Determination of bovine fetal sex by PCR using fetal fluid aspirated by transvaginal ultrasound-guided amniocentesis

Fetal sex can be determined by the polymerase chain reaction (PCR) using cells from fetal fluid collected by transvaginal ultrasound-guided amniocentesis. A total of 35 aspirates from 30 cows, 15 Holsteins and 15 Japanese Blacks at 59 to 250 d of pregnancy were used. Five cows were aspirated twice at a 10-d interval. A 5.0 MHz convex array transducer connected to a scanner was inserted into the vagina under caudal epidural anesthesia. The transducer was equipped with a 65-cm long, 21-g needle within the probe carrier. A bovine male-specific primer and a bovine gender-neutral primer were used. Fetal fluid was obtained from all except 2 cows in early pregnancy. Five animals aborted within 1 wk following aspiration. A total of 33 samples, 29 of amniotic fluid and 4 of allantoic fluid, was subjected to PCR analysis. Fetal gender was verified in 31 33 samples (18 females and 13 males). Gender was also determined by gross examination of external genitalia of offspring after calving or abortion. Fetal gender was correctly identified by PCR analysis of aspirated fetal fluid in 16 16 females and in 13 15 males. Transvaginal ultrasound-guided amniocentesis followed by PCR analysis of aspirated cell DNA can be used accurately to determine fetal sex in cows at 70 to 100 d of gestation. The procedure requires considerable skill and is not without some risk to fetal viability.     

Nonaspiration fine needle cytology. Application of a new technique to nodular thyroid disease

The basic principle underlying fine needle aspiration (FNA) is the aspiration of cellular material from target masses, often utilizing fairly high suction pressures. The procedure requires a needle and a syringe, advisedly held in a syringe holder, enabling single-handed suction to be exercised. Mastery of the technique is variable, with few operators acquiring consistent skill. A new technique, pioneered in France but essentially unpublicized, eliminates active aspiration, replacing it by the principle of capillary suction of fluid or semifluid material into a thin channel (a fine needle). This nonaspiration sampling method was tested in a consecutive series of 50 solid thyroid nodules. Simultaneously performed conventional FNAs served as controls. Cell samples were cytologically assessed as unsuitable, diagnostic/adequate or diagnostic/superior, without knowledge of the sampling method employed. Diagnostically superior specimens were obtained significantly more frequently by the nonaspiration technique in 36 benign lesions and 13 neoplasms. The method of nonaspiration fine needle cytology ("cytopuncture") is described and illustrated, and the implications for its use in other sites are discussed.     

Equine fetal kinetics: Allantoic-fluid shifts and uterine-horn closures

Uterine filling by the conceptus and uterine constrictions and allantoic-fluid shifts after filling were studied by daily ultrasound examinations on Days 40 to 74 in 10 mares. Allantoic fluid was first detected at the extremities of the uterus on the following days: 1) tip of umbilical-cord horn, Day 60.0 +/-1.9 (mean +/-SEM); 2) tip of noncord horn, Day 64.8 +/-1.3; and 3) near cervix, Day 48.5 +/-1.3. Transient uterine constrictions with expulsion of allantoic fluid were detected in 37% of 271 examinations. Other end points were studied by weekly ultrasound examinations from Day 40 to term. Height of cross-sections of the fetal body increased (P<0.01) progressively over Monthes 2 to 8, whereas the height of placental fluid minus fetal body decreased (P<0.05) after Month 3. An increasing (P<0.05) frequency of examinations with a reclosed uterine horn occurred between Months 3 and 4. The frequency of reclosed noncord horns continued to increase (P<0.05) after Month 4 and reached 96% at Month 7. The frequency of reclosure of cord horns plateaued (no significant differences) at 54% over Months 4 to 7 and then rapidly decreased (P<0.05). Both uterine horns were closed during the examination that preceded the permanent entry of the fetal hind limbs into the cord horn in all of 9 mares in which this temporal relationship was studied. After entry of the fetal hind limbs, the cord horn remained constricted upon the limbs until at least Month 10. The results provide rationale for the following hypotheses: 1) The gradual decrease in fetal mobility (location and presentation changes) previously reported for Months 4 to 8 is associated with growth of the fetus and a concomitant decrease in volume of allantoic fluid and number of open uterine segments; 2) Closure of both uterine horns near mid-pregnancy plays a role in final selection of cranial fetal presentation followed by entry of the fetal hind limbs into the cord horn.     

