Effect of alcohol intake and cigarette smoking on sperm parameters and pregnancy.

Much has been published about smoking and alcohol intake influencing male fertility, sperm parameters and reproductive outcome. However, there is no conclusive agreement about the effects of cigarette smoking and alcohol use on these outcomes and thus no generally accepted guidelines. The combined effect of cigarette smoking and alcohol intake, though, has not been rigorously investigated. Because alcohol consumption and smoking are often seen together, this study focuses on the effect of smoking and drinking habits separately and combined on semen parameters, such as volume, sperm count, motility and morphology, and on pregnancy outcome. These suggested toxic effects are studied in a group of subfertile, asthenozoospermic men (<10% motile spermatozoa), compared with a group of 'proven fertile', healthy men. The extreme asthenozoospermic group has especially been chosen because of the suspected effect, that is, oxidative stress, on sperm motility. In our study, we found that cigarette smoking and alcohol intake did not differ between the subfertile and fertile group. In conclusion, cigarette smoking and alcohol consumption do not appear to significantly affect sperm parameters, such as volume, sperm count, motility and morphology or pregnancy outcome in our study population.

Metabolomic profiling by near-infrared spectroscopy as a tool to assess embryo viability: a novel, non-invasive method for embryo selection.

BACKGROUND: The morphology of an embryo has a limited predictive value for assessing viability and ongoing pregnancy, therefore new selection tools are needed to maintain success rates with single-embryo transfer (SET). In this study, we investigated if metabolomic profiling of biomarkers of embryo culture medium by near-infrared (NIR) spectroscopy has a correlation with ongoing pregnancy in SET. METHODS: A total of 333 patients scheduled for in vitro fertilization (IVF) with SET were included in the study. Embryos were selected for transfer by morphological criteria on Days 2 and 3 of in vitro culture, and left over culture media samples were analyzed by NIR spectroscopy. RESULTS: The NIR spectral analysis produced unique metabolomic profiles that correlated to an embryo's reproductive potential. Resulting relative viability scores between positive and negative pregnancy outcomes were statistically significant (P < 0.03). A logistic regression of factors correlated to pregnancy outcomes showed that maternal age, percent fragmentation and relative viability scores all demonstrated a relationship. The extent of the correlation was determined by accuracy computation, where the accuracy of assessing viable embryos on Day 3 by metabolomic profiling was 53.6% and the accuracy of the morphological selection was 38.5%. In addition, the positive predictive value of metabolomic profiling was 0.365 and the negative predictive value was 0.830. CONCLUSIONS: NIR metabolomic profiling of spent embryo culture media was able to distinguish viable embryos from non-viable embryos for reproduction.

The postwash total progressively motile sperm cell count is a reliable predictor of total fertilization failure during in vitro fertilization treatment.

OBJECTIVE: To predict the chance of total fertilization failure (TFF) before the day of ovum pickup with known semen and female variables. DESIGN: A statistical model was constructed to predict TFF by retrospective analysis (2,366 couples) and subsequently tested on a new IVF population (917 couples). SETTING: Academic tertiary referral center. PATIENT(S): Three thousand three hundred eighty-three couples who underwent an IVF-ET treatment. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The ability to predict the probability of TFF in IVF. RESULT(S): Two variables-postwash total progressively motile sperm cell count (postwash TPMC) and number of follicles-were found to be significant. Taking a probability of 25% as an acceptable risk of TFF, we calculated that a postwash TPMC of <1.1 x 10(6) cells results in a risk of TFF of >25%. Low responders (<4 follicles) needed a postwash TPMC of >2.2 x 10(6) cells to reduce the risk of TFF to <25%. High responders (>15 follicles) needed only 0.35 x 10(6) postwash progressively motile spermatozoa. CONCLUSION(S): When postwash TPMC and number of follicles are known and an unacceptable TFF outcome is expected, one can propose an ICSI procedure a few days before the day of ovum pickup.

The probability of pregnancy after embryo transfer is affected by the age of the patient, cause of infertility, number of embryos transferred and the average morphology score, as revealed by multiple logistic regression analysis.

