Visualization and characterization of engineered nanoparticles in complex environmental and food matrices using atmospheric scanning electron microscopy.

Imaging and characterization of engineered nanoparticles (ENPs) in water, soils, sediment and food matrices is very important for research into the risks of ENPs to consumers and the environment. However, these analyses pose a significant challenge as most existing techniques require some form of sample manipulation prior to imaging and characterization, which can result in changes in the ENPs in a sample and in the introduction of analytical artefacts. This study therefore explored the application of a newly designed instrument, the atmospheric scanning electron microscope (ASEM), which allows the direct characterization of ENPs in liquid matrices and which therefore overcomes some of the limitations associated with existing imaging methods. ASEM was used to characterize the size distribution of a range of ENPs in a selection of environmental and food matrices, including supernatant of natural sediment, test medium used in ecotoxicology studies, bovine serum albumin and tomato soup under atmospheric conditions. The obtained imaging results were compared to results obtained using conventional imaging by transmission electron microscope (TEM) and SEM as well as to size distribution data derived from nanoparticle tracking analysis (NTA). ASEM analysis was found to be a complementary technique to existing methods that is able to visualize ENPs in complex liquid matrices and to provide ENP size information without extensive sample preparation. ASEM images can detect ENPs in liquids down to 30 nm and to a level of 1 mg L(-1) (9×10(8) particles mL(-1) , 50 nm Au ENPs). The results indicate ASEM is a highly complementary method to existing approaches for analyzing ENPs in complex media and that its use will allow those studying to study ENP behavior in situ, something that is currently extremely challenging to do.

Inhibition of sperm-egg interaction by specific antibody.

Treatment of hamster ova, in vitro, with ovary specific antibody interferes with sperm-egg interaction by inhibiting sperm attachment to the zona pellucida and subsequent zona penetration. This inhibition results from precipitation of the zona by the specific antibody.

Autosomal dominant inheritance of the Kabuki make-up (Niikawa-Kuroki) syndrome.

We report on three individuals (two sibs and their father) with the Kabuki make-up syndrome. The two sibs had congenital dislocation of the hips and all three individuals had short stature and the facial characteristics of the syndrome. To our knowledge this is the first report of familial occurrence of the Kabuki make-up syndrome.

What do we really know about autoantibody abnormalities and reproductive failure: a critical review.

CONDENSATION: The diagnosis and treatment of autoantibody-associated forms of reproductive failure is critically reviewed. OBJECTIVE: To critically evaluate the published literature in reference to autoantibody-associated forms of reproductive failure. LOCATION: Medical School-affiliated private Infertility Center. MATERIALS: A review of over 200 published papers reflecting on the topic. RESULTS: Autoantibody associated reproductive failure, characterized by a decrease in fecundity and an increase in the risk of pregnancy loss, appears established. Autoantibody abnormalities, as routinely detected by standard laboratory assays, are, however, neither immunologically nor biologically specific since cross reactivities between autoantibodies are frequent and a specific autoantibody may cause a biological effect in one but not in another affected individual. CONCLUSIONS: The evaluation of autoantibody abnormalities in all cases of suspected autoimmune-associated reproductive failure is valuable and will improve clinical care of affected patients. Clinicians need, however, to recognize the limitations of autoantibody testing and have to adjust their clinical management to the degree and quality of autoantibody evaluation available to them in their community.

Correlation between peripheral blood and follicular fluid autoantibodies and impact on in vitro fertilization.

