Short communication: Progressive motility of frozen-thawed canine semen is highest five minutes after thawing.

Progressive motility is usually estimated by visual inspection using a light contrast microscope at X 100 immediately after semen collection or immediately after thawing frozen semen. Standard operating procedures have never been established for this test. The objective of this experiment was to examine time-dependent changes of motility after thawing cryopreserved canine semen. Semen of 35 dogs was collected, and volume, concentration, progressive motility, morphology, membrane integrity and HOS test were evaluated. For cryopreservation, CaniPRO® Freeze A&B was used. Semen was thawed and diluted using CaniPRO® culture medium. After thawing, semen was evaluated as before. In addition, every sample was evaluated for progressively motile sperm cells 0, 5, 20 and 60 min after thawing. Progressive semen motility was significantly highest five minutes after thawing.

Prognostic value of a pre-freeze hypo-osmotic swelling test on the post-thaw quality of dog semen.

Throughout cryopreservation, sperm are exposed to major osmotic challenges. Only intact membranes of sperm cells are able to regulate these volumetric changes, which can be determined by the hypo-osmotic swelling test (HOS test). Correlations between the HOS test and conventional semen variables are inconsistent. Therefore, the objectives of this study were (1) to examine relationships between HOS test results and standard semen variables before freezing and after thawing and (2) to evaluate the prognostic value of the HOS assessments on post-thaw quality of dog semen. Semen of 35 dogs was collected and analyzed before freezing and after thawing following a 7-day freeze-thaw interval. Conventional semen variables such as sperm cell motility, membrane integrity morphology were evaluated and the HOS test was conducted with results from this test being recorded. In fresh semen the HOS test was positively correlated with progressive motility of sperm cells: r=0.52, sperm cell membrane integrity: r=0.50 and normal sperm cell morphology: r=0.46 (P<0.05). In frozen-thawed semen, the data obtained with the HOS test were positively correlated with progressive sperm cell motility: r=0.67 and membrane integrity: r=0.86 (P<0.05). The data obtained with the HOS test in fresh semen were positively correlated with sperm cell membrane integrity: r=0.50 normal sperm cell morphology: r=0.55 and data from the HOS test (r=0.43; P<0.05) with frozen-thawed semen. For the prediction of individual cryopreservation capacity, results from assessment of the fresh semen variables of good and poor semen quality were statistically compared. Based on these results, it is not possible to predict the quality of frozen-thawed dog semen using the HOS test.

A systematic review of studies performing the hypo-osmotic swelling test to evaluate the quality of canine spermatozoa.

The hypo-osmotic swelling test (HOS test) is a simple and inexpensive test to evaluate the functional integrity of sperm cell membranes. According to the existing literature, its simple applicability has turned it into a valuable additional parameter to standard canine semen analysis. In the recent years, much research has been conducted in this field. The aim of this systematic review was to evaluate the quality of published literature in canine reproduction concerning the HOS test. Using two distinguished databases, 38 articles were detected and analysed subsequently according to various aspects, for example study design, population, semen sampling and implementation concerning the HOS test. Although there are numerous articles available, the diagnostic value of the HOS test remains ambiguous. Until now, neither a recognized test protocol nor reliable reference values have been defined. Most of the trials evaluated show serious methodological flaws and therefore do not permit drawing reliable conclusions. According to our results, approximately half of the studies (n = 20) included a sample size of five or less animals. None of the studies examined the inter- or intraobserver agreement for the HOS test. Further research is warranted including appropriate statistical methods and a sufficient number of animals to establish a standardized test protocol as well as reliable reference values. Most importantly, it is required to clarify a correlation between the HOS test and the fertilizing capacity to determine the diagnostic value of the HOS test.

ADM3, TFF3 and LGALS3 are discriminative molecular markers in fine-needle aspiration biopsies of benign and malignant thyroid tumours.

