermatozoa which were sonicated by ultrasound (40% output; Vibra Cell). The effect of sonication time (3 × 5, 3 × 15, and 180 sec) on membrane isolation was investigated. Sonicated samples were centrifuged (500g, 5 min) and the supernatant was pipetted off. The supernatant of the centrifuged sample was layered on either a sucrose cushion (supernatant on 1.6 M sucrose) or a discontinuous sucrose gradient and centrifuged (100,000g, 1 hr). Contents of supernatants of sonicated samples and fractions (sucrose interfaces) were then fixed in 1.0% tannic acid and 2.5% buffered glutaraldehyde and examined electron microscopically using standard procedures.Results(1) The optimal sonification time was found to be 3 × 15 sec. (2) Membrane isolation using a sucrose cushion was found to be inadequate, showing significant cellular contamination. (3) Sperm membrane isolation from the sucrose interface between 0.75 and 1.05 M sucrose was found to be most effective.ConclusionThe advantage of this method is its simplicity. The drawback of this method is the large number of spermatozoa required for membrane purification.

Freeze-thaw of human follicular fluid: Influence on the biochemical profile

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