Development of mammalian embryos exposed to mixed-size nanoparticles.

Inhaled or ingested ultrafine nanoparticles and their effects on early pregnancy remain polemic. The objectives of the study were: (a) to determine the embryotoxic effects of nanoparticles at the 2-cell stage and (b) to localize the internalized nanoparticles in the blastocyst. Thawed mouse 2-cell embryos (no. = 128) were exposed to either mixed-size polystyrene-based nanoparticles (11 million/ml) or control G1.3 medium and assessed after 72 hours. Additionally, blastocysts (no. = 146) were exposed to nanoparticles and analyzed. The results showed that the nanoparticles did not inhibit 2-cell embryo development to the blastocyst stage (89.4 vs 96.8%; treated vs control). There were no differences in hatching (34.8 vs 43.5%), implantation (13.6 vs 24.2%) and degeneration (10.6 vs 3.2%). Delayed exposure to nanoparticles showed similar percent hatching (40.7 vs 47.3%) and implantation (17.6 vs 20.0%). Although nanoparticles were internalized, embryo development was not inhibited suggesting a lack of embryotoxicity. During hatching, the larger nanoparticles adhered to the extruding blastocyst, preferentially on trophoblasts, but interference was insignificant. Exposure to polystyrene-based nanoparticles at the concentration tested are not associated with embryonic loss.

Gonadotropin levels and secretory patterns in patients with typical and atypical polycystic ovarian disease.

Gonadotropin levels and secretory patterns were studied in 28 oligomenorrheic patients with various types of polycystic ovary disease (PCO). On the basis of ovarian morphology and histology, the patients PCOuld be separated into two distinct categories arbitarily designated "typical" (type I) and "atypical" (type II) PCO. Although no differences were noted in symptomatology or 17-ketosteroid, testosterone, or follicle-stimulating hormone levels, the 12 type I patients demonstrated widely fluctuating, but markedly elevated, luteinizing hormone (LH) levels, while the 16 type II patients demonstrated lower and less fluctuating LH levels which were comparable to those found during the normal follicular phase. It is likely that type I PCO is a distinct entity similar to that described by Stein and Leventhal, while type II co represents a heterogenous spectrum of disorders, many of which remain obscure.

Antibiotics: effect on cryopreserved-thawed human sperm motility in vitro.

OBJECTIVE: To analyze the motility and fertilizing capacity of sperm treated with different antibiotics. DESIGN: Prospective comparative study. SETTING: Clinical and academic research environment. PATIENT(S): Pooled cryopreserved donor sperm (n = 14). INTERVENTION(S): Sperm were washed with Percoll and resuspended in HEPES-buffered human tubal fluid medium containing either amoxicillin, ofloxacin, ciprofloxacin hydrochloride, nitrofurantoin monohydrate, doxycycline hyclate, cefuroxime axetil, or control medium. MAIN OUTCOME MEASURE(S): Sperm kinematic and fertilizing parameters. RESULT(S): Sperm hyperactivation was decreased in physiologic concentrations of ciprofloxacin hydrochloride and doxycycline hyclate over the course of 48 hours. At pharmacologic concentrations, ciprofloxacin hydrochloride, cefuroxime axetil, and nitrofurantoin monohydrate adversely affected motility with decreased rapid progression. Cessation of motility occurred in cefuroxime axetil and nitrofurantoin monohydrate. Sperm hyperactivation was also absent. Cefuroxime axetil decreased the percentage of intact acrosomes. In contrast, physiologic doses of ciprofloxacin hydrochloride or ofloxacin enhanced sperm fertilizing capacity. CONCLUSION(S): Ciprofloxacin affected hyperactivation by altering membrane properties, whereas doxycycline inhibited the capacitation process. Cessation of motility in cefuroxime axetil was linked to disrupted sperm head membranes. Sperm motility and fertilizing capacity were decreased in nitrofurantoin because of decreased metabolism. The positive effect of ofloxacin on fertilizing capacity did not involve changes in acrosome.