BACKGROUND: Previously, we reported a six-marker gene set, which allowed a molecular discrimination of benign and malignant thyroid tumours. Now, we evaluated these markers in fine-needle aspiration biopsies (FNAB) in a prospective, independent series of thyroid tumours with proven histological outcome. METHODS: Quantitative RT-PCR was performed (ADM3, HGD1, LGALS3, PLAB, TFF3, TG) in the needle wash-out of 156 FNAB of follicular adenoma (FA), adenomatous nodules, follicular and papillary thyroid cancers (TC) and normal thyroid tissues (NT). RESULTS: Significant expression differences were found for TFF3, HGD1, ADM3 and LGALS3 in FNAB of TC compared with benign thyroid nodules and NT. Using two-marker gene sets, a specific FNAB distinction of benign and malignant tumours was achieved with negative predictive values (NPV) up to 0.78 and positive predictive values (PPV) up to 0.84. Two FNAB marker gene combinations (ADM3/TFF3; ADM3/ACTB) allowed the distinction of FA and malignant follicular neoplasia with NPV up to 0.94 and PPV up to 0.86. CONCLUSION: We demonstrate that molecular FNAB diagnosis of benign and malignant thyroid tumours including follicular neoplasia is possible with recently identified marker gene combinations. We propose multi-centre FNAB studies on these markers to bring this promising diagnostic tool closer to clinical practice.

Distinct regulation of intrinsic apoptosis in benign and malignant thyroid tumours.

Aberrations in the control of apoptosis represent a central feature of thyroid carcinogenesis. However, little is known about the regulation of components of the intrinsic apoptosis pathway in the thyroid. Using a real-time PCR approach we investigated the mRNA expression levels of Caspase3, Caspase3 s, xIAP, Bad, and beta-actin in a panel of 79 thyroid tumours. Additionally, we assessed the activation status of Caspase3 by immunohistochemistry. In the present study, we provide first evidence for a deregulation of the intrinsic apoptosis pathway on the transcriptional and post-transcriptional level. Thus, malignant thyroid tumours revealed a significant downregulation of the proapoptotic Bad. In contrast Caspase3 s, an alternative splice variant of Caspase3 with anti-apoptotic characteristics, was upregulated in follicular and anaplastic cancers. Moreover, papillary thyroid tumours revealed a significant upregulation of Caspase3 mRNA. On the post-translational level, thyroid malignancies featured an impairment in the activation of Caspase3, since activated Caspase3 accumulated exclusively in the cytoplasm of thyroid cancer cells, whereas follicular adenoma and normal thyroid tissues showed no cytoplasmatic but nuclear Caspase3 distribution. Further knowledge on apoptosis-deregulation during thyroid carcinogenesis might confer diagnostic and therapeutic benefits in the management of thyroid cancer.

Impact of pregnancy on prevalence of goitre and nodular thyroid disease in women living in a region of borderline sufficient iodine supply.

An interplay of genetic, epigenetic, and environmental factors contributes to thyroid disease. In a cross-sectional study, we aimed to determine the influence of parity in combination with other risk factors on the prevalence of goitre and nodular thyroid disease (NTD) in women living in a region of previous overt iodine deficiency, which experienced a continuous improvement in alimentary iodine supply in the last two decades. Thyroid ultrasonography (7.5 MHz; Merck Thyromobil) was performed by the same investigator in 736 women living in Thuringia and Saxony. Age and BMI were documented and a comprehensive history on pregnancies, family history of thyroid disease, and past or present smoking was obtained. Goitre prevalence was 19.1%. Solitary thyroid nodules were detected in 21.5%, and multiple nodules in 23.8% of women. In a multivariate analysis, neither age nor parity was positively correlated with goitre prevalence and NTD. A significant correlation was detected between BMI and goitre and multinodular disease. Goitre was found in 25.3% of women with a positive family history for thyroid disease, as opposed to 16.1% goitre in women with a negative family history. Neither goitre nor NTD were associated with a history of smoking in the whole study population. Thyroid nodules and/or goitre are present in up to 45% of women in an area of previous overt iodine deficiency. Whereas BMI and family history are positively correlated with the presence of NTD and goitre, no such correlation could be detected for pregnancy and smoking after processing our data with multivariate analyses.

[Thyroid storm--thyrotoxic crisis: an update]. / Thyreotoxische Krise--ein Update.

Thyroid storm or thyrotoxic crisis is a rare but life-threatening condition requiring immediate treatment, preferably in an intensive care unit. Its incidence is about 1-2% among patients with overt hyperthyroidism. A thyrotoxic crisis occurs predominantly in the elderly and is three to five times more common in women than in men. The overall mortality is 10-20%. Even though the pathogenesis is still not fully understood, an increased sensitivity to catecholamines appears to be an important mechanism, and a number of endogenous and exogenous stress factors that can provoke the onset of a thyrotoxic storm have been identified. The diagnosis of a thyrotoxic crisis is made entirely on the clinical findings. Most importantly, there is no difference in thyroid hormone levels between patients with "uncomplicated" thyrotoxicosis and those undergoing a thyroid storm. Any delay in therapy, e.g. by awaiting additional laboratory results, must be strictly avoided, because the mortality rate may rise to 75%. Thus early thyroidectomy should be considered as the treatment of choice, if medical treatment fails to result in clinical improvement. Medical treatment is based on three principles: 1) counteracting the peripheral effects of thyroid hormones; 2) inhibition of thyroid hormone synthesis; and 3) treatment of systemic complications. These measures should bring about clinical improvement within 12-24 hours. If death occurs it is most likely to be cardiopulmonary failure, particularly in the elderly.