Choriocarcinoma metastatic to the lung. A cytologic study with identification of human choriogonadotropin with an immunoperoxidase technique

A case of choriocarcinoma metastatic to the lung following a previous hydatidiform mole is presented. It was possible to make definitive identification of trophoblastic elements on a needle aspiration biopsy using an immunoperoxidase staining technique, thus avoiding diagnostic thoracotomy prior to therapeutic intervention. A method of immunoperoxidase staining of previously fixed and Papanicolaou-stained needle aspiration biopsy specimens is also described, and other uses of the immunoperoxidase technique on needle biopsy specimens are discussed.     

Cytologic findings in a fetal intracranial teratoma. A case report

BACKGROUND: Fetal neoplasms are very rare. Recently we had the opportunity to examine the fine needle aspiration (FNA) biopsy of a fetal intracranial teratoma. CASE: The tumor was found in a 30-week-gestation fetus; the mother was 32 years old, gravida 4, para 1. She presented with a rapid increase in abdominal girth over a two-week period. An ultrasound scan showed severe fetal hydrocephalus and a massive intracranial tumor thought to be a teratoma because of variations in echogenicity and spotty calcification. An FNA biopsy was performed under ultrasound guidance. It showed mainly neuroepithelial cells, so a differential diagnosis of malignant neuroepithelial tumor was considered. At autopsy, several other tissue types were found in the tumor, consistent with a teratoma. CONCLUSION: Advances in diagnosis of fetal anomalies by ultrasound have been associated with an increase in the use of fetal interventions performed in utero. This includes the availability of fetal surgery in some centers. FNA biopsy of fetal lesions does not appear to be well described. Increased experience with this technique is necessary if its full potential is to be realized.     

Effect of delayed supplementation of fetal calf serum to culture medium on bovine embryo development in vitro and following transfer

Supplementation of synthetic oviduct fluid (SOF) medium plus amino acids and bovine serum albumin (BSA) with either fetal calf serum (FCS) or charcoal-treated FCS (CT-FCS) from Day 5 of development was investigated to determine if either in vitro or post-transfer development was altered. Development to the compact morula stage or beyond was similar for all 3 treatments. However, blastocyst development at Day 7 was accelerated when serum was added to the medium (21.6, 40.1 and 39.4% blastocysts from cleaved embryos for BSA, FCS and CT-FCS, respectively; P < 0.01), but cell number of the resulting embryos was unaffected. Furthermore, addition of CT-FCS decreased the between replicate variation in embryo development and produced more Grade 1 and 2 quality embryos (25.8%) than BSA supplementation (18.1%; P < 0.05). The transfer of Grade 1 and 2 embryos at Day 7 following culture resulted in similar pregnancy and embryo survival rates for the 3 treatments, with a tendency for lower embryo survival of embryos cultured in FCS (embryo survival at Day 50 = 37.7% vs 53.3% and 57.6% for FCS, BSA and CT-FCS, respectively; P = 0.1). Significant fetal loss from Day 50 to term occurred within all 3 treatments. There were no birth weight differences for calves amongst the 3 culture treatments; however, one of the sires produced calves that were significantly heavier than expected, suggesting a possible sire-by-embryo interaction. These results demonstrate that addition of FCS may promote blastocyst development; however, there was also a tendency for lower embryo survival. Thus charcoal treatment of FCS is recommended, because it decreases variability in embryo development between runs and results in embryo survival rates to term similar to that BSA-supplemented media.     

Effects of the histamine H1 antagonist chlorcyclizine on rat fetal palate development

BACKGROUND: The effects of histamine H1 antagonist chlorcyclizine on rat palate development were characterized following in utero exposure. METHODS: To identify the optimum dose for inducing cleft palate, pregnant rats were administered 30, 60, or 90 mg/kg chlorcyclizine on Gestation Days 11 to 14. Fetal palate gene expression was also assessed after 90 mg/kg chlorcyclizine at 8, 15 and 30 hours post‐dose on Gestation Day 14 using microarray and qRT‐PCR. RESULTS: Rats in the 60‐ and 90‐mg/kg groups exhibited adverse clinical signs and body weight loss. Rats in the 90‐mg/kg group also demonstrated increases in late resorptions and decreases in fetal weight. Effects in the low‐dose group were limited to decreases in body weight gain. Fetal assessment on Gestation Day 21 revealed that findings were limited to the 60‐ and 90‐mg/kg groups, and included cleft palate (80% of litters for both groups), high arched palate, small nose, micrognathia, high domed head, digits shortened/absent and small limb. The fetal incidence of cleft palate was higher at 90 mg/kg, thus this dose was selected to assess palate gene expression. The altered genes associated with chlorcyclizine‐induced cleft palate included Wnt5a, Bmp2, Bmp4, Fgf10, Fgfr2, Msx1, and Insig1 but the magnitude of the change was relatively small (1.5‐ to 2‐fold). CONCLUSIONS: Expression of several genes involved in palate, limb and digit development was altered in the fetal palate following in utero exposure to chlorcyclizine. The subtle perturbation and interplay of these genes may have profound effects on the dynamics of fetal palate development. Birth Defects Res (Part B) 89:474–484, 2010. © 2010 Wiley‐Liss, Inc.     

