Chemoautotrophy, symbiosis and sedimented diatoms support high biomass of benthic molluscs in the Namibian shelf.

The molluscs Lucinoma capensis, Lembulus bicuspidatus and Nassarius vinctus are highly abundant in Namibian oxygen minimum zone sediments. To understand which nutritional strategies allow them to reach such impressive abundances in this extreme habitat we investigated their trophic diversity, including a chemosymbiosis in L. capensis, focussing on nitrogen biochemical pathways of the symbionts. We combined results of bulk nitrogen and carbon (δ13C and δ15N) and of compound-specific isotope analyses of amino acid nitrogen (AAs-δ15NPhe and δ15NGlu), with 16S rRNA gene sequencing of L. capensis tissues and also with exploratory results of ammonium, nitrate and nitrite turnover. The trophic position (TP) of the bivalve L. capensis is placed between autotrophy and mixotrophy, consistent with its proposed symbiosis with sulfur-oxidizing Candidatus Thiodiazotropha sp. symbionts. The symbionts are here revealed to perform nitrate reduction and ammonium uptake, with clear indications of ammonium host-symbionts recycling, but surprisingly unable to fix nitrogen. The TP of the bivalve L. bicuspidatus is placed in between mixotrophy and herbivory. The TP of the gastropod N. vinctus reflected omnivory. Multiple lines of evidences in combination with current ecosystem knowledge point to sedimented diatoms as important components of L. bicuspidatus and N. vinctus' diet, likely supplemented at times with chemoautotrophic bacteria. This study highlights the importance of benthic-pelagic coupling that fosters the dietary base for macrozoobenthos in the OMZ. It further unveils that, in contrast to all shallow water lucinid symbionts, deeper water lucinid symbionts rely on ammonium assimilation rather than dinitrogen fixation to obtain nitrogen for growth.

The perplexing role of immuno-oncology drugs in osteosarcoma.

Osteosarcoma is a rare, primary tumour of bone. Curative treatment consists of multi-agent chemotherapy and complete surgical resection. Despite the use of multi-agent chemotherapy, the risk of recurrence is high. Survival outcomes for patients with osteosarcoma have not changed since the 1980's. Based on biologic rationale, there has been interest in adding immunotherapies to upfront curative intent chemotherapy, including mifamurtide (a macrophage activator) and interferon. However, results to date have been disappointing. In the metastatic setting, checkpoint inhibitors alone have not proven effective. Ongoing translational work is needed to further understand which patients may benefit from immune-oncology approaches with standard cytotoxic chemotherapy.

[A case of sole supportive care of severe neuroparalytic foodborne botulism due to homemade pesto]. / Fall einer rein supportiven Behandlung eines schweren neuroparalytischen Nahrungsmittelbotulismus durch hausgemachtes Pesto.

In this article, a case of severe foodborne botulism in a 78-year-old man due to homemade pesto is presented. His initial symptoms were gastrointestinal problems. Later he suffered of cranial nerve palsies, muscle weakness of the upper extremities and respiratory failure, so he had to be admitted to the intensive care unit for mechanical ventilation. The botulism was confirmed by serology and culture. Because of spontaneously improving neurological deficits, we decided not to treat with the botulism antitoxin and the patient had complete clinical remission.

Development of a marine fish model for studying in vivo molecular responses in ecotoxicology.

A protocol for fixation and processing of whole adult marine medaka (Oryzias melastigma) was developed in parallel with in situ hybridization (ISH) and immunohistochemistry (IHC) for molecular analysis of in vivo gene and protein responses in fish. Over 200 serial sagittal sections (5microm) can be produced from a single adult medaka to facilitate simultaneous localization and quantification of gene-specific mRNAs and proteins in different tissues and subcellular compartments of a single fish. Stereological analysis (as measured by volume density, V(v)) was used to quantify ISH and IHC signals on tissue sections. Using the telomerase reverse transcriptase (omTERT) gene, omTERT and proliferating cell nuclear antigen (PCNA) proteins as examples, we demonstrated that it is possible to localize, quantify and correlate their tissue expression profiles in a whole fish system. Using chronic hypoxia (1.8+/-0.2 mgO(2)L(-1) for 3 months) as an environmental stressor, we were able to identify significant alterations in levels of omTERT mRNA, omTERT protein, PCNA (cell proliferation marker) and TUNEL (apoptosis) in livers of hypoxic O. melastigma (p<0.05). Overall, the results suggest that O. melastigma can serve as a model marine fish for assessing multiple in vivo molecular responses to stresses in the marine environment.