Because the process of conception is affected by many variables, a multiple logistic regression analysis was performed to assess (i) the impact and relative weight of both patient and embryo variables and (ii) their possible effects on the probability of a vital pregnancy after embryo transfer. A statistical model was constructed predicting the probability of pregnancy after embryo transfer. The variables that contributed significantly to the predictive value of the model were the age of the patient, the cause of infertility, the number of embryos transferred and the average morphology score of the transferred embryos. Embryo variables appeared to have a significant but modest value in predicting the probability of pregnancy after embryo transfer. Other variables, such as the thickness of the endometrium, were found to have no prognostic value. Moreover, we found that their effect could be explained by the variables already included in the model.

A study of cells present in lymph draining from a contact allergic reaction in pigs sensitized to DNFB.

Pigs were skin-painted with the contact sensitizing agent 1-fluoro 2,4 dinitrobenzene (DNFB) and lymph cells coming from the site were collected. Half the animals were sensitive to DNFB and half were normal controls. Special attention was paid to cells belonging to the veiled cell series. At 20 hr after DNFB application, some blast cells--on morphological grounds belonging to the veiled cell series--could be observed in the lymph of presensitized animals. But the most predominant finding in these pigs was that, approximately 30 hr after painting, the total cell output began to increase, reaching 10 times the normal level at 70 hr. The increase in the output of lymphoblasts was the most marked, suggesting peripheral sensitization. Such changes were not observed in normal animals. Here the most striking alterations were seen in the veiled cells. Some of these cells were moving ponderously about, apparently in pursuit of other lymph-borne cells, and this activity resulted in the formation of large cellular aggregates. Since it is known that veiled cells are involved in antigen handling, this behaviour probably facilitates the induction of the T-cell immune response. By contrast, the veiled cells from presensitized animals behaved in a more normal manner and the proportion found in aggregates was only briefly increased. In these animals lymphocytes were seen to interact with veiled cells in a manner reminiscent of peripolesis, apparently recognizing antigenic signals on the surface of the veiled cells. It is discussed that this might result in deletion of the antigen-presenting veiled cells, thus controlling the magnitude of the immune response.

Quantitation of arthritis by 99mTc-uptake measurements in the mouse knee-joint: correlation with histological joint inflammation scores.

A method is described to detect and to quantitate inflammation in knee-joints of mice. Approximately 10 muCi 99mTechnetium pertechnetate (99mTc) is injected subcutaneously in the neck region and the accumulation of the short-lived isotope in the knee-joint is detected by external gamma counting. 99mTc-uptake values correlate well with histological grading of joint inflammation at various intervals after induction of the inflammation. The method can be used to detect changes in activity of unilateral as well as bilateral joint inflammation using either the ratio of 99mTc uptake in the right knee versus that in the left knee or absolute 99mTc-uptake values.

Flare-up of antigen-induced arthritis in mice after challenge with intravenous antigen: studies on the characteristics of and mechanisms involved in the reaction.

Intravenous (i.v.) administration of 300 micrograms methylated BSA (mBSA) to C57-Black mice with unilateral antigen-induced arthritis (AIA) of 4-6 weeks duration resulted in a reproducible flare-up of ongoing arthritis without signs of inflammation in the contralateral non-arthritic knee joint. In the present study we investigated characteristics of the flare-up phenomenon using histological grading and 99mtechnetium pertechnetate uptake measurements to detect and quantitate knee joint inflammation. At 6 h after intravenous mBSA administration flare-up of arthritis was demonstrable, showing histological characteristics of acute joint inflammation; later this progressed to chronic stages, but the inflammatory stage did not return to baseline values up to day 9 after i.v. challenge. Varying the dose of i.v. mBSA from 2 to 1000 micrograms showed that 10 micrograms was sufficient to elicit the phenomenon and also that the latter is dose-dependent. Using mice immunized with both mBSA and methylated HGG (mHGG) with either mBSA or mHGG-induced arthritis, we could show that 4-6 weeks after arthritis induction the flare-up phenomenon is antigen specific. Intra-articular injection of as little as 10 ng of mBSA resulted in exacerbation of inflammation in joints with chronic mBSA-induced arthritis, but induced no inflammation in the contralateral non-arthritic knee joints, indicating local hypersensitivity in the former joints. The flare-up phenomenon may be due to local hyper-reactivity of chronically inflamed joints to minimal amounts of circulating antigen entering the joint.

Only hydrosalpinges visible on ultrasound are associated with reduced implantation and pregnancy rates after in-vitro fertilization.