ASPIRATION OF BREAST CYSTS

In 1,364 cases of breast cyst aspiration reported in the literature, there is no note of a missed diagnosis of carcinoma.The author carried out needle aspiration in 80 patients with a definite mass in the breast as a therapeutic or diagnostic procedure.A diagnosis must be established for every definite mass in the breast and needle aspiration is a logical diagnostic procedure. If the needle encounters a solid mass, the mass must be removed for biopsy. If the needle encounters a cyst containing fluid, the fluid should be removed completely. A biopsy specimen then should be taken from the mass if (a) the fluid is bloody, (b) the mass does not entirely disappear, or (c) the mass recurs promptly. Adherence to these rules will keep the examining physician from missing a carcinoma within the cyst.Aspiration of breast cysts is a simple and safe diagnostic and therapeutic procedure that saves the patient distress and money.     

Changes in lung liquid dynamics induced by prolonged fetal hypoxemia

Our aim was to determine the effect of prolonged fetal hypoxemia, induced by reduced maternal uterine blood flow (RUBF), on fetal lung liquid secretion, flow, and volume. In chronically catheterized fetal sheep, lung liquid volume (VL) and the secretion rate of lung liquid (Vs) were measured before and after a 24-h period of either RUBF or normoxemia. Tracheal fluid flow and the incidence of fetal breathing movements (FBM) were measured before, during, and after the 24-h period. In normoxic control fetuses Vs was not significantly altered. After 24 h of RUBF, Vs was significantly (P less than 0.005) reduced compared with pre-RUBF values. During 24 h of RUBF the incidence of FBM declined initially but returned to control values after 12-16 h. In seven of eight fetuses, over the 12- to 24-h period of RUBF, large amounts of liquid (22.7-62.6 ml) were drawn into the lungs during FBM, resulting in a net movement of amniotic fluid into the lungs. During the 18- to 24-h period of RUBF, changes in the incidence of FBM were found to be significantly and positively correlated (r = 0.86, P less than 0.005) with the changes in VL that occurred over the 24-h period. Thus, prolonged RUBF can result in the inhalation of large volumes of amniotic fluid by the fetus, which could be a cause of in utero meconium aspiration.     

Generation of dwarf goat (Capra hircus) clones following nuclear transfer with transfected and nontransfected fetal fibroblasts and in vitro-matured oocytes

The developmental potential of caprine fetal fibroblast nuclei after in vitro transfection and nuclear transfer (NT) into enucleated, in vitro-matured oocytes was evaluated. Fetal fibroblasts were isolated from Day 27 to Day 30 fetuses from a dwarf breed of goat (BELE: breed early lactate early). Cells were transfected with constructs containing the enhanced green fluorescent protein (eGFP) and neomycin resistance genes and were selected with G418. Three eGFP lines and one nontransfected line were used as donor cells in NT. Donor cells were cultured in Dulbecco minimum Eagle medium plus 0.5% fetal calf serum for 4-8 days prior to use in NT. Immature oocytes were recovered by laparoscopic ovum pick-up and matured for 24 h prior to enucleation and NT. Reconstructed embryos were transferred as cleaved embryos into synchronized recipients. A total of 27 embryos derived from transgenic cells and 70 embryos derived from nontransgenic cells were transferred into 13 recipients. Five recipients (38%) were confirmed pregnant at Day 35 by ultrasound. Of these, four recipients delivered five male kids (7.1% of embryos transferred) derived from the nontransfected line. One recipient delivered a female kid derived from an eGFP line (7.7% of embryos transferred for that cell line). Presence of the eGFP transgene was confirmed by polymerase chain reaction, Southern blotting, and fluorescent in situ hybridization analyses. Nuclear transfer derivation from the donor cells was confirmed by single-strand confirmation polymorphism analysis. These results demonstrate that both in vitro-transfected and nontransfected caprine fetal fibroblasts can direct full-term development following NT.     