[Antioxidant vitamin supplementation in the prevention of cardiovascular disease]. / Antioxidanzien und Vitamine in der Prävention von kardiovaskulären Erkrankungen.

Oxidative stress, in particular oxidative modification of LDL-cholesterol, appears to be of great importance in the pathogenesis of atherosclerosis. Various observational epidemiological studies have suggested that antioxidant vitamin intake is associated with reduced cardiovascular morbidity and mortality. Also, experimental studies in animals have demonstrated that antioxidant vitamins slow the progression of atherosclerosis. However, prospective controlled clinical trials have failed to demonstrate a benefit of antioxidant vitamin supplementation in primary or secondary prevention of cardiovascular disease. Thus, the use of antioxidants and vitamin supplements as a preventive or therapeutic intervention can not be recommended.

Cultural recovery and determination of antimicrobial susceptibility in Helicobacter pylori by using commercial transport and isolation media.

BACKGROUND: Antimicrobial resistance of Helicobacter pylori is the main reason for eradication failure. We have studied the feasibility of a commercial transport medium for cultural recovery and subsequent drug susceptibility testing. PATIENTS AND METHODS: From March to December 2000, 79 consecutive gastric biopsies, positive in a rapid urease test, were transferred into a commercial transport medium and sent within 24 hours from the district hospital to the microbiological laboratory for culture and susceptibility testing. A commercial agar plate and an in-house Wilkins-Chalgren agar plate were used for culture. Susceptibility data were compared with data collected from 1992 to 2003 in the University Hospital of Zurich. RESULTS: Cultural recovery and susceptibility testing of H. pylori was successful in 55 of 79 patients. In 17 cases cultural recovery failed because of technical problems (n = 14), long transport time (n = 1) and unknown reason (n = 2). Failure of susceptibility testing (n = 7) was mainly due to fungal overgrowth. Resistance to metronidazole and clarithromycin was found in 15 (27%) and in 12 patients (22%), respectively; resistance to amoxicillin was not observed. Five patients (9%) showed resistance both to metronidazole and to clarithromycin. Eradication therapy failed in all patients with macrolide resistance. Resistance rates were higher in females than in males; 30% vs 12% for clarithromycin and 33% vs 20% for metronidazole. Resistance to metronidazole was significantly lower in Swiss patients (15%) than in non-Swiss patients (39%). CONCLUSION: Antimicrobial resistance data can reliably be obtained by sending the biopsy specimen in a commercial transport medium to a microbiological laboratory. This is especially important after eradication failure. Resistance to metronidazole and clarithromycin is highly prevalent and more common in women and non-Swiss patients.

Prenatal programming of postnatal obesity: fetal nutrition and the regulation of leptin synthesis and secretion before birth.

Exposure to either an increased or decreased level of intrauterine nutrition can result in an increase in adiposity and in circulating leptin concentrations in later life. In animals such as the sheep and pig in which fat is deposited before birth, leptin is synthesised in fetal adipose tissue and is present in the fetal circulation throughout late gestation. In the sheep a moderate increase or decrease in the level of maternal nutrition does not alter fetal plasma leptin concentrations, but there is evidence that chronic fetal hyperglycaemia and hyperinsulinaemia increase fetal fat mass and leptin synthesis within fetal fat depots. Importantly, there is a positive relationship between the relative mass of the 'unilocular' component of fetal perirenal and interscapular adipose tissue and circulating fetal leptin concentrations in the sheep. Thus, as in the neonate and adult, circulating leptin concentrations may be a signal of fat mass in fetal life. There is also evidence that leptin can act to regulate the lipid storage, leptin synthetic capacity and potential thermogenic functions of fat before birth. Thus, leptin may act as a signal of energy supply and have a 'lipostatic' role before birth. Future studies are clearly required to determine whether the intrauterine and early postnatal nutrient environment programme the endocrine feedback loop between adipose tissue and the central and peripheral neuroendocrine systems that regulate energy balance, resulting in an enhanced risk of obesity in adult life.

Effects of leptin on fetal plasma adrenocorticotropic hormone and cortisol concentrations and the timing of parturition in the sheep.

