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Despite the progress in assisted reproductive techniques, there is still a lack of rapid and minimally invasive in situ approaches for further enhancements of female fertility. Therefore, we synthesized clinically relevant liposome nanoparticles for ovarian intrafollicular injection to allow in vivo cellular imaging for future drug delivery, using the mare as an animal model. Ovarian follicles of living mares were injected in vivo with fluorescently labeled liposomes. Samples of the follicular wall (mural granulosa, theca interna, and theca externa), granulosa cells, and follicular fluid were harvested 24 h post-injection through the follicle wall biopsy (FWB), flushing, and aspiration techniques, respectively, using a transvaginal ultrasound-guided approach. In parallel, post-mortem dissected, and cultured porcine antral follicles were microinjected with doxorubicin-encapsulated liposomes to assess intracellular delivery potential. All injected mare and pig follicles were macroscopically healthy, and fluorescence imaging revealed successful intrafollicular binding to mural granulosa cells and progressive migration of liposomes to other follicle cell layers (theca interna, and theca externa), regardless of the follicle size. Intracellular delivery of doxorubicin was confirmed in all porcine follicle wall cell types. We conclude that the intrafollicular injection of nanomolecules is a promising approach for real-time monitoring of intrafollicular processes and potential utilization of in vivo cellular drug delivery to assist in follicle disease treatments and fertility improvement.

Liposomes are spherical vesicles with a membrane composed of at least one phospholipid bilayer. They have been successfully applied for intracellular drug or genetic material delivery in many biomedical areas. In agriculture, however, their use to produce transgenic farm animals through exogenous DNA transfection of spermatozoa remains unsatisfactory. Here, we tested a newly designed liposome preparation for effective and harmless interactions with boar spermatozoa. Extended fresh boar semen samples were gently centrifuged (N=3 independent replicates), and sperm pellets were resuspended in PBS. Sperm concentrations were then adjusted to 2×108 mL−1 and aliquoted in 0.5mL for labelling with various doses of fluorescent liposomes (0, 15, 30, or 60µg), for plasma membrane labelling (Experiment 1) or with nonfluorescent liposomes loaded with doxorubicin (0 or 60µg) having natural fluorescence property, for nucleus labelling (Experiment 2). After co-incubation for 45 to 60min at 37°C, sperm aliquots of each treatment were immediately evaluated for motility and morphology characteristics using a computer-assisted sperm analyzer. Remaining sperm-liposome mixtures were centrifuged to remove the excess of liposomes, and pelleted spermatozoa were imaged with the In vivo Imaging System (IVIS: PerkinElmer, Waltham, MA, USA) and fluorescence confocal laser microscope (LSCM; Zeiss, Oberkochen, Germany). Data were statistically analysed using one-way ANOVA or Student’s t-test, with P0.05). The microscope imaging revealed sperm-liposome interactions with displayed sperm membrane and nucleus fluorescence in Experiments 1 and 2, respectively. The proportion of stained cells in each experiment is in evaluation by flow cytometry. In conclusion, the newly designed liposomes were effective for simple interactions with the plasma membrane and molecule delivery to boar spermatozoa, while improving sperm motility. Further studies are ongoing with the replacement of doxorubicin with a DNA fragment. This work was supported by USDA-ARS Biophotonics Initiative #58-6402-3-018.

In asymptomatic infertility patients, no significant relationship was found between the presence of antisperm antibodies (ASA) in serum and in semen samples (IgG and/or IgA ASA), differentiated with the mixed antiglobulin reaction (MAR), and the microbial colonization of ejaculates covering a broad spectrum of microorganisms. Likewise, there was no significant association of ASA with microbial findings in patients' female partners, who also presented without symptoms of genital tract infection and were screened at the same time. Furthermore, ASA in semen (IgG and IgA) were not significantly related to several potential markers of subclinical male sexual gland infection or inflammation (leukocytes, PMN elastase, albumin, C3c) evaluated in aliquots of the same ejaculates used for immunological testing.