Development and efficacy of ultrasound-guided fetal fluid aspiration techniques for prenatal diagnosis of caprine beta-mannosidosis

Ultrasound-guided fetal fluid sampling was performed on 13 pregnant goats at Days 59 to 65 of gestation to establish safe techniques for accurate sampling and to determine the feasibility of prenatal diagnosis of beta-mannosidosis. Fluids were analyzed for electrolyte and creatinine content to assess accuracy of sampling. Values correlated well with previously reported concentrations for caprine and ovine fetal fluids at the same gestational stage. The single abortion which occurred following ultrasound-guided sampling was correlated with placentome penetration and aspiration of bloody fluids. Thin layer chromatography of amniotic and allantoic fluids was performed to detect oligosaccharides that accumulate in beta-mannosidosis. Abnormal accumulated oligosaccharides were identified in the allantoic but not amniotic fluid from a beta-mannosidase-deficient 62-d-old fetus. Thus, allantocentesis was shown to be an optimal, safe procedure for providing information at this gestational stage to diagnose caprine beta-mannosidosis.     

Conjugated and unconjugated oestrogens in fetal and maternal fluids of the cow throughout pregnancy.

The changes in concentration of oestrone, oestradiol (-17alpha and -17beta), oestrone sulphate and the oestradiol sulphates have been measured in allantoic and amniotic fluids and in maternal peripheral plasma throughout gestation. Oestrone sulphate was the major oestrone present in all of the fluids. It was measurable in allantoic fluid before Day 52 and reached a peak concentration of 475 ng/ml around Day 133. A lower peak occurred in the amniotic fluid around Day 110. The changes in oestradiol sulphates in allantoic fluid were similar to those of oestrone sulphate but at a much lower level. Considerable fluctuation was observed in the oestradiol sulphate concentrations in amniotic fluid. The ratio of oestradiol-17alpha sulphate to oestradiol-17beta sulphate was considerably higher in amniotic fluid than in allantoic fluid. Consistent changes in the levels of oestrone and the oestradiols were found in amniotic fluid but not in allantoic fluid during the second half of pregnancy. In maternal peripheral plasma oestrone sulphate was measurable before Day 72. In the limited number of samples analysed no difference in oestrogen concentration due to the sex of the fetus was evident in any of the fetal or maternal fluids.     

Arginine nutrition and fetal brown adipose tissue development in nutrient-restricted sheep

Intrauterine growth restriction is a significant problem worldwide, resulting in increased rates of neonatal morbidity and mortality, as well as increased risks for metabolic and cardiovascular disease. The present study investigated the role of maternal undernutrition and l-arginine administration on fetal growth and development. Embryo transfer was utilized to generate genetically similar singleton pregnancies. On Day 35 of gestation, ewes were assigned to receive either 50 or 100% of their nutritional requirements. Ewes received i.v. injections of either saline or l-arginine three times daily from Day 100 to Day 125. Fetal growth was assessed at necropsy on Day 125. Maternal dietary manipulation altered circulating concentrations of leptin, progesterone, and amino acids in maternal plasma. Fetal weight was reduced in nutrient-restricted ewes on Day 125 compared with 100% fed ewes. Compared with saline-treated underfed ewes, maternal l-arginine administration did not affect fetal weight but increased weight of the fetal pancreas by 32% and fetal peri-renal brown adipose tissue mass by 48%. These results indicate that l-arginine administration enhanced fetal pancreatic and brown adipose tissue development. The postnatal effects of increased pancreatic and brown adipose tissue growth warrant further study.     

Laparoscopic studies of ovulation, pregnancy diagnosis, and follicle aspiration in sheep

A technique for rapid and repeated laparoscopic ovarian examination was developed for use in the sheep. A midventral approach was used on ewes under general anesthesia on a 45-degree sloped table. Examination could be done as often as every other day.The technique was used for detection of ovulation in studies of gonadotropin stimulation in anestrous ewes. Ovulation was induced with 5 to 10 mg FSH and 100 to 2000 iu HCG. The use of laparoscopy in pregnancy diagnosis was also investigated. Accurate diagnosis could be made by Day 17 using both uterine and ovarian characteristics. Visualization through the laparoscope was utilized for aspiration of follicular oocytes using a 23 ga, 11 cm needle.     