We investigated whether leptin can suppress the prepartum activation of the fetal hypothalamus-pituitary-adrenal (HPA) axis and delay the timing of parturition in the sheep. First, we investigated the effects of a 4-day intravascular infusion of recombinant ovine leptin (n = 7) or saline (n = 6) on fetal plasma adrenocorticotropic hormone (ACTH) and cortisol concentrations, starting from 136 days gestation (i.e., at the onset of the prepartum activation of the fetal HPA axis. The effects of a continuous intrafetal infusion of leptin (n = 7) or saline (n = 5) from 144 days gestation on fetal plasma ACTH and cortisol concentrations and the timing of delivery were also determined in a separate study. There was an increase in fetal plasma ACTH (P < 0.01) and cortisol (P < 0.001) concentrations when saline was infused between 136-137 and 140-141 days gestation. Plasma ACTH and cortisol concentrations did not rise, however, when leptin was infused during this period of gestation. When leptin was infused after 144 days gestation, there was no effect of a 4- to 5-fold increase in circulating leptin on fetal ACTH concentrations. In contrast, leptin infusion from 144 days gestation suppressed (P < 0.05) fetal plasma cortisol concentrations by around 40% between 90 and 42 h before delivery. There was no difference, however, in the length of gestation between the saline- and leptin-infused groups (saline infused, 150.2 +/- 0.5 days; leptin infused, 149.8 +/- 1.0 days). In saline-infused fetuses, there was a significant negative relationship between the plasma concentrations of cortisol (y) and leptin (x) between 138 and 146 days gestation (y = 81.4 - 7.7x, r = 0.38, P < 0.005). This study provides evidence for an endocrine negative feedback loop between leptin and the HPA axis in fetal life.

An association between sex chromosomal aneuploidy in sperm and an abortus with 45,X of paternal origin: possible transmission of chromosomal abnormalities through ICSI.

BACKGROUND: Although it has been speculated that the increased de-novo chromosomal abnormalities in ICSI pregnancies may be associated with an increase of aneuploidy in sperm from infertile men, little direct evidence exists to support this claim. We studied sperm from an infertile man with an abortus from ICSI to determine if increased sex chromosomal aneuploidy in the sperm could have contributed to the karyotype of the abortus. METHODS: The couple underwent ICSI due to severe oligozoospermia. Spontaneous aborted material was subjected to cytogenetic and molecular tests to ascertain the existence, type and origin of a chromosomal abnormality. Sperm from the man were analysed by multi-coloured fluorescent in-situ hybridization (FISH) with probes specific for chromosomes X, Y and 18. RESULTS: At 8+ weeks after embryo replacement, the patient spontaneously miscarried. Both cytogenetic and comparative genomic hybridization analysis of aborted material showed a 45,X karyotype. Origin of the abnormality was established as a loss of the paternal X chromosome. FISH analysis of sperm revealed 19.6% (1990/10,164) nullisomy for a sex chromosome and 18.6% (1886/10,164) with XY disomy, which is significantly increased when compared to controls with 0.3% (58/20,429) and 0.1% (20/20,429) respectively (P<0.0001). CONCLUSIONS: This study indicates that the paternal origin of the 45,X abortus was likely the result of a high level of nullisomy in the sperm and provides evidence for the transmission of chromosomal abnormality from sperm to the conceptus through ICSI.

Cytogenetic and molecular study of a premature male infant with 46,XX derived from ICSI: case report.

We investigated the aetiology of the male phenotype in a premature infant derived from ICSI with a 46,XX karyotype. A karyotypically normal couple underwent ICSI because of obstructive azoospermia in the male partner. Sperm were retrieved by testicular sperm extraction (TESE), cryopreserved, and later used for ICSI. The pregnancy after ICSI ended at 20 weeks. A normal-appearing male was delivered but he did not survive. Umbilical cord blood and placenta were sampled and used for molecular and cytogenetic investigation. The 46,XX karyotype from G-banding in this male infant correlated to a balanced female comparative genomic hybridization (CGH) profile in placental tissue. No PCR amplification of SRY on the p arm of the Y chromosome was observed while fluorescence in-situ hybridization (FISH) with the SRY probe also could not detect the gene in cord blood or placental tissues. CGH and FISH, with X and Y centromeric probes, failed to detect mosaicism in the trophoblast, stroma and amnion. Skewed X-chromosome inactivation (81%) was found in the chorionic villi. The molecular and cytogenetic studies indicated a 46,XX male infant without the SRY gene or 46,XX/XY mosaicism. The possible mechanism in this SRY-negative XX male by ICSI is discussed.

