Sperm membrane integrity and stability after selection of cryopreserved ovine semen on colloidal solutions.

The aim of this study was to evaluate the effect of four methods of sperm selection, on the integrity and stability of the plasma membrane, integrity of the acrosomal membrane and spermatic morphology in frozen/thawed ovine semen. Two types of colloidal silica: colloidal silica-silane and colloidal silica-polyvinylpyrrolidone (PVP), and two aliquots: 1 and 4 ml, were used for sperm selection. Probes FITC-PSA and PI were used to measure the integrity of the plasma and acrosomal membranes. Plasma membrane stability was measured, using fluorescent probes M540 and YOPRO1. Effective reduction in the incidence of spermatozoa with acrosomal pathologies was only achieved using 1 ml colloidal silica-silane. All methods were efficient in select viable and unreacted spermatozoa. Only methods using 1 ml of silica were efficient in decrease spermatozoa stained by PI (death). Methods using silica colloidal-silane were more efficient to decrease apoptotic cells after selection when compared to silica colloidal-PVP. In conclusion, sperm selection in colloidal silica-silane and colloidal silica-PVP improved sperm quality when compared to the controls. The method using 1 ml of colloidal silica-silane is the preferred method because its effectiveness and lower cost.

Detection and isolation of Toxoplasma gondii from fresh semen of naturally infected dogs in Southern Brazil.

The aim of this study was to isolate Toxoplasma gondii and determine the viability of the parasite in fresh semen samples of clinically healthy adult dogs naturally infected. Eleven seropositive dogs with T. gondii IgG antibodies from southern Brazil were selected to confirm the presence and viability of T. gondii in fresh semen samples using in vitro isolation in Vero cell culture, polymerase chain reaction (PCR) and sequencing analysis. The presence of viable T. gondii was confirmed by in vitro isolation and PCR in five semen samples. The ITS1 region of the isolated protozoa (TG S4) was amplified and sequenced. The nucleotide sequence obtained was 99% compatible with the T. gondii DNA sequences stored in the GenBank. It has been shown that T. gondii tachyzoites may be isolated in vitro from fresh semen samples of clinically healthy dogs seropositive for T. gondii.

Radar detection of cloud-seeding effects.

The effects on precipitation of artificially seeding clouds with Dry Ice have been monitored from cloud to ground with a radar that has a wavelength of 8.6 millimeters.

Evaluation of stallion sperm motility with ImageJ using a cell phone camera and a light microscope / [Avaliação da motilidade espermática de garanhões com ImageJ usando-se câmera de celular e microscópio óptico]

This study aimed to determine the accuracy of assessing stallion sperm motility using a light microscope, a cell phone camera, and a free computer-assisted semen analysis (FCASA) package for ImageJ. The total motility of frozen (n=22) and cooled (n=48) equine semen was determined by FCASA and compared to the results of subjective visual analysis (SVA) by two technicians. Frozen samples were also evaluated by a commercial computer-assisted semen analysis (CCASA) system. The Friedman test revealed no significant differences (P>0.05) between cooled samples analyzed by FCASA (38.0) and SVA (technician 1, 40.0; technician 2, 40.0), nor between frozen samples analyzed by FCASA (23.36 ± 15.9), SVA (25.5 ± 18.8 and 25.8 ± 18.5), and CCASA (25.2 ± 18.3). However, mean FCASA results were underestimated by 7.2% compared with CCASA. The correlation between FCASA and CCASA was significant and strong (P<0.0001, r=0.95). Chi-squared tests indicated that FCASA provided similar results (P=0.14) to the reference method (CCASA), but SVA had lower accuracy (P=0.04). ImageJ analysis of cell phone videos captured under a light microscope can be used for estimation of stallion sperm motility with comparable accuracy to commercial systems.(AU)