Ofloxacin: the next generation of antibiotic in sperm and embryo cultures for assisted reproductive technologies.

OBJECTIVE: To analyze the effect of different concentrations of ofloxacin on sperm kinematic parameters and to determine the embryotoxicity of ofloxacin at physiologic and at 100x concentrations. DESIGN: Prospective comparative study. SETTING: Clinical and academic research environment. PATIENT(S): Pooled cryopreserved donor sperm (n = 7). INTERVENTION(S): Human sperm were processed through two-layer discontinuous Percoll gradients, and the resultant pellet was resuspended in either HEPES-buffered human tubal fluid medium containing different concentrations of ofloxacin or the control medium. After measuring the kinematic parameters, the percentages of apoptosis and viability were obtained. Next, the sperm DNA was extracted and polymerase chain reaction of beta-globin gene was performed followed by denaturing gradient gel electrophoresis. Mouse embryos recovered at the one-cell pronuclear or zygote stages were cultured in the presence or absence of ofloxacin up to the hatched blastocyst stage and differences in development were recorded. MAIN OUTCOME MEASURE(S): Sperm kinematic parameters, sperm beta-globin gene, and number of embryos reaching the hatched blastocyst stage. RESULT(S): The number of embryos exposed to control and physiologic ofloxacin concentrations showed comparable excellent growth. However, the 100x concentration significantly arrested development. Rates of sperm viability and apoptosis measured 48 hours after exposure to the above concentrations were not different from controls. No differences were noted in the sperm kinematic parameters of sperm exposed to ofloxacin concentrations (1x, 10x, and 100x) or control medium after 0, 4, and 48 hours of incubation. Denaturing gradient gel electrophoresis of beta-globin genes from DNA exposed to varying ofloxacin concentrations failed to show any point mutations. CONCLUSION(S): Ofloxacin was embryotoxic at pharmacologic concentrations (100x). At physiologic or higher concentrations, ofloxacin appears to be safe and does not affect sperm kinematic parameters when compared with controls. This may indicate that sperm motility parameters alone cannot be relied on to evaluate the effects of drugs on fertility and that in vitro embryologic studies are essential. Ofloxacin at any concentration did not alter the rates of sperm apoptosis or viability. Ofloxacin does not appear to be mutagenic as evidenced by the beta-globin gene analysis.

A convenient mixed immunobeads screen for antisperm antibodies during routine semen analysis.

OBJECTIVE: To determine if leftover unused IgG, IgA, and IgM immunobeads could be combined as a convenient screen for antisperm antibodies in unwashed sperm. For comparisons, the mixed antiglobulin reaction (SpermMar) and immunobead test were performed. DESIGN: Prospective comparative study. SETTING: Clinical and academic research environment. SUBJECT(S): Donor human sperm negative for antisperm antibodies. INTERVENTION(S): Sperm specimens (n = 9) were equally divided and sera with antisperm antibodies added to one portion (positive control). MAIN OUTCOME MEASURE(S): Assessment of antisperm antibodies in sperm. RESULT(S): The mixed immunobeads screen qualitative results were the same for both the SpermMar and immunobead tests. The mixed immunobeads screen (positive when > or =10% motile sperm was bound) had a sensitivity and specificity of 100%. The percentages of bound sperm for the mixed immunobeads screen correlated with the SpermMar but not the immunobead test. The mixed immunobeads screen intraassay and interassay coefficients of variation were 28.9% and 45.6%, respectively. CONCLUSION(S): Matching results between the mixed immunobeads screen and the SpermMar or immunobead test suggested that the screen could be used to detect antisperm antibodies in unwashed sperm. There was no advantage to washing the sperm, although the screen was flexible and applicable for both unwashed and washed sperm.

On the etiologic role of ureaplasma urealyticum (T-mycoplasma) infection in infertility.