Determinants of fetal leptin synthesis, fat mass, and circulating leptin concentrations in well-nourished ewes in late pregnancy.

We have investigated the factors regulating leptin synthesis, fat deposition, and circulating leptin concentrations in fetuses of well nourished ewes in late pregnancy. Vascular catheters were surgically inserted in 17 pregnant ewes and their fetuses at 103-120 d gestation (term = 147 +/- 3 d). Ewes were fed a diet providing either 100% (control; n = 9) or approximately 155% (well fed; n = 8) of the maintenance energy requirements and fetal perirenal and interscapular fat depots were collected at 139-141 d gestation. There was a significant relationship between the relative mass of fetal unilocular fat and fetal glucose (relative mass of unilocular fat, 1.14; fetal glucose, +0.16; r = 0.50; P < 0.04; n = 17), but not insulin, concentrations in the control and well-fed groups. In contrast to the controls, there was a positive relationship between the relative abundance of leptin mRNA and fetal insulin, but not glucose, concentrations in fetal perirenal adipose tissue in the well-fed group. A moderate increase in maternal nutrition also resulted in a strong reciprocal relationship between uncoupling protein 1 and leptin expression in fetal perirenal adipose tissue in late gestation (well-fed group: uncoupling protein 1 mRNA:18S rRNA, -0.51; leptin mRNA:beta-actin mRNA, +1.53; r = 0.80; P < 0.02; n = 8). These studies provide evidence that fetal glucose and insulin differentially regulate fetal fat deposition and leptin mRNA expression within the fetal perirenal fat depot in the well nourished animal during late gestation.

Leptin alters the structural and functional characteristics of adipose tissue before birth.

This study aimed to determine for the first time whether leptin can act to alter the structural and functional characteristics of adipose tissue before birth. Leptin (0.48 mg/kg/day) or saline was infused intravenously into fetal sheep for 4 days from either 136 or 137 days of gestation (term=147+/-3 days). Circulating leptin concentrations were increased approximately four- to fivefold by leptin infusion. Leptin infusion resulted in a significant increase in the proportion of smaller lipid locules present within fetal perirenal adipose tissue (PAT), and this was associated with a significant increase in the proportion of multilocular tissue and a significant decrease in the proportion and relative mass of unilocular tissue in fetal PAT. The relative abundance of leptin mRNA in fetal PAT was significantly lower in the leptin-infused group, and there was a positive correlation between the relative abundance of leptin mRNA and the proportion of unilocular adipose tissue in fetal PAT. The amount of uncoupling protein 1 tended to be higher (P=0.06) in leptin-infused compared with saline-infused fetuses. This is the first demonstration that leptin can act to regulate the lipid storage characteristics, leptin synthetic capacity, and potential thermogenic functions of fat before birth.

X-chromosome inactivation (XCI) patterns in placental tissues of a paternally derived bal t(X;20) case.

Non-random X-chromosome inactivation (XCI) is often seen in female carriers of balanced X-autosome translocations and is generally attributed to a selective growth of cells that inactivate the normal X chromosome. However, little is known concerning when in development the selection acts, and thus whether skewed XCI would also be seen in placental tissues. Furthermore, as males with X-autosome translocations are normally infertile, all translocations studied to date for XCI-skewing have been either maternal or de novo in origin. We now present an analysis of XCI status in cord blood, umbilical cord and four different extraembryonic tissues from a female carrier of a paternally derived balanced (X;20) translocation. Using methylation based assays to determine XCI status, we found preferential inactivation of the non-translocated X in cord blood, umbilical cord and amnion samples of the propositus. Remarkably, random XCI was evident in several placental tissues analyzed (chorion, and chorionic villi trophoblast and mesenchyme). While these findings support the hypothesis of strong selection against cells with an inactive translocated X-chromosome in most embryonic/fetal tissues, they also suggest weaker selective forces taking place during placental development. Additionally, the finding of normal placental development in the present case, rules out the possibility of a parental bias to XCI in human extraembryonic tissues as a requisite for normal development. The finding of hypomethylation in extraembryonic tissues for two out of three markers used in the study is consistent with previous findings demonstrating low levels of methylation in these tissues.

