The Importance of Neutrophil Gelatinase - Associated Lipocalin and Liver Type Fatty Acid Binding Protein in Acute Kidney Injury.

BACKGROUND: The most important factor in the diagnosis of AKI is to accurately and early detect the damage that occurs in the kidney before the filtration capacity of the kidney decreases. Therefore, we discussed the use of NGAL and L-FABP in the early diagnosis of acute kidney injury, evaluation of clinical severity and prognosis as well as prediction of hemodialysis decision in this prospective study. METHODS: We studied 82 participants which included 41 patients aged 18 years and older with the diagnosis of acute kidney injury. We compared the renal function tests collected at 0 and 6 hours with the plasma NGAL and LFABP levels measured using ELISA. Acute kidney injury was defined as serum creatinine increase of 0.3 mg/dL in the last 48 hours, or an increase more than 1.5 times, or an increase in the basal serum creatinine value in the last seven days, or less than 0.5/mL/kg of urine volume within six hours. We tested the power of these new biomarkers in the early diagnosis, and prediction of hemodialysis and survival of the patients with AKI using ROC analysis. RESULTS: Fifteen (36.6%) of the patients were anuric and 26 (63.4%) were oliguric. Twenty-one (51.2%) patients were KDIGO Stage 3. Seventeen (41.5%) patients underwent hemodialysis. In the patient group, the mean NGAL level was 289.7 ± 117.4 ng/mL and the mean L-FABP level was 232.7 ± 72.8. Eleven (26.9%) of 41 patients died within the first 24 hours. In the dead patients, the mean plasma NGAL level was statistically significantly high (p = 0.005). The mean NGAL level was found to be statistically increased in correlation with the severity of acute kidney injury in patients (p < 0.05). To predict acute kidney injury, the ROC analysis showed that the area under the curve (AUC) was 0.819 (95% confidence interval (CI): 0.729 - 0.909) (p < 0.001) for plasma NGAL level, and the area under the curve (AUC) was 0.891 (95% confidence interval (CI): 0.822 - 0.959) for plasma L-FABP level (p < 0.001). CONCLUSIONS: Our study provides evidence that NGAL and L-FABP are effective biomarkers for early detection of AKI as well as predicting clinical severity and hemodialysis.

Evaluation of patients with lympocele after renal transplantation / Evaluación de pacientes con linfocele luego de trasplante renal

Abstract Introduction: The lymphocele is a common complication following renal transplantation and may cause significant clinical problems especially when reachs to big volumes. The aim of this study is to present the clinical characteristics, diagnostic approaches, and therapeutic strategies of lymphocele formations in a group of Turkish patients. Methods: A total of 244 renal transplantations were included in this retrospective study. Data of patients who were diagnosed with lymphocele during the postoperative period were analyzed. Results: Ten (2.4%) patients have been diagnosed with lymphocele. There were six males and 4 females, with a mean age of 46 years. The median onset was 19 days posttransplantation. The median size of the lymphoceles was 53 mm. All lymphoceles were localizated between the lower pole of the transplanted kidney and urine bladder. On presentation, one patient had hydronephrosis and three patients had elevated serum creatinine while the remaining six ones were asymptomatic. Five patients were successfully treated by percutaneous aspiration whereas two patients required surgery. Three patients' lymphoceles dissolved spontaneously. Conclusion: Preventive strategies including preserving the lymphatics of the recipient, careful organ retrieval and 'back table' work are of great importance to reduce the incidence of lymphocele. Early decision of radiological or surgical intervention should be considered in patients with symptomatic lymphoceles in order to prevent further complications.