Total immunoglobulins, C3 and C4 complement, and autoantibodies to phospholipid antigens, histones, histone subfractions, and nucleotides were measured in serum and follicular fluids of 26 patients undergoing in vitro fertilization. Immunoglobulins and complement fractions appeared in statistically lower concentrations in follicular fluid than in serum. This gradient was clearly larger for immunoglobulin M (IgM) than for IgG, IgA, C3, or C4. Ten patients who were autoantibody positive to one or more of the evaluated antigens demonstrated statistically higher total IgG and IgM levels and lower C3 and C4 than 16 autoantibody negative patients. With regard to follicular fluid, positive patients had significantly elevated IgM levels in comparison with negative control patients, and significantly decreased C3 and C4 concentrations. Patients who were autoantibody positive in serum also exhibited uniformly abnormal follicular fluid autoantibodies. Positive patients had significantly higher IgG antiphospholipid antibodies in follicular fluid than controls, and demonstrated a similar trend for IgM and IgA antiphospholipid antibodies. Positive patients demonstrated no significant differences from negative patients in number of in vitro fertilization attempts, number of oocytes retrieved, fertilization rate, cleavage rate, and number of abnormal (granular) oocytes. However, positive patients experienced a lower pregnancy rate (5.4%) than negative patients (26.1%), although the difference did not reach statistical significance (P = .07). This observation strongly suggests that the presence of abnormal autoantibodies in the female may reduce the chance of pregnancy for in vitro fertilization patients. A similar effect of abnormal autoantibodies can then also be expected in attempts at spontaneous conception.

Penetration of bacteria and spermatozoa into bovine cervical mucus.

It has been reported that bacteria may attach to motile spermatozoa, be carried through the cervix and uterus to the fallopian tubes, and cause acute salpingitis. In an attempt to mimic these conditions in vitro, we incubated Escherichia coli, Streptococcus faecalis, Pseudomonas aeruginosa, and Staphylococcus aureus with motile spermatozoa which then were allowed to migrate through a capillary filled with bovine cervical mucus. After satisfactory sperm migration through the mucus, the capillaries were broken and cultured at different distances from the original insemination site. Mucus fractions in proximity to bacterial inocula grew varying amounts of the pathogens. More distal fractions of mucus columns were generally culture-negative even though they contained motile sperm which had been exposed to bacteria. Migration of spermatozoa exposed to bacteria through bovine cervical mucus did not result in enhanced bacterial penetration.

Inhibition of implantation in the golden hamster by zona-precipitating antibody.

Antisera prepared against saline extracts of hamster ovary, and made specific to ovary by absorption with small intestine, precipitated the zona pellucida of provulatory hamster ova and all early embryonic stages examined, including the pronuclear, 2-, 4-, 8-, and 16-cell, morula, and blastocyst stages; this indicates that (1) the zona antigen is present during oocyte maturation and early embryonic development and (2) the antigen does not change its ability to react with zonaprecipitating antibody. Embryos in the morula and blastocyst stages, treated in vitro with zona-precipitating antibody, pre-immunization rabbit serum, or control media were transferred into synchronized pseudopregnant recipients. Precipitation of zonae by specific antibody inhibited implantation by blocking the process known as zona shedding, and thereby prevented the embryo from making contact with the uterus.

The association of antithyroid antibodies in euthyroid nonpregnant women with recurrent first trimester abortions in the next pregnancy.

OBJECTIVE: To evaluate the prognostic value of antithyroid antibodies in euthyroid women with a history of recurrent first trimester abortions on future pregnancy loss. DESIGN: The sera of 42 euthyroid women with a history of three or more consecutive first trimester abortions were evaluated for the presence of antibodies to thyroglobulin and thyroid peroxidase before pregnancy and again as soon as the diagnosis of pregnancy was made. SETTING: Medical school-affiliated private infertility center. PATIENTS: Forty-two women with a history of three or more consecutive first trimester abortions who were planning to conceive again. MAIN OUTCOME MEASURE: The presence of antithyroid antibodies in the nonpregnant state and their association with pregnancy loss in the next gestation. RESULTS: Thirteen of 42 women (31%) were positive for the presence of antithyroid antibodies at the initial screening before pregnancy. All 13 maintained positivity by the time their next pregnancy was diagnosed. Only 12 of those 42 women (29%) experienced a first trimester abortion. Eight of these 12 women (67%) were positive for one or more antithyroid antibody. In contrast, among 30 nonaborting women, only 5 of 30 (17%) exhibited thyroid antibody positivity. The detection of thyroid antibodies before conception carried an increased risk of pregnancy loss in the next pregnancy (8 of 13, 62% versus 4 of 29, 14%). CONCLUSION: The presence of antithyroid antibodies in nonpregnant women with a history of recurrent abortion identifies a subgroup of women at significantly increased risk for yet another pregnancy loss in their next gestation. Because organ-specific autoantibodies thus demonstrate similar prognostic significance to nonorgan-specific autoantibodies, it is tempting to conclude that peripheral autoantibody abnormalities seen in habitual aborters only reflect an underlying T-lymphocyte defect, which may be the actual cause of pregnancy loss.