Two hundred consecutive infertility patients and sixty-seven controls subjects were studied for the incidence of infection with Ureaplasma urealyticum (T-mycoplasma). On the basis of a complete infertility investigation, the infertility patients were subdivided into those with explained infertility and those with unexplained infertility. Of the patients with unexplained infertility, 55% had a positive culture for T-mycoplasma as compared with a 32% incidence of positive cultures in the control population. The differences were statistically significant. The 6-month pregnancy rate following successful antibiotic treatment in patients with unexplained infertility was 42%. The 6-month pregnancy rate in a comparable group of patients with unexplained infertility, seen during a 3-year period prior to mycoplasma culture and treatment, was 32%. The difference in pregnancy rates between the two groups was not statistically significant. No correlation was found between a poor postcoital test and the presence of T-mycoplasma infection nor between T-mycoplasma infection and poor cervical mucus. The role of T-mycoplasma infection in infertility was neither proven nor disproven by this study.

An alternative medicine study of herbal effects on the penetration of zona-free hamster oocytes and the integrity of sperm deoxyribonucleic acid.

OBJECTIVE: To analyze the effects of certain herbs on sperm DNA and on the fertilization process. DESIGN: Prospective comparative study. SETTING: Clinical and academic research environment. PATIENT(S): Donor sperm specimens. INTERVENTION(S): Zona-free hamster oocytes were incubated for 1 hour in saw palmetto (Serenoa repens), echinacea purpura, ginkgo biloba, St. John's wort (Hypericum perforatum), or control medium before sperm-oocyte interaction. The DNA of herb-treated sperm was analyzed with denaturing gradient gel electrophoresis. MAIN OUTCOME MEASURE(S): Oocyte penetration and integrity of the sperm BRCAI exon 11 gene. RESULT(S): Pretreatment of oocytes with 0.6 mg/mL of St. John's wort resulted in zero penetration. A lower concentration (0.06 mg/mL) had no effect. High concentrations of echinacea and ginkgo also resulted in reduced oocyte penetration. Exposure of sperm to echinacea purpura and St. John's wort resulted in DNA denaturation. In contrast, saw palmetto and ginkgo had no effect. Sperm exposed to 0.6 mg/mL of St. John's wort showed mutation of the BRCA1 exon 11 gene. CONCLUSION(S): High concentrations of St. John's wort, echinacea, and ginkgo had adverse effects on oocytes. Saw palmetto had no effect. The data suggested that St. John's wort, ginkgo, and echinacea at high concentrations damage reproductive cells. St. John's wort was mutagenic to sperm cells.

Enhanced fertility after heat-induced hyperactivation.

OBJECTIVE: To determine sperm hyperactivation, kinematic parameters, and fertilizing capacity after pretreating sperm at 40 degrees C for 4 hours. DESIGN: Prospective study involving pooled donor sperm that were colloid washed and incubated at either 23 degrees C (control) or 40 degrees C (heat-treated) for 4 hours as pretreatment. After incubation, analyses were performed with a computer-assisted sperm analyzer, whereas separate portions of sperm were evaluated with the sperm penetration assay at 37 degrees C. SETTING: Clinical and academic research environment. PATIENT(S): Cryopreserved-thawed sperm from different donors (n = 5). MAIN OUTCOME MEASURE(S): Sperm kinematic and fertilizing parameters. RESULT(S): Heat pretreatment of sperm resulted in over 22 times higher hyperactive motility at hour 4 compared with the control. The other kinematic parameters were also different. The heat-pretreated sperm group had a significantly higher percent penetration of zona-free oocytes with more swollen sperm heads per oocyte and enhanced sperm binding. CONCLUSION(S): The results showed that hyperactivation was induced by pretreatment of sperm with 40 degrees C heat, suggesting the involvement of heat factors in hyperactivation. The fertilizing capacity of sperm may be improved by the mild heat pretreatment when marked by the presence of heat-induced hyperactivation.

Spermac stain analysis of human sperm acrosomes.