Resumen Introducción: El linfocele es una complicación frecuente luego de un trasplante renal y puede ocasionar problemas clínicos importantes, especialmente, cuando alcanza volúmenes elevados. El objetivo de este estudio es presentar las características clínicas, métodos de diagnóstico y estrategias para el tratamiento del linfocele en un grupo de pacientes turcos. Material y métodos: Se incluyeron 244 pacientes en este estudio retrospectivo. Se analizaron los datos de pacientes diagnosticados con linfocele durante el período postoperatorio. Resultados: Se diagnosticó linfocele a diez pacientes (2,4%). Eran seis hombres y cuatro mujeres con una edad promedio de 46 años. El comienzo promedio fue 19 días luego del trasplante. El tamaño medio de los linfoceles fue de 53 mm. Todos se encontraban entre el polo inferior del riñón trasplantado y la vejiga urinaria. En la consulta, un paciente presentó hidronefrosis, y tres pacientes, creatinina sérica elevada, mientras que los seis restantes eran asintomáticos. Cinco pacientes fueron tratados con éxito por aspiración percutánea; en cambio, otros dos pacientes requirieron cirugía. Tres pacientes mostraron disolución espontánea de los linfoceles. Conclusión: Las estrategias preventivas, que incluyen la preservación de los vasos linfáticos del receptor, la extracción cuidadosa de los órganos y la preparación de estos antes de realizar el trasplante, son de gran importancia para reducir la incidencia de linfocele. Debe considerarse tempranamente la intervención radiológica o quirúrgica en pacientes con linfoceles sintomáticos para prevenir complicaciones adicionales.

In Vitro Fertilization After Renal Transplantation: A Single-Center Experience.

INTRODUCTION: Although kidney transplantation often increases the chances of fertility, the rate of infertile patients is still high. In vitro fertilization promises successful results for infertile renal transplantation patients. The purpose of this study was to analyze the experience of a single center. METHODS: Patients were invited to complete a survey for their obstetric history. Documentation review included demographic and clinical characteristics of patients, like procedure records, follow-up complications, immunosuppression maintenance protocols, and pregnancy outcomes. RESULTS: Thirteen patients were reached to complete the survey. The mean age of patients was 33 ± 4 years at in vitro fertilization (IVF). The median duration of infertility was 2 years. Twenty-four IVF sessions were applied to these 13 women with renal transplantation. The procedure failed in 13 of these sessions; and 8 women achieved 11 clinical pregnancies. There were 3 miscarriages and 2 stillbirths. Six women had live births with no neonatal deaths. One patient had a graft rejection after the IVF procedure. Serum creatinine level increased more than 30% in 3 patients after the IVF procedure, while 9 patients had a minimal or no change. DISCUSSION: In our study, we evaluated the records of 13 patients with renal transplantation who had IVF procedures. Fortunately, more than half of these patients had live births with no neonatal deaths. In our opinion, our findings show that IVF procedures can be accepted as a promising method in patients with renal transplantation and need a therapy for fertility. Moreover, a 25% live-birth rate per procedure is also a satisfactory result.

Effect of bisphosphonate as an adjunct treatment for chronic periodontitis on gingival crevicuar fluid levels of nuclear factor-κB ligand (RANKL) and osteoprotegerin in postmenopausal osteoporosis.

Osteoporosis and periodontal disease are linked by an altered receptor activator of nuclear factor κB ligand and osteoprotegerin ratio (RANKL/OPG), and medical treatment with bisphosphonate (BP) may help control these molecules. The effect of BP on clinical findings and gingival crevicular fluid (GCF) values of RANKL and OPG using enzyme-linked immunosorbent assays was evaluated in postmenopausal women; 13 patients with both chronic periodontitis and osteoporosis (group A), 12 systemically healthy patients with chronic periodontitis (group B), 12 periodontally healthy patients with osteoporosis (group C), and 10 systemically and periodontally healthy individuals (group D). Recordings were repeated at the end of months 1, 6, and 12 in groups A, B, and C. At the baseline, groups A and B exhibited the lowest OPG values (P < 0.05). After periodontal treatment, OPG values were markedly increased at the end of 6th month in group A and 12th month in group B (P < 0.008). There was no significant difference in GCF RANKL values among groups (P > 0.05) or during the observation period (P > 0.008). The use of BP may be effective in preventing periodontal breakdown by controlling the levels of these markers in osteoporosis as an adjunct to periodontal treatment.