Pregnancy after intrauterine insemination with sperm retrieved from the rectum.

A pregnancy, which was achieved with semen retrieved from the rectum of a male with urethrorectal fistula, is reported. The pregnancy was established after IUI with repeatedly washed sperm. Semen maintains its fertilization capacity even after exposure to the rectal environment.

Intrauterine inseminations with washed human spermatozoa.

The success of intrauterine inseminations with washed human spermatozoa was evaluated in four groups of patients. The first two groups were couples in whom either the man or the woman had detectable circulating sperm-agglutinating antibodies. The third group included couples where the woman exhibited hostile or absent cervical mucus, whereas the fourth group represented couples with oligozoospermic males. The couples underwent 3.7 +/- 2.1 insemination cycles, with a 30% overall pregnancy rate for all four groups. Intrauterine inseminations of women with hostile cervical mucus yielded a 68% conception rate. Couples with either male or female serum sperm-agglutinating antibodies showed 25% and 40% pregnancy rates, respectively. However, none of the couples with oligozoospermic males achieved pregnancy. Intrauterine inseminations with washed human spermatozoa thus represent a satisfactory method of achieving pregnancy in women with hostile cervical mucus and in couples with circulating sperm agglutinating antibodies. Couples in whom oligozoospermia has been identified as the principal cause of infertility do not seem to benefit from this therapy.

Inhibition of sperm motility and agglutination of sperm cells by free fatty acids in whole semen.

The effects of a serial dilution of linoleic acid on human spermatozoa in whole semen was tested on 21 semen samples obtained from 11 normal volunteers. The minimal concentration of linoleic acid required to stop the movement of at least 75% of the moving sperm ranged from 1 to greater than 100 mg/dl. Fifteen of 21 (71%) of the semen samples were inhibited by added free fatty acids (FFA) concentrations that were less than or close to the physiologic concentration ranges of FFA in blood plasma (1 to 30 mg/dl). The immobilized sperm often formed aggregates similar to those formed by the action of autoantibodies against sperm cells. Preliminary studies conducted on a variety of other FFA have indicated that oleic acid (18/1) was less toxic than linoleic acid (18/2) and that linolenic acid (18/3) was more toxic than linoleic acid. The saturated FFA palmitic acid (16/0) and stearic acid (18/0) at concentrations up to 100 mg/dl showed little or no toxicity to sperm cells. It is suggested that FFA toxicity be included among physiologic factors that affect the motility and spontaneous aggregation of sperm cells.

Pooled sequential ejaculates: a way to increase the total number of motile sperm from oligozoospermic men.

We investigated the yield of total number of motile spermatozoa from oligozoospermic men by pooling two closely spaced sequential ejaculates. Semen characteristics were compared between sequential ejaculates (within a period of 1 to 4 hours) of 18 oligozoospermic males (sperm concentration less than 20 X 10(6)/mL and total sperm count less than 40 X 10(6) in the ejaculate) and a control group of 16 normozoospermic men. Whereas the median total number of motile sperm of normozoospermic males significantly decreased from 70 X 10(6) in the first ejaculate to 23 X 10(6) in the second sequential ejaculate, such a decrease was not detected in oligozoospermic males, 3.6 X 10(6) and 3.1 X 10(6), respectively. The percent of normozoospermic and oligozoospermic men who demonstrated a decreased (less than 50%), a comparable (50% to 150%), or an increased (greater than 150%) total motile sperm count in the second ejaculate in comparison with the first ejaculate were 69%, 31%, and 0 versus 39%, 28%, and 33%, respectively. Consequently, pooling of two sequential ejaculates significantly increased the median total number of motile sperm from normozoospermic males by 144% and from oligozoospermic males by 329%, (to 10.2 X 10(6]. We suggest that pooling of two sequential ejaculates from oligozoospermic males is a simple and cost effective method to increase significantly the total number of motile sperm for intrauterine insemination, in vitro fertilization, gamete intrafallopian transfer, or semen cryopreservation.