O objetivo deste estudo foi testar as configurações necessárias para avaliar a motilidade espermática total de garanhões, mediante o uso de ImageJ, microscópio óptico e câmera de celular. Os valores de motilidade total das amostras de sêmen equino congeladas (22) e refrigeradas (48) foram comparados por análise visual (SVA) e pelo plugin do ImageJ (CASAF). Amostras congeladas também foram comparadas por um CASA comercial (CCASA). O teste de Friedman não resultou em diferença estatística (P>0,05) entre as 48 amostras analisadas com CASAF (38,0) e SVA de dois avaliadores (40,0 e 40,0). A comparação das 22 amostras congeladas entre CASAF (23,36±15,9), SVA (25,5±18,8 e 25,8±18,5) e CCASA (25,2±18,3) também não resultou em diferença estatística, sendo que a média dos resultados obtidos com CASAF subestimou a obtida com o CCASA em 7,2%. A correlação entre CASAF e CCASA foi significativamente elevada (r=0,95, P<0,0001). O teste de qui-quadrado resultou em proporção de acertos semelhantes entre o CASAF e o CCASA (P=0,14), enquanto SVA resultou em proporção diferente (P=0,04), indicando menor acurácia. O uso de microscópio óptico e câmera de celular foi útil para obter vídeos de sêmen de garanhões a serem analisados com ImageJ, proporcionando resultados de motilidade total equiparáveis a sistemas comerciais.(AU)

Human dermal endothelial cells express membrane-associated mast cell growth factor.

Mast cell growth factor (MGF), a molecule that serves as a ligand for the receptor tyrosine kinase c-kit, is important in mast cell differentiation, migration, and activation. Previous studies of paraffin-embedded human skin using antibody to murine MGF and reverse transcription-polymerase chain reaction have demonstrated MGF protein and mRNA expression in keratinocytes and isolated dermal cells. We utilized a monoclonal antibody to human MGF to further define patterns of immunoreactivity in frozen specimens of neonatal and adult skin from normal individuals and from patients with urticaria pigmentosa. In addition to keratinocytes and isolated dermal cells in normal and urticaria pigmentosa skin, MGF was detected in cells lining superficial and mid-dermal vessels. Co-expression of MGF and the vascular antigen CD31, and immunoelectron microscopy, identified MGF-positive cells as endothelial cells. Patterns of endothelial MGF expression were not influenced by mast cell degranulation and endothelial E-selectin induction in vitro. By ultrastructure, unfixed specimens demonstrated MGF expression both within the endothelial cytoplasm and in association with lumenal, but not ablumenal, surfaces. Specimens fixed with Nakane's solution had diminished endothelial cytoplasmic MGF reactivity, but lumenal expression was maintained, suggesting persistence of a membrane-associated reactivity. MGF mRNA was also detected in cultured dermal microvascular endothelial cells using reverse transcription-polymerase chain reaction. These data establish human dermal endothelial cells as sites of MGF production and expression in human skin. Mast cell precursors must home to skin via vascular channels and differentiate in the immediate perivascular space. Thus, endothelial MGF may be an important determinant of adhesion and differentiation of mast cell progenitors expressing receptors for MGF.

Prenatal serum alpha-fetoprotein screening for neural tube defects.

Twenty-two cases of open neural tube defect were found in a population of 17,703 unselected pregnancies (1.2 per 1000) within the Long Island, New York, region. Voluntary screening of maternal serum alpha-fetoprotein levels identified 20 of the 22 cases (91%). Six hundred ninety-two participants demonstrated serial elevations in maternal serum alpha-fetoprotein. Of this group, which was designated at increased risk for open neural tube defect, 24% had underestimated gestational age, 13% had multiple gestations, and 53% were candidates for amniocentesis. In the amniocentesis group, the detection yield of neural tube defect was 20 per 365 (5.5%). Neither false-negative amniotic fluid evaluations nor termination of normal pregnancy due to false-positive amniotic fluid levels occurred. Perinatal outcome data, including pregnancy complications, date, mode of delivery, sex, birth weight, Apgar score, and congenital malformations of the neonate other than neural tube defect, are reported for the first 9300 consecutive participants of the 17,703 population study. These data identify a correlation between rate of perinatal loss and maternal serum alpha-fetoprotein levels.

Antenatal diagnosis and management of the small-for-gestational-age fetus.