OBJECTIVE: To study the association between low percentages of intact sperm acrosomes and fertilization failures in conventional IVF procedures. DESIGN: A retrospective study. SETTING: Clinical and academic research environment. PATIENT(S): Patients undergoing treatment of infertility. INTERVENTION(S): Sperm cells were fixed and stained using the Spermac stain. MAIN OUTCOME MEASURE(S): Percentages of intact acrosomes and fertilization. RESULT(S): There was a significant association between specimens with <40% intact acrosomes and failed conventional IVF procedures. Among the 29 cases with <40% intact acrosomes, 9 cases (31%) resulted in zero penetration of the oocytes. The mean (+/-SEM) percentage of fertilization was lower in the abnormal acrosome group (43.3% +/- 6.5%) than in the normal acrosome group (64.1% +/- 5.6%). The status of the sperm acrosome was not correlated with the results of fertilization in intracytoplasmic sperm injection procedures. CONCLUSION(S): Sperm with low percentages of intact acrosomes were associated with failed fertilization. The Spermac stain was useful for assessing acrosomes and identifying possible male factor infertility problems. The results suggested that a minimum percentage of sperm with intact acrosomes are needed for fertilization to occur in vitro.

A simple comet assay for archived sperm correlates DNA fragmentation to reduced hyperactivation and penetration of zona-free hamster oocytes.

OBJECTIVE: To correlate sperm variables with sperm DNA fragmentation, as assessed by using a modified alkaline comet assay for sperm smears. DESIGN: The comet assay was adapted for fixed sperm smears (59 cases), and the level of DNA fragmentation was determined. SETTING: Clinical and academic research environment. PATIENT(S): 59 patients undergoing fertility treatment. INTERVENTION(S): Sperm samples leftover from IVF procedures were fixed and processed for the comet assay. MAIN OUTCOME MEASURE(S): Sperm head DNA density and sperm variables. RESULT(S): A correlation was observed between increased sperm head DNA fragmentation and decreased penetration of zona-free hamster oocytes. Heat-induced hyperactive motility decreased as DNA fragmentation increased. The DNA fragmentation did not correlate with percentages of intact acrosome, normality, maturity, and strict normal morphology. CONCLUSION(S): The advantages of the comet assay for archived cells include simplicity, low intraassay coefficient of variation, and low performance cost; in addition, DNA analysis can be carried out at leisure. Low DNA damage was associated with higher hyperactivation and oocyte penetration, suggesting that failed fertilization was linked to compromised DNA integrity in the sperm. Exploration of compounds to repair damaged DNA is warranted.

Pulmonary dysfunction in patients with femoral shaft fracture treated with intramedullary nailing.

BACKGROUND: This study was undertaken to determine whether alveolar dead space increases during intramedullary nailing of femoral shaft fractures and whether alveolar dead space predicts postoperative pulmonary dysfunction in patients undergoing intramedullary nailing of a femoral shaft fracture. METHODS: All patients with a femoral shaft fracture were prospectively enrolled in the study unless there was evidence of acute myocardial infarction, shock, or heart failure. Arterial blood gases were measured at three consecutive time-periods after induction of general anesthesia: before intramedullary nailing and ten and thirty minutes after intramedullary nailing. The end-tidal carbon-dioxide level, minute ventilation, positive end-expiratory pressure, and percent of inspired and expired inhalation agent were recorded simultaneously with the blood-gas measurement. Postoperatively, all subjects were monitored for evidence of pulmonary dysfunction, defined as the need for mechanical ventilation or supplemental oxygen (at a fraction of inspired oxygen of >40%) in the presence of clinical signs of a respiratory rate of >20 breaths/min or the use of accessory muscles of respiration. RESULTS: Seventy-four patients with a total of eighty femoral shaft fractures completed the study. Fifty fractures (62.5%) underwent nailing after reaming, and thirty fractures (37.5%) underwent nailing with minimal or no reaming. The mean alveolar dead-space measurements before canal opening and at ten and thirty minutes after canal opening were 14.5%, 15.8%, and 15.2% in the total series of seventy-four patients (general linear model, p = 0.2) and 20.5%, 22.7%, and 24.2% in the twenty patients with postoperative pulmonary dysfunction (general linear model, p = 0.05). Of the twenty-one patients with an alveolar dead-space measurement of >20% thirty minutes after nailing, sixteen had postoperative pulmonary dysfunction. According to univariate and multivariate analysis, the alveolar dead-space measurement was strongly associated with postoperative pulmonary dysfunction. CONCLUSIONS: According to our data, intramedullary nailing of femoral shaft fractures did not significantly increase alveolar dead space, and the amount of alveolar dead space can predict which patients will have pulmonary dysfunction postoperatively.

