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.

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.

Estradiol/progesterone substitution in the luteal phase improves pregnancy rates in stimulated cycles--but only in younger women.

Innumerable studies have attempted to demonstrate that hormonal support of the luteal phase during ovulation induction cycles improves pregnancy rates. None has, however, so far been able to confirm the validity of such treatment conclusively, possibly because most studies only utilized progesterone substitution. Since luteal phase endometrium also requires estradiol support, this study attempted to investigate whether hormone substitution with progesterone and estradiol would be more successful in improving pregnancy rates. Amongst approximately 7500 consecutive ovulation induction cycles were identified prospectively which were characterized by a precipitous drop of luteal phase serum estradiol levels by more than 50% over a 48 hour period within 10 days from hCG administration. Those cycles were prospectively randomized to oral micronized estradiol substitution (Group I) or not (Group II), while both groups received routine progesterone substitution of the luteal phase. Cycles were then evaluated in regards to the occurrence of chemical, ectopic and clinical pregnancies. One hundred sixty-three Group I cycles resulted in 34 pregnancies (20.9%), which compared favorably to 21 pregnancies in 167 Group II patients (12.6%) (x2[1] = 4.06; p < 0.04). The advantage for Group I cycles (29/95 pregnancies, 31%) vs. Group II cycles (16/105, 15%) became even more pronounced when only women up to age 35 years were evaluated. Estradiol substitution maintained a significant advantage until age 38 (x2 [1] = 6.87; p < 0.009). While gravidity did not affect pregnancy success, estradiol substitution in Group I benefited nulliparous (23% pregnancy rate) over multiparous women (12% pregnancy rate) (x2 [2] = 6.86; p< 0.03). This association was, however, age-dependent. A combined estradiol and progesterone substitution of the luteal phase of ovulation induction cycles increases the overall pregnancy rate. Since estradiol substitution was initiated in this study only once a precipitous drop in serum estradiol levels had already taken place, an even larger improvement in pregnancy rates could conceivably be possible if earlier estradiol substitution of the luteal phase is initiated. A further expansion of investigations of similar protocols for routine ovulation induction and in vitro fertilization (IVF) cycles may be indicated, especially in women below age 38 years and in nulliparous females.

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.

The bubble test: a new tool to improve the diagnosis of endometriosis.

The objective of this study was to determine the clinical sensitivity and specificity of a bubbling phenomenon, including peritoneal surfaces, as a diagnostic test for endometriosis during laparoscopy. A prospectively controlled study of women with infertility of at least 1 year duration, who underwent laparoscopy, was conducted at a medical school-affiliated private infertility centre and research foundation. The study included 48 prospectively enrolled female infertility patients who underwent laparoscopy. Of these, 32 were found to suffer from endometriosis (group A) and 16 control patients did not show any evidence of disease (group B). The study involved the irrigation of the posterior cul-de-sac with short bursts of either saline or lactated Ringer's solution, utilizing a standard laparoscopic aspiration/irrigation system, and the subsequent observation for an excessive soap-like bubbling phenomenon (positive bubble test) in association with endometriosis. All 32 endometriosis patients (group A) demonstrated a positive bubble test. In contrast, only two of the 16 control patients (group B) were positive (P = 0.00242, Fisher's exact test; odds ratio, 8.000). A positive bubble test during laparoscopy was thus 100% sensitive and 88% specific for the diagnosis of endometriosis by laparoscopy, resulting in positive and negative predictive values of 94 and 100% respectively. Since the literature provides considerable evidence that the diagnosis of endometriosis during laparoscopy is frequently missed, a positive bubble test during laparoscopy therefore may be considered a reason to search further (possibly with biopsies) for endometriosis in the absence of obviously visible disease.(ABSTRACT TRUNCATED AT 250 WORDS)

[What do we really know about autoantibody abnormalities and reproductive infertility? A critical review]. / Que savons-nous vraiment des anomalies des auto-anticorps et de la maladie reproductive? Une revue critique.

UNLABELLED: 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 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.

Clinical significance of beta 2-glycoprotein I-dependent anticardiolipin antibodies in the reproductive autoimmune failure syndrome: correlation with conventional antiphospholipid antibody detection systems.

OBJECTIVE: Our purpose was to determine whether beta 2-glycoprotein I-dependent anticardiolipin antibodies may represent a superior marker of reproductive risk than do conventional antiphospholipid antibodies. STUDY DESIGN: The incidence of beta 2-glycoprotein I-dependent and beta 2-glycoprotein I-independent anticardiolipin antibodies and of six conventional antiphospholipid antibodies was statistically compared between study groups with and without autoantibody-associated features of reproductive failure. Sera from 356 women were randomly selected from the frozen sera bank at the Center for Human Reproduction, Chicago. They included sera from 259 patients with autoantibody-associated features of reproductive failure such as unexplained infertility, endometriosis, and repeated pregnancy loss and 97 infertile controls. Autoantibody levels by a modified enzyme-linked immunosorbent assay for beta 2-glycoprotein I-dependent and beta 2-glycoprotein I-independent anticardiolipin antibodies and a standard enzyme-linked immunosorbent assay for anticardiolipin antibody and five other antiphospholipid antibodies were then compared. RESULTS: Patients demonstrated a significantly higher incidence of beta 2-glycoprotein I-dependent anticardiolipin antibodies (5.4%) than did controls (0%) in a modified enzyme-linked immunosorbent assay (p = 0.01). No such difference was, however, noted for beta 2-glycoprotein I-independent anticardiolipin antibodies or any one of six antiphospholipid antibodies. Two or more among six antiphospholipid antibodies, especially if involving anticardiolipin antibodies, antiphosphatidylserine and antiphosphatidylinositol, as assayed by standard enzyme-linked immunosorbent assay, were significantly more often (p = 0.02) positive in the patients (5.0%) than in the controls (0%). Moreover, positivity in two of those three antiphospholipid antibodies correlated in 59% of cases to positivity in the beta 2-glycoprotein I-dependent anticardiolipin antibody. CONCLUSIONS: As a single test beta 2-glycoprotein I-dependent anticardiolipin antibody appears to be superior to cofactor-independent anticardiolipin antibody or any other single conventional antiphospholipid antibody for the detection of autoantibody-associated conditions of reproductive failure. A broadly based panel of conventional antiphospholipid antibodies, especially if inclusive of anticardiolipid antibody, antiphosphatidylserine, and antiphosphatidylinositol, may, however, achieve similar results.

beta 2-Glycoprotein I-dependent and -independent anticardiolipin antibodies in healthy pregnant women.