Cortisol in fetal fluids and the fetal adrenal at parturition in the tammar wallaby (Macropus eugenii)

Glucocorticoid hormones may play a critical role in initiating parturition in tammar wallabies. In this study, we investigated the concentration of cortisol in fetal fluids and cortisol production by fetal adrenals over the last 3 days of the 26-day pregnancy and within 24 h postpartum. The fetal adrenals almost doubled in size between Days 24 and 26 of pregnancy, and their cortisol content increased over 10-fold during this period, from 10 pg to over 100 pg per adrenal pair. After birth, neonatal adrenals continued to grow, but cortisol content fell dramatically to 20 pg. The prepartum increase in adrenal cortisol was reflected by a substantial rise in cortisol concentrations in yolk sac fluid, allantoic fluid, and fetal blood, which were below 10 ng/ml on Day 24 and rose to over 40 ng/ml by Day 26. Cortisol concentrations in neonatal blood decreased postpartum, mirroring decreased cortisol content in neonatal adrenals. Cortisol production by the fetal adrenal was stimulated in vitro by ACTH and prostaglandin E2, suggesting that the in vivo increase may be stimulated by release of ACTH from the fetal hypothalamic-pituitary axis and prostaglandin E2 from the placenta. These results indicate that increasing cortisol production by the fetal adrenal is a characteristic of late pregnancy in the tammar wallaby and support the suggestion that fetal cortisol may trigger the initiation of parturition in this marsupial species.     

Influence of maternal dietary zinc intake on in vitro tubulin polymerization in fetal rat brain

The hypothesis that one of the biochemical lesions underlying zinc deficiency-induced teratogenicity is altered microtubule formation was tested. Day 19 fetuses from zinc-deficient Sprague-Dawley dams were characterized by low brain supernate zinc concentrations and slow brain tubulin polymerization rates compared to controls. Brain supernate tubulin and protein concentrations were similar in zinc-deficient and control fetuses. In vitro brain tubulin polymerization rates were increased following addition of zinc to either control or zinc-deficient brain supernates; however, the stimulatory effect of added zinc on polymerization was significantly higher in brain supernates obtained from zinc-deficient fetuses compared to controls. These results support the idea that one effect of fetal zinc deficiency is a reduction in tubulin polymerization, which in turn may result in altered microtubule function.     

An Experience With Ultrasonically Guided Liver Aspirates From South India

Fine needle aspiration (FNA) was performed on 102 hepatic lesions under ultrasound control. Eighty-eight lesions were solid and 14 were cystic. Ninety aspirates provided adequate material for cytodiagnosis. Aspirates from the solid lesions were assessed for the presence or absence of 20 cytological features. It was found that on a univariate statistical analysis, 11 of the features were significantly associated with hepatocellular carcinomas. Five of these 11 were considered diagnostic of the lesion on a step-wise multiple linear regression analysis. Two other features (not included among the 11) were found to be significantly associated with metastatic neoplasms. Cystic lesions were diagnosed as being either abscesses, hydatid cysts or congenital cysts. the physical appearance of the cyst fluid proved to be of diagnostic value in many cases.     

Plastic-embedded semi-thin sections of fine needle aspiration biopsies with dibasic staining. Diagnostic and didactic applications

The diagnostic and didactic utility of plastic-embedded semi-thin sections of fine needle aspiration biopsies is presented using a case-study approach. The Spurr epoxy semi-thin sections were stained with a newly developed sequential basic fuchsin-methylene blue stain, which gives hematoxylin-and-eosin-like staining and simultaneously substitutes for a wide variety of special stains. The informational content of the sections can approach that of electron microscopy. The use of a direct off-the-slide "pop-off" technique in preparing the plastic-embedded sections allows for a direct comparison between similar groups of cells embedded in plastic and present on the routine aspiration slides; retrospective analysis can discern subtle, previously unrecognized morphologic features in the alcohol-fixed, Papanicolaou-stained slides. The limitations of this comparative approach, however, become manifest when the effects of alcohol fixation on cells are directly compared in plastic and at the ultrastructural level to aldehyde fixation.     

Endoscopic fine needle aspiration biopsy

A catheter assembly that can be passed through the biopsy channel of a standard fiberoptic gastroduodenoscope was devised to permit fine needle aspiration biopsy of gastrointestinal neoplasms under direct vision of the endoscope. This technique for endoscopic aspiration biopsy was performed in ten consecutive patients with esophageal and gastric carcinomas, along with the conventional endoscopic brushing and biopsy. Endoscopic aspiration biopsy gave a positive diagnosis in all ten cases while the other two techniques gave inconclusive results in one patient with an ulcerative growth. We feel that endoscopic aspiration biopsy can be used to obtain representative samples from gastrointestinal neoplasms, and it may add to the diagnostic accuracy of endoscopic biopsy and brushing cytology.