Impairment of sea urchin sperm quality by UV-B radiation: predicting fertilization success from sperm motility.

Sperm quality of the sea urchin, Anthocidaris crassispina, after exposure to environmentally realistic UV-B irradiances, was assessed by changes in sperm motility (measured by the computer-assisted sperm analysis (CASA) system), and related to subsequent fertilization success. Percentage motile sperm of A. crassispina declined significantly after exposure to a UV-B dose of 16.2 kJ m(-2), while sperm motion velocity as measured by curvilinear velocity (VCL), straight line velocity (VSL), and average path velocity (VAP) showed significant reduction after exposure to a UV-B dose of 5.4 kJ m(-2). A parallel study showed that fertilization success was significantly reduced after sperm were exposed to UV-B doses > or = 5.4 kJ m(-2). Notably, the four sperm motility parameters were strongly correlated with fertilization success (P < 0.001), followed the increasing order: VSL (r = 0.8) < % motile sperm (r = 0.804) < VCL (r = 0.912) < VAP (r = 0.928). Fertilization success is best predicted by VAP using the exponential model: y = 8.678 + 90.202/[1 + exp(82.83 - x)/10.27)] (r(2) = 0.95). Thus, impairment of sperm motility of sea urchin, as measured by the CASA method, can be used to predict reproductive success and ecological effects.

Circulating leptin concentrations are positively related to leptin messenger RNA expression in the adipose tissue of fetal sheep in the pregnant ewe fed at or below maintenance energy requirements during late gestation.

We have investigated the effects of maternal undernutrition during late gestation on maternal and fetal plasma concentrations of leptin and on leptin gene expression in fetal perirenal adipose tissue. Pregnant ewes were randomly assigned at 115 days of gestation (term = 147 +/- 3 days [mean +/- SEM]) to either a control group (n = 13) or an undernourished group (n = 16) that received approximately 50% of the control diet until 144-147 days of gestation. Maternal plasma glucose, but not leptin, concentrations were lower in the undernourished ewes. A significant correlation was found, however, between mean maternal plasma leptin (y) and glucose (x) concentrations (y = 2.9x - 2.4; r = 0.51, P < 0.02) when the control and undernourished groups were combined. Fetal plasma glucose and insulin, but not fetal leptin, concentrations were lower in the undernourished ewes, and no correlation was found between mean fetal leptin concentrations and either mean fetal glucose or insulin concentrations. A positive relationship, however, was found between mean fetal (y) and maternal (x) plasma leptin concentrations (y = 0.18x + 0.45; r = 0.66, P < 0.003). No significant difference was found in the relative abundance of leptin mRNA in fetal perirenal fat between the undernourished (0.60 +/- 0.09, n = 10) and control (0.70 +/- 0.08, n = 10) groups. Fetal plasma concentrations of leptin (y) and leptin mRNA levels (x) in perirenal adipose tissue were significantly correlated (y = 1.5x +/- 0.3; r = 0.69, P < 0.05). In summary, the capacity of leptin to act as a signal of moderate maternal undernutrition may be limited before birth in the sheep.

Cytogenetic investigation of fetuses and infants conceived through intracytoplasmic sperm injection.

OBJECTIVE: To determine the incidence of aneuploidy among fetuses and infants conceived through intracytoplasmic sperm injection (ICSI) in our clinic using umbilical cord blood samples. DESIGN: Follow-up study of the cytogenetic outcome of ICSI pregnancies. SETTING: University-based IVF clinic. PATIENT(S): Forty-six couples who underwent ICSI and conceived. INTERVENTION(S): Umbilical cord blood was taken after delivery of the infant for analysis. Samples of chorionic villi and chorion were taken for studies on the spontaneous abortuses. Amniocentesis was performed for couples that chose prenatal diagnosis. MAIN OUTCOME MEASURE(S): The cytogenetic chromosomal status of the pregnancy outcome. RESULT(S): Fifty pregnancies and 55 live births were recorded, with nine spontaneous abortions. Of 43 separate umbilical cord blood samples analyzed, 1 abnormality (2%) was found, 45, XX,+21. Nine births went through prenatal diagnosis alone, with four accepting both forms of analysis-no abnormalities were found. Origin of abnormality was established in two spontaneous abortion cases (45, XO and 45, XY,-21), and the maternal chromosome was lost in both cases. CONCLUSION(S): Using umbilical cord blood obtained after birth, we obtained karyotype results from 78% of the ICSI population in our clinic. Combined with results from five additional cases that underwent prenatal diagnosis but not umbilical cord blood sampling, a chromosomal result was obtained in 87% of our ICSI population. The use of umbilical cord blood for cytogenetic analysis substantially improves the ability to determine rates of chromosomal abnormalities in newborns produced via ICSI clinics.