Pentraxin 3, ischemia-modified albumin, and myeloperoxidase in predicting a cardiac damage in acute carbon monoxide poisoning.

BACKGROUND: Carbon monoxide (CO) poisoning is associated with cardiac injuries or manifestations, frequently attributing to direct hypoxic damage at cellular level. For this, the aims were to evaluate the role of serum pentraxin 3 (PTX 3), ischemia-modified albumin (IMA), and myeloperoxidase (MPO) as an early biomarker for cardiac damage when compared to cardiac troponin I (cTnI) and creatine kinase-MB fraction (CK-MB) in adult patients with acute CO poisoning. METHODS: Forty patients with acute CO poisoning admitted to the emergency department. The patients were divided into 2 main groups as follows: cardiac injury (group I, n=19) and nonsuspected cardiac injury (group II, n=21). Pentraxin 3, IMA, MPO, cTnI, CK-MB, and the other assays in the circulation were measured on admission. RESULTS: Upon measuring the serum PTX 3, IMA, MPO, cTnI, and CK-MB levels as well as large electrocardiography and echocardiography abnormalities of patients with cardiac injury on admission, no statistical difference for PTX 3, IMA, and MPO was found between the groups (P>.05). However, cTnI, CK-MB, and leukocyte count (white blood cell) were higher determined in patients of group I compared to group II (P<.05). Receiver operating characteristic curve was also performed to evaluate the diagnostic performance of these tests in patients with cardiac injury. CONCLUSIONS: Our results suggest that PTX, IMA, and MPO assays are not superior to cTnI and CK-MB in predicting a cardiac damage in patients with acute CO intoxication.

Reliable single sperm cryopreservation in Cell Sleepers for azoospermia management.

Conventional sperm freezing methods perform best when freezing sperm samples containing at least hundreds of spermatozoa. In this severe male factor infertility case series, we examined the reproductive outcomes in 12 intracytoplasmic sperm injection cases where spermatozoa used were frozen in Cell Sleepers. Cell Sleepers are novel devices in which individual spermatozoa can be frozen in microdroplets. The case series included five men with obstructive azoospermia, six with nonobstructive azoospermia and one with cryptozoospermia, in whom microscopic sperm retrievals from testicular sperm extraction (TESE), micro-TESE extracts and a centrifugation procedure resulted in less than 50 spermatozoa. A total of 304 microscopically retrieved spermatozoa were frozen in 20 Cell Sleepers using a rapid manual cryopreservation method. A total of 179 mature oocytes were injected with recovered thawed spermatozoa, resulting in a fertilisation rate of 65.9% (118 of 179), with no total fertilisation failures. In 10 cases, an embryo transfer was performed, three on day 3 and seven on day 5, resulting in a per cycle pregnancy rate of 58.3% (seven of 12). Four of the pregnancies have progressed past 20 gestation weeks. The recovery and use of spermatozoa that were frozen in Cell Sleepers was uncomplicated and effective and eliminated the need to perform any microscopic sperm retrieval procedures on the day of oocyte collection. Modification of the routine sperm cryopreservation methodology to include the use of Cell Sleepers increases the range of sperm samples that can be effectively cryopreserved, to include men with severe male factor fertility.

VEGF and GM-CSF levels in nodular thyroid diseases.