In a double-blind clinical study 90 women with vaginal candidosis were randomly assigned to treatment with either single-dose ketoconazole (400 mg or 800 mg) or placebo vaginal suppositories. Microscope checks of vaginal fluid and mycological cultures were performed one week and four weeks after the treatment. The four weeks observation period gave no evidence for any significant differences among the three groups regarding symptoms reported as well as clinical picture and microscopy findings. Cure rate after one week, as determined by fungal cultures, was significantly lower in the placebo group (43%) than in the 400 mg (60%) and 800 mg groups (76%; p = 0.031). The mycological cure rate after four weeks did no longer show a significant difference between the groups tested, being 47% in the placebo group and 67% in each of the ketoconazole groups. Treatment of the sexual partner as well as predisposing factors did not influence the therapeutic effect. Due to the low ketoconazole serum concentrations after absorption of single vaginal doses, topical ketoconazole treatment can be applied in pregnancy. It remains to be tested, however, whether repeated vaginal application over three to five days will render higher and longer-lasting cure rates.

The sperm-cervical mucus-penetration test (SCMPT) was used in vitro to examine the influence of heavy metals (mercury, cadmium and copper) on sperm function. 30 each of freshly obtained different sperm samples with varying starting sperm motility and oestrogen-stimulated cervical mucosa from women of known fertility were used. Mercury chloride (HgCl2; 0.005-0.5 mmol/l), cadmium chloride (CdCl2; 0.05-10 mmol/l) and copper sulphate (CuSO4; 0.0001-10 mmol/l) were added to the in-vitro system. After incubation, at 37 degrees C for 6 hours, linear progression, penetration depth and motility index of the sperm (cumulative SCMPT score) were analysed. An untreated control was run in parallel with each test. The added substances, especially CdCl2 and HgCl2, significantly reduced the quantitative and qualitative penetrability of sperm (P = 0.001), in step with the concentration. The concentrations which were used, especially those of mercury, were in the range of in-vivo. measured levels. These results make clear that heavy metals can influence human sperm function.

5-Bromo-2,9-bis(bromomethyl)-1,10-phenanthroline (5) has been synthesized starting from 5-bromo-2,9-dimethyl-1,10-phenanthroline (1) in overall yields of up to 53 % by two approaches. Next, calix[6]arene (7) was bridged with 5, giving the A,D-bridged bimacrocyle 9 in 42 % yield. Under application of Sonogashira reaction conditions, trimethylsilyl-protected ethyne was first coupled to the 5-position of the 1,10-phenanthroline, and the ethyne unit was then deprotected and connected with 1,3,5-triiodobenzene (12) as a trivalent core. A trimeric first-generation dendrimer 13 was formed, together with a Glaser dimer sideproduct, and could be isolated in 36 % yield. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)

2,9-Dimethyl-1,10-phenanthroline (neocuproine, 1) has been attached to dendritic structures through its 5-position providing a trimer 7 and the hexamers 11 and 12. The copper-binding capability of the hexamer 12 has been studied, and the 6:1 complex has been used to catalyze the Cu+-promoted substitution of an iodoarene 14 to give an aryl ether 15. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)

A macrobicylic ligand 1 formed by the A,D-bridging of a calix[6]arene with a 2,9-disubstituted 1,10-phenanthroline unit reacts with Yb(III) under basic conditions in dimethylformamide (dmf) to give a neutral, mononuclear complex characterised by X-ray crystallography as [Yb(1-3H)(dmf)] · 3dmf. The structure shows that although the metal can be considered to lie within a cavity formed by the “concave reagent” ligand, this does not entirely prevent its coordination to a dmf molecule between arms of the macrobicycle. Stability constant measurements indicate that 1 forms complexes of similar strength across the lanthanide(III) series.

2,9-Dimethyl-1,10-phenanthroline (neocuproine, 1) was functionalised selectively in the 5-position. Silylation of the methyl groups followed by bromination in the 5-position was carried out to give the bis(tert-butyldimethylsilyl)-substituted neocuproine 3 and 5-bromo-2,9-dimethyl-1,10-phenanthroline (4) after deprotection. Compounds 3 and 4 are versatile building blocks for the construction of 5-substituted neocuproines. Palladium-catalysed couplings (Suzuki, Sonogashira, Buchwald/Hartwig) can be utilized to connect different substituents to the 1,10-phenanthroline moiety (5–7). These substituents may carry additional functional groups that allow the further connection of the 5-substituted neocuproines to form other molecules. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)