Cytology and mRNA expression analysis of fine needle aspirates of thyroid nodules in an East German region with borderline iodine deficiency.

Fine needle aspiration cytology (FNAC) is widely recommended as an important tool for pre-operative identification of malignancy in patients with nodular thyroid disease. To assess the diagnostic contribution of FNAC and the potential of quantitative mRNA analysis in fine needle aspirates in daily practice, we conducted a prospective study in thyroid clinics (n=2) and endocrine practices (n=3), respectively in an East German region with borderline iodine deficiency. Two-hundred and forty-four consecutive FNACs were obtained over a period of 2 years (2002-2004) from euthyroid patients presenting for first evaluation of a solitary thyroid nodule. The mean nodule size for FNAC was 27 mm (range: 10-79 mm). In 55% of patients FNAC was performed after scintiscan detection of a cold or normal functioning thyroid nodule (CTN), while in the remainder FNAC was performed as a primary investigation. FNAC outcomes were: 57.8% benign, 22.1% indeterminate, 2.5% suspicious for malignancy, 17.6% non-diagnostic. Messenger RNA levels for a house keeping gene (beta-actin) and a thyroid specific marker (thyroglobulin, Tg) were studied as basic molecular markers using real-time PCR. Both in the IN VIVO and EX VIVO FNA series, beta-actin and Tg mRNA levels were positively correlated with the thyrocyte cell yield/respective FNA smear. However, subgroup analysis showed that FNAC with histologically confirmed follicular thyroid cancer and/or microfollicular adenoma exhibited significantly lower Tg mRNA expression despite high beta-actin levels. Sufficient mRNA quantities were obtained in >90% of FNA specimen to allow quantitative mRNA analysis of at least 5 further genes. In conclusion, quantitative mRNA analysis is feasible in FNA on a routine basis and provides a perspective for a molecular distinction of thyroid nodules, once specific marker genes have been defined for benign and malignant thyroid tumours respectively.

Deoxyribonucleic acid damage and spontaneous mutagenesis in the thyroid gland of rats and mice.

Thyroid tumors are a frequent finding not only in iodine-deficient regions. They are predominantly characterized by somatic genetic changes (e.g. point mutations or rearrangements). Because slow thyroid proliferation is a apparent contradiction to a high frequency of tumor initiation, we characterized mutational events in thyroid. First we studied the frequency of certain base exchanges in somatic TSH receptor (TSHR) mutations and determined the spontaneous mutation rate in thyroid and liver. Then we applied different protocols of the comet assay to quantify genomic DNA damage and conducted immunohistochemistry for 8-oxoguanine as a molecular marker for oxidative stress. Among 184 somatic mutations of the human TSHR found in thyroid tumors, C-->T transitions had a unexpectedly high frequency (>32%). The mutation rate in thyroid is 8-10 times higher than in other organs. The comet assay detected increased levels of oxidized pyrimidine (2- to 3-fold) and purine (2- to 4-fold) in thyroid, compared with liver and lung, and a 1.6-fold increase of oxidized purine, compared with spleen. Immunohistochemistry revealed high levels of 8-oxoguanine in thyroid epithelial cells. We have shown a strikingly high mutation rate in the thyroid. Furthermore, results of the comet assay as well as immunohistochemistry suggest that oxidative DNA modifications are a likely cause of the higher mutation rate. It is possible that free radicals resulting from reactive oxygen species in the thyroid generate mutations more frequently. This is also supported by the spectrum of somatic mutations in the TSHR because more frequent base changes could stem from oxidized base adducts that we detected in the comet assay and with immunohistochemistry.

Evaluation of peroxisome proliferator-activated receptor-gamma expression in benign and malignant thyroid pathologies.