VEGF is a specific mitogen for endothelial cells. GM-CSF is a key player in the regulation of steady-state functions. The aim of this study was to evaluate VEGF and GM-CSF levels in thyroid nodules >1 cm, which are negative for malignancy with fine needle aspiration biopsy. Age, serum VEGF, GM-CSF, TSH, fT3, fT4, anti-TG, anti-TPO, thyroid size, and thyroid volume were compared between 41 female patients and 20 healthy female volunteers. This study was performed with 41 female patients who were euthyroid and whose nodules were benign. Twenty healthy female volunteers were enrolled as the control group. VEGF and GM-CSF were assayed by ELISA; TSH, fT3, and fT4 were detected by electrochemiluminescence method and anti-TPO and anti-TG were detected by competitive immunoassay method. Only thyroid volume and anti-TG levels were significantly different between the two groups (p < 0.007 and p < 0.026, respectively). Other parameters including VEGF and GM-CSF were not significantly different. VEGF has a weak positive correlation only with anti-TPO levels in the patient group (r = 0.325, p = 0.036). There was a weak positive correlation between anti-TPO and anti-TG (r = 0.388, p = 0.007). There was a positive correlation between nodule size and thyroid volume (r = 0.464, p = 0.015). GM-CSF was not correlated with any parameters. VEGF and GM-CSF were not found to be increased in euthyroid patients with benign nodules and they do not seem to play a role in development of simple nodular goiter.

The comparison of the intestinal adaptation effects of subcutaneous and oral insulin in a rats with short bowel syndrome.

AIM: Insulin has been reported to have positive effects on intestinal adaptation after short bowel syndrome when applicated oral or subcutaneously. The purpose of this study is to compare the intestinal adaptation effects of subcutaneous and oral routes of insulin in rats with short bowel syndrome. MATERIALS AND METHODS: The short bowel syndrome (SBS) was performed through 70-75% of small intestinal resection and an end-to-end anastomosis. The control group rats underwent SBS only. In the second group, oral insulin (1 U/ml) was administrated twice-daily. In the last group, the insulin was administrated subcutaneously (1 U/kg) as in the control group. All rats were killed on day 15. Outcome parameters were weight of small intestine, the crypt length, villous depth, the blood levels of vascular endothelial growth factor (VEGF), and granolocyt-monocyst colony-stimulating factor (GMCSF). RESULTS: Intestinal weight was significantly more in oral insulin group and subcutaneous insulin group than in the control group (72.6 ± 4.3, 78.6 ± 4.8 and 59.7 ± 4.8) (P < 0.05). There was no difference between the groups according to villus length, crypt depth, and villous/crypt ratio both in proximal and distal parts of the resected bowel (P > 0.05). VEGF values were not statistically significant between the groups (200.3 ± 41.6, 178.9 ± 30.7 and 184.3 ± 52.2) (P > 0.05). GMCSF was statistically higher in the control group than in other groups (3.34 ± 1.34, 1.56 ± 0.44 and 1.56 ± 0.44) (P < 0.05). CONCLUSION: Insulin has positive effects on intestinal adaptation in short bowel syndrome. Subcutaneous administration is slightly more effective than the oral route.

Protective effects of cysteine, methionine and vitamin C on the stomach in chronically alcohol treated rats.

A chronic intake of high dose alcohol may cause oxidative stress and inflammation in the stomach. It is hypothesized that cysteine-methionine and vitamin C may neutralize harmful compounds while potentiating the antioxidant capacity of the cell or tissue. The experimental animals were fed regular diets and were maintained for 90 days in the control group, the alcoholic group, which was given 2.5 g of 50% ethanol kg(-1) body wt. administered intragastrically every other day, or the alcoholic with antioxidant supplement group, to whom 2.5 g of 50% ethanol kg(-1) body wt. + a solution that contained 200 mg vitamin C, 100 mg cysteine and 100 mg methionine was administered intragastrically every other day. After the treatments, the stomach was taken for pathological and biochemical analysis. The stomach of the alcoholic group rats had higher scores of pathological findings compared with the control group, whereas the scores of the antioxidant-supplemented group were lower than the alcoholic group. In addition, the oxidized protein and lipid content in the stomachs of the alcoholic group were significantly higher than the control, but antioxidant supplementation lowered the amount of oxidation in the antioxidant supplemented group. The amount of stomach glutathione in the alcoholic group was higher than that of the control and antioxidant-supplemented groups. Interestingly, the level of total thiol in the stomach tissue of rats with antioxidant supplement was statistically higher than that of the control and alcoholic groups. In conclusion, the scores of the pathological findings in the stomach of rats with the antioxidant supplement were lower than the chronic alcohol-treated rats, albeit the amount of total thiol was increased in this group. Moreover, chronic alcohol treatment led to an increase in the level of lipid and protein oxidation in the stomach tissue of rats. A simultaneous intake of ascorbate/l-cys/l-met along with ethanol attenuated the amount of oxidation which suggested that cysteine-methionine and vitamin C could play a protective role in the stomach against oxidative damage resulting from chronic alcohol ingestion.