The objective of this study was to determine the clinical significance of complement fraction C3 (C3c) in seminal plasma. Therefore 120 samples from randomly chosen subfertile males without signs of genital tract infection were screened for C3 and for seminal leucocytes as markers for subclinical infection/inflammation. A comprehensive semen evaluation included sperm analysis, sperm migration testing, immunocytochemical round cell differentiation to determine seminal leucocyte counts and the leucocyte ratio and semen cultures, in aliquots of the same ejaculates. C3 concentrations were significantly correlated with leucocyte counts per ml (P < 0.002) and per ejaculate (P < 0.001), and with the leucocyte ratio (P < 0.001). No association of C3 concentrations with semen quality or with the bacterial colonization of semen samples was found. The significant association with seminal leucocytes suggests that C3 might be used as an additional marker for silent male genital tract infection. In comparison with semen leucocytes, C3 screening does not reveal any further information about semen quality or infection/inflammation pathogenesis of the male genital tract.

The clinical significance of circulating antisperm antibodies (ASA) during infertility investigation is controversial. For detection of ASA in serum, enzyme-linked immunosorbent assays (ELISA) are widely used because of practical advantages. In the present prospective study, the prevalence of serum ASA, determined with a commercially available ELISA (Elias Inc., Freiburg) was compared in different groups: in a total of 260 subfertile patients (148 women and 112 men) with a median duration of infertility of five years, and in 190 controls (69 normal pregnant women who presented for routine pregnancy care, 41 virgins and 80 prostitutes). Evaluation of serum samples in the different groups offered median ASA-values of 11.9 U/ml in women with long-standing infertility, of 9.6 U/ml in subfertile males, of 10.6 U/ml in the normal pregnant women, of 12.2 U/ml in virgins and of 10.0 U/ml in prostitutes. Differences between patients and controls were not significant. When a cut-off at > 25 U/ml was used for definition of positivity, this ELISA revealed that 18.2% of the female and 15.2% of the subfertile male patients were ASA positive, and within the control groups, 11.6% of the normal pregnant women, 14.6% of the virgins and 21.3% of the prostitutes (not significant, also with regard to other cut-offs). Within the group of subfertile patients, no significant differences were found between ASA-positive and -negative patients with regard to medical history, results of clinical examination, causes of infertility, microbial colonisation, of genital secretions, outcome of sperm analysis, postcoital testing (PCT), and crossed in-vitro sperm cervical mucus penetration testing (SCMPT). Pregnancies were achieved within three months without any therapy in spite of high ASA-titres. The results of this study demonstrate that evaluation of serum samples of subfertile patients for antisperm antibodies by means of this commercial ELISA is without clinical relevance and cannot be recommended during infertility investigation.

In 136 women with a median age of 78 (60-98) years the serum concentrations of FSH, LH, prolactin, estradiol-17 beta, testosterone and DHEA-S were determined completed by GnRH and ACTH stimulation tests in a subgroup. This resulted in median values for FSH of 15.8 ng/ml, LH 6.4 ng/ml, prolactin 6.9 ng/ml, estradiol 16 pg/ml, testosterone 270 pg/ml and 306 ng/ml for DHEA-S. No correlation with age in this population was found for gonadotropins as well as the other hormones for an age level of up to 98 years. Determination of multiple general clinical and laboratory parameters demonstrate the significance particularly in a geriatric group of patients to consider these patient characteristics when interpreting the serum hormone levels. In summary, the results of this prospective study underline the importance for health, of the secretion of gonadotropic and sexual hormones even at a very advanced age.

Spermiograms, Sims-Huhner postcoital tests, in-vitro sperm penetration tests and hormone stimulation tests, including gonadotrophin-releasing hormone, thyrotrophin-releasing hormone and the adrenocorticotrophic hormone (ACTH) test, were obtained in 225 infertile men. The present paper deals with the influence which anamnestic factors have on the spermiogram and on hormone levels. Adrenocortical function decreased with age. Blue-collar workers had poorer sperm parameters than white-collar workers. Smokers exhibited hormone alterations without significant changes in the spermiogram. Regular alcohol consumption was associated with elevated dehydroepiandrosterone-sulphate and oestradiol concentrations and poor postcoital test results. Emotional stress had a negative influence on sperm parameters. Environmental influences warrant additional attention in the evaluation of men with abnormal spermiograms.