The small-for-gestational-age fetus continues to account for a large proportion of fetal demise in late pregnancy. Delivery at 37 to 38 weeks' gestation to preempt this is our recommended mode of management. Prenatal diagnosis was made in only 33% of cases in this study. The course of 101 growth-retarded pregnancies has been critically evaluated towards improving the rate of prenatal diagnosis.

Amniography in the prenatal diagnosis of neural tube defects.

Amniography for the visualization and confirmation of suspected neural tube defect was performed in 9 midtrimester gravidas. In all cases, amniotic fluid alpha-feto protein (AFP) was abnormally elevated. Four cases of anencephaly and one of spina bifida were demonstrated by amniography. These pregnancies were terminated and the defects were confirmed by gross pathologic examination. In 4 remaining cases, amniography was normal. Three of these pregnancies proceeded to term, culminating in the birth of a normal child. The fourth patient had spontaneous abortion of a normal fetus at 23 weeks of gestation. The experience reported here suggests that amniography is an important adjunctive diagnostic technique in the prenatal diagnosis of neural tube defect, and if used correctly, may significantly reduce the chance of false-positive diagnosis.

Maternal serum and amniotic fluid alpha-fetoprotein as a marker of acute fetal distress in a midtrimester abortion model.

Maternal serum (MS) and amniotic fluid (AF) alpha-fetoprotein (alpha-FP) levels were serially determined in 18 cases of elective midtrimester abortion. Prostaglandin F2alpha (PGF2alpha) and 20% NaCl were used as the abortifacients in 2 groups of 9 patients, respectively. The time from instillation to abortion (IAT) was accurately recorded in all cases. A marked 260-600% increase in MS alpha-FP occurred prior to fetal demise in both groups. Amniotic fluid alpha-FP content remained largely unchanged for the first 6 hours following intraamniotic prostaglandin injection. A 50% INCREASE WAS OBSERVED IN THE AF alpha-FP content in the group with 20% NaCl-induced abortion (after an initial dilutionary drop). The results of this investigation confirm the value of alpha-FP in MS as a marker of impending fetal demise. This rise is not caused by a prior alpha-FP change in the AF. The data suggest a major fetomaternal transplacental route for alpha-FP.

Origin of amniotic fluid alpha-fetoprotein in normal and defective pregnancies.

Alpha-fetoprotein was determined by electroimmunodiffusion and radioimmunoassay in 109 neonatal urine samples and 94 amniotic fluid samples. The samples were obtained from newborns and from pregnancies ranging in gestational age from 20 to 40 weeks. When alpha-fetoprotein values of neonatal urine and amniotic fluid were correspondingly correlated with gestational age, almost identical declining curves could be drawn. Twenty-one cerebrospinal fluid samples from newborns ranging from 25 to 40 weeks of gestation were similarly determined. No correlation between cerebrospinal fluid alpha-fetoprotein and gestational age could be demonstrated. It is concluded that fetal urine is the major source of alpha-fetoprotein in the amniotic fluid of normal pregnancy. In pregnancies associated with neural tube defects, alpha-fetoprotein elevation is probably not due to the leakage of cerebrospinal fluid into the amniotic cavity.

Correlation of fetal heart rate-uterine contraction patterns with fetal scalp blood pH.

The significance of fetal heart rate-uterine contraction (FHR-UC) monitoring as a means of predicting the condition of the fetus during labor was studied by correlating 460 fetal pH determinations obtained from 216 patients with the analysis of the 20-minute FHR-UC record preceding the fetal scalp blood sample. Both qualitative and quantitative analyses of the FHR-UC record were performed. The results indicate a less than 10% chance of fetal pH less than or equal to 7.250 with an normal baseline FHR and no periodic changes or with periodic accelerations, early decelerations, and uncomplicated baseline bradycardia or tachycardia. Variable or delayed decelerations with a total surface area (TSA) of 1-100 for the 20-min period were associated with a fetal pH less than or equal to 7.250 in 23 and 34% of cases, respectively. Variable or delayed decelerations with a TSA greater than 100 had a 35 or 47% chance of fetal pH less than or equall to 7.250. It is concluded that FHR-UC monitoring can be used to screen the innocuous from the ominous periodic change but that fetal scalp blood pH must be obtained to identify accurately the true from the false-positive ominous pattern.