Single-dose depot leuprolide is as efficient as daily short-acting leuprolide in ICSI cycles.

BACKGROUND: In this prospective randomized study, we aimed to compare the efficacy of a single depot (1.88 mg) reduced dose with a daily low dose (0.5 mg/day) of leuprolide for pituitary suppression as part of controlled ovarian hyperstimulation (COH) in an ICSI program. METHODS: The study population consisted of 103 patients randomized into two groups. Group 1 (n = 52) consisted of patients who had daily low-dose leuprolide injections. Group 2 (n = 51) consisted of patients who had the 1.88 mg single-dose leuprolide injection. RESULTS: The age of the patients, the number of metaphase II oocytes, the number and quality of embryos transferred were similar between the two groups. Although the length of gonadotrophin stimulation was significantly longer in group 2 (P < 0.01), the amount of gonadotrophins used was similar (P = 0.34). Leuprolide levels were significantly lower in group 2 within the first 8 h after injection (P < 0.05), but no difference was observed thereafter. Although LH levels on the day of hCG (P = 0.06) administration and estradiol levels on day 3 (P < 0.01) were lower in group 2, LH levels and progesterone levels 1 week after embryo transfer did not show any statistically significant difference. Clinical pregnancy rates per embryo transfer, implantation rates and first trimester abortion rates were also similar for both groups. CONCLUSIONS: A single reduced depot dose (1.88 mg) of leuprolide was found to be as effective as classical long multi-dose protocol for pituitary desensitization in COH for ICSI cycles.

Flushing the endometrium prior to the embryo transfer does not affect the pregnancy rate.

This study aimed to determine whether direct flushing of endometrial cavity with culture media just after cervical irrigation at the time of embryo transfer has any effect on pregnancy rates. A total of 240 women were prospectively randomised; one group of patients (group 1) underwent intrauterine direct flushing of endometrial cavity with culture media just after cervical irrigation at the time of embryo transfer as detected by transabdominal ultrasound and the other group of patients (group 2) had cervical flushing but did not undergo intrauterine flushing. Pregnancy (positive human chorionic gonadotrophin) rates were 57.5% and 62.9% for group 1 and group 2, respectively. Clinical pregnancy (positive fetal heart rate) rates were 45.2% and 51.4% for group 1 and group 2, respectively. Implantation rates were 20.0% and 21.2% for group 1 and group 2, respectively. Ongoing pregnancy (>12 weeks of pregnancy) rates were 47.9% and 47.2% for group 1 and group 2, respectively. There were no significant differences in pregnancy rates, clinical pregnancy rates, implantation rates and ongoing pregnancy rates between the two groups. In conclusion, even direct flushing of media into the uterine cavity neither improves nor adversely affects the pregnancy rate.

Endometrial polyps smaller than 1.5 cm do not affect ICSI outcome.