A retrospective analysis of clinical and laboratory data was made of all in-vitro fertilization (IVF) patients with tubal pathology who had their first ever embryo transfer cycle between January 1st, 1992 and September 1st, 1996. The aim of the study was to determine the effect of the presence of a hydrosalpinx, whether or not visible by ultrasound, on pregnancy, multiple pregnancy and implantation rates in our patient population. The IVF success rate was also analysed by calculating cumulative ongoing pregnancy rates of the same patient group using the lifetime table approach. In the presence of an ultrasound-visible hydrosalpinx, rates of pregnancy and multiple pregnancy appeared reduced, but the differences were not significant. The rates of implantation, clinical implantation and ongoing implantation were significantly lower in the presence of an ultrasound-visible hydrosalpinx (odds ratios 0.33-0.46, C.I. 0.21-0.96). The cumulative chance of achieving an ongoing pregnancy after one or more IVF cycles was significantly reduced in the presence of an ultrasound-visible hydrosalpinx (relative hazard 0.36, C.I. 0.22-0.59). In the presence of a hydrosalpinx not visible by ultrasound the IVF outcome was not reduced. This retrospective study confirms that patients with hydrosalpinges have an impaired IVF outcome. Unique to this study and previously unobserved is the finding that there is a subgroup of patients with hydrosalpinges, those with ultrasound-visible hydrosalpinges, which is exclusively responsible for this impaired outcome.

Flare of antigen-induced arthritis in mice after intravenous challenge. Kinetics of antigen in the circulation and localization of antigen in the arthritic and noninflamed joint.

Intravenous injection of methylated bovine serum albumin (mBSA) in mice with unilateral, chronic, mBSA-induced arthritis has been shown to cause a flare of smouldering arthritis without affecting the contralateral, noninflamed knee joint. We studied the kinetics of 125I-labeled mBSA in the blood, and the accumulation of antigen in both arthritic and noninflamed joints. The bulk of the antigen was eliminated from the blood within 10-30 minutes, and accumulation of antigen in the joints occurred mainly within this period. The amounts of antigen found in the arthritic joints were of the same order of magnitude as the amounts found in noninflamed joints. Autoradiography of whole joint sections revealed that, in arthritic joints, antigen was located primarily at the deep capillaries and large vessels, and in the noninflamed joints, antigen was located at the small superficial capillaries. Antigen was handled by granulocytes in the arthritic joint and by synovial lining cells in the noninflamed joint. Our data indicate that tiny amounts of antigen reach the synovial stroma in both normal and arthritic joints but cause inflammation only in the arthritic joints, because of local hyperreactivity.

Exacerbation of antigen-induced arthritis after challenge with intravenous antigen.

The occurrence and morphology of flare-up of arthritis after intravenous (i.v.) antigen administration was investigated in mice with on-going antigen-induced arthritis (AIA). Intravenous injection of 300 micrograms methylated bovine serum albumin (mBSA) in mice with unilateral arthritis induced by mBSA, elicited 6 weeks previously, caused clear flare-up of the smouldering joint inflammation without affecting the contralateral non-arthritic knee joint. Histological signs of the flare-up reaction were already present at 6 hr after i.v. challenge and lasted for at least 4 days. Characteristic features are the presence of large numbers of granulocytes just beneath the synovial lining layer, inflammatory foci in adjacent periarticular tissues, and deposits of fibrin-like material in the joint space. Variations in the severity of the preceding arthritis and in the interval (4-12 weeks) between AIA induction and i.v. challenge had no major influence on the occurrence and degree of the flare-up phenomenon. Since exacerbations of joint inflammation are seen in rheumatoid arthritis, this phenomenon may be of importance in this disease.

The involvement of dendritic cells in the handling of the immune stimulant C. parvum. A morphological investigation using immunoperoxidase techniques.