Octreotide is not useful for clomiphene citrate resistance in patients with polycystic ovary syndrome but may reduce the likelihood of ovarian hyperstimulation syndrome.

OBJECTIVE: To determine whether octreotide is effective for ovulation induction in patients with polycystic ovary syndrome (PCOS) and clomiphene citrate resistance or for reduction of the risk of ovarian hyperstimulation syndrome (OHSS) with gonadotropin therapy. DESIGN: Prospective, double-blind, placebo-controlled, crossover trial. SETTING: Private infertility practice. PATIENT(S): Twelve patients with PCOS undergoing therapy for infertility. INTERVENTION(S): The patients were assigned randomly to receive either octreotide or placebo. Those with clomiphene citrate-resistant PCOS received clomiphene citrate, 150 mg. Patients at risk for the development of OHSS received urinary FSH for ovulation induction. MAIN OUTCOME MEASURE(S): Ovulation, pregnancy, the development of OHSS, and levels of fasting insulin, insulin-like growth factor 1, insulin-like growth factor binding proteins 1 and 3, testosterone, androstenedione, DHEAS, E2, LH, and FSH. RESULT(S): Octreotide significantly reduced levels of fasting insulin, insulin-like growth factor 1, and LH in both clomiphene citrate- and urinary FSH-stimulated cycles. Levels of insulin-like growth factor binding protein 3 were increased. Two of six clomiphene citrate-stimulated cycles reached ovulation with the use of either octreotide or placebo. In urinary FSH-stimulated cycles, patients who received octreotide had significantly lower E2 levels at the time of hCG administration and fewer mature follicles. No cases of OHSS occurred in either group. One pregnancy occurred in each group. CONCLUSION(S): Octreotide was no more effective than placebo for clomiphene citrate resistance in patients with PCOS, but it did reduce E2 levels and follicle numbers when combined with urinary FSH. Thus, octreotide may reduce the incidence of OHSS in patients with PCOS.

Ovarian stimulation protocol for in vitro fertilization with gonadotropin-releasing hormone agonist widens the implantation window.

Pregnancy rates vary considerably with the type of ovarian stimulation used for in vitro fertilization and embryo transfer (IVF-ET). The window of implantation may represent one of the rate-limiting steps in IVF success. We therefore investigated estimated implantation times of 10 consecutive IVF singleton pregnancies, achieved using pituitary suppression with gonadotropin-releasing hormone agonist (GnRH-a) before and during ovarian stimulation with human menopausal gonadotropins (hMG), and compared those with 9 consecutive IVF pregnancies achieved by hMG stimulation only. Estimated implantation times were calculated by regression analysis of serial human chorionic gonadotropin (hCG) measurements between days 7 and 16 after ET. The GnRH-a/hMG pregnancies implanted between days 7 and 11, whereas hMG pregnancies implanted between days 7 and 9 after ET. The hCG regression curve for the GnRH-a/hMG pregnancies revealed a delay of 1.5 days in estimated implantation time compared with the hMG only group. There were no significant differences in pretransfer in vitro embryos development between the two groups. Thus, the delay in hCG rise probably reflects a delay in embryo implantation. We therefore conclude that a GnRH-a/hMG stimulation protocol appears to widen the implantation window in comparison with a hMG only protocol. This observation may at least in part explain the improved IVF pregnancy success with GnRH-a/hMG stimulation protocols.

Validation of methods for the detection and quantification of engineered nanoparticles in food.

The potential impact of nanomaterials on the environment and on human health has already triggered legislation requiring labelling of products containing nanoparticles. However, so far, no validated analytical methods for the implementation of this legislation exist. This paper outlines a generic approach for the validation of methods for detection and quantification of nanoparticles in food samples. It proposes validation of identity, selectivity, precision, working range, limit of detection and robustness, bearing in mind that each "result" must include information about the chemical identity, particle size and mass or particle number concentration. This has an impact on testing for selectivity and trueness, which also must take these aspects into consideration. Selectivity must not only be tested against matrix constituents and other nanoparticles, but it shall also be tested whether the methods apply equally well to particles of different suppliers. In trueness testing, information whether the particle size distribution has changed during analysis is required. Results are largely expected to follow normal distributions due to the expected high number of particles. An approach of estimating measurement uncertainties from the validation data is given.