This study aimed to determine whether the presence of endometrial polyps discovered during ovarian stimulation affects the outcomes of intracytoplasmic sperm injection (ICSI) cycles. This retrospective descriptive study was conducted in a private assisted reproductive technology unit. Medical records of ICSI cycles performed between January 2003 and December 2004 were reviewed. Patients were divided into three groups: patients with endometrial polyps discovered during ovarian stimulation (group 1, n=15), patients who underwent hysteroscopic polyp resection prior to their ICSI cycle (group 2, n=40) and patients without polyps (group 3, n=956). Main outcome measures were clinical pregnancy rates and implantation rates. Age of the patients, age of the husbands, body mass index, total amount of gonadotrophins used, length of stimulation, peak oestradiol concentrations, peak endometrial thickness and number of embryos replaced were not significantly different between the groups, nor were the pregnancy and implantation rates. Only one patient (12.5%) from the first group experienced miscarriage within 12 weeks of pregnancy. In conclusion, endometrial polyps discovered during ovarian stimulation do not negatively affect pregnancy and implantation outcomes in ICSI cycles.

Public opinion regarding oocyte donation in Turkey: first data from a secular population among the Islamic world.

INTRODUCTION: We aimed to reveal the general attitudes of Turkish people toward various aspects of oocyte donation. METHODS: This descriptive study was carried out in two separate districts of a large Turkish city. Four hundred participants were chosen by cluster sampling methods. The questionnaires were performed by 4th year medical students face to face with the participants. RESULTS: The participants consisted of 232 women (58%) and 168 men (42%); 65% were married, 5% were divorced; 64% had children, 15 (4%) had infertility problems, 263 (66%) were graduates of high school or university; 269 (67%) considered themselves religious. Only less than one-third of the respondents actually knew what oocyte donation meant. Approval of oocyte donation was high in our study sample. Only 61 (15%) respondents showed complete objection to oocyte donation and more men were in favour than women. Less than half of the participants thought that their religion would prevent oocyte donation if they needed it. More than half of the participants would prefer the use of oocyte donation treatment rather than adoption of a child. CONCLUSION: This is the first report on the attitudes towards oocyte donation from a country having a secular constitution and whose population is mainly Muslim. The most important conclusion to be drawn from the present study is the fact that most of the participants did not have any objection to oocyte donation treatments.

Alcohol-induced oxidative stress and reduction in oxidation by ascorbate/L-cys/ L-met in the testis, ovary, kidney, and lung of rat.

Chronic exposure to high doses of alcohol results in many pathophysiologic changes in cellular function caused by the alcohol itself and the effects of its metabolism (ie, generation of acetaldehyde, nicotinamide adenine dinucleotide [NADH], free radicals, and oxidative stress). However, the role of each of these effects on the testis, ovary, kidney, and lung in chronic alcoholism must be investigated. It is hypothesized that cysteine-methionine and vitamin C might neutralize harmful compounds and potentiate the antioxidant capacity of the cell or tissue. In this study, rats were fed regular diets and were maintained in the following groups for 90 days: control group; alcoholic group (2.5 g of 50% ethanol/kg body wt administered intragastrically every other day); and alcoholic with antioxidant supplement group (2.5 g of 50% ethanol plus a solution containing 200 mg vitamin C, 100 mg cysteine, and 100 mg methionine/kg body wt administered intragastrically every other day). After treatment had been completed, rat blood, testis, ovary, kidney, and lung were taken for biochemical analysis. Mean alcohol level in the alcoholic group was raised (by 40%) compared with that in the control group, but it was lower (by 30%) in the antioxidant-supplemented group than in the alcoholic group. In accordance with the levels of alcohol, oxidized protein and lipid content in the testis, ovary, kidney, and lung were low in the control group, higher in the antioxidant-supplemented group, and highest in the alcoholic group. It is interesting to note that levels of glutathione in the testis and lung of the alcoholic group were lower than those in both the control and antioxidant-supplemented groups. In conclusion, chronic alcohol administration led to a significant increase in the level of protein oxidation in the ovary and kidney of rats. Simultaneous intake of ascorbate/L-cys/L-met, along with ethanol, partly attenuated the amount of lipid and protein oxidation that occurred in tissues with oxidative stress caused by alcohol consumption.