In earlier experiments we showed that locally administered Corynebacterium parvum (C. parvum) stimulated the T-cell system and had a beneficial effect on the recurrence rate of surgically resected cancers of the uterine cervix. In this paper we report the use of an immunoperoxidase technique to trace C. parvum antigen in the draining lymph nodes. In a guinea pig model the popliteal lymph node was studied after the injection of 70 micrograms of C. parvum in the hind footpad. At 6 h, intact bacteria were detected in sinus histiocytes. A transient granuloma formation was apparent between days 2 and 6, originating in the subcapsular and interfollicular areas. Three antigen-positive cell types were observed in these granulomas: a) cytophagocytic macrophages which were weakly positive, the antigen being distributed in clumps; b) dendritic cells with a strong, fine-granular positivity and c) some epithelioid cells with a small positive cytoplasmic rim. The majority of epithelioid cells was negative. Antigen-positive dendritic cells were also observed just beyond the granulomas in the T-dependent paracortical area. These cells are known as interdigitating cells (IDC) and present antigen to T-cells. Ten days following C. parvum injection the lymph node follicles became positive and the antigen could be detected in the long cellular protrusions of the dendritic reticulum cells (DRC). DRC probably play a part in immunological memory by trapping antigen in the form of immune complexes.(ABSTRACT TRUNCATED AT 250 WORDS)

Flare-up of antigen-induced arthritis in mice after challenge with oral antigen.

Mice with unilateral chronic mBSA-induced arthritis were orally challenged with mBSA. Three hours after antigen challenge clear flare-up of the chronic arthritis was demonstrable as detected by an increase in the 99mTc uptake of the knee joint and the reaction continued for at least 2 days. The contralateral non-arthritic knee joint was not affected. The dose of mBSA needed to induce a flare-up in nearly all mice within a group was in the order of 20 mg. After oral challenge with 10 or 5 mg of mBSA the incidence was lower and flare-up reactions were only rarely observed after challenge with 2.5 or 1.25 mg mBSA. Histology of knee joints taken at 24 h after oral challenge of 20 mg mBSA revealed an increase in the number of cells in the infiltrate in the synovial tissue and exudate in the joint space, the most conspicuous sign being the increase of PMN. Passage of macromolecules through the gastrointestinal mucosa may be an important principle in the perpetuation of human chronic arthritis.

Antigen induced arthritis: antigen handling and chronicity of joint inflammation.

Data are summarized on antigen handling by the joint and the relevance of antigen retention for the chronicity of joint inflammation. Antigen retention may occur at collagenous structures of the joint, when antibodies are present in these tissues or simply when the charge of an antigen favours nonimmune binding at the highly charged collagenous tissues. Antigen retention will change with alterations in antibody load and fixed charge density of these tissues. Longterm retained antigen seems not inflammatory at the retention sites, but its importance for the chronicity of joint inflammation may lie in the continuous leakage of small amounts to other compartments, where it does behave as an inflammatory stimulus, thereby rendering the joint specifically hypersensitive to antigen. Such a joint reacts to minute amounts of antigen in the circulation leading to exacerbation of smouldering joint inflammation.

Extravasation of proteins in the joint tissue: differential behaviour of native BSA and cationic mBSA.

Mice were intravenously injected with 125I-labelled native BSA and cationic mBSA. The amount of 125I-protein in the extra- and intravascular compartment was determined in normal joints and in joints in which the vascular permeability was increased by histamine. The extravasation of BSA and mBSA is different. More BSA compared with mBSA accumulated in normal joints and the extravasation of BSA increased almost 3 times shortly after histamine injection, whereas histamine hardly affected the extravasation of mBSA.

Antibodies to 'second colloid antigen.' A study on the prevalence in sporadic forms of congenital hypothyroidism.

Antibodies against the so called 'second colloid antigen' (CA2 antibodies) occurred in 51% of the mothers of hypothyroid children detected by screening for neonatal congenital hypothyroidism in Quebec (N = 49) and in The Netherlands (N = 26). In The Netherlands where corresponding neonatal serum was available, 31% (8 of 26) of the infants with congenital hypothyroidism were positive for antibodies against the second colloid antigen. When during follow-up, 3 to 5 years after diagnosis, the mothers and their children were investigated, 46% (7 of 15) of the mothers were positive for antibodies against the second colloid antigen, whereas 29% (4 of 14) of the hypothyroid children were also positive. Various control groups did not show more than a 12% positivity. This presence of thyroid-reactive antibodies in a proportion of the hypothyroid children 3 to 5 years after diagnosis is not compatible with a mere transplacental passage; it indicates that the antibodies must be produced by the mothers and by the children themselves. We conclude that a thyroid autoimmune response occurs in a considerable part of infants with congenital hypothyroidism and their mothers and that this immune response seems to persist in both of them for years.