After screening a large series of infertile patients (n = 1000 couples), potentially pathogenic microorganisms were identified in genital secretions of the majority of couples. None of the patients displayed signs or symptoms of infection of the lower genital tract. In semen specimens and cervical swabs, mycoplasmas were found in 18 and 12%, potentially pathogenic aerobic bacteria in 50 and 31%, additionally commensal aerobes in 38% and 25%, respectively. The microbial pattern showed great variability with polymicrobial growth most common. The rate of cultures positive for potentially pathogenic anaerobes was markedly influenced by the transport conditions. In endocervical material, Herpes simplex virus (HSV) was identified in 4.5%. Yeasts were found in the posterior vaginal fornix in 10%. Concerning both partners, there were only 10 couples (1%) from whom microorganisms in genital secretions could not be isolated, not including lactobacilli in females. This high prevalence of microbes and the low number of leukocytes indicate colonization rather than infection. When the microbial findings from both partners were analysed with regard to the outcome of sperm-cervical mucus penetration testing in vitro, which was performed simultaneously, no significant correlation was found. Furthermore, microbial prevalence did not differ between couples with 'explained' and 'unexplained' infertility. The results of this study demonstrate that in patients without symptoms of genital tract infection, microbial colonization is of minor importance for sperm--mucus interaction and that extensive microbial screening should be preferentially performed in cases of poor sperm and/or mucus function.

The objective of the study was to investigate patient compliance with two different dosage regimens. Ethinyloestradiol 80 μg daily was prescribed to 35 female outpatients to be taken as 20 μg four times daily or 40 μg twice daily for 7 days. It was given to standardise cervical mucus before a sperm cervical mucus penetration-test (SCMPT) was performed. Sixty-five patients with primary infertility (mean age 29.9 y) completed the study. Compliance was assessed by microprocessor-based compliance monitoring. Besides compliance (percentage of prescribed doses taken), the adherence of the patients to the dosage schedule was evaluated — ‘regimen compliance’. The latter parameter of drug intake behaviour was calculated by the number of days in which 2 (BID) or 4 openings (QID) were recorded by the electronic monitor. As a third parameter, the deviation from the prescribed dosing intervals, i.e. 12 or 6 h, was also determined. Partial compliance was the predominant finding and only 9 patients (13.8%) were over-compliant. Mean compliance was 75.7% in all 65 patients as a group, range 7.1 to 143%. The mean compliance with the QID regimen was 67% compared to 85% with the BID regimen. ‘Regimen compliance’, the percentage of doses taken on schedule, was 36% and 63% for the QID and BID regimens, respectively. Drug-intake behaviour was more erratic with the QID than the BID regimen, as indicated by the 55% of opening intervals recorded which exceeded the range of 3–9 h (mean: 6 h), compared to only 19% exceeding 6–18 h intervals (mean: 12 h). The present study provides evidence that the prescribed dosage frequency has an impact on drug compliance by patients. From a practical point of view, it seems reasonable to use treatment regimens that are as simple as possible, provided that they are pharmacologically and clinically appropriate.

The aim of this study was to examine more closely the interaction between thyroid function and pituitary--ovarian axis in infertile women. In 185 infertile women without clinical signs of thyroid dysfunction, TRH-tests (TSH basal and 30 min after 200 micrograms TRH i.v.) were performed in the early follicular phase in addition to routine hormonal checks (gonadotrophins, oestradiol, DHEAS, testosterone, prolactin). The women were classified as euthyroid (n = 74; TSH stim 5-20 mU/l), latent hyperthyroid (n = 31; TSH stim less than 5 mU/l), and preclinical hypothyroid (n = 80; TSH stim greater than 20 mU/l). From frozen serum, the following determinations were performed: TSH IRMA, laevothyroxine (T4), thyroxine binding globulin (TBG), microsomal (Mab) and thyroglobulin (Tab) antibodies. Various correlations between the thyroid parameters and the pituitary--ovarian axis were demonstrated. With increasing TBG concentrations, the interval between menses decreased. Overall and spontaneous pregnancy rates were highest in women with normal (less than 75th perc.) basal and stimulated TSH, high (greater than 75th perc.) T4 and low (less than 25th perc.) Mab. Women with normal Tab or high TBG experienced the highest delivery rate (77 versus 30%), while in women with low Tab or high Mab abortion and tubal pregnancies were most frequent. As only 25 women exhibited elevated Mab (greater than 500 U/ml) or Tab (greater than 200 U/ml) which correlated with elevated TSH and normal T4, the routine determination of thyroid antibodies was not necessary. The TRH-test, however, should be included in infertility work-up.