Periarticular heterotopic ossification after multiple knee ligament reconstructions. A report of three cases.

Heterotopic ossification is a frequently encountered clinical and radiographic entity. There are no previous reports in the English literature of heterotopic ossification after arthroscopically assisted ligament reconstructions for knee dislocations. Further, a link between the PCL reconstruction and posterior capsular ossification has not been heretofore recognized. Our three cases should raise the clinical awareness of such an entity.

Biceps tendinitis and subluxation.

Since the 17th century, the long head of the biceps tendon as a source of shoulder pain and its functional significance has been a source of debate. Although the term tendinitis is commonly used, overuse tendon injuries infrequently demonstrate inflammatory cells; instead, degenerative changes resulting from the failure of self-repair usually are found. Bicipital tendinitis or bicipital tenosynovitis is most often secondary to impingement beneath the coracoacromical arch. Primary bicipital tendinitis and tendinitis secondary to instability are possible, however. Through a careful history, physical examination, and appropriate imaging studies, the clinician can establish the diagnosis of disorders of the biceps tendon Arthroscopic evaluation greatly improves the diagnosis and treatment of biceps tendon and related shoulder pathology. Although the exact functional role of the biceps tendon remains incompletely defined, a growing body of evidence supports its role as a stabilizer of the glenohumeral joint. This stabilizing function should be incorporated into the treatment of biceps tendon disorders. Routine tenodesis has been replaced by a more individualized approach, taking into consideration physiologic age, activity level, expectations, and exact shoulder pathology present. New repair techniques are under development, and preservation of the biceps-labral complex is now preferred when possible.

A follow-up expanded study of the correlation of sperm velocity in seminal plasma and offspring gender.

A preliminary study reported finding higher sperm velocity in seminal plasma in males of partners that conceived female offsprings. The null hypothesis was that sperm velocity was not related to the offspring gender. The objectives were: (a) to expand the previous study, and (b) to correlate offspring gender results with motility parameters determined through the computer-aided sperm analyzer (CASA) system. In combined fresh and frozen cycles (N = 187), sperm from cases with all female offsprings displayed higher curvilinear (48 +/- 1.0 mu/sec versus male 46 +/- 1.0, P < 0.05) and average path velocities (36 +/- 0.7 mu/sec versus male 34 +/- 0.7, P < 0.01). A criteria of less than 30 mu/sec or over 41 mu/sec average path velocity predicted 73 or 72% of the male or female offspring cases, respectively. A curvilinear velocity of less than 49 mu/sec or over 55 mu/sec predicted 58 or 59 % of the male or female offspring cases, respectively. Semen viscosity reflected in sperm velocity was linked to predominantly male or female sperm populations. Paracrine signals from the gender-skewed sperm precursor populations controlling viscosity merit further exploration.

Prevalence of negative-pressure pulmonary edema at an orthopaedic hospital.