Interaction of prostaglandin F(2alpha) and prostaglandin E(2) on progesterone production in human granulosa-luteal cells.

This study examined the effects of prostaglandin-F(2alpha) (PGF(2alpha)), prostaglandin-E(2) (PGE(2)) and their interactions on progesterone production in human granulosa-luteal cells (GLCs). Human GLCs collected from in vitro fertilization patients were cultured for 1 (D(1)) or 8 days (D(8)), followed by a 24-hour treatment period, after which media were collected and radioimmunoassayed for progesterone. Seven-point PGF(2alpha) and PGE(2) concentration-response curves were crossed into a matrix of 49 separate treatments. Responses were plotted in three dimensions and as two-dimensional "slices". In D(1) cultured human GLCs neither PGF(2alpha) nor PGE(2) alone had any effect on progesterone production, however two different combinations of these hormones led to at least a 3-fold increase in progesterone production. This stimulation was seen when cells were treated with 10(-6) M PGF(2alpha) plus 10(-9) M PGE(2), and when they were treated with 10(-10) M PGF(2alpha) plus 10(-9) M PGE(2). In D(8) GLCs, PGF(2alpha) stimulated progesterone production maximally at 10(-9) M, while the lowest (10(-11) M) and highest concentrations (10(-6) M) tested were ineffectual. On the contrary, in the presence of high concentrations of PGE(2) (10(-6) to 10(-7) M), PGF(2alpha)-mediated stimulation of progesterone production was attenuated. In a similar fashion to PGF(2alpha), PGE(2) also acted in a luteotrophic manner, although the maximal stimulation of progesterone production was seen at a higher concentration (10(-8) to 10(-7) M). Likewise, PGE(2)-mediated progesterone production was attenuated by the presence of high concentrations of PGF(2alpha) (10(-6) to 10(-7) M). In conclusion, in D(1) human GLCs neither PGF(2alpha) nor PGE(2) alone were luteotrophic, although specific combinations of these hormones were. Conversely, in D(8) GLCs both PGF(2alpha) and PGE(2) stimulated progesterone production in a biphasic manner, while the presence of a high concentration of either of these prostaglandins attenuated the luteotrophic effects of the other. Therefore, PGF(2alpha) and PGE(2) interacted in a concentration-dependent manner, resulting in a multimodal progesterone response, which was easily visualized using three-dimensional plots.

Intracytoplasmic sperm injection could minimize the incidence of prematurely condensed human sperm chromosomes.

OBJECTIVE: To compare the fertilization and prematurely condensed human sperm chromosomes (PCCs) rates between two intracytoplasmic sperm injection (ICSI) techniques. DESIGN: A retrospective study. SETTING: The data were obtained from the University of British Columbia in vitro fertilization (IVF) laboratory. PATIENT(S): ICSI cycles (n = 105) were performed for couples suffering from severe male-factor infertility and dysfunction of fertilization. INTERVENTION(S): Two types of ICSI techniques were used for ICSI procedures. MAIN OUTCOME MEASURE(S): Fertilization and pregnancy rates in group B using the improved ICSI technique were compared with those of group A using the standard ICSI technique. Unfertilized oocytes from the two groups were studied with cytogenetic methods. RESULT(S): Oocyte damage dropped from 14.8% in group A to 5.3% in group B. Normal fertilization for each group was 57.3% and 88.4%, respectively (P<.05). Pregnancy rate per egg retrieval was 15.6% in group A and 27.4% in group B (P<.05). PCCs occurred in 19.4% of unfertilized oocytes in group A and did not occur in group B. CONCLUSION(S): This study indicates that ICSI not only yields high fertilization rates, but also minimizes the incidence of PCCs. It may be directly related to two crucial steps (immobilization of sperm and aspiration of oocyte cytoplasm) used in ICSI procedures. This study also suggests that it is possible to overcome one cause of IVF failure resulting from the formation of PCCs by using the improved ICSI technique.