Sperm-mucus interaction under in-vitro or in-vivo conditions can be affected by local anti-sperm antibodies (ASA). In a prospective study, a mixed antiglobulin reaction (MAR) test, using immunoglobulin-coated red cells and spermatozoa, was used to detect the presence of IgG and/or IgA on the spermatozoal surface in 209 males of infertile partnerships. The results of direct MAR tests range from 0 to 100% and a significant correlation between MAR % IgG and MAR % IgA (r = 0.74) was found. MAR positive semen samples were significantly more frequent in the group of patients with an inadequate outcome of postcoital testing (PCT). The sperm-mucus interaction in vitro, evaluated by means of the in-vitro sperm-cervical mucus penetration test (SCMPT) with cervical mucus (CM) of patients' partners, was particularly related to spermatozoal surface antibodies of the IgA class: MAR IgA positive ejaculates were seen in 13.9% of males with inadequate SCMPT versus 4.4% when SCMPT was adequate. The significant correlation between MAR IgA and the sperm penetration ability in vitro could also be proven when donors' CM was taken for the SCMPT, but not when a non-human material was used for in-vitro penetration testing. Microbial colonization of semen specimens did not interfere with the outcome of MAR testing. The pregnancy rate after 12 months was 23% (48/209) and was significantly lower when greater than 30% of spermatozoa were covered with surface ASA of the IgG or IgA class.(ABSTRACT TRUNCATED AT 250 WORDS)

The objective of this study was to determine the clinical significance of complement fraction C3 (C3c) in seminal plasma. Therefore 120 samples from randomly chosen subfertile males without signs of genital tract infection were screened for C3 and for seminal leucocytes as markers for subclinical infection/inflammation. A comprehensive semen evaluation included sperm analysis, sperm migration testing, immunocytochemical round cell differentiation to determine seminal leucocyte counts and the leucocyte ratio and semen cultures, in aliquots of the same ejaculates. C3 concentrations were significantly correlated with leucocyte counts per ml (P0.002) and per ejaculate (P0.001), and with the leucocyte ratio (P0.001). No association of C3 concentrations with semen quality or with the bacterial colonization of semen samples was found. The significant association with seminal leucocytes suggests that C3 might be used as an additional marker for silent male genital tract infection. In comparison with semen leucocytes, C3 screening does not reveal any further information about semen quality or infection/inflammation pathogenesis of the male genital tract.

Constructing a full CAD model of a part requires feature descriptions from all sides; in this case we consider surface patches as the geometric primitives. Most previous research in surface patch extraction has concentrated on extracting patches from a single view. This leads to several problems with aligning and combining partial patch fragments in order to produce complete part models. We have avoided these problems by adapting our single view, range data segmentation program to extract patches, and thus models, directly from fully merged range datasets.

We present an 11-year-old girl with sensory and autonomic neurological dysfunction, and respiratory insufficiency caused by recurrent aspiration. The diagnosis of familial dysautonomia (FD) was confirmed by a missing axonal flare to histamine, miosis in response to conjunctival methacholine and homozygous polymorphic linked markers DS58(18) and DS159(7) on chromosome 9. Ashkenazi Jewish descent could not be ascertained by history. A variety of positive tests for autoantibodies were initially interpreted as evidence for systemic lupus erythematosus vs. overlap syndrome with pulmonary, cerebral, skin, and ocular involvement. The diagnosis of FD was delayed because of the rarity of this disorder in Germany (second case reported). We discuss possible explanations for the misleading immunological findings, including interference by antibodies binding to milk proteins used as blocking reagents in enzyme-linked immunoassays and circulating immune-complexes due to chronic aspiration pneumonitis.