Negative-pressure pulmonary edema (NPPE) occurs when a large, negative intrathoracic pressure is generated against an obstructed upper airway, causing fluid to shift into the lung interstitium. Young, healthy, athletic male patients appear to be at increased risk for this disorder, but the prevalence in orthopaedic surgery patients has been unknown. We retrospectively reviewed the charts of 14 patients (11 male, 3 female) with NPPE at our institution over a 15-year period. The patients had 11 different surgical procedures; 16,653 similar procedures were done during this time. The overall prevalence of NPPE (< 0.1%) was not significantly different between male and female patients. Patients with NPPE were significantly younger than those without NPPE. If NPPE is recognized promptly and treated appropriately with intravenous diuretic and oxygen therapy, most patients respond well. Physicians should be vigilant to the potential for NPPE in young, otherwise healthy patients after general anesthesia.

Modulation of pituitary responses to synthetic LH-RH by gonadal steroids in women with secondary amenorrhoea.

LH-RH stimulation tests were performed on two successive days in 25 women with secondary amenorrhoea of probable hypothalamic origin by intravenous injection of 150 mug of synthetic LH-RH. The patients were selected by exclusion of definite pituitary or ovarian disease and in the absence of clinical or laboratory evidence of androgen excess. Five women received successive LH-RH stimulation tests only without administration of steroid hormone. Twenty women were treated with gonadal steroids in addition to the double LH-RH stimulation test by intramuscular injection of the steroid 4 h after the first LH-RH injection. Of these 20, 9 received oestradiol (Oe2), 5 received progesterone (P) and 6 received Oe2 + P. Gonadotrophin responses to LH-RH in successive tests without steroid were not significantly different. Oe2 or P alone each produced suppression of LH and FSH responses to LH-RH in the second test as compared to the first. Combined Oe2 + P produced augmentation or suppression of the second gonadotrophin responses depending on the dose of Oe2 administered. The results demonstrate a direct effect of oestrogen or progesterone alone and in combination on the pituitary responsiveness to LH-RH. The effect of the combined steroids can be modified by variation in the oestrogen: progesterone ratio.

Oocyte comet assay of luteal phase sera from nonpregnant patients after assisted reproductive procedures.

PURPOSE: Serum factors in patients with recurrent spontaneous abortions (RSA) inhibit mouse embryo development in vitro. Serum factors affecting DNA integrity remain to be tested. The null hypothesis was that patient sera do not affect DNA integrity. The objectives were (a) to use the oocyte comet assay to assess DNA damage after exposure to patient sera and (b) to determine the effect of sera from gravidity 0 parity 0 patients to induce DNA apoptosis. METHODS: Luteal phase sera were drawn 1 week after embryo transfer following assisted reproductive procedures. Frozen-thawed hamster zona intact oocytes at metaphase II were incubated in groups of eight in either control medium or medium supplemented with 50% patient serum for 1.5 h at 37 degrees C in room air. The oocytes were fixed, stained in acridine orange, embedded in agarose, lysed, and alkaline electrophoresis performed. The intensities of the digitized fluorescent images were analyzed. RESULTS: The sera of nonpregnant patients (64%) caused significant fragmentation of hamster oocyte DNA when compared with pregnant patient sera. This difference was also observed when adjusted for patient age. Sera of patients that had never been pregnant also resulted in oocyte DNA fragmentation. CONCLUSIONS: The results suggested that sera from patients that did not conceive contained factors that did not support cell growth by causing DNA fragmentation and apoptosis. The level of the apoptotic factors varied from cycle to cycle. However, more studies are needed to determine if the sera factors actually reach the uterine environment to cause the undesirable effects.

Correlation between intact sperm acrosome assessed using the Spermac stain and sperm fertilizing capacity.

Accurate determination of sperm acrosomal status is important in fertility studies. The objective was to correlate the percentage of intact acrosome assessed using the new Spermac stain with the capacity of sperm to fertilize oocytes. Sperm specimens were processed either by centrifuge wash, 48:95 Percoll gradient or test yolk buffer (TYB) wash, and tested using the zona-free hamster oocyte assay. The results indicated a correlation between the percentage of sperm with intact acrosome reaction and the percentage of sperm penetrating the oocytes in the TYB-washed group. The data suggest the usefulness of the Spermac stain for assessing the acrosomal status and in predicting the fertilizing capacity of the sperm.