Correlation of the oxytocin challenge test with perinatal outcome.

A total of 234 oxytocin challenge tests (OCT) were performed on 100 high-risk patients. The results were negative (N) in 68 of these 100 patients, suspicious (S) in 22, and positive (P) in 10. The incidence of late decelerations during labor was N, 5%; S, 40%; P, 86%; and meconium staining of the amniotic fluid was N, 4%; S, 5%; and P, 30%. The cesarean section rate was N, 16%; S, 36%; and P, 60%; and of these the cesarean section rate for fetal indications was N, 9%; S, 25%; and P, 67%. The overall perinatal mortality in the study group was 2% (N, 1.5%; S, 0%; P, 10%). The results confirm the negative OCT as innocuous and positive OCT as the most ominous. They also indicate that the majority of patients with positive OCT can be delivered vaginally without endangering the fetus if fetal scalp blood pH determinations can be performed.

Sinusoidal fetal heart rate patterns in severe Rh disease.

Four patients with severe Rh isoimmunization are presented in whom sinusoidal fetal heart rate patterns were recorded. In 2 cases in which the pattern was seen intermittently in the postintrauterine transfusion period, the perinatal outcome was good. In the 2 cases in which the pattern was seen spontaneously and continuously, the outcome was poor, confirming the ominous and possibly agonal significance of the latter pattern.

Screening for SGA by the roll-over test.

The roll-over test (ROT) was evaluated in normal pregnant patients between 28 and 34 weeks' gestation and found to be an accurate method of screening for small-for-gestational age (SGA) infants and for preeclampsia. Patients with a positive ROT had a significant risk of SGA or preeclampsia or both. In patients with a positive ROT, the development of SGA may be further predicted by an examination of maternal prepregnancy weight and weight gain. Patients with a prepregnancy weight of 50 kg or less, total weight gain of 10 kg or less, and/or differential weight gain of 20% or less have a highly significant risk of developing an SGA fetus. In contrast, patients with a negative ROT have a significantly decreased risk for SGA and/or preeclampsia.

Saline instillation with oxytocin administration in midtrimester abortion.

PIP: Oxytocin was infused at 10 units per hour in 100 ml Ringers lactate after instilling saline for 108 midtrimester abortions in 107 patients. Amniocentesis was performed in the treatment room with drapes, cap, mask, gloves, local anesthetic, but without pelvic examination. Strict care was taken that clear amniotic fluid flowed before 20% saline was injected. The patients averaged 23 years old, 2.05 parity and 17.6 weeks since their last menstral period, but the 41 multiparas were significantly older than the 66 primiparas. In 7 amniocenteses more than one puncture was needed. The mean volume of saline instilled was 185 ml over a 10 minute interval. Although time before oxytocin and labor did not differ significantly, the mean length of labor was significantly shorter in multiparas (6.7 hours) than in primiparas (10.5). Thus the total mean time to abortion was 19 hours for multiparas compared with 26 hours for primiparas (p less than .0005). Frequency of incomplete abortion (34%) needing curettage (4.6%) rather than manual or sponge stick manipulation was high. Complications included 1 failure to abort, 20 instances of tachycardia during amniocentesis, 1 fever, and 1 grand mal seizure probably due to water intoxication.^ieng

Interlocking twins. Experience with four cases and suggested management.

Experience with the management of 4 pairs of locked twins is described and analyzed. In view of the high morbidity figures a proposed preemptive management is suggested.

Amniotic fluid alpha-fetoprotein as a marker in prenatal diagnosis of neural tube defects.