The concentration of interleukin (IL) -8 and IL-6 was determined in seminal plasma (SP) samples from 137 randomly chosen subfertile males to evaluate the relationship with other potential parameters of subclinical infection/inflammation such as seminal leukocytes, and with semen quality in a prospective study. All patients were asymptomatic for genital tract infection. A comprehensive semen evaluation included sperm analysis, sperm migration testing, antisperm antibody screening, immunocytochemical round cell differentiation to determine seminal leukocytes counts and the leukocyte ratio, complement fraction C(3) (C(3c)) determination, and semen cultures, in aliquots of the same ejaculates. The SP concentration of IL-8 was inversely related to semen quality, e.g. to the total number of motile spermatozoa or to the outcome of the sperm migration test (motile sperm harvested after a swim-up procedure). IL-8 concentrations were significantly correlated with leukocyte counts per ml (P < 0.0001) and per ejaculate (P < 0.0001), and with the leukocyte ratio (P < 0.001). All leukocytospermic samples had high IL-8 concentrations (< or =2 ng/ml). The SP concentration of IL-6 was much lower, but was significantly correlated with IL-8 (P < 0.0001). Both IL-8 and IL-6 were significantly related with the C(3c). No association of interleukin concentrations with the bacterial colonization of semen samples was found. The results indicate a marked relationship of some pro-inflammatory cytokines with semen quality. The significant association with seminal leukocytes and other potential inflammation markers suggests that IL-8 might be used as sensitive marker for silent male genital tract infection.

Utility of product variants is a nonlinear function of product features. Such a utility function can be represented by a multi-layer perceptron and embedded into the classical logit demand function. However, the utility (which is the output of the multi-layer perceptron to be learned) is not explicitly known. This is why the backpropagation learning rule has been extended to fit the demand function directly to observed market shares. Forecasts of market shares on the German automobile market with the help of a perceptron-based and classical logit model are compared. The perceptron-based model leads to a significant improvement of the forecast quality.

Chlamydia trachomatis infections are the most prevalent bacterial sexually transmitted infections (STI) recognized throughout the world. Worldwide, the magnitude of morbidity associated with sexually transmitted chlamydial infections is enormous. C.trachomatis is a common cause of urethritis and cervicitis, and sequelae include pelvic inflammatory disease (PID), ectopic pregnancy, tubal factor infertility, epididymitis, proctitis and reactive arthritis. The sharp worldwide increase in the incidence of PID during the past two decades has led to the secondary epidemics of tubal factor infertility and ectopic pregnancy. Chlamydial PID is the most important preventable cause of infertility and adverse pregnancy outcome. Chlamydial infections, like STI in general, are primarily a woman's health care issue since the manifestations and consequences are more damaging to the reproductive health in women than in men. Based on the available evidence, approximately 20% of women with chlamydial lower genital tract infection will develop PID, approximately 4% develop chronic pelvic pain, 3% infertility, and 2% adverse pregnancy outcome. However, these estimates are based on relatively weak evidence. Research on the link between C.trachomatis and male aspects of infertility has been much more limited. Currently recommended treatment regimens include azithromycin in a single dose or doxycycline for 7 days. These therapies are highly efficacious. Timely management of sex partners is essential for decreasing the risk for re-infection. Immunopathogenesis of C.trachomatis infection is one of the main focal points of current research into Chlamydia. Chlamydial infection fills the general prerequisites for disease prevention by screening, i.e. chlamydial infections are highly prevalent, usually asymptomatic, are associated with significant morbidity, can be reliably diagnosed, and are treatable. Screening programmes for C.trachomatis will be of paramount importance in the prevention of long-term sequelae. The cost of screening is only a fraction of the health care costs incurred due to complications resulting from undiagnosed and untreated chlamydial infections. Current strategies to control C.trachomatis still largely depend on clinic-based screening of symptomatic patients, and have not been successful. The development of highly sensitive and specific nucleic acid amplification tests for the diagnosis of chlamydial infections has been an important advance in the ability to conduct population-based screening programmes to prevent complications. Thus, the case for screening is clearly made, but much detail remains to be worked out.