Morphology of draining lymph nodes after local immune stimulation with C. parvum: comparison of pelvic nodes in carcinoma of cervix and popliteal and inguinal nodes of guinea-pig.

Morphological changes are described in pelvic lymph nodes excised 10 days after C. parvum (CP) treatment of patients with cervical carcinoma. Guinea-pig popliteal and inguinal lymph nodes were investigated from Days 1 to 10 after an injection of 70 micrograms CP into the footpad. Eosinophils were detected from the first few hours after stimulation, initially in the marginal sinus, then in the medullary sinuses and subsequently in the efferent lymphatics. From Day 2 to Day 6, histiocyte accumulations with the appearance of epithelioid cells were found mainly in subcapsular and interfollicular areas, and small granulomas were also seen in the paracortex. The granuloma formation in the lymph node was considered as an indication of the activation of histiocytes. Besides small granulomas in the paracortex, activated interdigitating cells, surrounded by scattered lymphoblasts and eosinophils, were also present. We considered this lymphoblastic response and eosinophilic accumulation as likely to be due to blastogenic factor and eosinophil stimulation promotor. Eight to 10 days after CP stimulation, the macrophage lymphoblast eosinophil response was replaced by a B-cell reaction: germinal-centre activation and medullary plasma cells. Such a B-cell reaction was also found in the human pelvic nodes removed at operation, but this reaction could not be attributed to CP treatment alone, since cervical-carcinoma patients not treated with CP also showed such reactions. In contrast, pelvic lymph nodes removed at necropsy from females killed in traffic accidents showed no predominance of either B- or T-cell stimulation.

Flare-up of antigen-induced arthritis in mice after challenge with intravenous antigen: effects of pre-treatment with cobra venom factor and anti-lymphocyte serum.

Intravenous injection of methylated bovine serum albumin (mBSA) into mice with unilateral chronic mBSA-induced arthritis (AIA) causes a flare-up of the joint inflammation without affecting the contralateral non-arthritic knee joint. We studied the mechanism of the flare-up by decomplementation with cobra venom factor (CoVF) and by treatment with anti-lymphocyte serum (ALS) prior to the induction of the flare-up. Treatment of mice with CoVF had no effect on the induction of the flare-up reaction whereas a reversed passive Arthus reaction (RPA) in the ear of similarly treated mice was clearly suppressed. The complement activity in the serum was zero at 2 h after CoVF treatment and remained for 24 h. This indicates that this type of flare-up reaction is not complement-dependent. On the other hand, the flare-up reaction was completely abolished after treatment with ALS. Control experiments revealed that ALS treatment diminished the number of lymphocytes in the peripheral blood and clearly suppressed a delayed hypersensitivity reaction in the ear, but had no effect on an RPA. These results suggest an important role of T lymphocytes in the mechanism of the flare-up of arthritis. T lymphocytes were demonstrated in the synovial tissue of chronically inflamed joints by immunofluorescence and appeared to be diminished after ALS treatment. Interaction between exogenous antigen and antigen reactive T lymphocytes present in chronically inflamed joints, may be an important principle in the exacerbation and propagation of joint inflammation.

Lower relapse rates after neighbourhood injection of Corynebacterium parvum in operable cervix carcinoma.

The effect of adjuvant immunotherapy with a single neighbourhood injection of 2 mg C. parvum (CP) was investigated in a randomized study involving 43 patients with carcinoma of the cervix uteri, all of whom were treated by radical surgery. All patients had carcinoma confined to the cervix, the upper part of the vagina or the parametrial region. When the malignancy had spread to the parametrial region, additional postoperative radiotherapy was given. 22 patients received immunotherapy 10 days before surgery, whereas the remaining 21 control patients received no immune stimulation. Only minor side effects of CP were encountered. Follow-up shows a relapse rate of 5% in the CP treated group and of 29% in the controls (P less than 0.05). A further 15 patients with more advanced malignancies were added to our studies. In these, CP stimulation had no effect on relapse rates, but the relapse-free intervals were longer after immune stimulation: control 3.5 months (mean) +/- 1.5 (s.d.), CP 13.0 months +/- 7.0 (P less than 0.05). The number of peripheral T cells and the ability to become sensitized to DNCB were increased after CP stimulation. A decrease was found in the number of blood monocytes and the number of monocytes capable of transforming into active macrophages, indicating a possible sequestration of these cells in the tissues.