The purpose of this study was to determine the association between beta 2-glycoprotein I (beta 2GPI)-dependent anticardiolipin antibodies (aCL) and beta 2GPI-independent aCL and their respective relevance to adverse pregnancy outcomes. Therefore, we prospectively studied 210 normal pregnant women, utilizing a modified enzyme-linked immunosorbent assay method for beta 2GPI-dependent and -independent aCL. Seven of the 210 pregnant women (3.3%) demonstrated evidence for beta 2GPI-independent immunoglobulin G (IgG)-aCL. Two patients, who also appeared positive for beta 2GPI-dependent IgG-aCL, were proven to be false positives. Amongst the 210 patients, not one was thus positive for beta 2GPI-dependent aCL. Women with beta 2GPI-independent aCL demonstrated no adverse pregnancy outcomes. These results suggest that the presence of beta 2GPI-independent aCL is not associated with the presence of beta 2GPI-dependent aCL, though it may give rise to false positive results. Since the presence of beta 2GPI-independent aCL does not appear to be associated with adverse pregnancy outcomes, beta 2GPI-dependent assays may represent better markers of miscarriage risk.

Autoantibody profiles and immunoglobulin levels as predictors of in vitro fertilization success.

OBJECTIVE: Our aim was to determine the predictive value of autoantibody and immunoglobulin determinations as indicators of the success of in vitro fertilization. STUDY DESIGN: This was a blinded study in which laboratory evaluations were performed on coded samples obtained from another institution. Codes were broken and data were analyzed after results of all laboratory tests had been reported out. One hundred five infertility patients who had undergone in vitro fertilization were randomly chosen. Among those, 46 were considered low responders (six or fewer oocytes were retrieved) and 59 as high responders (13 to 30 oocytes were retrieved). Total immunoglobulin G, M, and A levels and 15 autoantibody levels (6 antiphospholipids, 5 antihistones, and 4 antipolynucleotides) were determined separately for immunoglobulin G, immunoglobulin M, and immunoglobulin A isotypes. RESULTS: High and low responders demonstrated an unusual incidence of autoantibody (25% and 30%, respectively) and immunoglobulin (46% and 48%, respectively) abnormalities. They did not differ from each other, however, in either immunoglobulin or autoantibody parameters. Autoantibody and immunoglobulin abnormalities alone or in combination did not predict pregnancy success (24% vs 16%), incidence of chemical pregnancies (15% vs 24%), or clinical pregnancy loss (9% vs 11%) when such women were compared with those without either abnormality. However, the occurrence of hypergammaglobulinemias, in contrast to hypogammaglobulinemias, was associated with a significant decrease in the clinical pregnancy rate (6% vs 24%, p = 0.05). CONCLUSIONS: Neither autoantibody abnormalities nor total immunoglobulin abnormalities allow differentiation between high and low responders in in vitro fertilization cycles. The presence of autoantibody and total immunoglobulin abnormalities also does not predict low clinical pregnancy rates. Within a group of women with immunoglobulin abnormalities, those with hypergammaglobulinemias appear, however, at significant risk for low pregnancy rates with in vitro fertilization. This observation suggests that high total immunoglobulin levels may serve as a marker for an as yet to be determined immunologic factor that adversely affects the chance of conception. The evaluation of total immunoglobulin levels may be indicated as part of a routine infertility workup.

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.

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.

Antithyroid antibodies and the association with non-organ-specific antibodies in recurrent pregnancy loss.

OBJECTIVES: The purpose of our study was to evaluate the incidence of antithyroid antibodies and non-organ-specific antibodies in women who have had three or more recurrent spontaneous abortions. STUDY DESIGN: Sera from 45 women for the presence of antithyroid antibodies to thyroglobulin and thyroid peroxide and for the non-organ-specific autoantibodies to 6 phospholipids, 5 histones, and 4 polynucleotides were analyzed. Sera from 100 apparently health blood donors served as controls. RESULTS: The test results of 14 (31%) of 45 study subjects were positive for one or both antithyroid antibodies compared with 19 (19%) of controls. Five (11%) of 45 patients had positive test results for one or more non-organ-specific antibodies, and 4 (8%) of 45 had positive test results for the lupus anticoagulant by either activated partial thromboplastin, tissue thromboplastin time, or both. Only 3 (21%) of 14 subjects whose test results were positive for thyroid antibodies also demonstrated non-organ-specific autoantibodies. COMMENTS: The incidence of antithyroid antibodies in women who have had recurrent abortions appears not to be significantly increased compared with a normal random control population. Antithyroid antibodies do occur, however, with significantly greater frequencies in women with recurrent spontaneous abortions than non-organ-specific autoantibodies (p = 0.02). Organ-specific and non-organ-specific autoantibodies may serve as independent markers of risk for repeated pregnancy loss in patient populations where pregnancy loss is associated with abnormal autoimmune function.

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.

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.

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.

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.