The prenatal diagnosis of anencephaly and spina bifida (neural tube defect, NTD) through amniotic fluid analysis for alpha-fetoprotein (AFP) is gradually gaining clinical recognition. AFP concentrations were determined in 237 amniotic fluids from normal pregnancies ranging between 7 and 42 weeks of gestation. A steady decline in AFP from 26 mug/ml at 7-9 weeks to 155 ng/ml at term is observed. AFP concentration was determined in 35 amniotic fluids from 33 confirmed neural tube defective pregnancies. In 14 cases where amniotic fluid was examined prior to the 26th week of gestation. AFP was markedly elevated when compared with the normal range of the same gestational period. In 21 amniotic fluids past the 26th week, 17 cases (85-) had markedly elevated AFP levels; however, 2 cases of anencephaly, 1 of spina bifida, and 1 of hydrocephaly gave levels within the normal range. It is concluded that elevated AFP in the amniotic fluid is a reliable but nonspecific marker for open neural tube defects prior to the 26th week of pregnancy, but may become normal after the 26th week in a small percentage of patients.

PIP: A steady decline in alpha fetoprotein (AFP) levels was observed in single specimens of amniotic fluid (AF) from 237 patients, ranging from 26 mcg/ml at 7-9 weeks to 155 ng/ml at term. All pregnancies tested were normal. 35 AF specimens from 33 confirmed neural tube defective pregnancies were assayed for AFP. Very high levels of AFP were found in 13/14 fluids examined before Week 26 of gestation. A value of 23 mcg/ml was determined in 1 sample where the infant had skin-covered encephalocele. A fluid taken from the same patient at 34 weeks fell to 6.4 mcg of AFP. 21 AF samples from patients past the 26th week of pregnancy were analyzed. Of these, 1 case of spina bifida and 2 of anecephaly gave no detectable levels of AFP by electroimmunodiffusion. By radioimmunoassay, however, these samples measured 3700, 256, and 700 ng/ml. 1 case of hydroencephaly, examined at 33 weeks, had an AFP level of 1.5 mcg/ml. A sharp drop in AFP from 353.6 at 15 weeks to 10.4 mcg/ml at 29 weeks was noted in the only serially examined open neural tube defective pregnancy.

Priming of the uterine cervix with oral prostaglandin E2 in the term multigravida.

A double-blind study evaluating two dose schedules of oral prostaglandin E2 (PGE2) was conducted in 56 normal "unripe" multiparas at term. The effect of PGE2 was evaluated by comparing the change in Bishop score of the cervix and patient's response to oxytocin induction of labor after the prostaglandin priming. In the two study groups (one group on a fixed PGE2 dosage and the other on a variable drug regimen) an average change of more than three points in the Bishop score was observed. In control group no significant change in cervical ripeness was observed. Nine of the 32 patients (28%) receiving prostaglandin delivered during the priming phase of the study, whereas only 1 control patient (4.2%) delivered during priming. Six of 24 control patients (25%) failed to deliver, wheras only 3 of 32 study patients (9%) failed. In the successfully induced patients, the response to oxytocin, as expressed by the time to active phase, total time of labor, and mean dose of oxytocin required, did not vary significantly when control and study groups were compared. It is concluded that PGE2 at either dosage schedule is effective in changing the Bishop score of the cervix and that it is not feasible to titrate the PGE2 dose in order to effectively ripen the cervix without a very high incidence of induction of labor. In those cases where priming was completed, without accidental delivery, the induction phase was successful more than 90% of the time. No adverse effect to mother or baby was observed during this study.

Early prenatal diagnosis of soft-tissue malformations.

This study presents 3 cases of severe soft-tissue malformations of the fetus. Prenatal diagnosis was established early in the second trimester, and the accuracy of the diagnosis was confirmed by pathological examination in each instance. A clinically oriented work-up plan for early prenatal diagnosis is proposed.

Modern management of hypertensive disorders of pregnancy.

Hypertensive disorders of pregnancy contribute significantly to perinatal mortality. Successful application of modern tests of antepartum fetal status and appropriately timed delivery, with monitored labor and liberal use of cesarean section led to significantly improved fetal and neonatal salvage in 242 patients with hypertensive disorders of pregnancy between 1973 and 1975. The reduction in perinatal mortality to a corrected rate of 12/1000 in the years 1973-1975, compared to a previous rate of 75/1000 between 1970 and 1972, justifies the modern aggressive management of hypertensive disorders in pregnancy.