Bone resorption starts at 14 days of treatment with gonadotropin-releasing hormone agonist in in vitro fertilization cycles.

The effect of gonadotropin-releasing hormone agonist (GnRH-a) use on bone turnover was investigated in a prospective cohort study of female patients undergoing in vitro fertilization (IVF) treatment. In 46 couples diagnosed with male-factor infertility, the women underwent a long step-down ovulation induction protocol. Urinary cross-linked N-telopeptide (uNTx) level was used to demonstrate bone turnover rate and was measured at the first day of GnRH-a administration, the first day of gonadotropin administration, the day after human chorionic gonadotropin injection and 12 days after embryo transfer. Urinary NTx levels (mean+/-standard deviation (SD)) were 71+/-34, 81+/-40, 81+/-50 and 83+/-47 nmol BCE/mmol creatinine (BCE, bone collagen equivalents), respectively. There was no statistically significant difference between the four measurements (p = 0.28). In 19 women GnRH-a was administered for > or = 14 days. Urinary NTx values of this group and the remaining 27 patients after GnRH-a treatment were 96.2+/-40.7 and 71.5+/-36.8 nmol BCE/mmol creatinine (mean+/-SD), respectively. The difference between these groups was statistically significant (p=0.038). These findings suggest that < 14 days' use of GnRH agonist in IVF patients has no effect on bone metabolism. To our knowledge, this is the first study demonstrating that the effect of agonists on bone metabolism starts as soon as estradiol suppression has started.

Intra- and inter-laboratory variability in the assessment of sperm morphology by strict criteria: impact of semen preparation, staining techniques and manual versus computerized analysis.

We designed prospective studies to compare manual and computerized analysis of sperm morphology by strict criteria using different semen processing and staining techniques. A total of 54 semen samples were studied; slides were prepared from each subject from liquefied semen and after washing, and stained with Diff-Quik or Papanicolaou. An intra-laboratory, blind assessment was performed manually (two observers) and using a computerized analyser (two readings). This demonstrated a very good correlation between manual analysis of liquefied and washed samples with both staining techniques [intraclass coefficient (ICC) = 0.93 and 0.83]. Greater agreement was observed between computerized readings (washed samples) of Diff-Quik (ICC = 0.93) than of Papanicolaou-stained slides (ICC = 0.66). An excellent intra-laboratory correlation was observed for within-computer readings (ICC = 0.93). There was moderate agreement between inter-laboratory computer readings (two centres, ICC = 0.72). Although there was lower inter-laboratory agreement for manual and manual versus computer readings, overall results of all manual and computer analyses showed good agreement (ICC = 0.73). Diff-Quik staining is reliable for both manual (liquefied) and computer (washed) analysis of strict sperm morphology. Intra- and inter-computer analyses using this method reached satisfactory levels of agreement. There is still high inter-laboratory variability for the manual method.

Assessment of interlaboratory and intralaboratory sperm morphology readings with the use of a Hamilton Thorne Research integrated visual optical system semen analyzer.

OBJECTIVE: To evaluate the level of variance produced in a multicenter study with the use of a computer-assisted sperm morphology analyzer. DESIGN: A multicenter, prospective, blinded study. SETTING: Assisted reproduction research laboratories. PATIENT(S): Semen samples produced for assisted reproductive procedures. INTERVENTION(S): Hamilton Thorne Research (Beverly, MA) integrated visual optical system semen analyzers were used at five different centers to evaluate the same set of 30 slides that were prepared and numerically coded at Tygerberg Hospital in Tygerberg, South Africa. MAIN OUTCOME MEASURE(S): The percentage of normal sperm. RESULT(S): Interlaboratory coefficients of variation (CVs) ranged between 16.31% and 23.09%. One of the participating laboratories produced an approximately 14% (-6.5-7.7) limits of agreement analysis, with a CV of 11.36%, for its duplicate readings. The use of a 10% normal sperm morphology cutoff point to determine discordance levels produced rates ranging between 10% and 23.3% for the interlaboratory and intralaboratory readings. This level of discordance equates with < or = 7 of the corresponding readings from two laboratories falling into a different normal sperm morphology group (< or = 10% or >10%). CONCLUSION(S): The magnitudes of variation produced by the readings performed in our study reached the same level as for the manual evaluation of sperm morphology. A < 10% CV can be obtained if the correct quality control measures are implemented.