Impairment of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) function through a dominant negative PAX-8/PPAR-gamma fusion gene or other events resulting in wild-type PPAR-gamma downregulation has been implicated in malignant thyroid cell transformation. The aim of our study was to perform a systematic evaluation of PPAR-gamma mRNA and protein expression in normal thyroid tissue as opposed to benign thyroid pathologies of different functional status and thyroid malignancy, to gain further insights into a putative physiological role of PPAR-gamma in the thyroid and to define whether PPAR-gamma could serve as a marker of thyroid cell differentiation. Ten cold benign (CTN) and 10 toxic (TTN) thyroid nodules and corresponding normal thyroid tissues, 10 follicular thyroid cancers (FTC), 10 papillary thyroid cancers (PTC) and 8 Graves' disease (GD) thyroids were studied by real-time polymerase chain reaction (PCR), immunohistochemistry and reverse transcriptase (RT)-PCR (PAX-8/PPAR-gamma fusion gene). PPAR-gamma mRNA expression was demonstrated in all samples. When comparing benign nodular and normal thyroid tissue of the same patient no significant difference in PPAR-gamma mRNA expression was observed. PPAR-gamma mRNA levels were similar in CTN and FTC. In contrast, PPAR-gamma mRNA expression was downregulated in 9 of 10 PTC and all GD samples, whereby at least 4 fold downregulation (compared with normal and benign nodular thyroid tissues) was observed in the latter. Immunohistochemistry showed an increased, patchy PPAR-gamma nuclear staining in CTNs and TTNs and only faint staining in the corresponding normal thyroid tissues. A diffuse and weak PPAR-gamma staining pattern was observed in all GD samples. No PAX-8/PPAR-gamma rearrangements were detected in any of the 68 thyroid tissue samples. In conclusion PPAR-gamma mRNA and protein expression levels are not concordant in benign thyroid nodular disease. Furthermore there is no clear-cut association of PPAR-gamma mRNA expression with follicular thyroid tumorigenesis. Absence of a PAX-8/PPAR-gamma fusion gene in the series of 68 thyroid samples is in agreement with the suggestion of PAX-8/PPAR-gamma rearrangement being restricted to a subset of follicular thyroid cancers. The marked downregulation of PPAR-gamma in GD warrants further investigation and could be linked, for example, with changes in apoptosis.

Evaluation of insulin-like growth factor II, cyclooxygenase-2, ets-1 and thyroid-specific thyroglobulin mRNA expression in benign and malignant thyroid tumours.

OBJECTIVE: We evaluated three markers (insulin-like growth factor II (IGF-II), cyclooxygenase-2 (COX-2) and ets-1) of thyroid growth stimulation and cell transformation together with a thyroid-specific marker (thyroglobulin (Tg)) for their potential to differentiate benign and malignant follicular thyroid neoplasia (FN). DESIGN AND METHODS: mRNA expression levels were determined by real-time PCR in 100 snap-frozen thyroid samples: 36 benign thyroid nodules with different histology and function (19 cold (CTN) and 17 toxic thyroid nodules (TTN)), 36 corresponding normal thyroid tissues of the same patients, eight Graves' disease (GD) thyroids, 10 follicular thyroid carcinomas (FTC) and 10 papillary thyroid carcinomas (PTC). RESULTS: Mean IGF-II and COX-2 levels were not significantly altered between benign and malignant thyroid nodules (IGF-II) or nodular (FTC, TTN, CTN) and normal thyroid tissues (COX-2). In contrast, eight- to tenfold upregulation of ets-1 was observed in PTC and three- to fourfold upregulation of ets-1 was observed in FTC (and GD) compared with benign thyroid nodules and normal thyroid tissues. In addition, thyroglobulin mRNA expression was markedly downregulated (50- to 100-fold) in FTC, PTC and GD samples compared with benign nodular and normal thyroid tissues. Hence an ets-1/Tg ratio >20 distinguished differentiated thyroid cancer from benign nodular or normal thyroid tissue. We then studied ets1- and Tg mRNA expression levels in fine needle aspiration cytology (FNAC) samples. However, in a consecutive series of 40 FNAC samples only equivocal results were obtained on 38 benign and two malignant (FTC) thyroid tumour samples. CONCLUSIONS: Upregulation of ets-1 and downregulation of Tg mRNA expression occur in differentiated thyroid cancer and may facilitate pre-operative identification of thyroid malignancy depending on further evaluation of these potentially promising markers in a larger series of benign and malignant thyroid tumours and their FNAC samples.