Direct evidence for the involvement of carbohydrate sequences in human sperm-zona pellucida binding.

Several lines of evidence indicate that mammalian fertilization is initiated via a binding process that is dependent upon the recognition of oligosaccharide sequences associated with zona pellucida (ZP) glycoproteins. Here, specific chemical and enzymatic methods were employed to modify human ZP and to test their effects on sperm binding in the hemizona assay system (HZA). Periodate oxidation of human ZP under very mild conditions (10 min, 0 degrees C, 1 mM sodium m-periodate) that attacks only terminal sialic acid resulted in a 30% loss of human sperm binding in the HZA [hemizona index (HZI) = 70.2 +/- 10.9, n = 22; P < 0.05]. Periodate oxidation under mild conditions (1 h, 23 degrees C, 10 mM sodium m-periodate) caused a 40% decrease in binding (HZI = 60.8 +/- 10.3; n = 24; P< 0.01). Treatment of human ZP with neuraminidase caused a substantial increase in sperm binding to human ZP (HZI = 297 +/- 45, n = 22; P < 0.01). These findings indicate that there are sialic acid dependent binding sites coexisting with binding sites that are obscured by sialic acid. To determine the periodate sensitivity of these obscured sites, hemizona were first digested with neuraminidase and subsequently subjected to mild periodate oxidation. The combined enzymatic and chemical treatments caused a 79% decrease in sperm binding compared to control hemizona (HZI = 20.7 +/- 4.4, n = 16; P < 0.001). Human sperm-ZP interaction was also increased by digestion of human ZP with endo-beta-galactosidase (HZI = 710 +/- 232, n = 14; P < 0.01), indicating that potential binding sites for spermatozoa are also obscured by lactosaminoglycan sequences. These studies support a definitive role for the involvement of ZP-associated glycans in the binding of human spermatozoa to oocytes.

Increase of intracellular calcium is not a cause of pentoxifylline-induced hyperactivated motility or acrosome reaction in human sperm.

OBJECTIVE: To investigate the effects of the phosphodiesterase inhibitor pentoxifylline on hyperactivated motility and acrosome reaction in human sperm and to determine whether its stimulatory effects occur via increased intracellular calcium levels. DESIGN: Prospective study. SETTING: Academic tertiary care facility. PARTICIPANT(S): Healthy male donors. INTERVENTION(S): The effects of pentoxifylline on hyperactivated motility, acrosome reaction, and intracellular calcium were studied and compared with the effects of progesterone. Thapsigargin, a known mobilizer of intracellular calcium, also was used as positive control. MAIN OUTCOME MEASURE(S): Hyperactivated motility was assessed by computer-assisted sperm motion analysis using the HTM-IVOS, acrosome reaction was evaluated with the fluorescent probe fluorescein isothiocyanate-labeled Pisum sativum agglutinin, and intracellular calcium was determined by fura-2 using spectrofluorometry. RESULT(S): Pentoxifylline significantly increased both hyperactivated motility and acrosome reaction. Enhancement of hyperactivated motility by pentoxifylline in the capacitation medium persisted for up to 5 hours after pentoxifylline was washed from the medium. It also enhanced the percentage of acrosome-reacted spermatozoa after 4 hours of incubation. These effects occurred in the presence of a marginally significant decrease in intracellular calcium. CONCLUSION(S): Pentoxifylline stimulates hyperactivated motility and acrosome reaction in spermatozoa from fertile men. Its stimulatory effects occur through mechanism(s) other than increase in intracellular calcium.